NZ719771B2 - Methods and compositions for sustained immunotherapy - Google Patents
Methods and compositions for sustained immunotherapy Download PDFInfo
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- NZ719771B2 NZ719771B2 NZ719771A NZ71977114A NZ719771B2 NZ 719771 B2 NZ719771 B2 NZ 719771B2 NZ 719771 A NZ719771 A NZ 719771A NZ 71977114 A NZ71977114 A NZ 71977114A NZ 719771 B2 NZ719771 B2 NZ 719771B2
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Abstract
This disclosure provides compositions and methods for promoting the formation, expansion and recruitment of TR1 cells and /or Breg cells in an antigen-specific manner and treating autoimmune diseases and disorders in a subject in need thereof. The compositions comprise antigen-MHC class II-nanoparticle complexes. In an embodiment the antigen-MHC class II complex is linked to the nanoparticle via a PEG linker. PEG functionalized iron oxide nanoparticles can be prepared by thermally decomposing iron acetyl acetonate in the presence of functionalized PEG molecules, with or without benzyl ether. This simplifies the production of these nanoparticles. cle complexes. In an embodiment the antigen-MHC class II complex is linked to the nanoparticle via a PEG linker. PEG functionalized iron oxide nanoparticles can be prepared by thermally decomposing iron acetyl acetonate in the presence of functionalized PEG molecules, with or without benzyl ether. This simplifies the production of these nanoparticles.
Description
METHODS AND COMPOSITIONS FOR SUSTAINED IMMUNOTHERAPY
CROSS-REFERENCE TO RELATED APPLICATION
This application claims priority under 35 U.S.C. § 119(e) to US. Provisional Patent
Application No. 61/899,826, filed November 4, 2013, the entire content of which is hereby
incorporated by reference into the present disclosure.
FIELD OF DISCLOSURE
This sure is directed to compositions and methods related to immunotherapy
and medicine.
BACKGROUND
Throughout and within this disclosure are technical and patent publications,
referenced by an identifying citation or by an Arabic number. The full bibliographic citation
corresponding to the Arabic number is found in the specification, preceding the claims. The
disclosures of all references cited herein are incorporated by reference into the present
application to more fully describe the state of the art to which this invention ns.
Autoimmune diseases are caused by an attack of self-tissues by the immune .
An ideal therapy would be one capable of selectively blunting the autoimmune se
(against all antigenic epitopes targeted in that disease) without impairing systemic immunity
(immune responses to foreign antigens). Unfortunately, the lymphocyte specif1cities ed
in any one autoimmune disease are many and incompletely defined, making this a
challenging goal.
SUMMARY
In se to this need in the art, described herein are therapeutic compositions
useful in ng mune disorders. One aspect relates to a method for expanding and/or
developing populations of anti-pathogenic autoreactive T cells and/or B-cells in a subject in
need thereof, which method comprises, or consists essentially of, or yet further consists of,
administering to that subject an antigen-MHC class II-nanoparticle (“NP”) complex (“NP-
x”), wherein the antigen is an autoimmunity related antigen or tigen. In some
aspects all the antigens on the particular NP are identical or they can be different. In another
aspect, the antigens on the NP have different amino acid sequences but are isolated from the
same antigenic protein. In a fiarther aspect, the antigens on the NP are from different
antigens. In r aspect, the MHCII are the same or different.
In one aspect, this disclosure provides a NP-complex comprising, or alternatively
ting essentially of, or yet further consisting of, a nanoparticle; a MHC class II protein
and a e-relevant antigen that can be in the form of an antigen/MHCII complex, for use
in expanding and/or developing one or more populations of B-regulatory cells and TRl cells
(e.g., TRl and CD4+ cells), in a t, wherein the nanoparticle has a diameter selected
from the group of: from about 1 nm to about 100 nm in diameter; from about 1 nm to about
50 nm in diameter or from about 1 nm to about 20 nm or from about 5 nm to about 20 nm in
diameter and the ratio of the number of antigen-MHCII complexes to nanoparticles is from
about 10:1 to about 1000: 1. In one aspect, the complex has a MHC class II density from
about 0.05 pMHCII/lOO nm2 NP surface area (including coating) to about 25 pMHCII/lOO
nm2 NP surface area (including coating). The antigen is an autoantigen involved in an
autoimmune response or mimic thereof such as, for example, pre-diabetes, diabetes, multiple
sis (“MS”) or a multiple sclerosis-related disorder, and optionally wherein when the
disease is pre-diabetes or diabetes, the autoantigen is an epitope from an n expressed by
atic beta cells or the autoantigen IGRP, Insulin, GAD or IA-2 protein. In another
aspect, the MHC class II component comprises all or part of a HLA-DR, , or HLA-
DP. The antigen-MHC class II complex is covalently or valently linked to the
nanoparticle. The nanoparticle can be orbable and/or biodegradable.
In a further aspect, the nanoparticle is non-liposomal and/or has a solid core,
preferably a gold or iron oxide core. When covalently linked, the antigen-MHC class II
complex is covalently linked to the nanoparticle through a linker less than 5 kD in size. In
one aspect, the linker comprises polyethylene glycol (PEG). The pMHC can be linked to the
nanoparticle or the nanoparticle coating by any structure, including but not limited to linkers
or by cross-linking. In one aspect, the MHC is linked to the nanoparticle or the coating
directionally through the C-terminus.
ant has discovered that the density of the n-MHC class II complexes
on the nanoparticle contributes to the therapeutic benefit. Thus as disclosed herein, the
antigen-MHCII nanoparticle complex can have a defined y in the range of from about
0.05 MHC molecules per 100 nm2 of surface area of the nanoparticle (the e area
measured to e any coating), assuming at least 2 MHCII, or alternatively at least 8, or
alternatively at least 9, or atively at least 10, or alternatively at least 11, or alternatively
at least 12, MHCII complexed to the nanoparticle. In one aspect the complex has a density of
MHCII from about 0.01 MHCII per 100 nm2 (0.05 100 nmz) to about 30 MHCII/100
nmz, or alternatively from 0.1 100 nm2 to about 25 MHCII/100 nmz, or atively
from about 0.3 MHCII/100 nm2 to about 25 MHCII/100 nmz, or alternatively from about 0.4
MHCII/100 nm2 to about 25 MHCII/100 nmz, or alternatively from about 0.5 MHCII/100
nm2 to about 20 MHCII/100 nmz, or alternatively from 0.6 MHCII/100 nm2 to about 20
MHCII/100 nmz, or atively from about 1.0 MHCII/100 nm2 to about 20 MHCII/100
nmz, or alternatively from about 5.0 MHCII/100 nm2 to about 20 MHCII/100 nm2, or
alternatively from about 10.0 MHCII/100 nm2 to about 20 MHCII/100 nmz, or alternatively
from about 15 MHCII/100 nm2 to about 20 MHCII/100 nmz, or alternatively at least about
0.5, or alternatively at least about 1.0, or alternatively at least about 5.0, or alternatively at
least about 10.0, or alternatively at least about 15.0 MHCII/100 nm2, the nm2 surface area of
the nanoparticle to include any coating. In one aspect, when 9 or at least 9 MHCII are
complexed to a nanoparticle, the density range is from about 0.3 MHCII/100 nm2 to about 20
MHCII/100 nmz.
This disclosure also provides a composition comprising a therapeutically effective
amount of the NP-complex as described herein and a carrier, e.g., a pharmaceutically
able carrier. In one aspect, all plexes in the composition are identical. In
another aspect, the NP-complexes of the composition include diverse or different MHC-
antigen complexes.
Methods to make the complexes and compositions are further provided herein. The
method can comprise, or alternatively consist essentially of, or yet fiarther consist of, non-
covalently coating or covalently complexing antigen-MHC complexes (e. g., MHCII
complexes) onto a nanoparticle.
l and stic methods are also provided. In one aspect, a method is
provided for promoting the formation, expansion and recruitment of B-regulatory cells and/or
TRl cells (e. g., TRl and CD4+ cells) in an antigen-specific manner in a subject in need
thereof, comprising, or alternatively ting essentially of, or yet r consisting of,
stering to the subject an effective amount of the NP-complex or composition as
described herein.
In r aspect, a method for treating or preventing an autoimmune disease or
disorder as described herein, e. g., MS, a MS-related er, diabetes or pre-diabetes, in a
subject in need thereof is provided, the method comprising, or alternatively consisting
essentially of, or yet further consisting of, stering to the subject an ive amount of
the NP-complex or composition as bed herein, n the autoantigen is disease-
relevant for the disease to be treated, e.g., for the prevention or treatment of diabetes, the
antigen is a diabetes-relevant antigen. In a further aspect, the autoimmune disease is MS or a
MS-related disorder and the antigen is evant.
Kits are also ed. The kits comprise, or alternatively t essentially of, or
yet fiarther consist of a NP-complex as bed herein or a composition and instructions for
use.
In one aspect, provided herein is a method of making nanoparticles comprising
thermally decomposing or heating a nanoparticle precursor. In one embodiment, the
nanoparticle is a metal or a metal oxide nanoparticle. In one embodiment, the nanoparticle is
an iron oxide nanoparticle. In one embodiment, the nanoparticle is a gold nanoparticle. In
one embodiment, provided herein are the nanoparticles prepared in accordance with the
t technology. In one embodiment, provided herein is a method of making iron oxide
nanoparticles comprising a l decomposition reaction of iron acetyl acetonate. In one
embodiment, the iron oxide nanoparticle obtained is water-soluble. In one aspect, iron oxide
nanoparticle is suitable for protein conjugation. In one embodiment, the method comprises a
single-step thermal osition reaction.
In one aspect, the thermal decomposition occurs in the presence of fianctionalized
PEG molecules. Certain non-limiting examples of fianctionalized PEG linkers are shown in
Table 1.
In one , the thermal decomposition ses heating iron acetyl acetonate.
In one embodiment, the thermal decomposition comprises heating iron acetyl acetonate in the
presence of fianctionalized PEG molecules. In one embodiment, the thermal decomposition
comprises heating iron acetyl acetonate in the presence of benzyl ether and functionalized
PEG molecules.
Without being bound by theory, in one embodiment, functionalized PEG molecules
are used as reducing reagents and as surfactants. The method of making nanoparticles
provided herein simplifies and improves conventional methods, which use surfactants that are
difficult to be displaced, or are not displaced to tion, by PEG molecules to render the
particles water-soluble. tionally, surfactants can be expensive (e.g., phospholipids) or
toxic (e.g., Oleic acid or oleilamine). In another aspect, without being bound by theory, the
method ofmaking rticles obviates the need to use conventional tants, thereby
achieving a high degree of molecular purity and water solubility.
In one embodiment, the thermal decomposition involves iron acetyl acetonate and
benzyl ether and in the absence of tional surfactants other than those employed herein.
In one embodiment, the ature for the thermal decomposition is about 80 to
about 300°C, or about 80 to about 200°C, or about 80 to about 150°C, or about 100 to about
250°C, or about 100 to about 200°C, or about 150 to about 250°C, or about 150 to about
250°C. In one embodiment, the thermal decomposition occurs at about 1 to about 2 hours of
time .
In one embodiment, the method of making the iron oxide nanoparticles comprises a
purification step, such as by using Miltenyi Biotec LS magnet column.
In one embodiment, the nanoparticles are stable at about 4°C in phosphate buffered
saline (PBS) without any detectable degradation or aggregation. In one embodiment, the
nanoparticles are stable for at least 6 months.
In one aspect, provided herein is a method of making nanoparticle complexes
comprising contacting pMHC with iron oxide rticles ed herein. Without being
bound by theory, pMHC encodes a Cysteine at its carboxyterminal end, which can react with
the maleimide group in filnctionalized PEG at about about pH 6.2 to about pH 6.5 for about
12 to about 14 hours.
In one , the method of making nanoparticle xes comprises a
purification step, such as by using Miltenyi Biotec LS magnet column.
DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to
further demonstrate certain aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in ation with the detailed
description of specific embodiments presented herein.
FIGS. lA-lC show schematics ofNP-complexes. is a schematic of a
single-chain pMHC-class I expression construct (top) and a representative flow cytometric
profile of the binding of the corresponding pMHC tetramer (fluorochrome-labeled) to
cognate CD8+ T-cells. is a schematic showing the linkers and two dimensional
structure ofNP-complexes. As can be seen, one NP can contain the same antigen complexed
to the nanoparticle core through various chemical linkers. shows maleimide-
functionalized NPs conjugated to NPs.
shows the structure of a typical pMHC class II monomer (top) and a
representative FACS profile of cognate CD4+ T-cells d with the ponding pMHC
tetramer or left unstained.
FIGS. 3A-3B show different TlD-relevant pMHC class II-NPs reverse
hyperglycemia in newly diabetic NOD mice. shows dual mouse blood
glucose curves. Mice were considered ‘cured’ when stably normoglycemic for 4 wk, after
which treatment was withdrawn. HEL14_22, a foreign antigen, was used as l.
shows incidence of disease reversal.
shows intraperitoneal e-tolerance tests (IPTGTTs) and insulin-
production ty in long-term cured mice. IDDM, diabetic untreated mice; Cured, mice
with normoglycemia at 50 wk of age (>30 wk after treatment withdrawal); Control, age-
matched non-diabetic untreated mice (50 wk-old).
shows that TlD-relevant pMHC class II-NPs expand cognate autoreactive
CD4+ T-cells. Data correspond to mice treated with 2.5mi/I-Ag7-NPs. Bottom right,
expansion is specific for the pMHC on the NPs, as mice treated with 2.5mi/I-Ag7-NPs did
not show increased percentages of two other autoreactive CD4+ T-cell specif1cities. PLN,
pancreatic lymph nodes; MN, eric lymph nodes; BM, bone marrow (a reservoir of
memory T-cells).
shows that TlD-relevant pMHC class II-NPs expand cognate autoreactive
CD4+ T-cells. Expansion is shown for spleen but similar patterns are seen in the pancreative
lymph nodes, blood and marrow. “Onset” correspond to pre-treatment values; “Cured” are
mice ed normoglycemic with pMHC-NP (analyzed at >3Owk of treatment withdrawal);
“IDDM” are mice that relapsed upon ent awal (~25%); “50 wk-old” corresponds
to age-matched untreated abetic controls.
shows that TlD-relevant pMHC class II-NPs expand cognate memory-like
latory-l (“Trl or TRl”) cells.
shows that the autoreactive CD4+ T-cells expanded by pMHC class II-NP
are IL-lO producers. IGRP126_145/I-Ag7 tetramer+ cells from mice treated with IGRP126_145/I-
Ag7-NPs or control NPs were , challenged with cognate and non-cognate peptides and
the sups assayed for cytokine content with luminex technology.
shows that pMHC class II-NPs reverse hyperglycemia in an IL-10 and
TGFb-dependent manner. shows ability of IGRP4_22/IAg7-NPs to restore
lycemia (top), expand cognate Trl cells (bottom left) and ss autoantigen
presentation in the PLNs (to IGRP206_214-reactive CD8+ T-cells; bottom right) of mice treated
with cytokine blocking dies ). Anti-ILlO and anti-TGFB Abs partially restore
autoantigen tation and inhibit the therapeutic effect of pMHC-NPs, without impairing
Trl cell expansion.
FIGS. 10A-10B show that pMHC class II-NP therapy does not compromise
systemic immunity. A shows that pMHC-NP-treated NOD mice can y clear an
acute viral (vaccinia virus) infection (bottom, compare day 4 versus day 14 after infection)
despite systemic expansion of autoregulatory Trl CD4+ T-cells (top). B shows that
pMHC-NP-treated mice (10 doses) can mount antibody ses against KLH-DNP upon
immunization in CFA, as ed to untreated and unvaccinated mice.
shows that pMHC class II-NP therapy reduces the severity of established
EAE in C57BL/6 mice. B6 mice were immunized with pMOG35-55 in CFA and d with
pertussis toxin i.v. Mice were scored for signs of EAE using ished criteria over a 15-
point scale. Affected mice were treated with two weekly doses of 7.5-22.5 ug of pMOGgg-
49/IAb-coated NPs, beginning 21 days after zation.
FIGS. 12A-12C show structure and properties ofpMHC class II-NPs. A is
a cartoon depicting the different chemistries that can be used to covalently coat pMHCs onto
functionalized, biocompatible iron oxide NPs. FIG. IZB is a transmission electron
micrograph ofpMHC-coated NPs. C shows Dynamic Light Scattering profiles of
pMHC-coated vs. uncoated NPs.
FIGS. 13A-13C show expansion and differentiation of cognate B-cells into Breg
cells in pMHC class II-NP-treated mice. In A, l:l mixtures of PKH26-labeled/pulsed
with 2.5mi peptide B-cells (bottom) (or dendritic cells, top) plus CFSE-labeled/GPI peptide-
pulsed B-cells (bottom) (or tic cells, top) were injected into 2.5mi/IAg7-NP-treated
NOD mice. Seven days later, the hosts were analyzed for presence of both subsets of B-cells
(bottom) or dendritic cells (top). Left panels show representative results and Right histograms
show a summary of the results obtained over several experiments. The data indicate that
2.5mi-peptide-pulsed B-cells (but not DCs) expand in 2.5mi/IAg7-NP-treated NOD mice. In
B (left panel), Applicant compared the B-cell content in the pancreatic (PLN) and mesenteric
(MLN) lymph nodes ofNOD mice treated with 2.5mi/IAg7-NPs versus NPs coated with
l (diabetes-irrelevant) pMHC-NPs. Data show increased recruitment of B-cells in the
. In B (right panel), Applicant compared the recruitment of B-cells to the PLNs as a
function of Trl cell recruitment. Data were obtained using l different pMHC-NP
ations. Data show a statistically-significant correlation between recruitment ofpMHC-
NP-expanded TRl cells and B-cell recruitment to the PLNs. In B, Applicant
ered B-cells, pulsed with 2.5mi or control peptides, from ILlO-eGFP knock-in NOD
mice into several different donor mouse types (top labels). After 7 days, spleens were
analyzed for conversion of the transfused B-cells into ILlO-producing (eGFP+) B-cells
expressing high levels of CDld and CD5 (B-regulatory cells). Data show robust expansion
and conversion of cognate (2.5mi-loaded) B-cells into B-reg cells only in 2.5mi/IAg7-NP-
treated hosts.
shows synthesis of surface onalized iron oxide nanoparticle by
thermal decomposition of iron acetylacetonate and bioconjugation.
DETAILED DESCRIPTION
It is to be understood that this invention is not limited to particular embodiments
bed, as such may, of course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments only, and is not intended to be
limiting, since the scope of the present invention will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the ar forms
“ 3, (C
, an”, and “the” include plural referents unless the t y dictates otherwise.
Thus, for example, reference to “an excipient” es a plurality of excipients.
I. DEFINITIONS
Unless defined ise, 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. As used herein the ing terms have the following meanings.
As used herein, the term “comprising” or “comprises” is intended to mean that the
compositions and methods include the recited ts, but not ing others.
“Consisting essentially of” when used to define compositions and methods, shall mean
excluding other elements of any essential significance to the combination for the stated
purpose. Thus, a ition consisting essentially of the elements as defined herein would
not exclude other materials or steps that do not materially affect the basic and novel
characteristic(s) of the claimed invention, such as itions for treating or preventing
multiple sclerosis. sting of” shall mean excluding more than trace elements of other
ingredients and ntial method steps. Embodiments defined by each of these transition
terms are within the scope of this invention.
An “auto-reactive T cell” is a T cell that recognizes an “auto-antigen”, which is a
molecule produced and contained by the same individual that contains the T cell.
A genic T cell” is a T cell that is harmful to a subject containing the T cell.
Whereas, a non-pathogenic T cell is not substantially harmful to a subject, and an anti-
pathogenic T cells reduces, ameliorates, inhibits, or negates the harm of a pathogenic T cell.
As used herein the terms regulatory B-cells or B-regulatory cells gs”) intend
those cells that are responsible for the anti-inflammatory effect, that is characterized by the
expression of CDld, CD5 and the secretion of IL-10. B-regs are also identified by
expression of Tim-l and can be induced through Tim-l on to promote tolerance. The
ability of being B-regs was shown to be driven by many stimulatory factors such as toll-like
receptors, CD40-ligand and others. However, filll terization of B-regs is ongoing. B-
regs also express high levels of CD25, CD86, and TGF-B. This subset of B cells is able to
suppress Thl proliferation, thus contributing to the maintenance of self-tolerance. The
potentiation of B-reg function should become the aim ofmany immunomodulatory drugs,
contributing to a better control of autoimmune diseases. See for example:
ncbi.nlm.nih.gov/pubmed/23707422, last accessed on October 31, 2013.
2014/003014
T Regulatory 1 cells (Trl) are a subset of CD4+ T cells that have tory
properties and are able to suppress antigen-specific immune responses in vitro and in vivo.
These T-regulatory 1 (Trl) cells are defined by their unique profile of cytokine production
and make high levels of IL-10 and TGF-beta, but no IL-4 or IL-2. The IL-10 and TGF-beta
produced by these cells mediate the inhibition of primary naive T cells in vitro. There is also
eVidence that Tr1 cells exist in vivo, and the presence of high IL-lO-producing CD4(+) T
cells in ts with severe combined deficiency who have received allogeneic stem-
cell transplants have been documented. Tr1 cells are involved in the regulation of peripheral
tolerance and they could potentially be used as a cellular therapy to modulate immune
responses in vivo. See for example: ncbi.nlm.nih.gov/pubmed/10887343 last accessed on
October 31, 2013,
Type-1 T regulatory (Trl) cells are defined by their ability to produce high levels of
IL-10 and TGF-beta. Tr1 cells specific for a variety of antigens arise in vivo, but may also
differentiate from naive CD4+ T cells in the presence of IL-10 in vitro. Tr1 cells have a low
proliferative capacity, which can be overcome by IL-15. Tr1 cells suppress naive and
memory T helper type 1 or 2 responses via production of IL-10 and TGF-beta. Further
characterization of Tr1 cells at the molecular level will define their mechanisms of action and
clarify their relationship with other subsets of Tr cells. The use of Tr1 cells to identify novel
s for the development ofnew therapeutic agents, and as a cellular y to modulate
peripheral tolerance, can be foreseen. See for example, ncbi.nlm.nih.gov/pubmed/l1722624,
last accessed on October 31, 2013.
The terms “inhibiting,” “reducing,” or “prevention,” or any variation of these terms,
when used in the claims and/or the specification includes any able se or
complete inhibition to achieve a desired result.
Throughout this application, the term “about” is used to indicate that a value
es the standard deviation of error for the device or method being employed to
determine the value. The term “about” when used before a cal designation, e.g.,
temperature, time, amount, and concentration, including range, indicates approximations
whichmayvaryby(+)or(—)10%,5%,or1%.
By "biocompatible", it is meant that the components of the delivery system will not
cause tissue injury or injury to the human biological system. To impart patibility,
polymers and excipients that have had history of safe use in humans or with GRAS
2014/003014
(Generally Accepted As Safe) status, will be used preferentially. By biocompatibility, it is
meant that the ingredients and excipients used in the composition will ultimately be
"bioabsorbed" or cleared by the body with no e effects to the body. For a composition
to be biocompatible, and be ed as non-toxic, it must not cause toxicity to cells.
Similarly, the term “bioabsorbable” refers to nanoparticles made from materials which
undergo bioabsorption in viva over a period of time such that long term accumulation of the
al in the patient is d. In a preferred embodiment, the biocompatible nanoparticle
is bioabsorbed over a period of less than 2 years, preferably less than 1 year and even more
preferably less than 6 months. The rate of bioabsorption is related to the size of the particle,
the al used, and other factors well recognized by the skilled artisan. A mixture of
bioabsorbable, biocompatible materials can be used to form the nanoparticles used in this
invention. In one embodiment, iron oxide and a biocompatible, bioabsorbable polymer can
be combined. For example, iron oxide and PGLA can be combined to form a nanoparticle.
An antigen-MHC-nanoparticle complex (“NP-complex”) refers to tation of a
peptide, carbohydrate, lipid, or other antigenic segment, fragment, or epitope of an antigenic
molecule or protein (i.e., self peptide or autoantigen) on a surface, such as a biocompatible
biodegradable nanosphere. en" as used herein refers to all, part, fragment, or segment
of a molecule that can induce an immune response in a t or an ion of anti-
pathogenic cells.
A "mimic" is an analog of a given ligand or peptide, wherein the analog is
substantially similar to the ligand. "Substantially similar" means that the analog has a
binding profile similar to the ligand except the mimic has one or more fianctional groups or
modifications that collectively accounts for less than about 50%, less than about 40%, less
than about 30%, less than about 20%, less than about 10%, or less than about 5% of the
molecular weight of the ligand.
The term "anti-pathogenic autoreactive T cell" refers to a T cell with anti-
pathogenic properties (i.e., T cells that ract an autoimmune disease such as MS, a MS-
related disease or disorder, or abetes). These T cells can include anti-inflammatory T
cells, effector T cells, memory T cells, low-avidity T cells, T helper cells, autoregulatory T
cells, cytotoxic T cells, natural killer T cells, TRl cells, CD4+ T cells, CD8+ T cells and the
like.
The term “anti-inflammatory T cell” refers to a T cell that es an anti-
inflammatory response. The anti-inflammatory fianction of the T cell may be accomplished
through production and/or secretion of anti-inflammatory proteins, cytokines, chemokines,
and the like. Anti-inflammatory proteins are also intended to encompass anti-proliferative
signals that suppress immune ses. Anti-inflammatory proteins include IL-4, IL-lO, IL-
13, IL-21, IL-23, IL-27, IFN—u, TGF-B, IL-lra, G-CSF, and soluble receptors for TNF and
IL-6. Accordingly, aspects of the sure relate to methods for treating, in a patient, an
autoimmune disorder, such as MS, a MS-related disorder, diabetes or abetes, the
method comprising, consisting essentially of or yet further consisting of administering to that
patient an antigen-MHCII-nanoparticle complex, n the antigen is a disease-relevant
antigen.
The term “IL-10” or “Interleukin-10” refers to a ne encoded by the IL-lO
gene. The IL-lO sequence is represented by the GenBank Accession No.: NM_000572.2
(mRNA) and NP_000563.1 (protein).
The term “TGF-B” or “Transforming growth factor beta” refers to a protein that can
have an anti-inflammatory effect. TGF-B is a secreted protein that exists in at least three
isoforms called TGF-Bl, TGF-B2 and . It was also the original name for TGF-Bl,
which was the founding member of this family. The TGF-B family is part of a superfamily of
proteins known as the transforming growth factor beta superfamily, which includes inhibins,
activin, anti-mullerian e, bone morphogenetic protein, decapentaplegic and Vg-l.
A "an effective amount" is an amount sufficient to e the intended purpose,
non-limiting examples of such include: tion of the immune se, modulation of the
immune response, suppression of an inflammatory response and modulation of T cell activity
or T cell populations. In one aspect, the effective amount is one that functions to achieve a
stated therapeutic purpose, e.g., a eutically effective amount. As described herein in
detail, the effective amount, or dosage, depends on the purpose and the composition,
component and can be determined according to the present disclosure.
The use of the word "a" or "an" when used in conjunction with the term
"comprising" in the claims and/or the cation may mean "one," but it is also consistent
with the meaning of "one or more, at least one," and "one or more than one."
By phere," "NP," or “nanoparticle” herein is meant a small discrete particle
that is administered singularly or pluraly to a subject, cell specimen or tissue specimen as
appropriate. In certain embodiments, the rticles are substantially spherical in shape.
In certain embodiments, the nanoparticle is not a me or viral particle. In further
embodiments, the nanoparticle is solid or has a solid core. The term "substantially spherical,"
as used herein, means that the shape of the particles does not e from a sphere by more
than about 10%. s known antigen or peptide complexes of the invention may be
applied to the particles. The nanoparticles of this invention range in size from about 1 nm to
about 1 um and, preferably, from about 1 nm to about 100 nm or alternatively from about 1
nm to about 50 nm, or alternatively from about 5 to 50 nm or alternatively from about 5 nm
to 100 nm, and in some aspects refers to the average or median diameter of a plurality of
nanoparticles when a plurality of nanoparticles are intended. r nanosize les can
be obtained, for example, by the process of fractionation whereby the larger particles are
allowed to settle in an aqueous solution. The upper n of the solution is then recovered
by methods known to those of skill in the art. This upper portion is enriched in smaller size
particles. The process can be repeated until a desired e size is generated.
The use of the term "or" in the claims is used to mean "and/or" unless explicitly
indicated to refer to alternatives only or the alternatives are mutually exclusive, although the
disclosure supports a definition that refers to only alternatives and "and/or."
As used herein the phrase "immune response" or its equivalent "immunological
response" refers to the development of a cell-mediated response ted by antigen-
specific T cells or their ion products). A cellular immune response is elicited by the
presentation of polypeptide epitopes in association with Class I or Class II MHC molecules,
to treat or prevent a viral infection, expand antigen-specific Breg cells, TCl, CD4+ T helper
cells and/or CD8+ cytotoxic T cells and/or disease generated, autoregulatory T cell and B cell
y” cells. The response may also involve activation of other components.
The terms matory response" and "inflammation" as used herein indicate the
complex biological response of vascular tissues of an individual to harmful stimuli, such as
pathogens, damaged cells, or irritants, and includes secretion of cytokines and more
particularly of pro-inflammatory cytokines, i.e. cytokines which are produced predominantly
by activated immune cells and are involved in the amplification of inflammatory reactions.
ary pro-inflammatory cytokines include but are not limited to IL-1, IL-6, IL-lO, TNF-
2014/003014
a, IL-l7, IL21, IL23, IL27 and TGF-B. Exemplary inflammations include acute inflammation
and c inflammation. Acute ation indicates a short-term process characterized
by the classic signs of inflammation (swelling, redness, pain, heat, and loss of filnction) due
to the infiltration of the tissues by plasma and leukocytes. An acute inflammation typically
occurs as long as the ous stimulus is present and ceases once the stimulus has been
removed, broken down, or walled off by scarring (fibrosis). Chronic inflammation indicates
a ion characterized by concurrent active inflammation, tissue destruction, and attempts
at repair. Chronic inflammation is not characterized by the classic signs of acute
inflammation listed above. Instead, chronically inflamed tissue is characterized by the
infiltration of mononuclear immune cells (monocytes, hages, lymphocytes, and
plasma cells), tissue destruction, and attempts at healing, which include angiogenesis and
fibrosis. An inflammation can be inhibited in the sense of the present disclosure by affecting
and in particular inhibiting any one of the events that form the complex biological response
associated with an inflammation in an dual.
An autoimmune disorder may include, but is not limited to, diabetes melitus, pre-
diabetes, transplantation rejection, multiple sclerosis,a le-sclerosis related disorder,
premature ovarian failure, scleroderm, Sjogren's disease, lupus, vilelego, alopecia (baldness),
polyglandular failure, Grave's disease, yroidism, polymyosititis, pemphigus, s
disease, colititis, autoimmune hepatitis, hypopituitarism, myocardititis, Addison's disease,
autoimmune skin diseases, uveitis, pernicious anemia, hypoparathyroidism, and/or
rheumatoid arthritis. In n s, a peptide component of an antigen/MHCII/particle
complex is derived or designed from an autoantigen or an autoantigen epitope, or a mimic
thereof, involved in the mune response to be probed, modulated, or blunted by the
treatment. In particular aspects, the autoantigen is a peptide, carbohydrate, or lipid. In
certain aspects, an autoantigen is a fragment, epitope, or peptide of a protein, carbohydrate, or
lipid expressed by n cells of a subject, such as pancreatic beta cells, and e, but is
not limited to a fragment of IGRP, Insulin, GAD or IA-2 protein. Various such proteins or
epitopes have been identified for a variety of autoimmune conditions. The autoantigen may
be a peptide, carbohydrate, lipid or the like derived from a second endocrine or neurocrine
component, such as peri-islet Schwann cell or the like.
As used , the term “disease-relevant” n s an antigen or fragment
thereof selected to treat a selected disease. For e, a diabetes-relevant antigen is an
antigen or fragment thereof that will treat diabetes. A MS-relevant antigen is selected to treat
MS. A diabetes-relevant antigen would not be selected to treat MS. Similarly, an
autoimmunity related antigen is an antigen that is relevant to an autoimmune e and
would not be selected for the treatment of a disorder or disease other than autoimmunity, e.g.,
As used herein, the term “diabetes” intends a variable disorder of carbohydrate
metabolism caused by a combination of hereditary and environmental factors and is usually
characterized by inadequate secretion or utilization of insulin, by excessive urine production,
by excessive amounts of sugar in the blood and urine, and by thirst, hunger, and loss of
weight. Diabetes is characterized by Type 1 diabetes and Type 2 diabetes. The se
ic (“NOD”) mouse is an accepted animal model for the study and treatment of diabetes.
Type 1 Diabetes (TlD) in mice is associated with autoreactive CD8+ T-cells. Nonobese
diabetic (NOD) mice develop a form of TlD, closely resembling human TlD, that results
from selective destruction of pancreatic B cells by T-cells recognizing a growing list of
autoantigens. Although initiation of T1D clearly requires the contribution of CD4+ cells,
there is compelling ce that TlD is CD8+ T-cell-dependent. It has been discovered that
a significant fraction of islet-associated CD8+ cells in NOD mice use CDR3-invariant VOLl7-
J(142+ TCRs, referred to as ‘8.3-TCR—like’. These cells, which recognize the mimotope
NRP-A7 (defined using combinatorial peptide ies) in the context of the MHC molecule
Kd, are already a significant component of the earliest NOD islet CD8+ infiltrates, are
ogenic, and target a peptide from islet-specific glucosephosphatase catalytic subunit-
related protein , a n of unknown on. The CD8+ cells that recognize this
peptide (IGRP206_214, similar to NRP-A7) are unusually frequent in the circulation (>l/200
CD8+ cells). Notably, progression of insulitis to diabetes in NOD mice is invariably
accompanied by cyclic expansion of the ating IGRP206_214-reactive CD8+ pool, and by
avid maturation of its islet-associated counterpart. More ly, it has been shown that
islet-associated CD8+ cells in NOD mice recognize multiple IGRP epitopes, ting that
IGRP is a dominant autoantigen for CD8+ cells, at least in murine TlD. NOD islet-
associated CD8+ cells, particularly those found early on in the disease process also recognize
an insulin epitope (Ins B15_23).
Association studies have suggested that certain HLA class I s (i.e., HLA-
) afford susceptibility to human TlD. Pathology studies have shown that the insulitis
lesions of newly diagnosed patients consist mostly of (HLA class I-restricted) CD8+ s,
which are also the predominant cell population in patients d by transplantation with
pancreas isografts (from identical twins) or allografts (from related donors).
Insulin is a key target of the antibody and CD4+ response in both human and murine
TlD. The human insulin B chain epitope hInsB10_1g is ted by HLA-A*0201 to
autoreactive CD8+ cells both in islet transplant recipients and in the course of spontaneous
disease. In addition, four onal peptides have been identified from mouse pre-proinsulin
l or 2 that are recognized by islet-associated CD8+ T-cells from HLA-A*0201-transgenic
mice in the context of HLA-A*0201.
As used herein, the term “pre-diabetes” intends an asymptomatic period ing
a diabetic condition characterized by subclinical beta cell damage wherein the patient exhibits
normal plasma glucose levels. It also is characterized by the presence of islet cell
autoantibodies (ICAs) and, when close to the onset of clinical symptoms, it may be
anied by rance to glucose.
As used herein, the term “multiple sclerosis” or “MS” intends the autoimmune
disorder in which the body's immune system eats away at the protective sheath that covers
nerves. This interferes with the communication between the brain and the rest of the body.
Ultimately, this may result in deterioration of the nerves themselves, a process that is not
reversible.
As used herein, the term “multiple sis-related disorder” intends a disorder that
co-presents with a susceptibility to MS or with MS. Non-limiting examples of such include
neuromyelitis optica (NMO), uveitis, athis pain sis, sclerosis,
arteriosclerosis, sclerosis disseminata systemic sclerosis, spino-optical MS, primary
ssive MS (PPMS), and relapsing remitting MS (RRMS), progressive systemic
sclerosis, and ataxic sclerosis,
The terms "epitope" and "antigenic determinan " are used interchangeably to refer
to a site on an antigen to which B and/or T cells respond or recognize. B-cell epitopes can be
formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by
tertiary folding of a n. Epitopes formed from contiguous amino acids are typically
retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are
typically lost on treatment with denaturing solvents. An epitope typically includes at least 3,
-l6-
and more usually, at least 5 or 8-20 amino acids in a unique spatial conformation. Methods
of determining spatial conformation of epitopes include, for example, x-ray crystallography
and 2-dimensional r magnetic resonance. See, e. g., Glenn E. , Epitope Mapping
Protocols (1996). T-cells recognize uous epitopes of about nine amino acids for CD8
cells or about 13-15 amino acids for CD4 cells. T cells that recognize the epitope can be
identified by in Vitro assays that measure antigen-dependent proliferation, as determined by
midine incorporation by primed T cells in response to an epitope (Burke et al., J. Inf.
Dis., 170:1 1 10-1 1 19, 1994), by antigen-dependent killing (cytotoxic T cyte assay,
Tigges et al., J. Immunol., 156(10):3901-3910, 1996) or by cytokine secretion. The presence
of a cell-mediated immunological response can be ined by proliferation assays (CD4+
T cells) or CTL (cytotoxic T lymphocyte) assays.
Optionally, an antigen or preferably an epitope of an antigen, can be chemically
conjugated to, or sed as, a fusion protein with other ns, such as MHC and MHC
related proteins.
As used herein, the terms “patient” and ct” are used synonymously and refer
to a mammal. In some ments the patient is a human. In other embodiments the
patient is a mammal commonly used in a laboratory such as a mouse, rat, simian, canine,
feline, bovine, equine, or ovine.
As used in this ation, the term "polynucleotide" refers to a nucleic acid
molecule that either is recombinant or has been isolated free of total genomic nucleic acid.
Included within the term "polynucleotide" are oligonucleotides (nucleic acids 100 residues or
less in length), recombinant vectors, including, for example, plasmids, cosmids, phage,
Viruses, and the like. Polynucleotides include, in certain aspects, regulatory sequences,
ed ntially away from their naturally occurring genes or protein encoding
sequences. Polynucleotides may be RNA, DNA, analogs thereof, or a combination thereof A
nucleic acid encoding all or part of a polypeptide may contain a contiguous nucleic acid
sequence encoding all or a portion of such a polypeptide of the following lengths: 10, 20, 30,
40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,
240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,
420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580,
590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760,
770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940,
950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1095,
1100, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000,
9000, 10000, or more tides, nucleosides, or base pairs. It also is contemplated that a
particular polypeptide from a given species may be encoded by nucleic acids containing
natural variations that have slightly different nucleic acid sequences but, nonetheless, encode
the same or substantially similar protein, polypeptide, or peptide.
A polynucleotide is composed of a specific ce of four nucleotide bases:
adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the
polynucleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical
entation of a polynucleotide molecule. This alphabetical representation can be input
into databases in a computer having a central processing unit and used for bioinformatics
applications such as functional genomics and homology searching.
The term “isolated” or “recombinant” as used herein with respect to nucleic acids,
such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, tively
that are present in the natural source of the macromolecule as well as polypeptides. The term
“isolated or recombinant nucleic acid” is meant to include nucleic acid fragments which are
not naturally ing as fragments and would not be found in the l state. The term
“isolated” is also used herein to refer to polynucleotides, polypeptides and proteins that are
ed from other cellular proteins and is meant to ass both purified and
recombinant ptides. In other embodiments, the term “isolated or recombinant” means
separated from constituents, cellular and otherwise, in which the cell, , polynucleotide,
peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated
in nature. For example, an isolated cell is a cell that is separated from tissue or cells of
dissimilar phenotype or genotype. An isolated cleotide is separated from the 3 ’ and 5 ’
contiguous tides with which it is normally associated in its native or natural
environment, e.g., on the some. As is apparent to those of skill in the art, a non-
naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s)
thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart.
A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region)
having a certain percentage (for example, 80%, 85%, 90%, or 95%) of “sequence identity” to
another sequence means that, when aligned, that percentage of bases (or amino acids) are the
same in comparing the two sequences. The alignment and the percent homology or sequence
identity can be determined using software programs known in the art, for example those
bed in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement
, section 7.7.18, Table 7.7. l. Preferably, default parameters are used for alignment. A
preferred ent program is BLAST, using default parameters. In particular, red
programs are BLASTN and BLASTP, using the following default ters: Genetic code =
rd; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62;
Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank
+ EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + te + PIR.
Details of these programs can be found at the following Internet address:
ncbi.nlm.nih.gov/cgi-bin/BLAST.
It is to be inferred without explicit recitation and unless otherwise intended, that
when the present invention relates to an antigen, ptide, protein, polynucleotide or
antibody, an equivalent or a biologically equivalent of such is intended within the scope of
this invention. As used herein, the term "biological equivalent thereof ’ is intended to be
synonymous with "equivalent thereof’ when referring to a nce antigen, protein,
antibody, fragment, polypeptide or nucleic acid, and intends those having minimal homology
while still maintaining desired structure or functionality. Unless specifically recited herein, it
is contemplated that any polynucleotide, polypeptide or protein mentioned herein also
includes equivalents thereof In one aspect, an equivalent polynucleotide is one that
hybridizes under stringent conditions to the cleotide or ment of the
polynucleotide as bed herein for use in the described methods. In another aspect, an
equivalent antibody or antigen binding polypeptide intends one that binds with at least 70 % ,
or alternatively at least 75 % or alternatively at least 80 % or alternatively at least 85 %, or
, ,
alternatively at least 90 %, or alternatively at least 95 % affinity or higher affinity to a
reference antibody or antigen binding nt. In another aspect, the equivalent thereof
competes with the binding of the antibody or antigen binding fragment to its antigen under a
competitive ELISA assay. In another aspect, an equivalent intends at least about 80 %
gy or identity and alternatively, at least about 85 %, or alternatively at least about
90 %, or alternatively at least about 95 %, or alternatively 98 % percent homology or identity
and exhibits ntially equivalent biological activity to the reference protein, polypeptide
or nucleic acid.
"Hybridization" refers to a reaction in which one or more polynucleotides react to
form a complex that is stabilized via hydrogen bonding between the bases of the tide
residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding,
or in any other sequence-specific manner. The complex may comprise two s forming a
duplex structure, three or more strands forming a multi-stranded complex, a single selfhybridizing
strand, or any combination of these. A hybridization reaction may constitute a
step in a more extensive process, such as the tion of a PC reaction, or the enzymatic
cleavage of a polynucleotide by a ribozyme.
Examples of stringent hybridization conditions include: incubation temperatures of
about 25°C to about 37°C; hybridization buffer concentrations of about 6x SSC to about 10x
SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4x
SSC to about 8x SSC. Examples of moderate hybridization conditions include: incubation
temperatures of about 40°C to about 50°C; buffer concentrations of about 9x SSC to about 2x
SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5x
SSC to about 2x SSC. Examples of high ency conditions include: tion
temperatures of about 55°C to about 68°C; buffer concentrations of about lx SSC to about
0. lx SSC; formamide concentrations of about 55% to about 75%; and wash solutions of
about lx SSC, 0.lx SSC, or deionized water. In general, hybridization incubation times are
from 5 minutes to 24 hours, with l, 2, or more washing steps, and wash tion times are
about 1, 2, or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that
equivalents of SSC using other buffer systems can be employed.
“Homology” or ity” or “similarity” refers to sequence similarity between two
peptides or between two nucleic acid les. Homology can be determined by comparing
a position in each sequence which may be aligned for purposes of comparison. When a
position in the compared sequence is occupied by the same base or amino acid, then the
molecules are homologous at that position. A degree of homology between sequences is a
function of the number of ng or homologous positions shared by the sequences. An
“unrelated” or “non-homologous” sequence shares less than 40% ty, or alternatively
less than 25% identity, with one of the ces of the present invention.
"Homology" or ity" or "similarity" can also refer to two nucleic acid
molecules that hybridize under ent conditions.
As used herein, the terms "treating," "treatment" and the like are used herein to
WO 63616
mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be
therapeutic in terms of a partial or complete cure for a disorder and/or adverse effect
attributable to the disorder. In one aspect, treatment indicates a ion in the signs of the
disease using an established scale.
IGRP, which is encoded by a gene ed on chromosome 2q28-32 that ps a
TlD susceptibility locus, IDDM7 (2q3 1), has also been recently fied as a ell
autoantigen of potential relevance in human TlD. Two HLA-A*O20l-binding epitopes of
human IGRP (hIGRP228_236 and hIGRP265_273) are recognized by islet-associated CD8+ cells
from murine MHC class I-deficient NOD mice expressing an HLA-A*0201 transgene. Non-
limited examples of IGRP antigens binding to the murine MHC class II molecule (IAg7)
include for example, IGRP206_214, which comprises the antigenic peptide VYLKTNVFL and
IGRP“; which comprises the antigenic peptide LHRSGVLIIHHLQEDYRTY or an
equivalent thereof, and IGRP128_145 ,which comprises the antigenic peptide
TAALSYTISRMEESSVTL or an equivalent thereof.
“To prevent” intends to prevent a disorder or effect in vitro or in vivo in a system or
subject that is predisposed to the disorder or effect.
A “composition” is intended to mean a combination of active agent and another
compound or composition, inert (for example, a detectable agent or label) or active, such as
an adjuvant. In certain embodiments, the composition does not contain an adjuvant.
A “pharmaceutical composition” is intended to include the ation of an active
agent with a r, inert or active, making the ition suitable for diagnostic or
therapeutic use in vitro, in vivo or ex vivo.
The term "fianctionally equivalent codon" is used herein to refer to codons that
encode the same amino acid, such as the six codons for arginine or serine, and also refers to
codons that encode ically equivalent amino acids (see below Table).
Codon Table
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As used herein, a "protein" or "polypeptide" or "peptide" refers to a molecule
comprising at least five amino acid residues.
Other objects, features and advantages of the present invention will become
apparent from the following detailed description. It should be understood, r, that the
detailed description and the specific examples, while indicating specific embodiments of the
invention, are given by way of illustration only, since various changes and modifications
within the spirit and scope of the invention will become apparent to those skilled in the art
from this detailed description.
11. DESCRIPTIVE EMBODIMENTS
There is currently no therapeutic platform that enables te suppression of
polyclonal autoimmune responses without compromising systemic immunity. Applicant’s
disclosure described herein enables the design of autoimmune disease-specific nes that
turn active disease-specific CD4+ T-cells and B-cells into e, mono-specific
tory CD4+ T-cells and B-cells that coordinately suppress all other autoreactive T and
B-cell responses of the host, regardless of their fine nic specificity, and yet with
exquisite disease-specificity and without impairing ic immunity.
The autoantigenic complexity of Type 1 Diabetes (TlD).
TlD is caused by a chronic autoimmune se that progressively erodes the
pancreatic Beta-cell mass. B-cell destruction in both humans and NOD mice is ed by T-
cells recognizing many autoantigens (Tsai, S. et al. (2008) Adv. Immunol. 100:79-124;
Lieberman, S. et al. (2003) Tissue Antigens -377). Although the precise sequence of
events remains ill defined, current evidence suggests that TlD requires CD4+ and CD8+
cells; that active T cells entiate into killers by engaging B-cell antigens on local
APCs; and that these T-cells target a wide repertoire of autoantigens (Tsai, S. et al. (2008)
Adv. Immunol. 100:79-124; Santamaria, P. (2010) Immunity 32:437-445).
It has been shown that soluble peptides can induce peptide-specific T-cell tolerance
in viva, but cannot blunt poly-specific autoimmune responses (Han et al. (2005) Nature
Medicine ll(6):645-652). Unexpectedly, it was found that, unlike therapy with soluble
peptide, therapy with NPs coated with a single levant pMHC class I (originally used
as a negative contol) blunted the progression of TlD in pre-diabetic NOD mice and restored
normoglycemia in diabetic animals (Tsai, S. et al. (2010) Immunity 32:568-5 80). Subsequent
work led to the unexpected discovery that pMHC-NP y ons by expanding, in an
epitope-specific manner, autoantigen-experienced autoreactive CD8+ cells that suppressed
the recruitment of other autoantigenic T cell cities by inhibiting and killing
autoantigen-loaded APCs. More recently, Applicant has found that this therapeutic platform
can be harnessed for the in vivo ion of active T-regulatory CD4+ cells.
Specifically, Applicant discovered that NPs coated with individual TlD-relevant pMHC class
II expand disease-specific TRl CD4+ T-cells, expressing the TRl markers CD49b and LAG3
(Gagliani, N. et al. (2013) Nature Medicine 19:739-746) and producing the cytokines ILlO
and TGF-B (see below).
Collectively, these observations t a new paradigm in the progression of
autoimmunity, stating that chronic stimulation of naive autoreactive CD8+ or CD4+ T cells
by nous epitopes triggers their differentiation into memory-like autoreactive
regulatory T cells; and that these memory autoreactive regulatory cells suppress the activation
of both cognate and non-cognate high-avidity autoreactive T cell specificities by suppressing
and/or killing autoantigen-loaded APCs (Tsai, S. et al. (2010) Immunity 32:568-5 80).
Importantly, and without being bound by theory, any single epitope (pMHC) city
involved in an autoimmune disease (among many) can be used, when coated as a ligand onto
NPs, to blunt complex autoimmune responses. It is Applicant’s belief that these NP
preparations cannot activate na'1've T-cells, hence induce or T-cell responses, because
they lack key co-stimulatory molecules, such as CD80 and CD86. In fact, cognate naive and
effector autoreactive cells are deleted by this approach. Therefore, the therapeutic approach
that enabled its discovery provide a platform for a new class of therapeutics in autoimmunity,
potentially capable of resolving polyclonal autoimmune responses in a e- and organspecif1c
manner without compromising systemic immunity.
III. METHODS
Medical and diagnostic methods are also provided. In one aspect, a method is
provided for promoting the ion, expansion and recruitment of B-regulatory cells and/or
TRl cells (e. g., TRl and CD4+ cells) in an antigen-specific manner in a subject in need
thereof, comprising, or alternatively ting essentially of, or yet further consisting of,
stering to the subject an effective amount of the NP-complex or composition as
described herein.
In another aspect, a method for treating or preventing an autoimmune disease or
disorder as described herein, e. g., MS, a MS-related disorder, diabetes or pre-diabetes, in a
subject in need thereof is provided, the method comprising, or alternatively consisting
essentially of, or yet further consisting of, administering to the subject an effective amount of
the NP-complex or composition as bed herein, n the autoantigen is disease-
relevant for the e to be treated, e.g., for the tion or treatment of diabetes, the
antigen is a diabetes-relevant antigen. In a further aspect, the autoimmune disease is
multiple-sclerosis or a multiple-sclerosis d disorder and the antigen is MS-relevant.
Peptide antigens for the treatment or prevention of pre-diabetes or es, include,
but are not limited to hInsB10_1g (HLVEALYLV), hIGRP228_236 LWSV), hIGRP265_273
(VLFGLGFAI), IGRP206_214 (VYLKTNVFL), NRP-A7 (KYNKANAFL), NRP-I4
(KYNIANVFL), NRP-V7 NVFL), YAI/Db (FQDENYLYL) and/or INS 315,23
(LYLVCGERG), GAD65114_123, VMNILLQYVV; 36_545, RMMEYGTTMV;
GFAP143_151, NLAQTDLATV; GFAP214_222, QLARQQVHV; IA-2172_180, SLSPLQAEL; IA-
2482490, SLAAGVKLL; IA-2805_813, VIVMLTPLV; ppIAPP5_13, KLQVFLIVL; ppIAPP9_17,
FLIVLSVAL; IGRP152_160, MLI; IGRP211_219, NLFLFLFAV; IGRP215_223,
FLFAVGFYL; IGRP222_230, YLLLRVLNI; IGRP228_236, LNIDLLWSV; IGRP265_273,
VLFGLGFAI; IGRP293_301, TSL; Pro-insulinL2_10, ALWMRLLPL; Pro-insulinL3_11,
LWMRLLPLL; sulinL6_14, RLLPLLALL; Pro-insulinBs_14, HLCGSHLVEA; Pro-
insulinBlo_1g, YLV; sulinBl4_22, ALYLVCGER; Pro-insulin315_24,
ERGF; Pro-insulinBl7_25, LVCGERGFF; Pro-insulinBlg_27, VCGERGFFYT; Pro-
insulin1320_27, GERGFFYT; sulin1321_29, ERGFFYTPK; Pro-insulin1325_01, FYTPKTRRE;
Pro-insulinBz7_cs, TPKTRREAEDL; Pro-insulinczo_28, SLQPLALEG; Pro-insulin025_33,
ALEGSLQKR; Pro-insulin029_A5, SLQKRGIVEQ; Pro-insulinA1_10, GIVEQCCTSI; Pro-
insulinA2_10, IVEQCCTSI; Pro-insulinA12_20, SLYQLENYC or equivalents and/or
combinations f. Additional examples include ProIns 76-90, SLQPLALEGSLQKRG,
ProIns 79-89, PLALEGSLQKR, ProIns 90-109, GIVEQCCTSICSLYQLENYC, ProIns 94-
105, QCCTSICSLYQL, GAD 247-266, NMYAMMIARFKMFPEVKEKG, GAD 255-265,
RFKMFPEVKEK, GAD 555-567, NFFRMVISNPAAT, IGRP 13-25, QHLQKDYRAYYTF,
IGRP 8-27, GVLIIQHLQKDYRAYYTFLN, ProIns B24-C36, FFYTPMSRREVED and
equivalents of each thereof.
Whent the method is directed to the treatment of MS or MS-related disorders, the
complex includes antigens related to multiple sclerosis. Such antigens include, for example,
those disclosed in US. Patent Publication No. 2012/0077686, and ns derived from
myelin basic protein, myelin associated glycoprotein, myelin oligodendrocyte protein,
proteolipid n, oligodendrocyte myelin oligoprotein, myelin associated oligodendrocyte
basic protein, endrocyte specific protein, heat shock proteins, oligodendrocyte specific
ns NOGO A, glycoprotein Po, peripheral myelin protein 22, and 2'3'-cyclic nucleotide
3'-phosphodiesterase. In certain embodiments, the antigen is derived from Myelin
Oligodendrocyte Glycoprotein (MOG). Non-limited examples include, for example,
MAG287_295, SLLLELEEV; MAG509_517, LMWAKIGPV; MAG556_564, VLFSSDFRI; MBP110_
118, SLSRFSWGA; MOG114_122, KVEDPFYWV; MOG166_175, RTFDPHFLRV; MOG172_180,
FLRVPCWKI; MOG179_188, KITLFVIVPV; MOG188_196, VLGPLVALI; MOG181_189,
PVL; MOG205_214, RLAGQFLEEL; PLP80_88, FLYGALLLA or equivalents or
ations thereof.
Additional non-limiting examples of antigens that can be used in this invention
comprise polypeptides comprising, or atively consisting essentially of, or yet r
ting of the polypeptides of the group: MOG35_55, MEVGWYRSPFSRVVE-H..YRNG K;
MOG36_55, EVGWYRSPFSRVVI-HXRNGK; MAG287_295, SLLLELEEV; MAG509_517,
LMWAKIGPV; MAG556_564, VLFSSDFRI; MBPImng, SLSRFSWGA; _122,
KVEDPFYWV; MOG166_175, RTFDPHFLRV; MOG172_180, WKI; MOG179_188,
KITLFVIVPV; _196, VLGPLVALI; MOG181_189, TLFVIVPVL; MOG205_214,
RLAGQFLEEL; PLP80_88, FLYGALLLA, or an equivalent of each thereof, or combinations
thereof.
Methods to determine and monitor the therapy are known in the art and briefly
described herein. When red in vitro, administration is by contacting the composition
with the tissue or cell by any appropriate method, e.g., by administration to cell or tissue
culture medium and is useful as a screen to determine if the y is appropriate for an
individual or to screen for alternative therapies to be used as a substitute or in combination
with the disclosed compositions. When stered in vivo, stration is by systemic
or local administration. In vivo, the methods can be ced on a non-human animal to
screen alternative therapies to be used as a substitute or in combination with the disclosed
compositions prior to human administration. In a human or non-human , they are
also useful to treat the disease or disorder.
The above methods require stration of an effective amount of a NP-complex.
The MHC of the antigen-MHC-nanoparticle complex can be MHC I, MHC II, or
assical MHC but preferably MHCII. MHC proteins are described herein. In one
embodiment, the MHC of the antigen-MHC-nanoparticle complex is a MHC class I. In
another embodiment, the MHC is a MHC class II. In other embodiments, the MHC
component of the antigen-MHC-nanoparticle complex is MHC class II or a non-classical
MHC molecule as described herein. In one aspect, the antigen comprises, or alternatively
consists essentially of, or yet further consists of the polypeptide GWYRSPFSRVVH or an
equivalent of GWYRSPFSRVVH.
The size of the nanoparticle can range from about 1 nm to about 1 um. In certain
embodiments, the nanoparticle is less than about 1 um in diameter. In other embodiments,
the nanoparticle is less than about 500 nm, less than about 400 nm, less than about 300 nm,
less than about 200 nm, less than about 100 nm, or less than about 50 nm in diameter. In
further embodiments, the nanoparticle is from about 1 nm to about 10 nm, 15 nm, 20 nm, 25
nm, 30 nm, 40 nm, 50 nm, 75 nm, or 100 nm in diameter. In specific embodiments, the
nanoparticle is from about 1 nm to about 100 nm, about 1 nm to about 50 nm, about 1 nm to
about 20 nm, or about 5 nm to about 20 nm.
The size of the complex can range from about 5 nm to about 1 um. In certain
embodiments, the complex is less than about 1 um or alternatively less than 100 nm in
diameter. In other embodiments, the complex is less than about 500 nm, less than about 400
nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, or less than
about 50 nm in diameter. In further embodiments, the complex is from about 5 nm or 10 nm
to about 50 nm, or about 5 nm to about 75 nm, or about 5 nm to about 50 nm, or about 5 nm
to about 60 nm, or from about 10 nm to about 60 nm, or in one aspect about 55 nm.
Applicant has discovered that the density of the antigen-MHC complexes on the
nanoparticle contributes to the therapeutic benefit. Thus, as disclosed herein the antigen-
MHC nanoparticle x can have a defined density in the range of from about 0.05 MHC
molecules per 100 nm2 of surface area of the nanoparticle including the complex, assuming at
least 2 MHC, or alternatively at least 8, or alternatively at least 9, or atively at least 10,
or alternatively at least 11, or alternatively at least 12, MHC complexed to the nanoparticle.
In one aspect the complex has a y of MHC from about 0.01 MHC per 100 nm2 (0.05
MHC/100 nmz) to about 30 MHC/100 nm2, or alternatively from 0.1 MHC/100 nm2 to about
0 nm2, or alternatively from about 0.3 MHC/100 nm2 to about 25 MHC/100
nmz, or alternatively from about 0.4 MHC/100 nm2 to about 25 MHC/100 nmz, or
alternatively from about 0.5 MHC/100 nm2 to about 20 MHC/100 nm2, or alternatively from
0.6 MHC/100 nm2 to about 20 MHC/100 nmz, or atively from about 1.0 MHC/100 nm2
to about 20 MHC/100 nmz, or alternatively from about 5.0 MHC/100 nm2 to about 20
MHC/100 nmz, or alternatively from about 10.0 MHC/100 nm2 to about 20 MHC/100 nmz,
or alternatively from about 15 MHC/100 nm2 to about 20 MHC/100 nm2, or alternatively at
least about 0.5, or atively at least about 1.0, or atively at least about 5.0, or
alternatively at least about 10.0, or alternatively at least about 15.0 MHC/100 nm2 .
In one
aspect, When 9 or at least 9 MHC are complexed to a nanoparticle, the y range is from
about 0.3 MHC/100nm2 to about 20 MHC/100nm2.
In one of its method aspects, there is provided a method for accumulating B-
tory cells and/or anti-inflammatory T cells in a t in need thereof. In a further
embodiment, the T cell is a CD4+ or CD8+ T cell. In a related embodiment, the T cell
secretes IL-10 or TGFB. The method comprises, consists essentially of, or yet further
consists of administering to a patient in need thereof an effective amount of the antigen-MHC
nanoparticle x as described .
In one embodiment, the compositions and methods described herein are for treating
an autoimmune disorder such as MS, MS-associated disorder, diabetes or pre-diabetes. The
method comprises, consists essentially of, or yet further consists of administering to a t
in need thereof an effective amount of the antigen-MHCII nanoparticle complex as described
herein.
Details regarding modes of administration in vitro and in vivo are described within.
This disclosure also provides use of the NP-complexes for the ation of
ments for the treatment and/or prevention of diseases and disorders as described
herein.
IV. ANTIGEN-MHC-NANOPARTICLE COMPLEXES
A. Polypeptides and Polynucleotides
Further aspects relate to an isolated or purified polypeptide antigens, comprising, or
consisting essentially of, or yet further consisting of, the amino acid sequences as described
herein, or a polypeptide having at least about 80% sequence identity, or alternatively at least
85 %, or alternatively at least 90%, or alternatively at least 95 %, or alternatively at least 98
% sequence identity to the amino acid sequences of the ns, or polypeptides encoded by
polynucleotides having at about 80% sequence identity, or alternatively at least 85 %, or
alternatively at least 90%, or alternatively at least 95 %, or alternatively at least 98 %
sequence identity to the polynucleotide ng the amino acid sequences of the antigen, or
its complement, or a polypeptide encoded by a polynucleotide that hybridizes under
conditions of moderate to high stringency to a polynucleotide encoding the amino acid
ce of the antigens, or its complement. Also provided are ed and purified
polynucleotides encoding the antigen polypeptides disclosed herein, or amino acids having at
least about 80% sequence identity thereto, or atively at least 85 %, or atively at
least 90%, or alternatively at least 95 %, or alternatively at least 98 % sequence identity to the
disclosed sequences, or an equivalent, or a polynucleotide that hybridizes under stringent
conditions to the polynucleotide, its equivalent or its complement and ed or purified
polypeptides encoded by these polynucleotides. The polypeptides and polynucleotides can be
ed with non-naturally occurring nces with which they are not associated with in
, e. g., carriers, pharmaceutically acceptable rs, vectors and MHC molecules,
nanoparticles as known in the art and as described herein.
ns, including segments, fragments and other molecules derived from an
antigenic species, including but not limited to peptides, carbohydrates, lipids or other
molecules presented by classical and non-classical MHC molecules of the invention are
typically complexed or operatively coupled to a MHC molecule or derivative f.
Antigen recognition by T lymphocytes is major ompatibility complex (MHC)-
restricted. A given T lymphocyte will recognize an antigen only when it is bound to a
particular MHC molecule. In general, T lymphocytes are stimulated only in the presence of
self -MHC molecules, and antigen is recognized as fragments of the antigen bound to self
MHC les. MHC restriction def1nes T lymphocyte city in terms of the antigen
recognized and in terms of the MHC molecule that binds its antigenic fragment(s). In
particular aspects n ns will be paired with certain MHC molecules or polypeptides
derived therefrom.
The term "operatively coupled" or "coated" as used , refers to a situation
where individual polypeptide (e. g., MHC) and antigenic (e.g., peptide) components are
combined to form the active complex prior to binding at the target site, for example, an
immune cell. This includes the situation where the individual polypeptide complex
components are synthesized or recombinantly expressed and subsequently isolated and
combined to form a complex, in vitro, prior to administration to a subject; the situation where
a chimeric or fusion polypeptide (i.e., each discrete protein component of the complex is
ned in a single polypeptide chain) is synthesized or recombinantly expressed as an
intact x. Typically, polypeptide complexes are added to the nanoparticles to yield
nanoparticles with adsorbed or coupled polypeptide complexes having a ratio of number of
molecules:number of nanoparticle ratios from about, at least about or at most about about 0.1,
0.5, 1, 3, 5, 7, 10, 15, 20, 25, 30, 35, 40, 50, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325,
350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500 or more to:1, more
typically 0.1 :1, 1:1 to 50:1 or 300: 1. The polypeptide content of the nanoparticles can be
determined using standard techniques.
B. MHC Molecules
ellular and extracellular antigens present quite different challenges to the
immune system, both in terms of recognition and of appropriate response. Presentation of
2014/003014
antigens to T cells is mediated by two distinct classes of molecules MHC class I (MHC-I) and
MHC class II (MHC-II) (also fied as “pMHC” ), which utilize distinct antigen
processing pathways. Peptides derived from intracellular antigens are presented to CD8+ T
cells by MHC class I molecules, which are expressed on virtually all cells, while extracellular
antigen-derived peptides are presented to CD4+ T cells by MHC-II molecules. However,
there are certain exceptions to this dichotomy. Several studies have shown that peptides
generated from endocytosed particulate or soluble ns are presented on MHC-I
molecules in macrophages as well as in dendritic cells. In certain embodiments of the
invention, a particular antigen is identified and presented in the antigen-MHC-nanoparticle
complex in the t of an appropriate MHC class I or II polypeptide. In certain aspects,
the genetic makeup of a t may be assessed to determine which MHC polypeptide is to
be used for a particular patient and a particular set of peptides. In certain embodiments, the
MHC class 1 component comprises all or part of a HLA-A, HLA-B, HLA-C, HLA-E, HLA-
F, HLA-G or CD-l molecule. In embodiments wherein the MHC component is a MHC class
II component, the MHC class II component can se all or a part of a HLA-DR, HLA-
DQ, or .
Non-classical MHC molecules are also plated for use in MHC complexes of
the invention. Non-classical MHC molecules are non-polymorphic, conserved among
species, and possess narrow, deep, hydrophobic ligand binding pockets. These binding
pockets are capable of presenting glycolipids and phospholipids to Natural Killer T (NKT)
cells or certain subsets of CD8+ T-cells such as Qal or restricted CD8+ T-cells.
NKT cells represent a unique lymphocyte population that co-express NK cell markers and a
semi-invariant T cell receptor (TCR). They are implicated in the regulation of immune
responses associated with a broad range of diseases.
C. Antigenic Components
Certain aspects of the invention include methods and compositions concerning
antigenic itions including segments, fragments, or epitopes of polypeptides, peptides,
nucleic acids, ydrates, lipids and other molecules that provoke or induce an antigenic
response, lly referred to as antigens. In particular, antigenic segments or fragments of
antigenic determinants, which lead to the ction of a cell via an autoimmune response,
can be fied and used in making an antigen-MHC-nanoparticle complex described
herein. Embodiments of the invention include compositions and methods for the
modulation of an immune response in a cell or tissue of the body.
Antigenic polypeptides and peptides of the invention may be modified by various
amino acid deletions, insertions, and/or substitutions. In particular embodiments, modified
ptides and/or peptides are capable of modulating an immune response in a subject. In
some embodiments, a wild-type version of a protein or peptide are employed, r, in
many embodiments of the invention, a modified protein or polypeptide is employed to
generate an antigen-MHC-nanoparticle complex. An antigen-MHC-nanoparticle complex
can be used to generate an nflammatory immune response, to modify the T cell
population of the immune system (i.e., re-educate the immune system), and/or foster the
recruitment and lation of anti-inflammatory T cells to a particular tissue. The terms
described above may be used interchangeably herein. A "modified protein" or "modified
polypeptide" or "modified e" refers to a protein or polypeptide whose chemical
structure, particularly its amino acid sequence, is altered with respect to the wild-type protein
or polypeptide. In some embodiments, a modified n or polypeptide or peptide has at
least one modified activity or function (recognizing that proteins or polypeptides or peptides
may have le ties or filnctions). It is cally contemplated that a modified
protein or polypeptide or peptide may be altered with respect to one ty or function yet
retains a wild-type activity or function in other respects, such as immunogenicity or ability to
interact with other cells of the immune system when in the context of an MHC-nanoparticle
Non-limiting examples, of peptide antigens e, but are not limited to hInsB10_1g
(HLVEALYLV), hIGRP22g_236 (LNIDLLWSV), hIGRP265_273 GFAI), IGRP206_214
(VYLKTNVFL), NRP-A7 (KYNKANAFL), NRP-I4 (KYNIANVFL), NRP-V7
(KYNKANVFL), YAI/Db (FQDENYLYL) and/or INS B1543 (LYLVCGERG), as well as
peptides and proteins disclosed in US. Patent Application Publication No. 2005/0202032 and
lents and/or combinations thereof.
In certain aspects, a peptide antigen for treatment of T1D is GAD65114_123,
VMNILLQYVV; GAD65536_545, RMMEYGTTMV; GFAP143_151, NLAQTDLATV; GFAP214_
222, QLARQQVHV; 2_1go, SLSPLQAEL; IA-24g2_490, SLAAGVKLL; IA-2805_813,
VIVMLTPLV; 5_13, KLQVFLIVL; ppIAPP9_17, FLIVLSVAL; IGRP152_160,
FLWSVFMLI; IGRP211_219, FAV; IGRP215_223, FLFAVGFYL; IGRP222_230,
YLLLRVLNI; IGRP228_236, LNIDLLWSV; IGRP265_273, VLFGLGFAI; IGRP293_301,
RLLCALTSL; Pro-insulinL2_10, ALWMRLLPL; Pro-insulinL3_11, LWMRLLPLL; Pro-
insulinL6_14, RLLPLLALL; Pro-insulin135_14, HLCGSHLVEA; Pro-insulin1310_1g,
HLVEALYLV; Pro-insulinBl4_22, ALYLVCGER; Pro-insulin315_24, ERGF; Pro-
insulinBl7_25, LVCGERGFF; Pro-insulinBlg_27, VCGERGFFYT; Pro-insulinBzo_27,
GERGFFYT; Pro-insulin1321_29, ERGFFYTPK; sulin1325_01, FYTPKTRRE; Pro-
insulint7_Cs, EAEDL; Pro-insulinczo_28, SLQPLALEG; Pro-insulin025_33,
ALEGSLQKR; Pro-insulin029_A5, SLQKRGIVEQ; Pro-insulinA1_10, GIVEQCCTSI; Pro-
insulinA2_10, IVEQCCTSI; Pro-insulinA12_20, SLYQLENYC or equivalents and/or
combinations thereof.
Additional miting examples of antigens include MS and MS-relevant or
related antigens that can be used in this invention comprise polypeptides comprising, or
alternatively consisting essentially of, or yet fiarther consisting of the polypeptides of the
group: MOG35_55, MEVGWYRSE’FSRVVHLYEKN’GK; MOG36_55,
EV’SWWRSPFSRVVE-ELYRNGK; MAG287_295, EEV; _517, LMWAKIGPV;
MAG556_564, VLFSSDFRI; MBP1110_118, SLSRFSWGA; MOG114_122, KVEDPFYWV;
MOG166_175, RTFDPHFLRV; MOG172_180, FLRVPCWKI; MOG179_1gg, KITLFVIVPV;
MOG188_196, VLGPLVALI; MOG181_189, TLFVIVPVL; MOG205_214, LEEL; PLP80_
gg, FLYGALLLA, or an equivalent of each thereof, or ations thereof.
In still filther aspects peptide antigens for the treatment of MS and MS-related
disorders include without limitation: MOG35_55, MEV’GWY RSPFSRVVH LYRNGK; MOG36_
55, ‘WYRSPFSRVVHLYRNGK; MAG287_295, SLLLELEEV; MAG509_517,
LMWAKIGPV; MAG556_564, VLFSSDFRI; MBP110_118, SLSRFSWGA; MOG114_122,
KVEDPFYWV; MOG166_175, RTFDPHFLRV; MOG172_180, FLRVPCWKI; MOG179_1gg,
KITLFVIVPV; _196, VLGPLVALI; MOG181_189, TLFVIVPVL; MOG205_214,
RLAGQFLEEL; 88, LLA MAG287_295, SLLLELEEV; MAG509_517,
LMWAKIGPV; MAG556_564, VLFSSDFRI, and equivalents and/or combinations f
Antigens for the treatment ofMS and ated disorders include, those disclosed
in US. Patent Application Publication No. 2012/0077686, and antigens derived from myelin
basic protein, myelin associated glycoprotein, myelin endrocyte protein, proteolipid
protein, oligodendrocyte myelin rotein, myelin associated oligodendrocyte basic
protein, oligodendrocyte specific protein, heat shock ns, oligodendrocyte specific
proteins NOGO A, glycoprotein Po, peripheral myelin n 22, and 2'3'-cyclic nucleotide
3'-phosphodiesterase. In certain embodiments, the antigen is derived from Myelin
Oligodendrocyte rotein (MOG).
In certain embodiments, the size of a protein or polypeptide (wild-type or
modified), including any x of a protein or peptide of st and in ular a MHC-
e filSlOIl, may comprise, but is not limited to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,
230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625,
650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200,
1300, 1400, 1500, 1750, 2000, 2250, 2500 amino molecules or greater, including any range
or value derivable therein, or derivative thereof. In certain aspects, 5, 6, 7, 8, 9, 10 or more
contiguous amino acids, including derivatives thereof, and fragments of an antigen, such as
those amino acid sequences disclosed and referenced herein, can be used as antigens. It is
contemplated that polypeptides may be d by truncation, rendering them shorter than
their corresponding wild-type form, but also they might be altered by fusing or conjugating a
heterologous protein sequence with a ular function (e.g., for presentation as a protein
complex, for enhanced immunogenicity, etc.).
Proteinaceous compositions may be made by any technique known to those of skill
in the art, including (i) the expression of proteins, polypeptides, or peptides through standard
molecular biological techniques, (ii) the isolation of proteinaceous compounds from l
sources, or (iii) the chemical synthesis of proteinaceous materials. The nucleotide as well as
the protein, polypeptide, and peptide sequences for various genes have been previously
sed, and may be found in the recognized computerized databases. One such database is
the National Center for Biotechnology Information's GenBank and GenPept databases (on the
World Wide Web at ncbi.nlm.nih.gov/). The all or part of the coding regions for these genes
may be amplified and/or expressed using the techniques disclosed herein or as would be
known to those of ordinary skill in the art.
Amino acid sequence variants of autoantigenic epitopes and other polypeptides of
these itions can be substitutional, insertional, or deletion variants. A modification in a
pfideoftheinvenfionrnayaflmctl,2,3,4,5,6,7,8,9,lO,ll,l2,l3,l4,lS,l6,l7,
18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,4l,42,
43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,6l,62,63,64,65,66,67,
68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,9l,92,
93,94,95,96,97,98,99,lOO,lOO,lOl,lOZ,103,104,105,106,107,108,109,110,11L
112,113,ll4,llS,ll6,ll7,118,ll9,120,121,122,123,124,125,126,127,128,129,
130,l3l,l32,l33,l34,l35,l36,l37,138,139,140,141,l42,l43,l44,l45,l46,l47,
148,149,150,151,152,153,154,155,156,157,158,159,l60,l6l,l62,l63,l64,l65,
166,167,l68,l69,l70,l7l,l72,l73,174,175,176,177,178,179,180,181,182,183,
184,185,186,187,188,189,l90,l9l,192,193,194,195,l96,l97,l98,l99,200,201,
202,203,204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219,
220,221,222,223,224,225,226,227,228,229,230,23l,232,233,234,235,236,237,
9,240,241,242,235,236,237,238,239,240,24l,242,243,244,245,246,247,
248,249,250,251,252,253,254,255,256,257,258,259,260,26l,262,263,264,265,
7,268,269,270,271,272,273,274,275,276,277,278,279,280,281,282,283,
284,285,286,287,288,289,290,291,292,293,294,295,296,297,298,299,300,301,
302,303,304,305,306,307,308,309,310,311,312,3l3,3l4,315,316,317,318,3l9,
320,321,322,323,324,325,326,327,328,329,330,33l,332,333,334,335,336,337,
9,340,341,342,343,344,345,346,347,348,349,350,351,352,353,354,355,
356,357,358,359,360,361,362,363,364,365,366,367,368,369,370,371,372,373,
374,375,376,377,378,379,380,381,382,383,384,385,386,387,388,389,390,39L
392,393,394,395,396,397,398,399,400,401,402,403,404,405,406,407,408,409,
410,411,412,413,414,415,416,417,418,419,420,421,422,423,424,425,426,427,
428,429,430,431,432,433,434,435,436,437,438,439,440,44l,442,443,444,445,
446,447,448,449,450,451,452,453,454,455,456,457,458,459,460,461,462,463,
464,465,466,467,468,469,470,471,472,473,474,475,476,477,478,479,480,481,
482,483,484,485,486,487,488,489,490,491,492,493,494,495,496,497,498,499,500
or more non-contiguous or contiguous amino acids of a peptide or polypeptide, as compared
to wild-type.
Deletion variants typically lack one or more residues of the native or Wild-type
amino acid ce. Individual residues can be deleted or a number of contiguous amino
acids can be deleted. A stop codon may be introduced (by substitution or insertion) into an
encoding nucleic acid sequence to generate a truncated protein. Insertional mutants typically
2014/003014
involve the addition of material at a non-terminal point in the polypeptide. This may include
the insertion of one or more residues. Terminal additions, called fusion proteins, may also be
generated.
Substitutional variants typically contain the exchange of one amino acid for another
at one or more sites within the protein, and may be designed to modulate one or more
properties of the ptide, with or without the loss of other functions or properties.
Substitutions may be conservative, that is, one amino acid is ed with one of similar
shape and charge. Conservative substitutions are well known in the art and include, for
example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or
histidine; aspartate to glutamate; ne to serine; glutamine to asparagine; glutamate to
aspartate; e to proline; histidine to asparagine or glutamine; isoleucine to leucine or
valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine;
phenylalanine to tyrosine, leucine or methionine; serine to ine; threonine to serine;
tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or
leucine. Alternatively, substitutions may be non-conservative such that a fianction or actiVity
of a polypeptide or peptide is affected, such as aVidity or affinity for a cellular receptor(s).
Non-conservative changes typically involve substituting a residue with one that is chemically
dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and
vice versa.
Proteins of the invention may be inant, or sized in Vitro.
Alternatively, a recombinant protein may be isolated from ia or other host cell.
It also will be understood that amino acid and nucleic acid sequences may include
additional residues, such as additional N— or C-terminal amino acids, or 5' or 3' nucleic acid
ces, respectively, and yet still be ially as set forth in one of the sequences
disclosed herein, so long as the sequence meets the criteria set forth above, including the
maintenance of biological n actiVity (e.g., genicity). The addition of terminal
sequences particularly applies to nucleic acid sequences that may, for example, include
various non-coding sequences g either of the 5' or 3' portions of the coding region.
It is plated that in compositions of the invention, there is between about
0.001 mg and about 10 mg of total protein per ml. Thus, the concentration of protein in a
composition can be about, at least about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3,
0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0,
8.5, 9.0, 9.5, 10.0, 50, 100 ug/ml or mg/ml or more (or any range derivable n). Of this,
about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 1,12,13,14,15,16,17,18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,
94, 95, 96, 97, 98, 99, 100% may be antigen-MHC-nanoparticle x.
In addition, US. Patent No. 4,554,101 (Hopp), which is incorporated herein by
reference, teaches the identification and preparation of epitopes from primary amino acid
sequences on the basis of hydrophilicity. Through the methods disclosed in Hopp, one of
skill in the art would be able to identify potential epitopes from within an amino acid
sequence and confirm their immunogenicity. us ific publications have also
been devoted to the prediction of secondary structure and to the identification of epitopes,
from es of amino acid sequences (Chou & Fasman, Adv. Enzymol., 47:45-148, 1978;
Chous and Fasman, Annu, Rev. Biochem., 47:251-276, 1978, Chou and Fasman,
Biochemistry, 13(2):211-222, 1974; Chau and Fasman, Biochemistry, 13(2):222-245, 1974,
Chou and Fasman, Biophys. J of these may be used, if desired,
., 26(3):385-399, 1979). Any
to supplement the teachings of Hopp in US. Patent No. 4,554,101.
For any given autoimmune e the antigen MHC compex can be identified and
pre-selected using known methods in the art. Algorithms exist - d from a set of aligned
peptides known to bind to a given MHC molecule, which can be used as a predictor of both
peptide-MHC binding and T-cell es. See, e.g., Reche and Reinherz (2007) Methods
Mol. Biol. 409: 185-200.
Molecules other than peptides can be used as antigens or antigenic fragments in
complex with MHC molecules, such molecules include, but are not limited to carbohydrates,
lipids, small molecules, and the like. Carbohydrates are major components of the outer
surface of a variety of cells. Certain carbohydrates are teristic of different stages of
differentiation and very often these carbohydrates are recognized by specific antibodies.
Expression of distinct carbohydrates can be restricted to specific cell types.
D. Substrates/Nanoparticles
In certain aspect, antigen/MHC complexes are ively coupled to a substrate
which can be bound covalently or non-covalently to the substrate. A substrate can be in the
form of a nanoparticle that optionally comprises a biocompatible and/or bioabsorbable
material. Accordingly, in one embodiment, the nanoparticle is biocompatible and/or
bioabsorbable. In r aspect, the nanoparticle has a solid core and/or is not a liposome.
A substrate can also be in the form of a nanoparticle such as those described usly in
US. Patent Publication No. 2009/0155292. rticles can have a structure of variable
dimension and known variously as a nanosphere, a nanoparticle or a biocompatible
biodegradable nanosphere or a patible biodegradable rticle. Such particulate
formulations containing an antigen/MHC complex can be formed by covalent or non-
covalent coupling of the complex to the nanoparticle.
The nanoparticles typically consist of a substantially spherical core and optionally
one or more . The core may vary in size and composition. In addition to the core, the
nanoparticle may have one or more layers to provide functionalities appropriate for the
applications of interest. The thicknesses of layers, if present, may vary depending on the
needs of the c applications. For example, layers may impart useful optical properties.
Layers may also impart al or biological fianctionalities, referred to herein as
chemically active or biologically active layers, and for these functionalities the layer or layers
may typically range in thickness from about 0.001 micrometers (l nanometer) to about 10
micrometers or more ding on the d nanoparticle diameter), these layers typically
being applied on the outer surface of the nanoparticle.
The compositions of the core and layers may vary. Suitable materials for the
particles or the core include, but are not limited to polymers, ceramics, glasses, minerals, and
the like. Examples include, but are not limited to, standard and specialty glasses, silica,
polystyrene, polyester, polycarbonate, acrylic polymers, polyacrylamide, polyacrylonitrile,
polyamide, fluoropolymers, silicone, celluloses, silicon, metals (e.g., iron, gold, ),
minerals (e. g., ruby), nanoparticles (e.g., gold nanoparticles, colloidal particles, metal oxides,
metal sulfides, metal des, and magnetic materials such as iron oxide), and composites
thereof. The core could be of homogeneous composition, or a composite of two or more
s of material depending on the properties desired. In certain aspects, metal
nanoparticles will be used. These metal les or rticles can be formed from Au, Pt,
Pd, Cu, Ag, Co, Fe, Ni, Mn, Sm, Nd, Pr, Gd, Ti, Zr, Si, and In, precursors, their binary alloys,
their ternary alloys and their intermetallic compounds. See US. Patent No. 6,712,997. In
certain embodiments, the compositions of the core and layers may vary provided that the
rticles are biocompatible and bioabsorbable. The core could be of homogeneous
composition, or a composite of two or more classes of material depending on the properties
d. In certain aspects, metal nanospheres will be used. These metal rticles can be
formed from Fe, Ca, Ga and the like. In certain embodiments, the nanoparticle comprises a
core sing metal or metal oxide such as gold or iron oxide.
As previously stated, the nanoparticle may, in addition to the core, include one or
more layers. The nanoparticle may include a layer consisting of a biodegradable sugar or
other polymer. Examples of biodegradable layers include but are not limited to dextran;
poly(ethylene glycol); poly(ethylene oxide); mannitol; poly(esters) based on polylactide
(PLA), ycolide (PGA), polycaprolactone (PCL); poly(hydroxalkanoate)s of the PHB-
PHV class; and other modified poly(saccharides) such as starch, cellulose and chitosan.
Additionally, the rticle may include a layer with suitable surfaces for attaching
chemical onalities for chemical binding or coupling sites.
Layers can be produced on the nanoparticles in a y of ways known to those
skilled in the art. Examples e sol-gel chemistry techniques such as described in Iler,
Chemistry of Silica, John Wiley & Sons, 1979; Brinker and Scherer, Sol-gel Science,
Academic Press, (1990). Additional approaches to producing layers on nanoparticles include
surface chemistry and encapsulation techniques such as described in Partch and Brown, J.
Adhesion, 67:259-276, 1998; Pekarek et al., Nature, 367:258, (1994); Hanprasopwattana,
Langmuir, 12:3173-3179, (1996); Davies, Advanced Materials, 10: 270, (1998); and
references therein. Vapor deposition techniques may also be used; see for example Golman
and Shinohara, Trends Chem. Engin., 6:1-6, (2000); and US. Pat. No. 6,387,498. Still other
approaches e layer-by-layer self-assembly techniques such as described in Sukhorukov
et al., Polymers Adv. Tech., 9(10-11):759-767, (1998); Caruso et al., Macromolecules,
2317-2328, (1998); Caruso et al., J.Amer. Chem. Soc., 121(25):6039-6046, (1999);
US. Pat. No. 6,103,379 and nces cited therein.
Nanoparticles may be formed by contacting an aqueous phase containing the
antigen/MHC/co-stimulatory le complex and a polymer and a nonaqueous phase
followed by evaporation of the nonaqueous phase to cause the coalescence of particles from
the aqueous phase as taught in US. Patent No. 330 or 542. Preferred polymers
for such preparations are natural or synthetic copolymers or polymers selected from the group
consisting of gelatin agar, starch, arabinogalactan, albumin, collagen, polyglycolic acid,
polylactic acid, glycolide-L(-) lactide poly(episilon-caprolactone, poly(epsilon-caprolactone-
tic acid), poly(epsilon-caprolactone-CO-glycolic acid), poly(B-hydroxy butyric acid),
poly(ethylene oxide), polyethylene, poly(alkylcyanoacrylate), poly(hydroxyethyl
rylate), polyamides, poly(amino acids), poly(2-hydroxyethyl DL-aspartamide),
poly(ester urea), poly(L-phenylalanine/ethylene glycol/l,6-diisocyanatohexane) and
poly(methyl rylate). Particularly preferred polymers are polyesters, such as
polyglycolic acid, polylactic acid, glycolide-L(-) lactide pisilon-caprolactone),
poly(epsilon-caprolactone-CO-lactic acid), and poly(epsilon-caprolactone-CO-glycolic acid).
Solvents useful for dissolving the polymer include: water, hexafluoroisopropanol,
methylenechloride, tetrahydrofuran, , benzene, or hexafluoroacetone sesquihydrate.
The size of the nanoparticle can range from about 1 nm to about 1 um. In certain
embodiments, the rticle is less than about 1 um in diameter. In other embodiments,
the rticle is less than about 500 nm, less than about 400 nm, less than about 300 nm,
less than about 200 nm, less than about 100 nm, or less than about 50 nm in diameter. In
further embodiments, the nanoparticle is from about 1 nm to about 10 nm, 15 nm, 20 nm, 25
nm, 30 nm, 40 nm, 50 nm, 75 nm, or 100 nm in diameter. In specific embodiments, the
nanoparticle is from about 1 nm to about 100 nm, about 1 nm to about 50 nm, about 1 nm to
about 20 nm, or about 5 nm to about 20 nm.
The size of the complex can range from about 5 nm to about 1 um. In certain
embodiments, the complex is less than about 1 um or alternatively less than 100 nm in
diameter. In other embodiments, the complex is less than about 500 nm, less than about 400
nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, or less than
about 50 nm in diameter. In further embodiments, the complex is from about 10 nm to about
50 nm, or about 20 nm to about 75 nm, or about 25 nm to about 60 nm, or from about 30 nm
to about 60 nm, or in one aspect about 55 nm.
E. Coupling n-MHC Complex with the Nanoparticle
In order to couple the substrate or nanospheres to the antigen-MHC complexes the
following techniques can be applied.
The binding can be generated by chemically modifying the substrate or nanoparticle
which typically involves the generation of ional groups" on the surface, said fianctional
groups being capable ofbinding to an antigen-MHC complex, and/or linking the optionally
chemically modified surface of the substrate or nanoparticle with covalently or non-
ntly bonded so-called "linking molecules," followed by reacting the antigen-MHC
complex with the nanoparticles obtained.
The term "linking le" means a substance capable of linking with the
substrate or nanoparticle and also capable of g to an antigen-MHC complex. In certain
embodiments, the antigen-MHC complexes are coupled to the rticle by a linker. Non-
limiting examples of suitable linkers include dopamine (DPA)-polyethylene glycol (PEG)
linkers such as DPA-PEG-NHS ester, DPA-PEG-orthopyridyl-disulfide (OPSS) and/or DPA-
PEG-Azide. Other linkers e peptide s, ethylene glycol, biotin, and strepdavidin.
The term "fianctional groups" as used herein before is not restricted to ve
chemical groups forming covalent bonds, but also includes chemical groups leading to an
ionic interaction or hydrogen bonds with the antigen-MHC complex. er, it should be
noted that a strict distinction between "fianctional groups" generated at the surface and linking
molecules bearing "functional groups" is not possible, since sometimes the modification of
the surface requires the reaction of r linking molecules such as ethylene glycol with the
nanosphere surface.
The functional groups or the linking molecules bearing them may be ed from
amino groups, carbonic acid groups, thiols, hers, disulfides, guanidino, hydroxyl
groups, amine , l diols, aldehydes, alpha-haloacetyl groups, mercury organyles,
ester , acid halide, acid thioester, acid anhydride, isocyanates, isothiocyanates, sulfonic
acid halides, imidoesters, diazoacetates, diazonium salts, l,2-diketones, phosphonic acids,
phosphoric acid esters, sulfonic acids, azolides, imidazoles, indoles, N—maleimides, alpha-
beta-unsaturated carbonyl compounds, arylhalogenides or their derivatives.
Non-limiting examples for other g molecules with higher lar weights
are nucleic acid molecules, polymers, copolymers, polymerizable coupling agents, silica,
proteins, and chain-like molecules having a surface with the opposed polarity with respect to
the substrate or nanoparticle. Nucleic acids can provide a link to affinity molecules
containing themselves nucleic acid molecules, though with a complementary sequence with
t to the g molecule.
A specific example of a covalent linker includes poly(ethylene) glycol (PEG) such
as functionalized PEGs. As used herein, “functionalized PEGs” refer to PEG moieties
including terminal functional group, non-limiting examples of which include amino,
mercapto, thioether, carboxyl, and the likes. Non-limiting examples of functionalized PEG
linkers on various nanoparticle cores are provided in Tables 1 and 2 attached hereto, e.g., the
PEG linker thiol-PEG-NHZ linker.
In certain embodiments, the linker as described herein has a defined size. In some
embodiments, the linker is less that about 10 kD, less than about 5 kD, less than about 4.5
kD, less than about 4 kD, less than about 3.5 kD, less than about 3 kD, less than about 2.5
kD, less than about 2 kD, or less than about 1 kD. In further embodiments, the linker is from
about 0.5 kD to about 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, or 1 kD. In yet further embodiments, the
linker is from about 1 to about, 4.5, 4, 3.5, 3, 2.5, 2, or 15 kD.
As examples for polymerizable coupling agents, diacetylene, styrene ene,
vinylacetate, te, acrylamide, vinyl compounds, styrene, silicone oxide, boron oxide,
phosphorous oxide, borates, pyrrole, polypyrrole and phosphates can be cited.
The surface of the substrate or nanoparticle can be chemically modified, for
instance by the binding of phosphonic acid derivatives having functional reactive groups.
One example of these phosphonic acid or phosphonic acid ester tes is imino-
bis(methylenphosphono) ic acid which can be synthesized according to the "Mannich-
Moedritzer" on. This binding reaction can be performed with substrate or here
as directly obtained from the ation process or after a pre-treatment (for instance with
trimethylsilyl bromide). In the first case the phosphonic acid (ester) derivative may for
instance displace components of the reaction medium which are still bound to the surface.
This displacement can be enhanced at higher temperatures. Trimethylsilyl e, on the
other hand, is believed to dealkylate alkyl group-containing orous-based complexing
agents, thereby creating new binding sites for the phosphonic acid (ester) derivative. The
phosphonic acid (ester) derivative, or g molecules bound thereto, may y the same
functional groups as given above. A r e of the surface treatment of the substrate
or nanosphere involves heating in a diole such as ethylene glycol. It should be noted that this
treatment may be redundant if the synthesis already proceeded in a diol. Under these
circumstances the synthesis product directly obtained is likely to show the ary
functional . This treatment is however applicable to substrate or nanoparticle that were
produced in N— or P-containing complexing agents. If such substrate or particle are subjected
to an after-treatment with ethylene glycol, ingredients of the reaction medium (e.g.
complexing agent) still binding to the surface can be replaced by the diol and/or can be
dealkylated.
It is also possible to replace N-containing complexing agents still bound to the
particle surface by primary amine derivatives having a second fianctional group. The surface
of the substrate or nanoparticle can also be coated with silica. Silica allows a relatively
simple chemical conjugation of organic molecules since silica easily reacts with organic
linkers, such as triethoxysilane or chlorosilane. The nanoparticle surface may also be coated
by homo- or copolymers. Examples for polymerizable coupling agents are N—(3-
ropyl)mercaptobenzamidine, 3-(trimethoxysilyl)propylhydrazide and 3-
trimethoxysilyl)propylmaleimide. Other non-limiting es of polymerizable ng
agents are mentioned herein. These coupling agents can be used singly or in combination
depending on the type of copolymer to be generated as a coating.
r surface modification technique that can be used with substrates or
nanoparticles containing oxidic transition metal nds is conversion of the oxidic
transition metal compounds by chlorine gas or organic chlorination agents to the
corresponding oxychlorides. These oxychlorides are e of ng with nucleophiles,
such as hydroxy or amino groups as often found in biomolecules. This technique allows
generating a direct conjugation with proteins, for instance-via the amino group of lysine side
chains. The conjugation with proteins after surface modification with oxychlorides can also
be effected by using a ctional linker, such as maleimidopropionic acid hydrazide.
For non-covalent g techniques, type molecules having a polarity or
charge opposite to that of the substrate or nanosphere surface are particularly le.
Examples for linking molecules which can be non-covalently linked to core/shell
nanospheres involve anionic, cationic or zwitter—ionic surfactants, acidic or basic proteins,
polyamines, ides, polysulfone or polycarboxylic acid. The hydrophobic interaction
between substrate or nanosphere and amphiphilic reagent having a functional reactive group
can te the necessary link. In particular, chain-type les with amphiphilic
character, such as phospholipids or derivatized polysaccharides, which can be crosslinked
with each other, are useful. The absorption of these molecules on the surface can be achieved
by coincubation. The binding between ty molecule and substrate or nanoparticle can
also be based on non-covalent, self-organising bonds. One example thereof involves simple
2014/003014
detection probes with biotin as linking molecule and - or strepdavidin-coupled
molecules.
Protocols for coupling reactions of functional groups to biological molecules can be
found in the literature, for instance in "Bioconjugate Techniques" (Greg T. Hermanson,
Academic Press 1996). The ical molecule (e.g., MHC molecule or derivative thereof)
can be d to the g molecule, covalently or non-covalently, in line with standard
procedures of organic chemistry such as oxidation, halogenation, alkylation, acylation,
addition, tution or amidation. These methods for coupling the covalently or non-
covalently bound linking molecule can be d prior to the coupling of the linking
molecule to the substrate or nanosphere or thereafter. Further, it is possible, by means of
incubation, to effect a direct binding of molecules to correspondingly pre-treated substrate or
nanoparticle (for instance by trimethylsilyl bromide), which display a modified surface due to
this pre-treatment (for instance a higher charge or polar surface).
F. Protein Production
The present invention describes polypeptides, peptides, and proteins for use in
various embodiments of the present invention. For example, specific peptides and their
complexes are assayed for their ies to elicit or modulate an immune se. In
specific embodiments, all or part of the peptides or proteins of the invention can also be
synthesized in solution or on a solid support in accordance with conventional techniques.
Various automatic synthesizers are commercially available and can be used in accordance
with known protocols. See, for example, Stewart and Young, Solid Phase Peptide Synthesis,
2Ild Ed., Pierce Chemical Co.l, (1984); Tam et al., J. Am. Chem. Soc., 105:6442, (1983);
ield, Science, 232(4748):34l-347, (1986); and Barany and Merrifield, The Peptides,
Gross and Meinhofer (Eds.), Academic Press, NY, 1-284, (1979), each orated herein
by reference. Alternatively, recombinant DNA technology may be employed wherein a
tide sequence which encodes a peptide of the invention is inserted into an expression
vector, transformed or transfected into an appropriate host cell and cultivated under
conditions suitable for expression.
One embodiment of the invention includes the use of gene transfer to cells,
including rganisms, for the production of ns. The gene for the protein of interest
may be transferred into appropriate host cells followed by culture of cells under the
appropriate conditions. A nucleic acid ng virtually any polypeptide may be employed.
The generation of recombinant expression vectors, and the elements included therein, are
known to one skilled in the art and are briefly sed herein. Examples of mammalian
host cell lines include, but are not limited to ero and HeLa cells, other B- and T-cell lines,
such as CEM, 721.221, H9, Jurkat, Raji, as well as cell lines of e hamster ovary
(CHO), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cells. In addition, a host
cell strain may be chosen that modulates the expression of the inserted sequences, or that
modifies and processes the gene product in the manner desired. Such modifications (e. g.,
glycosylation) and processing (e.g., cleavage) of n products may be important for the
function of the protein. Different host cells have characteristic and specific mechanisms for
the post-translational processing and modification of proteins. Appropriate cell lines or host
systems can be chosen to ensure the correct modification and processing of the foreign
protein expressed.
A number of selection systems may be used including, but not limited to HSV
thymidine kinase, hypoxanthine-guanine oribosyltransferase, and adenine
oribosyltransferase genes, in tk-, hgprt— or aprt-cells, respectively. Also, anti-
metabolite resistance can be used as the basis of selection: for dhfr, which confers ance
to trimethoprim and methotrexate; gpt, which confers ance to mycophenolic acid; neo,
which confers resistance to the lycoside G418; and hygro, which confers resistance to
hygromycin.
G. Nucleic Acids
The present invention may include recombinant polynucleotides encoding the
proteins, polypeptides, es of the invention, such as those encoding antigenic peptides.
In particular ments, the invention concerns isolated nucleic acid segments
and recombinant vectors orating nucleic acid sequences that encode an autoantigen
and/or a MHC molecule. The term "recombinant" may be used in conjunction with a
polypeptide or the name of a ic polypeptide, and this generally refers to a polypeptide
produced from a nucleic acid molecule that has been manipulated in vitro or that is a
replication product of such a molecule.
The nucleic acid segments used in the present invention, regardless of the length of
the coding sequence itself, may be combined with other nucleic acid sequences, such as
promoters, polyadenylation signals, additional ction enzyme sites, multiple cloning sites,
other coding ts, and the like, such that their overall length may vary considerably. It
is therefore contemplated that a nucleic acid fragment of almost any length may be employed,
with the total length preferably being d by the ease of preparation and use in the
ed recombinant nucleic acid protocol. In some cases, a nucleic acid sequence may
encode a polypeptide sequence with additional logous coding sequences, for example
to allow for purification of the polypeptide, transport, secretion, post-translational
modification, or for therapeutic benefits such as targeting or efficacy. A tag or other
heterologous polypeptide may be added to the modified polypeptide-encoding sequence,
wherein "heterologous" refers to a polypeptide that is not the same as the modified
polypeptide.
V. PHARMACEUTICAL COMPOSITIONS AND ADMINISTRATION
Provided herein are pharmaceutical compositions useful for the treatment of
disease.
A. ceutical Compositions
The antigen-MHC nanoparticle complexes can be administred alone or in
combination with a r, such as a pharmaceutically acceptable carrier in a composition.
Compositions of the invention may be conventionally administered parenterally, by injection,
for example, intravenously, aneously, or intramuscularly. Additional formulations
which are suitable for other modes of administration include oral formulations. Oral
formulations include such normally employed excipients such as, for example,
pharmaceutical grades of mannitol, lactose, , magnesium stearate, sodium saccharine,
cellulose, magnesium carbonate and the like. These compositions take the form of solutions,
suspensions, tablets, pills, capsules, sustained release formulations or powders and contain
about 10% to about 95% of active ient, preferably about 25% to about 70%. The
ation of an aqueous ition that contains an antigen-MHC-nanoparticle complex
that modifies the subj ect's immune condition will be known to those of skill in the art in light
of the t disclosure. In certain embodiments, a composition may be inhaled (e.g., US.
Patent No. 6,651,655, which is specifically orated by nce in its entirety). In one
embodiment, the antigen-MHC-nanoparticle complex is administered systemically.
Typically, compositions of the invention are administered in a manner compatible
with the dosage formulation, and in such amount as will be therapeutically effective and
immune modifying. The quantity to be administered depends on the subject to be treated.
Precise amounts of active ient required to be administered depend on the judgment of
the practitioner. However, suitable dosage ranges are of the order of ten to l hundred
nanograms or micrograms antigen-MHC-nanoparticle complex per administration. Suitable
s for initial administration and boosters are also variable, but are typified by an initial
administration followed by subsequent strations.
In many ces, it will be desirable to have multiple administrations of a peptide-
MHC-nanoparticle complex, about, at most about or at least about 3, 4, 5, 6, 7, 8, 9, 10 or
more. The administrations will normally range from 2 day to twelve week intervals, more
usually from one to two week intervals. Periodic boosters at intervals of 025-5 years, y
two years, may be desirable to maintain the condition of the immune system. The course of
the administrations may be followed by assays for inflammatory immune responses and/or
autoregulatory T cell activity.
In some embodiments, pharmaceutical compositions are stered to a t.
Different aspects of the present invention involve administering an effective amount of a
antigen-MHC-nanoparticle complex ition to a subject. Additionally, such
compositions can be administered in combination with modifiers of the immune system.
Such itions will generally be dissolved or dispersed in a pharmaceutically acceptable
carrier or aqueous medium.
The phrases "pharmaceutically acceptable" or "pharmacologically acceptable" refer
to molecular entities and compositions that do not produce an adverse, allergic, or other
untoward reaction when administered to an animal, or human. As used herein,
"pharmaceutically acceptable carrier" includes any and all solvents, dispersion media,
coatings, antibacterial and antifilngal agents, ic and absorption delaying agents, and the
like. The use of such media and agents for pharmaceutical active substances is well known in
the art. Except insofar as any conventional media or agent is incompatible with the active
ingredients, its use in immunogenic and therapeutic compositions is contemplated.
The pharmaceutical forms suitable for able use include sterile aqueous
solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene
glycol; and sterile powders for the extemporaneous preparation of sterile inj ectable ons
or dispersions. In all cases the form must be e and must be fluid to the extent that it may
be easily injected. It also should be stable under the ions of manufacture and storage
and must be preserved against the inating action of microorganisms, such as bacteria
and fiangi.
The compositions may be formulated into a neutral or salt form. ceutically
acceptable salts, include the acid on salts (formed with the free amino groups of the
protein) and which are formed with inorganic acids such as, for example, hydrochloric or
phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
Salts formed with the free carboxyl groups can also be derived from nic bases such as,
for example, sodium, ium, ammonium, calcium, or ferric hydroxides, and such organic
bases as isopropylamine, trimethylamine, histidine, procaine and the like.
The carrier may be a solvent or dispersion medium containing, for example, water,
ethanol, polyol (for example, glycerol, propylene glycol, and liquid thylene glycol),
and the like, suitable mixtures f, and vegetable oils. The proper y can be
ined, for example, by the use of a coating, such as lecithin, by the maintenance of the
required particle size in the case of dispersion, and by the use of surfactants. The prevention
of the action of microorganisms can be brought about by various antibacterial and antifungal
agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for example, sugars or sodium
chloride. Prolonged absorption of the inj ectable compositions can be t about by the
use in the itions of agents ng absorption, for example, aluminum monostearate
and gelatin.
e injectable solutions are prepared by incorporating the active compounds in
the ed amount in the appropriate solvent with various of the other ingredients
enumerated above, as required, followed by sterilization. Sterilization of the solution will be
done in such a way as to not diminish the therapeutic properties of the antigen-MHC-
nanoparticle complex. Generally, dispersions are prepared by incorporating the various
sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium
and the required other ients from those enumerated above. In the case of sterile
powders for the preparation of sterile inj ectable solutions, the preferred methods of
preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the
active ingredient, plus any additional desired ingredient from a previously sterilized solution
thereof. One such method of sterilization of the solution is sterile filtration, however, this
invention is meant to e any method of sterilization that does not significantly decrease
the therapeutic properties of the antigen-MHC-nanoparticle complexes. Methods of
ization that involve intense heat and re, such as aVing, may compromise the
tertiary structure of the complex, thus significantly sing the therapeutic properties of
the antigen-MHC-nanoparticle xes.
An ive amount of therapeutic ition is determined based on the
intended goal. The term "unit dose" or "dosage" refers to physically discrete units suitable
for use in a subject, each unit containing a predetermined quantity of the composition
calculated to produce the desired responses discussed above in ation with its
administration, i.e., the appropriate route and regimen. The quantity to be administered, both
according to number of treatments and unit dose, depends on the result and/or protection
desired. Precise amounts of the ition also depend on the nt of the practitioner
and are ar to each indiVidual. Factors affecting dose include physical and clinical state
of the subject, route of administration, intended goal of treatment (alleviation of symptoms
versus cure), and potency, stability, and toxicity of the particular composition. Upon
formulation, solutions will be administered in a manner compatible with the dosage
formulation and in such amount as is therapeutically or prophylactically effective. The
formulations are easily administered in a variety of dosage forms, such as the type of
injectable solutions described above.
B. Combination Therapy
The compositions and related methods of the present invention, particularly
administration of an antigen-MHC-nanoparticle x, may also be used in combination
with the administration of traditional therapies. These e, but are not limited to, Avonex
(interferon beta-la), Betaseron (interferon beta-lb), Copaxone ramer acetate),
Novantrone (mitoxantrone), Rebif (interferon beta-la), Tysabri (natalizumab), Gilenya
(fingolimod), Glatiramer, steroids, Cytoxan, Imuran, Baclofen, deep brain stimulation,
Ampyra (dalfampridine), acupuncture, and physical therapy.
When ation therapy is employed, various combinations may be employed,
for example antigen-MHC-nanoparticle complex administration is "A" and the additional
agent is "B":
A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B
B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A/ B/B/A/A
B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A
Administration of the peptide-MHC complex compositions of the present ion
to a patient/subj ect will follow general protocols for the administration of such nds,
taking into account the toxicity, if any. It is expected that the treatment cycles would be
repeated as necessary. It also is contemplated that various standard therapies, such as
hydration, may be applied in combination with the described therapy.
C. In Vitro 0r Ex Vivo Administration
As used herein, the term in vitro stration refers to manipulations med
on cells d from or outside of a subject, including, but not limited to cells in culture.
The term ex vivo administration refers to cells which have been manipulated in vitro, and are
subsequently administered to a subject. The term in viva administration includes all
manipulations performed within a subject, including administrations.
In certain aspects of the t invention, the compositions may be administered
either in vitro, ex vivo, or in vivo. In certain in vitro embodiments, autologous T cells are
ted with compositions of this invention. The cells or tissue can then be used for in
vitro analysis, or alternatively for ex vivo administration.
VI. EXAMPLES
The ing examples are given for the purpose of illustrating various
embodiments of the invention and are not meant to limit the present invention in any n.
One skilled in the art will appreciate readily that the present invention is well adapted to carry
out the objects and obtain the ends and advantages mentioned, as well as those objects, ends
and advantages inherent herein. The present es, along with the methods described
herein are tly representative of embodiments and are exemplary, and are not intended
as limitations on the scope of the invention. Changes therein and other uses which are
encompassed within the spirit of the invention as defined by the scope of the claims will
occur to those skilled in the art.
Example 1. Preparation and analysis of pMHC nanoparticles.
pMHC production
Two ent methods were used to express recombinant pMHC class I complexes.
The first involved re-folding MHC class I heavy and light chains expressed in bacteria in the
presence of peptide, followed by purification via gel filtration and anion exchange
chromatography, as described (Garboczi, D.N. et al. (1992) Proc Natl. Acad Sci USA
89:3429-3433; Altman, JD. et al. (1996) Science 274:94-96). The second involved
expressing MHC class I xes at high yields in lentiviral-transduced freestyle CHO cells
as single chain constructs in which the peptide-coding sequence, the MHC class I light and
heavy chains are sequentially tethered with flexible GS linkers (Yu, Y.Y. et al. (2002) J
Immunol 168:3145-3149) followed by a carboxyterminal linker encoding a BirA site, a 6xHis
tag ending with a free Cys. The secreted proteins were d from culture supematants
using nickel columns and anion exchange chromatography and used directly for NP coating
or biotinylated to produce pMHC tetramers using hrome-conjugated streptavidin.
Tetramers generated using entative -chain pMHC complexes encoding the
IGRP206_214 autoantigenic peptide or its mimic NRP-V7 ntly bind to cognate
monoclonal autoreactive CD8+ T-cells but not to their polyclonal counterparts (not ,
as determined by flow cytometry.
Recombinant pMHC class II monomers were initially purified from Drosophila
SC2 cells transfected with constructs encoding I-AB and I-Ad chains carrying c-Jun or c-Fos
leucine zippers, respectively, and a BirA and 6xHis tags as previously described (Stratmann,
T. et al. (2000) J Immunol 165:3214-3225, Stratmann, T. et al. (2003) J. Clin. Invest.
14-3225). As the yields of this approach were generally low and time-consuming,
Applicant developed an expression system in freestyle CHO cells transduced with
lentiviruses encoding a monocistronic message in which the peptide-IAB and IA0L chains of
the complex are ted by the me skipping P2A sequence (Holst, J. et al. (2006) Nat
Protoc 1:406-417). As with the single chain pMHC class I constructs described above, a
linker encoding a BirA site, a 6xHis tag and a free Cys was added to the carboxyterminal end
of the construct. The self-assembled pMHC class II xes were purified from the cell
culture supernatants by nickel chromatography followed by anion exchange and used for
coating onto NPs or processed for biotinylation and tetramer formation as described above.
pMHC class II tetramers generated using a representative pMHC class II complex encoding
the 2.5mi autoantigenic peptide are specifically and efficiently bound by cognate monoclonal
autoreactive CD4+ T-cells, as determined by flow cytometry.
pMHC tetramer staining
PE-conjugated TUM-
and BDC2.5mi/IAg7 tetramers were prepared using biotinylated pMHC monomers as
described (Stratmann, T. et al. (2000) J l 165:3214-3225; Stratmann, T. et al. (2003)
J. Clin. Invest. 112:3214-3225; Amrani, A. et al. (2000) Nature 406:739-742). Peripheral
blood mononuclear cells, splenocytes and lymph node CD8+ or CD4+ T-cells were stained
with er (5 ug/mL) in FACS buffer (0.1% sodium azide and 1% PBS in PBS) for 1 h at
4°C, washed, and incubated with FITC-conjugated D80L or anti-CD4 (5 ug/mL) and
PerCP-conjugated anti-B220 (2 ug/mL; as a 'dumb' gate) for 30 min at 4°C. Cells were
washed, fixed in 1% PFA/PBS and analyzed by FACS.
NP sis
Gold nanoparticles (GNPs) were sized using chemical reduction of gold
chloride with sodium e as described (Perrault, S.D. et al. (2009) Nano Lett 9: 1909-
1915). , 2 mL of 1% of HAuCl4 (Sigma Aldrich) was added to 100 mL H20 under
vigorous ng and the solution heated in an oil bath. Six (for 14 nm GNPs) or two mL (for
40 nm GNPs) of 1% Na Citrate were added to the boiling HAuCl4 solution, which was stirred
for an additional 10 min and then cooled down to room temperature. GNPs were stabilized by
the addition of 1 uMol of thiol-PEG linkers (Nanocs, MA) functionalized with —COOH or —
NHz groups as acceptors ofpMHC (Tables 1 and 2). Pegylated GNPs were washed with
water to remove fiee thiol-PEG, concentrated and stored in water for further analysis. NP
density was via spectrophotometry and calculated according to Beer’s law.
The SFP series iron oxide NPs (SFP IONPs) were produced by thermal
decomposition of iron acetate in organic solvents in the presence of surfactants, then rendered
solvent in aqueous buffers by pegylation (Xie, J. et al. (2007) Adv Mater 19:3163; Xie, J. et
al. (2006) Pure Appl. Chem. 78: 1003-1014; Xu, C. et al. (2007) Polymer International
56:821-826). Briefly, 2 mMol Fe(acac)3 (Sigma Aldrich, le, ON) were ved in a
mixture of 10 mL benzyl ether and oleylamine and heated to 100°C for 1 hr followed by
300°C for 2 hr with reflux under the protection of a en blanket. Synthesized NPs were
precipitated by on of ethanol and resuspended in hexane. For pegylation of the IONPs,
100 mg of different 3.5 kDa DPA-PEG linkers (81-85 in Table 1; Jenkem Tech USA) were
dissolved in a e of CHC13 and HCON(CH3)2 (DMF). The NP solution (20 mg Fe) was
then added to the DPA-PEG solution and stirred for 4 hr at room temperature. ted SFP
NPs were precipitated ght by addition of hexane and then resuspended in water. Trace
amounts of aggregates were removed by high-speed centrifiagation 0 xg, 30 min), and
the sperse SFP NPs were stored in water for fiarther characterization and pMHC
conjugation. The concentration of iron in IONP products was determined by
spectrophotometry at A410 in 2N HCL. Based on the molecular structure and diameter of
SFP NPs (Fe304; 8:1 nm diameter) (Xie, J. et al. (2007) Adv Mater 19:3163; Xie, J. et al.
(2006) Pure Appl. Chem. 78: 1003-1014), Applicant estimates that SFP solutions containing 1
mg of iron contain 5x1014 NPs.
Applicant subsequently developed a new IONP design that allowed the formation,
also by thermal decomposition but in a single step, of pegylated IONPs in the complete
absence of surfactants (PF series IONPs). In this novel design, PEG molecules were used
both as reducing reagents and as surfactants. In a typical reaction, 3 g PEG (2 kDa) were
melted slowly in a 50 mL round bottom boiling flask at 100°C and then mixed with 7 mL of
benzyl ether and 2 mMol c)3. The reaction was vigorously stirred for one hr and heated
to 260°C with reflux for an additional two hr. The reaction mixture was cooled down to room
temperature, transferred to a centrifilgation tube and mixed with 30 mL water. Insoluble
materials were removed by centrifilgation at 2,000xg for 30 min. The free PEG molecules
were removed by ultrafiltration through Amicon-15 filters (MWCO 100 kDa, Millipore,
Billerica, MA). ant was able to generate IONPs with most, albeit not all of the PEG
molecules tested (Table 1, P1-P5). The size of the IONPs varied depending on the onal
groups of the PEG linkers used in the thermal decomposition reactions (Tables 1 and 2). The
NPs could be readily purified using magnetic (MACS) columns (Miltenyi Biotec, Auburn,
CA) or an IMag cell tion system (BD BioSciences, Mississauga, ON). The purified
IONPs were stored in water or in various buffers (pH 5-10) at room temperature or at 4°C
without any able aggregation. NP density was ated as described above for SFP
NPs.
pMHC conjugation of NPs
pMHC conjugation to NPs produced with PEG linkers carrying distal -NH2 or —
COOH groups was achieved via the formation of amide bonds in the presence of l-Ethyl
ethylamin0pr0pyl]carbodiimide hydrochloride (EDC). NPs (GNP-C, SFP-C and PF-C,
Table 2) with —COOH groups were first dissolved in 20 mM MES buffer, pH 5.5. N-
hydroxysulfosuccinimide sodium salt (sulpha-NHS, Thermo scientific, Waltham, MA, final
concentration 10 mM) and EDC (Thermo scientific, Waltham, MA, final concentration 1
mM) were added to the NP solution. After 20 min of ng at room temperature, the NP
solution was added drop-wise to the solution containing pMHC monomers dissolved in 20
mM borate buffer (pH 8.2). The mixture was stirred for additional 4 hr. To conjugate pMHCs
to NHz-functionalized NPs (GNP-N, SFP-N and PF-N, Table 2), pMHC complexes were
first dissolved in 20 mM MES buffer, pH 5.5, containing 100 mM NaCl. Sulpha-NHS (10
mM) and EDC (5 mM) were then added to the pMHC solution. The activated pMHC
molecules were then added to the NP solution in 20 mM borate buffer (pH 8.2), and stirred
for 4 hr at room temperature.
To conjugate pMHC to maleimide-functionalized NPs (SFP-M and PF-M, Table 2
and ), pMHC molecules were first ted with Tributylphospine (TBP, 1 mM)
for 4 hr at room temperature. pMHCs engineered to encode a free carboxyterminal Cys
residue were then mixed with NPs in 40 mM phosphate buffer, pH 6.0, containing 2 mM
EDTA, 150 mM NaCl, and incubated overnight at room temperature. pMHCs were
covalently bound with NPs via the formation of a carbon-sulfide bond between ide
groups and the Cys e.
Click chemistry was used to conjugate pMHC or avidin to NPs onalized with
azide groups (SFP-Z, Table 2). For this reaction, pMHC or avidin molecules were first
incubated with dibenzocyclooctyl (DBCO, Click try Tools, Scottdale, AZ) t for
2 hr at room temperature. Free DBCO molecules were removed by dialysis overnight.
pMHC- 0r -DBCO conjugates were then incubated with SFP-Z for 2 hr, resulting in
formation of le bonds between pMHCs or avidin molecules and NPs.
Unconjugated pMHC complexes in the different pMHC-NP conjugating reactions
were removed by extensive dialysis against PBS, pH 7.4, at 4°C though 300 kDa molecular
weight cut off membranes (Spectrum labs). Alternatively, pMHC-conjugated IONPs were
purified by magnetic separation. The conjugated NPs were concentrated by ultrafiltration
through Amicon 15 units (100 kDa MWCO) and stored in PBS.
Electron microscopy, dynamic light scattering, DLS and small angle o beam
ction
The core size and dispersity of unconjugated and pMHC-conjugated NPs were first
assessed via transmission electron microscopy (TEM, Hitachi H7650). Dynamic light
scattering (DLS) was used to determine the pMHC-NPs’ hydrodynamic size, zeta potential
and monodisperity using a ZetaSizer ment (Malvem, UK). The chemical nature of the
iron oxide core of the PF series ofNPs was evaluated using small angle electro beam
diffraction (SEBD).
r Transformation Infrared spectroscopy
The surface chemical properties of the PF-series IONP designs were evaluated
using Fourier Transformation Infrared spectroscopy (FTIR). The FTIR spectra of control
PEG and PEG anchored on the PF-NP surface were obtained using a Nicolet FTIR
ophotometer on an ATR (attenuated total reflection) mode. Each of the spectra was
ed as the average of 256 scans at 4 cm"1 spectral resolution. The stretching vibration
signatures of the PEG backbone C-O-C groups and their distal pMHC-acceptor filnctional
groups were identified.
Agarose gel ophoresis
To quickly evaluate changes on the NP charge as a function of pegylation or pMHC
coating, NPs were subjected to electrophoresis on 0.8% agarose gels. Pegylated NPs migrated
to ve or ve poles depending on the overall surface charge. Coomassie blue
staining was done to confirm co-migration of the pMHCs with the NPs.
Native and denaturing polyacrylamide gel electrophoresis
pMHC conjugated NPs were subjected to native-PAGE (10%) and SDS-PAGE
(12%) analyses to confirm absence of free (unconjugated pMHC) in the pMHC-NP
preparations and to confirm presence of intact trimolecular pMHC complexes on the NP’s
surface.
pMHC valency measurements
To evaluate the number ofpMHC monomers conjugated onto individual NPs
(pMHC valency), we measured the pMHC concentration of the pMHC-NP preps using
different approaches, including Bradford assay (Thermo ific), amino acid analysis
(HPLC-based fication of 17 different amino acids in yzed pMHC-NP
preparations) (University of Toronto), dot-ELISA and signature peptide analysis by mass
spectrometry) and the values converted to ratios of pMHC molecular number to NP number.
Briefly, in the “dot-ELISA” approach, pMHC-conjugated and unconjugated NPS and pMHC
monomer solutions (as standards) were serially diluted in PBS and then absorbed to a PVDF
ne in a ell filter plate (PALL Corporation). The plate was d to partially
dry at room temperature and then ted with pMHC specific primary antibodies (i.e.,
ZM and anti-Kd antibodies for pMHC class I-coated NPs, clones 2M2 and SFl-l .l,
BioLegend, San Diego, CA), followed by HRP- or AP-conjugated secondary antibodies.
Upon development of the enzymatic color reactions, the contents of the wells were
transferred to wells in a conventional ELISA plate and their absorbances measured at 450 nm
using a plate reader. For the signature e mass ometry approach, pMHC-specific
trypsin es (signature peptides TWTAADTAALITR for Kd complexes and
AQNSELASTANMLR for I-Ag7 complexes) were identified via mass spectrometry. The
corresponding synthetic peptides were labeled with stable isotopes (AQUA peptide synthesis,
Sigma Aldrich). The isotope-labeled peptides were then serially diluted to defined
concentrations and mixed with pMHC-conjugated NPs for trypsin digestion. The mixtures
were subjected to mass spectroscopy (Agilent QTOF6520) to quantify the ratios of isotope-
labeled versus unlabeled signature peptides, as a read-out ofpMHC concentration. Since the
values generated by these different methods were similar, the Bradford assay (using
unconjugated NPs as blanks) became the method of choice for ease and simplicity.
Agonistic activity of pMHC-NPs in vitro
FACS-sorted splenic CD8+ cells from TCR-TG mice (2.5 x105 cells/mL) were
incubated with serially diluted pMHC conjugated or control NPs for 24-48 h at 37°C. The
supernatants were d for IFNy by ELISA. The cultured cells were pulsed with l mCi of
[3H]-thymidine and harvested after 24 h to e [3H] incorporation.
pMHC-NP therapy
Cohorts of 10 wk-old female NOD mice were injected iv. with pMHC-coated NPs
in PBS twice a week for 5 wk (10 doses in total). Increases in the size of tetramer+ CD8+ or
CD4+ T-cell pools in blood, spleen, lymph nodes and/or marrow, as well as their phenotypic
properties, were assessed by flow cytometry as described (Tsai, S. et al. (2010) Immunity
32:568-5 80) (and Clemente-Casares et al., ted). In other experiments, mice displaying
blood glucose levels >11 mM for 2 days were d i.v. twice a wk with pMHC-NP and
monitored for hyperglycemia until stably lycemic (for 4 wk). Animals were also
assessed daily for glycosuria and given human insulin isophane (l IU per day) so. if 3+.
Statistical analyses
Data were compared by two-tailed Student’s t, Mann-Whitney U, Chi-Square, or
two-way ANOVA tests. Statistical cance was assumed at P < 0.05.
Mice
NOD/Lt mice were from the Jackson Lab (Bar Harbor, ME). 17.40L/8.3 (8.3-NOD),
17.6u/8.3u NOD) and BDC2NOD mice have been described (Katz, J.D. et al. (1993)
Cell 74:1089-1100; Verdaguer, J. et al. (1997) J Exp Med 186:1663-1676; Han, B. et al.
(2005) J Clin Invest 115:1879-1887).
e 2. Production of TlD-relevant pMHC class II.
Several different TlD-relevant and irrelevant (i.e., negative control) peptide/I-Ag7
complexes were produced in eukaryotic (S2 or CHO cells). Studies using ers
generated from these monomer preps confirm that these rs are ed into the
supernatant as ly folded pMHC complexes. provides an example.
Reversal of hyperglycemia in NOD mice by treatment with TlD-relevant pMHC class
II-NPs
Diabetic NOD mice were treated twice a wk with 7.5 ug ofpMHC class II-coated-
NPs. Mice were considered cured when lycemic for 4 wk, at which point treatment
was withdrawn. As shown in Fig. 3, whereas 2.5mi/I-Ag7-, IGRP128_145/I-Ag7-, and IGRP4_22/I-
Ag7-NPs reversed hyperglycemia in 90-100% of mice (n=29 mice), treatment with HEL14_22/IAg7-NPs
(a foreign pMHC) had no effect. Intraperitoneal glucose tolerance tests (IPGTTs) in
cured mice >30 wk after treatment withdrawal yielded curves that were very similar to those
in age-matched non-diabetic untreated controls and cantly different than those ed
in untreated acutely diabetic NOD mice (. Thus, NPs coated with levant
pMHC class II restore glucose homeostasis in diabetic mice.
TlD-relevant pMHC class II-NPs expand cognate memory TR1 gulatory CD4+ T
cells
Studies of blood, spleens, pancreatic lymph nodes (PLNs), mesenteric lymph nodes
(MLNs) and bone marrow of 50 wk-old diabetic mice that had been rendered normoglycemic
by treatment with 2.5mi/I-Ag7-NPs ed significantly increased tages of 2.5mi/I-
Ag7 tetramer+ CD4+ cells, as compared to mice studied at diabetes onset or age-matched non-
ic untreated s (. CD4+ T-cell expansion was antigen-specific (.
The tempo, magnitude and distribution of expansion were similar for the three TlD-relevant
pMHC class II-NPs tested (. Phenotypic analyses of the NP-expanded tetramer+ cells
vs. tetramer— cells in all these cohorts revealed a memory-like TR1 phenotype ( top)
with co-expression of the ecific markers described recently (Gagliani, N. et al. (2013)
Nature Medicine 19:739-746) ( bottom): CD6210W/CD44high/ ICOS+/CD25’/FoxP3’
/surface TGFBV CD49b+/LAG3+. That these cells were not FoxP3+ was confirmed in NOD
mice expressing FoxP3 promoter-eGFP, in which all pMHC-NP-expanded cells were eGFP-
negative (not shown).
Consistent with these phenotypic data, tetramer+ CD4+ cells sorted from pMHCNP-treated
mice responded to DCs pulsed with cognate peptide by almost exclusively
secreting IL-10 and, to a lower extent, IFNy (and not shown). Importantly, purified
CD4+ but not CD8+ T cells from pMHC-NP-treated donors ted T1D in NOD.scz'd mice
transferred with diabetogenic splenocytes and hosts treated with pMHC class II-NPs were
100% protected for >100 days (not shown).
These pMHC class II-NP-expanded tetramer+ cells, unlike their er—
counterparts, inhibited the proliferation of non-cognate T-cells to peptide-pulsed DCs
(presenting the peptides targeted by both the responder and tetramer+ TR1 cells). on of
an anti-IL10 or anti-TGFB mAbs to the cultures partially inhibited the suppression, versus
es receiving anti-IFNy or rat-IgG (not shown). Most importantly, studies of diabetic
mice treated with IGRP4_22 or 2.5mi/I-Ag7-NPs and blocking anti-IL-lO, anti-TGFB or anti-
IFNy mAbs or rat-IgG ( indicate that restoration of normoglycemia by pMHC class
II-NPs requires IL-10 and TGFB but not IFNy. However, studies in spontaneously diabetic
I 07/7 and NOD.Ifng’/’ mice suggest that expression of both IL-lO and IFNy are
necessary for development of the TRl cells that expand in response to pMHC class II-NPs; in
these mice, P therapy expanded Th2-like cells (NOD.Ifng’/’) or IFNy+/IL-4+/IL10’
cells (NOD.IZI OT/Tmice). Studies in ic IGRP’/’ NOD mice (unable to prime IGRP-
reactive T cells) showed that these mice did not respond to IGRP4_22/I-Ag7-NPs (there was no
T cell expansion or restoration of normoglycemia) because these mice lacked IGRP4_22-
primed cells. In contrast, all the diabetic IGRP’P NOD mice treated with 2.5mi/I-Ag7-NPs
cured (not shown). Thus, pMHC class II-NPs, like pMHC class I-NPs, operate by expanding
disease-primed regulatory , but cannot prime these ses de novo because they
lack co-stimulatory signals.
Lastly, studies with vaccinia virus (rVV) showed that pMHC class II-NP-treated
NOD mice can readily clear an acute viral infection (A). In agreement with this,
d mice can mount antibody responses t a model antigen in adjuvant (B).
Example 3. Monospecific pMHC class II-NPs decrease the severity of EAE.
Applicant then tested the therapeutic potential of a pMHC class II-based
nanomedicine in Experimental Autoimmune Encephalomye-litis (EAE). This model was
utilized in the most stringent test possible: to igate ifpMHC-NPs can reverse
ished EAE as opposed to prevent or blunt its development. This is not a trivial issue. A
recent review of interventions in EAE shows that <l% of over 400 studies initiated treatment
21 days after EAE induction , J. et al. (2006) Nat Protoc 1:406-417); the reported data
were obtained in mice in which treatment was initiated 21 days after EAE induction and
improved disease scores in a dose-dependent manner ().
Example 4. Synthesis and quality control of pMHC class II-coated NPs.
Applicant developed an optimized iron oxide NP design that does not employ
surfactants for synthesis and yields highly stable, monodispersed ations that can be
loaded with optimal pMHC loads. Although several different pMHC-coating chemistries can
be used (A), Applicant regularly uses NPs functionalized with maleimide-conjugated
PEGs, which accept high valencies ofpMHCs engineered to encode a free Cys at their
carboxyterminal end (up to more than 60 pMHCs/NP). These pMHC class II-NPs are
processed h l quality control checks to define pMHC valencies per NP (dot-
ELISA, amino acid analysis), NP density, NP charge and NP size (metal core, as defined by
WO 63616
TEM; and hydrodynamic diameter, as defined via dynamic light scattering (DLS)). FIG. IZB
shows a representative TEM image and C shows DLS profiles ofpMHC-uncoated
vs. coated NPs. A typical dosing regimen involves the administration of 1-50 ug of total
pMHC ated) per dose (about 2 uL of the preparation diluted in 100 uL of PBS).
Example 5. Treatment with pMHC class II-coated NPs.
The above data are consistent with data Applicant previously obtained in mice
treated with pMHC class I-NPs: pMHC class II-NPs expand cognate memory regulatory T
cells (in this case TRl) that ss the presentation of other autoantigenic peptides by local
autoantigen-loaded APCs (Amrani, A. et al. (2000) Nature 406:739-742).
Human TRl CD4+ T-cell clones have been reported to kill certain subsets of
professional APCs, such as dendritic cells (DCs) (Amrani, A. et al. (2000) Nature 406:739-
742). Applicant therefore investigated whether the antigen-specific TRl cells that expand in
response top MHC class II-NP therapy suppressed autoimmunity by killing tigen-
loaded APCs. This was done by tranfusing 1:1 mixtures of DCs pulsed with 2.5mi or GPI
peptides and labeled with PKH26 (2.5mi-pulsed DCs) or CFSE (GPI-pulsed DCs), into NOD
mice that had received 10 doses of 2.5mi/1Ag7-NPs during the ing 5 weeks, or NOD
mice that had not received any treatment. The hosts were sacrificed 7 days later to compare
the ratios of PKH26+ vs CFSE+ cells in the two different hosts. As shown in A (top
panels), no differences were observed, suggesting that the TRl CD4+ T-cells that expanded
in response to P therapy do not kill n-expressing DCs.
To investigate whether this was a peculiarity of the type ofAPC used (a DC) or a
general feature of other APC types, the above ments were ed but using splenic B-
cells as opposed to DCs. Unexpectedly, it was found that the numbers of 2.5mi-pulsed B-
cells expanded (rather than decreased) in hosts that had been treated with 2.5mi/IAg7-NPs-
coated NPs (A, bottom panels). This was cted because, based on the state-of-
the-art, it was expected just the opposite outcome (a selective and specific decrease of 2.5mi-
pulsed B-cells as compared to their GPI-pulsed rparts).
Applicant then ascertained whether such a B-cell-expanding effect ofpMHC class
II-NP treatment could be nted by comparing the absolute numbers and percentages of
B-cells in the pancreas-draining (PLN) and non-draining (MLN) lymph nodes of mice treated
with 2.5mi/IAg7-NPs versus untreated controls. As shown in B, pMHC class II-NP-
treated NOD mice had a marked increase in the percentage of B-cells in the PLN but not
MLN. No such differences were seen in the PLN vs MLN of untreated NOD mice, indicating
that these effects were a consequence ofpMHC-NP y. Notably, there was a statistically
significant correlation between the frequency of 2.5mi-specif1c TRl CD4+ T-cells in the
PLNs of individual mice and the frequency of PLN-associated B-cells, suggesting that such
an increased recruitment of B-cells to the PLNs of the pMHC-NP-treated NOD mice was
driven by the 2.5mi-specif1c TRl CD4+ T-cells that expanded in se to MHC-NP
therapy.
Collectively, these data raised the possibility that the B-cells that expanded in
response to MHC-NP therapy might be B-regulatory cells, that is B-cells that acquire the
capacity to produce IL-lO in response to cognate interactions with the P-expanded
TRl CD4+ T-cells. This case scenario posits that 2.5mi-specif1c TRl CD4+ T-cells would
induce the differentiation and expansion of undifferentiated chromogranin A-specif1c B-cells
(chromogranin A is the natural antigenic source of the 2.5mi epitope) that have captured
chromogranin A and therefore present the corresponding 2..’5mi/IAg7 pMHC complexes on
their surface, to IL-lO-producing Breg cells.
To test this hypothesis, Applicant transfused 2.5mi- or GPI-pulsed B-cells (labeled
with PKH26) from a strain ofNOD mice in which one of its two ILlO loci carries a ed
insertion of an IRES-eGFP cassette n the stop codon and polyadenylation signal of
exon 5 (ll), into 2.5mi/1Ag7-NP-treated or untreated NOD hosts.
Seven days after transfer, the flow cytometric phenotype of the donor PKH26+ B-
cells in the hosts (C, top panel) was determined. As shown in C e and
bottom panels), a significant fraction of the donor B-cells expressed ILlO-encoded eGFP and
were both CD5+ and gh. These are three key markers of Breg cells (Xie, J. et al.
(2007) Adv Mater 19:3163; Xie, J. et al. (2006) Pure Appl. Chem. 78: 014). This was
only seen with s pulsed with 2.5mi, but not with B-cells pulsed with a negative control
e (GPI), and it only occurred in pMHC-NP-treated mice. Importantly, this effect was
mediated, at least in part, by the IL-lO pMHC-NP-expanded TRl CD4+ s, because no
such response was observed in IL-lO-deflcient NOD hosts.
Taken together, these data demonstrate that pMHC class II-NP therapy induces the
differentiation and expansion of antigen-specific B-cells into B-regulatory cells.
The description of data suggesting the existence of B lymphocytes with regulatory
properties can be found in literature dating back to 1974. Similarly to the TRl CD4+ T-cells
that expand in response to pMHC class II-NP therapy, Breg cells express immunosuppressive
cytokines, including IL-10 and TGFb, as well as other molecules that can inhibit pathogenic
autoreactive T- and B-cells in an antigen-dependent and highly specific manner, via cognate,
pMHC class II-driven cell-to-cell interactions (Xu, C. et al. (2007) Polymer International
56:821-826). Although ent stimuli have been shown to be able to induce Breg
formation in vitro, and to a much lesser extent in vivo, to the best of Applicant’s knowledge
there is currently no therapeutic approach capable of ng and expanding antigen-specific
Breg cells in vivo. By eliciting highly disease-specific TR1 CD4+ T-cells, Applicant
trates that pMHC class II-based nanomedicines also elicit disease-specific Breg cells.
Since Breg cells can also promote the differentiation of effector into TR1 CD4+ T-cells,
pMHC class II-based nanomedicines unleash a profound and sustained immunosuppressive
response that is highly n-specific and therefore capable of selectively suppressing
autoimmune responses without compromising systemic immunity.
Example 6. Synthesis of surface functionalized iron oxide nanoparticle by thermal
decomposition of iron acetonate, and bioconjugation thereof
PEG is melted. Benzyl ether and iron acetyle acetonate is added. After 1 hr of
heating at 105°C, the ature is increased to 2600C and refluxed. After about 2 hr, iron
rticles form and the color of the solution turns black. The reaction is cooled down to
room temperature and some water added to extract nanoparticles from the reaction vessel.
The nanoparticles are purified by Miltenyi Biotec LS magnet column. The making of iron
oxide nanoparticle protein ates include adding n and the iron nanoparticle at a
buffered pH of 6.2-6.5 (0.15M NaCl and 2mM EDTA), stirring at room temperature for 12-
14 hours, and purifying protein conjugated particle by Miltenyi Biotec LS magnet column.
It should be tood that although the present invention has been specifically
disclosed by preferred embodiments and optional features, modification, improvement and
variation of the ions embodied therein herein disclosed may be ed to by those
skilled in the art, and that such modifications, improvements and ions are considered to
be within the scope of this ion. The materials, methods, and examples provided here
are representative of preferred embodiments, are exemplary, and are not intended as
tions on the scope of the invention.
The ion has been described broadly and generically herein. Each of the
narrower species and subgeneric groupings falling within the generic disclosure also form
part of the ion. This includes the generic description of the invention with a proviso or
negative limitation removing any subject matter from the genus, regardless of whether or not
the excised material is specifically d herein.
In addition, where features or aspects of the invention are described in terms of
Markush groups, those d in the art will recognize that the invention is also thereby
described in terms of any individual member or subgroup of s of the Markush group.
The use of the term “or” in the claims is used to mean “and/or” unless explicitly
indicated to refer to alternatives only or the alternatives are mutually exclusive, although the
disclosure supports a definition that refers to only atives and “and/or.”
As used in this specification and claim(s), the words “comprising” (and any form of
comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as
“have” and “has”), “including” (and any form of including, such as “includes” and “include”)
or ining” (and any form of containing, such as “contains” and “contain”) are inclusive
or open-ended and do not exclude additional, unrecited elements or method steps.
Throughout this disclosure, various publications, patents and hed patent
specifications are referenced by an identifying citation. All publications, patent applications,
patents, and other references mentioned herein are expressly incorporated by reference in
their ty, to the same extent as if each were incorporated by reference dually. In
case of conflict, the present specif1cation, including def1nitions, will control.
Table 1. onalized PEG linkers
Nana-n afiéfie-
-. \ a
makmgtcmfi ‘ "
Mai—FES— wuse
Slap-\m‘nniv PES-
nwéiufi-fise
' " \cpaxitie
Table 2. Nanoparticle designs and pMHC-binding capacity.
iflfi Syméaesas mag-E: {ma-aisugaiésm
RFEEEi§3§a§fi=fi§
figags’egatian
we Rammm
agifiw
'1‘) m 3%E}
(-1!
Claims (15)
1. A method for making PEG functionalized iron oxide nanoparticles comprising thermally decomposing iron acetyl acetonate in the presence of functionalized PEG molecules or the ce of functionalized PEG molecules and benzyl ether, and wherein the temperature of the thermal decomposition is n 80 to 300° C, and wherein the functionalized PEG is a maleimide functionalized PEG.
2. The method of claim 1, wherein the iron oxide nanoparticle is soluble.
3. The method of claim 1, wherein the thermal decomposition comprises a single-step on.
4. The method of claim 3, wherein the thermal decomposition is carried out in the presence of benzyl ether.
5. The method of claim 1, n the temperature for the thermal osition is 80 to about 200°C.
6. The method of claim 1, wherein the thermal decomposition is carried out for about 1 to about 2 hours.
7. The method of claim 1, wherein the nanoparticles are stable at about 4°C in PBS without any detectable degradation or aggregation.
8. The method of claim 7, wherein the rticles are stable for at least 6 months.
9. The method of any one of claims 1-8, wherein the method further comprises ing the nanoparticles with a magnetic column.
10. A method for making major histocompatibility complex peptide (pMHC) nanoparticle complexes comprising: a) thermally decomposing iron acetyl acetonate in the ce of functionalized PEG molecules or the presence of functionalized PEG molecules and benzyl ether, wherein the temperature for the thermal decomposition is between 80 to 300 ºC, thereby providing PEG functionalized iron oxide nanoparticles; and b) contacting the PEG functionalized iron oxide nanoparticles with pMHC thereby providing pMHC nanoparticle complexes, and wherein the functionalized PEG is a maleimide functionalized PEG.
11. The method of claim 10, wherein the method further comprises purifying the nanoparticles with a magnetic .
12. The method of claim 1, wherein the temperature for the thermal decomposition is 80 to about 150°C.
13. The method of claim 1, n the temperature for the thermal decomposition is about 100 to about 250°C.
14. The method of claim 1, wherein the temperature for the thermal decomposition is about 100 to about 200°C.
15. The method of claim 1, wherein the temperature for the thermal decomposition is about 150 to about 250°C W0
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201361899826P | 2013-11-04 | 2013-11-04 | |
US61/899,826 | 2013-11-04 | ||
PCT/IB2014/003014 WO2015063616A2 (en) | 2013-11-04 | 2014-11-03 | Methods and compositions for sustained immunotherapy |
Publications (2)
Publication Number | Publication Date |
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NZ719771A NZ719771A (en) | 2020-11-27 |
NZ719771B2 true NZ719771B2 (en) | 2021-03-02 |
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