U.S. patent application number 17/542820 was filed with the patent office on 2022-07-28 for therapeutically triggering an innate immune response in a target tissue.
The applicant listed for this patent is Qu Biologics Inc.. Invention is credited to Mark Bazett, Momir Bosiljcic, Harold David Gunn, Boyko Traychev Kabakchiev, Shirin Kalyan, Dermot McGovern, David W. Mullins, Ho Pan Sham, Marcel Thalen, Monan Angela Zhang.
Application Number | 20220236284 17/542820 |
Document ID | / |
Family ID | |
Filed Date | 2022-07-28 |
United States Patent
Application |
20220236284 |
Kind Code |
A1 |
Gunn; Harold David ; et
al. |
July 28, 2022 |
Therapeutically Triggering an Innate Immune Response in a Target
Tissue
Abstract
The invention provides therapeutic compositions that present an
artificial repertoire of mammalian pattern recognition receptor
(PRR) agonists, so that the pattern of PRR agonists recapitulates a
distinct portion of a PRR agonist signature of a mammalian
pathogen. The artificial repertoire of PRR agonists may be
formulated together in a therapeutic vehicle for combined
presentation to an innate immune cell resident in a target tissue
in a mammalian host, and the vehicle adapted to deliver the PRR
agonists to the target tissue, so as to modulate an immune
response.
Inventors: |
Gunn; Harold David;
(Vancouver, CA) ; Mullins; David W.; (Norwich,
VT) ; Kalyan; Shirin; (Burnaby, CA) ;
Bosiljcic; Momir; (Burnaby, CA) ; Zhang; Monan
Angela; (Burnaby, CA) ; Bazett; Mark;
(Burnaby, CA) ; Thalen; Marcel; (Burnaby, CA)
; McGovern; Dermot; (Los Angeles, CA) ;
Kabakchiev; Boyko Traychev; (Guelph, CA) ; Sham; Ho
Pan; (Burnaby, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Qu Biologics Inc. |
Burnaby |
|
CA |
|
|
Appl. No.: |
17/542820 |
Filed: |
December 6, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16096120 |
Oct 24, 2018 |
11226340 |
|
|
PCT/CA2017/050513 |
Apr 26, 2017 |
|
|
|
17542820 |
|
|
|
|
62327953 |
Apr 26, 2016 |
|
|
|
62385798 |
Sep 9, 2016 |
|
|
|
62395783 |
Sep 16, 2016 |
|
|
|
62421511 |
Nov 14, 2016 |
|
|
|
62442759 |
Jan 5, 2017 |
|
|
|
62457618 |
Feb 10, 2017 |
|
|
|
62472394 |
Mar 16, 2017 |
|
|
|
International
Class: |
G01N 33/68 20060101
G01N033/68; C12Q 1/6883 20060101 C12Q001/6883 |
Claims
1-8. (canceled)
9. The method according to claim 36, wherein the therapeutic
vehicle further comprises one or more of: GMCSF, vitamin D, NOHA,
alph1 antitrypsin, glutathione, an isoprenoid, or
.alpha.-galactosylceramide.
10-19. (canceled)
20. The method according to claim 36, wherein therapeutic vehicle
is administered at an administration site that is the skin or
subcutaneous tissue.
21-24. (canceled)
25. The method according to claim 36, wherein the therapeutic
vehicle is administered in a plurality of doses over a dosage
duration, and the dosage duration is at least two weeks.
26. The method according to claim 25, wherein the doses are
administered subcutaneously every day, or every other day.
27. (canceled)
28. The method according to claim 36, wherein the patient is
immunosuppressed or immunocompromised.
29. The method according to claim 36, wherein the patient is a
geriatric patient.
30. The method according to claim 36, wherein the patient is a
pediatric patient.
31-32. (canceled)
33. The method of claim 36, wherein the Klebsiella spp. is a K.
variicola or K. pneumonia that is a pathogenic strain of K.
variicola or K. pneumonia.
34-35. (canceled)
36. A method of treating neutropenia in a human patient, comprising
administering to the patient an effective amount of a therapeutic
vehicle, comprising a whole killed or attenuated cell of a
Klebsiella spp.
37. The method of claim 36, wherein the neutropenia is caused by a
myelosuppressive chemotherapy.
38-73. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/096,120 filed Oct. 24, 2018, now issued as
U.S. Pat. No. 11,226,340, which application is a U.S. National
Stage Application of PCT Application No. PCT/CA2017/050513 filed
Apr. 26, 2017, which application, pursuant to 35 U.S.C. .sctn.
119(e), claims priority to the filing dates of U.S. Provisional
Application No. 62/327,953 filed Apr. 26, 2016, U.S. Provisional
Application No. 62/385,798 filed Sep. 9, 2016, U.S. Provisional
Application No. 62/395,783 filed Sep. 16, 2016, U.S. Provisional
Application No. 62/421,511 filed Nov. 14, 2016, U.S. Provisional
Application No. 62/442,759 filed Jan. 5, 2017, U.S. Provisional
Application No. 62/457,618 filed Feb. 10, 2017, and U.S.
Provisional Application No. 62/472,394 filed Mar. 16, 2017, which
applications are incorporated herein by reference in its
entirety.
FIELD
[0002] Innovations are disclosed in the field of medical and
veterinary science, relating to preparations that contain
immunogens, such as microbial components. The preparations are
formulated for medical purposes, and methods of using the
preparations in therapy are provided.
BACKGROUND
[0003] There is growing recognition that immunological
dysregulation, an imbalance between immune response and immune
tolerance, is not only a primary factor in allergic and autoimmune
disease, it also has an underlying mechanistic role in a wide
variety of pathologies, including cancer (see Mills et al., 2016,
Cancer Res; 76(3); 1-4), metabolic disease (obesity, diabetes),
degenerative disease (Alzheimer's, Parkinson's, Amyotrophic Lateral
Sclerosis, osteoporosis), respiratory and cardiovascular disease
(see Immune Rebalancing, 1st Edition: The Future of
Immunosuppression, 2016, Boraschi and Penton-Rol Eds, Academic
Press).
[0004] In vertebrates, an important aspect of immunological
regulation involves the concerted activity of the innate immune
system and the adaptive immune system. This concerted activity
involves metabolic, enzymatic and molecular genetic changes within
immune cells, orchestrating an elaborate system of cellular,
cytokine and chemokine communication pathways mediating the
coordinated activity of the disparate components of these
complementary systems (see Iwasaki & Madzhitov, 2015, Nature
Immunology 16:343-353; WO0209748; WO03051305; Turner et al., 2014,
BBA-Molecular Cell Research 1843:11 2563-2582). An aspect of this
coordinated activity underlies the recognition that ligands of the
pattern recognition receptors (PRRs) of the innate immune system
may be used as vaccine adjuvants to improve an adaptive immune
response (see Maisonneuve et al., 2014, PNAS 111(34), 12294-9;
WO2007035368).
[0005] Immunological memory, involving the recognition of specific
antigens by B and T cell receptors, is a long recognized and
central feature of the adaptive immune system, and the basis for
vaccine efficacy (see Nature Immunology, Focus on immunological
memory: June 2011, Volume 12 No 6 pp 461-575). Innate immune memory
is a more recently recognized and less well understood
characteristic of the immune system (see Netea et al., 2015, Nature
Immunology 16, 675-679; and Bordon, 2014, Nature Reviews Immunology
14, 713).
[0006] A wide variety of innate and adaptive immune cells are
understood to be resident in non-lymphoid tissues, with diverse
roles in tissue homeostasis (see Nature Immunology, Focus on
tissue-resident leukocytes, October 2013, Volume 14 No 10 pp
977-1100). The complexities of this homeostasis are evident in the
observation that even the ontogeny of tissue resident immune cells
may in some cases be distinct from the ontogeny of similar immune
cells that are not tissue resident (Italiani and Boraschi,
Frontiers in Immunology, October 2014, Vol 5, article 514).
SUMMARY
[0007] Immunomodulatory or immunogenic compositions are provided
that constitute an artificial repertoire of mammalian pattern
recognition receptor (PRR) agonists. The PRR agonist repertoire is
selected so that it in effect recapitulates a distinct portion of a
PRR agonist signature of a microbial pathogen, and more
specifically a pathogen that is pathogenic in a selected target
tissue. The PRR agonist signature is distinct in the sense that it
is different from PRR agonist signatures of microbes that are not
pathogenic in the target tissue, and it is also distinct in the
sense that it is different from the native PRR agonist signature of
the wild-type pathogen. This distinct artificial repertoire of PRR
agonists may then be formulated so that the PRR agonists are
presented together in a therapeutic vehicle, for example so that
the PRR agonist repertoire may be presented in combination. The
therapeutic vehicle may for example be a recombinant microbe, a
cellular fraction of a microbial cell, a microparticle or a
liposome. The composition may for example comprise microbial
agonists for at least a minimum number of distinct mammalian PRRs,
for example at least 5, as described in more detail herein. The
vehicle may then be delivered, for example systemically, so that
the PRR agonist repertoire is presented to an innate immune cell
resident in the target tissue in a host, such as a mammalian host.
The therapeutic vehicle may for example aggregate the artificial
repertoire of PRR agonists, so that the proximity of the plurality
of PRR agonists is maintained during systemic distribution in a
host. Compositions of this kind may be used to treat a wide variety
of diseases characterized by immune dysregulation, including
neoplastic diseases and auto-immune diseases.
[0008] Aspects of the innovation involve the use of an immunogenic
composition in methods of treating an immune dysregulation in a
target tissue in a mammalian host, wherein the composition
comprises the foregoing artificial repertoire of mammalian PRR. The
artificial repertoire of PRR agonists may be formulated together in
a therapeutic vehicle for combined presentation following
administration to a mammalian host. Composition may for example
include components of the microbial mammalian pathogen that are
agonists for a select number of distinct mammalian PRRs, as
discussed in more detail below, for example at least 5.
Compositions may for example be adapted for use so as to modulate
an innate immune response in the target tissue. The therapeutic
vehicle may for example include a recombinant microbe, a cellular
fraction of the recombinant microbe, a cellular fraction of a
microbial cell, a microparticle or a liposome, each comprising
components of the microbial mammalian pathogen that provide the PRR
agonists that together make up the artificial repertoire of PRR
agonists. A recombinant microbe may for example include a
recombinant gene encoding a component of at least one of the PRR
agonists. In select aspects, the therapeutic vehicle may for
example include a whole killed or attenuated cell of the
recombinant microbe. Alternatively, the cellular fraction of the
microbial mammalian pathogen may be used, for example, a bacterial
outer membrane fraction; a bacterial inner membrane fraction; a
pellet from a gradient centrifugation of microbial cell components;
or chromosomal DNA. The therapeutic vehicle may for example be
formulated for use for delivering the PRR agonists to the target
tissue.
[0009] In select embodiments, the PRRs and the corresponding PRR
agonists may for example be selected from the group consisting
of:
TABLE-US-00001 PRR PRR Agonist TLR2 Microbial cell wall
components/preparations, Pam2C- Aca-Benzyl-Murabutide (Pam2C-
conjugated murabutide) TLR3 Polyadenylic-polyuridylic acid,
Polyinosine-polycytidylic acid TLR4 Lipopolysaccharide,
Monophosphoryl Lipid A TLR5 Flagellin TLR7/8 Single-stranded RNAs,
Nucleoside analogs, Imidazoquinolines/Thiazoquinolines TLR9
unmethylated CpG DNA motifs NOD1 iE-DAP, Acylated iE-DAP, D-gamma-
Glu-mDAP, L-Ala-gamma-D-Glu-mDAP NOD2 MDP (MurNAc-L-Ala-D-isoGln,
muramyl dipeptide), N-glycolylated muramyldipeptide,
N-Acetyl-muramyl- L-Alanyl-D-Glutamin-n-butyl-ester,
MurNAc-Ala-D-isoGln-Lys, N- Acetylmuramyl-L-Alanyl-D- Isoglutamine
(L-D isoform), 6-O- stearoyl-N-Acetyl-muramyl-L-alanyl-D-
isoglutamine, Pam2C-Aca-Benzyl- Murabutide, TLR2/NOD2
Pam2C-conjugated murabutide NOD1/NOD2 PGN, Pam2C-conjugated
murabutide RIG1/MDA5 5' triphosphate double stranded RNA
(18-20mer), polyriboinosinic:polyribocytidylic acid DAI, LRRFIP1,
AIM2, dsDNA, poly(dA-dT).cndot.poly(dT-dA) RIG1 Dectin-1
Beta-glucan peptide, fungal cell wall preparations Mincle damaged
microbial cells, fungus, yeast and mycobacteria, Trehalose-6,6-
dibehenate, trehalose-6,6-dimycolate STING Cyclic dinucleotides
(c-di-nucleotides), xanthenone derivatives, 3'3'-cGAMP, 2'3'-cGAMP,
2'2'-cGAMP, 2'2'-cGAMP, c-di-AMP (cyclic di-adenylate
monophosphate), c-di-GMP, c-di-IMP, c-di-UMP, c-di-AMP RIG-I
PPP-ssRNA (PPP-ssRNA, ssRNA with a 5'-triphosphate group), RNA with
base pairing and polyl:C MDA5 Long dsRNA LGP2 dsRNA DDX41 B-form
DNA and CDNs (cyclic dinucleotides) DHX9 DNA, RNA, CpG-A
oligodeoxynucleotids and CpG-B ODNs DDX3 Viral RNA DHX36 DNA, RNA,
CpG-A oligodeoxynucleotids and CpG-B oligodeoxynucleotids
DDX1-DDX21-DDX36 RNA and polyl:C DDX60 ssRNA, dsRNA and dsDNA KU70
DNA cGAS DNA STING CDNs (c-di-GMP and c-di-AMP) NOD2 ssRNA NLRP3
ssRNA, dsRNA, bacterial mRNA and oxidized mitochondrial DNA AIM2
DNA IFI16 dsDNA LRRFIP1 B-form DNA, Z-form DNA and dsRNA DAI DNA
IFIT1, 2, 3 and 5 PPP-ssRNA
[0010] The therapeutic vehicle may for example include additional
therapeutic moieties, such as one or more of: GMCSF, vitamin D,
NOHA, alph1 antitrypsin, glutathione, an isoprenoid, or
.alpha.-galactosylceramide. In alternative embodiments, the
therapeutic vehicle further comprises an antigen, such as a cancer
antigen. Alternatively, the therapeutic vehicle may further include
a heterologous PRR agonist, such as a PRR agonist that is not a
component of the microbial mammalian pathogen.
[0011] The subject of treatment, such as a mammalian host or human
patient, may for example be suffering from a disease or condition
characterized by the immune dysregulation in the target tissue,
such as a cancer or an inflammatory disorder.
[0012] The composition may be adapted for use in an amount
effective to modulate a biomarker, for example one or more of PD1,
PDL1, IP-10, MIG, RANTES, neutrophils, Ly6C monocytes, and NKG2D.
In select embodiments, the composition may for example be adapted
for use in an amount effective to down-regulate PD1 and/or PDL1
expression in cells present in the target tissue. The composition
may accordingly be adapted for use so as to modulate an adaptive
immune response in the host, for example as a concomitant of
modulating an innate immune response.
[0013] The therapeutic vehicle is for administration at an
administration site that is not the target tissue, and the site may
for example be the skin, subcutaneous tissue, the respiratory
tract. Administration may be enteric, or non-enteric. The
therapeutic vehicle may be formulated for systemic distribution of
the PRR agonists following administration at a localized
administration site. The therapeutic vehicle may be administered in
a plurality of doses over a dosage duration, and the dosage
duration may for example be at least two weeks, or any of other
wide range of dosage regimens disclosed herein or known in the
art.
[0014] In select embodiments, human patient treated in accordance
with the invention may for example be immunosuppressed or
immunocompromised, or may be geriatric or pediatric patients.
[0015] The therapeutic uses recited herein are reflected in
corresponding methods of treatment, and vice versa.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a schematic timeline of a site specific
immunotherapy (SSI) in accordance with one aspect of the invention,
illustrating intra-tracheal (IT) instillation of a K. pneumoniae
(KPN) whole killed cell SSI at day -31, and subcutaneous (SQ)
injections of SSI or saline (placebo) every other day starting on
day -10, with intravenous (IV) Lewis lung carcinoma (LLC)
administration on day 0, followed by sacrifice (sac) on day 18.
[0017] FIG. 2 is a graph illustrating therapeutic efficacy of
alternative SSI formulations in a murine cancer model.
[0018] FIG. 3 is a schematic timeline illustrating a murine
pre-infection model of SSI-mediated anti-tumour efficacy.
[0019] FIG. 4 is a graph illustrating anti-cancer efficacy of an
SSI after pre-infection in a murine Lewis lung carcinoma (LLC)
cancer model.
[0020] FIG. 5 is a line graph illustrating tumour volume over time
for alternative SSI therapies in a murine B16 skin cancer model.
10.times.QBSAU and 1.times.QBSAU are denoted as QBSAUR and QBSAU,
respectively, herein.
[0021] FIG. 6 is a bar graph illustrating tumour volume at day 7
for alternative SSI therapies in a murine B16 skin cancer
model.
[0022] FIG. 7 is a bar graph illustrating tumour volume at day 8
for alternative SSI therapies in a murine B16 skin cancer
model.
[0023] FIG. 8 is a bar graph illustrating tumour volume at day 10
for alternative SSI therapies in a murine B16 skin cancer
model.
[0024] FIG. 9 is a bar graph illustrating tumour volume at day 12
for alternative SSI therapies in a murine B16 skin cancer
model.
[0025] FIG. 10 is a bar graph illustrating tumour volume at day 14
for alternative SSI therapies in a murine B16 skin cancer
model.
[0026] FIG. 11 is a schematic illustration, top panel, showing an
SSI administration schedule, and a bar graph, bottom panel,
illustrating therapeutic efficacy of an SSI in a murine cancer
model.
[0027] FIG. 12 is a chart illustrating the efficacy of various SSI
co-formulations in a murine cancer model.
[0028] FIG. 13 is an alternative bar graph representation of the
efficacy of various SSI co-formulations in a murine cancer
model.
[0029] FIG. 14 is a series of graphs illustrating efficacy of SSI
treatment in alternative model animals in the colitis model: a
logarithmic Y axis scale illustrating relative levels of IFN-gamma
(A) and IL-17A expression (B), and cumulative data for IL-17A
expression (C), as well as site-specific evidence of QBECO efficacy
in increasing IL-18 gene expression in colon tissue, compared to
QBKPN (D).
[0030] FIG. 15 is a series of graphs illustrating efficacy of SSI
treatment in alternative model animals in the colitis model:
microbiome (A and B) and histology (C).
[0031] FIG. 16 is a bar graph illustrating efficacy of an SSI in a
murine asthma/allergy model.
[0032] FIG. 17 includes two bar graphs illustrating efficacy of an
SSI in a murine asthma/allergy model, showing counts of A)
Eosinophils, B) Lymphocytes.
[0033] FIG. 18 includes two bar graphs illustrating efficacy of an
SSI in a murine asthma/allergy model, showing A) IL-4 and B) IL-5
concentrations.
[0034] FIG. 19 is a bar graph illustrating results of ex-vivo
imaging of Cy5.5 labelled KPN SSI (QBKPN) measured in organs
(heart, lungs and spleen) 24 hours after a third SQ SSI
injection.
[0035] FIG. 20 is a bar graph illustrating house dust mite
(HDM)-specific IgE responses following saline or HDM exposure,
treated with either Placebo or QBKPN.
[0036] FIG. 21 is a series of bar graphs (A-E) illustrating aspects
of an anti-inflammatory SSI treatment for asthma from an animal
model, particularly BAL cell counts and differentials in Saline or
HDM exposed mice treated with Placebo or QBKPN SSI.
[0037] FIG. 22 includes two bar graphs illustrating aspects of an
anti-inflammatory SSI treatment for asthma from an animal model,
particularly serum (A) and BAL (B) mediators that are linked to
eosinophilia.
[0038] FIG. 23 is a series of bar graphs illustrating aspects of an
anti-inflammatory SSI treatment for asthma from an animal model,
particularly Th1 (A) and Th2 (B and C) lung gene expression
following HDM exposure and QBPKN treatment.
[0039] FIG. 24 is a series of bar graphs illustrating aspects of an
anti-inflammatory SSI treatment for asthma from an animal model,
particularly the effects of HDM exposure and QBKPN treatment on
Th1- (A-C) and Th2- (D-F) mediated BAL fluid cytokine levels.
[0040] FIG. 25 is a graph illustrating aspects of an
anti-inflammatory SSI treatment for asthma from an animal model,
particularly a principal component analysis (PCA) of BAL cytokines
showing partial normalization of overall cytokine profile.
[0041] FIG. 26 is a bar graph illustrating aspects of an
anti-inflammatory SSI treatment for asthma from an animal model,
particularly illustrating airway goblet cell quantification
following HDM exposure and QBPKN treatment.
[0042] FIG. 27A is a bar graph illustrating aspects of an
anti-inflammatory SSI treatment for COPD from an animal model,
particularly BAL cell differential. FIG. 27B reflects this data,
illustrating that a KPN SSI intervention attenuated cigarette smoke
exposure induced increases in lung macrophages and lymphocytes but
not total cells or neutrophils. FIG. 27B illustrates BAL cell
counts and differentials following placebo and KPN SSI treatment in
filtered air or cigarette smoke-exposed groups: (a) BAL total
cells, (b) lymphocytes, (c), macrophages (d), and neutrophils.
*p<0.05 comparing to the groups relative control; #p<0.05
comparing KB group to relative placebo control. Data are
means.+-.SD of 9-10 mice per group.
[0043] FIGS. 28A-28D: FIG. 28A illustrates data showing that a KPN
SSI intervention attenuated cigarette smoke exposure induced
increases Th1-skewed lung inflammatory responses, as follows. BAL
supernatant fluid analysis following placebo and KB treatment in
filtered air or cigarette smoke-exposed groups. (a) IFN.gamma., (b)
CXCL9, (c) CXCL10, (d) CCL5, (e) IL-6, (f) G-CSF, (g) CXCL1, (h)
IL-17. *p<0.05 comparing to the groups relative control;
#p<0.05 comparing KPN SSI group to relative placebo control.
Data are means.+-.SD of 10 mice per group. FIG. 28B provides data
illustrating that KPN SSI intervention differentially modulates
cigarette smoke exposure induced changes in serum immune mediators,
as follows. Serum analysis following placebo and KPN treatment in
filtered air or cigarette smoke-exposed groups: (a) VEGF, (b)
IL-1.beta., (c) CCL2, (d) CXCL9, (e) CXCL10 and (f) CCL5.
*p<0.05 comparing to the groups relative control; #p<0.05
comparing KB group to relative placebo control. Data are
means.+-.SD of 9-10 mice per group. FIG. 28C provides data
illustrating that KPN SSI intervention increased blood and lung
Ly6CHI monocytes and neutrophils, as follows. Flow cytometric
analysis of blood (a-b) and lung (c-d) Ly6C.sup.HI monocytes and
neutrophils following placebo and KB treatment in filtered air or
cigarette smoke-exposed groups. *p<0.05 comparing to the groups
relative control. #p<0.05 comparing KB group to relative placebo
control. Data are means.+-.SD of 10 mice per group. FIG. 28D is
series of bar graphs (A-C) illustrating aspects of an
anti-inflammatory SSI treatment for COPD from an animal model,
particularly select lung gene expression profiles.
[0044] FIG. 29 is a series of bar graphs (A-G) illustrating aspects
of an anti-inflammatory SSI treatment for COPD from an animal
model, particularly select BAL cytokine expression profiles.
[0045] FIG. 30 is a series of bar graphs (A-C) illustrating aspects
of an anti-inflammatory SSI treatment for COPD from an animal
model, particularly serum cytokine expression profiles.
[0046] FIG. 31 is a bar graph illustrating reduced tumour burden in
a B16 melanoma model of metastases to the lung using a Klebsiella
variicola SSI.
[0047] FIG. 32 is a bar graph illustrating QBKPN SSI efficacy in
reducing lung nodules in the absence of CD25 positive cells.
[0048] FIG. 33 includes three bar graphs: FIG. 33A is a bar graph
illustrating delta C.sub.t (cycle threshold) values associated with
a KPN SSI formulation (QBKPN) administered in a B16 melanoma model
of metastases to the lung, with progressive dilutions of the KPN
SSI (10.times., 100.times. and 1000.times.). Delta C.sub.t values
are inversely proportional to the amount of target nucleic acid in
the sample. As illustrated, tumour burden increased with increasing
dilution of the SSI. FIG. 33B is a bar graph illustrating a similar
dose-dependent effect of the KPN SSI as measured by the number of
B16 tumour nodules in the lung. FIG. 33C is a bar graph
illustrating that a variety of dosing regimes provide a therapeutic
effect, with intervals between injections varying from 1 to 7 days
all providing a therapeutic effect.
[0049] FIG. 34 includes two bar graphs illustrating that the
proportion of cells that express Rae-1 was inversely correlated
with tumour burden in a B16 melanoma model of metastases to the
lung (A) and this is dependent on NKG2D expression (B).
[0050] FIG. 35 is a bar graph illustrating that a QBKPN SSI
provided a markedly stronger effect in reducing tumour nodules in
the lung in a Lewis lung carcinoma (LLC)-RFP model.
[0051] FIG. 36 is a bar graph illustrating a concomitant reduction
in the number of LLC-RFP cells in the lungs at day 15 after
inoculation with LLC.
[0052] FIG. 37 is a line graph showing that a QBECO SSI conferred a
greater survival advantage than did either QBKPN or QBSAU in an
MC38 colon cancer model.
[0053] FIG. 38 is a bar graph illustrating that mice treated with a
QBKPN SSI, but not 10.times.QBSAU, exhibited elevated lung-specific
Rae-1 expression in a skin and lung tumour model.
[0054] FIG. 39 is a bar graph illustrating decreased PD-1
expression in the lung of QBKPN-treated mice as compared to
placebo-treated mice in the skin and lung tumour model.
[0055] FIG. 40 is a bar graph illustrating that treating mice with
10.times.QBSAU, but not QBKPN, led to a decrease in the skin tumour
burden as compared to placebo control in the B16 skin and lung
tumour model.
[0056] FIG. 41 is a bar graph illustrating that both intravenous
(IV) SSI and subcutaneous (SQ) SSI treatments provide therapeutic
benefit in a B16 lung metastasis model.
[0057] FIG. 42 is a schematic time line illustrating the study
design for an example based on efficacy of QBKPN in a treatment and
prophylaxis of cancer in a B16 lung cancer model.
[0058] FIG. 43 includes 4 bar graphs illustrating efficacy of QBKPN
in a treatment and prophylaxis of cancer in a B16 lung cancer
model.
[0059] FIG. 44 is a bar graph illustrating aspects of the efficacy
of QBKPN in a treatment and prophylaxis of cancer in a B16 lung
cancer model.
[0060] FIG. 45 is a bar graph illustrating aspects of how quickly
SSI therapies have detectable therapeutic effects involving myeloid
cell populations, particularly neutrophils.
[0061] FIG. 46 is a bar graph illustrating aspects of how quickly
SSI therapies have detectable therapeutic effects involving myeloid
cell populations, particularly Ly6C monocytes.
[0062] FIGS. 47A and 47D are a series of graphs illustrating
efficacy of alternative cellular fractions in a B16 melanoma model
in the lung, including dose-dependent and site-specific efficacy.
FIG. 47A includes three bar graphs illustrating that both 1.times.
and 0.01.times.KPN outer membrane fractions (i) were efficacious,
in a dose-dependent manner, with the 1.times. fraction having
comparable efficacy to the whole killed cell formulation, as were
the 1.times. and 4.times.DNA fractions (ii), while the inner
membrane fraction showed a dose dependent trend that lacked strong
statistical significance (iii). FIG. 47B is a bar graph
illustrating results following 10 injections of outer membrane SSI,
showing that Rae-1 expression was elevated by the outer membrane
fraction in a dose dependent manner. FIG. 47C includes two bar
graphs illustrating OM dose-dependent elevated neutrophil and
monocyte blood counts after 4 injections of QBKPN SSI, placebo, or
various concentrations of OM fraction (0.01.times., 1.times.,
10.times. or 20.times.) in blood collected 2 days prior to tumour
implant. FIG. 47D is a column scatter graph plot illustrating the
site-specific efficacy of KPN fractions compared to E. coli
fractions in the B16 lung cancer model.
[0063] FIG. 48 is a bar graph illustrating that a QBKPN SSI
increases NCI-H358 cancer cell death at high doses (1/20, 1/200
dilution) in a 24-hour killing assay.
[0064] FIG. 49 is a bar graph illustrating that a KPN SSI increases
.gamma..delta. T cell mediated killing of NCI-H358 cancer cells at
alternative doses (1/20 dilution, 1/200 dilution) in a 24 hour cell
killing assay.
[0065] FIG. 50 is a bar graph illustrating that a KPN SSI (QBKPN)
potentiated the effect of zoledronate in inducing .gamma..delta. T
cell mediated cancer cell lysis, at 1/200 and 1/2000 dilutions in a
24 hour cell killing assay.
[0066] FIG. 51 is a line graph illustrating the therapeutic
efficacy of a QBECO SSI in a MC-38 colon cancer model.
[0067] FIG. 52 is a line graph illustrating that NKG2D expression
is correlated with QBECO efficacy in a MC-38 colon cancer model
using NKG2D knockout mice.
[0068] FIG. 53 is a schematic representation of a treatment schema
in a model neutropenia system.
[0069] FIG. 54 is a series of 4 graphs that depict the results of
flow cytometry in the neutropenia model, illustrating counts of
particular cell populations from lung samples, gated on live,
CD45+CD11b+ cells.
[0070] FIG. 55 includes two column scatter graph plots illustrating
the proportion (A) and number (B) of neutrophils in lung samples in
the neutropenia model.
[0071] FIG. 56 includes two plots illustrating the proportion (A)
and number (B) of neutrophils in spleen samples in the neutropenia
model.
[0072] FIG. 57 is a bar graph illustrating the proportion of cells
having the denoted characteristics in blood samples from lung
cancer patients segregated into a neoplastic patient population and
a pre-neoplastic patient population, showing elevated PDL1 and PD1
expression in the neoplastic patient population compared to the
pre-neoplastic patients.
[0073] FIG. 58 is a bar graph illustrating the relative number of
cells having the denoted characteristics in blood samples from lung
cancer patients segregated into a neoplastic patient population and
a pre-neoplastic patient population, showing elevated PDL1 and PD1
expression in the neoplastic patient population compared to the
pre-neoplastic patients.
[0074] FIGS. 59A and 59B include two bar graphs illustrating the
SSI mediated reduction of PD-L1 expression in neoplastic lung
cancer in two patients, Patient 01-001 (FIG. 59A) and Patient
01-002 (FIG. 59B), at: week 1, day 4 (W1D4); week 1, day 5 (W1D5);
week 2 (W2); week 4 (W4), week 12 (W12) and week 16 (W16)
[0075] FIGS. 60A and 60B include two bar graphs illustrating the
SSI mediated reduction in PD-1 expression in two neoplastic lung
cancer patients, Patient 01-001 (FIG. 60A) and Patient 01-002 (FIG.
60B).
[0076] FIGS. 61A and 61B include two bar graphs illustrating the
increase in the proportion of M1 macrophages in two neoplastic lung
cancer patients, Patient 01-001 (FIG. 61A) and Patient 01-002 (FIG.
61B).
[0077] FIGS. 62A and 62B include two bar graphs, showing RT-qPCR
fold changes in (FIG. 62A) GzmA, GzmB, Prf1, and (FIG. 62B) Tyr in
lungs of B16 inoculated mice with differing QBKPN doses. Data
points are mean+/-SD. Significance was calculated using a one-way
Tukey's multiple comparison ANOVA test. **p<0.01, p<0.001 and
****p<0.0001.
[0078] FIG. 63 includes three bar graphs illustrating the
activation of pattern recognition receptors in HEK cells after
QBECO or QBKPN stimulation, showing respectively: A) Toll-like
receptor (TLR) activation as measured by NK-.kappa.B activation; B)
NOD2 and C-type lectin receptors (CTL) as measured by NK-.kappa.B
activation; and, C) RLR (Rig-1-like receptors) as measured by IRF3
activation.
[0079] FIG. 64 is a PRR repertoire fingerprint bar graph, in which
a PRR fingerprint was constructed for QBECO and QBKPN SSIs from the
1/10 dilution data, after subtracted the negative control data.
Bars, in order, represent TLR2, 3, 4, 5, 7, 8, 9, NOD1, NOD2,
Dectin 1a, Dectin 1b and Mincle. RIG-1 and MDA5 are not shown. The
positive control is specific for each PRR (ie LPS for TLR4).
[0080] FIG. 65 is a PRR fingerprint radar graph, in which a PRR
fingerprint was constructed for QBECO and QBKPN SSIs from the 1/10
dilution data, after subtracted the negative control data, and
plotted on a radar graph.
[0081] FIG. 66 is a bar graph illustrating neutrophil levels in the
blood at day 7 after treatment with Placebo, QBKPN or Rabies
Vaccine. Neutrophil levels were measured by flow cytometry and were
assessed as the percentage of neutrophils (Ly6G+) of total CD45+
cells. N=4-5 mice per group. * is P<0.05 compared to placebo as
assessed by Student's t-test. Average.+-.standard deviation shown.
QBKPN is a bacterial SSI derived from Klebsiella. Rabies is the
Imrab 3TF Rabies Vaccine which contains killed rabies virus.
[0082] FIG. 67 is a bar graph illustrating Ly6C.sup.HI monocyte
levels in the blood at day 7 after treatment with Placebo, QBKPN or
Rabies Vaccine. Ly6C.sup.HI monocyte levels were measured by flow
cytometry and were assessed as the percentage of Ly6C.sup.HI
monocyte (Ly6C.sup.HILy6G.sup.-) of total CD45+ cells. N=4-5 mice
per group. * is P<0.05 compared to placebo as assessed by
Student's t-test. Average.+-.standard deviation shown. QBKPN is a
bacterial SSI derived from Klebsiella. Rabies is the Imrab 3TF
Rabies Vaccine which contains killed rabies virus. Fel-O-Vac is
Feline Rhinotracheitis-Calici-Panleukopenia Vaccine which contains
the three killed viruses. Nobivac is Canine Influenza H3H8 which
contains killed influenza H3H8.
[0083] FIG. 68 is a column scatter plot illustrating cancer antigen
potentiation using QBKPN to potentiate the effect of the
melanoma-associated antigen gp100. The anti-tumour efficacy of
QBKPN SSI in combination with gp100 is compared to the irrelevant
control antigen OVA (SIINFEKL), including OVA adjuvanted with
CpG.
[0084] FIG. 69 is a column scatter plot illustrating surface
metastatic-like tumour nodules in mice challenged with B16
melanoma, evidencing enhanced efficacy of the microbial SSI QBKPN
augmented with an additional PRR agonist, the STING agonist
2'2'-cGMAP.
[0085] FIG. 70 is a column scatter plot illustrating
treatment-induced IFN-.gamma. levels in plasma in mice challenged
with B16 melanoma, evidencing enhanced IFN-.gamma. levels when the
microbial SSI QBKPN is augmented with a STING agonist.
[0086] FIG. 71A is a violin plot representing the log distribution
of risk scores, comparing last recorded response for all CD
subjects using risk scores based on 112 IBD SNPs (P-value:
2.430E-05).
[0087] FIG. 71B is a violin plot representing the log distribution
of risk scores, comparing last recorded response for all CD
subjects using risk scores based on 3 IBD SNPs (P-value:
1.385E-04).
[0088] FIG. 72 is a violin plot representing the log distribution
of risk scores, comparing last recorded response for all UC
subjects using risk scores based on 84 IBD SNPs (P-value:
1.255E-02).
[0089] FIG. 73 is a violin plot representing the log distribution
of risk scores, comparing last recorded response for all CD and UC
subjects using risk scores based on 112 IBD SNPs (P-value:
8.184E-07).
[0090] FIG. 74 is a graph illustrating the change in serum IL-18
levels in patients treated with QBECO vs. Placebo.
[0091] FIG. 75 is a set of 4 graphs illustrating serum immune
cytokine changes with QBECO treatment that associated with clinical
response.
[0092] FIG. 76 is a set of 3 graphs illustrating baseline levels of
Eotaxin-1, IL-10 and IL-12p40 by patient response to QBECO.
[0093] FIG. 77 is a graph illustrating the change in body weight
over time in a murine DSS colitis model.
[0094] FIG. 78 is a graph illustrating change in disease activity
index over time in a murine DSS colitis model.
[0095] FIG. 79 is a graph illustrating change in the FITC-dextran
assay over time in a murine DSS colitis model.
[0096] FIG. 80 is a graph illustrating blood neutrophil levels in
disease free mice, over time, with or without an initial QBECO SSI
treatment (mean+/-SEM, n=10 mice per group).
[0097] FIG. 81 is a collection of three graphs illustrating blood
cytokine levels in disease free mice, over time, with or without an
initial QBECO SSI treatment (mean+/-SEM, n=10 mice per group).
[0098] FIG. 82 is a graph illustrating the pharmacokinetics of
QBKPN, in which QBKPN SSI was fluorescently labelled and
subcutaneously injected into disease-free mice. Mice were bled at
different timepoints over 48 hours and the blood cell count was
quantified.
[0099] FIG. 83: includes 6 bar graphs, illustrating gene expression
in the lung tissues for CXCL10 (IP-10), CCL2 (MCP-1) and CCR2. Mice
were treated every second day for 10 days with Placebo, QBKPN or
QBECO before B16F10 tumour implantation into the lungs via tail
vein injection. Treatment continued every second day after tumour
inoculation. Mice were euthanized on day 5 (A, C, E) or day 17 (B,
D, F).
DETAILED DESCRIPTION
[0100] In the following detailed description, various examples are
set out of particular embodiments, together with experimental
procedures that may be used to implement a wide variety of
modifications and variations in the practice of the present
invention. For clarity, a variety of technical terms are used
herein in accordance with what is understood to be the commonly
understood meaning, as reflected in definitions set out below.
General Definitions
[0101] An "immunogen" refers to a molecule, or a composition
comprising the molecule, that is capable of eliciting an immune
response by an organism's immune system. An "antigen" refers to a
molecule that is capable of binding to the product of an immune
response.
[0102] "Pathogenic" agents are agents, such as microbes, such as
bacteria or viruses, which are known to cause infection in a host
in nature, and in this sense, "pathogenic" is used in the context
of the present invention to mean "naturally pathogenic". Although a
wide variety of microbes may be capable of causing infection under
artificial conditions, such as artificial inoculations of a microbe
into a tissue, the range of microbes that naturally cause infection
is necessarily limited, and well established by medical
practice.
[0103] An "infection" is the state or condition in which the body
or a part of it is invaded by a pathogenic agent (e.g., a microbe,
such as a bacterium) which, under favorable conditions, multiplies
and produces effects that are injurious (Taber's Cyclopedic Medical
Dictionary, 14th Ed., C. L. Thomas, Ed., F. A. Davis Company, PA,
USA). An infection may not always be apparent clinically and may
result in only localized cellular injury. Infections may remain
subclinical, and temporary if the body's defensive mechanisms are
effective. Infections may spread locally to become clinically
apparent as an acute, a subacute, or a chronic clinical infection
or disease state. A local infection may also become systemic when
the pathogenic agent gains access to the lymphatic or vascular.
Infection is usually accompanied by inflammation, but inflammation
may occur without infection.
[0104] "Inflammation" is the characteristic tissue reaction to
injury (marked by swelling, redness, heat, and pain), and includes
the successive changes that occur in living tissue when it is
injured. Infection and inflammation are different conditions,
although one may arise from the other (Taber's Cyclopedic Medical
Dictionary, supra). Accordingly, inflammation may occur without
infection and infection may occur without inflammation (although
inflammation typically results from infection by pathogenic
bacteria or viruses). Inflammation is characterized by the
following symptoms: redness (rubor), heat (calor), swelling
(tumour), pain (dolor). Localized visible inflammation on the skin
may be apparent from a combination of these symptoms, particularly
redness at a site of administration.
[0105] Various subjects may be treated or assayed or sampled in
accordance with alternative aspects of the invention. As used
herein, a "subject" is an animal, for e.g., a vertebrate or a
mammal. Accordingly, a subject may be a patient, e.g., a human,
suffering from an immune dysregulation. A subject may also be an
experimental animal, e.g., an animal model of an immune
dysregulation. In some embodiments, the terms "subject" and
"patient" may be used interchangeably, and may include a human, a
non-human mammal, a non-human primate, a rat, mouse, or dog. A
healthy subject may be a human who is not suffering from a disease,
such as a cancer or immune dysfunction, or suspected of having the
disease, or who is not suffering from a chronic disorder or
condition. A "healthy subject" may also be a subject who is not
immunocompromised. By immunocompromised is meant any condition in
which the immune system functions in an abnormal or incomplete
manner. Immunocompromisation may be due to disease, certain
medications, or conditions present at birth. Immunocompromised
subjects may be found more frequently among infants, the elderly,
and individuals undergoing extensive drug or radiation therapy.
[0106] A "sample" from a subject may include any relevant
biological material, including for example a cell, tissue or bodily
fluid sample taken from a patient. For example, a sample may
conveniently include samples of skin, cheek, blood, stool, hair or
urine. Sample nucleic acids for use in diagnostic and prognostic
methods can for example be obtained from a selected cell type or
tissue of a subject. For example, a subject's bodily fluid (e.g.
blood) can be obtained by known techniques. Alternatively, nucleic
acid tests can be performed on dry samples (e.g., hair or
skin).
[0107] The term "polymorphism" refers to a location within a
biological sequence, such as a genomic sequence, which varies
within a population. Polymorphisms are comprised of different
"alleles". The term "genotype" refers to the specific alleles in a
genome, for example in a cell, tissue sample or an individual. The
location of a polymorphism may be identified by its position, for
example within the genome or within a sequence such as a protein
that is reflective of a genomic locus. This may for example be
provided in the form of a characterization of the different amino
acids or bases that are found at a designated location. For diploid
genomes, the genotype is typically comprised of at least two
alleles, which may be the same (homozygous) or different
(heterozygous). Individual polymorphisms are typically assigned
unique identifiers in the art (such as "Reference SNP", "refSNP" or
"rs#"), for example in the Single Nucleotide Polymorphism Database
(dbSNP) of Nucleotide Sequence Variation available on the NCBI
website.
[0108] Characterization of polymorphisms, alleles or a genotype may
be performed by any of very wide variety of methods. These methods
may for example variously involve hybridization, labeling, cloning,
sequencing and/or amplification of nucleic acids, such as genomic
DNA, for example using PCR, LCR, xMAP, invader assays, mass
spectrometry, pyrosequencing, selective oligonucleotide
hybridization, selective amplification, selective primer extension
or probes. In this context, the term "probes" includes naturally
occurring or recombinant single- or double-stranded nucleic acids
or chemically synthesized nucleic acids. A probe can for example be
a polynucleotide of a length suitable for selective hybridization
to a nucleic acid containing a polymorphic region. Labeled probes
also can be used in conjunction with amplification of a
polymorphism. DNA microarray technologies, sometimes referred to as
DNA chips or gene chips, may for example be used for genomic
characterization, for example to characterize point mutations,
single nucleotide polymorphisms (SNPs), and/or short tandem repeats
(STRs). For example, several probes capable of hybridizing
specifically to an allelic variant may be attached to a solid phase
support by a variety of processes, including lithography.
Additional methods include laser capture microdissection (LCM),
comparative genomic hybridization (CGH) and chromatin
immunoprecipitation (ChiP). Allele specific hybridization may for
example make use of probes overlapping the polymorphic site and
having about 5, or alternatively 10, or alternatively 20, or
alternatively 25, or alternatively 30 nucleotides around the
polymorphic region. Alternatively, the presence of the specific
allele in DNA from a subject can in some case be characterized by
restriction enzyme analysis. Similarly, protection from cleavage
agents (such as a nuclease, hydroxylamine or osmium tetroxide) can
be used to detect mismatched bases in RNA/RNA DNA/DNA, or RNA/DNA
heteroduplexes, using technique that may be described as "mismatch
cleavage" assays. Alterations in electrophoretic mobility may be
used to characterize allelic variants, for example to detect single
strand conformation polymorphisms.
[0109] Many of the methods described herein may be performed using
kits, for example comprising at least one probe or primer nucleic
acid, or one of more of the compositions described herein and
instructions for use of the kit. Kits can for example comprise at
least one probe or primer which is capable of specifically
hybridizing to a polymorphic region or adjacent to the polymorphic
region, so that the oligonucleotides are "specific for" the
polymorphic region. Kits may also comprise at least one reagent
necessary to perform a particular assay. Kits can also include
positive controls, negative controls, sequencing markers, or
antibodies, for example for determining a subject's genotype or
biological marker profile.
[0110] An "immune response" includes, but is not limited to, one or
more of the following responses in a mammal: induction or
activation of antibodies, neutrophils, monocytes, macrophages
(including both M1-like macrophages and M2-like macrophages as
described herein), B cells, or T cells (including helper T cells,
natural killer cells, cytotoxic T cells, gamma-delta
(.gamma..delta.) T cells), such as induction or activation by one
or more immunogens in an immunogenic composition, following
administration of the composition. An immune response to a
composition thus generally includes the development in the host
animal of a cellular and/or antibody-mediated response to the
composition. In some embodiments, the immune response is such that
it will also result in slowing or stopping the progression of an
immune dysregulation, or a disease characterized by immune
dysregulation. An immune response may accordingly include one or
both of a cellular immune response and/or a humoral immune
response, and may be an adaptive response or an innate immune
response.
[0111] "Immune dysregulation" is an inappropriately regulated
immune response, such as an inappropriately restrained or
inappropriately robust immune response. The immune dysregulation
may for example be in the context of an autoimmune, inflammatory,
or degenerative disease (such as rheumatoid arthritis, Crohn's
disease, inflammatory bowel disease, multiple sclerosis,
neurodegenerative disease, or allergies) or a neoplastic disease,
such as a cancer, or a host defense against pathogens. Inflammatory
bowel disease (IBD) is a name frequently given to a group of
inflammatory conditions of the colon and small intestine, generally
characterized by similar symptoms of immune dysregulation and
indeterminate etiology. Major sub-types of IBD are recognized
clinically as Crohn's disease and ulcerative colitis. In addition
to Crohn's disease and ulcerative colitis, IBD may also include
conditions recognized as any one of the following: collagenous
colitis, lymphocytic colitis, ischaemic colitis, diversion colitis,
Behget's syndrome or indeterminate colitis. The difference between
these conditions relate primarily to the location and nature of the
inflammatory changes in the gastrointestinal tract (GIT). Crohn's
disease, for example, is generally recognized as potentially
affecting any part of the gastrointestinal tract, from mouth to
anus, with a majority of the cases marked by relapsing and
remitting granulomatous inflammation of the alimentary tract in the
terminal ileum and colon. Ulcerative colitis, in contrast, is
generally considered to be restricted to the colon and the rectum.
The various regions of the gastrointestinal tract in which these
inflammatory conditions may exhibit symptoms include: the bowel or
intestine, including: the small intestine (which has three parts:
the duodenum, the jejunum, and the ileum); the large intestine
(which has three parts: the cecum, the colon, which includes the
ascending colon, transverse colon, descending colon and sigmoid
flexure; and the rectum); and, the anus.
[0112] A "site specific immunotherapy" (SSI) is an immunomodulatory
treatment that is effective to therapeutically or prophylactically
alter an aspect of the immune state, or immune system physiology,
at an anatomical site or sites, such as an organ or tissue. In some
instances, for example, an SSI may be adapted to ameliorate an
immune dysregulation, or to treat a condition characterized by an
immune dysregulation.
[0113] A "cancer" or "neoplasm" is any unwanted growth of cells
serving no physiological function. In general, a cancer cell has
been released from its normal cell division control, i.e., a cell
whose growth is not regulated by the ordinary biochemical and
physical influences in the cellular environment. Thus, "cancer" is
a general term for diseases characterized by abnormal uncontrolled
cell growth. In most cases, a cancer cell proliferates to form
clonal cells that are malignant. The lump or cell mass, "neoplasm"
or "tumour," is generally capable of invading and destroying
surrounding normal tissues. By "malignancy", as used herein, is
meant as an abnormal growth of any cell type or tissue that has a
deleterious effect in the organism having the abnormal growth. The
term "malignancy" or "cancer" includes cell growths that are
technically benign but which carry the risk of becoming malignant.
Cancer cells may spread from their original site to other parts of
the body through the lymphatic system or blood stream in a process
known as "metastasis." Many cancers are refractory to treatment and
prove fatal. Examples of cancers or neoplasms include, without
limitation, transformed and immortalized cells, tumours,
carcinomas, in various organs and tissues as described herein or
known to those of skill in the art.
[0114] Most cancers fall within three broad histological
classifications: carcinomas, which are the predominant cancers and
are cancers of epithelial cells or cells covering the external or
internal surfaces of organs, glands, or other body structures (for
e.g., skin, uterus, lung, breast, prostate, stomach, bowel), and
which tend to metastasize; carcinomas, which are derived from
connective or supportive tissue (for e.g., bone, cartilage,
tendons, ligaments, fat, muscle); and hematologic tumours, which
are derived from bone marrow and lymphatic tissue. Carcinomas may
be adenocarcinomas (which generally develop in organs or glands
capable of secretion, such as breast, lung, colon, prostate or
bladder) or may be squamous cell carcinomas (which originate in the
squamous epithelium and generally develop in most areas of the
body). Sarcomas may be osteosarcomas or osteogenic sarcomas (bone),
chondrosarcomas (cartilage), leiomyosarcomas (smooth muscle),
rhabdomyosarcomas (skeletal muscle), mesothelial sarcomas or
mesotheliomas (membranous lining of body cavities), fibrosarcomas
(fibrous tissue), angiosarcomas or hemangioendotheliomas (blood
vessels), liposarcomas (adipose tissue), gliomas or astrocytomas
(neurogenic connective tissue found in the brain), myxosarcomas
(primitive embryonic connective tissue), or mesenchymous or mixed
mesodermal tumours (mixed connective tissue types). Hematologic
tumours may be myelomas, which originate in the plasma cells of
bone marrow; leukemias which may be "liquid cancers" and are
cancers of the bone marrow and may be myelogenous or granulocytic
leukemia (myeloid and granulocytic white blood cells), lymphatic,
lymphocytic, or lymphoblastic leukemias (lymphoid and lymphocytic
blood cells) or polycythemia vera or erythremia (various blood cell
products, but with red cells predominating); or lymphomas, which
may be solid tumours and which develop in the glands or nodes of
the lymphatic system, and which may be Hodgkin or Non-Hodgkin
lymphomas. In addition, mixed type cancers, such as adenosquamous
carcinomas, mixed mesodermal tumours, carcinosarcomas, or
teratocarcinomas also exist.
[0115] Cancers named based on primary site may be correlated with
histological classifications. For example, lung cancers are
generally small cell lung cancers or non-small cell lung cancers,
which may be squamous cell carcinoma, adenocarcinoma, or large cell
carcinoma; skin cancers are generally basal cell cancers, squamous
cell cancers, or melanomas. Lymphomas may arise in the lymph nodes
associated with the head, neck and chest, as well as in the
abdominal lymph nodes or in the axillary or inguinal lymph nodes.
Identification and classification of types and stages of cancers
may be performed by using for example information provided by the
Surveillance, Epidemiology, and End Results (SEER) Program of the
National Cancer Institute, which is an authoritative source of
information on cancer incidence and survival in the United States
and is recognized around the world. The SEER Program currently
collects and publishes cancer incidence and survival data from 14
population-based cancer registries and three supplemental
registries covering approximately 26 percent of the US population.
The program routinely collects data on patient demographics,
primary tumour site, morphology, stage at diagnosis, first course
of treatment, and follow-up for vital status, and is the only
comprehensive source of population-based information in the United
States that includes stage of cancer at the time of diagnosis and
survival rates within each stage. Information on more than 3
million in situ and invasive cancer cases is included in the SEER
database, and approximately 170,000 new cases are added each year
within the SEER coverage areas. The incidence and survival data of
the SEER Program may be used to access standard survival for a
particular cancer site and stage. For example, to ensure an optimal
comparison group, specific criteria may be selected from the
database, including date of diagnosis and exact stage (for example,
in the case of the lung cancer example herein, the years were
selected to match the time-frame of the retrospective review, and
stage 3B and 4 lung cancer were selected; and in the case of the
colon cancer example herein, the years were also selected to match
the time-frame of the retrospective review, and the stage 4 colon
cancer was selected).
[0116] Cancers may also be named based on the organ in which they
originate i.e., the "primary site," for example, cancer of the
breast, brain, lung, liver, skin, prostate, testicle, bladder,
colon and rectum, cervix, uterus, etc. This naming persists even if
the cancer metastasizes to another part of the body that is
different from the primary site. With the present invention,
treatment is directed to the site of the cancer, not type of
cancer, so that a cancer of any type that is symptomatic or
etiologically located in the lung, for example, would be treated on
the basis of this localization in the lung.
PRR Ligands
[0117] Aspects of the invention relate to the use of PRR ligands.
PRR ligands may for example be available commercially, for example
in widely available preparations of attenuated or killed
recombinant bacteria, which may for example be ligands for TLR2,
TLR4 and TLR5. Compositions of pathogen-associated molecular
patterns (PAMPs) may include PAMPS that are recognized by PRRs,
including: Toll-like receptors (TLRs), NOD-like receptors (NLRs),
RIG-1-like receptors (RLRs), C-type lectin receptors (CLRs)
including Dectin-1, cytosolic dsDNA sensors (CDSs) and NLRs
involved in the formation of inflammasomes.
[0118] Toll-like receptor 2 (TLR2) is involved in the recognition
of a wide array of microbial molecules representing broad groups of
species including Gram-positive and Gram-negative bacteria, as well
as mycoplasma and yeast. TLR2 recognizes cell-wall components such
as peptidoglycan, lipoteichoic acid and lipoprotein from
Gram-positive bacteria, lipoarabinomannan from mycobacteria, and
zymosan from the yeast cell wall. Toll-like receptor 3 (TLR3)
recognizes double-stranded RNA (dsRNA). Bacterial
lipopolysaccharide (LPS) is recognized by Toll-like receptor 4
(TLR4) which interacts with at least three different extracellular
proteins: LPS-binding protein (LBP), CD14 and, myeloid
differentiation protein 2 (MD-2), to induce a signaling cascade
leading to the activation of NF-.kappa.B and the production of
proinflammatory cytokines. LPS generally consists of a
polysaccharide region that is anchored in the outer bacterial
membrane by a carbohydrate lipid moiety: lipid A, which is largely
responsible for the immunostimulatory activity of LPS. Particularly
active forms of lipid A contain six fatty acyl groups, as for
example may be found in pathogenic bacteria that are strains of
Escherichia coli or Salmonella spp. Toll-like receptor 5 (TLR5)
recognizes flagellin from both Gram-positive and Gram-negative
bacteria. Toll-like receptor 7 (TLR7) and TLR8 recognize single
stranded RNAs and small synthetic molecules such as
imidazoquinolines and nucleoside analogs. Toll-like receptor 9
(TLR9) recognizes specific unmethylated CpG motifs prevalent in
microbial but not vertebrate genomic DNA.
[0119] NLRs are a family of at least 22 cytoplasmic innate immune
sensors, including NOD1 (CARD4) and NOD2 (CARD15) which are
intracellular pattern-recognition receptors involved in the
recognition of peptidoglycan (PGN). These receptors detect specific
motifs within PGN. NOD1 senses the diaminopimelatic acid
(DAP)-containing muropeptide (specifically d-Glu-meso-DAP dipeptide
"iE-DAP" dipeptide) which is found primarily in PGN of
Gram-negative bacteria, as well as certain Gram-positive bacteria.
NOD2 recognizes the muramyl dipeptide (MDP) structure found in
almost all bacterial PGN.
[0120] The RIG-1-Like receptors (RLRs), particularly RIG-1 and
MDA-5, detect viral RNA species.
[0121] CLR ligands include Dectin-1 and Mincle
(macrophage-inducible C-type lectin) agonists. Dectin-1 is a
specific receptor for .beta.-glucans, which are glucose polymers
found in the cell walls of fungi. Mincle is a multi-tasking danger
signal receptor that recognizes a wide variety of ligands such as
damaged cells, fungal components, yeast components and components
of mycobacteria.
[0122] Cytosolic DNA Sensors (CDS) bind intracellular DNA from
pathogens, and there are multiple CDSs which may display contextual
preferences for the recognition of particular DNAs.
[0123] Cyclic dinucleotides (CDNs) and xanthenone derivatives, such
as DMXAA, bind to and activate STING (STimulator of INterferon
Genes).
[0124] The inflammasome is a multi-protein complex involved in the
production of mature IL-1.beta., specifically through cleavage of
pro-IL-1.beta. and pro-IL-18 into active and secretable forms.
Inflammasomes may be segregated into NLRP1, NLRP3, NLRC4 and AIM2
subtypes, which are activated by a wide variety of microbial
molecules, danger signals and crystalline substances.
TABLE-US-00002 TABLE 1 PRR Receptors and their Ligands PRR Ligand
TLR2 Microbial cell wall components/preparations, Pam2C-
Aca-Benzyl-Murabutide (Pam2C- conjugated murabutide) TLR3
Polyadenylic-polyuridylic acid, Polyinosine-polycytidylic acid TLR4
Lipopolysaccharide, Monophosphoryl Lipid A TLR5 Flagellin TLR7/8
Single-stranded RNAs, Nucleoside analogs,
Imidazoquinolines/Thiazoquinolines TLR9 unmethylated CpG DNA motifs
NOD1 iE-DAP, Acylated iE-DAP, D-gamma- Glu-mDAP, L-Ala-gamma-D-Glu-
mDAP NOD2 MDP (MurNAc-L-Ala-D-isoGln, muramyl dipeptide),
N-glycolylated muramyldipeptide, N-Acetyl-muramyl- L-
Alanyl-D-Glutamin-n-butyl-ester, MurNAc-Ala-D-isoGln-Lys, N-
Acetylmuramyl-L-Alanyl-D- Isoglutamine (L-D isoform), 6-O-
stearoyl-N-Acetyl-muramyl-L-alanyl-D- isoglutamine,
Pam2C-Aca-Benzyl- Murabutide, TLR2/NOD2 Pam2C-conjugated murabutide
NOD1/NOD2 PGN, Pam2C-conjugated murabutide RIG1/MDA5 5'
triphosphate double stranded RNA (18-20mer),
polyriboinosinic:polyribocytidylic acid DAI, LRRFIP1, AIM2, RIG1
dsDNA, poly(dA-dT).cndot.poly(dT-dA) Dectin-1 Beta-glucan peptide,
fungal cell wall preparations Mincle damaged microbial cells,
fungus, yeast and mycobacteria, Trehalose- 6,6-dibehenate,
trehalose-6,6- dimycolate STING Cyclic dinucleotides
(c-di-nucleotides), xanthenone derivatives, 3'3'-cGAMP, 2'3'-cGAMP,
2'2'-cGAMP, 2'2'- cGAMP, c-di-AMP (cyclic di-adenylate
monophosphate), c-di-GMP, c-di-IMP, c-di-UMP, c-di-AMP
TABLE-US-00003 TABLE 2 Cytosolic nucleic acid-sensing PRRs and
their Ligands (Broz & Monack, 2013, Nature Reviews Immunology
13, 551-565). PRR Ligands RIG-I PPP-ssRNA (PPP-ssRNA, ssRNA with a
5'- triphosphate group), RNA with base pairing and polyl:C MDA5
Long dsRNA LGP2 dsRNA DDX41 B-form DNA and CDNs (cyclic
dinucleotides) DHX9 DNA, RNA, CpG-A oligodeoxynucleotids and CpG-B
ODNs DDX3 Viral RNA DHX36 DNA, RNA, CpG-A oligodeoxynucleotids and
CpG-B oligodeoxynucleotids DDX1-DDX21- RNA and polyl:C DDX36 DDX60
ssRNA, dsRNA and dsDNA KU70 DNA cGAS DNA STING CDNs (c-di-GMP and
c-di-AMP) NOD2 ssRNA NLRP3 ssRNA, dsRNA, bacterial mRNA and
oxidized mitochondrial DNA AIM2 DNA IFI16 dsDNA LRRFIP1 B-form DNA,
Z-form DNA and dsRNA DAI DNA IFIT1, 2, 3 and 5 PPP-ssRNA
[0125] Aspects of the invention accordingly involve using PRR
agonists derived from a selected microbial pathogen. For example,
peptidoglycan (PGN) may be obtained from a bacteria or bacterial
strain that is pathogenic in a selected target tissue or organ, for
use as a NOD1/NOD2 agonist. Similarly, cell wall components may be
obtained from a bacteria or bacterial strain that is pathogenic in
a selected target tissue or organ, for use as a TLR32 agonist.
Similarly, DNA, including double stranded DNA, particularly
repetitive double stranded DNA, may be obtained from a microbial
pathogen, such as a bacteria or bacterial strain that is pathogenic
in a selected target tissue or organ, for use as a DAI, LRRFIP1,
RIG1, TLR9, AIM2 or cytosolic DNA sensor (CDS) agonist. Beta-glucan
peptides may be obtained from fungi or yeast that are pathogenic in
a selected target tissue or organ, for use as a Dectin-1 agonists.
Cyclic dinucleotides may be obtained from a microbial pathogen that
is pathogenic in a selected target tissue or organ, for use as a
STING agonist.
[0126] Aspects of the invention involve compositions that have a
distinct PRR agonist signature, which connotes a repertoire of PRR
agonists that are together collected in a therapeutic vehicle, so
that the selected collection of PRR agonists is distinct. A
"therapeutic vehicle" in this context is a formulation that
aggregates and retains the PRR agonists, for example in a
pharmaceutically acceptable particle or vesicle, such as a
recombinant microbe. For example, the PRR agonist signature may be
different from a reference PRR agonist signature, for example
different from the collection of PRR agonists that would be present
on a microbe that is not pathogenic in the target tissue. The PRR
signature may also be distinct in the sense that it is different
than a native PRR agonist signature of the microbial mammalian
pathogen, for example altered by way of the recombinant expression
of genes that alter what would otherwise be the wildtype PRR
agonist signature of the pathogen. For purposes of determining the
distinctiveness of a PRR agonist signature, the levels or kinds of
PRR agonist may be directly measured, or may be measured for
example by determining the activation or inhibition of a signalling
pathway in a cell consequent to PRR agonist/receptor binding.
Recombinant Embodiments
[0127] Various genes and nucleic acid sequences of the invention
may be recombinant sequences. The term "recombinant" means that
something has been recombined, so that when made in reference to a
nucleic acid construct the term refers to a molecule that is
comprised of nucleic acid sequences that are joined together or
produced by means of molecular biological techniques. Nucleic acid
"constructs" are accordingly recombinant nucleic acids, which have
been generally been made by aggregating interoperable component
sequencers. The term "recombinant" when made in reference to a
protein or a polypeptide refers to a protein or polypeptide
molecule which is expressed using a recombinant nucleic acid
construct created by means of molecular biological techniques. The
term "recombinant" when made in reference to the genetic
composition or an organism or cell refers to new combinations of
alleles that did not occur in the parental genomes. Recombinant
nucleic acid constructs may include a nucleotide sequence which is
ligated to, or is manipulated to become ligated to, a nucleic acid
sequence to which it is not ligated in nature, or to which it is
ligated at a different location in nature. Referring to a nucleic
acid construct as "recombinant" therefore indicates that the
nucleic acid molecule has been manipulated using genetic
engineering, i.e. by human intervention (so that it is
anthropogenic). Recombinant nucleic acid constructs may for example
be introduced into a host cell by transformation. Such recombinant
nucleic acid constructs may include sequences derived from the same
host cell species or from different host cell species, which have
been isolated and reintroduced into cells of the host species.
Recombinant nucleic acid construct sequences may become integrated
into a host cell genome, either as a result of the original
transformation of the host cells, or as the result of subsequent
recombination and/or repair events.
[0128] Recombinant constructs of the invention may include a
variety of functional molecular or genomic components, as required
for example to mediate gene expression or suppression in a
transformed plant. In this context, "DNA regulatory sequences,"
"control elements," and "regulatory elements," refer to
transcriptional and translational control sequences, such as
promoters, enhancers, polyadenylation signals, terminators, and
protein degradation signals that regulate gene expression, as well
as epigenetic regulatory signals for example involving methylation
or acetylation of histones (e.g. histone methyltransferase or
acetyltransferase), leading to conformational changes in the
transcriptional landscape and gene expression differences. In the
context of the present disclosure, "promoter" means a sequence
sufficient to direct transcription of a gene when the promoter is
operably linked to the gene. The promoter is accordingly the
portion of a gene containing DNA sequences that provide for the
binding of RNA polymerase and initiation of transcription. Promoter
sequences are commonly, but not universally, located in the 5'
non-coding regions of a gene. A promoter and a gene are "operably
linked" when such sequences are functionally connected so as to
permit gene expression mediated by the promoter. The term "operably
linked" accordingly indicates that DNA segments are arranged so
that they function in concert for their intended purposes, such as
initiating transcription in the promoter to proceed through the
coding segment of a gene to a terminator portion of the gene. Gene
expression may occur in some instances when appropriate molecules
(such as transcriptional activator proteins) are bound to the
promoter. Expression is the process of conversion of the
information of a coding sequence of a gene into mRNA by
transcription and subsequently into polypeptide (protein) by
translation, as a result of which the protein is said to be
expressed. As the term is used herein, a gene or nucleic acid is
"expressible" if it is capable of expression under appropriate
conditions in a particular host cell.
[0129] An "isolated" nucleic acid or polynucleotide as used herein
refers to a component that is removed from its original environment
(for example, its natural environment if it is naturally
occurring). An isolated nucleic acid or polypeptide may contain
less than about 50%, less than about 75%, less than about 90%, less
than about 99.9% or less than any integer value between 50 and
99.9% of the cellular or biological components with which it was
originally associated. A polynucleotide amplified using PCR so that
it is sufficiently distinguishable (on a gel from example) from the
rest of the cellular components is, for example, thereby
"isolated". The polynucleotides of the invention may be
"substantially pure," i.e., having the high degree of isolation as
achieved using a purification technique.
[0130] In the context of biological molecules "endogenous" refers
to a molecule such as a nucleic acid that is naturally found in
and/or produced by a given organism or cell. An "endogenous"
molecule may also be referred to as a "native" molecule.
Conversely, in the context of biological molecules "exogenous"
refers to a molecule, such as a nucleic acid, that is not normally
or naturally found in and/or produced by a given organism or cell
in nature.
[0131] As used herein to describe nucleic acid or amino acid
sequences, the term "heterologous" refers to molecules or portions
of molecules, such as DNA sequences, that are artificially
introduced into a particular host cell, for example by
transformation. Heterologous DNA sequences may for example be
introduced into a host cell by transformation. Such heterologous
molecules may include sequences derived from the host cell.
Heterologous DNA sequences may become integrated into the host cell
genome, either as a result of the original transformation of the
host cells, or as the result of subsequent recombination
events.
[0132] Various aspects of the present disclosure encompass nucleic
acid or amino acid sequences that are homologous to other
sequences. As the term is used herein, an amino acid or nucleic
acid sequence is "homologous" to another sequence if the two
sequences are substantially identical and the functional activity
of the sequences is conserved (as used herein, sequence
conservation or identity does not infer evolutionary relatedness).
Nucleic acid sequences may also be homologous if they encode
substantially identical amino acid sequences, even if the nucleic
acid sequences are not themselves substantially identical, for
example as a result of the degeneracy of the genetic code.
[0133] With reference to biological sequences "substantial
homology" or "substantial identity" is meant, in the alternative, a
homology of greater than 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% or 99% up to 100% sequence
identity. Homology may refer to nucleic acid or amino acid
sequences as the context dictates. In alternative embodiments,
sequence identity may for example be at least 75%, at least 90% or
at least 95%. Optimal alignment of sequences for comparisons of
identity may be conducted using a variety of algorithms, such as
the local homology algorithm of Smith and Waterman (1981) Adv.
Appl. Math 2: 482, the homology alignment algorithm of Needleman
and Wunsch (1970) J. Mol. Biol. 48:443, the search for similarity
method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:
2444, and the computerized implementations of these algorithms
(such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics
Software Package, Genetics Computer Group, Madison, Wis., U.S.A.).
Sequence identity may also be determined using the BLAST algorithm,
described in Altschul et al. (1990), J. Mol. Biol. 215:403-10
(using the published default settings). Software for performing
BLAST analysis may be available through the National Center for
Biotechnology Information (NCBI) at their Internet site. The BLAST
algorithm involves first identifying high scoring sequence pairs
(HSPs) by identifying short words of length W in the query sequence
that either match or satisfy some positive-valued threshold score T
when aligned with a word of the same length in a database sequence.
T is referred to as the neighborhood word score threshold. Initial
neighborhood word hits act as seeds for initiating searches to find
longer HSPs. The word hits are extended in both directions along
each sequence for as far as the cumulative alignment score can be
increased. Extension of the word hits in each direction is halted
when the following parameters are met: the cumulative alignment
score falls off by the quantity X from its maximum achieved value;
the cumulative score goes to zero or below, due to the accumulation
of one or more negative-scoring residue alignments; or the end of
either sequence is reached. The BLAST algorithm parameters W, T and
X determine the sensitivity and speed of the alignment. The BLAST
program may use as defaults a word length (W) of 11, the BLOSUM62
scoring matrix (Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci.
USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10,
M=5, N=4, and a comparison of both strands. One measure of the
statistical similarity between two sequences using the BLAST
algorithm is the smallest sum probability (P(N)), which provides an
indication of the probability by which a match between two
nucleotide or amino acid sequences would occur by chance. In
alternative embodiments, nucleotide or amino acid sequences are
considered substantially identical if the smallest sum probability
in a comparison of the test sequences is less than about 1, less
than about 0.1, less than about 0.01, or less than about 0.001.
[0134] An alternative indication that two amino acid sequences are
substantially identical is that one peptide is specifically
immunologically reactive with antibodies that are also specifically
immunoreactive against the other peptide. Antibodies are
specifically immunoreactive to a peptide if the antibodies bind
preferentially to the peptide and do not bind in a significant
amount to other proteins present in the sample, so that the
preferential binding of the antibody to the peptide is detectable
in an immunoassay and distinguishable from non-specific binding to
other peptides. Specific immunoreactivity of antibodies to peptides
may be assessed using a variety of immunoassay formats, such as
solid-phase ELISA immunoassays for selecting monoclonal antibodies
specifically immunoreactive with a protein (see Harlow and Lane
(1988) Antibodies, A Laboratory Manual, Cold Spring Harbor
Publications, New York).
[0135] An alternative indication that two nucleic acid sequences
are substantially identical is that the two sequences hybridize to
each other under moderately stringent, or stringent, conditions.
Hybridization to filter-bound sequences under moderately stringent
conditions may, for example, be performed in 0.5 M NaHPO.sub.4, 7%
sodium dodecyl sulfate (SDS), 1 mM EDTA at 65.degree. C., and
washing in 0.2.times.SSC/0.1% SDS at 42.degree. C. (see Ausubel, et
al. (eds), 1989, Current Protocols in Molecular Biology, Vol. 1,
Green Publishing Associates, Inc., and John Wiley & Sons, Inc.,
New York, at p. 2.10.3). Alternatively, hybridization to
filter-bound sequences under stringent conditions may, for example,
be performed in 0.5 M NaHPO.sub.4, 7% SDS, 1 mM EDTA at 65.degree.
C., and washing in 0.1.times.SSC/0.1% SDS at 68.degree. C. (see
Ausubel, et al. (eds), 1989, supra). Hybridization conditions may
be modified in accordance with known methods depending on the
sequence of interest (see Tijssen, 1993, Laboratory Techniques in
Biochemistry and Molecular Biology--Hybridization with Nucleic Acid
Probes, Part I, Chapter 2 "Overview of principles of hybridization
and the strategy of nucleic acid probe assays", Elsevier, N.Y.).
Generally, stringent conditions are selected to be about 5.degree.
C. lower than the thermal melting point for the specific sequence
at a defined ionic strength and pH. The term "a polynucleotide that
hybridizes under stringent (low, intermediate) conditions" is
intended to encompass both single and double-stranded
polynucleotides although only one strand will hybridize to the
complementary strand of another polynucleotide. Washing in the
specified solutions may be conducted for a range of times from
several minutes to several days and those skilled in the art will
readily select appropriate wash times to discriminate between
different levels of homology in bound sequences.
[0136] It is well known in the art that some modifications and
changes can be made in the structure of a polypeptide without
substantially altering the biological function of that peptide, to
obtain a biologically equivalent polypeptide. As used herein, the
term "conserved amino acid substitutions" refers to the
substitution of one amino acid for another at a given location in
the peptide, where the substitution can be made without any
appreciable loss or gain of function, to obtain a biologically
equivalent polypeptide. In making such changes, substitutions of
like amino acid residues can be made on the basis of relative
similarity of side-chain substituents, for example, their size,
charge, hydrophobicity, hydrophilicity, and the like, and such
substitutions may be assayed for their effect on the function of
the peptide by routine testing. Conversely, as used herein, the
term "non-conserved amino acid substitutions" refers to the
substitution of one amino acid for another at a given location in
the peptide, where the substitution causes an appreciable loss or
gain of function of the peptide, to obtain a polypeptide that is
not biologically equivalent.
[0137] In some embodiments, conserved amino acid substitutions may
be made where an amino acid residue is substituted for another
having a similar hydrophilicity value (e.g., within a value of plus
or minus 2.0), where the following hydrophilicity values are
assigned to amino acid residues (as detailed in U.S. Pat. No.
4,554,101): Arg (+3.0); Lys (+3.0); Asp (+3.0); Glu (+3.0); Ser
(+0.3); Asn (+0.2); Gln (+0.2); Gly (0); Pro (-0.5); Thr (-0.4);
Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Val (-1.5); Leu
(-1.8); Ile (-1.8); Tyr (-2.3); Phe (-2.5); and Trp (-3.4).
Non-conserved amino acid substitutions may be made were the
hydrophilicity value of the residues is significantly different,
e.g. differing by more than 2.0.
[0138] In alternative embodiments, conserved amino acid
substitutions may be made where an amino acid residue is
substituted for another having a similar hydropathic index (e.g.,
within a value of plus or minus 2.0). In such embodiments, each
amino acid residue may be assigned a hydropathic index on the basis
of its hydrophobicity and charge characteristics, as follows: lie
(+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9);
Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr
(-1.3); Pro (-1.6); His (-3.2); Glu (-3.5); Gln (-3.5); Asp (-3.5);
Asn (-3.5); Lys (-3.9); and Arg (-4.5). Non-conserved amino acid
substitutions may be made were the hydropathic index of the
residues is significantly different, e.g. differing by more than
2.0.
[0139] In alternative embodiments, conserved amino acid
substitutions may be made where an amino acid residue is
substituted for another in the same class, where the amino acids
are divided into non-polar, acidic, basic and neutral classes, as
follows: non-polar: Ala, Val, Leu, lie, Phe, Trp, Pro, Met; acidic:
Asp, Glu; basic: Lys, Arg, His; neutral: Gly, Ser, Thr, Cys, Asn,
Gin, Tyr. Non-conserved amino acid substitutions may be made were
the residues do not fall into the same class, for example
substitution of a basic amino acid for a neutral or non-polar amino
acid.
Microorganisms
[0140] Most animals are colonized to some degree by microorganisms,
such as bacteria, which exist in symbiotic or commensal
relationships with the host animal.
[0141] Thus, many species of normally harmless bacteria are found
in healthy animals, and are usually localized to the surface of
specific organs and tissues. Often, these microbial communities aid
in the normal functioning of the body, as members of what is termed
the microbiome. Microbes that are generally harmless, such as
Escherichia coli, can cause infection in healthy subjects, with
results ranging from mild infection to death. Whether or not a
microorganism is pathogenic (i.e., causes infection) depends on
factors such as: the route of entry and access to specific host
cells, tissues, or organs; the intrinsic virulence of the
microorganism; the amount of the microorganism present at the site
of potential infection; or the health of the host animal. Thus,
microorganisms that are normally harmless can become pathogenic
given favorable conditions for infection, and even the most
virulent microorganism generally requires specific circumstances to
cause infection. Accordingly, microbial species that are members of
the normal flora can be pathogens when they move beyond their
normal ecological role in the endogenous flora. For example,
endogenous species can cause infection outside of their ecological
niche in regions of anatomical proximity, for example by contiguous
spread. When this occurs, these normally harmless endogenous
bacteria are pathogenic.
[0142] Specific microbial species are known to cause infections in
specific cells, tissues, or organs in otherwise healthy subjects.
Examples of bacteria and viruses that commonly cause infections in
specific organs and tissues of the body are listed below; and these
examples are not limiting in the sense that a skilled person would
be able to recognize and identify infectious or pathogenic bacteria
that cause infections, or commonly cause infections, in various
organs and tissues in otherwise healthy organisms (and recognize
the relative frequency of infection with each bacterial species)
based on the knowledge in the field as represented, for example, by
the following publications: Manual of Clinical Microbiology 8th
Edition, Patrick Murray, Ed., 2003, ASM Press American Society for
Microbiology, Washington D.C., USA; Mandell, Douglas, and Bennett's
Principles and Practice of Infectious Diseases 5th Edition, G. L.
Mandell, J. E. Bennett, R. Dolin, Eds., 2000, Churchill
Livingstone, Philadelphia, Pa., USA, all of which are incorporated
by reference herein.
[0143] Infections of the skin are commonly caused by the following
bacterial species: Staphylococcus aureus, Beta hemolytic
streptococci group A, B, C or G, Corynebacterium diptheriae,
Corynebacterium ulcerans, or Pseudomonas aeruginosa; or viral
pathogens: rubeola, rubella, varicella-zoster, echoviruses,
coxsackieviruses, adenovirus, vaccinia, herpes simplex, or parvo
B19.
[0144] Infections of the soft tissue (e.g., fat and muscle) are
commonly caused by the following bacterial species: Streptococcus
pyogenes, Staphylococcus aureus, Clostridium perfringens, or other
Clostridium spp.; or viral pathogens: influenza, or
coxsackieviruses.
[0145] Infections of the breast are commonly caused by the
following bacterial species: Staphylococcus aureus, or
Streptococcus pyogenes.
[0146] Infections of the lymph nodes of the head and neck are
commonly caused by the following bacterial species: Staphylococcus
aureus, or Streptococcus pyogenes; or viral pathogens:
Epstein-Barr, cytomegalovirus, adenovirus, measles, rubella, herpes
simplex, coxsackieviruses, or varicella-zoster.
[0147] Infections of the lymph nodes of the arm/axillae are
commonly caused by the following bacterial species: Staphylococcus
aureus, or Streptococcus pyogenes; or viral pathogens: measles,
rubella, Epstein-Barr, cytomegalovirus, adenovirus, or
varicella-zoster.
[0148] Infections of the lymph nodes of the mediastinum are
commonly caused by the following bacterial species: viridans
streptococci, Peptococcus spp., Peptostreptococcus spp.,
Bacteroides spp., Fusobacterium spp., or Mycobacterium
tuberculosis; or viral pathogens: measles, rubella, Epstein-Barr,
cytomegalovirus, varicella-zoster, or adenovirus.
[0149] Infections of the pulmonary hilar lymph nodes are commonly
caused by the following bacterial species: Streptococcus
pneumoniae, Moraxella catarrhalis, Mycoplasma pneumoniae,
Klebsiella pneumoniae, Haemophilus influenza, Chlamydophila
pneumoniae, Bordetella pertussis or Mycobacterium tuberculosis; or
viral pathogens: influenza, adenovirus, rhinovirus, coronavirus,
parainfluenza, respiratory syncytial virus, human metapneumovirus,
or coxsackievirus.
[0150] Infections of the intra-abdominal lymph nodes are commonly
caused by the following bacterial species: Yersinia enterocolitica,
Yersinia pseudotuberculosis, Salmonella spp., Streptococcus
pyogenes, Escherichia coli, Staphylococcus aureus, or Mycobacterium
tuberculosis; or viral pathogens: measles, rubella, Epstein-Barr,
cytomegalovirus, varicella-zoster, adenovirus, influenza, or
coxsackieviruses.
[0151] Infections of the lymph nodes of the leg/inguinal region are
commonly caused by the following bacterial species: Staphylococcus
aureus, or Streptococcus pyogenes; or viral pathogens: measles,
rubella, Epstein-Barr, cytomegalovirus, or herpes simplex.
[0152] Infections of the blood (i.e., septicemia) are commonly
caused by the following bacterial species: Staphylococcus aureus,
Streptococcus pyogenes, coagulase-negative staphylococci,
Enterococcus spp., Escherichia coli, Klebsiella spp., Enterobacter
spp., Proteus spp., Pseudomonas aeruginosa, Bacteroides fragilis,
Streptococcus pneumoniae, or group B streptococci; or viral
pathogens: rubeola, rubella, varicella-zoster, echoviruses,
coxsackieviruses, adenovirus, Epstein-Barr, herpes simplex, or
cytomegalovirus.
[0153] Infections of the bone are commonly caused by the following
bacterial species: Staphylococcus aureus, coagulase-negative
staphylococci, Streptococcus pyogenes, Streptococcus pneumoniae,
Streptococcus agalactiae, other streptococci spp., Escherichia
coli, Pseudomonas spp., Enterobacter spp., Proteus spp., or
Serratia spp.; or viral pathogens: parvovirus B19, rubella, or
hepatitis B.
[0154] Infections of the joint are commonly caused by the following
bacterial species: Staphylococcus aureus, coagulase-negative
staphylococci, Streptococcus pyogenes, Streptococcus pneumoniae,
Streptococcus agalactiae, other streptococci spp., Escherichia
coli, Pseudomonas spp., Enterobacter spp., Proteus spp., Serratia
spp., Neisseria gonorrhea, salmonella species, Mycobacterium
tuberculosis, Hemophilus influenza; or viral pathogens: parvovirus
B19, rubella, hepatitis B; or fungal pathogen: Scedosporium
prolificans
[0155] Infections of the meninges are commonly caused by the
following bacterial species: Haemophilus influenzae, Neisseria
meningitidis, Streptococcus pneumoniae, Streptococcus agalactiae,
or Listeria monocytogenes; or viral pathogens: echoviruses,
coxsackieviruses, other enteroviruses, or mumps.
[0156] Infections of the brain are commonly caused by the following
bacterial species: Streptococcus spp. (including S. anginosus, S.
constellatus, S. intermedius), Staphylococcus aureus, Bacteroides
spp., Prevotella spp., Proteus spp., Escherichia coli, Klebsiella
spp., Pseudomonas spp., Enterobacter spp., or Borrelia burgdorferi;
or viral pathogens: coxsackieviruses, echoviruses, poliovirus,
other enteroviruses, mumps, herpes simplex, varicella-zoster,
flaviviruses, or bunyaviruses.
[0157] Infections of the spinal cord are commonly caused by the
following bacterial species: Haemophilus influenzae, Neisseria
meningitidis, Streptococcus pneumoniae, Streptococcus agalactiae,
Listeria monocytogenes, or Borrelia burgdorferi; or viral
pathogens: coxsackieviruses, echoviruses, poliovirus, other
enteroviruses, mumps, herpes simplex, varicella-zoster,
flaviviruses, or bunyaviruses.
[0158] Infections of the eye/orbit are commonly caused by the
following bacterial species: Staphylococcus aureus, Streptococcus
pyogenes, Streptococcus pneumoniae, Streptococcus miller,
Escherichia coli, Bacillus cereus, Chlamydia trachomatis,
Haemophilus influenza, Pseudomonas spp., Klebsiella spp., or
Treponema pallidum; or viral pathogens: adenoviruses, herpes
simplex, varicella-zoster, or cytomegalovirus.
[0159] Infections of the salivary glands are commonly caused by the
following bacterial species: Staphylococcus aureus, viridans
streptococci (e.g., Streptococcus salivarius, Streptococcus
sanguis, Streptococcus mutans), Peptostreptococcus spp., or
Bacteroides spp., or other oral anaerobes; or viral pathogens:
mumps, influenza, enteroviruses, or rabies.
[0160] Infections of the mouth are commonly caused by the following
bacterial species: Prevotella melaninogenicus, anaerobic
streptococci, viridans streptococci, Actinomyces spp.,
Peptostreptococcus spp., or Bacteroides spp., or other oral
anaerobes; or viral pathogens: herpes simplex, coxsackieviruses, or
Epstein-Barr.
[0161] Infections of the tonsils are commonly caused by the
following bacterial species: Streptococcus pyogenes, or Group C or
G B-hemolytic streptococci; or viral pathogens: rhinoviruses,
influenza, coronavirus, adenovirus, parainfluenza, respiratory
syncytial virus, or herpes simplex.
[0162] Infections of the sinuses are commonly caused by the
following bacterial species: Streptococcus pneumoniae, Haemophilus
influenza, Moraxella catarrhalis, a-streptococci, anaerobic
bacteria (e.g., Prevotella spp.), or Staphylococcus aureus; or
viral pathogens: rhinoviruses, influenza, adenovirus, or
parainfluenza.
[0163] Infections of the nasopharynx are commonly caused by the
following bacterial species: Streptococcus pyogenes, or Group C or
G B-hemolytic streptococci; or viral pathogens: rhinoviruses,
influenza, coronavirus, adenovirus, parainfluenza, respiratory
syncytial virus, or herpes simplex.
[0164] Infections of the thyroid are commonly caused by the
following bacterial species: Staphylococcus aureus, Streptococcus
pyogenes, or Streptococcus pneumoniae; or viral pathogens: mumps,
or influenza.
[0165] Infections of the larynx are commonly caused by the
following bacterial species: Mycoplasma pneumoniae, Chlamydophila
pneumoniae, or Streptococcus pyogenes; or viral pathogens:
rhinovirus, influenza, parainfluenza, adenovirus, corona virus, or
human metapneumovirus.
[0166] Infections of the trachea are commonly caused by the
following bacterial species: Mycoplasma pneumoniae; or viral
pathogens: parainfluenza, influenza, respiratory syncytial virus,
or adenovirus.
[0167] Infections of the bronchi are commonly caused by the
following bacterial species: Mycoplasma pneumoniae, Chlamydophila
pneumoniae, Bordetella pertussis, Streptococcus pneumoniae, or
Haemophilus influenzae; or viral pathogens: influenza, adenovirus,
rhinovirus, coronavirus, parainfluenza, respiratory syncytial
virus, human metapneumovirus, or coxsackievirus.
[0168] Infections of the lung are commonly caused by the following
bacterial species: Streptococcus pneumoniae, Moraxella catarrhalis,
Mycoplasma pneumoniae, Klebsiella pneumoniae, or Haemophilus
influenza; or viral pathogens: influenza, adenovirus, respiratory
syncytial virus, or parainfluenza.
[0169] Infections of the pleura are commonly caused by the
following bacterial species: Staphylococcus aureus, Streptococcus
pyogenes, Streptococcus pneumoniae, Haemophilus influenzae,
Bacteroides fragilis, Prevotella spp., Fusobacterium nucleatum,
peptostreptococcus spp., or Mycobacterium tuberculosis; or viral
pathogens: influenza, adenovirus, respiratory syncytial virus, or
parainfluenza.
[0170] Infections of the mediastinum are commonly caused by the
following bacterial species: viridans streptococci, Peptococcus
spp., Peptostreptococcus spp., Bacteroides spp., Fusobacterium
spp., or Mycobacterium tuberculosis; or viral pathogens: measles,
rubella, Epstein-Barr, or cytomegalovirus.
[0171] Infections of the heart are commonly caused by the following
bacterial species: Streptococcus spp. (including S. mitior, S.
bovis, S. sanguis, S. mutans, S. anginosus), Enterococcus spp.,
Staphylococcus spp., Corynebacterium diptheriae, Clostridium
perfringens, Neisseria meningitidis, or Salmonella spp.; or viral
pathogens: enteroviruses, coxsackieviruses, echoviruses,
poliovirus, adenovirus, mumps, rubeola, or influenza.
[0172] Infections of the esophagus are commonly caused by the
following bacterial species: Actinomyces spp., Mycobacterium avium,
Mycobacterium tuberculosis, or Streptococcus spp.; or viral
pathogens: cytomegalovirus, herpes simplex, or
varicella-zoster.
[0173] Infections of the stomach are commonly caused by the
following bacterial species: Streptococcus pyogenes or Helicobacter
pylori; or viral pathogens: cytomegalovirus, herpes simplex,
Epstein-Barr, rotaviruses, noroviruses, or adenoviruses.
[0174] Infections of the small bowel are commonly caused by the
following bacterial species: Escherichia coli, Clostridium
difficile, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides
thetaiotaomicron, Clostridium perfringens, Salmonella enteriditis,
Yersinia enterocolitica, or Shigella flexneri; or viral pathogens:
adenoviruses, astroviruses, caliciviruses, noroviruses,
rotaviruses, or cytomegalovirus.
[0175] Infections of the colon/rectum are commonly caused by the
following bacterial species: Escherichia coli, Clostridium
difficile, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides
thetaiotaomicron, Clostridium perfringens, Salmonella enteriditis,
Yersinia enterocolitica, or Shigella flexneri; or viral pathogens:
adenoviruses, astroviruses, caliciviruses, noroviruses,
rotaviruses, or cytomegalovirus.
[0176] Infections of the anus are commonly caused by the following
bacterial species: Streptococcus pyogenes, Bacteroides spp.,
Fusobacterium spp., anaerobic streptococci, Clostridium spp.,
Escherichia coli, Enterobacter spp., Pseudomonas aeruginosa, or
Treponema pallidum; or viral pathogens: herpes simplex.
[0177] Infections of the perineum are commonly caused by the
following bacterial species: Escherichia coli, Klebsiella spp.,
Enterococcus spp., Bacteroides spp., Fusobacterium spp.,
Clostridium spp., Pseudomonas aeruginosa, anaerobic streptococci,
Clostridium spp., or Enterobacter spp.; or viral pathogens: herpes
simplex.
[0178] Infections of the liver are commonly caused by the following
bacterial species: Escherichia coli, Klebsiella spp., Streptococcus
(anginosus group), Enterococcus, spp. other viridans streptococci,
or Bacteroides spp.; or viral pathogens: hepatitis A, Epstein-Barr,
herpes simplex, mumps, rubella, rubeola, varicella-zoster,
coxsackieviruses, or adenovirus.
[0179] Infections of the gallbladder are commonly caused by the
following bacterial species: Escherichia coli, Klebsiella spp.,
Enterobacter spp., enterococci, Bacteroides spp., Fusobacterium
spp., Clostridium spp., Salmonella enteriditis, Yersinia
enterocolitica, or Shigella flexneri.
[0180] Infections of the biliary tract are commonly caused by the
following bacterial species: Escherichia coli, Klebsiella spp.,
Enterobacter spp., enterococci, Bacteroides spp., Fusobacterium
spp., Clostridium spp., Salmonella enteriditis, Yersinia
enterocolitica, or Shigella flexneri; or viral pathogens: hepatitis
A, Epstein-Barr, herpes simplex, mumps, rubella, rubeola,
varicella-zoster, cocsackieviruses, or adenovirus.
[0181] Infections of the pancreas are commonly caused by the
following bacterial species: Escherichia coli, Klebsiella spp.,
Enterococcus spp., Pseudomonas spp., Staphylococcal spp.,
Mycoplasma spp., Salmonella typhi, Leptospirosis spp., or
Legionella spp.; or viral pathogens: mumps, coxsackievirus,
hepatitis B, cytomegalovirus, herpes simplex 2, or
varicella-zoster.
[0182] Infections of the spleen are commonly caused by the
following bacterial species: Streptococcus spp., Staphylococcus
spp., Salmonella spp., Pseudomonas spp., Escherichia coli, or
Enterococcus spp.; or viral pathogens: Epstein-Barr,
cytomegalovirus, adenovirus, measles, rubella, coxsackieviruses, or
varicella-zoster.
[0183] Infections of the adrenal gland are commonly caused by the
following bacterial species: Streptococcus spp., Staphylococcus
spp., Salmonella spp., Pseudomonas spp., Escherichia coli, or
Enterococcus spp.; or viral pathogens: varicella-zoster.
[0184] Infections of the kidney are commonly caused by the
following bacterial species: Escherichia coli, Proteus mirabilis,
Proteus vulgatus, Providentia spp., Morganella spp., Enterococcus
faecalis, or Pseudomonas aeruginosa; or viral pathogens: BK virus,
or mumps.
[0185] Infections of the ureter are commonly caused by the
following bacterial species: Escherichia coli, Proteus mirabilis,
Proteus vulgatus, Providentia spp., Morganella spp., or
Enterococcus spp.
[0186] Infections of the bladder are commonly caused by the
following bacterial species: Escherichia coli, Proteus mirabilis,
Proteus vulgatus, Providentia spp., Morganella spp., Enterococcus
faecalis, or Corynebacterium jekeum; or viral pathogens:
adenovirus, or cytomegalovirus.
[0187] Infections of the peritoneum are commonly caused by the
following bacterial species: Staphylococcus aureus, Streptococcus
pyogenes, Streptococcus pneumoniae, Escherichia coli, Klebsiella
spp., Proteus spp., enterococci, Bacteroides fragilis, Prevotella
melaninogenica, Peptococcus spp., Peptostreptococcus spp.,
Fusobacterium spp., or Clostridium spp.
[0188] Infections of the retroperitoneal area are commonly caused
by the following bacterial species: Escherichia coli, or
Staphylococcus aureus.
[0189] Infections of the prostate are commonly caused by the
following bacterial species: Escherichia coli, Klebsiella spp.,
Enterobacter spp., Proteus mirabilis, enterococci spp., Pseudomonas
spp., Corynebacterium spp., or Neisseria gonorrhoeae; or viral
pathogens: herpes simplex.
[0190] Infections of the testicle are commonly caused by the
following bacterial species: Escherichia coli, Klebsiella
pneumoniae, Pseudomonas aeruginosa, Staphylococcus spp.,
Streptococcus spp., or Salmonella enteriditis; or viral pathogens:
mumps, coxsackievirus, or lymphocytic choriomeningitis virus.
[0191] Infections of the penis are commonly caused by the following
bacterial species: Staphylococcus aureus, Streptococcus pyogenes,
Neisseria gonorrhoeae, or Treponema pallidum; or viral pathogens:
herpes simplex.
[0192] Infections of the ovary/adnexae are commonly caused by the
following bacterial species: Neisseria gonorrhoeae, Chlamydia
trachomatis, Gardenerella vaginalis, Prevotella spp., Bacteroides
spp., Peptococcus spp. Streptococcus spp., or Escherichia coli.
[0193] Infections of the uterus are commonly caused by the
following bacterial species: Neisseria gonorrhoeae, Chlamydia
trachomatis, Gardenerella vaginalis, Prevotella spp., Bacteroides
spp., Peptococcus spp., Streptococcus spp., or Escherichia
coli.
[0194] Infections of the cervix are commonly caused by the
following bacterial species: Neisseria gonorrhoeae, Chlamydia
trachomatis, or Treponema pallidum; or viral pathogens: herpes
simplex.
[0195] Infections of the vagina are commonly caused by the
following bacterial species: Gardenerella vaginalis, Prevotella
spp., Bacteroides spp., peptococci spp., Escherichia coli,
Neisseria gonorrhoeae, Chlamydia Trachomatis, or Treponema
pallidum; or viral pathogens: herpes simplex.
[0196] Infections of the vulva are commonly caused by the following
bacterial species: Staphylococcus aureus, Streptococcus pyogenes,
or Treponema pallidum; or viral pathogens: herpes simplex.
[0197] Bacterial species are classified operationally as
collections of similar strains (which generally refers to groups of
presumed common ancestry with identifiable physiological but
usually not morphological distinctions, and which may be identified
using serological techniques against bacterial surface antigens).
Thus, each bacterial species (e.g., Streptococcus pneumoniae) has
numerous strains (or serotypes), which may differ in their ability
to cause infection or differ in their ability to cause infection in
a particular organ/site. For example, although there are at least
90 serotypes of Streptococcus pneumoniae, serotypes 1, 3, 4, 7, 8,
and 12 are most frequently responsible for pneumococcal disease in
humans.
[0198] Certain strains of Escherichia coli, referred to as
extraintestinal pathogenic E. coli(ExPEC), are more likely to cause
urinary tract infection or other extraintestinal infections such as
neonatal meningitis, whereas other strains, including
enterotoxigenic E. coli(ETEC), enteropathogenic E. coli(EPEC),
enterohemorrhagic E. coli(EHEC), Shiga toxin-producing E.
coli(STEC), enteroaggregative E. coli(EAEC), enteroinvasive E.
coli(EIEC) and diffuse adhering E. coli(DAEC) are more likely to
cause gastrointestinal infection/diarrhea. Even among the
sub-category of ExPEC strains, specific virulence factors (e.g.,
production of type-1 fimbriae) enable certain strains to be more
capable of causing infection of the bladder, while other virulence
factors (e.g., production of P fimbriae) enable other strains to be
more capable of causing infection in the kidneys. In accordance
with the present invention, an ExPEC strain(s) that is more likely
to cause infection in the bladder may be chosen for a formulation
to target immune dysregulation in the bladder cancer, whereas an
ExPEC strain(s) that is more likely to cause infection in the
kidney may be chosen for a formulation to target immune
dysregulation in the kidney cancer. Likewise, one or more of an
ETEC, EPEC, EHEC, STEC, EAEC, EIEC or DAEC strains of E. coli(i.e.,
strains that cause colon infection), may be chosen for a
formulation to treat immune dysregulation in the colon.
[0199] Similarly, there may be numerous subtypes of specific
viruses. For example, there are three types of influenza viruses,
influenza A, influenza B and influenza C, which differ in
epidemiology, host range and clinical characteristics. For example,
influenza A is more likely to be associated with viral lung
infection, whereas influenza B is more likely to be associated with
myositis (i.e., muscle infection). Furthermore, each of these three
types of influenza virus have numerous subtypes, which also may
differ in epidemiology, host range and clinical characteristics. In
accordance with the present invention, one may choose an influenza
A subtype most commonly associated with lung infection to target
immune dysregulation in the lung, whereas one may choose an
influenza B strain most commonly associated with myositis to treat
immune dysregulation in the muscle/soft tissues.
[0200] There are specific microbiota associated with some
pathological tissue states, for example microbiota of specific
tumours. For example, Fusobacterium and Providencia have been
associated with colorectal cancer.
[0201] The compositions of the invention include immunogens of
pathogenic microbial species (bacterial, viral or fungal) that are
pathogenic in a specific tissue or organ, in which the immunogens
are provided in the form of an artificial repertoire of mammalian
PRR agonists that recapitulate a distinct portion of the PRR
agonist signature of the microbial mammalian pathogen that is
pathogenic in the target tissue. In select embodiments, the portion
of the PRR agonist signature is distinct in the sense that it is
both: different from a reference PRR agonist signature of a microbe
that is not pathogenic in the target tissue; and, different than
the native PRR agonist signature of the microbial mammalian
pathogen. This distinct artificial repertoire of mammalian PRR
agonists are formulated together in a therapeutic vehicle for
combined presentation to an innate immune cell resident in the
target tissue in the mammalian host.
Formulations and Therapeutic Vehicles
[0202] Compositions of the invention may be provided alone or in
combination with other compounds (for example, nucleic acid
molecules, small molecules, peptides, or peptide analogues), in the
presence of a liposome, an adjuvant, or any pharmaceutically
acceptable carrier, in a form suitable for administration to
mammals, for example, humans (a "therapeutic vehicle"). As used
herein "pharmaceutically acceptable carrier" or "excipient"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like that are physiologically compatible.
The carrier can be suitable for any appropriate form of
administration, including subcutaneous, intradermal, intravenous,
parenteral, intraperitoneal, intramuscular, sublingual,
inhalational, intratumoural or oral administration.
Pharmaceutically acceptable carriers include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. The use
of such media and agents for pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or
agent is incompatible with the active compound (i.e., the specific
bacteria, bacterial antigens, or compositions thereof of the
invention), use thereof in the pharmaceutical compositions of the
invention is contemplated. Supplementary active compounds can also
be incorporated into the compositions.
[0203] Aspects of the invention involve the use of nanoparticle
(NP) formulations. For example, virus-like particles (VLPs) are in
essence empty viral particles with an intact protein hull and, in
some embodiments, membrane envelopes. In general, VLPs lack genetic
material. Production of VLPs may for example be by expression of
viral proteins in mammalian, avian, insect, plant, yeast, or
bacterial cells. Alternatively, fully synthetic VLPs may be
produced. Alternative nanoparticle formulations emulsions,
liposomes alginates, chitosan, and polylactide-coglycolide (PLGA)
NPs. Examples of NP/TLR ligand preparations that may be adapted for
use to induce immune responses are ligands for TLR2 (Pam(3)Cys),
TLR9 (Poly I:C), TLR4 (3-O-desacyl-4 0-monophosphoryl lipid A
(MPL)), TLR7 (9-benzyl-8-hydroxyadenine), TLR7/8 (resiquimod,
R848), and TLR9 (CpG DNA).
[0204] In addition to selected co-formulations, a wide variety of
adjuvants may be used to potentiate a desired immune response (see
Levast et al., 2014, Vaccines, 2, 297-322).
[0205] Treatment with PRR ligands according to the invention may be
combined with more traditional and existing therapies. For cancer,
for example, these may include chemotherapy, radiation therapy,
surgery, etc., or with a therapy that stimulates the immune system,
reduces inflammation or otherwise benefits the subject, such as
nutrients, vitamins and supplements. For example, vitamin A,
vitamin D, vitamin E, vitamin C, vitamin B complex, selenium, zinc,
co-enzyme Q10, beta carotene, fish oil, curcumin, green tea,
bromelain, resveratrol, ground flaxseed, garlic, lycopene, milk
thistle, melatonin, other antioxidants, cimetidine, indomethacin,
or COX-2 Inhibitors (e.g., Celebrex.TM. [celecoxib] or Vioxx.TM.
[rofecoxib]) may be also be administered to the subject.
[0206] Conventional pharmaceutical practice may be employed to
provide suitable formulations or compositions to administer the
compounds to subjects. Alternative routes of administration may be
employed, for example, parenteral, intravenous, intradermal,
subcutaneous, intramuscular, intracranial, intraorbital,
ophthalmic, intraventricular, intracapsular, intraspinal,
intrathecal, intracisternal, intraperitoneal, intranasal,
inhalational, aerosol, topical, intratumoural, sublingual or oral
administration. Therapeutic formulations may be in the form of
liquid solutions or suspensions; for oral administration,
formulations may be in the form of tablets or capsules; for
intranasal formulations, in the form of powders, nasal drops, or
aerosols; and for sublingual formulations, in the form of drops,
aerosols or tablets.
[0207] Methods well known in the art for making formulations are
found in, for example, "Remington's Pharmaceutical Sciences" (20th
edition), ed. A. Gennaro, 2000, Mack Publishing Company, Easton,
Pa. Formulations for parenteral administration may, for example,
contain excipients, sterile water, or saline, polyalkylene glycols
such as polyethylene glycol, oils of vegetable origin, or
hydrogenated napthalenes. Biocompatible, biodegradable lactide
polymer, lactide/glycolide copolymer, or
polyoxyethylene-polyoxypropylene copolymers may be used to control
the release of the compounds. Other potentially useful parenteral
delivery systems for include ethylene-vinyl acetate copolymer
particles, osmotic pumps, implantable infusion systems, and
liposomes. Formulations for inhalation may contain excipients, for
example, lactose, or may be aqueous solutions containing, for
example, polyoxyethylene-9-lauryl ether, glycocholate and
deoxycholate, or may be oily solutions for administration in the
form of nasal drops, or as a gel. For therapeutic or prophylactic
compositions, the pathogenic bacterial species are administered to
an individual in an amount effective to stop or slow progression or
metastasis of the cancer, or to increase survival of the subject
(relative to, for example, prognoses derived from the SEER
database) depending on the disorder.
[0208] Pharmaceutical compositions or formulations may be packaged
in a variety of ways depending upon the method used for
administering the drug. For example, an article of manufacture or
package may include a container having deposited therein the
pharmaceutical formulation in an appropriate form. Suitable
containers may for example include materials such as bottles
(plastic and glass), sachets, ampoules, plastic bags, metal
cylinders, and vials. The container may have a sterile access port,
for example the container may be an intravenous solution bag or a
vial having a stopper pierceable by a hypodermic injection needle.
The package or container may also include a tamper-proof or
multi-use mechanism adapted to control access to the contents of
the package or the container, for example a multi dose vial adapter
matched to a vial contained in the package. The container or
package may include a label, for example a label that describes the
contents of the container, for example a drug label identifying the
pharmaceutical composition therein and/or specifying modes or
routes of administration. The label may also include appropriate
warnings, for example specifying storage conditions for the
container or package, or setting out contraindications or adverse
effects of a mode of treatment. Articles of manufacture may
accordingly take the form of a "kit" comprising pharmaceutical
compositions or accessories adapted to facilitate use of
pharmaceutical compositions. Kits may include a label or package
insert, where the term "package insert" is used to refer to
instructions customarily included in commercial packages of
therapeutic products, that contain information about the
indications, usage, dosage, administration, contraindications
and/or warnings concerning the use of such therapeutic products.
Kits may further include accessories associated with use of the
pharmaceutical composition, including buffers, diluents, filters,
needles, and syringes. Kits may also be adapted for the delivery of
selected dosage forms of a pharmaceutical composition, for example
including a number of unit dosages. Such kits can include a memory
aid or mechanism, in the form of a physical or written indication
of the intended timing of a treatment schedule in which the dosages
are to be used.
[0209] A "companion diagnostic" may be associated with a
pharmaceutical treatment or composition. Companion diagnostics are
assays that facilitate the associated treatment, by providing
diagnostic or prognostic information, typically in the form of a
diagnostic test to determine the applicability of a treatment to a
specific patient. Point-of-care companion diagnostics may for
example involve providing diagnostic compositions and/or articles
of manufacture in conjunction with providing a pharmaceutical
formulation, for example as part of a kit. Alternatively, companion
diagnostics may be separately provided, as assays to monitor the
therapy of subjects or to predict the therapeutic efficacy of an
intended treatment. A companion diagnostic may for example take the
form of a medical device, such as an imaging tool, or a process
carried out by such a device, for example for conducting assays in
vitro, which provides information that is relevant for the safe and
effective use of a corresponding drug or biological product.
Companion diagnostics may be used with therapies disclosed herein
so as to provide diagnostic or prognostic information about
therapeutic efficacy or evidence of undesirable side effects or
risks. The use of a companion diagnostic with a particular
therapeutic may be stipulated in instructions, for example on the
labeling of a diagnostic device and/or the labeling of the
corresponding therapeutic product. Types of companion diagnostic
tests may for example include: screening and detection, in form of
tests that screen for genetic patterns, such as genetic SSI
response markers; prognosis and theranostics, such as assays for
biochemical SSI response markers that help to predict the future
course of a disease, or indicate a patient's response to a therapy;
monitoring, for example to evaluate the effectiveness and
appropriate dosing of a prescribed therapy; or, recurrence,
involving tests that analyze the patient's risk for a recurrence of
the disease.
[0210] An "effective amount" of a composition according to the
invention includes a therapeutically effective amount or a
prophylactically effective amount. A "therapeutically effective
amount" refers to an amount effective, at dosages and for periods
of time necessary, to achieve the desired therapeutic result, such
as reduction or elimination of the immune dysregulation. A
therapeutically effective amount of a composition may vary
according to factors such as the disease state, age, sex, and
weight of the individual, and the ability of the compound to elicit
a desired response in the individual. Dosage regimens may be
adjusted to provide the optimum therapeutic response. A
therapeutically effective amount may also be one in which any toxic
or detrimental effects of the composition are outweighed by the
therapeutically beneficial effects. A "prophylactically effective
amount" refers to an amount effective, at dosages and for periods
of time necessary, to achieve the desired prophylactic result, such
as amelioration of immune dysregulation. Typically, a prophylactic
dose is used in subjects prior to or at an earlier stage of cancer,
so that a prophylactically effective amount may be less than a
therapeutically effective amount.
[0211] For any particular subject, the timing and dose of
treatments may be adjusted over time (e.g., timing may be daily,
every other day, weekly, monthly) according to the individual need
and the professional judgment of the person administering or
supervising the administration of the compositions. For example, in
the context of subcutaneous or intradermal administration, the
compositions may be administered every second day. An initial dose
of approximately 0.05 ml may be administered subcutaneously,
followed by increases from 0.01-0.02 ml every second day until an
adequate skin reaction is achieved at the injection site (for
example, a 1 inch to 2 inch diameter delayed reaction of visible
redness at the injection site). Once this adequate immune reaction
is achieved, this dosing is continued as a maintenance dose. The
maintenance dose may be adjusted from time to time to achieve the
desired visible skin reaction (inflammation) at the injection site.
Dosing may be for a dosage duration, for example of at least 1
week, 2 weeks, 2 months, 6 months, 1, 2, 3, 4, or 5 years or
longer.
[0212] Oral dosages may for example range from 4 times per day,
daily or weekly. Dosing may be for a dosage duration, for example
of at least 1 week, 2 weeks, 2 months, 6 months, 1, 2, 3, 4, or 5
years or longer. In some embodiments, the invention may include
compositions administered sublingually or by inhalation, or
administered to one or more epithelial tissues (i.e., skin by
intradermal or subcutaneous injection; lung epithelium by
inhalation; gastrointestinal mucosa by oral ingestion; mouth mucosa
by sublingual administration) simultaneously or sequentially.
Accordingly, in some embodiments the compositions of the invention
are administered so as to provoke an immune response in an
epithelial tissue. In some embodiments, one or more epithelial
routes of administration may be combined with one or more
additional routes of administration, such as intratumoural,
intramuscular or intravenous administration.
[0213] In the case of immunogenic formulations, an immunogenically
effective amount of a composition of the invention can be provided,
alone or in combination with other compounds, for example with an
immunological adjuvant. The composition may for example include
compounds linked with a carrier molecule, such as bovine serum
albumin or keyhole limpet hemocyanin to enhance immunogenicity. An
immunogenic composition is a composition that includes materials
that elicit a desired immune response. An immunogenic composition
may select, activate or expand, without limitation: memory B, T
cells, neutrophils, monocytes or macrophages of the immune
system.
[0214] An antigenic composition comprising killed recombinant
bacteria for administration by injection may be made as follows.
The bacteria may be grown in suitable media, and washed with
physiological salt solution. The bacteria may then be centrifuged,
resuspended in saline solution, and killed with heat. The
suspensions may be standardized by direct microscopic count, mixed
in required amounts, and stored in appropriate containers, which
may be tested for safety, shelf life, and sterility in an approved
manner. In addition to the pathogenic bacterial species and/or
antigens thereof, a killed bacterial vaccine suitable for
administration to humans may include 0.4% phenol preservative
and/or 0.9% sodium chloride. The bacterial vaccine may also include
trace amounts of brain heart infusion (beef), peptones, yeast
extract, agar, sheep blood, dextrose, sodium phosphate and/or other
media components.
[0215] In select embodiments, medicaments may be administered at an
administration site in successive doses given at a dosage interval
of between one hour and one month, over a dosage duration of at
least one week. Optionally, the medicament may be administered
intradermally or subcutaneously. Optionally, the medicament may be
administered in a dose so that each dose is effective to cause a
visible localized inflammatory immune response at the
administration site. Optionally, the medicament may be administered
so that visible localized inflammation at the administration site
occurs within 1 to 48 hours. However, a visible localized
inflammatory immune response may not always be present in all
circumstances despite an immune response being initiated. There are
other methods by which the mounting of an immune response can be
monitored. For example, the profile (and relative change in
characterization) of immune cells from a subject undergoing an
immune reaction can be compared with those from a subject that is
not undergoing an immune reaction.
[0216] In another aspect, a method of monitoring efficacy of a
treatment regime in an individual being treated for an immune
dysfunction in a specific organ or tissue is provided. The method
involves measuring a characteristic of an immune response in a
past-treatment immune sample obtained from the specific organ or
tissue after the individual has been subject to the treatment
regime for a period of time.
[0217] In some embodiments, PRR agonists derived from bacteria that
are members of the endogenous flora of a particular region of the
GIT may be used to formulate immunogenic compositions of the
invention. The rows of Table 3 list a number of bacterial species,
together with the biological regions in which each species may form
a part of the endogenous flora. For example, Abiotrophia spp. are
typically members of the endogenous flora of the mouth.
TABLE-US-00004 TABLE 3 Human Bacterial Normal Flora (Endogenous
Bacterial Human Pathogens) Duodenum/ Bacterial species Mouth
Stomach Jejunum Ileum Colon CFU/mL 10{circumflex over ( )}5
10{circumflex over ( )}2 10{circumflex over ( )}5 10{circumflex
over ( )}8 10{circumflex over ( )}11 Abiotrophia spp + Acholeplasma
+ laidlawii Acidaminococcus + + + + fermentans Acinetobacter + + +
+ spp. Actinobacillus + spp. Actinobaculum + + + + spp. Actinomyces
+ + + + spp. Aeromonas spp. + + + Anaerorhabdus + + furcosus
Anaerococcus + + hydrogenalis Anaerococcus + + lactolyticus
Anaerococcus + + prevotii Atopobium spp. + + + + Bacillus spp. + +
Bacteroides + + caccae Bacteroides + + distasonis Bacteroides + +
eggerthii Bacteroides + + fragilis Bacteroides + + merdae
Bacteroides + + ovatus Bacteroides + + splanchnicus Bacteroides + +
thetaiotaomicron Bacteroides + + vulgatus Bifidobacterium + + +
adolescentis Bifidobacterium + + + bifidum Bifidobacterium + + +
breve Bifidobacterium + + + catenulatum Bifidobacterium + + + +
dentium Bifidobacterium + + + longum Bilophila + + + + wadsworthia
Burkholderia + + + cepacia Butyrivibrio + + + fibrisolvens
Campylobacter + + + concisus Campylobacter + + + curvus
Campylobacter + + + gracilis Campylobacter + + + jejuni
Campylobacter + + + rectus Campylobacter + + + + showae
Campylobacter + sputorum Capnocytophaga + granulosum Capnocytophaga
+ gingivalis Campylobacter + haemolytica Capnocytophaga + + + +
ochracea Capnocytophaga + sputigena Cardiobacterium + hominis
Cedecea spp + Centipeda + periodontii Citrobacter + + + freundii
Citrobacter + + + koseri Clostridium spp. + + + Corynebacterium +
accolens Corynebacterium + afermentans Desulfomonas + + + pigra
Dysgonomonas + + + spp. Eikenella + + + + corrodens Enterobacter +
+ + aerogenes Enterobacter + + + cloacae Enterobacter + + +
gergoviae Enterobacter + + + sakazakii Enterobacter + + + taylorae
Enterococcus + + + spp. Escherichia coli + + + Escherichia + + +
fergusonii Escherichia + + + hermannii Escherichia + + + vulneris
Eubacterium spp. + + + + Ewingella + americana Finegoldia + + +
magnus Fusobacterium + alocis Fusobacterium + + + gonidiaformans
Fusobacterium + + + mortiferum Fusobacterium + + + naviforme
Fusobacterium + + + + necrophorum Fusobacterium + + nucleatum
Fusobacterium + sulci Fusobacterium + + + russii Fusobacterium + +
+ varium Gardnerella + + + vaginalis Gemella + haemolysans Gemella
+ + + + morbillorum Globicatella spp. + + Granulicatella + spp.
Haemophilus + spp. Hafnia alvei + + + Helcococcus kunzii
Helicobacter spp. + + + Kingella spp. + Klebsiella spp. + + + +
Lactobacillus + + + + + acidophilus Lactobacillus + breve
Lactobacillus + casei Lactobacillus + + + + + fermentum
Lactobacillus + + + + reuteri Lactobacillus + + + + + salivarius
Leclercia + + + adecarboxylata Leminorella spp. + + + Leptotrichia
+ buccalis Megasphaera + + + elsdenii Micrococcus + luteus
Micrococcus + lylae Micromonas + micros Mitsuokella + + +
multiacidus Mobiluncus + + + curisii Mobiluncus + + + mulieris
Moellerella + + + wisconsensis Moraxella + catarrhalis other
Moraxella + spp. Morganella + + + morganii Mycoplasma + buccale
Mycoplasma + fermentans Mycoplasma + hominis Mycoplasma +
lipophilum Mycoplasma + orale Mycoplasma + pneumoniae Mycoplasma +
salivarium Pantoea + + + agglomerans Pasteurella + multocida
Pediococcus + + spp. Peptoniphilus + + + asaccharolyticus
Peptostreptococcus + + + + anaerobus Peptostreptococcus + + +
productus Porphyromonas + + + + asaccharolytica Porphyromonas + +
catoniae Porphyromonas + + endodontalis Porphyromonas + +
gingivalis Prevotella + + buccae Prevotella + + buccalis Prevotella
+ +
corporis Prevotella + + dentalis Prevotella + + denticola
Prevotella + + enoeca Prevotella + + heparinolytica Prevotella + +
intermedia Prevotella + + loescheii Prevotella + + melaninogenica
Prevotella + + nigrescens Prevotella oralis + + Prevotella oris + +
Prevotella + + oulorum Prevotella + + tannerae Prevotella + +
veroralis Prevotella + + zoogleoformans Propionibacterium +
propionicum Proteus mirabilis + + Proteus penneri + + Proteus
vulgaris + + Providencia + + rettgeri Providencia + + + stuartii
Pseudomonas + + + aeruginosa Retortamonas + + + intestinalis Rothia
+ dentocariosa Rothia + mucilaginosa Ruminococcus + + + productus
Selenomonas + spp. Serratia + + liquefaciens Serratia + +
marcescens Serratia odorifera + + Staphylococcus + aureus
Staphylococcus + epidermidis Streptococcus + + + agalactiae
Streptococcus + + + + anginosus Streptococcus + + + bovis
Streptococcus + + + + constellatus Streptococcus + criceti
Streptococcus + crista Streptococcus + equisimilis Streptococcus +
gordonii Streptococcus + + + intermedius Streptococcus + + mitis
Streptococcus + mutans Streptococcus + oralis Streptococcus +
parasanguis Streptococcus + + pyogenes Streptococcus + + salivarius
Streptococcus + + sanguis Streptococcus + sobrinus Streptococcus +
vestibularis Group C + G + + Streptococci Succinivibrio + + +
dextrinosolvens Sutterella spp. + + + Suttonella + indologenes
Tissierella + + + praeacuta Treponema + denticola Treponema +
maltophilum Treponema + socranskii Treponema + vincentii Ureaplasma
+ urealyticum Veillonella spp. + + + +
[0218] Endogenous microbial flora, such as bacteria, have access to
tissues for pathogenesis either through contiguous spread or
bacteremic spread. Under favorable conditions, endogenous organisms
can become pathogenic and invade locally and spread by contiguous
spread to adjacent tissues and organs. Endogenous bacterial flora
of the skin, mouth and colon are species that are understood to
also be amenable to bacteremic spread. Bacteria that are members of
a particular endogenous flora domain may therefore cause infection
in tissues or organs to which these bacteria may spread.
Accordingly, one aspect of the invention involves the use of PRR
agonists derived from endogenous microbial pathogens to treat an
immune dysregulation having symptoms localized to a region of the
GIT in which the endogenous bacteria may spread to cause infection.
The columns of Table 2 list domains for endogenous flora. The rows
of Table 4 list regions of the GIT within which immune
dysregulation may be symptomatic or etiologically located.
Accordingly, one aspect of the invention involves the use of PRR
agonists derived from endogenous microbial pathogens to formulate
immunogenic compositions for treating an immune dysregulation
symptomatic or etiologically located in the region of the GIT to
which the pathogen may spread to cause an infection. Accordingly,
in alternative embodiments, an immune dysregulation that is
symptomatic in the region listed in the first column of Table 2 may
be treated with immunogenic compositions comprising an artificial
repertoire of mammalian PRR agonists that recapitulates a distinct
portion of a PRR agonist signature of a microbial mammalian
pathogen that is a member of the endogenous flora of one or more of
the endogenous flora domains listed in the first row of Table 2 and
indicated with an X or a check mark in the appropriate row.
TABLE-US-00005 TABLE 4 Tissue/Organ Pathogenicity of Endogenous
Flora organ site Duo-denum/ Tissue Mouth Stomach Jejunum Ileum
Colon Oral x Tonsil x Nasopharynx/Sinus x Esophagus x Stomach x
Small bowel x x Colon/Rectum x Anus x
[0219] In accordance with the combined information in Tables 1 and
2, an immune dysregulation manifest in a particular region of the
GIT set out in column 1 of Table 2 may be treated with antigenic
compositions comprising an artificial repertoire of mammalian PRR
agonists that recapitulates a distinct portion of a PRR agonist
signature of a microbial mammalian pathogen that is one of the
corresponding bacterial species of Table 1, so that the column
headings in Table 2 are in effect replaced with the bacterial
species of Table 1.
[0220] In some embodiments, PRR agonists may be derived from
exogenous bacterial pathogens. For example, PRR agonists derived
from the organisms listed in Table 5 may be used in an artificial
repertoire of PRR agonists to treat an immune dysregulation that is
symptomatic in the region of the GIT listed with the relevant
organism in Table 5. In some embodiments, PRR agonists derived from
both endogenous and exogenous microbial species may be used in
combination.
TABLE-US-00006 TABLE 5 Exogenous Bacterial Human Pathogens, and
their Sites of Infection in the GIT. Bacterial Species Region of
the GIT Aerobacter spp. small bowel, colon, Bacillus anthracis
oral, small bowel, colon, hematological Bacillus cereus colon,
other Bacillus spp. colon, stomach, small bowel Brucella spp. small
bowel, colon Campylobacter coli small bowel, colon Campylobacter
colon jejuni Campylobacter small bowel, colon sputorum Clostridium
small bowel, colon, stomach bifermentans Clostridium colon, small
bowel botulinum Clostridium difficile colon Clostridium indolis
small bowel, colon, stomach, Clostridium small bowel, colon,
stomach mangenolii Clostridium small bowel, colon, stomach
perfringens Clostridium sordellii small bowel, colon, stomach
Clostridium small bowel, colon, stomach sporogenes Clostridium
small bowel, colon, stomach subterminale Edwarsiella tarda small
bowel, colon Francisella small bowel tularensis Helicobacter pylori
stomach Leptospirosis spp. oral Listeria small bowel, colon
monocytogenes Mycobacterium colon, small bowel bovis Mycobacterium
small bowel, colon tuberculosis Pediococcus spp. colon Plesiomonas
small bowel, colon shigelloides Rickettsia small bowel rickettsiae
Salmonella spp. stomach, small bowel, colon Shigella boydii colon
Shigella colon dysenteriae Shigella flexneri colon Shigella sonnei
colon other Spirillum spp. colon Streptococcus small bowel
zooepidemicus Treponema oral, anus pallidum Tropheryma small bowel,
colon whipplei Vibrio cholerae colon, small bowel Vibrio fluvialis
small bowel, colon Vibrio furnissii small bowel, colon Vibrio
hollisae small bowel, colon Vibrio colon, small bowel
parahaemolyticus Yersinia small bowel, colon enterocolitica
Yersinia small bowel, colon pseudotuberculosis
[0221] In some embodiments, PRR agonists for use in the invention
may be derived from viral pathogens. Table 6 provides an exemplary
list of viral pathogens together with the tissue and organ sites
for which each viral species is reportedly a pathogen. Accordingly,
one aspect of the invention involves utilizing immunogenic
compositions of PRR agonists derived from the named viruses to
treat an immune dysregulation that is symptomatic in the region of
the GIT that is identified adjacent to the name of the virus in
Table 6.
TABLE-US-00007 TABLE 6 Viral Human Pathogens and Their Sites of
Infection Virus Region of the GIT Herpes Simplex rectum, anus virus
(1 and 2) Cytomegalovirus small bowel, colon/rectum Epstein-Barr
virus oral Adenovirus oral, small bowel, colon Human anus, oral
papillomavirus Orthoreoviruses small bowel, colon, oral
Coltiviruses oral Rotaviruses small bowel, colon Alphaviruses small
bowel, colon, Coronaviruses oral, small bowel, colon Toroviruses
small bowel, colon Parainfluenza oral viruses Respiratory syncytial
oral virus Human oral, small bowel, colon metapneumovirus Vesicular
stomatitis oral, small bowel, colon virus Rabies virus oral
Influenza virus oral Hantaviruses oral Machupo virus small bowel,
colon Junin virus small bowel, colon Poliovirus small bowel, colon
Coxsackieviruses small bowel, colon Echoviruses oral, small bowel,
colon Hepatitis A virus small bowel, colon Rhinoviruses oral
Noroviruses and small bowel, colon other Caliciviruses Astroviruses
small bowel, colon Picobirnaviruses small bowel, colon Hepatitis E
virus small bowel, colon
[0222] In some embodiments, the pathogen from which PRR agonists
are derived for use in immunogenic compositions of the invention
may be one that is a common cause of acute infection in the region
of the GIT in which the immune dysregulation to be treated is
symptomatic. Table 7 identifies bacterial and viral pathogens of
this kind, together with the region of the GIT in which they
commonly cause infection. Accordingly, in selected embodiments, an
immune dysregulation that is symptomatic in a region of the GIT
identified in the first column of Table 7 may be treated with an
immunogenic composition that comprises an artificial repertoire of
mammalian PRR agonists that recapitulates a distinct portion of the
PRR agonist signature of a pathogenic organism listed in the second
column of Table 7.
TABLE-US-00008 TABLE 7 Common causes of acute infection (bacteria
and viruses) for selected regions of the GIT Selected regions of
the GIT Common Bacterial or Viral Pathogens Oral Prevotella
melaninogenicus, anaerobic streptococci, viridans streptococci,
Actinomyces spp., Peptostreptococcus spp., Bacteroides spp., and
other oral anaerobes herpes simplex, coxsackieviruses, Epstein-Barr
Stomach Streptococcus pyogenes, Helicobacter pylori
cytomegalovirus, herpes simplex, Epstein-Barr, rotaviruses,
noroviruses, adenoviruses Small Escherichia coli, Clostridium
difficile, Bacteroides fragilis, bowel Bacteroides vulgatus,
Bacteroides thetaiotaomicron, Clostridium perfringens, Salmonella
enteriditis, Yersinia enterocolitica, Shigella flexneri
adenoviruses, astroviruses, caliciviruses, noroviruses,
rotaviruses, cytomegalovirus Colon/ Escherichia coli, Clostridium
difficile, Bacteroides fragilis, Rectum Bacteroides vulgatus,
Bacteroides thetaiotaomicron, Clostridium perfringens, Salmonella
enteriditis, Yersinia enterocolitica, Shigella flexneri
adenoviruses, astroviruses, caliciviruses, noroviruses,
rotaviruses, cytomegalovirus Anus Streptococcus pyogenes,
Bacteroides spp., Fusobacterium spp., anaerobic streptococci,
Clostridium spp., E. coli, Enterobacter spp., Pseudomonas
aeruginosa, Treponema pallidum herpes simplex
[0223] Humans are hosts to a wide range of gastrointestinal
parasites, including various protozoa and helminths, which for
purposes of the present invention constitute pathogens of the GIT
(Schafer, T. W., Skopic, A. Parasites of the small intestine. Curr
Gastroenterol Reports 2006; 8:312-20; Jernigan, J., Guerrant, R.
L., Pearson, R. D. Parasitic infections of the small intestine. Gut
1994; 35:289-93; Sleisenger & Fordtran's Gastrointestinal and
liver disease. 8th ed. 2006; Garcia, L. S. Diagnostic medical
parasitology. 5th ed. 2007). Compositions of the invention may
accordingly include PRR agonists of various protozoa, including for
example: Giardia lamblia, Cryptosporidium parvum, Cryptosporidium
hominus, Isospora belli, Sarcocystis species, Coccidian like bodies
(Cyclospora species), Enterocytozoon bieneusi, Entamoeba
histolytica, Entamoeba dispar, Entamoeba coli, Entamoeba hartmanni,
Endolimax nana, Iodamoeba butschlii, Dientameoba fragilis,
Blastocystis hominus, Cyclospora cayetanensis, Microsporidia,
Trypanosoma cruzi, Chidomastix mesnili, Pentatrichomonas hominis,
Balantidium coli. Similarly, compositions of the invention may
include antigenic components of various helminths, including for
example: Cestodes (tapeworms), Taenia saginata, Taenia solium,
Diphyllobothrium species, Hymenolepis nana, Hymenolepis diminuta,
Dipylidium caninum, Nematodes (round worms), Ascaris lumbricoides,
Strongyloides stercoralis, Necator americanus, Ancylostoma
duodenale, Ancylostoma caninum, Tichuris trichiura, Capillaria
philippinensis, Trichostrongylus species, Trichinella species,
Necator americanus, Anisakis and related species, Angiostrongylus
costaricensis, Enterobius vermicularis, Trematodes (flukes),
Fasciolopsis buski, Heterophyes species, Echinostoma species,
Clonorchis sinensis, Opisthorchis species, Fasciola species,
Metagonimus yokogawi, Schistosoma mansoni, Schistosoma japonicum,
Schistosoma mekongi, Schistosoma intercalatum, Echinostoma species
and Paragonimus species.
[0224] In accordance with the foregoing, in various aspects, the
invention may involve the treatment of an immune dysregulation with
formulations of an artificial repertoire of mammalian PRR agonists
that recapitulates a distinct portion of a PRR agonist signature of
a microbial pathogen that is an: Acidaminococcus fermentans;
Acinetobacter spp.; Actinobaculum spp.; Actinomyces spp.; Aeromonas
spp.; Anaerorhabdus furcosus; Anaerococcus hydrogenalis;
Anaerococcus lactolyticus; Anaerococcus prevotii; Atopobium spp.;
Bacillus spp.; Bacteroides caccae; Bacteroides distasonis;
Bacteroides eggerthii; Bacteroides fragilis; Bacteroides merdae;
Bacteroides ovatus; Bacteroides splanchnicus; Bacteroides
thetaiotaomicron; Bacteroides vulgatus; Bifidobacterium
adolescentis; Bifidobacterium bifidum; Bifidobacterium breve;
Bifidobacterium catenulatum; Bifidobacterium dentium;
Bifidobacterium longum; Bilophila wadsworthia; Burkholderia
cepacia; Butyrivibrio fibrisolvens; Campylobacter concisus;
Campylobacter curvus; Campylobacter gracilis; Campylobacter jejuni;
Campylobacter rectus; Campylobacter showae; Capnocytophaga
ochracea; Cedecea spp; Citrobacter freundii; Citrobacter koseri;
Clostridium spp.; Desulfomonas pigra; Dysgonomonas spp.; Eikenella
corrodens; Enterobacter aerogenes; Enterobacter cloacae;
Enterobacter gergoviae; Enterobacter sakazakii; Enterobacter
taylorae; Enterococcus spp.; Escherichia coli; Escherichia
fergusonii; Escherichia hermannii; Escherichia vulneris;
Eubacterium spp.; Finegoldia magnus; Fusobacterium gonidiaformans;
Fusobacterium mortiferum; Fusobacterium naviforme; Fusobacterium
necrophorum; Fusobacterium nucleatum; Fusobacterium russii;
Fusobacterium varium; Gardnerella vaginalis; Gemella morbillorum;
Globicatella spp.; Hafnia alvei; Helicobacter spp.; Klebsiella
spp.; Lactobacillus acidophilus; Lactobacillus fermentum;
Lactobacillus reuteri; Lactobacillus salivarius; Leclercia
adecarboxylata; Leminorella spp.; Megasphaera elsdenii; Mitsuokella
multiacidus; Mobiluncus curisii; Mobiluncus mulieris; Moellerella
wisconsensis; Morganella morganii; Pantoea agglomerans; Pediococcus
spp.; Peptoniphilus asaccharolyticus; Peptostreptococcus anaerobus;
Peptostreptococcus productus; Porphyromonas asaccharolytica;
Proteus mirabilis; Proteus penneri; Proteus vulgaris; Providencia
rettgeri; Providencia stuartii; Pseudomonas aeruginosa;
Retortamonas intestinalis; Ruminococcus productus; Serratia
liquefaciens; Serratia marcescens; Serratia odorifera;
Streptococcus agalactiae; Streptococcus anginosus; Streptococcus
bovis; Streptococcus constellatus; Streptococcus intermedius; Group
C+G Streptococci; Succinivibrio dextrinosolvens; Sutterella spp.;
Tissierella praeacuta; Veillonella spp.; Aerobacter spp.; Bacillus
anthracis; Bacillus cereus; other Bacillus spp.; Borrelia
recurrentis; Brucella spp.; Campylobacter coli; Campylobacter
fetus; Campylobacter jejuni; Campylobacter sputorum; Clostridium
bifermentans; Clostridium botulinum; Clostridium difficile;
Clostridium indolis; Clostridium mangenolii; Clostridium
perfringens; Clostridium sordellii; Clostridium sporogenes;
Clostridium subterminale; Edwarsiella tarda; Francisella
tularensis; Listeria monocytogenes; Mycobacterium bovis;
Mycobacterium tuberculosis; Pediococcus spp.; Plesiomonas
shigelloides; Rickettsia rickettsiae; Salmonella spp.; Shigella
boydii; Shigella dysenteriae; Shigella flexneri; Shigella sonnei;
other Spirillum spp.; Streptococcus zooepidemicus; Tropheryma
whipplei; Vibrio cholerae; Vibrio fluvialis; Vibrio fumissii;
Vibrio hollisae; Vibrio parahaemolyticus; Yersinia enterocolitica;
Yersinia pseudotuberculosis; Herpes Simplex virus (1 and 2);
Cytomegalovirus; Adenovirus; Orthoreoviruses; Rotaviruses;
Alphaviruses; Coronaviruses; Toroviruses; Human metapneumovirus;
Vesicular stomatitis virus; Machupo virus; Junin virus; Poliovirus;
Coxsackieviruses; Echoviruses; Hepatitis A virus; Noroviruses and
other Caliciviruses; Astroviruses; Picobimaviruses; or Hepatitis E
virus.
[0225] In alternative aspects, the invention may involve the
treatment of an immune dysregulation with formulations of an
artificial repertoire of mammalian PRR agonists that recapitulates
a distinct portion of a PRR agonist signature of a microbial
mammalian pathogen that is a common small and larger bowel
pathogens, for example: Escherichia coli, Clostridium difficile,
Bacteroides fragilis, Bacteroides vulgatus, Bacteroides
thetaiotaomicron, Clostridium perfringens, Salmonella enteriditis,
Yersinia enterocolitica, Shigella flexneri; adenoviruses,
astroviruses, caliciviruses, noroviruses, rotaviruses, and
cytomegalovirus.
[0226] In selected embodiments, the invention involves diagnostic
steps to assess a patient's previous exposure to an organism. For
example, the diagnostic steps may include taking a medical history
of exposure to selected pathogens, and/or evaluating a patient's
immune response to a selected pathogen. For example, a serology
test may be conducted to detect antibodies to selected pathogens in
a patient's sera. In connection with this aspect of the invention,
antigenic determinants of a selected pathogen may be chosen for use
in an immunogenic composition on a selected patient based on a
diagnostic indication that the patient has had one or more prior
exposure(s) to the pathogen, for example by virtue of the presence
of antibodies to antigenic determinants of that pathogen in the
patient's sera.
[0227] In further selected embodiments, the invention involves
diagnostic steps to assess a patient's immunological response to
treatment with a selected immunogenic composition. For example, the
diagnostic steps may include evaluating a patient's immune response
to the immunological determinants of that immunogenic composition,
for example using a serological test to detect antibodies to those
immunogenic determinants. In connection with this aspect of the
invention, a treatment with a selected immunogenic composition may
be continued if the evaluation indicates that there is an active
immunological response to the immunogenic determinants of that
composition, and the treatment may be discontinued, and an
alternative treatment with a different immunogenic composition may
be initiated, if the evaluation indicates that there is not a
sufficiently active immunological response to the immunogenic
determinants of the immunogenic composition.
[0228] The immunomodulatory properties of formulations of the
invention can be employed for use in the treatment of a variety of
diseases characterized by pathological immune dysregulation, for
example using PRR agonists derived from endogenous pathogens or
exogenous pathogens that are pathogenic in the tissue or organ
within which the immune dysregulation is symptomatic or manifest,
including bacterial, viral and fungal pathogens. Table 8 lists
diseases characterized by immune dysregulation, which may be
treated in accordance with alternative aspects of the
invention.
TABLE-US-00009 TABLE 8 List of Diseases of Immune Dysregulation.
Acne vulgaris Acute disseminated encephalomyelitis Acute
hemorrhagicleukoencephalitis Addison's Disease Agammaglobulinemia
Allergies Alopecia areata Alzheimer's Amyotrophic Lateral Sclerosis
Anaemia, autoimmune hemolytic Anaemia, pernicious Ankylosing
spondylitis Anti-GBM/TBM Nephritis Antiphospholipid syndrome
Antisynthetase syndrome Arteritis, temporal (also known as "giant
cell arteritis") Arthritis, juvenile Arthritis, psoriatic
Arthritis, reactive (Reiter's syndrome, rea) Arthritis, rheumatoid
Asthma Atherosclerosis Atopic allergy Atopic dermatitis Autoimmune
enteropathy Autoimmune aplastic anemia Balo disease/Balo concentric
sclerosis Bartter syndrome Bechets Syndrome Berger's disease
Bickerstaff's encephalitis Blau syndrome Bronchitis, chronic
Bullous pemphigoid Bursitis Cardiomyopathy, autoimmune Castleman's
disease Celiac disease Chronic fatigue syndrome Chronic
inflammatory demyelinating polyneuropathy Chronic recurrent
multifocal osteomyelitis Churg-Strauss syndrome
Cicatricialpemphigoid Cirrhosis, primary biliary Cogan syndrome
Cold agglutinin disease Colitis Complement component 2 deficiency
Connective tissue disease, mixed Connective tissue disease,
undifferentiated COPD (chronic obstructive lung disease) Cranial
arteritis CREST syndrome Cryoglobulinemia Cushing's Syndrome
Cutaneous leukocytoclasticangiitis Cystitis, interstitial
Dacryadenitis Dego's disease Dercum's disease Dermatitis Dermatitis
herpetiformis Dermatitis, autoimmune progesterone Dermatomyositis
Diabetes Diabetes insipidus, nephrogenic Diabetes mellitus type 1
Diffuse cutaneous systemic sclerosis Discoid lupus erythematosus
Diverticulitis Dressier's syndrome Dysmenorrhea (menstrual
cramps/pain) Eczema Eczema Endometriosis Enthesitis-related
arthritis Eosinophilic fasciitis Eosinophilic gastroenteritis
Epidermolysisbullosaacquisita Erythema nodosum Essential mixed
cryoglobulinemia Evan's syndrome
Fibrodysplasiaossificansprogressiva Fibromyalgia Fibrosingaveolitis
Fungal infections (tinea pedis, onchomycosis, etc.) Gastritis,
atrophic Gastritis, atrophic Gastrointestinal pemphigoid Giant cell
arteritis Glomerulonephritis Glomerulonephritis Goodpasture's
syndrome Gout, acute Gout, arthritic Graves' disease Guillain-Barre
syndrome (GBS) Haemolytic anaemia Hashimoto's encephalitis
Hashimoto's thyroiditis Hemolyticanemia, autoimmune
Henoch-Schonleinpurpura Hepatitis, autoimmune Hepatitis, viral
Herpes gestationis Hypogammaglobulinemia Idiopathic Inflammatory
Demyelinating Diseases Idiopathic pulmonary fibrosis Iga
nephropathy Ileus (bowel obstruction) Inclusion body myositis
Inflammatory bowel disease, Crohn's disease Inflammatory bowel
disease, ulcerative colitis Inflammatory demyelinating
polyneuopathy Inner ear disease, autoimmune Interstitial cystitis
Irritable bowel syndrome (IBS) Juvenile idiopathic arthritis
Juvenile rheumatoid arthritis Kawasaki's Disease Kidney stones
Lambert-Eaton myasthenic syndrome Leukocytoclasticvasculitis Lichen
planus Lichen sclerosus Linear iga disease (LAD) Lou Gehrig's
disease (Also Amyotrophic lateral sclerosis) Lupoid hepatitis Lupus
Lupus erythematous Lymphoproliferative syndrome, autoimmune Majeed
syndrome Meniere's disease Meningitis Metabolic Syndrome
Microscopic polyangiitis Miller-Fisher syndrome Morphea
Mucha-Habermann disease Multiple sclerosis Myasthenia gravis
Myositis Myositis, inclusion body Nephritis Nephrotic syndrome
Neuromyelitisoptica (Also Devic's Disease) Neuromyotonia Occular
cicatricial pemphigoid Ocular inflammation (acute and chronic
non-bacterial inflammation of the anterior part of the eyes)
Opsoclonus myoclonus syndrome Ord thyroiditis Osteoarthritis
Osteoporosis Paget's disease of bone Palindromic rheumatism
Pancreatitis, autoimmune PANDAS (pediatric autoimmune
neuropsychiatric disorders associated with streptococcus)
Paraneoplastic cerebellar degeneration Parkinsonism Paroxysmal
nocturnal hemoglobinuria (PNH) Parry Romberg syndrome Pars planitis
Parsonnage-Turner syndrome Pelvic inflammatory disease Pemphigus
Pemphigus vulgaris Pericarditis, non-rheumatic Peripheral
neuropathy, autoimmune Perivenous encephalomyelitis POEMS syndrome
Polyarteritisnodosa Polychondritis, relapsing Polycystic ovary
syndrome (PCOS) Polyendocrine syndrome, autoimmune Polymyalgia
rheumatica Polymyalgia rheumatica Polymyositis Primary sclerosing
cholangitis Progressive inflammatory neuropathy Prostatitis,
chronic Pseudogout Psoriasis Psoriasis Pure red cell aplasia
Pyodermagangrenosum Rasmussen's encephalitis Raynaud phenomenon
Reiter's syndrome Restless leg syndrome Retinopathy of prematurity
Retroperitoneal fibrosis Rheumatoid fever Rhinitis, allergic
Sarcoidosis Schmidt syndrome Schnitzler syndrome Scleritis
Scleroderma Sclerosis, systemic Sjogren's syndrome
Spondyloarthropathy Still's disease Subacute bacterial endocarditis
(SBE) Susac's syndrome Sweet's syndrome Sydenham chorea Sympathetic
ophthalmia Takayasu's arteritis Temporomandibular joint disorder
(TMJD or TMD), or TMJ syndrome Thrombocytopenic purpura, autoimmune
Thrombocytopenic purpura, idiopathic Tolosa-Hunt syndrome Toxic
Shock Syndrome Transplant rejection Transverse myelitis
Undifferentiated spondyloarthropathy Urticaria Uveitis, autoimmune
Valvular disease, non -rheumatic Vasculitis Vitiligo Wegener's
granulomatosis
[0229] As provided in the Table above, arthritis is a chronic
inflammatory disease. In particular, arthritis is understood to be
a description of inflammation of one or more joints. There are many
types of arthritis, or conditions that have arthritic symptoms,
which include (but are not limited to) the following: Ankylosing
spondylitis, Behcet's disease, Ehlers-Danlos Syndrome, Familial
Mediterranean fever, Fibromyalgia, Fifth disease, Giant cell
arteritis, Gout, Haemochromatosis, Henoch-Schonleinpurpura,
Hyperimmunoglobulinemia D with recurrent fever, Inflammatory bowel
disease arthritis (including Crohn's Disease and Ulcerative
Colitis), Juvenile rheumatoid arthritis, Juvenile
spondyloarthropathy, Lyme disease, Marfan syndrome, Osteoarthritis,
Pseudo-gout, Psoriatic arthritis, Reactive Arthritis (Reiter's
syndrome), Rheumatoid arthritis, Sarcoidosis, Scleroderma, SEA
syndrome (seronegativity, enthesopathy, arthropathy), Sjogren's
syndrome, Still's disease, Systemic lupus erythematosus (SLE), TNF
receptor associated periodic syndrome, and Wegener's granulomatosis
(and other vasculitis syndromes).
Screening
[0230] Patients may advantageously be screened for disorders of
innate immunity, such as genetic disorders, for example by primary
sequence analysis or by analysis of epigenetic changes. A variety
of genetic disorders have for example been identified that are
associated with gene products involved in innate immunity (see
Mogensen T., 2009, Clinical Microbiology Reviews, Vol. 22, No. 2,
p. 240-273), such as TLR genes (TLR2, TLR3, TLR4, TLR5, TLR7, and
TLR9), signalling protein genes (MyD88, Mal, IRAK1, IRAK4, NEMO,
I.kappa.B.alpha., IRF5), NLR genes (NOD2, NALP1, NALP3) and others
(CD14, UNC93B). Patients identified as having a Medelian primary
immunodeficiency associated with impaired TLR signaling or
NF-.kappa.B activation may for example not benefit from some
embodiments, or may require an approach adapted to their condition.
Patients having polymorphisms in genes encoding components of
innate signalling pathways may also be identified prior to
treatment with an SSI, for example having mutations in the gene
encoding TIR-domain-containing adaptor-inducing beta interferon
(TRIF).
[0231] There are a variety of microbial strategies for evading the
innate immune system (Mogensen T., 2009, Clinical Microbiology
Reviews, Vol. 22, No. 2, p. 240-273), and embodiments of the
invention may accordingly be adapted to avoid the inhibitory effect
of such strategies on the triggered innate response. Select
embodiments provide recombinant microorganisms that lack virulence
factors that impede TLR signalling, such as recombinant E. coli
that lack TIR domain-containing proteins (Cirl, C. et al., 2008,
Nat. Med. 14:399-406). Gram negative bacterial formulation may
advantageously comprise an LPS that is recognized by a TLR, such as
TLR4, rather than a form of LPS that is not recognized by a TLR
(Homef, M. W. et al., 2002, Nat. Immunol. 3:1033-1040). Similarly,
bacterial formulations may advantageously include a class of
flagellin that activates a TLR, such as TLR5, rather than one that
does not (Andersen-Nissen, E. et al., 2005, Proc. Natl. Acad. Sci.
USA 102-9247-9252). In some embodiments, it may be advantageous to
exclude peptidases that proteolytically degrade important
components of the triggered innate response, such as the
amastigote-specific cysteine peptidases of Leishmania mexicana that
proteolytically degrade I.kappa.B and NF-.kappa.B (Cameron, P. et
al., 2004, J. Immunol. 173:3297-3304). In alternative embodiments,
these undesirable components may be removed from a formulation by
an appropriate step of manufacturing, for example to wash or
fractionate a microbial preparation so as to remove a
component.
[0232] Patients may be genotyped, for example by identifying
polymorphisms in PRR genes (see WO 2009003905). Genes associated
with inflammation and immune related diseases and disorders may for
example be the subject of screening, such as:
[0233] AIDS (KIR3DL1, NKAT3, NKB1, AMB11, KIR3DS1, IFNgamma,
CXCL12, SDF1); Autoimmune lymphoproliferative syndrome (TNFRSF6,
APT1, FAS, CD95, ALPS1A); Combined immunodeficiency, (IL2RG,
SCIDX1, SCIDX, IMD4); HIV-1 (CCL5, SCYA5, D17S136E, TCP228), HIV
susceptibility or infection (IL10, CSIF, CMKBR2, CCR2, CMKBR5,
CCCKR5 (CCR5)); Immunodeficiencies (CD3E, CD3G, AICDA, AID, HIGM2,
TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSF5, CD40LG, HIGM1, IGM, FOXP3,
IPEX, AIID, XPID, PIDX, TNFRSF14B, TACI); Inflammation (IL-10, IL-1
(IL-1a, IL-1b), IL-13, IL-17 (IL-17a (CTLA8), IL-17b, IL-17c,
IL-17d, IL-17f), 11-23, Cx3cr1, ptpn22, TNFa, NOD2/CARD15 for IBD,
IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3cl1); Severe combined
immunodeficiencies (SCIDs)(JAK3, JAKL, DCLRE1C, ARTEMIS, SCIDA,
RAG1, RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG, SCIDX1,
SCIDX, IMD4). Alternatively, genes involved in selected signalling
pathways may for example be screened, identifying for example
patients that are more or less susceptible to an SSI treatment,
such as: GM-CSF Signaling (LYN; ELK1; MAPK1; PTPN11; AKT2; PIK3CA;
CAMK2A; STAT5B; PIK3CB; PIK3C3; GNB2L1; BCL2L1; MAPK3; ETS1; KRAS;
RUNX1; PIM1; PIK3C2A; RAF1; MAP2K2; AKT1; JAK2; PIK3R1; STAT3;
MAP2K1; CCND1; AKT3; STAT1); IL-10 Signaling (TRAF6; CCR1; ELK1;
IKBKB; SP1; FOS; NFKB2; MAP3K14; MAPK8; MAPK13; RELA; MAPK14; TNF;
IKBKG; RELB; MAP3K7; JAK1; CHUK; STAT3; NFKB1; JUN; IL1R1; IL6);
Toll-like Receptor Signaling (IRAK1; EIF2AK2; MYD88; TRAF6; PPARA;
ELK1; IKBKB; FOS; NFKB2; MAP3K14; MAPK8; MAPK13; RELA; TLR4;
MAPK14; IKBKG; RELB; MAP3K7; CHUK; NFKB1; TLR2; JUN).
[0234] In addition, patients may for example be genotyped for SNPs
located in the non-coding regions of the genome that are linked to
inflammatory disorders, such as SNP's identified through publicly
available GWAS datasets, for example SNPs in genomic regions linked
to sequences which serve a regulatory role in
immune-function-related gene expression.
Alternative Embodiments
[0235] Although various embodiments of the invention are disclosed
herein, many adaptations and modifications may be made within the
scope of the invention in accordance with the common general
knowledge of those skilled in this art. Such modifications include
the substitution of known equivalents for any aspect of the
invention in order to achieve the same result in substantially the
same way. Numeric ranges are inclusive of the numbers defining the
range, and inclusive of all numbers and fractions subsumed within
the respective ranges. The word "comprising" is used herein as an
open-ended term, substantially equivalent to the phrase "including,
but not limited to", and the word "comprises" has a corresponding
meaning. Terms such as "consisting essentially of" and "consists
essentially of" allow for elements not explicitly recited, but
exclude elements that are found in the prior art or that affect a
basic or novel characteristic of the invention. As used herein, the
singular forms "a", "an" and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "a thing" includes more than one such thing. Citation
of references herein is not an admission that such references are
prior art to the present invention. Any priority document(s) and
all publications, including but not limited to patents and patent
applications, cited in this specification are incorporated herein
by reference as if each individual publication were specifically
and individually indicated to be incorporated by reference herein
and as though fully set forth herein. The invention includes all
embodiments and variations substantially as hereinbefore described
and with reference to the examples and drawings. Nothing herein is
intended as a promise of any specific utility for all
embodiments.
[0236] The term "about" or "approximately" as used herein when
referring to a measurable value such as a parameter, an amount, a
temporal duration, and the like, is meant to encompass variations
of +/-20% or less, preferably +/-10% or less, more preferably +/-5%
or less, and still more preferably +/-1% or less of and from the
specified value, insofar such variations are appropriate to perform
in the disclosed invention. It is to be understood that the value
to which the modifier "about" or "approximately" refers is itself
also specifically, and preferably, disclosed.
[0237] All references cited in the present specification are hereby
incorporated by reference in their entirety. In particular, the
teachings of all references herein specifically referred to are
incorporated by reference, along with all documents cited in
documents that are cited herein.
[0238] Standard reference works setting forth the general
principles of recombinant DNA technology include Molecular Cloning:
A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989;
Current Protocols in Molecular Biology, ed. Ausubel et al., Greene
Publishing and Wiley-Interscience, New York, 1992 (with periodic
updates) ("Ausubel et al. 1992"); the series Methods in Enzymology
(Academic Press, Inc.); Innis et al., PCR Protocols: A Guide to
Methods and Applications, Academic Press: San Diego, 1990; PCR 2: A
Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor
eds. (1995); Harlow and Lane, eds. (1988) Antibodies, a Laboratory
Manual; and Animal Cell Culture (R. I. Freshney, ed. (1987).
General principles of microbiology are set forth, for example, in
Davis, B. D. et al., Microbiology, 3rd edition, Harper & Row,
publishers, Philadelphia, Pa. (1980).
[0239] Reference throughout this specification to "one embodiment"
or "an embodiment" means that a particular feature, structure or
characteristic described in connection with the embodiment is
included in at least one embodiment of the present invention. Thus,
appearances of the phrases "in one embodiment" or "in an
embodiment" in various places throughout this specification are not
necessarily all referring to the same embodiment, but may.
Furthermore, the particular features, structures or characteristics
may be combined in any suitable manner, as would be apparent to a
person skilled in the art from this disclosure, in one or more
embodiments. Furthermore, while some embodiments described herein
include some but not other features included in other embodiments,
combinations of features of different embodiments are meant to be
within the scope of the invention, and form different embodiments,
as would be understood by those in the art. For example, in the
appended claims, any of the claimed embodiments can be used in any
combination.
[0240] In this description of the invention, reference is made to
the accompanying drawings that form a part hereof, and in which are
shown by way of illustration only of specific embodiments in which
the invention may be practiced. It is to be understood that other
embodiments may be utilized and structural or logical changes may
be made without departing from the scope of the present invention.
The description, therefore, is not to be taken in a limiting sense,
and the scope of the present invention is defined by the appended
claims.
[0241] Preferred statements (features) and embodiments may be
combined with any other features or embodiments unless clearly
indicated to the contrary. In particular, any feature indicated as
being preferred or advantageous may be combined with any other
feature or features or statements indicated as being preferred or
advantageous.
[0242] In some embodiments, the invention excludes steps that
involve medical or surgical treatment. Similarly, in some
embodiments, the invention disclaims naturally occurring
embodiments, so that aspects of the invention relate only to
anthropogenic compositions. Further, in select aspects of the
invention, previously known products, process of making products,
or methods of using products are hereby disclaimed.
General Codes and Abbreviations
[0243] SSI Site Specific Immunomodulator [0244] MC-38 Murine Colon
Adenocarcinoma cell line [0245] PD1 Programmed cell death 1 [0246]
OD Optical density [0247] IP Intraperitoneal [0248] SC Subcutaneous
[0249] SOP Standard operating protocol [0250] RPM Revolutions per
minute [0251] EDTA Ethylenediaminetetraacetic acid [0252] ANOVA
Analysis of variance [0253] Ly6G Lymphocyte antigen 6 complex,
locus G [0254] Ly6C Lymphocyte antigen 6 complex, locus C [0255]
CD45 Cluster of differentiation 45 [0256] SD Standard deviation
[0257] NA No value; not applicable; not present [0258] Rael
Ribonucleic acid export 1 [0259] CD3 Cluster of differentiation 3
[0260] CD11b Cluster of differentiation molecule 11B [0261] KO
knockout [0262] PBS Phosphate Buffered Saline [0263] NKG2D Natural
killer group 2, member D [0264] g Gram [0265] .mu.M micrometre
[0266] .mu.L Microliter [0267] hr Hours [0268] min Minute [0269]
QBECO Escherichia coli whole killed cell SSI [0270] QBKPN
Klebsiella pneumoniae phylogroup III (also known as K. variicola)
whole killed cell SSI [0271] QBSAU Staphylococcus aureus whole
killed cell SSI
EXAMPLES
Example 1: Recombinant Microbes
[0272] A family of virulence factors in Escherichia coli and
Brucella melitensis, named TIR domain-containing proteins, impede
TLR signalling through direct binding to MyD88, thus suppressing
innate immunity and increasing bacterial virulence. Aspects of the
invention accordingly provide recombinant bacteria that lack
expression of TIR domain-containing proteins, or other virulence
factors that interfere with an innate host immune response to the
pathogen.
Staphylococcus aureus
[0273] In select embodiments, compositions may be prepared from
recombinant S. aureus strains. For example, strains of sequence
type ST-291, having the following alleles, or homologous sequences
being at least 99% identical thereto: arcc-3, aroe-37, glpf-19,
gmk-2, pta-20, tpi-26, and yqil-32 (Larsen et al., 2012, O. J.
Clin. Micobiol 50(4): 1355-1361). Strains may totally lack
resistance genes to the following classes of antibiotic:
aminoglycoside, beta-lactam, fluoroquinolone, fosfomycin, fusidic
acid, MLS--macrolide, lincosamide and streptogramin B,
nitroimidazole, oxazolidinone, phenicol, rifampicin, sulphonamide,
tetracycline, trimethoprim, and glycopeptide. Alternatively,
strains may have one or more resistance genes, such as the blaZ
beta-lactam resistance gene (accession AP004832). Similarly,
strains may or may not include one or more virulence factor genes
(Cosentino et al., 2013, PLoS ONE 8(10):e77302), having for example
at least 90%, 95%, 99% or 100% identity to selected database
sequences (identified by accession number in the following tables).
[Recite strains lacking leukotoxins, particularly targeting the
innate immune system]
TABLE-US-00010 TABLE 9 S. aureus - Adherence Virulence Factors
Virulence Accession factor Protein function number eap
extracellular adherence protein CP002110.1 fnbB fibronectin-binding
protein B AM990992.1 fnbB fibronectin-binding protein B AM990992.1
icaA intercellular adhesion protein A CP003808.1 atl bifunctional
autolysin Atl AM990992.1 eap extracellular adherence protein
CP002114.2 icaB intercellular adhesion protein B AM990992.1 vwb von
Willebrand factor-binding CP002643.1 protein spa spa immunoglobulin
G binding BA000033.2 protein A spa spa immunoglobulin G binding
AM990992.1 protein A vwb von Willebrand factor-binding AM990992.1
protein vwb von Willebrand factor-binding AM990992.1 protein icaC
intercellular adhesion protein C CP003808.1 ebpS cell surface
elastin binding CP001996.1 protein clfA fibrinogen-binding protein
A, AM990992.1 clumping factor icaR intercellular adhesion regulator
AJ938182.1 sdrC Ser-Asp rich fibrinogen-binding CP001996.1 protein
C spa spa immunoglobulin G binding CP002110.1 protein A efb
extracelular fibrinogen-binding CP003045.1 protein fib
fibrinogen-binding protein CP003045.1 sdrH Ser-Asp rich
fibrinogen-binding CP003194.1 portein H
TABLE-US-00011 TABLE 10 S. aureus - Toxin Virulence Factors
Virulence Accession factor Protein function number SEntA putative
enterotoxin type A CP003194.1 hlgA gamma-hemolysin chain II
CP002110.1 precursor hlgC gamma-hemolysin component BX571856.1 C
hlgB gamma-hemolysin component HE681097.1 B precursor lukE
leukotoxin LukE AJ938182.1 hlgB gamma-hemolysin component
FR821779.1 B precursor eta exfoliative toxin A AM990992.1 SExo
exotoxin CP003045.1 SExo exotoxin CP003045.1 SExo exotoxin
CP003045.1 hla alpha-hemolysin precursor AM990992.1 SExo exotoxin
CP003045.1 hlb beta-hemolysin CP003166.1 lukF-PV LukF-PV BX571856.1
set16 exotoxin homolog BA000033.2 SExo exotoxin CP003045.1 SExo
exotoxin CP003045.1 SExo exotoxin CP003045.1 set1 superantigen-like
protein BX571856.1 set5 superantigen-like protein 5 CP003045.1 set4
superantigen-like protein AM990992.1 hld delta-hemolysin HE681097.1
set26 exotoxin homolog BA000033.2
TABLE-US-00012 TABLE 11 S. aureus - Exoenzyme Virulence Factors
Virulence Accession factor Protein function number geh glycerol
ester hydrolase HE681097.1 hysA hyaluronate lyase BA000018.3 nuc
thermonuclease BA000018.3 sspA serine V8 protease FR821779.1 sspB
cysteine protease BX571856.1 sspC cysteine protease CP003808.1 nuc
thermonuclease CP003808.1 splA serine protease splA CP003194.1 splC
serine protease splC BX571856.1 splB serine protease splB
CP002110.1 sak staphylokinase CP000253.1 scn complement inhibitor
SCIN CP002120.1 splE serine protease splE BX571856.1 splF serine
protease splF CP002110.1 geh glycerol ester hydrolase AM990992.1
sspB cysteine protease CP002110.1 hysA hyaluronate lyase
AM990992.1
TABLE-US-00013 TABLE 12 S. aureus - Host Immune Evasion Virulence
Factors Virulence Accession factor Protein function number capP
capsular polysaccharide CP001996.1 synthesis enzyme capP capO
capsular polysaccharide CP003808.1 synthesis enzyme capO capN
capsular polysaccharide FR821779.1 synthesis enzyme capN capM
capsular polysaccharide CP003808.1 synthesis enzyme capM cap5L
capsular polysaccharide CP002120.1 biosynthesis protein cap5L cap5K
capsular polysaccharide CP003045.1 biosynthesis protein cap5K cap5I
capsular polysaccharide AM990992.1 biosynthesis protein cap5I cap5H
capsular polysaccharide AM990992.1 biosynthesis protein cap5H capG
capsular polysaccharide CP003808.1 synthesis enzyme capG capF
capsular polysaccharide CP003808.1 synthesis enzyme capF capE
capsular polysaccharide CP003808.1 synthesis enzyme capE capD
polysaccharide biosynthesis CP003808.1 protein capD cap5C capsular
polysaccharide CP001844.2 biosynthesis protein cap5C capB capsular
polysaccharide HE681097.1 biosynthesis protein capB cap8A truncated
capsular BA000033.2 polysaccharide synthesis enzyme cap5A isb
IgG-binding protein SBI BX571856.1 capD polysaccharide biosynthesis
AP009351.1 protein capD capC capsular polysaccharide AM990992.1
synthesis protein capC cap1B capsular polysaccharide CP002120.1
biosynthesis protein cap1B cap1A capsular polysaccharide CP002110.1
biosynthesis protein cap1A cap5M capsular polysaccharide AJ938182.1
biosynthesis protein cap5M
TABLE-US-00014 TABLE 13 S. aureus - Secretion system Virulence
Factors Virulence Accession factor Protein function number esxB
virulence factor EsxB family FR821777.2 protein esaC EsaC protein
within ESAT-6 CP000730.1 gene cluster essC type VII secretion
protein EssC BA000018.3 esaC EsaC protein within ESAT-6 CP003166.1
gene cluster essB putative secretion system CP003808.1 component
EssB esaB Putative secretion accessory CP001844.2 protein EsaB/YukD
essA protein secretion system EssA CP001844.2 esaA type VII
secretion protein EsaA CP001844.2 esxA ESAT-6/WXG100 family
CP002120.1 secreted protein EsxA/YukE
[0274] Recombinant strains may include one or more plasmids
(Carattoli et al., 2014, Antimicrobial Agents and Chemotherapy
58(7): 3895-3903), for example having 90%, 95%, 99% or 100%
identity to plasmid rep5 (accession NC005011) or plasmid rep16
(accession CP002115.1).
Klebsiella spp.
[0275] In select embodiments, compositions may be prepared from
recombinant Klebsiella strains, such as K. pneumoniae or K.
variicola (formerly identified as K. pneumoniae). For example,
strains of a sequence type having the following alleles, or
homologous sequences being at least 99% identical thereto: gapa16,
infb24, mdh30, pgi40, phoe92, rpob17, tonb67. Strains may totally
lack resistance genes to the following classes of antibiotic:
aminoglycoside, beta-lactam, fluoroquinolone, fosfomycin, fusidic
acid, MLS--macrolide, lincosamide and streptogramin B,
nitroimidazole, oxazolidinone, phenicol, rifampicin, sulphonamide,
tetracycline, trimethoprim, and glycopeptide. Alternatively,
strains may have one or more resistance genes, such as the blaLEN24
beta-lactam resistance gene (accession AM850914). Similarly,
strains may or may not include one or more virulence factors
identified in the following table (see Leticia et al., 2014, BMC
Biology 12:41).
TABLE-US-00015 TABLE 14 KPN Virulence Factors Virulence-factor
Function rmpA Regulator of capsule expression Aerobactin
Siderophore Enterobactin Siderophore Yersiniabactin Siderophore
Colibactin Genotoxin T4SS (virB) Conjugative machinery/protein
secretion T2SS Protein secretion T6SS Protein secretion Pld-family
Lipid metabolism Sel1 lipoproteins Unknown cOMP Putative cytotoxin
Igg-like Binding to extra cellular matrix compounds SEFIR-domain
Potentially hijack IL17R signaling pathways Bcl Binding to
hydrophobic ligands/putative regulation of homeostasis and
immunity
Escherichia coli. (Prostate)
[0276] In select embodiments, compositions may be prepared from
recombinant E. coli strains specifically adapted for therapy of
prostate immune dysfunction. For example, strains of a sequence
type having the following alleles, or homologous sequences being at
least 99% identical thereto: adk-37, fumc-38, gyrb-19, icd-37,
mdh-151, pura-11, reca-26 (sequence type 1231). Strains may totally
lack resistance genes to the following classes of antibiotic:
aminoglycoside, beta-lactam, fluoroquinolone, fosfomycin, fusidic
acid, MLS--macrolide, lincosamide and streptogramin B,
nitroimidazole, oxazolidinone, phenicol, rifampicin, sulphonamide,
tetracycline, trimethoprim, and glycopeptide. Similarly, strains
may or may not include one or more virulence factor genes, having
for example at least 90%, 95%, 99% or 100% identity to selected
database sequences (identified by accession number in the following
tables). The strain may also lack stx holotoxin virulence
factors.
TABLE-US-00016 TABLE 15 E. coli - Virulence factors Virulence
Accession factor Protein function number iroN Enterobactin
siderophore CP000243 receptor protein sfaS S-fimbriae minor subunit
CP000243 senB Plasmid-encoded enterotoxin CP000038 iss Increased
serum survival CU928160 gad Glutamate decarboxylase CP002167 cnf1
Cytotoxic necrotizing factor CP002167 ccl Cloacin DQ298019
[0277] The serotype of the E. coli strain may for example be
O18ac:H7, for example representing the presence of H type serotype
gene fliC (accession AF228492, and O type serotype genes wzx
(accession GU299793), and wzy (accession GU299793).
[0278] Recombinant strains may include one or more plasmids, for
example having 90%, 95%, 99% or 100% identity to plasmid IncFIB
(accession AP001918) and/or plasmid IncFII(29) (accession
CP003035), and/or plasmid CoIRNAI (accession DQ298019) and/or
plasmid Col156 (accession NC009781).
[0279] The recombinant E. coli may for example be, or be derived
from an E. coli strain having at least 80%, 90% or 95% sequence
identity to E. coli UT189 (see Chen et al., 2006, Proc Natl Acad
Sci USA 103:5977-82).
Escherichia coli. (Colon)
[0280] In select embodiments, compositions may be prepared from
recombinant E. coli strains specifically adapted for therapy of
colon immune dysfunction. For example, strains of a sequence type
having the following alleles, or homologous sequences being at
least 99% identical thereto: adk-76, fumc-43, gyrb-9, icd-36,
mdh-404, pura-14, reca-10 (sequence type ST-5292). Strains may
totally lack resistance genes to the following classes of
antibiotic: aminoglycoside, beta-lactam, fluoroquinolone,
fosfomycin, fusidic acid, MLS--macrolide, lincosamide and
streptogramin B, nitroimidazole, oxazolidinone, phenicol,
rifampicin, sulphonamide, tetracycline, trimethoprim, and
glycopeptide. Alternatively, strains may have one or more
resistance genes, such as the strB or strA aminoglycoside
resistance genes (accession numbers M96392 or AF321551), and/or
sul1 sulphonamide resistance gene (accession AY224185), and/or sul2
sulphonamide resistance gene (accession GQ421466), and/or dfrA5
trimethoprim resistance (accession X12868). Similarly, strains may
or may not include one or more virulence factor genes, having for
example at least 90%, 95%, 99% or 100% identity to selected
database sequences (identified by accession number in the following
tables). The strain may also have a gene that is at least 95% or
99% or 100% identical to the stx holotoxin virulence factor gene
stx1 (accession M19437).
TABLE-US-00017 TABLE 16 E. coli - Virulence factors Virulence
Accession factor Protein function number Gad Glutamate
decarboxylase CP001671 lha Adherence protein AE005174 Gad Glutamate
decarboxylase CP001671 senB Plasmid-encoded enterotoxin CP000038
sigA Shigella IgA-like protease CP000038 homologue stx1B Shiga
toxin 1, subunit B, AM230663 variant a stx1A Shiga toxin 1, subunit
A, EF079675 variant a astA EAST-1 heat-stable toxin AB042002
[0281] The serotype of the E. coli strain may for example be
O117:H7, for example representing the presence of H type serotype
gene fliC (accession AF228492, and O type serotype genes wzx
(accession EU694096).
[0282] Recombinant strains may include one or more plasmids, as set
out in the following table.
TABLE-US-00018 TABLE 17 E. coli plasmids Accession Plasmid Note
number IncFII(pRSB107) pRSB107 AJ851089 IncB/O/K/Z CU928147
Col(BS512) NC_010656 IncFIB(AP001918) AP001918 IncB/O/K/Z GU256641
Col156 NC_009781 IncFII AY458016
[0283] The recombinant E. coli(colon) may for example be, or be
derived from an E. coli strain having at least 80%, 90% or 95%
sequence identity to E. coli SE15 or any O117:H7 serotype E.
coli.
Example 2: Minimal SSI Formulations
[0284] This Example illustrates modest efficacy in a minimal SSI
formulation comprising TLR agonists and a microbial antigen. In
this Example, the TLR agonists are a TLR2/6 agonist, diacylated
lipoprotein (InVivogen Pam2CSK4) and a TLR4 agonist (LPS, Sigma
L1519). The microbial antigen was a recombinant outer membrane
protein A (ompA) from Klebsiella pneumoniae (CUSABIO
CSB-EP340587KBG). These components were formulated together in a
liposomal vehicle, and administered in a murine model of SSI
therapy in accordance with the treatment timeline illustrated in
FIG. 1. As illustrated in FIG. 2, TLR-only liposomes did not have
the same degree of activity as the TLR+Ag liposome constructs,
illustrating that the engagement of alternative immunogenic
pathways can augment an SSI effect. It is important to note in the
context of this data that the antigenic component of these minimal
formulations was recombinantly expressed in E. coli, with a purity
given by the manufacturer of >90% (SDS-PAGE) with the attendant
characteristic that the antigen preparations included additional
bacterial components, including additional PRR agonists, that act
as additional innate immunogens.
Example 3: SSI Mediates Anti-Tumour Activity in MyD88-/- Mice
[0285] Pathogen recognition and inflammatory signalling in innate
immune defenses involves a number of pathways, including MyD88
dependent and MyD88 independent pathways (Mogensen, 2009, Clinical
Microbiology Reviews, 22(2): 240). In this Example, using a B16
melanoma model in a commercial strain of MyD88-/- mice, it was
observed that B16 tumour burden is enhanced in MyD88 mice, relative
to B6 mice (genetically matched except for the MyD88 knockout).
This is consistent with literature reports to the effect that MyD88
knockouts are inherently less able to control disease. In the model
of SSI therapy, with a Klebsiella SSI ("KPN SSI"), a significantly
reduced tumour burden was observed following treatment with KPN SSI
in both WT B6 and MyD88-knockout mice. This is evidence that SSIs
can work in a MyD88-independent manner, at least in part,
indicating that alternative PRR signalling pathways, beyond what
are generally considered classic TLR signalling, are involved in
the SSI mediated innate immune responses. This is consistent with
another exemplary observation, that the NOD-like receptor NLRP3 is
up-regulated in cultured cells treated with KPN SSI and ECO SSI.
Alternatively, in some embodiments, SSIs may mediate classical TLR
signalling via an adaptor other than MyD88 downstream of TLR, such
as TRIF. Furthermore, in additional studies it has been shown that
SSI therapies can engage the MyD88 signalling pathway. In
combination, these results are evidence of the robust and diverse
PRR signalling pathways that may be engaged by SSI therapies.
Example 4: Pre-Exposure to Related Strains Potentiates SSI
Efficacy
[0286] This Example illustrates that in some embodiments,
pre-exposure of an organism to a microbial pathogen potentiates
subsequent SSI efficacy.
[0287] In an animal model of SSI therapy, treated in accordance
with the treatment timeline illustrated in FIG. 3, pre-infection
with Klebsiella pneumoniae potentiated KPN SSI efficacy using a
distinct strain of Klebsiella sp., whereas pre-infection with S.
pneumoniae failed to induce KPN SSI efficacy (in models of cancer
in the lungs). The result, as illustrated in FIG. 4, indicates that
an SSI response may require or benefit from pre-exposure of an
organism to at least closely-related pathogens. This is consistent
with another observation, of KPN SSI activity in mice from colonies
that test negative for K. pneumoniae, but may carry K. oxytoca, a
strain that's commonly found in research animal colonies,
indicating that in some cases pre-exposure to K. oxytoca is
adequate to induce responsiveness to KPN SSI. Differences in
pre-exposure in mice from Jackson Labs (JAX) compared to mice from
Taconic (TAC) are evident in FIG. 4, which illustrates that even
without pre-infection, S. pneumoniae (SPN) did not elicit the same
therapeutic efficacy that QBKPN did in mice sourced from JAX. In
mice sourced from TAC, pre-infection was needed for QBKPN to show
efficacy.
Example 5: Killed Klebsiella pneumoniae Treatment Reduces Tumour
Burden in Murine Melanoma Model
[0288] This Example illustrates Klebsiella-mediated anti-cancer
efficacy in metastatic-like B16 melanoma using heat-killed
Klebsiella pneumoniae SSI. Subcutaneous injection of KPN SSI
significantly reduced tumour burden. Furthermore, subcutaneous
treatment with heat-killed E. coli(ECO SSI) or Staph. aureus (SAU
SSI) did not have a therapeutic effect in the lung tumour model.
This illustrates that subcutaneous immune induction using a
lung-specific pathogen activates a lung-specific antitumour
response. To illustrate the effects of pre-exposure to K.
pneumoniae, mice were exposed to live K. pneumoniae via
intratracheal infection prior to subcutaneous injections with KPN
SSI. Pre-exposure to K. pneumoniae significantly enhanced KPN
SSI-induced anti-tumour immunity and control of metastatic-like B16
melanoma in the lungs. The anti-tumour efficacy in exposed mice
correlated with an influx of monocytes and neutrophils, but did not
correlate with an influx of T cells into the lungs. Collectively,
these data illustrate that pre-exposure to K. pneumoniae may, in
some embodiments, induce tissue-specific immunologic memory, for
example an innate immunological memory, that mediates tumour
cytolysis.
Example 6: Site Specificity in a Skin Cancer Model
[0289] This Example illustrates site specificity of a S. aureus SSI
in a murine skin cancer model. This Example involved the use of a
concentrated S. aureus SSI (10.times.QBSAU which is designated
"QBSAUR" herein), as well as a Klebsiella sp. SSI, an E. coli SSI
and placebo, in a B16 skin tumour model in which .about.100,000 B16
melanoma cells were injected into the right flank of C57BL/6 mice
in a volume of 100 ul on Day 0. SSI treatment started on Day -10
and continued till Day +12. Tumour volumes were monitored starting
on Day 7, and the endpoint reached at Day 14. The tumour volume
results of FIGS. 5-10 illustrate dramatically the site specificity
of the S. aureus SSI formulation.
Example 7: Use of SSI Potentiates a Cancer Antigen Response
[0290] This Example illustrates the effect of combining an SSI
(such as Klebsiella spp. SSI, abbreviated as "KPN SSI") with a
model cancer antigen (in this case OVA, or hen egg ovalbumin)
expressed by transformed cancer cells, in this case in a B16
melanoma animal model. OVA does not naturally occur in mammals or
bacteria, and is immunogenic in C57Bl/6 mice, with known CD4- and
CD8-associated epitopes. In this Example, B16 cells have been
transfected to express cytoplasmic OVA protein, leading to
presentation of the OVA epitopes in the tumour's MHC, thereby
allowing OVA-specific T cell recognition of the melanoma. In some
studies, whole protein was used; evidencing host immune system
processing of the protein into relevant antigens. In other studies,
purified OVA antigen (SIINFELK, a class I-restricted, CD8-specific
antigen) was used. Some studies also used OT1 cells (transgenic CD8
cells that specifically recognize SIINFEKL through the T cell
receptor) as a readout system.
[0291] The data generated provide evidence of enhanced efficacy of
the model cancer antigen admixed with an SSI. In one result,
decreased tumour nodule counts were found in conjunction with
treatment using a Klebsiella spp. SSI+OVA, compared to PBS+OVA
(assessed photographically and by quantitative PCR for Tyrp1, a
melanoma-specific gene). Consistent with this, KPN SSI+OVA
increased the proportion of OVA-specific CD8 T cells in the lungs.
Similarly, expression of genes associated with effective
anti-tumour immunity (granzyme B and IFN-gamma) was increased by
KPN SSI+OVA vs. PBS+OVA. The same study showed survival data in
lung cancer models, in which Klebsiella sp. SSI+OVA showed extended
survival compared with control; and an E. coli SSI+OVA showed much
improved survival.
[0292] In an alternative study, it was observed that SSI induced or
enhanced the process of epitope spreading (see FIG. 11; and for
background see Vanderlugt & Miller, 2002, Nature Reviews
Immunology 2, 85-95). In this study, mice were treated with
Klebsiella sp. SSI, then challenged with B16 melanoma (IV). 5 days
later, mice received an adoptive transfer of TCR transgenic cells
(Pmel T cells) specific for a natural cancer antigen in melanoma.
Mice were also treated with FTY-720 (fingolimod), to prevent T cell
egress from lymph nodes and allow recovery of cells from the
draining lymph node of a tumour. The activation status of the
recovered pmel T cells was assessed, and it was found that SSI
enhanced the proportion of T cells in the draining lymph node that
were activated (i.e., antigen-exposed). This provides evidence that
an SSI may be adapted to augment the processing and presentation of
immunogenic cancer antigens, including self antigens and
exogenously-administered cancer antigens.
Example 8: Component Formulations
[0293] This Example relates to the fractionation of microbial
preparations for the purpose of formulating alternative SSIs. In
alternative embodiments, fractions may for example be prepared
from: bacterial outer membrane (for example from Gram negative
spp.); bacterial inner membrane; the pellet of a gradient
centrifugation (for example from a sucrose gradient); chromosomal
DNA; a capsular glycoprotein fraction; or, a peptidoglycan
fraction, such as peptidoglycan ghosts. In alternative embodiments,
engineered or recombinant organisms may be used in SSIs, in which
genes involved in pathways relevant to particular cellular
fractions have been modified, in particular genes involved in
determining the composition of the foregoing fractions.
[0294] For cell fraction preparations, bacteria may for example be
grown and heat-inactivated. Cell fractions may for example be
resuspended in sterile saline+0.4% phenol. Inner membranes may for
example be collected using the 2-step sucrose density gradient, as
for example described in Methods in Enzymology, Vol 125:309-328,
1986. The bacterial pellet obtained after cultivation of 250 mls of
cells may be resuspended in 20% sucrose, 10 mM Tris-HCl pH 8.0 and
50 ug/ml DNase 1. Cells may be incubated at 23.degree. C. for 10
min. Cells may then be placed on ice and lysed two times through a
French pressure cell at 15,000 psi; unbroken cells may be removed
by centrifugation at 5,000.times.g for 10 min at 4.degree. C.
Supernatants may be layered onto a 2-step sucrose gradient (60% and
70%) and centrifuged in a SW28 swinging bucket rotor at 23,000 rpm
for 18 hours at a temperature of 4.degree. C. The inner membranes
may be collected at the junction between the 20% and 60% sucrose.
Sucrose may be diluted to below 20% with sterile distilled water
and the membranes may be pelleted in an ultracentrifuge at 41,000
rpm at 4.degree. C. for 1 hour. The inner membranes may be washed
once with sterile water, and then resuspended in sterile
saline+0.4% phenol. Crude outer membrane preparations may also be
collected from the junction between the 60% and 70% sucrose
gradient steps.
[0295] Chromosomal DNA, for example for Klebsiella pneumoniae, may
be prepared using a Qiagen Blood and Tissue midi kit. Cells from 15
or 40 mls of broth culture from each strain may be harvested. The
manufacture's protocol for purification of total DNA may then be
followed.
[0296] The efficacy of a component formulation was demonstrated
with a Klebsiella pneumoniae (KPN) outer membrane fraction in the
B16 lung cancer model, in which SSIs were injected every other day
beginning 10 days before tumour cell inoculation by intravenous
injection. Three SSIs were compared, whole killed cell KPN (QBKPN),
a 1.times. outer membrane (OM) fraction (having an outer membrane
concentration that approximated the outer membrane concentration of
the whole cell formulation) and a 0.01.times. dilution of the outer
membrane fraction. As illustrated in FIG. 47A, both 1.times. and
0.01.times.KPN outer membrane fractions (i) were efficacious in the
B16 melanoma model in the lung, in a dose-dependent manner, with
the 1.times. fraction having comparable efficacy to the whole
killed cell formulation, as were the 1.times. and 4.times.DNA
fractions (ii), while the inner membrane fraction showed a dose
dependent trend that lacked strong statistical significance
(iii).
[0297] As illustrated in FIG. 47B, following 10 injections of outer
membrane SSI, Rae-1 expression was elevated by the 1.times. outer
membrane fraction in a dose dependent effect.
[0298] Of note, higher concentrations of the membrane fraction
caused pathology in animals prior to inoculation with tumour cells.
In particular, 10.times. and 20.times. outer membrane fractions
elicited strong toxicity in mice as evidenced by highly elevated
innate cell (monocyte and neutrophil) recruitment to the blood with
attendant deteriorating health conditions (e.g. dramatic weight
loss, gait, hunched posture, eye conditions). Similarly, in some
embodiments, concentrated whole cell preparations did not elicit
toxicity. FIG. 47C illustrates elevated neutrophil and monocyte
blood counts after 4 injections of SSI or placebo, in blood
collected 2 days prior to tumour implant.
[0299] As illustrated in FIG. 470, the KPN fractions illustrate
site specific preferential lung activity compared to E. coli
fractions in the B16 lung cancer model. In particular, compared to
placebo control, whole QBKPN was efficacious in reducing lung
tumour burden. A whole killed E. coli formulation (QBECO) was not
as efficacious as QBKPN. QBKPN fractions (OM or DNA alone) were
efficacious. When combined (OM+DNA), QBKPN fractions were
approximately as efficacious as whole QBKPN. QBECO fractions (OM,
DNA, or OM+DNA) did not show the same efficacy as QBKPN fractions.
Together, this illustrates site specificity associated with QBKPN
fractions, particularly combined DNA (4.times.) and OM (1.times.)
fraction.
Example 9: Co-Formulations and Co-Administration
[0300] This Example illustrates embodiments in which an SSI is
co-formulated with or co-administered with additional therapeutic
components.
[0301] One class of additional therapeutic components comprises
molecules or compositions for activating or recruiting innate
immune cells, and these include: [0302] GMCSF (particularly for
cancer), for example in an amount that synergistically recruits and
promotes the production of neutrophils and potentiates the
SSI-induced innate immune response. [0303] Vitamin D (for
inflammatory disease, such as IBD, and cancer), for example in an
amount that is effective to differentiate and activate monocytes
and play a role in regulating innate immune function. In
alternative embodiments, the vitamin D used in conjunction with
SSIs may for example be one or more of vitamin D.sub.3, D.sub.2 or
calcitriol (1,25-dihydroxycholecalciferol). In some embodiments,
vitamin D.sub.3 and/or D.sub.2 may for example be given locally at
a dosage that is effective to provide a locally effective amount of
calcitriol at the site of SSI and vitamin D administration. For
example, vitamin D precursors (D.sub.3 and/or D.sub.2) may be
administered in an amount that is locally effective once it is
converted into the calcitriol active form by local monocytes and/or
macrophages (expressing CYP27B1) at the site of administration. In
alternative embodiments, calcitriol may be administered in dose
that is locally effective at the site of SSI administration, and
this may for example be dose that is less than the dose required
for other systemic effects.
[0304] An additional class of therapeutic components for
co-formulation or co-administration comprise molecules or
compositions that relieve immunosuppression: [0305] NOHA
(N(omega)-hydroxy-nor-L-arginine), an Arginase inhibitor--Arginase
degrades arginine needed for immune activation. NOHA may for
example be used in an amount effective to relieve immune
suppression by making available free arginine. [0306] Alpha1
antitrypsin--for example in an amount effective to relieve immune
suppression mediated by neutrophils secreting proteases.
[0307] An additional class of therapeutic components for
co-formulation or co-administration comprise molecules or
compositions that prevent oxidative damage and improve immune
function under stress: [0308] Glutathione and other antioxidants,
particularly for fibrotic diseases (such as IBD).
[0309] An additional class of therapeutic components for
co-formulation or co-administration comprise co-stimulatory
molecules for innate cytotoxic lymphocytes (for example for
anticancer treatments): [0310] Phospho-antigens (isoprenoid
molecules, such as isopentenyl pyrophosphate)--recognized by human
peripheral blood V.gamma.9V.delta.2 T cells which play a central
role in anticancer responses, for example in amounts effective for
activating and differentiating monocytes working in concert with NK
cells to target both solid and liquid cancers. In exemplary
embodiments, it has been found that SSIs in co-formulation or
co-administration with zoledronate increase markers of activation,
for example CD25 and CD69, on human peripheral blood
V.gamma.9V.delta.2 T cells. [0311] Glycolipid molecules recognized
by Type I NKT cells (such as synthetic
.alpha.-galactosylceramide)
[0312] As set out in Table 18, and FIGS. 12 and 13, in an in vivo
demonstration of SSI co-formulations that improve anti-cancer
effects using the LLC model, co-formulations with GMCSF and Vitamin
D (D.sub.3) show the best performance, followed by NOHA (arginase
inhibitor) and alpha1-antitrypsin.
TABLE-US-00019 TABLE 18 Co-Formulations - comparing the mean
differences in tumour count vs placebo Mean Dunnett's multiple
comparisons test Diff. 95% CI of diff. Placebo vs. QBKPN 26.44
8.035 to 44.85 ** Placebo vs. QBKPN + Glutathione 27.41 8.998 to
45.82 ** Placebo vs. QBKPN + alpha-1-antitrypsin 29.33 10.92 to
47.74 *** Placebo vs. QBKPN + NOHA 30.31 11.90 to 48.72 ***
(arginase inhibitor) Placebo vs. QBKPN + Vitamin D 33.50 15.09 to
51.91 **** Placebo vs. QBKPN + GM-CSF 34.73 16.32 to 53.14 ****
Example 10: Colitis Animal Model, Anti-Inflammatory Efficacy
[0313] This Example illustrates results from a mouse spontaneous
colitis model (Muc2 knockout "KO" mice) that mimics the underlying
immune system defect and chronic bacterial infection associated
with Crohn's disease and ulcerative colitis. IBD patients typically
display structural and/or functional defects in their normally
protective colonic mucosal barriers. The mucus barrier is largely
dependent on the release of goblet cell-derived mucin (Muc2) which
prevents microbes and luminal antigens from contacting the
epithelial surface in the gastrointestinal tract. Muc2 KO mice are
healthy just after weaning (1 month old), as they age, they develop
progressive diarrhea and sporadic rectal prolapse. Histological
analysis of colonic tissue shows crypt hyperplasia, crypt
abscesses, inflammatory cell infiltration and submucosal edema.
Accordingly, the Muc2 KO mice have a defective gastrointestinal
mucosal barrier and after time spontaneously develop colitis,
resembling ulcerative colitis in humans. In this model, young (2
month old) Muc2 KO mice have less severe colitis, and older (3
month old) Muc2 KO mice have more severe colitis.
[0314] Results from this animal model, as shown in FIGS. 14A, 14B
and 14C illustrate that an E. coli SSI (QBECO) decreases
pro-inflammatory markers in the colon (using qPCR gene expression
data). FIG. 14D illustrates the site specific activity of QBECO in
increasing IL-18 gene expression in the colon, compared to QBKPN.
The IFN-gamma expression data in particular illustrates how SSI
efficacy can be affected by the stage of colitis (comparing
expression data in old vs young mice). IL17A data, relating to a
cytokine that is produced by activated T-cells (a marker of IBD
inflammation), illustrates a significant decrease in this marker of
inflammation after E. coli SSI treatment. Accordingly, QBECO
treatment substantially improved all components of the
histopathology score, including infiltration, integrity,
hyperplasia, and edema. The infiltration of T lymphocytes in the
colonic tissue, a hallmark of IBD in patients and mouse models, was
markedly decreased with QBECO treatment. Accordingly, this Example
illustrates that an SSI, such as QBECO, may be used to
significantly decrease disease severity in IBD model, including so
as to substantially dampen adaptive immune system
over-response.
[0315] QBECO was also shown to have a positive impact on the
gastrointestinal microbiome. Alterations in bacterial species in
the intestinal microbiome can either be detrimental (`unhealthy`
bacteria) or therapeutic (`healthy` bacteria) in IBD patients (and
mouse models). Some bacteria promote a healthy immune environment
and can improve symptoms (for example, Lactobacillus species),
whereas others (for example, .gamma.-proteobacteria) can have
detrimental effects in IBD. We analyzed the intestinal microbiome
before and after QBECO SSI treatment. As illustrated in FIGS. 15A
and 15B, QBECO SSI improved dysbiosis in the colon of Muc2 mice,
increasing the relative proportion of Lactobacillus (healthy
bacterial species) and decreasing the relative proportion of
gamma-proteobacteria (unhealthy bacterial species). As illustrated
in FIG. 15C, QBECO SSI also reduced all aspects of the histological
inflammation/damage score (infiltration, integrity, hyperplasia and
edema) in the colon of MUC2 spontaneous colitis mice. These results
illustrate that an SSI treatment using a formulation derived from a
GI pathogen, such as QBECO, has a therapeutic effect on the
gastrointestinal microbiome. Accordingly, aspects of the invention
involve the use of an SSI, such as an E. coli derived SSI, for
treating dysbiosis in IBD.
[0316] To summarize, QBECO treatment significantly improved the
overall histological score and reduced T cell infiltration in the
colonic tissues. Furthermore, a reduction in pro-inflammatory
mediators in the colon (IL-17A) and serum (KC) was observed. QBECO
treatment did not impact regulatory T cell marker (FoxP3) and
anti-inflammatory growth factor (TGF-.beta.) expressions in
affected tissues. In addition, SSI treated mice demonstrated
reduced levels of the antimicrobial lectins RegIII-.mu. and
RegIII-.gamma.. The changes in antimicrobial lectins brought on by
QBECO allowed for a modulation of the gut microbiome causing a
reduction in gamma-proteobacteria and a significant increase in
lactobacilli.
Example 11: SSI Efficacy in Asthma/Allergy
[0317] This Example provides animal model data illustrating the
efficacy of an SSI therapy, KPN SSI, in treating asthma/allergy. As
shown in FIG. 16, KPN SSI decreases total BAL cell count in
asthmatic mice. As shown in FIG. 17, KPN SSI decreases eosinophil
and lymphocyte counts in the BAL: A) Eosinophils, B) Lymphocytes.
As shown in FIG. 18, KPN SSI decreases TH2 cytokines in the BAL
supernatant: A) IL-4, B) IL-5.
Example 12: Systemic Distribution of SSI Administered SubQ
[0318] This example illustrates systemic distribution of a KPN SSI
administered subcutaneously in a murine model; using cyanine dye
(Cy5.5) labeled whole killed KPN cells and optical in-vivo dorsal
and ventral whole-body imaging. After a first injection, imaging
(at 1 hr, 3 hr, 6 hr, 24 hr and 47 hr) revealed systemic
distribution with the highest concentrations of the SSI at the
injection site. Following the first injection, the SSI was cleared
from circulation within approximately 24 hours. Subsequent
injections took place at alternative injection sites, and imaging
(at 1 hr, 3 hr, 6 hr and 24 hr) revealed systemic distribution with
highest concentrations seen at the new sites of injection and,
surprisingly, at previous sites of injection. This provides an
illustration of preferential SSI delivery/retention at sites of
inflammation following systemic dispersal of locally administered
formulations. Microscopic evaluation of blood samples confirmed
that the Cy5.5 fluorescence detected in the blood was not free dye.
As illustrated in FIG. 19, the distribution of SSI in organs after
24 hours showed a preferential accumulation of KPN SSI in the
lungs, compared to the heart and the spleen.
Example 13: Surgical Wound Treatment
[0319] In this Example, a topical formulation of an SSI is
formulated for administration to wounds, for example surgical
wounds, partial-thickness burns, lacerations, chronic wounds, or
vascular ulcers. The topical SSI formulation may for example
include PPR agonists derived from microbes that are skin pathogens,
formulated in an ointment or gel.
Example 14: Durability of Treatment Response in IBD
[0320] This Example illustrates that efficacious treatment for
Crohn's disease may be carried out over an extended period of
periodic dosing of an SSI. In particular, in a phase 1/2,
randomized, placebo-controlled, double-blinded clinical trial
involving adults with moderate to severe Crohn's disease, the
Crohn's Disease Activity Index (CDAI, Best et al., 1976,
Gastroenterology 70 (3): 439-444) declined on average by
significantly more on week 16 of treatment compared to week 8. More
specifically, by week 8, the average reduction in CDAI score in SSI
treated patients was approximately 80 points; by week 16, the
average reduction in CDAI score was approx 120 points. This
illustrates continued clinical improvement through 16 weeks of SSI
treatment.
[0321] This example involved use of a whole killed E. coli SSI
preparation, administered every second day by subcutaneous
injection. The dose was individualized to the patient by adjusting
the dose so that each dose was effective to cause a visible
localized inflammatory immune response at the administration site
(a 1 inch to 2 inch diameter delayed reaction of visible redness at
the injection site).
[0322] Accordingly, aspects of the invention involve use of an SSI
over an extended duration period, with dosage intervals and dosage
duration adapted to provide an increased therapeutic benefit over
the entire dosage duration, such as a progressive reduction of CDAI
score in Crohn's patients over a duration period of at last 16
weeks.
Example 15: Lung Inflammation--Asthma
[0323] This example illustrates therapeutic efficacy of an SSI (KPN
SSI) in a murine House Dust Mite (HDM)-induced asthma model,
explifying the underlying mechanistic basis for the use of SSIs in
treating asthmatic inflammation. In this Example, BALB/c mice were
exposed intranasally to HDM for two weeks. Mice were treated
subcutaneously with either KPN SSI or placebo for one week prior to
HDM exposure and throughout the two week exposure period. 24 hours
after the last exposure, lungs were analysed for inflammatory cell
infiltrate, gene expression, cytokine levels, goblet cell
metaplasia, and serum was analysed for allergen-specific serum IgE
levels.
Methods
Animals
[0324] Female mice (BALB/c) age 6-8 weeks old were purchased from
Jackson Laboratory (Bar Harbor, Me.). 10 mice per group were used.
Mice were housed in environmentally controlled specific pathogen
free conditions with a 12:12 hour light/dark cycle for the duration
of the study.
Allergen Exposure Protocol
[0325] Mice were exposed to saline (35 .mu.L) or house dust mite
(HDM, Dermatophagoides pteronyssimus, Greer Laboratories, Lenoir,
N.C.), intranasally, 25 .mu.g in 35 .mu.L of saline, under
isoflurane anesthesia. HDM or saline nasal exposure was done for 5
consecutive days in week 1 and 4 consecutive days in week 2
(experimental days: 1-5; 8-11, FIG. 1). Mice were euthanized 24
hours after the last exposure.
Klebsiella Intervention Strategy
[0326] KPN SSI was derived from Klebsiella originally isolated from
a patient infection, with whole heat killed cells suspended in
physiological saline containing 0.4% phenol as a preservative for a
final OD.sub.600 of 5.0. Placebo was physiological saline
containing 0.4% phenol. KB or placebo was prophylactically given on
day -7, -5, -3 of the experiment, and treatment was continued on
experimental days 1, 3, 5, 8, 10. 30 .mu.L of placebo or KB was
injected subcutaneously at alternative sites access skin in lower
right and left quadrant of the abdomen and upper right and left
quadrant of the chest.
Blood Collection, Bronchoalveolar Lavage (BAL), and Cytospin
Analysis for BAL Cell Differentials.
[0327] BAL cell differential counts were performed by examining
cytospins according to cell morphology and Wright-Giemsa staining.
A total of 100 cells per mouse were differentiated by a blinded
observer.
Quantification of HDM-Specific Immunoglobulins by ELISA
[0328] HDM was coated onto 96-well plates (2.5 ug/well) and
incubated overnight at 4.degree. C. After blocking with 5% FBS in
PBS, undiluted serum was added and incubated overnight at 4.degree.
C. After washing, biotin anti-mouse IgE (BD Bioscience--San Jose,
Calif., USA) was added and incubated at 37.degree. C. for one hour.
Streptavidin-HRP/TMB substrate was used to visualize levels and
absorbance was recorded at 450 nm.
Gene Expression
[0329] Right lung tissue was lysed by homogenizing with a
TissueLyser LT (Qiagen--Toronto, Ontario, Canada) and RNA isolation
performed using a PureLink RNA Mini Kit (Life
Technologies--Carlsbad, Calif., USA). iScript cDNA Synthesis
Kit-170-8891 was used for cDNA synthesis (Biorad). Gene expression
was done by quantitative RT-PCR on a StepOnePLus RT-PCR machine
(Applied Biosystems--Foster City, Calif., USA) using TaqMan Fast
Advanced Master Mix (Applied Biosystems) with Taqman probes for
IL-4 (Mn00445259_m1), IL-13 (Mn00434204_m1) and IFN-.gamma.
(Mn01168134_m1).
Cytokine and Chemokine Analysis of BAL and Serum Samples
[0330] 31 cytokine/chemokine/growth factor biomarkers were
quantified simultaneously using a Milliplex Mouse
Cytokine/Chemokine kit (Millipore, St. Charles, Mo., USA) according
to the manufacture's protocol. The multiplex was performed by using
the Bio-Plex.TM. 200 system (Bio-Rad Laboratories, Inc., Hercules,
Calif., USA). The 31-plex consisted of eotaxin, G-CSF, GM-CSF,
IFN.gamma., IL-1.alpha., IL-1.beta., IL-2, IL-3, IL-4, IL-5, IL-6,
IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17,
IP-10, KC, LIF, MCP-1, M-CSF, MIG, MIP-1.alpha., MIP-1.beta.,
MIP-2, RANTES, TNF.alpha., and VEGF. The assay sensitivities of
these markers range from 0.1-33.3 pg/mL. As IL-13 levels in the
multiplex were mainly below detection, IL-13 protein levels were
measured in the BAL fluid by an ELISA (eBioscience San Diego,
Calif., USA).
Histology
[0331] Lungs were dissected and inflated with 5 mL of 10% formalin.
Tissues were embedded with paraffin and sectioned at 3 .mu.m.
Sections were stained with Periodic acid-Schiff to quantify
mucus-containing goblet cells. Stained sections were scanned at
60.times. magnification using an Aperio Slidescanner (Vista,
Calif.), version 11.1.2.760. Positively stained pixels were
identified by colour segmentation in a cross-sectional manner in
the lung airway using Aperio Image Scope software to express the
number of strong positive pixels (Periodic acid-Schiff) normalized
to basement membrane length (.mu.M).
Data Analysis
[0332] Data were analysed using GraphPad Prism and are expressed as
mean.+-.SD. Multi-group comparisons were made by one-way ANOVA
followed by Sidak post-hoc test. Four experimental group
combinations were compared; Saline-placebo vs. Saline-Klebsiella,
Saline-placebo vs. HDM-placebo, Saline-Klebsiella vs.
HDM-Klebsiella, HDM-placebo vs. HDM-Klebsiella. For the purpose of
statistical analysis, any value that was below the lowest value of
the standard was recorded as half the lowest value of the standard.
Principal component analysis (PCA) was performed for the BAL
multiplex data, with and without the IL-13 ELISA. PCA was completed
in R (version 3.2.4) using the prcomp command. (R Core Team (2016)
R: A language and environment for statistical computing. R
Foundation for Statistical Computing, Vienna, Austria. URL
https://www.R-project.org/).
Results
[0333] FIG. 20 is a graph illustrating HDM specific IgE responses
following saline or HDM exposure, treated with either Placebo or
QBKPN. *P<0.05 between HDM treated mice and their appropriate
control (HDM Placebo vs Saline Placebo and HDM QBKPN vs Saline
QBKPN).
[0334] FIG. 21 is a series of graphs illustrating BAL cell counts
and differentials in Saline or HDM exposed mice treated with
Placebo or QBKPN: A) BAL total cells; B) BAL neutrophils; C) BAL
lymphocytes; D) BAL macrophages; Panel (E); BAL eosinophils. Data
are means.+-.SEM of 10 mice per group (*=p<0.05). Data are
means.+-.SD of 10 mice per group. *P<0.05 between HDM treated
mice and their appropriate control. #P<0.05 between HDM QBKPN
treated mice and HDM Placebo treated mice.
[0335] FIG. 22 includes two graphs illustrating BAL and serum
mediators that are linked to eosinophilia: Serum IL-5 (A) and BAL
eotaxin (B). Data are means.+-.SD of 10 mice per group
(*=p<0.05) between HDM treated mice and their appropriate
control. #P<0.05 between HDM QBKPN treated mice and HDM Placebo
treated mice.
[0336] FIG. 23 is a series of graphs illustrating Th1 and Th2 lung
gene expression following HDM exposure and QBPKN treatment: A)
Th-1-mediated response IFN-.gamma. cytokine gene expression, B)
Th-2-mediated response IL-4 cytokine gene expression, and C) IL-13
cytokine gene expression (data are means.+-.SD of 10 mice per
group; *P<0.05 between HDM treated mice and their appropriate
control).
[0337] FIG. 24 is a series of bar graphs illustrating the effects
of HDM exposure and QBKPN treatment on Th1- and Th2-mediated BAL
fluid cytokine levels: A) IFN-.gamma. cytokine level; B) IL-2
cytokine level; C) TNF-.alpha. cytokine level; D) IL-4 cytokine
level; E) IL-5 cytokine level; F) IL-13 cytokine level (data are
means.+-.SD of 10 mice per group; *P<0.05 between HDM treated
mice and their appropriate control).
[0338] FIG. 25 is a graph illustrating a principal component
analysis (PCA) of BAL cytokines showing partial normalization of
overall cytokine profile. This exemplifies overall BAL cytokine
profile changes between groups, using a principle component
analysis (PCA) based on all multiplex data. The different
experimental groups clustered separately based on the 1st principle
component (PC1). Within the placebo treated mice, saline-exposed
mice clustered separately from HDM exposed mice. KB treatment
minimized the separation of HDM exposed mice from saline controls.
Within the saline control group, KB and placebo treated mice were
similar--as shown by their clustering together. As PC1 appeared to
best differentiate the mice into different groups, the cytokines
that had the greatest contribution to PC1 were identified. The top
5 cytokines that determined PC1 were LIF (Lekemia Inhibitory
Factor; 8.2%), IL-5 (8.2%), Eotaxin (8.1%), IL-4 (7.5%) and CXCL10
(7.3%). Completing the principle component analysis with an
additional asthma markers (IL-13) measured by ELISA provided
similar clustering with the top 5 cytokines that determined PC1
were IL-5 (7.3%), eotaxin (7.2%), LIF (Leukemia Inhibitory Factor;
7.2%), IL-4 (6.8%) and IL-13 (6.7%).
[0339] FIG. 26 is a bar graph illustrating airway goblet cell
quantification following HDM exposure and QBPKN treatment. Goblet
cell quantification expressed as number of strong positive
pixels/basement membrane length. Data are means.+-.SD of 10 mice
per group. P<0.05 between HDM treated mice and their appropriate
control. #P<0.05 between HDM QBKPN treated mice and HDM Placebo
treated mice.
TABLE-US-00020 TABLE 19 BAL cytokine changes Statistical
Statistical Statistical Statistical significance significance
significanceSaline + significanceSaline + HDM + Saline + Placebo
vs. Placebo vs. Placebo vs. QBKPN vs. Saline + HDM + HDM + HDM +
Analyte QBKPN Placebo QBKPN QBKPN G-CSF ns ns ns ns GM-CSF ns ns ns
ns IFNg ns ns ns ns IL-1a ns ** ns ns IL-1b ns ns ns ns IL-2 ns ns
ns ns IL-3 ns ns ns ns IL-4 ns **** ** ns IL-5 ns **** *** ** IL-6
ns ns ns ns IL-7 ns ns ns ns IL-9 ns * ns ns IL-10 ns ** ns ns
IL-12p40 ns ns ns ns IL-12p70 ns ns ns ns IL-13 ns ns ns ns IL-15
ns ns ns ns IL-17 ns ns ns ns IP-10 ns **** * * KC ns ns ns ns LIX
- CXCL5 ns ns ns ns MCP-1 - CCL2 ns ns ns ns M-CSF ns ns ns ns MIG
- CXCL9 ns **** * ns MIP-1a-CCL3 ns ns ns ns MIP-1b - CCL4 ns ns ns
ns RANTES - CCL5 ns ns * ns TNFa ns ns ns ns VEGF ns * ns ns MIP-2
- CXCL2 ns ns ns ns
TABLE-US-00021 TABLE 20 SERUM Cytokine Levels Statistical
Statistical Statistical Statistical significance significance
significanceSaline + significanceSaline + HDM + Saline + Placebo
vs. Placebo vs. Placebo vs. QBKPN vs. Saline + HDM + HDM + HDM +
Analyte QBKPN Placebo QBKPN QBKPN G-CSF ns ** *** **** GM-CSF ns ns
ns ns IFNg ns ns ns ns IL-1a ns ns ns ns IL-1b ns ns ns ns IL-2 ns
ns ns ns IL-3 ns ns ns ns IL-4 ns ns ns ns IL-5 ns **** ** ns IL-6
**** ns * ** IL-7 ns ns ns ns IL-9 ** ns ns ns IL-10 ns ns ns ns
IL-12p40 ns ns ns ns IL-12p70 ns ns ns ns IL-13 ns ns ns ns IL-15
ns ns ns ns IL-17 * ns ns ns IP-10 * ns ** ns KC ns ns ns ns LIX -
CXCL5 ns ns ns ns MCP-1 - CCL2 ns ns ns ns M-CSF ns ns ns ns MIG -
CXCL9 ns ns ns ns MIP-1a - CCL3 ns ns ns ns MIP-1b - CCL4 ns ns ns
ns RANTES - CCL5 ns ns ns ns TNFa ns ns ns ns VEGF ns ns ns ns
MIP-2 - CXCL2 ns ns ns *
[0340] As this example illustrates, in asthma QBKPN SSI: decreases
the BAL total cells, neutrophils, lymphocytes, macrophages and
eosinophils; decreases mediators of eosinophilia including serum
IL-5 and BAL eotaxin; decreases Th2 cytokines in the BAL (IL-4 and
IL-5); and, reduces goblet cell hyperplasia. In particular, in
summary, this Example illustrated that HDM exposed mice developed
classical symptoms of experimental allergic asthma including goblet
cell hyperplasia, elevated allergen-specific serum IgE, airway
eosinophilia, and a concomitant increase in T.sub.H2 cytokines
including IL-4, IL-13 and IL-5. Treatment with KPN SSI attenuated
HDM-mediated airway eosinophilia, total BAL cell numbers,
bronchoalveolar lavage (BAL) T.sub.H2 cytokine production, and
goblet cell metaplasia. This Example demonstrates that treatment
with KPN SSI attenuated HDM-induced T.sub.H2-skewed airway
inflammatory responses and the associated goblet cell metaplasia.
An aspect of the invention accordingly provides a treatment, such
as subcutaneous treatment, with microbial biologics, such as
compositions derived from bacterial lung pathogens, as a treatment
for allergic airway disease, for example so as to attenuate an
allergen-induced T.sub.H2-skewed airway inflammatory response,
and/or an associated goblet cell metaplasia. These results
accordingly indicate that an SSI treatment with a biologic is
capable of simultaneously targeting two of the key molecular
components that promote airway eosinophilia, in a process that is
independent of the regulation of allergen specific IgE. In
particular, a KPN SSI may be administered in a dosage and for a
time that is efficacious at attenuating HDM-induced T.sub.H2 skewed
allergic airway inflammation, and/or airway eosinophilia, and/or
mucus content in goblet cells, and this may for example be
independent of modulating allergen-specific IgE levels.
Example 16: Lung Inflammation--COPD
[0341] This example illustrates anti-inflammatory efficacy in a
murine model of COPD, a short term (3 week) smoking model. In this
model, mice are pre-treated (with placebo or KPN SSI) every other
day 3 times (Monday, Wednesday and Friday of week 1). Mice are then
exposed to smoke over days 8-25 (air or cigarette smoke exposure
was done for 5 consecutive week days for the first 2 weeks and for
4 consecutive week days in week 3 (with no treatment or exposure on
weekends) with continued treatment (placebo or KPN SSI) every other
week day (Monday, Wednesday and Friday). On day 26, mice were
euthanized 24 hours after the last air/cigarette smoke exposure and
samples are collected.
[0342] Briefly, cigarette smoke exposure (Kentucky Research Grade
Cigarettes) was done by placing mice into plexiglass nose only
exposure chambers, ensuring the nose extends from main chamber.
Cigarettes were placed into smoking machine and lit with a lighter
and vacuum in the fume hood. The 20 cc syringes in the smoking
machine were filled with smoke, automatic valve was turned followed
by smoke injection into the nose only exposure chambers. Each
smoking puff cycle took 1.5 minutes. Each mouse smoked 3 cigarettes
per day for a total of 45 minutes of exposure. Control air exposure
mice were restrained for a similar duration without exposure to
smoke. Animals were monitored throughout the smoke exposure
procedure and for 30 minutes post smoke exposure.
[0343] The heat-killed Klebsiella strain (originating from a
patient infection) was administered as follows. KPN SSI or placebo
vehicle (physiological saline containing 0.4% phenol) was
prophylactically administered 3.times. every other day, and the
regimen continued therapeutically throughout the period of smoke
administration. Subcutaneous injections of 30 .mu.L placebo or KPN
SSI were administered into the lower right abdomen, the lower left
abdomen, the upper right chest, and the upper left chest, rotating
clockwise for each injection.
[0344] Cytospins were performed and evaluated based on morphology
and Wright-Giemsa staining. BAL cell differentials were then
counted using the prepared cytospin slide with 100 cells per mouse
counted in a blinded fashion.
[0345] Immune mediator profiling of BAL and serum samples was
performed as follows. Soluble mediator analysis in BAL and serum
was performed using a 31 cytokine/chemokine/growth factor multiplex
kit according to the manufacture's protocol (Eve Technologies Corp,
Calgary, AB, Canada) using the Bio-Plex.TM. 200 system (Bio-Rad
Laboratories, Inc., Hercules, Calif., USA). The 31-plex assay
included the following mediators: Eotaxin, G-CSF, GM-CSF,
IFN.gamma., IL-1.alpha., IL-1.beta., IL-2, IL-3, IL-4, IL-5, IL-6,
IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17,
IP-10 (CXCL10), KC (CXCL1), LIF, MCP-1 (CCL2), M-CSF, MIG (CXCL9),
MIP-1.alpha. (CCL3), MIP-1.beta. (CCL4), MIP-2 (CXCL2), RANTES
(CCL5), TNF.alpha., and VEGF. The assay sensitivities of these
markers range from 0.1-33.3 pg/mL.
[0346] Flow cytometric analysis of blood Ly6CHI
monocytes/macrophages and neutrophils was performed as follows.
Blood was collected in EDTA coated tubes (BD Microtainer) to
prevent clotting and stored on ice prior to staining. Blood was
stained with CD11b-FITC, Ly6G-PE, CD11c-PerCPCy5.5 and Ly6C-APC
before red blood cell lysis (BD lysis buffer). Flow cytometry was
run on a FACSCalibur (BD Bioscience). Analysis was completed on a
FlowJo V10.1. Neutrophils were defined as Ly6G.sup.+CD11b.sup.+
cells. Ly6CHI monocytes/macrophage were defined as
Ly6C.sup.HILy6G.sup.-CD11b.sup.+ cells.
[0347] Data Analysis was performed as follows. GraphPad Prism 6
Software (GraphPad Software, San Diego, Calif.) was used to perform
statistical analysis of the results. Data are expressed as
mean.+-.SD. One-way ANOVA analysis followed by multiple comparisons
using a Sidak post-hoc test was performed on the selected group
comparisons. Four experimental group combinations were compared;
air-placebo vs. air-KB, air-placebo vs. cigarette smoke-placebo,
air-KB vs. cigarette smoke-KB, cigarette smoke-placebo vs.
cigarette smoke-KB.
[0348] Body weight and clinical scores (e.g. hunched posture,
interaction with other animals, activity levels) were used to
monitor the overall health of mice exposed to filtered air or
cigarette smoke in the presence or absence KPN. Body weight was
normalized to the starting weight of each animal. No changes in
body weight were recorded in air-exposed group treated with placebo
or KPN (p>0.05). However, cigarette smoking exposed mice had a
prominent loss in body weight (p<0.05) and KPN treatment did not
reverse this detrimental effect (p>0.05). No observed changes
for clinical scores were observed for any groups.
[0349] Total BAL cell counts and cellular differentials were
analysed to assess airway lung inflammation. FIG. 27A is a bar
graph illustrating BAL cell differential, showing that QBKPN
decreases the total BAL cell count after smoke exposure through
reduction in lymphocyte and macrophage populations. In placebo
treated animals, cigarette smoke exposure induced an elevation in
total cell number in the BAL that was not attenuated with KPN
intervention (FIG. 27B (a), p<0.05). The cigarette smoke
exposure induced increase in BAL total cells was attributed to
lymphocytes, macrophages, and neutrophils (FIG. 27B (b)-(d),
p<0.05) but not eosinophils (p>0.05, data not shown). KPN
intervention attenuated the increase in lymphocytes and macrophages
in the cigarette smoke exposure group (p<0.05), although
macrophages remained elevated relative to air+KPN (FIG. 27B
(b)-(c). PN intervention had no impact on cigarette smoke induced
increases in neutrophils (FIG. 27B(d)).
[0350] Previous reports have indicated that mouse cigarette smoke
exposure models result in a TH1 skewed inflammatory response. This
Example illustrates that KPN SSI intervention attenuated cigarette
smoke exposure-induced TH1-skewed lung inflammatory responses, as
evidenced by multiplex analysis of 31 cytokines, chemokines, and
growth factors that included TH1 and non-TH1 mediators. Cigarette
smoke exposure induced 15 of 31 (46.7%) mediators measured in BAL
fluid that included IFN.gamma., CXCL9, CXCL10, CCL5, IL-6, IL-17,
G-CSF, CXCL1, LIF, CCL2, CCL3, CCL4, TNF.alpha., eotaxin, and VEGF
(p<0.05). IL-17 was elevated with cigarette smoke exposure in 4
of 10 samples at values close to the level of detection of this
mediator (0.64 pg/ml). KPN intervention attenuated cigarette
smoke-induced increases in IFN.gamma., CXCL9, CXCL10, CCL5, IL-6,
G-CSF, and IL-17 (FIG. 28A), all mediators that are associated with
a TH1 skewed inflammatory response. KPN SSI intervention had no
impact on air-exposed animals for any mediator measured.
[0351] The serum immune mediator protein expression profile was
minimally impacted by cigarette smoke exposure but is augmented by
KPN intervention, as evidenced by the same multiplex assay of 31
mediators applied to the serum from the four experimental groups.
Cigarette smoke exposure induced an increase in only VEGF, which
had elevated levels relative to air exposed control (FIG. 28B(a),
p<0.05). The KPN SSI intervention did not reverse the cigarette
smoke exposure-induced elevation in serum VEGF. KPN intervention in
air exposed animals decreased only 1 mediator, IL12p40, relative to
air+placebo, while the levels of IL-1.beta., CCL2, CXCL9, and
CXCL10 (FIG. 28B (b)-(d) p<0.05) were increased. In the smoke
exposed mice, KPN intervention increased the levels of CXCL9,
CXCL10, and CCL5 relative to the cigarette smoke+placebo group.
Collectively these serum data illustrate that the SSI intervention
induced a systemic impact independent of cigarette smoke exposure
that may administered so as to be efficacious for the local
suppression of cigarette smoke-induced lung inflammation.
[0352] To illustrate a systemic cellular immune response in this
COPD model, flow cytometry was used to assess the levels of
Ly6C.sup.HI monocytes/macrophages, an inflammatory monocyte
population, and neutrophils, in the blood after cigarette smoke
exposure and KPN intervention. Cigarette smoke exposure induced no
increase in blood Ly6C.sup.HI monocytes/macrophages or neutrophils
(FIG. 28C (a)-(b)). Surprisingly, KPN SSI intervention increased
the blood Ly6C.sup.HI monocytes/macrophages and neutrophils in the
cigarette smoke exposure groups (p<0.05) and the neutrophils in
the air-exposed animals. The increase in systemic Ly6C.sup.HI
monocytes/macrophages and neutrophils was correlated with similar
patterns for a local increase in the lung tissue (FIG. 28C
(c)-(d)), where KB induced an increase in these cell types, which
was further exacerbated by cigarette smoke exposure
(p<0.05).
[0353] FIG. 28D includes three bar graphs for select lung gene
expression profiles, illustrating that QBKPN decreases the
expression of three important inflammatory cytokine genes (IL-6,
IL-1beta, and IL-17A) in the lung tissue after smoke exposure.
[0354] FIG. 29 illustrates select BAL cytokine expression profiles,
with six bar graphs illustrating that QBKPN caused a significant
decrease in G-CSF, IL-6 and IP-10 in the COPD model, and a
downwards trend in IL-4, KC, MIG, TNFalpha.
[0355] FIG. 30 illustrates serum cytokine expression profiles,
identifying a number of markers for SSI efficacy, particularly
elevated serum levels of IP-10, MIG and RANTES. These serum markers
may accordingly be used as a biomarker for SSI efficacy, for
example to identify responders or non-responders to a particular
SSI, or as a marker of efficacious dosing in a dose adjustment
protocol.
[0356] This Example illustrates that a QBKPN SSI decreased a number
of markers of an inflammatory environment in a COPD model, in
particular: decreased BAL total cells, lymphocytes and macrophages;
decreased gene expression of cytokines that are usually elevated in
COPD including IL-6, IL-1beta and IL-17A; and decreased levels of
cytokines of importance in the BAL in COPD including IL-6, IP-10
and G-CSF. More particularly, these results demonstrate that KPN
treatment attenuated cigarette smoke-induced TH1-skewed lung
inflammation and BAL cellularity. In control air-exposed and
experimental cigarette smoke-exposed animals, KPN SSI induced a
systemic immune response that included immune mediator production,
and mobilization of monocytes and neutrophils, which was mirrored
in the local lung environment with an increase in Ly6C.sup.HI
monocytes/macrophages and neutrophils. This Example therefore
indicates that interventions with microbial components that enhance
certain aspects of an immune response, rather than generally
suppressing the immune responses, may be used to alter the course
of cigarette smoke exposure related COPD pathogenesis.
[0357] COPD has many underlying pathways with other inflammatory
diseases, including asthma and inflammatory bowel disease (IBD).
IBD and COPD share common observations including an altered
microbiome, immune dysfunction, altered epithelial cell function,
and chronic inflammation. There is also significant overlap between
asthma and IBD including altered respiratory microbiome and immune
dysfunction. Over all, the similarities between COPD and other
inflammatory disease that benefit from SSIs, as evidenced herein,
indicates that enhancing aspects of the immune response with a
repertoire of PRR agonists, such as microbial products or synthetic
formulations, may be employed as a therapeutic approach to
COPD.
[0358] In this Example of acute cigarette smoke exposure-induced
inflammation, we observe an elevation in IFN-.gamma., CXCL9,
CXCL10, CCL-5, IL-6, G-CSF and IL-17 in the BAL that is attenuated
with KPN treatment. This reduction in TH1-skewed inflammatory
mediators was associated with a concomitant reduction in lung
macrophage and lymphocyte recruitment, with KPN treatment
attenuating the quantity of BAL lymphocytes and macrophages.
Interestingly, systemically, KPN induced a T.sub.H1-skewed
chemokine signature (CXCL9, CXCL10, CCL-5) in both the air-exposed
and cigarette smoke exposed animals, similar to what is seen in
infection. In effect, in parallel to the attenuation of T.sub.H1
lung inflammation, KPN treatment induced a systemic immune
activation with increases in Ly6C.sup.HI monocytes/macrophages and
neutrophils. This Example accordingly indicates that KPN SSI
actively stimulates aspects of an immune response that may be
adapted to lead to mobilization and recruitment of TH1-skewed
immune cells systemically, but a reduction locally in the BAL.
[0359] Systemically, this Example indicates that KPN SSI
administration increased pro-inflammatory cytokines (e.g.
IL-1.beta.) and blood inflammatory monocytes (defined as
Ly6C.sup.HI) and neutrophils, similar to the response seen with an
acute infection. We further identified an increase in the
inflammatory monocytes and neutrophils in the lung tissue by flow
cytometry. In the lung inflammation examples, Examples 15 and 16,
in control mice (air exposed in the COPD study and saline exposed
in asthma study) QBKPN SSI increases cytokine levels in the serum.
These are accordingly available as biomarkers for efficacy,
particularly IP-10 (CXCL10) which was increased in both the asthma
and COPD examples in the QBKPN SSI treated control mice.
Example 17: Klebsiella variicola SSI
[0360] In a murine B16 melanoma model of metastases to the lung, an
SSI formulated with whole killed cells of Klebsiella phylogroup III
(K. variicola) was effective in reducing tumour burden, as
illustrated in FIG. 31 (in which the K. variicola is identified as
"QBKPN").
Example 18: CD25 Depletion
[0361] CD25 is expressed on activated T cells, activated B cells,
T.sub.regs and resting memory T cells (cells involved in adaptive
immunity). Utilizing an anti-CD25 antibody, this example
illustrates QBKPN SSI efficacy in reducing lung nodules in the
absence of CD25 positive cells, as shown in FIG. 32. This
illustrates that aspects of SSI efficacy are independent of CD25
positive adaptive immune cells in the B16 melanoma model (SSI was
administered prophylactically, with mice challenged with B16
melanoma cells injected IV and tumour foci counted 18 days post B16
injection). Accordingly, aspects of the invention relate to
modulating an immune response that is not dependent upon CD25.sup.+
cells, for example an innate immune response.
Example 19: Dose Dependency and Rae-1 Expression
[0362] In a murine B16 melanoma model of metastases to the lung,
the dilution of a KPN SSI progressively reduced efficacy (with
tumour burden measured by QPCR quantification of Trp-1 expression).
FIG. 33A shows the C.sub.t (cycle threshold) values associated with
a KPN SSI formulation (QBKPN), and progressive dilutions of the KPN
SSI (10.times., 100.times. and 1000.times.), on day 5 following B16
challenge. C.sub.t values accordingly indicate the number of PCR
cycles required for the fluorescent signal to cross the threshold
(i.e. to exceed background level). Delta C.sub.t values took into
account of C.sub.t values of a housekeeping gene, and the levels
are accordingly inversely proportional to the amount of target
nucleic acid in the sample. As illustrated, tumour burden increased
with increasing dilution of the SSI. As shown in FIG. 33B, this
dose dependency is also reflected in an assay of the number of B16
tumour nodules in the lung. FIG. 33C is a bar graph illustrating
that a variety of dosing regimes provide a therapeutic effect, with
intervals between injections varying from 1 to 7 days all providing
a therapeutic effect.
[0363] Further analysis, as shown in FIG. 34, illustrated that the
proportion of cells that express Rae-1 was inversely correlated
with tumour burden, evidencing the fact that SSIs increase target
tissue Rae-1 expression in a dose-dependent manner. The increased
Rae-1 signal would facilitate immune stimulation through NKG2D (see
below) receptors on innate lymphoid cells, such as NK cells,
leading to increased cancer cell killing and the reduced tumour
burden evidenced in this example. In effect, high SSI induced Rae-1
expression leads to decreased cancer burden.
[0364] As shown in FIG. 34(B), in NKG2D (natural-killer group 2,
member D) knockout mice, the therapeutic efficacy of QBKPN in the
B16 lung metastasis model is abrogated. This illustrates the
significant role of NKG2D signalling in various aspects of a
therapeutic SSI response, reinforcing the significance of the
evidence of increased Rae-1 expression in target tissues.
Example 20: Site Specificity
Lung
[0365] In a murine Lewis lung carcinoma expressing red fluorescent
protein (LLC-RFP), the efficacy of a KPN SSI (QBKPN) was compared
to E. coli (QBECO) and Staphylococcus aureus (QBSAU) SSIs (KPN
being a lung pathogen in mice while ECO and SAU are not). As
illustrated in FIG. 35, QBKPN provided a markedly stronger effect
in reducing tumour nodules in the lung. As illustrated in FIG. 36,
there was a concomitant reduction in the number of LLC-RFP cells in
the lungs at day 15 after inoculation with LLC.
[0366] Alternative data illustrates that while immune infiltrates
with QBKPN and QBECO may be comparable at early time points in some
systems, neutrophil levels are enhanced at day 7 (flow data) with
QBKPN compared to QBECO. Also, gene array analyses evidences
prolonged persistence of innate infiltrates in QBKPN vs QBECO
(.about.72 hrs). These data further indicate that the ongoing
immune response in lungs is different in response to QBKPN vs
QBECO.
Colon
[0367] In an MC38 colon cancer model, QBECO conferred a greater
survival advantage than did either QBKPN or 10.times. concentrated
QBSAU (QBSAUR), as illustrated in FIG. 37.
Skin
[0368] In a B16 melanoma model, 100,000 B16 melanoma cells were
injected into the right flank of C57BL/6 mice in a volume of 100
.mu.l on Day 0, SSI treatment started on Day -10 and continued till
Day +12. Tumour volume was monitored starting on Day 7, with the
endpoint reached at Day 14. As shown in FIG. 5, 10.times.
concentrated QBSAU or QBSAUR was much more effective than either
QBKPN or QBECO at reducing B16 tumour volume in the skin.
Skin and Lung
[0369] The B16 melanoma model was used to seed lung tumours by IV
administration, and to seed a skin tumour by subcutaneous dorsal
injection, so that each animal has both cancer situated in the skin
and cancer situated in the lung. In this study, mice (N=5/group) in
the experimental group were injected SQ with the placebo (30
.mu.l), QBKPN (30 .mu.l of 4.91 OD.sub.600), or 10.times.QBSAU (30
.mu.l of 8.6 OD.sub.600) every other day on day -8, -6, -4 and -2
prior to being implanted with the B16 melanoma cells
(1.times.10.sup.5 cells/100 .mu.l/mouse) both IV and SQ. Mice
(N=5/group) in the 4 control groups were injected SQ with either
QBKPN or 10.times.QBSAU on day -8, -6, -4, and -2: 2 groups of
these control mice were inoculated with the B16 tumour either IV
(QBKPN single positive control) or SQ (10.times.QUSAU single
positive control) on day 0, serving as single positive controls,
whereas 2 groups of these control mice did not receive any tumour
inoculation, serving as negative controls. SQ administration of
either the SSI treatment or the placebo control was given to mice
continuously every other day until the experiment was terminated on
day 5 post tumour implant. Tumour burden in the lung and the skin
were enumerated on day 5.
[0370] Mice treated with QBKPN, but not 10.times.QBSAU, exhibited
elevated lung-specific Rae-1 expression (FIG. 38) and recruitment
of monocytes and neutrophils to the lung. There was also decreased
PD-1 expression in the lung of QBKPN-treated mice as compared to
placebo-treated mice in the same model (FIG. 39). In contrast,
PD-L1 expression was not different among the groups in the lung.
Treating mice with 10.times.QBSAU, but not QBKPN, led to a decrease
in the skin tumour burden as compared to placebo control in the B16
skin and lung tumour model (FIG. 40).
[0371] Accordingly, QBKPN demonstrated site specificity in the lung
by elevating Rae-1 expression and the recruitment of monocytes and
neutrophils in animals having both skin and lung tumours.
Similarly, 10.times.QBSAU demonstrated site-specific efficacy by
reducing skin tumour in these animals.
Example 21: Dosing Routes and Schedules
[0372] Intravenous SSI vs. Subcutaneous SSI
[0373] In this example, a KPN SSI (QBKPN) was administered either
IV or SQ in a B16 lung metastasis model. On day 0, B16 cells were
administered IV to seed tumours. On days 1, 3, 5, and 7, KPN SSI
was administered (IV or SQ). On day 9, the endpoint was reached and
tumour counts measured. As illustrated in FIG. 41, both routes of
administration provide therapeutic benefit.
Prophylaxis Vs Treatment Schedules
[0374] In this example, the scheduling of SSI treatment, either
before challenge with cancer cells (prophylaxis) or after challenge
(treatment) was compared. This example also demonstrates immune
correlates linked with efficacy, particularly the M1/M2 ratio of
macrophages. FIG. 42 is a schematic illustration of the study
design, based on efficacy of QBKPN in a treatment versus
prophylactic regimen in the B16 lung cancer model. As illustrated
in FIG. 43, while the prophylactic regimen provided earlier
therapeutic benefit, by day 17 the treatment regimen shows a very
strong trend of efficacy. The efficacy of both prophylactic and
treatment regimens was reflected in common correlates of efficacy
in the M1/M2 macrophage ratios in the lung with alternative M2
marker CD206, as illustrated for the treatment group at day 10 and
the prophylactic group at day 17 in FIG. 44.
Early Time Point Blood Analysis
[0375] This example illustrates aspects of how quickly SSI
therapies have detectable therapeutic effects involving myeloid
cell populations, providing examples of therapeutic biomarkers. As
illustrated in FIG. 45, neutrophils increase at all time points for
QBKPN and QBECO SSIs, with significant increases seen even within 3
hours post injection. As illustrated in FIG. 46,
Ly6C.sup.HILy6G.sup.+CD11b.sup.+ cells (Ly6C monocytes) were
significantly increased at both 3 and 7 hours, with a decreasing
trend that falls back to placebo levels by around 24 hours. The
cellular immune response provoked by an SSI therapy may accordingly
be characterized by a rapid onset, within hours, followed by a
resolution within days. This pattern of cellular response supports
a dosing schedule with repeated administrations at an interval that
is measured in days, for example one administration every 1, 2, 3,
4, 5, 6, or 7 days.
Example 22: SSI Cytotoxicity
[0376] This example illustrates that an SSI (QBKPN) can directly
cause an increase in cancer cell death at high doses. NCI-H358
cells (a human lung cell line) were incubated in vitro with
successive dilutions of QBKPN for 24 hours. Efficacy was assessed
using a carboxyfluorescein succinimidyl ester (CFSE) labelling
assay (a green fluorescent cell staining dye to label target cells)
with the red live/dead viability dye 7-AAD (7-aminoactinomycin D)
used to identify the killed/dead cells present in the cytotoxicity
assay sample. As illustrated in FIG. 48, QBKPN increases NCI-H358
cancer cell death at high doses (1/20, 1/200 dilution) in this 24
hour killing assay. Using the same assay, it was also shown that
the KPN SSI increases .gamma..delta. T cell mediated killing of the
NCI-H358 cancer cells at similar doses (1/20 dilution, 1/200
dilution) in the 24 hour killing assay, as illustrated in FIG. 49.
In addition, the KPN SSI (QBKPN) potentiated the effect of
zoledronate in inducing .gamma..delta. T cell mediated cancer cell
lysis, at 1/200 and 1/2000 dilutions, as shown in FIG. 50.
[0377] As this example illustrates, in select embodiments, SSIs can
be administered directly to cancerous tissues, for example at the
site of surgical resection of a cancer. For example, an SSI, such
as QBSAU, may be applied topically to a melanoma in the skin or to
the site of a surgical excision of a skin melanoma.
Example 23: NKG2D Knockout Mice in the MC-38 IP Injection Model
[0378] This example illustrates the involvement of the NKG2D
receptor in mediating a therapeutic response to an SSI. This was
shown in a murine survival study after IP injection of MC-38 cells
(a murine adenocarcinoma cell line derived from a primary mouse
colon carcinoma). The tumour cells were injected intraperitoneally
in order to allow the tumour cells to seed the gut, creating a
MC-38 cell colon cancer model. QBECO treatment was compared to
placebo in wildtype mice (C57BL/6 mice) and NKG2D knockout mice (on
a C57BL/6 background). Wildtype and NKG2D mice were treated with
either QBECO or placebo (10 per group) for 10 days every second day
before MC-38 injection. Treatment was continued throughout the
survival study every second day.
[0379] As illustrated in FIG. 51, this study confirmed the
therapeutic efficacy of QBECO in the MC-38 colon cancer model,
showing in the wildtype mice a statistically significant increase
in survival with QBECO treatment compared to placebo treatment. As
illustrated in FIG. 52, NKG2D expression is correlated with QBECO
efficacy, as there was no statistical difference in survival
between NKG2D knockout mice treated with either QBECO or placebo.
Immunophenotyping confirmed that the NKG2D knockout mice had
reduced levels of NKG2D positive cells. Interestingly, QBECO caused
a decrease in NKG2D at each time point within the wildtype mice
(illustrating the use of NKG2D expression as a biomarker for SSI
efficacy). Immunophenotyping also showed a characteristic increase
in neutrophils by day -9 and monocytes by day -1 in both wildtype
and NKG2D KO mice treated with QBECO compared to placebo. There was
also an increase in PDL1+ after QBECO treatment, with QBECO causing
an increase in PDL1+ cells in the blood throughout the experiment,
with significance in wildtype mice at day -9 and -1, and a similar
by attenuated pattern in NKG2D knockout mice. In wildtype mice,
QBECO caused an increase in PD1+ cells in the blood in non-tumour
bearing mice (Day -9, Day -1), while in tumour bearing mice QBECO
induced an initial increase in PD1+ cells which was attenuated by
Day 11 in a trend that continued so that at survival QBECO caused a
decrease in PD1+ cells in the blood. NKG2D knockout mice follow
this trend with less magnitude and no significance. Together, this
data illustrates that PD1 and PDL1 may be used as biomarkers
indicative of SSI efficacy.
Example 24: Treating Neutropenia
[0380] This example illustrates the use of an SSI to treat
neutropenia in a mouse model. Neutrophil populations were assessed
in spleen (in mouse, representative of circulation) and lungs in
response to SSI treatment (QBKPN) and neutrophil-depleting
(anti-Ly6G) monoclonal antibody, as illustrated in the treatment
schema in FIG. 53. In indicated mice, SSI treatment was performed
every two days from Day -10 to Day +10. SSI (QBKPN) was injected SC
in alternating sites at a dose of 0.03 ml of an OD5.0 solution. In
indicated mice, anti-Ly6G treatment was performed every three days
from Day -1 to Day +11. Antibody (Bio-X-Cel clone 1A8) was injected
IP at a dose of 200 .mu.g/mouse. All mice were IV injected with B16
melanoma (200,000 cells/mouse) at Day 0. On Day +12, mice were
sacrificed. Single cell suspensions were generated from spleen and
lungs. Neutrophil populations (CD45+CD11b+Ly6G-hi
Ly6C-intermediate) were assessed by flow cytometry, using
monoclonal antibodies (BioLegend) and a Miltenyi MacsQuant
cytometer, and analyzed using FlowJo software. Representative
staining data from lungs is illustrated in FIG. 54, from lung
samples, gated on live, CD45+CD11b+ cells. The proportions of
neutrophils in lungs (FIG. 55A, showing % of live CD45+CD11b+
cells) and spleen (FIG. 56A) were calculated from primary cytometry
data. Numbers of neutrophils (CD45+CD11b+Ly6G+Ly6C+ cells) in the
lungs (FIG. 55B) and spleen (FIG. 56B) were calculated by
multiplying the proportion of cells by the total cellularity.
[0381] In both lungs and spleen, QBKPN treatment significantly
increased the proportion (FIG. 55A and FIG. 56A) of neutrophils.
QBKPN treatment significantly increased the number of neutrophils
in spleen (FIG. 56B). These data illustrate that QBKPN SSI induces
an expansion in the proportion and number of circulating
neutrophils.
[0382] Parallel cohorts of mice were treated with anti-Ly6G
monoclonal antibody. Absent QBKPN treatment, anti-Ly6G completely
depleted the neutrophils in lungs and spleen, both in terms of
proportions and numbers (FIGS. 55 and 56). However, neutrophil
populations in lung (FIG. 55) and spleen (FIG. 56) remained at high
levels in QBKPN-treated mice, despite anti-Ly6G monoclonal antibody
treatment. As neutrophils were detected using a
fluorescently-labeled anti-Ly6G antibody (indicating expression of
the antigen), the data indicate that the QBKPN treatment did not
render neutrophils resistant to anti-Ly6G-mediated depletion. Thus,
these data illustrate QBKPN SSI-induced expansion of the neutrophil
compartment in a model of neutropenia.
[0383] A number of common therapies, including chemotherapy drugs
used to treat cancers, suppress bone marrow function and reduce
neutrophil counts, causing neutropenia. As illustrated herein, an
SSI may accordingly be given so as to restore neutrophil counts.
There are additional therapeutic benefits available in SSI
therapies of this kind. In addition to treating neutropenia, in the
context of treating an underlying disease, the selection of a
targeted SSI, with a PRR agonist signature that recapitulates a
distinct portion of a PRR agonist signature of a microbial pathogen
that is pathogenic in the target tissue, results in site specific
restoration of innate immune function in the target tissue. This
may for example involve an anti-cancer immune response, or an
anti-inflammatory immune response mediated by the SSI (in addition
to the effect of treating the neutropenia).
[0384] Chemotherapy commonly produces myelosuppression, of which
the most clinically relevant component is neutropenia occurring
between 2-10 days post-chemotherapy. The clinical implications of
this are serious, interfering with the ability to maintain a
chemotherapeutic dose and schedule, and giving rise to the risk of
neutropenic sepsis. Accordingly, an SSI may be given to patients
undergoing a myelosuppressive chemotherapy as a prophylaxis or
treatment for neutropenia, for example being administered every
other day between cycles of chemotherapy.
Example 25: PD1 and PDL1 Markers in Patients
[0385] This Example provides clinical data illustrating the
efficacy of an SSI acting to down-regulate PD1 and PDL1 expression
in neoplastic disease. This also illustrates the use of PD1 and
PDL1 as markers of SSI efficacy, augmenting the NKG2D mouse model
data in Example 23. This is significant, given the understanding
that PD1 (expressed on T-cells) and it's ligand PD-L1 play a role
in preventing T-cell activation and mediating pathological
immunosuppression.
[0386] In a lung cancer clinical trial of a KPN SSI, 6 patients
presented in two distinct disease groups: pre-neoplastic and
neoplastic (2 of the 6 patients were neoplastic). This Example
provides data obtained from blood samples, analyzed by flow
cytometry.
[0387] As shown in Table 21, the 2 neoplastic patients presented
with elevated PDL1 and PD1 expression compared to the
pre-neoplastic patients. This is shown in the bar graph of FIG. 57
as a percentage of the distinct cell populations, and in FIG. 58 as
the relative number of cells having the denoted characteristics.
The neoplastic patients (01-001 and 01-002) express higher levels
of PD1 and PDL1, ad have a lower level of M4 macrophages than the
pre-neoplastic patients.
TABLE-US-00022 TABLE 21 PD1 and PDL1 Markers in Patients Prior to
SSI Treatment Subject # Pre- Pre- Pre- Pre- neoplastic neoplastic
neoplastic neoplastic Neoplastic Neoplastic 01-007 01-006 01-005
01-004 01-002 01-001 % of CD45.sup.+ 6.9 4.9 12.8 53.0 44.8 83.9
PDL1.sup.+ cells % of CD3.sup.+ 7.6 10.7 19.0 14.2 36.3 70.8
PD1.sup.+ cells M1 2.9 1.8 6.0 3.2 1.0 0.0 macrophages % of cell
CD45.sup.+ CD14.sup.+ HLA- DR.sup.+ CD86.sup.+ M2 85.4 96.7 80.8
87.8 79.9 99.1 macrophages % of CD14.sup.+ cells CD163.sup.+
[0388] With SSI treatment, the neoplastic patients showed a
significant decrease in PD1 and PDL1 expression, as well as a
significant increase in the percentage of M1 (CD45.sup.+ CD14.sup.+
HLA-DR.sup.+ CD86.sup.+) macrophage cells. FIGS. 59A-59B illustrate
the reduction of PD-L1 expression in Patient 01-001 (panel A) and
Patient 01-002 (panel B), at: week 1, day 4 (W1D4); week 1, day 5
(W1D5); week 2 (W2); week 4 (W4), week 12 (W12) and week 16 (W16);
during the course of SSI treatment every other day. FIGS. 60A-60B
illustrate the reduction in PD-1 expression in these patients at
these time points. FIGS. 61A-61B illustrate the increase in the
proportion of M1 macrophages in these patients at these time
points. As illustrated, with SSI treatment, over time, PDL1
expression decreases on CD45+ cells (all white blood cells), PD1
expression decreases in CD3+ cells (lymphocytes), and M1
macrophages increase in the blood (CD45.sup.+ CD14.sup.+
HLA-DR.sup.+ CD86.sup.+). In these patients, SSI treatment was
discontinued at week 12 (W12), and the assays indicated that the
relative M2 macrophage populations (CD14+CD163+) generally
decreased until the cessation of treatment, and then rebounded.
Example 26: Granzyme and Perforin Expression
[0389] This Example illustrates the relationship between SSI
dosage, tumour load, and expression of granzyme A, granzyme B, and
perforin in B16 melanoma mouse lung models, providing evidence that
eficatious SSI therapy elevates granzyme and perforin levels while
reducing tumour load.
[0390] Mice were intravenously injected with B16 melanoma cells to
provide a mouse lung cancer model. Five groups of mice (n=5) were
subcutaneously injected as follows: placebo (saline),
1.times.QBKPN, 1/50.times.QBKPN, 1/500.times.QBKPN, or
1/5000.times.QBKPN (a series of dilutions of the 1.times.KPN SSI).
SSIs were administered prophylactically ten days prior to tumour
inoculation at every second day. Mice were continually injected
with SSIs every two days until they were euthanized at fourteen
days post-tumour injection. The right lung post-caval lobe was
removed and stored in RNAlater.RTM..
[0391] The entire mouse right lung post-caval lobe was homogenized
in lysis buffer by a small bead mill (Qiagen, Cat No. 85600). All
of the homogenate was extracted for RNA using the PureLink.RTM. RNA
Mini Kit (ThermoFisher Scientific, Cat No. 12183018A). A
Nanodrop.TM. spectrophotometer was used to quantify the RNA
concentrations and purity. One microgram of RNA was reverse
transcribed into cDNA using the iScript cDNA Synthesis Kit
(Bio-Rad, Cat no. 170-8891). For quantitative PCR, fifty nanograms
of cDNA were loaded into each well of the reaction plate. In
addition to cDNA, the wells contained TaqMan.RTM. Fast Advanced
Master Mix (ThermoFisher Scientific, Cat No. 4444554), and
TaqMan.RTM. Gene Expression Assays probes. Samples were quantified
for granzyme A (ThermoFisher Scientific, Mm01304452_m1), granzyme B
(ThermoFisher Scientific, Mm00442837_m1), perforin (ThermoFisher
Scientific, Mm00812512_m1), tyrosinase (ThermoFisher Scientific,
Mm00495817_m1), and GAPDH (ThermoFisher Scientific, Mm99999915_g1)
as the housekeeping gene. Two technical replicates were plated for
the genes of interest (GOI), and in singlicate for the housekeeping
gene.
[0392] The ddC.sub.t method was used to calculate gene expression
fold changes. Technical replicates for the GOI were averaged, and
biological replicates were tested for outliers using the ROUT
method on GraphPad Prism 7.00 at 95% confidence. The technical
outliers for GzmB: 1/500x-3 and Prf1: 1/500x-3 were removed from
further analyses. The dC.sub.t value was calculated by subtracting
C.sub.t,GOI by C.sub.t,GAPDH. The ddC.sub.t values were calculated
by subtracting dC.sub.t of the sample by average dC.sub.t of the
placebo group. Fold change was calculated by taking the negative
exponent of ddC.sub.t with base two (2-ddC.sub.t). The average fold
change of each treatment group was analyzed for significance using
a one-way Tukey's multiple comparison ANOVA test at 95%
confidence.
[0393] FIGS. 62A-62B illustrate the results of the foregoing
assays, showing RT-qPCR fold changes in (A) GzmA, GzmB, Prf1, and
(B) Tyr in lungs of B16 inoculated mice euthanized on day 14 with
differing QBKPN doses. Taqman.RTM. Gene Expression Assays were
performed on 50 ng of cDNA isolated from the right lung post-caval
lobe of mice. Values are normalized to GAPDH, and relative to the
gene expression of the placebo group, which were mice injected with
saline. Data points are mean+/-SD. All data points have n=5 except
GzmB-1/500X and Prf1-1/500X, which have n=4 after removal of
dC.sub.t outliers. Significance was calculated using a one-way
Tukey's multiple comparison ANOVA test. **p<0.01, *p<0.001
and ****p<0.0001.
[0394] In accordance with one aspect of this Example, SSIs may be
formulated and administered in a dosage regime that is effective in
a target organ or tissue to mediate increased expression of one or
more granzyme or perforin, such as of granzyme A, granzyme B, and
perforin.
Example 27: Distinct SSIs Agonize Distinct PRRs
[0395] This Example illustrates that both QBECO and QBKPN SSIs
activate multiple PRRs, and QBECO and QBKPN each activate different
PRRs, with different PRR repertoire fingerprints being identified
for each SSI.
[0396] This data in this Example was obtained from assays of QBKPN
and QBECO PRR activation in cell lines that have a single PRR. The
cell lines used were HEK293 cells lines that express a single human
Toll-Like Receptor (TLR2, 3, 4, 5, 7, 8 and 9), NOD-Like Receptor
(NOD1 and NOD2), C-Type Lectin (Dectin 1a, Dectin 1b, and Mincle)
or RIG-1-like receptor (RIG-1 and MDA5).
[0397] As illustrated in FIG. 63, two TLRs were highly activated by
both QBECO and QBKPN (TLR 2 and TLR4). One TLR was highly activated
by just QBKPN (TLR5). 1 PRR was moderately activated by bother
QBECO and QBKPN (NOD2). 4 were moderately activated by only QBECO
(TLR3, TLR7, TLR8, TLR9) while 2 were moderately activated by only
QBKPN (Dectin 1a, Dectin 1b). NOD1, Mincle, RIG-1 and MDA5 were not
activated by either QBECO or QBKPN.
[0398] TLR2 and TLR4 are localized on the plasma membrane and
primarily recognize lipoprotein and LPS respectively. TLR5 is a
plasma membrane receptor that responds to Flagellin. Of the RNA/DNA
recognition TLR's, TLR3 was only slightly activated by QBECO (and
not by QBKPN). TLR3 is primarily a dsRNA receptor for viral RNA.
TLR7 and 8, which are located in the endolysosome and also
recognize RNA (bacterial and viral) were activated by only QBECO.
Finally, TLR9 which recognized CpG-DNA and is located in the
endolysosome was also activated by only QBECO. In this context, it
is relevant that HEK cells are not known to highly uptake bacteria
in endolysosomes. Therefore, the lack of QBKPN activation for TLR
7, 8 and 9 may be due to no interaction of the DNA/RNA with these
receptors. Nod-Like Receptors (NLR) are cytoplasmic receptors. NOD1
was not activated by either QBECO or QBKPN, but NOD2, which
recognizes muramyl dipeptide (MDP) was activated by both QBECO and
QBKPN. The other cytoplasmic receptors, RIG-1 and MDA5 which
recognize short dsRNA and long dsRNA respectively, were not
activated. The C-type lectin receptors (CLR) are located in the
plasma membrane and primarily recognize carbohydrates. Mincle was
not increased by either QBKPN or QBECO. Dectin 1a and Dectin 1b are
primarily fungi receptors for beta-Glucans but can also see
bacteria carbohydrates.
[0399] When graphed as either bar graphs (FIG. 64) or radar graphs
(FIG. 65), an overall PRR repertoire fingerprint appears. These
results are all derived from the 1/10 dilution of the relevant SSI,
with the negative control subtracted from the absorbance value.
Example 28: Viral SSIs
[0400] This Example illustrates that viral SSIs induce immune
changes are similar to bacterial SSIs, as evidenced by immune
correlates in the blood after 7 days of Viral SSI treatment
compared to QBKPN SSI treatment. Mice were treated with Placebo,
QBKPN, or three viral SSI models: Rabies Vaccine, Feline
Rhinotracheitis-Calici-Panleukopenia Vaccine and Canine Influenza
Vaccine. 30 .mu.L of the treatments were injected subcutaneously
every second day. The endpoint was 24 hours after the 4th SSI
injection (day 7). At endpoint, blood was collected and stained for
flow cytometry to determine the numbers of neutrophils and
Ly6C.sup.HI monocytes in the blood as a percentage of CD45.sup.+
cells.
[0401] As illustrated in FIG. 66, Rabies vaccine (killed rabies
virus) increased neutrophil levels when compared to placebo, to
levels comparable to levels seen with QBKPN treatment. As
illustrated in FIG. 67, treatment with Rabies vaccine, Fel-O-Vac
(Feline Rhinotracheitis-Calici-Panleukopenia virus) and Nobivac
(Canine Influence H3H8 virus) all had similar increases in
Ly6C.sup.HI monocytes, comparable to QBKPN treatment.
[0402] This data illustrates that SSIs produced from viral
compositions induce similar immune response to SSIs produced from
bacterial compositions. Viral SSIs are demonstrated to provide an
equivalent response in neutrophil and Ly6C.sup.HI monocyte levels
in the blood as does QBKPN.
Example 29: Cancer Antigen Potentiation
[0403] This Example illustrates that SSIs potentiate an immune
response when used in combination with cancer antigens. As set out
below in more detail, the lung-targeted SSI QBKPN mediated a
reduction in tumour burden, and when used in combination therapy
with a melanoma-associated antigen (gp100) further reduced tumour
burden. This effect was specific to the use of the cancer antigen,
as evidenced by the fact that an irrelevant (immunogenic but non
tumour-associated) antigen did not impact tumour burden. The SSI
cancer antigen combination therapy was effective both as
co-formulated compositions and as separate injections of SSI and
antigen. This evidences the use of an SSI as a adjuvant to drive
immune responses to immunogenic cancer antigens.
[0404] The anti-tumour efficacy of QBKPN SSI in combination with
the melanoma-specific antigen gp100 was compared to the irrelevant
control antigen OVA (also called SIINFEKL) in C57Bl/6 mice sourced
from Jackson Laboratories. On .about.day -31, mice were infected
with K. pneumoniae (2.5.times.10.sup.5 cells/mouse, by
oropharyngeal instillation after being anaesthetized with
isofluorane), then rested. Within 5-7 days, all mice were fully
recovered from K. pneumoniae challenge. Starting on day -10, some
mice were S.C. injected with QBKPN SSI; mice were injected every
other day from Day -10 to day +12. Injections were performed in
rotating sites (in accordance with the typical protocol in the
Examples herein) at a dose of 0.03 ml of a 5.0 OD suspension.
[0405] On days -10, -6, and -4, some mice were also treated with
indicated antigens and/or adjuvants, either co-mixed with SSI or
s.c. in a distal site (nape). Peptide vaccines consisted of the
melanoma-specific antigen gp100.sub.25-33 (KVPRNQDWL, 100
.mu.g/mouse) or the immunogenic control antigen from OVA
(OVA.sub.257-264, SIINFEKL, 100 .mu.g/mouse). Adjuvant consisted of
commercial CpG (ODN 1585 VacciGrade, 30 .mu.g/mouse s.c). On day 0,
mice were challenged with B16 melanoma (3.times.10.sup.5
cells/mouse, i.v.). On day +14, mice were sacrificed, surface
metastases enumerated, and spleens and blood collected. Splenocytes
were pooled among each group. Splenocytes (1.times.10.sup.6
cells/well) were cultured with gp100.sub.25-33, OVA.sub.257-264, or
control peptide (influenza NP366-374) (all peptides at 10 .mu.g/ml)
for 5 days, then supernatants assessed for IFN-.gamma. (by specific
ELISA) as a readout of immunogenicity. Blood was collected into
heparin-containing tubes, then centrifuged to remove cells. ELISA
(RND Systems DuoSet DY485, limit of detection 31.2 .mu.g/ml) was
performed per the manufacturer's protocol. ELISA was performed with
technical replicates (n=3) on all samples to generate a cytokine
value for each culture condition (restimulated splenocytes) or
animal (serum analyses); cytokine data are reported as group
mean+/-standard deviation. Statistical differences were evaluated
by unpaired T test (GraphPad PRISM).
[0406] As illustrated in FIG. 68, QBKPN treatment of K.
pneumoniae-pre-exposed mice significantly (p<0.0001) reduced
metastatic-like B16 melanoma in the lungs. Administration of cancer
antigen (gp100) without adjuvant had no significant effect, in
keeping with the relative paucity of response to non-adjuvanted
vaccines in murine systems. Administration of cancer antigen with
adjuvant (CpG) reduced tumour burden (p=0.0023), to a lesser extent
that SSI alone. Combination of QBKPN SSI with gp100
(tumour-associated antigen) significantly (p<0.05) enhanced
anti-tumour efficacy, compared with QBKPN alone. There was no
statistical difference in anti-tumour efficacy between SSI/antigen
combination used as a coformulation vs separate injection.
Combining the SSI with a non-specific antigen (OVA) did not enhance
anti-tumour efficacy, beyond the level of QBKPN SSI alone.
[0407] These data illustrate that QBKPN SSI can act as an adjuvant
to induce/enhance the efficacy of cancer vaccines, and this
adjuvant effect may be utilized either as a coformulation or
separate administration. These results evidence an anti-cancer
effect in which the SSI (alone or with antigen) was superior to
adjuvanted antigen (gp100+CpG). Consistent with this, SSI
treatment, without or with antigen, enhanced circulating levels of
IFN-.gamma..
Example 30: STING Agonists and SSIs
[0408] This Example illustrates enhanced efficacy of a microbial
SSI augmented with an additional PRR agonist, in this case a STING
agonist. These formulations constitute a class of artificial PRR
agonist repertoires in which a microbial PRR agonist repertoire is
augmented with one or more additional heterologous PRR
agonists.
[0409] The anti-tumour efficacy of QBKPN SSI in combination with
the STING agonist 2'2'-cGMAP (inVivoGen) was evidenced in C57BV/6
mice, as follows. On .about.day -31, mice were infected with K.
pneumoniae (2.5.times.10.sup.5 cells/mouse) by oropharyngeal
instillation after being anaesthetized with isofluorane, then
rested. Within 5-7 days, all mice were fully recovered from K.
pneumonia challenge. Starting on day -10, some mice were S.C.
injected with QBKPN SSI; mice were injected every other day from
Day -10 to day +12. Injections were performed in rotating sites at
a dose of 0.03 ml of a 5.0 OD suspension. On days -10, -6, and -4,
some mice were also treated with STING agonist (SC injection of 10
.mu.g/mouse in 20 .mu.l of saline), either co-mixed with SSI or
s.c. in a distal site (nape). On Day 0, mice were challenged with
B16 melanoma by tail-vein (I.V.) injection of single cell
suspensions of tumour cells (2.0.times.10.sup.5 cells/mouse). On
day +14, plasma was collected for ELISA, then mice were sacrificed
and visual metastases counted and recorded. Serum was collected
into heparin-containing tubes, then centrifuged to remove cells.
ELISA (RND Systems DuoSet DY485, limit of detection 31.2 pg/ml) was
performed per the manufacturer's protocol. ELISA was performed with
technical replicates (n=3) on all plasma samples to generate a
cytokine value for each animal; cytokine data are reported as group
mean+/-standard deviation (n=7 mice/group). Statistical differences
were evaluated by unpaired T test (GraphPad PRISM).
[0410] As illustrated in FIG. 69, QBKPN treatment of K.
pneumoniae-pre-exposed mice significantly (p<0.0001) reduced
metastatic-like B16 melanoma in the lungs. Administration of STING
agonist alone also reduced tumour burden (p=0.0038). The
combination of SSI and STING agonist further reduced tumour burden;
the number of tumour nodules was significantly reduced following
coinjection of SSI and agonist, compared with untreated
(p<0.0001), SSI alone (p=0.0206), or STING agonist alone
(p=0.0006) (FIG. 1). Likewise, simultaneous therapy (separate
injection sites) with SSI and STING agonist reduced tumour burden,
compared with untreated (p<0.0001) or agonist alone
(p=0016).
[0411] As shown in FIG. 70, both SSI treatment and STING agonist
treatment enhanced circulating levels of IFN-.gamma. (p<0.0001
and p=0.0038, respectively). The combination of SSI and STING
agonist further increased cytokine levels, even though STING
agonist treatment had not occurred for 18 days. Cytokine levels
following combination treatment were statistically greater than
single agent treatment. There was a significant inverse correlation
between tumour burden and plasma IFN-.gamma. levels. In sum, this
data illustrate effective combination therapy using a STING agonist
and an SSI.
Example 31: Genetic Markers for SSI Therapy Response
[0412] This Example provides a genetic analysis of subjects with
IBD undergoing treated with an SSI therapy, illustrating the use of
genetic markers associated with IBD to identify patient populations
amenable to SSI treatments. In this Example there were 48 subjects
with IBD and approximately 2.4 million single nucleotide
polymorphisms (SNPs) which were the subject of analyses following
genotyping on the Infinium Omni2.5-8 bead chip. The end-points used
for these genetic analyses were varied and encompassed both
clinical response and also the use of object markers of disease
activity. Following standard quality control measures (including
call frequency, minor allele frequency, and Hardy-Weinberg
equilibrium test) a total of 1,271,655 SNPs were available for
analyses. 113 known IBD loci were represented on the chip and
passed quality control. Of the study subjects there were 31 Crohn's
disease (CD) and 12 ulcerative colitis (UC) cases included.
[0413] A number of IBD-associated SNPs are associated with SSI
treatment outcomes in IBD cases, using p=0.05 as a nominal
significance, for example:
CD Phenotype and IBD-Associated SNPs
[0414] Last recorded response in CD (response vs no response)--a
SNP tagging the FASLG, TNFSF18 genes was the top association
(p=0.0033). [0415] The same FASLG, TNFSF18 locus was also
associated with drop in CDAI in CD cases (p=0.018). [0416] CD drop
in calprotectin was associated with a number of SNPs tagging 4
loci: [0417] NEXN, FUBP1, DNAJB4, GIPC2, MGC27382; [0418] ATF4,
TAB1; [0419] IL23R; [0420] IL8, CXCL1, CXCL6, CXCL3, PF4, CXCL5,
CXCL2 (all p<0.05). [0421] 8 week drop in CRP was associated
with a SNP tagging NOTCH2 (p=0.002).
UC Phenotype and IBD-Associated SNPs
[0421] [0422] Mayo score drop at 16 weeks associated with SNPs
tagging: [0423] HNF4A [0424] IRFI [0425] GPR12 [0426] nd FOXO1 (all
p<0.05) [0427] HNF4A, and GPR12 are also associated with drop in
CRP in UC after 16 weeks of treatment.
IBD Phenotype and IBD-Associated SNPs
[0427] [0428] Last response in all IBD cases combined showed
associations with SNPs that tagged loci tagging FASLG, TNFSF18
(p=0.02) and also JAK2 (p=0.04).
[0429] An analyses of all the SNPs across the chip against the
phenotypes listed above revealed a number of associations as
summarized in the Table 22:
TABLE-US-00023 TABLE 22 Unbiased analyses of all SNPs across the
genotyping platform SNP ID P value Genes kgp10600643 0.00037 BMPR1B
rs1998639 0.00043 CD1D, KIRREL rs9578586 0.00046 SGCG, SACS
rs1467073 0.00055 DENND3, SLC45A4 rs12364461 0.00062 P2RY2, P2RY6,
ARHGEF17, FCHSD2 kgp8836175 0.00069 ZFHX3
[0430] Using a cumulative gene-risk score (GRS; see Jostins et al.,
(2013) PLoS ONE 8(10): e76328) based on all known IBD associated
SNPs, a highly significant association was identified with CD
responders to SSI treatment having higher GRS developed from 112
IBD-associated SNPs (listed below) than non-responders
(p=2.43.times.10.sup.-5), as illustrated in FIG. 71A. Using just 3
of these SNPs, with raw p-values <0.05 (rs9286879, rs7517810,
rs17391694), also evidenced a significant association (P-value:
1.385E-04) with CD responders, as illustrated in FIG. 71B.
Similarly there was an association with higher GRSs observed in UC
responders than non-responders, as illustrated in FIG. 72
(p=0.012), providing an independent verification of the CD
phenotype and GRS finding summarized above. Given that the
overwhelming majority of IBD-associated loci are shared between CD
and UC combining these data as cumulative GRS in all IBD cases is
valid. Despite the small number of cases there is a very
significant association between GRS and response at last follow up,
as illustrated in FIG. 73 (p=8.18.times.10.sup.-7).
[0431] The remarkable association of the cumulative GRS with last
documented response in CD, UC, and combined IBD patient populations
indicates that individuals with IBD that are genetically enriched
for genetic markers associated with IBD are more likely to respond
to SSI. Furthermore, since the majority of these genetic variants
are associated with other immune-mediated diseases, this indicates
that this approach may be extended to other patient cohorts beyond
IBD when treated with an SSI. These findings indicate that it is
possible to identify subjects, such as IBD subjects, more likely to
respond to an SSI treatment. Accordingly, an aspect of the present
invention involves the provision of companion diagnostic genetic
testing assays in association with an SSI therapy. SNPs and genetic
loci that may be used in such assays are set out below. [0432] List
of 243 IBD susceptibility SNPs: rs1748195, rs34856868, rs11583043,
rs6025, rs10798069, rs7555082, rs11681525, rs4664304, rs3116494,
rs7556897, rs111781203, rs35320439, rs113010081, rs616597,
rs724016, rs2073505, rs4692386, rs6856616, rs2189234, rs395157,
rs4703855, rs564349, rs7773324, rs13204048, rs7758080, rs1077773,
rs2538470, rs17057051, rs7011507, rs3740415, rs7954567, rs653178,
rs11064881, rs9525625, rs3853824, rs17736589, rs9319943, rs7236492,
rs727563, rs17391694, rs6679677, rs3897478, rs9286879, rs1728918,
rs10865331, rs6716753, rs12994997, rs6837335, rs13126505,
rs10065637, rs7702331, rs17695092, rs12663356, rs9264942,
rs9491697, rs13204742, rs212388, rs10486483, rs864745, rs7015630,
rs6651252, rs3764147, rs16967103, rs2066847, rs2945412, rs2024092,
rs4802307, rs516246, rs2284553, rs10797432, rs6426833, rs2816958,
rs1016883, rs17229285, rs9847710, rs3774959, rs11739663, rs254560,
rs6927022, rs798502, rs4722672, rs4380874, rs4728142, rs483905,
rs561722, rs28374715, rs11150589, rs1728785, rs7210086, rs1126510,
rs6088765, rs6017342, rs12103, rs35675666, rs12568930, rs11209026,
rs2651244, rs4845604, rs670523, rs4656958, rs1801274, rs2488389,
rs7554511, rs3024505, rs6545800, rs925255, rs10495903, rs7608910,
rs6740462, rs917997, rs2111485, rs1517352, rs2382817, rs3749171,
rs4256159, rs3197999, rs2472649, rs7657746, rs2930047, rs11742570,
rs1363907, rs4836519, rs2188962, rs6863411, rs11741861, rs6871626,
rs12654812, rs17119, rs9358372, rs1847472, rs6568421, rs3851228,
rs6920220, rs12199775, rs1819333, rs1456896, rs9297145, rs1734907,
rs38904, rs921720, rs1991866, rs10758669, rs4743820, rs4246905,
rs10781499, rs12722515, rs1042058, rs11010067, rs2790216,
rs10761659, rs2227564, rs1250546, rs6586030, rs7911264, rs4409764,
rs907611, rs10896794, rs11230563, rs4246215, rs559928, rs2231884,
rs2155219, rs6592362, rs630923, rs11612508, rs11564258, rs11168249,
rs7134599, rs17085007, rs941823, rs9557195, rs194749, rs4899554,
rs8005161, rs17293632, rs7495132, rs529866, rs7404095, rs26528,
rs10521318, rs3091316, rs12946510, rs12942547, rs1292053,
rs1893217, rs7240004, rs727088, rs11879191, rs17694108, rs11672983,
rs6142618, rs4911259, rs1569723, rs913678, rs259964, rs6062504,
rs2823286, rs2836878, rs7282490, rs2266959, rs2412970, rs2413583,
rs2641348, rs7517810, rs1260326, rs7438704, rs10061469, rs2503322,
rs5743289, rs6667605, rs1440088, rs3774937, rs477515, rs1182188,
rs17780256, rs11083840, rs3766606, rs13407913, rs6708413,
rs2457996, rs10051722, rs4976646, rs7746082, rs38911, rs13277237,
rs2227551, rs7097656, rs12778642, rs11229555, rs174537, rs568617,
rs2226628, rs566416, rs11054935, rs3742130, rs1569328, rs2361755,
rs3091315, rs1654644, rs4243971, rs6087990, rs6074022, rs5763767.
[0433] Subset of 112 SNPs which together generated the GRS of FIG.
71A: rs10065637, rs1016883, rs1042058, rs10521318, rs10758669,
rs1077773, rs10781499, rs10865331, rs10896794, rs11054935,
rs11083840, rs11150589, rs11168249, rs11209026, rs11583043,
rs11672983, rs11739663, rs11742570, rs1182188, rs1260326,
rs12778642, rs13204048, rs13277237, rs1517352, rs1569723,
rs1654644, rs17085007, rs17119, rs17229285, rs1728918, rs1734907,
rs17391694, rs1748195, rs17780256, rs1801274, rs1847472, rs1893217,
rs194749, rs2024092, rs2111485, rs212388, rs2155219, rs2188962,
rs2189234, rs2227551, rs2231884, rs2413583, rs2472649, rs2641348,
rs2651244, rs26528, rs2816958, rs2823286, rs2836878, rs2930047,
rs3024505, rs35320439, rs3742130, rs3764147, rs3766606, rs38904,
rs395157, rs4243971, rs4409764, rs4692386, rs4728142, rs477515,
rs4802307, rs4836519, rs483905, rs4976646, rs516246, rs559928,
rs564349, rs566416, rs568617, rs6017342, rs6088765, rs616597,
rs6426833, rs6651252, rs6667605, rs6856616, rs6863411, rs6920220,
rs7097656, rs7134599, rs7210086, rs7236492, rs7240004, rs724016,
rs7282490, rs7495132, rs7517810, rs7702331, rs7758080, rs864745,
rs917997, rs921720, rs925255, rs9264942, rs9286879, rs9297145,
rs9319943, rs941823, rs9491697, rs9847710, rs12199775, rs12654812,
rs1292053, rs2227564, rs3197999, rs6074022.
[0434] The foregoing subset of 112 SNPs exhibited varying degrees
of association with response to SSI therapy, as set out in Tables
23A and 236, which identifies the relevant allele for each SNP and
the odds ratio reflecting the association of that allele with SSI
response. In Table 23A, odds ratios greater than 1 indicate that
the designated allele is positively associated with response to SSI
therapy, odds ratios less 1 indicate that alternative allele is
positively associated with response to SSI therapy and the allele
set out in the Table is negatively associated with response to SSI
therapy. In Table 23B, the odds ratios that are negative in Table
23A have been converted to positive odds ratios for the alternative
allele, so that all odds ratios are greater than one and the
Response Allele is the allele associated with response to SSI
therapy.
TABLE-US-00024 TABLE 23A SNP alleles associated (or negatively
associated) with Response to SSI Therapy rsID Response Allele Odds
Ratio rs7517810 G 19.83 rs17391694 G 12.00 rs2413583 G 5.67
rs13204048 G 3.84 rs11209026 G 3.64 rs1734907 G 3.56 rs212388 G
3.19 rs11739663 G 3.19 rs3742130 G 3.00 rs11672983 G 2.99 rs1801274
G 2.94 rs559928 G 2.68 rs1042058 G 2.68 rs9847710 G 2.66 rs4802307
C 2.49 rs4836519 G 2.37 rs194749 G 2.31 rs4243971 C 2.28 rs10781499
G 2.26 rs26528 G 1.99 rs864745 G 1.90 rs516246 G 1.87 rs2472649 G
1.87 rs12654812 G 1.85 rs3764147 G 1.85 rs2155219 C 1.79 rs12199775
G 1.69 rs4728142 G 1.63 rs1182188 G 1.63 rs17119 G 1.60 rs2189234 C
1.59 rs483905 G 1.58 rs925255 G 1.49 rs7702331 G 1.48 rs564349 G
1.44 rs35320439 G 1.43 rs10865331 G 1.37 rs7495132 G 1.33 rs1016883
G 1.33 rs1292053 G 1.29 rs1260326 G 1.27 rs724016 G 1.25 rs9264942
G 1.23 rs11742570 G 1.21 rs3024505 G 1.20 rs11083840 C 1.20
rs6863411 T 1.18 rs11150589 G 1.16 rs2188962 G 1.15 rs38904 G 1.15
rs2231884 G 1.14 rs568617 G 1.14 rs566416 C 1.09 rs941823 G 1.09
rs2930047 G 1.08 rs1748195 G 1.06 rs2227564 G 1.05 rs9491697 G 1.00
rs7240004 G 0.98 rs3766606 C 0.96 rs2227551 C 0.95 rs11054935 G
0.95 rs7758080 G 0.95 rs477515 G 0.94 rs1847472 C 0.93 rs10896794 G
0.92 rs6426833 G 0.91 rs1893217 G 0.90 rs4409764 C 0.89 rs13277237
G 0.86 rs6017342 C 0.82 rs1517352 C 0.82 rs11583043 G 0.81
rs4692386 G 0.79 rs2823286 G 0.79 rs2111485 G 0.77 rs395157 G 0.76
rs17780256 C 0.76 rs7210086 C 0.76 rs921720 G 0.75 rs616597 C 0.74
rs10521318 G 0.71 rs9319943 G 0.71 rs7282490 G 0.70 rs1569723 C
0.69 rs4976646 G 0.68 rs9297145 C 0.67 rs6074022 G 0.67 rs7097656 G
0.66 rs1077773 G 0.66 rs11168249 G 0.63 rs10758669 C 0.62 rs1728918
G 0.61 rs2651244 G 0.59 rs12778642 C 0.58 rs17229285 G 0.56
rs2836878 G 0.56 rs6667605 G 0.50 rs1654644 C 0.48 rs10065637 G
0.48 rs2641348 G 0.44 rs2816958 G 0.43 rs7134599 G 0.41 rs6651252 G
0.40 rs917997 G 0.38 rs6088765 C 0.38 rs2024092 G 0.38 rs3197999 G
0.34 rs7236492 G 0.32 rs17085007 G 0.24 rs6920220 G 0.21 rs9286879
G 0.05
TABLE-US-00025 TABLE 23B SNP alleles associated with Response to
SSI Therapy rsID Response Allele Odds Ratio rs9286879 A 19.83
rs7517810 G 19.83 rs17391694 G 12.00 rs2413583 G 5.67 rs6920220 A
4.87 rs17085007 A 4.20 rs13204048 G 3.84 rs11209026 G 3.64
rs1734907 G 3.56 rs212388 G 3.19 rs11739663 G 3.19 rs7236492 A 3.08
rs3742130 G 3.00 rs11672983 G 2.99 rs3197999 A 2.98 rs1801274 G
2.94 rs559928 G 2.68 rs1042058 G 2.68 rs2024092 A 2.67 rs6088765 T
2.66 rs9847710 G 2.66 rs917997 A 2.64 rs6651252 A 2.52 rs4802307 C
2.49 rs7134599 A 2.46 rs4836519 G 2.37 rs2816958 A 2.33 rs194749 G
2.31 rs2641348 A 2.29 rs4243971 C 2.28 rs10781499 G 2.26 rs10065637
A 2.10 rs1654644 T 2.06 rs26528 G 1.99 rs6667605 A 1.99 rs864745 G
1.90 rs516246 G 1.87 rs2472649 G 1.87 rs12654812 G 1.85 rs3764147 G
1.85 rs2836878 A 1.80 rs17229285 A 1.79 rs2155219 C 1.79 rs12778642
T 1.73 rs2651244 A 1.71 rs12199775 G 1.69 rs1728918 A 1.65
rs4728142 G 1.63 rs1182188 G 1.63 rs10758669 T 1.60 rs17119 G 1.60
rs2189234 C 1.59 rs483905 G 1.58 rs11168249 A 1.58 rs1077773 A 1.53
rs7097656 A 1.51 rs6074022 A 1.50 rs925255 G 1.49 rs9297145 T 1.48
rs7702331 G 1.48 rs4976646 A 1.46 rs1569723 T 1.45 rs564349 G 1.44
rs35320439 G 1.43 rs7282490 A 1.42 rs9319943 A 1.41 rs10521318 A
1.40 rs10865331 G 1.37 rs616597 T 1.35 rs7495132 G 1.33 rs1016883 G
1.33 rs921720 A 1.33 rs17780256 T 1.32 rs7210086 T 1.32 rs395157 A
1.32 rs2111485 A 1.30 rs1292053 G 1.29 rs1260326 G 1.27 rs2823286 A
1.27 rs4692386 A 1.26 rs724016 G 1.25 rs11583043 A 1.23 rs9264942 G
1.23 rs1517352 T 1.21 rs6017342 T 1.21 rs11742570 G 1.21 rs3024505
G 1.20 rs11083840 C 1.20 rs6863411 T 1.18 rs13277237 A 1.16
rs11150589 G 1.16 rs2188962 G 1.15 rs38904 G 1.15 rs2231884 G 1.14
rs568617 G 1.14 rs4409764 T 1.13 rs1893217 A 1.11 rs6426833 A 1.10
rs566416 C 1.09 rs941823 G 1.09 rs10896794 A 1.09 rs2930047 G 1.08
rs1847472 T 1.07 rs1748195 G 1.06 rs477515 A 1.06 rs7758080 A 1.06
rs11054935 A 1.05 rs2227564 G 1.05 rs2227551 T 1.05 rs3766606 T
1.05 rs7240004 A 1.02 rs9491697 G 1.00
[0435] Within the foregoing subset of 112 SNPs, a number were
individually associated with particular markers of clinical
efficacy, and these SNPs are in turn spacially associated with
genes, so that alternative markers, such as SNPs, associated with
these genes may also serve as markers of SSI efficacy, as set out
in Table 24.
TABLE-US-00026 TABLE 24 Select SNPs and associated Genes Efficacy
Metric SNP Raw p-value Important genes in area CD - rs9286879
3.32E-03 TNFSF18, TNFSF4, FASLG Response rs7517810 3.32E-03
TNFSF18, TNFSF4, FASLG score - rs17391694 4.06E-02 DNAJB4 (HSP-40
family member), Comparing GIPC2, NEXN, FUBP1, MGC27382 last
recorded response for all CD subjects CD - CDAI: rs1734907 1.43E-02
EPHB4, EPO, GNB2, TFR2, ZAN, Comparing POP7, ACTL6B, GIGYF drop in
CADI rs9286879 1.84E-02 See CD - response after 8 weeks rs7517810
1.84E-02 See CD - response of treatment for rs4836519 4.37E-02 all
CD subjects CD - rs17391694 7.21E-03 See CD - response
Calprotectin: rs2413583 1.09E-02 MAP3K7IP1, PDGFB, RPL3, Comparing
SYNGR1, SNORD43, SNORD83A, drop in fecal SNORD83B, FLJ23865, TAB1,
calprotectin ATF4 after 8 weeks rs11209026 1.62E-02 IL12RB2, IL23R
of treatment for rs2472649 2.85E-02 CXCL3, PF4, PPBP, CXCL5, all CD
subjects PPBPL2, IL8, CXCL1, CXCL6, CXCL2 CD - CRP: rs2641348
2.20E-03 NOTCH2, ADAM30, REG4, NBPF7 Comparing drop in CRP after 8
weeks of treatment for all CD subjects UC - Mayo: rs17085007
1.81E-02 GPR12 Comparing rs2024092 2.50E-02 CNN2, GPX4, POLR2E,
STK11, drop in Mayo ABCA7, SBNO2, HMHA1 score after 16 rs6017342
2.50E-02 HNF4A, SERINC3, PKIG, TTPAL, weeks of R3HDM treatment for
rs2188962 3.65E-02 IRF1, SLC22A4, SLC22A5, C5orf56 all UC subjects
rs941823 3.71E-02 LOC646982, FOX01 UC-CRP: rs17085007 1.95E-02 see
UC-Mayo Comparing rs2024092 1.95E-02 see UC-Mayo drop in CRP
rs6017342 3.81E-02 see UC-Mayo after 16 weeks rs17229285 3.96E-02
of treatment for all UC subjects
[0436] The foregoing IBD associated SNPs are spacially associated
with genes (Liu et al., Nature Genetics. 47.9 (September 2015): p
979), so that alternative markers, such as SNPs, associated with
these genes may also serve as markers of SSI efficacy, as set out
in Table 25:
TABLE-US-00027 TABLE 25 Additional SNPs and associated Genes SNP
Candidate Gene GRAIL gene rs1748195 USP1 rs34856868 BTBD8
rs11583043 SLC30A, EDG1 EDG1 rs6025 SELP, SELE, SELL SELP, SELE,
SELL NA (rs10798069) PTGS2, PLA2G4A NA (rs7555082) PTPRC rs11681525
-- rs4664304 MARCH7, LY75, PLA2R1 LY75 rs3116494 ICOS, CD28, CTLA4
ICOS, CD28, CTLA4 rs7556897, CCL20 CCL20 rs111781203 rs35320439
PDCD1, ATG4B PDCD1, ATG4B rs113010081 FLJ78302, LTF, FLJ78302, LTF,
CCR1/2/3/5 CCR1, CCR3, CCR5 rs616597 NFKBIZ NFKBIZ rs724016 --
rs2073505 HGFAC rs4692386 -- rs6856616 -- rs2189234 -- rs395157
OSMR, FYB, LIFR OSMR, FYB rs4703855 -- rs564349 C5orf4, DUSP1 DUSP1
rs7773324 IRF4, DUSP22 IRF4, DUSP22 rs13204048 -- rs7758080
MAP3K7IP2 MAP3K7IP2 rs1077773 AHR AHR rs2538470 CNTNAP2 rs17057051
PTK2B, TRIM35, EPHX2 PTK2B rs7011507 -- rs3740415 NFKB2, TRIM8,
TMEM180 NFKB2 rs7954567 CD27, TNFRSF1A, LTBR CD27, TNFRSF1A, LTBR
rs653178 SH2B3, ALDH2, ATXN2 SH2B3 rs11064881 PRKAB1 rs9525625
AKAP1, TNFSF11 TNFSF11 rs3853824 -- rs17736589 -- rs9319943 --
rs7236492 NFATC1, TST NFATC1 rs727563 TEF, NHP2L1, PMM1, L3MBTL2,
CHADL
[0437] The foregoing IBD associated SNPs are spacially associated
with genes (Jostins, et al., Nature. 2012; 491: 119-124), so that
alternative markers, such as SNPs, associated with these genes may
also serve as markers of SSI efficacy, as set out in Table 26:
TABLE-US-00028 TABLE 26 Further SNPs and associated Genes SNP
IC_SNP Key Genes (N) rs17391694 rs17391694 (5) rs6679677 rs6679677
PTPN22, (8) rs3897478 rs2641348 ADAM30, (6) rs9286879 rs7517810
TNFSF18, FASLG rs1728918 rs1260326 UCN, (22) rs10865331 rs10865331
(3) rs6716753 rs6716753 SP140, (5) rs12994997 rs12994997 ATG16L1,
(8) rs6837335 rs7438704 TEC, TXK, SLC10A4, (3) rs13126505
rs13126505 (1) rs10065637 rs10065637 IL6ST, IL31RA, (2) rs7702331
rs10061469 (4) rs17695092 rs17695092 CPEB4, (2) rs12663356
rs12663356 (2) rs9264942 rs9264942 HLA-C, PSORS1C1, (1) rs9491697
rs2503322 (3) rs13204742 rs13204742 (2) rs212388 rs212388 (6)
rs10486483 rs10486483 (2) rs864745 rs864745 CREB5, JAZF1 rs7015630
rs7015630 RIPK2, (4) rs6651252 rs6651252 (0) rs3764147 rs3764147
LACC1, FLJ38725, (2) rs16967103 rs16967103 RASGRP1, SPRED1, (2)
rs2066847** rs5743289 NOD2, (?) rs2945412 rs2945412 LGALS9, NOS2,
(4) rs2024092 rs2024092 APC2, GPX4, (21) rs4802307 rs4802307 (11)
rs516246 rs516246 DBP, IZUMO1, FUT2, SPHK2, (22) rs2284553
rs2284553 IFNGR2, IFNAR1, IL10RB, TMEM50B, IFNAR2, GART, (7)
[0438] The foregoing IBD associated SNPs are spacially associated
with genes (Jostins, eta, Nature. 2012; 491: 119-124), so that
alternative markers, such as SNPs, associated with these genes may
also serve as markers of SSI efficacy, as set out in Table 27a:
TABLE-US-00029 TABLE 27a Further Select SNPs and associated Genes
SNP IC_SNP All Genes rs17391694 rs17391694 NEXN, FUBP1, DNAJB4,
GIPC2, MGC27382 rs6679677 rs6679677 MAGI3, PHTF1, RSBN1, PTPN22,
BCL2L15, AP4B1, DCLRE1B, HIPK1, OLFML3 rs3897478 rs2641348 PHGDH,
HMGCS2, REG4, NBPF7, ADAM30, NOTCH2 rs9286879 rs7517810 FASLG,
TNFSF18 rs1728918 rs1260326 SLC5A6, C2orf28, CAD, SLC30A3, DNAJC5G,
TRIM54, UCN, MPV17, GTF3C2, EIF2B4, SNX17, ZNF513, PPM1G, FTH1P3,
NRBP1, KRTCAP3, IFT172, FNDC4, GCKR, C2orf16, ZNF512, CCDC121,
GPN1, SUPT7L rs10865331 rs10865331 COMMD1, B3GNT2, TMEM17 rs6716753
rs6716753 FBXO36, SLC16A14, SP110, SP140, SP140L, SP100 rs12994997
rs12994997 NGEF, NEU2, INPP5D, ATG16L1, SCARNA5, SCARNA6, SAG,
DGKD, USP40 rs6837335 rs7438704 TXK, TEC, SLAIN2, SLC10A4, ZAR1,
FRYL rs13126505 rs13126505 BANK1 rs10065637 rs10065637 IL31RA,
IL6ST, ANKRD55 rs7702331 rs10061469 FCHO2, TMEM171, TMEM174, FOXD1
rs17695092 rs17695092 CPEB4, C5orf47, HMP19 rs12663356 rs12663356
CDKAL1, SOX4, FLJ22536 rs9264942 rs9264942 HCG22, C6orf15,
PSORS1C1, CDSN, PSORS1C2, CCHCR1, TCF19, POU5F1, PSORS1C3, HCG27,
HLA-C, HLA-B, MICA, HCP5, HCG26, MICB, MCCD1, DDX39B, SNORD117,
SNORD84, ATP6V1G2, NFKBIL1 rs9491697 rs2503322 RSPO3, RNF146,
ECHDC1 rs13204742 rs13204742 THEMIS, PTPRK rs212388 rs212388 EZR,
OSTCP1, C6orf99, RSPH3, TAGAP, FNDC1 rs10486483 rs10486483 C7orf71,
SKAP2 rs864745 rs864745 JAZF1, LOC100128081, CREB5 rs7015630
rs7015630 RIPK2, OSGIN2, NBN, DECR1, CALB1 rs6651252 rs6651252
rs3764147 rs3764147 ENOX1, CCDC122, LACC1, LINC00284 rs16967103
rs16967103 SPRED1, FAM98B, RASGRP1, C15orf53 rs2066847** rs5743289
ADCY7, BRD7, NKD1, SNX20, NOD2, CYLD rs2945412 rs2945412 WSB1,
LOC440419, KSR1, LGALS9, NOS2 rs2024092 rs2024092 MED16, R3HDM4,
KISS1R, ARID3A, WDR18, GRIN3B, C19orf6, CNN2, ABCA7, HMHA1, POLR2E,
GPX4, SBNO2, STK11, C19orf26, ATP5D, MIDN, CIRBP-AS1, CIRBP,
C19orf24, EFNA2, MUM1 rs4802307 rs4802307 IGFL3, IGFL2,
DKFZp434J0226, IGFL1, HIF3A, PPP5C, CCDC8, PNMAL1, PNMAL2 rs516246
rs516246 GRWD1, KCNJ14, CYTH2, LMTK3, SULT2B1, FAM83E, SPACA4,
RPL18, SPHK2, DBP, CA11, SEC1, NTN5, FUT2, MAMSTR, RASIP1, IZUMO1,
FUT1, FGF21, BCAT2, HSD17B14, PLEKHA4, PPP1R15A, TULP2, NUCB1, DHDH
rs2284553 rs2284553 C21orf54, IFNAR2, IL10RB, IFNAR1, IFNGR2,
TMEM50B, DNAJC28, GART, SON, DONSON, CRYZL1, ITSN1
[0439] The correlation coefficient between pairs of loci may be
reflected by the term r-squared (r.sup.2), which may be used a
measure of the degree to which alternative genetic markers provide
similar diagnostic or prognostic information. The value of r.sup.2
ranges between 0 and 1 (1 when two markers provide identical
information, and 0 when they are in perfect equilibrium).
Conventionally, markers with r.sup.2>0.8 may be considered to be
in high linkage disequilibrium, so that they may provide similar
diagnostic or prognostic information. Accordingly, an aspect of the
assays described herein involves the use of makers that are in
linkage disequilibrium with the markers identified above, having
for example r.sup.2>0.7, r.sup.2>0.8, r.sup.2>0.9 or
r.sup.2>0.95. In addition, markers that provide related
information may be characterized by physical proximity in the
genome, for example being within 1 Mbp of each other, for example
within 50 Kb, 60 Kb, 70 Kb, 80 Kb, 90 Kb, 100 Kb, 200 Kb, 300 Kb,
400 Kb or 500 Kb of each other.
[0440] In accordance with the foregoing, a "genetic SSI response
marker" means a genetic biomarker, the presence of which is
correlated with the probability of response to a treatment with an
SSI. Exemplary genetic SSI response markers are disclosed in this
Example, evidencing a correlation with response to an SSI in IBD
patients. Genetic SSI response markers may be detected by a wide
range of genomic assays, and may also be detected by assays that
interrogate the transcription or translation products of a genome,
for example protein isoforms associated with a particular genomic
allele. Similarly, "biochemical SSI response markers" are disclosed
herein that provide a biochemical indication of response to an SSI
therapy, these for example include temporal or special changes in
cellular populations or in the abundance or concentration of
biologically relevant molecules. Biochemical and genetic SSI
response markers may be used as diagnostic or prognostic indicators
in the context of an SSI treatment, for example for IBD in general,
or for specific forms of IBD such as Crohn's Disease and ulcerative
colitis. Exemplary genetic SSI response markers are set out in
Table 27b, as well as Tables 23 to 26.
TABLE-US-00030 TABLE 27b Genetic SSI Response Markers Response SNP
Allele SNP related allele (or isoform) IBD rs9286879 A TNFSF18,
TNFSF4, FASLG Crohn's rs7517810 G TNFSF18, TNFSF4, FASLG Crohn's
rs17391694 G DNAJB4 (HSP-40 family member), Crohn's GIPC2, NEXN,
FUBP1, MGC27382 rs17085007 A GPR12 UC rs2024092 A CNN2, GPX4,
POLR2E, STK11, UC ABCA7, SBNO2, HMHA1 rs6017342 T HNF4A, SERINC3,
PKIG, TTPAL, UC R3HDM
Example 32: PRR Receptor Targets
[0441] This Example provides an analysis of the PRR receptors that
are the targets for alternative SSIs.
TABLE-US-00031 TABLE 28 List of PRRs stimulated by select SSIs,
including QBKPN, QBECO and QBSAU. Where a PRR is "Optional", this
indicates that some embodiments may be designed to include agonists
for the specificed PRR. Pattern Recognition Receptor Major Agonists
QBECO QBKPN QBSAU TLRs (Toll-Like Receptors) TLR1 Triacyl
lipoprotein/peptidoglycan Yes Yes Yes TLR2 Glycolipds, Lipoprotein,
Yes Yes Yes lipopeptides, lipoteichoic acid, others TLR3 dsRNA
(viral) No No No TLR4 Lipopolysaccharide (LPS), heat Yes Yes No
shock proteins, others TLR5 Flagellin, Profilin Yes No No TLR6
Diacyl lipoprotein Yes Yes Yes TLR7 ssRNA No No No TLR9 CpG-DNA Yes
Yes Yes TLR10 Unclear Optional Optional Optional CLR (C-Type Lectin
Receptors) (PMID 21616435) Mannose Mannose, N-acetylglucosamine
Optional Optional Optional Receptor (MR) and fucose on glycans
DEC-205 Promiscuous antigen receptor - Optional Optional Optional
Class B CpG-DNA (Lahoud et al. 2012. PNAS) Macrophage .alpha.- or
.beta.-N-acetylgalactosamine Optional Optional Optional
galactose-type (GalNAc, Tn) residues of N- lectin (MGL) and
O-glycans carried by glycoproteins and/or glycosphingolipids (PMID
15802303) DC-SIGN (CD- High-mannose-containing Optional Optional
Optional 209) glycoproteins Langerin (CD207) Similar to CD-209
Optional Optional Optional Mannose Binding Mannose and N- Optional
Optional Optional Lectin (MBL) acetylucosamine Myeloid DAP12-
Unclear, dengue viral particles Optional Optional Optional
associating lectin (PMC3204838) (MDL-1/CLEC5A) Dectin1/CLEC7A B
glucans on fungi, Optional Optional Optional mycobacteria
DNGR1/CLEC9A Actin filaments (no microbial Optional Optional
Optional ligands identified) SIGNR3 Mycobacterium tuberculosis
Optional Optional Optional CLEC4B1 Not Determined Optional Optional
Optional CLEC4B2 Not Determined Optional Optional Optional CLEC2
Endogenous (prodoplanin), Optional Optional Optional snake venom,
HIV CLEC12B Not Determined Optional Optional Optional CLEC12A Not
Determined Optional Optional Optional DCIR/CLEC4A HIV-1 Optional
Optional Optional Dectin 2/CLEC6A Mannose-type carbohydrates
Optional Optional Optional CLEC4C Unclear Optional Optional
Optional CLEC4E (Mincle) Fungal a-mannose and others Optional
Optional Optional NLR (Nod-Like Receptors) NOD1 diaminopimelatic
acid (DAP)- Optional Optional Optional containing muropeptide NOD2
muramyl dipeptide (MDP) Yes Yes Optional moieties universal to all
bacterial peptidoglycan NLRC3 (NOD3) Cytosolic DNA, cyclic di-GMP,
Optional Optional Optional DNA viruses (PMID 24560620) This is an
inhibitory PRR. NLRC4 (NOD4) Flagellin, components of the Optional
Optional Optional type three secretion system, others NLRC6 Unclear
Optional Optional Optional NLRX1 (NOD5) Unclear Optional Optional
Optional NALP1-14 Pathway unclear (Anthrax and Optional Optional
Optional muramyldipeptide for NALP1) NAIP Unclear Optional Optional
Optional CIITA (NLRA) Unclear (does not directly bind Optional
Optional Optional DNA) RLR (Rig-1 Like Receptors) RIG-1 dsRNA
(viral), maybe bacterial Optional Optional Optional MDA5 dsRNA
(viral) Optional Optional Optional LGP2 dsRNA (viral) Optional
Optional Optional Others DAI (DNA- DNA Optional Optional Optional
dependent activator of IRFs) (PMID 20098460) AIM2 (PMID dsDNA
Optional Optional Optional 20098460) Caspase 11 LPS Optional
Optional Optional (PMID 25145754) LBP LPS Optional Optional
Optional (Lipopolysaccharide Binding Protein CD14 LPS Optional
Optional Optional Scavenger Receptors LPS Optional Optional
Optional Beta2 Integrins LPS Optional Optional Optional
Peptidoglycan Peptidoglycan Minor Minor Major receptor proteins (4
different receptors)
TABLE-US-00032 TABLE 29 PRR agonists in select fractionated SSIs,
particularly in the DNA fractions Examplified herein. DNA Fractions
Component QBECO QBKPN DNA TLR9 TLR9 AIM2 AIM2 DAI DAI RIG-1 RIG-1
DEC205 DEC205 NLRC3 NLRC3
TABLE-US-00033 TABLE 30 PRR agonists in select fractionated SSIs,
particularly in the outer membrane fractions as Examplified herein.
Outer Membrane Fractions Component QBECO QBKPN LPS TLR4 TLR4 LBP
LBP CD14 CD14 Caspase 11 Caspase 11 Other Scavenger Other Scavenger
Receptors Receptors Lipoprotein TLR1 TLR1 TLR2 TLR2 TLR6 TLR6
Flagellin TLR5 N/A NOD4 NOD4 Peptidoglycan NOD2 NOD2 Capsule N/A
TLRs and CLRs Other Collection of CLRs
[0442] Accordingly, in select embodiments, SSI therapies are
provided that target a select subset of PRRs, using microbial PRR
agonists derived from microbial pathogens of a target tissue. For
example, an immunogenic composition is provided that comprises
microbial agonists for at least a select number of distinct PRRs,
for use so as to illicit an innate response in a target tissue,
wherein the PRR agonists are microbial components from a single
species of microbe that is selectively pathogenic in the target
tissue. The number of distinct PRRs targeted by the agonists may
for example be a number from 5 to 25, or at least a number within
that range of integers, for example at least 5, 6, etc. The
distinct PRRs may for example be selected from the PRRs set out in
Tables 28, 29 and/or 30.
Example 33: Cytokine Markers of SSI Therapy
[0443] This Example provides an indication of cytokine markers
indicative of various facets of SSI therapies. This data reflects
the analysis of 42 cytokines/chemokines from a cohort of Crohn's
Disease patients undergoing SSI therapy with QBECO, at baseline,
week 4, week 8, week 16, and week 24, of a randomized
placebo-controlled trial involving 68 patients.
Cytokines Changes with QBECO Exposure
[0444] QBECO exposure increased IL-18 and IP-10 at both the 8 week
and 16 week time points. Serum levels of IL-18 showed the most
significant differences between patients treated with QBECO vs.
Placebo at week 8 (median change 24 pg/mL, adjusted p=0.0256) (FIG.
74). This increase in IL-18 was evident at the week 16 time point
as well. The second serum biomarker to show significant differences
was IFN.gamma.-inducible protein 10 (IP-10, also known as CXCL10)
which showed greater increases in QBECO exposed patients at both
week 8 (median change 7 pg/mL, adjusted p=0.036) and week 16
(median change 22 pg/mL, adjusted p=0.0151).
[0445] Vascular endothelial growth factor A (VEGF-A) showed some
increase in the QBECO group at the week 8 mark (median change 14
pg/mL, adjusted p=0.0483), but this difference was lost at the end
of the week 16 treatment point. A number of other immune factors
showed strong trends in being increased from baseline to 8 weeks of
QBECO exposure; these included: granulocyte colony stimulating
factor (GCSF), IFN.gamma., IL-17A, IL-6, IL-7, and transforming
growth factor-.alpha. (TGF.alpha.).
[0446] None of the serum immune factors remained elevated after
patients were taken off all treatment after week 16 and evaluated
again at week 24, illustrating that these biomarkers are most
helpful to assess the immune responsiveness to QBECO while on
treatment.
Serum Biomarker Cytokine Concentration Changes that Associate with
Clinical Response
[0447] A sub-analysis was performed in patients exposed to QBECO
(N=42, including those initially randomized to QBECO and those who
were switched from placebo at week 8) to assess whether any of the
immune changes over time associated with clinical outcome. Il-18
increased less among those with clinical response and remission,
compared to non responders. IP-10 increased less among those with
clinical response and remission, compared to non responders.
IFN.gamma., IL-12p70, IL-17A and TGF.alpha. showed a significant
difference in increase over time for responders compared to non
responders. In particular, IFN.gamma., IL-12p70, IL-17A and
TGF.alpha., had greater increases over-time (adjusted p=0.0344) in
patients who experienced a clinical response to QBECO in comparison
to non-responders at week 8 (FIG. 75).
Baseline Serum Immune Factors that Associate with Clinical
Response
[0448] Lower Eotaxin 1 was a predictive biomarker for remission in
response to QBECO treatment. In particular, baseline serum levels
of Eotaxin-1 (C-C chemokine 11) had the strongest link to clinical
remission (adjusted p=0.0016), with patients who had higher levels
at baseline being less likely to go into clinical into clinical
remission by week 8 with QBECO treatment (FIG. 76). Although not
reaching statistical significance after correcting for multiple
comparisons, patients with higher baseline IL-10 and IL-12p40 were
also less likely to have a clinical response to QBECO treatment by
week 8 (FIG. 76).
[0449] Trial results indicate that patients who had been previously
exposed to TNF.alpha. inhibitors, such as Remicade.TM. or
Humera.TM., were less likely to experience clinical remission or
response after 8 weeks of QBECO treatment. This more difficult to
treat group may have more severe immune dysfunction, due to their
exposure to these immunosuppressive drugs and/or by virtue of the
nature of their condition. Stratifying the mean baseline serum
levels of the immune factors that associated with clinical outcome
by previous TNF.alpha. inhibitor exposure provides evidence to
support this. The baseline serum immune factors that inversely
associated with patient response to QBECO, Eoxtaxin-1, IL-10 and
IL-12p40, were higher in patients previously exposed to
anti-TNF.alpha. therapy relative to unexposed patients (Table
31).
TABLE-US-00034 TABLE 31 Mean baseline serum levels of Eotaxin-1,
IL-10 and IL- 12p40 stratified by previous TNF.alpha. inhibitor
exposure Previous 95% anti- Mean Confidence TNF.alpha. Mean .+-.
Difference .+-. Interval of the therapy N* SD SD Difference
Eotaxin-1 No 39 84 .+-. 43 -17 .+-. 12 (-41, 7) Yes 26 100 .+-. 54
IL-10 No 28 6 .+-. 14 -5 .+-. 5 (-14, 4) Yes 17 11 .+-. 17 IL-12p40
No 36 19 .+-. 67 -52 .+-. 36 (-123, 20) Yes 24 70 .+-. 198 *20
reads from the IL-10 assay and 5 reads from the IL-12p40 were out
of range of the assay or unreliable
High Response and Remission Rates in Anti-TNF.alpha. Naive
Patients
[0450] In anti-TNF.alpha. naive patients, treatment with QBECO SSI
for 8 weeks resulted in a statistically significant response rate
of 64% compared to 27% in the placebo control (p=0.041). Clinical
remission rates after 8 weeks of treatment were also impressive at
50%, more than double the placebo rate of 23% (p=0.16). Clinical
response and remission rates were assessed using the standard
Crohn's Disease Activity Index (CDAI), defined as a decrease in
CDAI of .gtoreq.70 points (response) and CDAI score .ltoreq.150
points (remission). Anti-TNF.alpha. naive patients include, for
example, patients who have not been treated with the
immunosuppressive drugs Remicade.RTM., Humira.RTM., Cimzia.RTM. and
Simponi.RTM.. In patients previously been treated with TNF.alpha.
inhibitors who completed 16 weeks of SSI treatment, 40% were in
remission, indicating that this more challenging patient group may
respond to QBECO SSI with longer treatment.
Building a Composite Prediction Model to Assess Likelihood of
Patient Response to QBECO by 8 Weeks of Therapy
[0451] Using a Regularized Logistic Regression modelling approach,
which simultaneously selects variables with the strongest
association with response and optimally weights them to generate a
prediction score, a composite prediction model was built including
both the baseline biomarker measures (i.e. the 42 immune factors
including cytokines, chemokines and growth factors) and baseline
clinical and demographic characteristics. The variables available
for the latter included enrollment year, age at randomization, age
at diagnosis, time from diagnosis to randomization, sex, race
(Caucasian or not), site (Vancouver or not), prior anti-TNF.alpha.
therapy, baseline Crohn's Disease Activity Index (CDAI) score,
baseline fecal calprotectin levels, and baseline C-reactive protein
levels.
[0452] An "optimism-adjusted" area-under the receiver operating
curve (AUROC) was made to correct for the potential over-estimation
of the model fit. This "optimism-adjusted" AUROC can thus be more
readily reliably applied to future independent data.
[0453] As shown in the analysis in this Example, high baseline
serum Eotaxin-1 was the strongest negative biomarker predictor for
clinical response after 8 weeks of QBECO treatment and was included
in all models generated. Of the clinical/demographic variables--sex
(females were more likely to respond to QBECO treatment) and
previous anti-TNF.alpha. therapy (those previously exposed less
likely to respond to QBECO) were the strongest predictors. Table 32
summarizes the different models generated. Typical commercial
biomarker standards require an AUROC >7 for commercial viability
of a prediction model. After optimism-adjustment, the composite
model generated from this data achieved this level of predictive
value with the inclusion of the following variables: sex, prior
TNF.alpha. therapy, and baseline levels of Eotaxin-1, GRO.alpha.
(also called CXCL1--a neutrophil chemokine), IL-10, PDGF AA and
RANTES (also called CCL5--a chemokine for activated T cells,
eosinophils, basophils). Alternatively, a predictive model may also
be developed using Eotaxin, GRO.alpha., IL10, PDGF AA, RANTES, Sex
and prior aTNF.alpha., predicting response with high
confidence.
TABLE-US-00035 TABLE 32 Performance of four prediction models for
clinical response and clinical remission following 8 weeks of QBECO
treatment Clinical Response @ 8 Weeks Clinical Remission @ 8 Weeks
Optimism- Optimism- Variables Raw Adjusted Variables Raw Adjusted
Candidates Included AUROC AUROC Included AUROC AUROC Reliable
Eotaxin 1 0.737 0.591 Eotaxin 1 0.846 0.644* Cytokines PDGF AA
(0.59, 0.88) (0.44, 0.73) GRO .alpha. (0.72, 0.97) (0.52, 0.77)
Only PDGF AA All Eotaxin 1 0.753 0.588 Eotaxin 1 0.842 0.612
Cytokines IL 10 (0.60, 0.90) (0.43, 0.73) GRO .alpha. (0.71, 0.97)
(0.50, 0.74) PDGF AA PDGF AA Clinical/ Sex Prior 0.760 0.642 Sex
0.674 0.627 Demographic aTNF.alpha. (0.62, 0.90) (0.50, 0.78)
(0.53, 0.81) (0.48, 0.76) Variables All Eotaxin 1 0.858 0.700*
Eotaxin 1 0.881 0.707* Cytokines GRO .alpha. (0.75, 0.97) (0.59,
0.81) GRO .alpha. (0.77, 0.99) (0.60, 0.82) and All IL 10 PDGF AA
Clinical/ PDGF AA RANTES Demographic RANTES Sex Variables Sex Prior
aTNF.alpha. *significant at 0.05 level.
Analysis
[0454] Cytokine change with QBECO exposure: IL-18 (adjusted p=0.011
@ 8 weeks and 0.067 @ 16 weeks) and IP-10 (adjusted p=0.036 @ 8
weeks and 0.015 @16 Weeks) demonstrated a substantial and
statistically significant increase with exposure to QBECO. These
two cytokines also demonstrated significantly different
trajectories for Clinical Responders vs Non-Responders (adjusted
p=0.0328 for both) and those in and not in Clinical Remission
(adjusted p=0.0368 for both) at week 8. Further, IL-18 demonstrated
significantly different trajectory for those randomized to QBECO vs
Placebo (adjusted p=0.0256).
[0455] Cytokine Association with Outcome: Baseline Eotaxin-1
concentration was most strongly associated with clinical outcome
among QBECO exposed subjects; those with higher Eotaxin-1
concentration at baseline were more likely to achieve Clinical
Remission (adjusted p=0.0016) following 8 weeks of QBECO
exposure.
[0456] Composite Biomarker: Baseline concentration of Eotaxin-1,
GRO-.alpha., IL-10, PDGF AA and RANTES, combined with clinical
variables Sex, and Prior anti-TNFA.alpha. exposure provided
predictions of 8-week clinical outcomes that were significantly
better than chance (optimism-adjusted AUROC=0.70, 95% CI [0.59,
0.81] for Response and 0.71, 95% CI [0.60,0.82] for Remission).
This model had some observable predictive ability for subjects in
the Placebo group (AUROC=0.67 95% CI [0.45, 0.89] for Response and
0.70 [0.47, 0.93] for Remission.
Summary
[0457] QBECO SSI therapy provokes a biological response by
increasing certain cytokines (IL-18 and IP-10) over time.
Surprisingly, although both cytokines are increased after QBECO
treatment, patients who were responders increased less. Treatment
protocols, such as dosing, may accordingly be adjusted to achieve
this result.
[0458] IFNg, IL-12P70, IL-17A and TGFa increased more in responders
than non responders. Treatment protocols, such as dosing, may
accordingly be adjusted to achieve this result. TGFa may for
example be used as a marker of mucosal healing.
[0459] Lower Eotaxin 1 levels may be used as an indicator of
patients more amenable to SSI treatment.
[0460] In conclusion: [0461] an increase in serum IL-18 from
baseline to week 8 and 16 of treatment was the best biomarker (of
the 42 assessed) for QBECO exposure/activity; [0462] a subsequent
rise in serum levels of IFN.gamma., IL-12p70, IL-17A and TGF.alpha.
after 8 weeks of QBECO treatment associated with clinical response;
[0463] Crohn's patients with higher baseline levels of Eotaxin-1
(and to a lesser extent, IL-10 and IL-12p40) were less likely to
experience a clinical response or remission to QBECO after 8 weeks
of treatment; previous anti-TNF.alpha. therapy may predispose to
having higher levels of these factors, and anti-TNF.alpha. naive
patients represent a distinct Crohn's patient population amenable
to QBECO SSI therapy; [0464] a composite model that includes
baseline serum biomarkers and clinical/demographic data would be
able to predict, after optimism-adjustment (AUROC .gtoreq.7), a
patient's likelihood to respond to 8 weeks of QBECO treatment; the
variables in the final model includes sex, previous anti-TNF.alpha.
therapy and baseline serum levels of Eotaxin-1, GRO.alpha., IL-10,
PDGF AA and RANTES.
[0465] This biomarker analysis illustrates the formulation of a
viable predictive composite model that can provide personalized
treatment for Crohn's disease. This biomarker analysis maybe useful
alone, or in combination with the genetic analysis exemplified
herein, which showed significant stratification between responders
and non-responders based on a derivation of a gene score.
Example 34: DSS Colitis Model
[0466] This Example illustrates results from a mouse model of
chemically induced colitis, used to assess the efficacy of QBECO
SSI therapy. Mice were given dextran sodium sulfate (DSS) in
drinking water to induce colitis that mimics human ulcerative
colitis. In the disease model, one cohort of mice was exposed to
DSS for 7 days, a second cohort was exposed to DSS for 7 days
followed by 3 days of water. Mice were given SSI injections every
other day during a 10 day period prior to DSS exposure. The SSI
injections continued every other day during DSS exposure. The
results, as illustrated, indicate that SSI treatment with QBECO
ameliorates disease severity by limiting weight loss (FIG. 77),
lowering disease severity (FIG. 78) and maintaining mucosal barrier
function (FIG. 79). The pharmacodynamics of QBECO SSI treatment in
this model are illustrated by the blood neutrophil (FIG. 80) and
blood cytokine (FIG. 81) levels in disease-free mice treated with
QBECO or placebo, with the pharmacokinetics of QBKPN (FIG. 82) used
to model the pharmacokinetics of SSIs in general, including QBECO
(QBKPN SSI was fluorescently labelled and subcutaneously injected
into disease-free mice, mice were bled at different timepoints over
48 hours).
Example 35: Tissue Biomarkers
[0467] This Example illustrates results from a mouse cancer model,
showing a tissue-specific biomarker response to SSI therapy. As
illustrated in FIG. 83, gene expression in the lung tissues
evidences tissue-specific SSI responses for CXCL10 (IP-10), CCL2
(MCP-1) and CCR2. In this Example, mice were treated every second
day for 10 days with Placebo, QBKPN or QBECO before B16F10 tumour
implantation into the lungs via tail vein injection. Treatment
continued every second day after tumour inoculation. Mice were
euthanized on day 5 (A, C, E) or day 17 (B, D, F). Accordingly, in
alternative embodiments, CXCL10 (IP-10), CCL2 (MCP-1) and/or CCR2
may be used as biochemical SSI response markers, for example in
biopsy tissue sample assays.
* * * * *
References