IR BioSciences Holdings, Inc.

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Homspera® - Cancer Therapy

Executive Summary
Homspera® is a synthetic, 11 amino acid peptide analog of Substance P (SP), an endogenous tachykinin. The
actions of Homspera are mediated predominately through interactions with the Neurokinin-1 receptor located on the
plasma membrane of many cell types. Substance P binds to all three neurokinin receptors (NK-1, NK-2 and NK-3),
though SP preferentially interacts at the NK-1R to mediate its biological effects [ 1]. In contrast to SP, Homspera is
highly selective for NK-1R and shows no activity in NK-2 and NK-3 receptor biological activity assays while
eliciting greater than 3-times the biological activity as SP in NK-1R-specific tissues [2]. The two-fold greater
binding affinity Homspera shows for the NK-1R compared to SP (0.8 ± 0.3 nM vs. 1.6 ± 0.4 nM, respectively) may
explain the observed increases in biological activity [3]. In most of these assays, Homspera behaves similarly to
Substance P, albeit with greater bioactivity. Studies sponsored by ImmuneRegen evaluating hematopoietic stem cell
activation have revealed an effect similar to, yet more potent than, Substance P.
In addition to effects on hematopoietic stem cells, Homspera acts directly on relevant cells of the immune system to
enhance their functionality. Antigen and adjuvant do not need to be co-administered, or even use the same route of
administration, as evidenced by studies utilizing a novel Influenza vaccine and Homspera as an adjuvant. As
summarized below, Homspera elicits a number of critical effects that enhance the efficacy of a vaccine for cancer.
Homspera prolongs immune cell (Dendritic cell) survival through NK1-R activation
1. Dendritic Cells (DCs) cultured with Homspera (Sar9, Met (O2)11 - Substance P, SarSP) showed increased
survival when adoptively transferred into syngeneic mice. DCs are maestro cells of the immune system and
the most important cells for generating immunity. [pg. Error: Reference source not found  (‘Ctrl’ +
click to follow)]
2. The reduced activation-induced apoptosis (AIA) of DCs cultured with Homspera ex-vivo is mediated
through the PI3K/AKT signaling pathway. This is an intracellular signaling pathway linking the NK1
receptor to NF-kB and involved in triggering the release of cytokines that can mediate the immune
response. [pg. Error: Reference source not found]
This prolonged survival is retained when exposed cells are returned into experimental animals
3. The increased survival of ex-vivo exposed DCs extends to in vivo survival when adoptively transferred into
syngeneic mice. Knowing that these cells survive in the lymph nodes supports the idea that a stronger
immune response will be generated. [pg. Error: Reference source not found]
Mechanisms of action are additive to other processes that utilize the NF-kB pathway, as does Homspera
4. Homspera acts independently as well as synergistically with CD40 ligation. Many researchers are using
CD40 activation to extend cell survival and Homspera enhances this treatment. [pg. Error: Reference
source not found]
Activation by Homspera includes the generation of antigen-specific helper T cells
5. Homspera cultured DCs produce enhanced Ag-specific CD4+ T helper function. CD4+ T helper cells are
crucial for a long term immune response and continued therapeutic efficacy against cancer. [pg. Error:
Reference source not found]

1 Tousignant, C., Guillemette, G., Drapeau, Telemaque, S., Dion, S., G., Regoli, D., Brain Res. 524 (1990) 263-270.

2 Tousignant, C., Guillemette, G., Drapeau, G. and Regoli, D., Neuropeptides, 14 (1989) 275-283.

3 Sagan, S. et al., J. Pharmacology and Experimental Therapeutics 276 (1996) 1039-1048.

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Homspera® - Cancer Therapy

Homspera increases the ability of genetic vaccines to produce their antigenic payload which stimulates the
immune response to the vaccine
6. Homspera therapy increased expression of antigenic transgene in gene-gun (GG) studies. By increasing
expression of the transferred gene, the vaccine is likely to be stronger. [pg. Error: Reference source not
found]
Homspera makes the activated Dendritic cells more effective at getting to the lymph nodes, makes the lymph
nodes more receptive to the DCs, and increases the responsiveness of DC in the lymph nodes
7. Homspera therapy increases trafficking of skin Langerhans’ cells (LCs) from the skin to skin draining
lymph nodes (sDLNs) and alters the physiology of sDLNs by increasing their vascularity and expression of
the DC-homing molecule CCL21. The activated cells need to interact with other immune cells in the lymph
nodes. Many cancer vaccines lack adequate stimulatory activity. [pg. Error: Reference source not found]
8. Homspera induces mixed subtype hypercellularity and the number of DCs in dLNs and also activates these
DCs as shown by increased MHC-II molecules. By attracting many different types of cells the immune
response will be more robust and effective. [pg. Error: Reference source not found]
Homspera increases the antibody response of the immune system as well as the release of cytokines that drive
the immune response
9. The net effect of Homspera therapy upon the immune response shows that Homspera therapy increases
antigen-specific antibodies and cytotoxic T cell number and function as well as Interferon-γ (IFN-γ)
expression by antigen-specific CD4+ and CD8+ cells. In addition to increasing both the antibody and
cellular immune responses, Homspera increases IFN-γ, an important anti-cancer molecule that can directly
kill cancer cells or direct other cells to do so. [pg. Error: Reference source not found]
The result of the multi-pronged immuno-stimulation by Homspera and antigen is a long-lasting immune
response to the administered antigen
10. Homspera therapy increased antigen-specific CD4 T-helper response as demonstrated by dramatically
enhanced delayed type hypersensitivity (DTH). The fact that this response lasts longer than 72 hours says
the immune response is very strong and effective. [pg. Error: Reference source not found]
The prolonged targeted immune response results in an inhibition in the growth of cancer transferred into the
treated animals
11. Homspera increases effector cellular immune responses and reduces and delays the growth of melanoma in
genetic vaccines. The responses described above lead to reduced cancer growth in animals. [pg. Error:
Reference source not found]
12. Homspera significantly reduces and delays the growth of melanoma, prolongs mouse survival and induces
long lasting immunity. The slower tumor growth results in increased survival and continual immunity to
melanoma. [pg. Error: Reference source not found]
In summary, the addition of Homspera to cancer vaccines (or to DC culture, antigen stimulation and loading
procedures for ex vivo stimulation) could produce more effective DCs primed to traffic to LNs and survive for
extended periods of time for an enhanced therapeutic immune response against the patient’s cancer.

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Substance P – Neuropeptide and Parent Compound of Homspera
Substance P is a relatively small (1,348 Daltons), endogenous peptide first discovered in 1931 and characterized
chemically about 40 years later [4]. Neuropeptides, such as SP, were originally discovered as being distributed
throughout the peripheral and central nervous systems. However, SP has since been shown to be produced in non-
neuronal cells such as human endothelial cells, Leydig cells, enterochromaffin cells, epithelial cells, fibroblasts,
keratinocytes, intestinal and airway smooth muscle cells, inflammatory and immune cells, and in cells of the female
reproductive system [5]. Park et al. have reported every mammal produces SP, with the exception of the African
naked mole-rat [6], evidence of the significance of this peptide throughout the animal kingdom.
Historically, Substance P has been recognized as a mediator of nonadrenergic, noncholinergic (NANC) excitatory
neurotransmission, and as playing a role in the transmission of pain [ 7]. More recent studies have called into
question Substance P’s role in pain and pain perception, however, and SP antagonists do not affect pain or pain
perception.
Modern research has brought forth additional functions for SP including the following: vasodilation, smooth muscle
contraction, submucosal gland secretion; increased vascular permeability; stimulation of mast cells, B- and T-
lymphocytes and macrophages; modulation of chemo-attraction of eosinophils and neutrophils, and modulation of
vascular adhesion of neutrophils [8]. The ability of SP to impact immune cells is of special interest to ImmuneRegen
given the immune-modulating properties observed for Homspera in animal studies.

Homspera – A Unique Analog of Substance P

A few years after the discovery of the Substance P amino acid sequence, numerous synthetic analogues were
produced to aid in characterization of the mechanism of action of SP. One particular analogue was created by
modifying two of the eleven amino acids in the SP sequence. These modifications included replacing glycine (Gly)
with sarcosine (Sar or N-methyl glycine) at the ninth position and introducing an oxidized form of methionine
[Met(O2)] at the eleventh position (as seen in and Figure 1), resulting in the peptide Sar9, Met (O2)11-SP (1,393.6
Daltons).

4 Chang, M., Leeman, S., J. Biol. Chem. 245 (1970) 4784-4790.

5 Pennefather, J., Lecci, A., Candenas, M., Patak, E., Pinto, F., Maggi, C., Life Sciences 74 (2004) 1445-1463.

6 Park, T., et al. PLoS Biol. 6 (2008).

7 Almeida, T., Rojo, J., Nieto, P., Pinto, F., Hernandez, M., Martin, J., Candenas, M., Curr. Med. Chem. 11 (2004) 2045-2081.

8 De Swert, K., and Joos, G. E. J. Phar. 533 (2006) 171-181.

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Figure 1: Structure of Homspera. Red indicates the modifications to Substance P that define Homspera.

As seen in below, the primary amino acid sequences of SP and Sar9, Met (O2)11-SP are distinct from the tachykinins
Neurokinin A (NKA) and Neurokinin B (NKB) at the N-terminus (referenced by the start of the amino acid
sequence as seen in below). These differences result in NKA and NKB binding with significantly less affinity to the
Neurokinin-1 receptor (NK-1R) than do SP and Sar9, Met (O2)11-SP, which share an identical N-terminal sequence
[9]. The C-terminal penta-peptide, Phe-Phe/Val-Gly-Leu-Met-NH2, is conserved between all natural tachykinins and
is required for receptor activation. Amidation of the C-terminal methionine is vital for peptide function as without
this modification tachykinins are unable to activate their corresponding mammalian receptors [10]. The specific C-
terminal modifications that separate Sar9, Met (O2)11-SP from endogenous SP contribute to differences in bioactivity
and confer NK-1 receptor specificity (via the modification of the Gly9), as the ligand-receptor interactions are
changed (Figure 1) [11].

Table 1: Amino acid sequences of Sar9, Met (O2)11 – SP and endogenous tachykinins.

Homspera and Substance P – Mechanistic Similarities

The actions of Homspera are mediated predominately through interactions with the Neurokinin-1 receptor located
on the plasma membrane of many cell types. Substance P binds to all three neurokinin receptors (NK-1, NK-2 and
NK-3), though SP preferentially interacts at the NK-1R to mediate its biological effects [1]. In contrast to SP,
Homspera is highly selective for NK-1R and shows no activity in NK-2 and NK-3 receptor biological activity assays
[1], while eliciting greater than 3-times the biological activity as SP in NK-1R-specific tissues [2]. The two-fold

9 Tousignant, C., Guillemette, G., Drapeau, G. and Regoli, D., Neuropeptides, 14 (1989) 275-283.

10 Nassel, D., Peptides 20 (1999) 141-158.

11 Zhang, Y., Berger, A., Milne, C., Paige, C., Curr. Drug Targets 7 (2006) 1011-1020.

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Homspera® - Cancer Therapy

greater binding affinity Homspera shows for the NK-1R compared to SP (0.8 ± 0.3 nM vs. 1.6 ± 0.4 nM,
respectively) may explain the observed increases in biological activity [12]. In most of these assays, Homspera
behaves similarly to Substance P, albeit with greater bioactivity. Studies sponsored by ImmuneRegen evaluating
hematopoietic stem cell activation and wound healing have revealed an effect similar to, yet more potent than,
Substance P.
The interaction of both Homspera and SP with the NK-1R induces secondary messenger signaling events, which
originate from the activated receptor and then rapidly cascade throughout the cell. Water-soluble messengers, like
Ca2+ and cyclic AMP (cAMP), diffuse throughout the cytosol, while the hydrophobic lipid-soluble messengers like
diacylglycerol (DAG) diffuse into the plasma membrane. In some tissues, activation of water-soluble messengers
can lead to signaling events in adjacent cells via gap junctions, thus leading to a broad multi-cellular response from
the stimulation of a single cell [13].
SP and Homspera are capable of utilizing both phosphatidylinositol (PI) hydrolysis and cAMP as second messenger
signaling systems (mediating changes in intracellular Ca2+ mobilization), and do so with high potency. Using an in
vitro model (Chinese Hamster Ovary cells transfected with human NK-1R) Sagan et al. have reported an EC50 of 1.0
± 0.6 nM for SP in stimulating phosphatidylinositol (PI) hydrolysis whereas Sar9, Met (O2)11-SP was reported to
have an EC50 of 0.40 ± 0.01 nM in stimulating PI hydrolysis [3] . The same group reported an EC50 of 8 ± 4 nM for
SP in stimulating cAMP formation whereas Sar9, Met (O2)11-SP was reported to have an EC50 of 16 ± 7 nM.

Neurokinin-1 Receptors and their Distribution

Substance P binds to all three neurokinin receptors (NK-1, NK-2 and NK-3), though SP preferentially interacts at
the NK-1R to mediate its biological effects [14]. The neurokinin receptors belong to “family 1” (rhodopsin-like) of
the G protein-coupled receptors. Like many G protein-coupled receptors, the NK-1R consists of seven putative α-
helical transmembrane segments, an intracellular carboxyl tail, and an extracellular amino-terminus. At the
extracellular amino terminus there is an N-glycosylation site, while many serine and threonine residues at the
intracellular carboxyl terminus are potential phosphorylation sites [15].
The Neurokinin-1 receptor has been identified in stem cell lines as well as cells derived from human placental cord
blood, rich in hematopoietic stem and progenitor cells [16]. Thus it is not unexpected that the NK-1R has been
identified in a multitude of tissues and cell types. The NK-1R is expressed in immune cells such as T and B
lymphocytes, monocytes/macrophages, neutrophils, and mast cells [17]. Non-immune cells like vascular endothelial
cells, bone marrow stromal cells, muscle cells, astrocytes, adipocytes, keratinocytes, and fibroblasts also express the
NK-1R [18, 19]. It appears the NK-1R receptor is involved in a number of physiological systems that may be of
significance to the immune system, and the cells that support the immune system.

12 Sagan, S. et al., J. Pharmacology and Experimental Therapeutics 276 (1996) 1039-1048.

13 Alberts, B. et al., Molecular Biology of the Cell (4th Ed.) 2002.

14 Tousignant, C., Guillemette, G., Drapeau, Telemaque, S., Dion, S., G., Regoli, D., Brain Res. 524 (1990) 263-270.

15 Cottrell, G. et al., J. Biol. Chem. 281 (2006) 27773-27783.

16 Li, Y., Douglas, S., Ho., W. J. Hematother. & Stem Cell Res. 9 (2000) 445-452.

17 Liu, J. et al., Brit. J. Derm. (2006) 155:657-662.

18 Lai, J., Ho., W., Kilpatrick, L., Wang, X., Tuluc, F., Korchak, H., Douglas, S., PNAS 103 (2006) 7771-7776.

19 Greco, S., Corcoran, K., Cho, KJ., Rameshwar, P., Frontiers in BioScience 9 (2004) 1782-1793.

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Why use Homspera as a vaccine adjuvant?
The first generation adjuvants are the aluminum salts, which predominantly act via their depot effect, increasing
immunogenicity of co-administered compounds. Second generation adjuvants might include Toll-Like Receptor
(TLR) ligands, oil/water emulsions and amphipathic agents, alone, in combinations or formulated into specific 3-
dimensional structures (e.g., ISCOMs). As a third generation adjuvant, Homspera acts directly on relevant cells of
the immune system to enhance their functionality. Antigen and adjuvant do not need to be co-administered, or even
use the same route of administration.
The addition of Homspera to cancer vaccines (or to DC culture, antigen stimulation and loading procedures for ex
vivo stimulation) could produce more effective DCs primed to traffic to LNs and survive for extended periods of
time for an enhanced therapeutic immune response against the patient’s cancer.

Dendritic cell (DC) stimulatory properties of Homspera enhance

antigen-presentation components, stimulated B- and T-cells and
ultimately immune responses

In vitro studies on Homspera:

Note: All figures in this section were derived from Janelsins et. al., Blood. 2009;113:3017-3026)

Dendritic Cells (DCs) cultured with Homspera (sometimes referred to as

SarSP below) showed increased survival when adoptively transferred into
syngeneic mice

This rescue from apoptosis was mediated through activation of the NK1R expressed on DCs.

Figure 2: Proinflammatory tachykinins prevent apoptosis of BMDCs by NK1R signaling. (A) Comparison of
the antiapoptotic effects of Homspera, Substance P, and HK-1 at physiological (10-9 mol/L) and nonphysiological
(10-5 mol/L) concentrations on CD11c+ BMDCs as determined by FACS analysis of annexin V and PI labeling. (B,
C) Bar-diagrams representing the antiapoptotic effect of Homspera on CD11c+ BMDCs is abrogated by
preincubation with the highly selective NK1R antagonist WIN-51708 or (C) in NK1R-/-KO BMDCs. ***P < .001.
(A-C) Means (+ SD) of 3 independent experiments are illustrated. (D) Dot-plots illustrating the percentage of
apoptosis (numbers in dot-plots) of CD11c+ BMDCs signaled with NK1R antagonist WIN-51708 alone. Data are
representative of 4 independent experiments.

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The reduced activation-induced apoptosis (AIA) of DCs cultured with
Homspera ex-vivo is mediated through the PI3K/AKT signaling pathway

AIA of stimulated DCs is a natural feedback and immune regulatory loop employed by the immune system to limit
an immune response. This apoptosis can be Ag-dependent or independent. This same mechanism likely limits the
immune response generated from ex-vivo loaded DCs for cancer immunotherapy. Extended survival of DCs post
adoptive transfer into vaccine recipients would allow for extended time for interaction of immune cells and a greater
antigen-specific immune response. Studies demonstrated that this increased survival as well as other effects upon
draining LNs (dLNs). Homspera’s AIA-protection properties on DCs are comparable to that of GM-CSF and IL-4
co-culture and likely synergistic based on signaling pathway analysis.

Figure 3: Signaling BMDCs via NK1R prevents apoptosis through activation of the PI3K-Akt cascade.
(A) Bar diagram representing the effect of intracellular pathway blockade on the anti-apoptotic effect exerted by
SarSP on CD11c+ BMDCs. Before culture with or without SarSP, BMDCs were left untreated (control) or
incubated with the cell-permeable inhibitors LY294002 (PI3K), PD98059 (ERK), SN-50 (NFκB), or JNK
inhibitor II (JNK-AP1). ***P < .001. (B) FACS analysis showing the level of pAktSer473 expression by CD11c +
BMDCs cultured in the presence of SarSP (gray empty histogram) or medium alone (gray filled histogram).
(C) Comparative analysis of the effects of LY294002 and Akt inhibitor X (pAktSer473 inhibitor) on the anti-
apoptotic effect of SarSP on BMDCs. ***P < .001. (D) FACS analysis illustrating the level of Bcl-2,
pBadSer136, and active caspase 3 expression by CD11c+ BMDCs cultured in the presence of SarSP (black empty
histogram), GM and IL-4 (gray empty histogram), or medium alone (gray filled histogram). (A, C) Means (+
SD) of 4 independent experiments are shown. (B, D) Data are representative of 4 independent experiments.
Numbers in histograms indicate percentage of CD11c+ BMDCs.

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The increased survival of ex-vivo exposed DCs extends to in vivo survival when
adoptively transferred into syngeneic mice

The ex vivo stimulated DCs were labeled with carboxyfluorescein succinimidyl ester (CFSE) to allow for tracking
post transfer. TN-DCs are BM derived DCs that were haptenized with 1 mM TNBS (trinitrobenzene sulfonic acid)
for 25 minutes before CFSE staining and adoptive transfer. DCs were unhaptenized DC cells. Homspera co-culture
was performed with a dose of 10-9 mol/L during the preceding 24 hrs before subsequent adoptive transfer.

Figure 4: Agonistic signaling via NK1R prolongs BMDC survival in vivo. (A) Contour-plots illustrating the
percentage of SarSP-DCs or control DCs (CD11c+ CFSE+) in dLNs after their adoptive transference into B6 mice
and quantification by FACS analysis at different time points. Data are representative of 3 independent experiments.
(B) Comparative analysis of the percentages of CD11c+ CFSE+ BMDCs in dLNs after adoptive transference of TN-
DCs, SarSP-TN-DCs, SarSP-DCs, or untreated DCs as quantified by FACS analysis at different time points.
***P .001. Means (+ SD) of 3 independent experiments are displayed.

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Homspera acts independently as well as synergistically with CD40 ligation

This synergy results in up-regulation Bcl-2 expression for as long as 96-hours post exposure. These results indicate
that the prolonged viability of transplanted DCs homed to dLNs was likely caused by a synergistic effect of NK1R
and CD40 signaling.

Figure 5: NK1R and CD40 signaling act synergistically to promote BMDC survival. (A) Bar-diagram
representing the percentage of CD11c+ BMDCs expressing Bcl-2 after culture in medium alone or with SarSP for
different time points. Agonistic CD40 mAb was added at 24 hours to cultures, and the percentage of CD11c +
BMDCs expressing Bcl-2 was analyzed at each time point. *P.05, **P.01, and ***P.001. Means (+ SD) of 3
independent experiments are shown. (B) Contour-plots illustrating the presence of SarSP-TN-DCs or TN-DCs
(CD11c+ CFSE+) in DLNs 5 days after adoptive transference of DCs in B6 mice treated with or without the CD154
(CD40L) neutralizing MR1 mAb. Data are representative of 3 independent experiments.

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Homspera cultured DCs produce enhanced Ag-specific CD4+ T helper function

The data above indicate that the extended survival of stimulated DCs in dLNs likely augments the immune response
through enhanced interaction between DCs and effector T and B cells. One important enhancer of Ag-specific
immunity is CD4+ T helper effect. To evaluate this question researchers evaluated the ability of TN-DCs and SarSP-
TN-DCs to induce DTH responses. Note that Homspera alone does not increase DTH indicating reduced chance of
unexpected immune-mediated side-effects.

Figure 6: Signaling BMDCs via NK1R prolongs effector cellular immune responses. (A) Ear thickness increase
of mice sensitized with adoptively transferred TN-DCs, SarSP-TNDCs, CD40-TN-DCs, or control (nonhaptenized)
SarSP-DCs or untreated DCs, as analyzed at different time points after DTH elicitation. Means (+ SD) of 6 mice per
experimental group are illustrated. ***P < .001

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In vivo effects of Homspera therapy:
Note: Figures in this section were derived from Mathers et. al., Journal of Immunology, 2007, 178:7006–7017.

The reduced AIA of DCs with Homspera therapy results in enhanced therapeutic immune response including
enhanced expression of MHC-II and antigen proteins in gene-gun studies, more efficient trafficking to dLNs,
enhanced TH1 and TH2 responses and elevated CD4 T helper function as determined by DTH response.

Homspera therapy increased expression of antigenic transgene in gene-gun

(GG) studies

Figure 7: GG immunizations with the NK1R agonist triggers skin acute inflammation and increases the
expression of transgene (tg) Ag. (A–D), Cross-sections of skin (abdomen) obtained from mice untreated (A) and
experimental animals 24 h after administration (i.d.) of Homspera (B), immunization with GG (C), or treatment with
GG plus Homspera (D). The arrows indicate the localization of the acute cellular infiltrate composed mainly of
PMN cells. The insets detail the characteristics of the acute inflammatory infiltrate. H&E staining: magnification,
X200; insets, X1000. E and F, Expression of transgene Luc in the skin (E) and sDLNs (F) 24 h following GG
delivery of pCMV-Luc in the presence (or not; control) of Homspera (an NK1R agonist) or the NK1R antagonist.
Data are representative of four independent experiments. E and F, Results are expressed as mean RLU + 1 SD of
the fold increase compared with background levels obtained from nonimmunized control mice. N.D., none detected.

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Homspera therapy increases trafficking of skin Langerhans’ cells (LCs) from
the skin to skin-draining lymph nodes (sDLNs) and alters the physiology of
sDLNs by increasing their vascularity and expression of the DC-homing
molecule CCL21

Figure 8 Effects of skin genetic immunizations with the GG and the NK1R agonist on migration of epidermal
LCs. (A–D), Distribution of DEC-205+ LCs (in red) in epidermal sheets (ear) obtained from nonimmunized control
mice (A) and from animals treated (24 h prior) with the NK1R agonist (i.d.) (B), GG (pCMV-Luc) (C), or GG plus
NK1R agonist (D). The few LCs remaining in the epidermis after treatment became roundshaped (arrows), a feature
indicative of LC migration. Immunofluorescence: magnification, X200. (E), Quantification of the density of DEC-
205+ LCs in epidermal sheets (ear) 24 h post-treatment. Data represent the means + 1 SD from three independent
experiments performed. (F), Comparison of the expression of LYVE-1 (red) and the chemokine CCL21/SLC
(green) in the sDLNs 24 h following the indicated treatments. Immunofluorescence: magnification, X100.

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Homspera induces mixed subtype hypercellularity and the number of DCs in
dLNs and also activates these DCs as shown by increased MHC-II molecules

Figure 9: Homspera enhances the abilities of the GG to promote sDLN inflammation and homing of activated
LCs. (A–D), Structure of sDLNs (inguinal) excised from nontreated control mice (A) and from animals treated (24 h
prior) with: Homspera (i.d.) (B), GG (pCMV-Luc) (C), or GG plus Homspera (D). sDLNs from treated mice show
sinus hyperplasia characterized by the presence of cells with abundant pale cytoplasm (some with morphological
features of DCs indicated by arrows and detailed in the insets) located mainly within the subcortical and paracortical
areas (dotted lines). Additionally, 1-µ m gold beads were detected in the cytoplasm of DCs 24 h after GG
treatments alone or in combination with Homspera (illustrated in insets of C and D and indicated by asterisks). H&E
stain: magnification, X400; insets, X1000. E–H, Identification of the population of LCs co-expressing langerin
(CD207 (green)) and CD11c (red) (arrows) in the paracortical areas of sDLNs (inguinal) excised from
nonimmunized control mice (E) and from animals treated (24 h prior) with Homspera (i.d.) (F), GG (pCMV-Luc)
(G), or GG plus Homspera (H). Insets show CD11c+ langerin+ LCs at higher magnification. Immunofluorescence:
magnification, X200; insets, X1000. (I), Quantification by flow cytometry of CD11c+ CD11b+ MHC-II (I-Ab)high
langerin- epidermal LCs and CD11c+ CD11b+ MHC-II (I-Ab) high langerin+ dermal DCs in the sDLNs (inguinal) of
non-immunized control mice and animals treated (24 h prior) with Homspera (i.d.), GG (pCMV-Luc), or GG plus
Homspera. Data are representative of two independent experiments. (J), Comparative analysis of transgene Luc
expression in sDLNs 24 h after GG delivery of pNFκB-Luc or pCMV-Luc in the presence (or not; control) of
Homspera. Means + 1 SD of the fold increase of RLU compared with background levels are illustrated. Three
independent experiments were performed.

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Homspera® - Cancer Therapy

The net effect of Homspera therapy upon the immune response shows that
Homspera therapy increases antigen-specific antibodies and cytotoxic T cell
number and function as well as Interferon – γ (IFN-γ) expression by antigen-
specific CD4+ and CD8+ cells

Figure 10: Skin a GG immunization in the presence of NK1R signaling enhances IFN-γ secretion by Ag-
specific T cells. (A and B), Detection by ELISA of total OVA-specific serum IgG. (A), OD450 at a 1/2000 serum
dilution; (B), OVA-specific serum IgG titer at an OD450 of 0.2. (C) and (D), Detection by ELISPOT assays of IFN-γ
and IL-5 secreted by CD4+ (C) and CD8+ (D) T cells isolated from spleens of B6 mice left untreated (control) or GG
immunized with pCMV-OVA-TR in the presence (or not; control) of Homspera. Results are expressed as the means
of spot-forming cells + 1 SD of triplicates. Data are representative of three experiments. Insets are representative
images of the IFN-γ ELISPOT wells. (E), Ag-specific cytotoxic in vivo killing assays performed in B6 mice pre-
immunized with GG on the abdominal skin with pCMV-OVA-TR and one boosting in the presence (or not; control)
of the NK1R agonist. Means +1 SD of the specific cell lysis determined by the percentage of splenic cells, CFSEhigh
and CFSElow, analyzed by flow cytometry. * indicates a p < 0.05 compared with GG immunizations alone.

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IR BioSciences Holdings, Inc.
8777 E Via de Ventura, Suite 280
Scottsdale, Arizona 85258
(480) 922-3926

Homspera® - Cancer Therapy

Homspera therapy increased antigen-specific CD4 T-helper response as
demonstrated by dramatically enhanced delayed-type hypersensitivity (DTH)
Importantly, the DTH response with Homspera therapy showed significantly increased F4/80 + macrophages, CD4+
and CD8+ T cells compared with naïve mice.

Figure 11: Administration of the NK1R agonist increases the ability of the GG to promote T cell responses.
(A) Ear thickness of mice immunized with the GG in the presence or not of Homspera and analyzed following the
elicitation of DTH assays. Data are representative of three independent experiments. * indicates a p < 0.05 compared
with the non-sensitized animals (naive). ** indicates p < 0.05 compared with GG immunizations alone at each time
point. B–E, Analysis of the cellular infiltrate in the skin (right ear) from non-sensitized control mice (B), control
animals sensitized with the irrelevant control plasmid encoding no transgene (C), and mice sensitized with the
pCMV-OVA-TR alone (D) or GG in the presence of Homspera (E). The skin was analyzed 72 h after sensitization.
The mononuclear cell infiltrate is detailed in the insets. Epidermis is indicated by arrows and dermis by arrowheads.
Red arrows indicate the thickening of the skin (excluding the hypodermis). H&E staining: magnification, X200;
insets, X1000. F–K, Cellular infiltrate composed of CD4+ and CD8+ T cells (F–I) and F4/80+ macrophages (J and
K) in the presence (G, I, and K) or absence (F, H, and J) of Homspera. Immunofluorescence: magnifications, F, G,
J, and K, X200; H and I, X400. L, Quantification of the cellular infiltrate in tissue sections by microscopy is
expressed as means + 1 SD of cell numbers from at least 20 high-power fields (HPF: magnification, X400).

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IR BioSciences Holdings, Inc.
8777 E Via de Ventura, Suite 280
Scottsdale, Arizona 85258
(480) 922-3926

Homspera® - Cancer Therapy

Protective Anti-melanoma Effect of Homspera with a melanoma vaccine
Collaborators working with ImmuneRegen have generated confirmatory data showing that the changes to the
immune response generated by Homspera is effective at preventing the growth of melanoma in mice vaccinated
against the weak self-antigen TRP2 (Figure 12). Importantly, increased DTH responses seen in the previous studies
correlated with an effective anti-tumor effect in these mice. Animals that received Homspera showed greater DTH
for up to 72hrs and a dramatic slowing of tumor growth by day 22 (Figure 12A). A single vaccination with
Homspera showed dramatically reduced growth and was almost as effective as two vaccinations in preventing
melanoma growth (Figure 12B).

Homspera increases effector cellular immune responses and reduces and

delays the growth of melanoma in genetic vaccines

Figure 12: (A) DTH assays illustrating % of ear thickness increase in mouse previously
immunized with pCMV-TRP2 in the presence or not of SarSP at 21 days post-injection (p<0.001).
(B) Picture of mice treated as in A and sacrificed 22d after i.d. injection of B16 melanoma cells.
Mice were genetically immunized with GG encoding the melanoma self Ag TRP2 in the presence
or in the absence of SarSP or the NK1R antagonist L733060 and injected with B16/F0 malignant
melanoma cells. Control groups included non-immunized mice and mice immunized with
irrelevant transgenic Ag (luciferase).

Not only was primary transplanted melanoma tumor growth significantly reduced in mice treated with Homspera
(Figure 13A) but these mice showed statistically significant increased survival. By day 50 post transplantation all
mice that were naïve, or vaccinated without Homspera were dead while 45-50% of mice receiving Homspera were
still alive (Figure 13B). This survival advantage continued for the duration of the experiment of 100 days implying
that an effective immune response was able to moderate and control the melanoma in these animals. This survival

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IR BioSciences Holdings, Inc.
8777 E Via de Ventura, Suite 280
Scottsdale, Arizona 85258
(480) 922-3926

Homspera® - Cancer Therapy

advantage was seen in animals who received one or two vaccinations with Homspera indicating that Homspera is
highly effective in boosting and anti-cancer immune response in these animals.

Homspera significantly reduces and delays the growth of melanoma, prolongs

mouse survival and induces long lasting immunity

Figure 13: (A) Mice were genetically immunized with GG encoding the melanoma self Ag TRP2
in the presence or in the absence of SarSP. Control groups included non-immunized mice and
mice immunized with irrelevant transgenic Ag (luciferase) and injected with B16/F0 malignant
melanoma cells. Data represents means± 1SE of tumor area (mm2) of 6 mice treated per
experimental group. Both GG TRP2 and GG TRP2 plus NK1R agonist treatments inhibit
significantly tumor growth compared to GG encoding irrelevant Ag and non-immunized mice
injected with B16/F0 cells (**p<0.0001). There was a significant inhibition on the tumor growth
in mice treated with GG pCMV-TRP2 plus NK1R agonist compared to GG TRP2 alone
(*p<0.01). (B) Survival curve of mice genetically immunized as described. An indefinite survival
of 45% and of 50% of mice injected either with one dose or with two doses of SarSP respectively.
Control groups included mice immunized with irrelevant transgenic Ag (luciferase) and non-
immunized mice injected with B16/F0 malignant melanoma cells or injected with the NK1R
agonist in the absence of GG immunization to TRP2. Data represents number of mice that
survived out of 6 mice treated per experimental group. GG-TRP2 plus one or two doses of the
NK1R agonist prolonged significantly the survival of mice compared to GG-TRP2 alone or with
GG encoding irrelevant Ag and non-immunized mice injected with B16/F0 cells (**p<0.0001).
(C) DTH assay of mice surviving from melanoma tumors and challenged with GG delivered

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8777 E Via de Ventura, Suite 280
Scottsdale, Arizona 85258
(480) 922-3926

Homspera® - Cancer Therapy

pCMV-TRP2 in the dorsal side of the ears 100d following the first injection. Surviving mice were
sacrificed 120d after immunizations and remain tumor free.

These results indicate that Homspera is able to prevent the growth of primary and metastatic melanoma in these
animal models. The strong immune modulating properties of Homspera likely mediate the anti-tumor effects
described below.

Anti-Cancer Properties of Homspera

Studies performed by ImmuneRegen Scientists utilized the well established B16-lung metastasis model to
investigate Homspera’s effect upon metastatic melanoma. Tail vein injection of B16 melanoma cells into syngeneic
mice was followed by Homspera treatments for 7 days. This resulted in a 63% reduction of lung metastases assessed
14 days after tumor cell injection [from 105 (S.D. +/-12) to 39 (+/-4), n=4], as well as a 24% increase in survival
(from 25+/-3 days to 31+/-4 days). To determine whether Homspera therapy was effective against established lung
metastases, B16 cells were injected but therapy was not applied until days 7-14 after injection. This nonetheless
resulted in a 97% reduction of lung metastases (from 30 (+/-5) to 1 (+/-0.3), n=6). These studies demonstrated anti-
tumor efficacy even in well-established melanoma metastatic disease.

Additionally, studies by ImmuneRegen show that Human melanoma cell lines A375 and A2058 are not stimulated
by Homspera. Furthermore, chemotherapeutic sensitivity of OVCAR-3 (human ovarian carcinoma), SK-OV-3
(human ovarian adenocarcinoma) and MDA-MD-231 (human breast adenocarcinoma) cell lines is not inhibited by
Homspera exposure. Thus, in oncology one finds an area in which many of Homspera’s actions focus and which
might provide the most exciting area for exploration.
As described above, Homspera has been shown in an accepted metastatic melanoma model to reduce lung
metastases by 63% if administered immediately after a heavy tumor burden (1 x 10 5 cells i.v.) and 97% when fewer
cells were injected (1 x 104) but therapy was delayed for 7 days, enough time to firmly establish lung metastases.

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