Document:

Exhibit
10.3

 

AGREEMENT
BY AND BETWEEN REGEN BIOPHARMA, INC. AND SANTOSH KESARI 

Agreement
by and between Santosh Kesari (“Consultant”) , a natural person whose address is at 3525 Del Mar Heights Road #133,
San Diego CA 92130 and Regen Biopharma, Inc. (“Company”) , a Nevada corporation whose address is 4700 Spring Street,
St 304, La Mesa, California 91942. 

It
is agreed as follows:

1.
SCOPE OF SERVICES 

	 	(a)	Consultant
    shall complete all experiments, procedures, studies and tasks described within that document entitled “ Dr. Kesari Lab-Regen
    BioPharma Collaborative Project-dCellVax FDA Requested Studies” (“Proposal”) attached to this Agreement
    as EXHIBT A and incorporated herein.

 

	 	(b)	Consultant
    shall assist the Company in the preparation of an Investigational New Drug Application (“IND”) to be submitted
    to the United States Food and Drug Administration (“FDA”) with regard to the marketing of the Company’s
    proprietary product “DCellVax” as a treatment for gliomas such a assistance to be provided for a period of no
    less than twelve months from the execution date of this Agreement.

2.
COMPENSATION 

	 	(a)	Consultant
    shall receive that number of common shares of the company, valued as of the closing price on the OTCBB as of the date of execution
    of this Agreement, which shall equal $66,000 USD (“Signing Shares”). In the event that the date of execution of
    this Agreement is not a Trading Day then the Signing Shares shall be valued as of the closing price on the OTCBB as of the
    latest complete Trading Day immediately prior to the execution of this Agreement. “Trading Day” shall mean any
    day on which the common shares of the Company are tradable for any period on the OTBB.

 

	 	(b)	One
    half of the Signing Shares to be issued shall be registered under the Securities Act of 1933 on Form S-8.

 

	 	(c)	Signing
    Shares shall be issued to the Consultant on or prior to the expiration of a two week period following the execution of this
    Agreement.

 

	 	(d)	Upon
    completion of the studies described within Section 3 of the Proposal and successful demonstration of silencing of indolamine
    2,3 deoxygenase in human dendritic cells Consultant shall be entitled to receive that number of common shares of the company,
    valued as of the closing price on the OTCBB as of the date that successful demonstration of silencing is presented to the
    Company by the Consultant (“Milestone Date”) , which shall equal $66,000 USD (“Milestone Shares”).
    In the event that the Milestone Date is not a Trading Day then the Milestone Shares shall be valued as of the closing price
    on the OTCBB as of the latest complete Trading Day immediately prior to the Milestone Date

 

	 	(e)	One
    half of the Milestone Shares to be issued shall be registered under the Securities Act of 1933 on Form S-8.

 

	 	(f)	Milestone
    Shares shall be issued to the Consultant on or prior to the expiration of a two week period following the Milestone Date.

 

	 	(g)	Upon
    the date of submission to the FDA of a response, prepared by the Consultant, providing evidence of vitro and/or in vivo confirmation
    of efficacy of the human siRNA sequences proposed for the clinical trial with regard to IND# 16200 for a proposed Phase I/II
    clinical trial assessing safety with signals of efficacy of the dCellVax gene silenced dendritic cell immunotherapy for treating
    breast cancer ( “Response Date”) Consultant shall be entitled to receive that number of common shares of the company,
    valued as of the closing price on the OTCBB as of the Response Date which shall equal $66,000 USD (“Response Date Shares”).
    In the event that the Response Date is not a Trading Day then the Response Date Shares shall be valued as of the closing price
    on the OTCBB as of the latest complete Trading Day immediately prior to the Response Date.

 

	 	(h)	One
    half of the Response Date Shares to be issued shall be registered under the Securities Act of 1933 on Form S-8.

 

	 	(i)	Response
    Date Shares shall be issued to the Consultant on or prior to the expiration of a two week period following the Response Date.

3.
NON DISCLOSURE

	 	(a)	All
    information, whether in oral, written, graphic, electronic or other form, disclosed by the Company to the Consultant shall
    be deemed to be “Proprietary Information.” In particular, Proprietary Information includes, without limitation,
    any trade secrets, confidential information, ideas, inventions or research and development information; matters of a technical
    nature, including technology; notes, products, know-how, engineering or other data (including test data and data files); specifications,
    processes, techniques, formulae or work-in-process; manufacturing, planning or marketing procedures, clinical data and regulatory
    strategies or information; accounting, financial or pricing procedures or information, budgets or projections, or personnel
    or salary structure/compensation information; information regarding suppliers, clients, customers, employees, contractors,
    investors or investigators of the Company, information which has been designated in writing as confidential by the Company;
    programs, procedures (including operating procedures), processes, methods, guidelines, policies, proposals or contracts; computer
    software, data bases or programming; and any other information which, if divulged to a third party, could have an adverse
    impact on the Company, or on any third party to which it owes a confidentiality obligation. In addition, “Proprietary
    Information” includes any of the foregoing relating to the past, present or future operations, organization, projects,
    finances, business interests, methodology or affairs of any third party to which the Company owes a duty of confidentiality
    including, without limitation, the mere fact that the Company is or may be working with or for any client.

 

	 	(b)	The
    obligations of confidentiality shall not apply to any Proprietary Information that was known by the Consultant at the time
    of disclosure to it by such Company, or that is independently developed or discovered by the Consultant after disclosure by
    such Company, without the aid, application or use of any item of such Company’s Proprietary Information, as evidenced
    by written records; now, or subsequently becomes, through no act or failure to act on the part of the Consultant, generally
    known or available; is disclosed to the Consultant by a third party authorized to disclose it; or is required by law or by
    court or administrative order to be disclosed; provided, that the Consultant shall have first given prompt notice to such
    Company of such required disclosure.

 

	 	(c)	Consultant
    shall exercise due care to prevent the unauthorized use or disclosure of the Company’s Proprietary Information, and
    shall not, without the Company’s prior written consent, disclose or otherwise make available, directly or indirectly,
    any item of the Company’s Proprietary Information to any person or entity other than those employees, independent contractors
    or agents of the Consultant (collectively, “Representatives”), to the extent such Representatives reasonably need
    to know the same in order to evaluate such Proprietary Information, to participate in the business relationship between the
    parties, or to make decisions or render advice in connection therewith. Consultant shall advise its Representatives who have
    access to the Company’s Proprietary Information of the confidential and proprietary nature thereof, and agrees that
    such Representatives shall be bound by terms of confidentiality and restrictions on use with respect thereto that are at least
    as restrictive as the terms of this Agreement.

 

	 	(d)	Consultant
    shall exercise due care to prevent the unauthorized use or disclosure of the Company’s Proprietary Information, and
    shall not, without the Company’s prior written consent, disclose or otherwise make available, directly or indirectly,
    any item of the Company’s Proprietary Information to any person or entity other than those employees, independent contractors
    or agents of the Consultant (collectively, “Representatives”), to the extent such Representatives reasonably need
    to know the same in order to participate in any business relationship between the parties, or to make decisions or render
    advice in connection therewith. Consultant shall advise its Representatives who have access to the Company’s Proprietary
    Information of the confidential and proprietary nature thereof, and agrees that such Representatives shall be bound by terms
    of confidentiality and restrictions on use with respect thereto that are at least as restrictive as the terms of this Agreement.

 

	 	(e)	Consultant
    shall use the Company’s Proprietary Information solely for the purposes of performing his duties pursuant to this Agreement
    and shall not make any other use of the Company’s Proprietary Information without the Company’s specific written
    authorization.

 

	 	(f)	All
    Proprietary Information of the Company (including all copies thereof) shall be and at all times remain the property of such
    Company, and all non-oral Proprietary Information of the Company which is then in the Consultant’s possession or control
    shall be destroyed or returned to the Company promptly upon its request at any time, and in any event, no later than 60 days
    following any expiration or termination of this Agreement.

 

	 	(g)	Nothing
    in this Agreement shall be construed, by implication or otherwise, as a grant of any right or license to trademarks, inventions,
    copyrights or patents, as a grant of a license to either Consultant to use any of the Company’s Proprietary Information
    except as expressly set forth herein.

 

	 	(h)	The
    provisions of Section 3 of this Agreement shall survive until such time as all Confidential Information disclosed hereafter
    becomes publically known and made generally available through no action or inaction of Consultant.

 

4.
REPRESENTATIONS AND WARRANTIES OF COMPANY

 

	 	(a)	Company
    is a corporation duly organized, validly existing and in good standing under the laws of the state its incorporation and has
    the requisite corporate power and authority to enter into and perform its obligations under this Agreement without the consent,
    approval or authorization of, or obligation to notify, any person, entity or governmental agency which consent has not been
    obtained.

 

	 	(b)	The
    execution, delivery and performance of this Agreement by Company does not and shall not constitute Company’s breach
    of any statute or regulation or ordinance of any governmental authority, and shall not conflict with or result in a breach
    of or default under any of the terms, conditions, or provisions of any order, writ, injunction, decree, contract, agreement,
    or instrument to which the Company is a party, or by which Company is or may be bound.

 

5.
REPRESENTATIONS AND WARRANTIES OF CONSULTANT

 

	 	(a)	Consultant
    has the requisite power and authority to enter into and perform his obligations under this Agreement without the consent,
    approval or authorization of, or obligation to notify, any person, entity or governmental agency which consent has not been
    obtained.

 

	 	(b)	The
    execution, delivery and performance of this Agreement by Consultant does not and shall not constitute Consultant’s breach
    of any statute or regulation or ordinance of any governmental authority, and shall not conflict with or result in a breach
    of or default under any of the terms, conditions, or provisions of any order, writ, injunction, decree, contract, agreement,
    or instrument to which the Consultant is a party, or by which Company is or may be bound.

 

6.
RESTRICTED SECURITIES ACKNOWLEDGMENT

Consultant
acknowledges that any securities issued pursuant to this Agreement that shall not be registered pursuant to the Securities Act
of 1933 shall constitute “restricted securities” as that term is defined in Rule 144 promulgated under the Securities
Act of 1933, and shall contain the following restrictive legend:

“THESE
SECURITIES HAVE NOT BEEN REGISTERED UNDER THE SECURITIES ACT OF 1933, AS AMENDED (THE “ACT”), OR SECURITIES LAWS OF
ANY STATE AND MAY NOT BE OFFERED, SOLD, ASSIGNED, PLEDGED, TRANSFERRED OR OTHERWISE DISPOSED OF IN THE ABSENCE OF AN EFFECTIVE
REGISTRATION STATEMENT UNDER THE ACT AND APPLICABLE STATE SECURITIES LAWS OR PURSUANT TO AN AVAILABLE EXEMPTION FROM REGISTRATION
UNDER THE ACT OR SUCH LAWS AND, IF REQUESTED BY THE COMPANY, UPON DELIVERY OF AN OPINION OF COUNSEL REASONABLY SATISFACTORY TO
THE COMPANY THAT THE PROPOSED TRANSFER IS EXEMPT FROM THE ACT OR SUCH LAWS.” 

7.
SPECIFIC PERFORMANCE 

Any
breach of this Agreement may result in irreparable damage to Company for which Company will not have an adequate remedy at law.
Accordingly, in addition to any other remedies and damages available, Consultant acknowledges and agrees that Company may immediately
seek enforcement of this Agreement by means of specific performance or injunction, without any requirement to post a bond or other
security.

8.
EXECUTION 

This
Agreement may be executed in two or more counterparts, all of which when taken together shall be considered one and the same Agreement
and shall become effective when counterparts have been signed by each party and delivered to the other party, it being understood
that both parties need not sign the same counterpart. In the event that any signature is delivered by facsimile transmission,
such signature shall create a valid and binding obligation of the party executing (or on whose behalf such signature is executed)
with the same force and effect as if such facsimile signature page were an original thereof.

 9.
ENTIRE AGREEMENT 

This
Agreement constitutes a final written expression of all the terms of the Agreement between the parties regarding the subject matter
hereof, are a complete and exclusive statement of those terms, and supersedes all prior and contemporaneous Agreements, understandings,
and representations between the parties.

10.
SEVERABILITY 

If
any provision of this Agreement is held to be invalid or unenforceable in any respect, the validity and enforceability of the
remaining terms and provisions of this Agreement shall not in any way be affected or impaired thereby and the parties will attempt
to agree upon a valid and enforceable provision that is a reasonable substitute therefore, and upon so agreeing, shall incorporate
such substitute provision in this Agreement

11.
GOVERNING LAW, VENUE, WAIVER OF JURY TRIAL

All
questions concerning the construction, validity, enforcement and interpretation of this Agreement shall be governed by and construed
and enforced in accordance with the internal laws of the State of California, without regard to the principles of conflicts of
law thereof. Each party hereby irrevocably submits to the exclusive jurisdiction of the state and federal courts sitting in California
for the adjudication of any dispute hereunder or in connection herewith or with any transaction contemplated hereby or discussed
herein and hereby irrevocably waives, and agrees not to assert in any suit, action or proceeding, any claim that it is not personally
subject to the jurisdiction of any such court, that such suit, action or proceeding is improper or inconvenient venue for such
proceeding. If either party shall commence an action or proceeding to enforce any provisions of this Agreement, then the prevailing
party in such action or proceeding shall be reimbursed by the other party for its attorneys’ fees and other costs and expenses
incurred with the investigation, preparation and prosecution of such action or proceeding.

IN
WITNESS WHEREOF, the parties hereto have caused this Agreement to be duly executed by their respective authorized signatories
as of the date first indicated above. 

	Company 	Consultant
	By:/s/
    David R. Koos 	By:/s/Santosh
    Kesari 
	David
    R. Koos	Dr.
    Satosh Kesari
	Chairman
    & CEO 	 
	Regen
    Biopharma, Inc. 	 
	Date:
    June 8, 2015	Date:
    May 13, 2015 

 

	 

 

EXHIBIT
A

 

Dr.
Kesari Lab-Regen BioPharma Collaborative Project

 

 

 

 

dCellVax
FDA Requested Studies

 

 

 

April
2015

 

 

 

 

Background

Regen
BioPharma has purchased a US patent and licensed Benitec Biopharma’s ddRNAi technology for the field of use of inhibiting
indolamine 2,3 deoxygenase (IDO) in dendritic cells (DC) for the purpose of immune stimulation in cancer patients.

Through
silencing of murine IDO in DC, Regen scientist Dr. Wei-Ping Min demonstrated inhibition of melanoma and breast cancer in the B16[1]
and 4T1 models[2], respectively.

Based
on these preclinical data, Regen BioPharma submitted an IND for treatment of breast cancer in patients that have failed conventional
therapy. The FDA responded to this IND requesting “in vitro and/or in vivo confirmation of efficacy of the human siRNA sequences
proposed for the clinical trial” before the clinical trial can be initiated.

The
experiments proposed below are designed to demonstrate in vitro efficacy of human IDO siRNA in the human DC in vitro model. Upon
completion of these experiments, the results will be submitte to the FDA for allowance to initiate clinical trials.

Objectives

	 	•	Development
    and optimization of sensitive molecular methods (reverse transcriptase polymerase chain reaction (RT-PCR)) for detecting the
    IDO gene, initially in human cancer cells[3], which then will be transferred to human dendritic cells

 

	 	•	Development
    and optimization of short interfering RNA (siRNA) sequences for effective silencing of IDO expression in cancer cells.

 

	 	•	Application
    of IDO detection and silencing methodology to human DC (in vitro dCellVax).

 

	 	•	Demonstration
    of in vitro augmentation of immunogenicity by IDO-silenced DC as compared to control DC

 

	 	o	Mixed
    lymphocyte reaction

 

	 	o	Cytokine
    Production (IL-4, IL-10, IL-12, IFN-gamma)

 

	 	o	Increased
    Tryptophan, decreased kyneurinine in culture media

Rationale

Although
the main role of the immune system is to protect the body from foreign pathogens, the immune system also requires a “regulatory”
arm in order to prevent immune responses from destroying not only the pathogen, but also the body. Examples of unrestrained immune
responses include autoimmune conditions, in which the immune system starts attacking the pancreas (Type 1 Diabetes), the nervous
system (multiple sclerosis) or collagen protein in the joints (rheumatoid arthritis). A more severe example of unrestrained immune
response is sepsis, where the immune activation against blood borne infections results in death of the patient. The immune system
possesses regulatory cells, whose role in the body is to restrain immunity. One specific type of regulatory cell is the immature
DC. While mature DC are known to be one of the most potent activators of immunity, in the immature state, DC actively suppress
T cell activation. Immature dendritic cells are found in pregnancy to protect the fetus from maternal immune attack against proteins
found on the fetus that are inherited from the father[4]. Immature DC are also found in tumors, and contribute to tumor
escape from the immune system[5]. Other conditions associated with immune regulation such as viral infections[6],
bacterial infections[7], and remission from autoimmune diseases[8] are associated with immature DC.

One
of the main molecular mechanisms by which immature DC suppress the immune system is through high expression of the enzyme indolamine
2,3 deoxygenase (IDO). IDO was originally discovered in 1967 in the rabbit intestine and has been the object of renewed attention
by immunologists in view of its capacity to act as an inducible negative regulator of T cell viability, proliferation, and activation
during inflammation. In addition to potential in direct effects by IDO on APC function, IDO has been proposed to suppress T cells
by degrading tryptophan and increasing the level of tryptophan degradation products (kynureneria and quinolinate). Both of these
activities suppress T cell response by inducing T cell apoptosis.

It
has been shown that immune suppression in cancer[9], pregnancy[10], and viral infection[11] can
be overcome by chemical inhibition of IDO. Unfortunately, chemical inhibitors of IDO, to date, do not possess specificity towards
IDO, and are associated with various side effects[12]. Additionally, administration of chemical inhibitors is not selective
to immune cells, given that IDO possesses numerous functions, systemic administration of inhibitors is not likely to induce sufficient
suppression of the enzyme where it is needed, in the tumor associated DC.

Induction
of RNA interference (RNAi) by administration of siRNA represents a highly specific and efficient mechanism of blocking gene expression.
As an endogenous viral defense mechanism, RNA interference (RNAi) uses a specific subset of enzymes that can bind and cleave homologous
transcripts within mammalian and plant cells. Dr. Wei-Ping Min from the University of Western Ontario has previous used siRNA
to silence immune-associated genes for the purpose of inducing immunomodulation in a publication with Dr. David Koos CEO of Regen
BioPharma[13]. Whereas in the mentioned publication the gene for IL-12 was silenced in DC in order to inhibit immunity,
Regen BioPharma believes that silencing of IDO in DC will stimulate cancer immunity.

Goals 

	 	1.	Establishment
    of assay system to detect IDO gene expression in human cancer cell lines

 

Cancer
cell lines such as HeLa (cervical)[14] and MDA-MB-231 (breast)[15] have been published to express human
IDO. RT-PCR methodology methodology with primers specific to human IDO in these cell lines. This methodology will allow for detection
of the IDO gene. It is important to qualitatively establish detection of this gene so that we can quantify the level of gene silencing
that is achieved by the siRNA sequences.

 

	 	2.	Identification
    of novel human siRNA sequences silencing IDO

 

Sequences
will be developed using bioinformatics algorithms, as well as based on prior literature. Approximately 10-20 sequences will be
utilized to identify optimal silencing effect on cancer cell lines. (The use of cancer cell lines is to safe costs on given
optimizing the detection and efficacy of silencing, dendritic cells are not ideal for screening experiments due to difficulty
in expanding them).

 

	 	3.	Application
    of optimized sequences to human DC

 

Once
ideal sequences for detection of IDO and silencing elucidated, they will be applied to human DC. Gene silencing efficacy will
be assessed by RT-PCR and immune stimulating ability will be assessed by ability to induce proliferation of allogeneic lymphocytes.
This will be the human preclinical data that goes into the IND package.

 

Proposed
Protocols

 

Stage
1 of Project: Quantification of IDO mRNA Detection

MDA-MB-231
human breast cancer cells will be obtained from American Type Tissue Culture (ATCC: Manassas, VA) and grown under fully humidified
5% CO2 environment with DMEM supplemented with 10% FBS, 2% sodium pyruvate, non-essential amino acids (2 mM), penicillin (100
units/ml), streptomycin (100 μg/ml), and glutamine (4 mM) (Gibco-BRL). HeLa cells will be grown under the same conditions
with the exception that MEM will be used as the base media. Cells are passaged by trypsinization twice weekly or as needed based
on 75% confluency. For some experiments IDO induction will be achieved by pretreatment with interferon gamma at a concentration
of 1 IU/ml.

Total
RNA will be isolated using the RNeasy Mini Kit (QIAGEN). Specifically, cells will be trypsinized and harvested at a concentration
of 5-10 x 106 cells, as a cell pellet after washing in PBS an appropriate volume of Buffer RLT Plus will be added and the cells
will be vortexted for 30 seconds. This will result in lysis of the cells, with the lysate then being spun at 3 minutes at 15000g.
The supernatant is then removed and applied to a gDNA Eliminator spin column which is then placed in a 2 ml collection tube. Subsequently,
the collected material is centrifuged for 30 s at ≥8000 x g (≥10,000 rpm). The column is discarded and the flow-through
is saved. The same volume (usually 350 μl or 600 μl) of 70% ethanol is added to the flow-through that has been collected.
Up to 700 μl of the sample, including any precipitate, is then added to an RNeasy spin column placed in a 2 ml collection tube
and the tube is centrifuged for 15 s at ≥8000 x g. The flow-through is discarded. 700 μl of Buffer RW1 is then added
to the RNeasy Mini spin column (in a 2 ml collection tube) and centrifuged for 15 s at ≥8000 x g. 500 μl of Buffer
RPE is added to the RNeasy spin column and centrifuged for 15 s at ≥8000 x g. Subsequently 500 μl of Buffer RPE is
added to the RNeasy spin column and centrifuged for 2 min at ≥8000 x g (≥10,000 rpm). The RNeasy spin column will
then be placed in a new 1.5 ml collection tube. Approximately 30–50 μl RNase-free water is added directly to the spin
column membrane and centrifuged for 1 min at ≥8000 x g to elute the RNA. Reverse transcription performed using Moloney
murine leukemia virus reverse transcriptase (Promega) following the manufacturer's instructions. Reverse-transcribed products
will be analyzed on a Mastercyler Ep Realplex (Eppendorf) using the QuantiFast SYBR Green PCR Kit (QIAGEN) according to the manufacturer's
instructions.

As
gene-specific primers, the following oligo-DNAs will be assessed

	 	a)	Forward
    (5′-3′): IDO, GGTCATGGAGATGTCCGTAAGGT

 

	 	b)	Forward
    5′-GGAAATAGCAGCTGCTTCTGCA-3′; IDO reverse, 5′-CTCCTCAGGGAGACCAGAGCTT3′

 

	 	c)	Forward
    5′-tgccaaatccacaggaaaat-3′, reverse: 5′-gtttgccaagacacagtctg-3′;

 

	 	d)	Forward
    5′-caaatccacgatcatgtgaacc-3′, reverse: 5′-agaacccttcatacaccagac-3′,

 

	 	e)	Forward
    5′-GGAAATAGCAGCTGCTTCTGCA-3′; reverse 5′-CTCCTCAGGGAGACCAGAGCTT-3′

 

	 	f)	Forward
    5’ TTCAGTGCTTTGACGTCCTG; reverse 5’ TGGAGGAACTGAGCAGCAT 

As
an internal control β-actin mRNA was also amplified using the following primers: β-actin forward, 5′-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3′;
β-actin reverse, 5′-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3

PCR
products will be size-separated on a 1.5% agarose gel; expression levels
normalized to the GAPDH mRNA product, and will be visualized by SYBR Safe DNA gel staining (Invitrogen).

A
minimum of 3 independent experiments will be performed per sequence assessed. If inducible expression is not observed, sequences
will be modified using internal algorithms, as well as variations in cycle number and annealing/reannealing temperatures.

Anticipated
Results: Sequences from the proposed sequences will possess varying ability to detect mRNA. This will be used as the test system
for human IDO expression. We anticipate significantly higher expression of IDO mRNA in cells that have been pretreated for 48
hours with Interferon Gamma, based on previous publications[16].

Stage
2 of Project: Identification of novel human siRNA sequences silencing IDO.

HeLa
and MDA cells either growing in stable conditions or pretreated with 1 IU/ml of interferon gamma for 48 hours under conditions
identified in stage 1 of the project to induce maximal expression of the IDO gene.

Various
siRNA sequences will be tested that have been previously shown to inhibit IDO or have been generated based on gene-specific algorithms.
To prepare the modified siRNA duplexes, complementary strands were mixed at equal concentrations, then heated at 70° for 1
min and allowed to anneal at 37° for 30 min. Successful annealing will be assessed with polyacrlamide gel electrophoresis.

Specific
sequences tested will include:

sense
strand

	5′-TAATACGACTCACTATAGCCGTGAGTTTGTCCTTTCAA-3′
	5′-TTGAAAGGACAAACTCACGGCTATAGTGAGACGTATTA-3′
	5′-UCACCAAAUCCACGAUCAUUU-3’
	5′-UUUCAGUGUUCUUCGCAUAUU-3’
	5′-GUAUGAAGGGUUCUGGGAAUU-3′
	5′-GAACGGGACACUUUGCUAAUU-3′

DNA
oligonucleotides for the anti-sense strand

	5′-TAATACGACTCACTATAGAAAGGACAAACTCACGGACT-3′
	5′-AGTCCGTGAGTTTGTCCTTTCTATAGTGAGACGTATTA-3′
	5′-PUAUGCGAAGAACACUGAAAUU-3′
	5′-PUAUGCGAAGAACACUGAAAUU-3′
	5′-PUUCCCAGAACCCUUCAUACUU-3′
	5′-PUUAGCAAAGUGUCCCGUUCUU-3′

Cells
will be transfected using the Amaxa Nucleofector Kit V (Amaxa biosystems, Koeln, Germany). Briefly, 3 * 105 cells
were resuspended in 100 μl of the nucleofector solution V and mixed with 1.5 μg of siRNA, then electroporated using
program V005. Alternatively, lipofectamine based transfection may be utilized depending on efficacy.

Suppression
of IDO gene expression will be assessed using the method identified as most sensitive from Project Stage 1.

Anticipated
Results: These experiments will provide ideal siRNA sequences for silencing of the IDO gene. These sequences will be utilized
as the basis of silencing IDO in DC in the next Stage of the project.

Stage
3 of Project: Application of optimized sequences to human DC.

DC
will be generated from peripheral blood of healthy volunteers. Peripheral blood mononuclear cells (PBMC) will be purified from
heparinized blood on lymphoprep cushions (LSM; Organon Teknilra Corp., Rockville, MD) and resuspended in RPMI-10% FCS, and allowed
to adhere to 6-well plates (Costar Corp., Cambridge, MA). After 2 h incubation at 37 Celsius, the nonadherent cells will be removed
and the adherent cells will subsequently be washed in phosphate buffered saline (PBS), followed by detachment by incubation with
Mg 2+ and Ca 2+ free PBS containing 0.5 mM EDTA at 37 Celsius. The adherent fraction is subsequently cultured at 3 x 10(6)/ml
in RPMI-10% FCS supplemented with 50 ng/ml GM-CSF and 1,000 U/ml IL-4. Media is changed every 2 days for a total of 8 days culture.
DC will be isolated by positive selection for CD83 and subsequently exposed to IFN-gamma on day 6 of culture. The siRNA sequences
identified in Stage 2 of the project will be utilized. IDO gene expression will be assessed by RT-PCR using primers identified
in Stage 1 of the project.

Successfully
silenced DC will be assessed for immunological responsiveness by ability to stimulate mixed lymphocyte reaction

Dendritic
Cell Isolation from Human Blood – Adherent Method

Materials:

	Reagent	Vendor	Cat#	Amount	Price
	Recombinant
    Human IL-4 CF	RND
    Systems	204-IL-010/CF	1
    mg	$4,300
    
	 	 	 	10
    μg	$205
    
	 	 	 	50
    μg	$679
    
	Recombinant
    Human GM-CSF	RND
    Systems	215-GM-010/CF	10
    μg	$219
    
	 	 	 	50
    μg	$799
    
	RPMI
    1640 with L glutmine and 25mM HEPES buffer	Life
    Technologies	22400-105	10x
    500ml	$271
    
	RPMI
    1640 with L glutamine	Life
    Technologies	11875-119	10x
    500ml	$188
    
	HEPES
    Buffer	Life
    Technologies	15630-106	20
    ml	$18.50
    
	Beta-mercaptoethanol	Sigma	M3148-25ML	25
    ml	$15.30
    
	6
    well plates	Fisher
    Scientific	08-772-1B	50
    plates	$118.03
    
	50
    ml Centrifuge Tube	Fisher
    Scientific	1443222	500
    tubes	$333.65
    
	Penicillin-Streptomycin
    (100x)	Life
    Technologies	15140-122	100
    ml	$18.91
    
	Human
    Albumin Solution 25%	Gemeni
    Bio Products	800-120	50
    ml	$95
    
	Human
    Serum AB (Heat Inactivated)	Gemeni
    Bio Products	100-512	100
    ml	$159
    
	Trypan
    BLue 0.4%	Sigma	T8154-20ML	20
    ml	$5

 

Reagents
to Prepare: 

IL-4
Reconstitution:

Reconstitute
in PBS with 8% Human Albumin Solution to a final concentration of 20μg/ml.

(Example:
We reconstituted 10μg of IL-4 in 500μL of solution)

GM-CSF
Reconstitution:

Reconstitute
in PBS with 8% Human Albumin Solution to a final concentration of 20μg/ml.

(Example:
We reconstituted 10μg of IL-4 in 500μL of solution)

Dendritic
Cell Culture Media (keep at 37C for culture):

RPMI
1640 w/ L-glutamine

25mM
HEPES

1%
Pen Strep

5%
Human Serum AB

50
μM BME (optional)

 

Before
culture make sure to filter with a 0.22μm filter to remove bacterial contaminants

200ml
PBS (1x) – for washing platelets out of supernatant following ficoll separation

Procedure:

	 	1.	Obtain 10ml
    of blood sample

 

	 	2.	Dilute 1:1
    in PBS warmed to RT

 

	 	3.	Place remaining
    ~180ml PBS on ice

 

	 	4.	Underlay
    10ml Ficoll

 

	 	a.	Slow down
    pipette aid to minimize disruptance to interphase layer.

 

	 	b.	Be careful
    of air bubbles that could disrupt layer

 

	 	5.	Carefully
    transfer tube into centrifuge being careful not to disrupt interphase layer

 

	 	a.	Spin at
    2000RPM, 20min, no brakes @RT

 

	 	b.	After spin
    is completed set temperature on centrifuge to 4C

 

	 	6.	Remove interphase
    layer and transfer to new 50ml conical

 

	 	a.	Optional
    tips: removing the upper layer (containing PRP) for easier access to interphase layer – this allows you to use a P1000
    pipette instead of a pipette aid for greater accuracy

 

	 	7.	Add
    PBS (on ice or 4C) to solution containing interphase layer to bring the total volume to 50ml

 

	 	8.	Centrifuge
    at 1500RPM, 10min, brakes on @4C

 

	 	9.	Inspect
    clarity of supernatant, repeat wash cycles (900RPM, 10min, no brakes, @4C) until supernatant is clear (typically 3 wash cycles
    completed)

 

	 	10.	Aspirate
    supernatant and resuspend in 1ml DC Culture Media at 4C

 

	 	11.	Take
    10μL of cell suspension and count using a hemocytometer to determine the number of wells necessary for culture (approximately
    2 million mononuclear cells per well)

 

	 	a.	Add trypan
    blue to check cell viability

 

	 	12.	Plate 6
    million cells per well with 3mL warmed DC media per well

 

	 	13.	Place 6
    well plate in incubator at 37C, 5% CO2 for 1 hour

 

	 	14.	After
    hour incubation is complete, aspirate supernatant leaving behind adherent cells, wash 4 times with warm PBS

 

	 	15.	Replace
    with fresh DC Culture Media with 3ml per well

 

	 	16.	IL-4 concentration

 

	 	a.	Desired
    final concentration 10ng/ml

 

	 	b.	Therefore
    given a stock solution of 20μg/ml, alloquat 3μL per well

 

	 	17.	GM-CSF concentration

 

	 	a.	Desired
    final concentration 50ng/ml

 

	 	b.	Therefore
    given a stock solution of 20μg/ml, alloquat 15μL per well

 

	 	18.	Place 6
    well plate back in incubator at 37C with 5% CO2

 

	 	19.	On day 2
    after isolation procedure, replace cytokines

 

	 	20.	On day 4
    after isolation procedure, replace cytokines

 

	 	21.	On 5th
    day, add maturation signal for 2 days

 

	 	a.	10 ng/ml
    Lipopolysaccharides for 24 hours

 

[1]
Zheng et al. J Immunol. 2006 Oct 15;177(8):5639-46.

[2]
Zheng et al. Int J Cancer. 2013 Feb 15;132(4):967-77.

[3]
Cancer cell lines are initially used to allow for rapid screening without the need for continual supply of blood

[4]
Bartmann et al. Am J Reprod Immunol. 2014 Feb;71(2):109-19.

[5]
Klatka et al. Oncol Rep. 2012 Jul;28(1):207-17

[6]
Jin et al. J Virol. 2009 May;83(10):4984-94.

[7]
Latchumanan et al. Tuberculosis (Edinb). 2005 Sep-Nov;85(5-6):377-83.

[8]
Pellegrini et al. Neuroimmunomodulation. 2005;12(4):220-34.

[9]
Chen et al. J Immunol. 2008 Oct 15;181(8):5396-404.

[10]
Munn et al. Science. 1998 Aug 21;281(5380):1191-3.

[11]
Fox et al. J Gen Virol. 2013 Jul;94(Pt 7):1451-61

[12]
Serbecic et al. Exp Res. 2006 Mar;82(3):416-26.

[13]
Li et al. J Transl Med. 2012 Jan 31;10:19.

[14]
Beatty et al. Infect Immun. 1994 Sep;62(9):3705-11.

[15]
Basu et al. J Immunol. 2006 Aug 15;177(4):2391-402.

[16]
Gu et al. Cancer Res. 2010 Jan 1;70(1):129-38.nrom_ex1010.htm

 

 

  

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