PI: Wagner, David H Title: Developing a small peptide to control autoimmune inflammation in type 1 

diabetes

Received: 09/02/2016 FOA: PA16-303 Council: 01/2017

Competition ID: FORMS-D FOA Title: PHS 2016-02 OMNIBUS SOLICITATION OF THE NIH FOR SMALL 

BUSINESS TECHNOLOGY TRANSFER GRANT APPLICATIONS (PARENT STTR 

[R41/R42])

1 R41 AI131784-01 Dual: DK,HD Accession Number: 3965986

IPF: 10020801 Organization: OP-T-MUNE, INC.

Former Number: Department:

IRG/SRG: ZRG1 EMNR-W (10)B AIDS: N Expedited: N

Subtotal Direct Costs 

(excludes consortium F&A) 

Year 1:

Animals: Y 

Humans: N 

Clinical Trial: N

Current HS Code: 10

HESC: N

New Investigator: N

Early Stage Investigator: N

Senior/Key Personnel: Organization: Role Category:

David Wagner Op-T-Mune, Inc. PD/PI 

Martin Yussman Op-T-Mune, Inc Other (Specify)-Medical Advisor

Always follow your funding opportunity's instructions for application format. Although this application

demonstrates good grantsmanship, time has passed since the grantee applied. The samples may not 

reflect the latest format or rules. NIAID posts new samples periodically:

https://www.niaid.nih.gov/grants-contracts/sample-applications

The text of the application is copyrighted. The awardee provided express permission for NIAID to post

this grant application and summary statement for educational purposes. The awardee allows you to use

the material (e.g., data, writing, graphics) they shared in the applications for nonprofit educational 

purposes only, provided the material remains unchanged and the principal investigators, awardee 

organizations, and NIH NIAID are credited.

Freedom of Information Act (FOIA). NIAID is strongly committed to protecting the integrity and

confidentiality of the peer review process. When NIH responds to FOIA requests for grant applications and

summary statements, the material will be subject to FOIA exemptions and include substantial redactions. 

NIH must protect all confidential commercial or financial information, reviewer comments and

deliberations, and personal privacy information.

Note on Section 508 Conformance and Accessibility. We have reformatted this sample to improve

accessibility for people with disabilities and users of assistive technology. If you have trouble accessing the 

content, contact the NIAID Office of Knowledge and Educational Resources at deaweb@niaid.nih.gov.
OMB Number: 4040-0001

Expiration Date: 06/30/2016

Tracking Number: GRANT12240201 Funding Opportunity Number: PA-16-303 . Received Date:

2016-09-02T13:28:53.000-04:00

APPLICATION FOR FEDERAL ASSISTANCE

SF 424 (R&R)

3. DATE RECEIVED BY STATE State Application Identifier

1. TYPE OF SUBMISSION* 4.a. Federal Identifier

❍ Pre-application ● Application ❍ Changed/Corrected

Application

b. Agency Routing Number

2. DATE SUBMITTED Application Identifier c. Previous Grants.gov Tracking Number

5. APPLICANT INFORMATION Organizational DUNS*: 016344134

Legal Name*: Op-T-Mune, Inc.

Department:

Division:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*: 

ZIP / Postal Code*: -

Person to be contacted on matters involving this application

Prefix: Dr. First Name*: David Middle Name: Last Name*: Wagner Suffix: 

Position/Title: Chief Scientific Officer

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*: STATES

ZIP / Postal Code*: -

Phone Number*: Fax Number: Email: 

6. EMPLOYER IDENTIFICATION NUMBER (EIN) or (TIN)*

7. TYPE OF APPLICANT* R: Small Business

Other (Specify):

Small Business Organization Type ❍ Women Owned ❍ Socially and Economically Disadvantaged

8. TYPE OF APPLICATION* If Revision, mark appropriate box(es).

● New ❍ Resubmission ❍ A. Increase Award ❍ B. Decrease Award ❍ C. Increase Duration

❍ Renewal ❍ Continuation ❍ Revision ❍ D. Decrease Duration ❍ E. Other (specify) :

Is this application being submitted to other agencies?* ❍Yes ●No What other Agencies?

9. NAME OF FEDERAL AGENCY*

National Institutes of Health

10. CATALOG OF FEDERAL DOMESTIC ASSISTANCE NUMBER

TITLE:

11. DESCRIPTIVE TITLE OF APPLICANT'S PROJECT*

Developing a small peptide to control autoimmune inflammation in type 1 diabetes

12. PROPOSED PROJECT

Start Date* Ending Date*

04/01/2017 03/31/2018

13. CONGRESSIONAL DISTRICTS OF APPLICANT

CO-001
Contact PD/PI: Wagner, David

Tracking Number: GRANT12240201 Funding Opportunity Number: PA-16-303 . Received Date:

2016-09-02T13:28:53.000-04:00

SF 424 (R&R) APPLICATION FOR FEDERAL ASSISTANCE Page 2

14. PROJECT DIRECTOR/PRINCIPAL INVESTIGATOR CONTACT INFORMATION

Prefix: Dr. First Name*: David Middle Name: Last Name*: Wagner Suffix: 

Position/Title: Chief Scientific Officer

Organization Name*: Op-T-Mune, Inc.

Department:

Division:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*: 

ZIP / Postal Code*: -

Phone Number*: Fax Number: Email*: 

15. ESTIMATED PROJECT FUNDING

a. Total Federal Funds Requested* $

b. Total Non-Federal Funds* $0.00

c. Total Federal & Non-Federal Funds* $

d. Estimated Program Income* $0.00

16. IS APPLICATION SUBJECT TO REVIEW BY STATE

EXECUTIVE ORDER 12372 PROCESS?*

a. YES ❍ THIS PREAPPLICATION/APPLICATION WAS MADE

AVAILABLE TO THE STATE EXECUTIVE ORDER 12372

PROCESS FOR REVIEW ON:

DATE:

b. NO ● PROGRAM IS NOT COVERED BY E.O. 12372; OR

❍ PROGRAM HAS NOT BEEN SELECTED BY STATE FOR

REVIEW

17. By signing this application, I certify (1) to the statements contained in the list of certifications* and (2) that the statements herein

are true, complete and accurate to the best of my knowledge. I also provide the required assurances * and agree to comply with

any resulting terms if I accept an award. I am aware that any false, fictitious, or fraudulent statements or claims may subject me to

criminal, civil, or administrative penalties. (U.S. Code, Title 18, Section 1001)

● I agree*

* The list of certifications and assurances, or an Internet site where you may obtain this list, is contained in the announcement or agency specific instructions.

18. SFLLL or OTHER EXPLANATORY DOCUMENTATION File Name: 

19. AUTHORIZED REPRESENTATIVE

Prefix: Dr. First Name*: David Middle Name: Last Name*: Wagner Suffix: 

Position/Title*: Chief Scientific Officer

Organization Name*: Op-T-Mune, Inc.

Department:

Division:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*: 

ZIP / Postal Code*: -

Phone Number*: Fax Number: Email*: 

Signature of Authorized Representative*

David Wagner

Date Signed*

09/02/2016

20. PRE-APPLICATION File Name: Mime Type:

21. COVER LETTER ATTACHMENT File Name:1235-CoverSTTRDiabetes.pdf Mime Type: application/pdf
424 R&R and PHS-398 Specific

Table Of Contents Page Numbers

SF 424 R&R Cover Page----------------------------------------------------------------------------------------- 1

 Table of Contents------------------------------------------------------------------------- 3

Performance Sites--------------------------------------------------------------------------------------------- 4

Research & Related Other Project Information------------------------------------------------------------------ 5

 Project Summary/Abstract(Description)----------------------------------------------------- 6

 Project Narrative------------------------------------------------------------------------- 7

 Facilities & Other Resources-------------------------------------------------------------- 8

 Equipment--------------------------------------------------------------------------------- 9

 Other Attachments------------------------------------------------------------------------- 10

 1241-SBC_000405651---------------------------------------------------- 10

Research & Related Senior/Key Person-------------------------------------------------------------------------- 11

Research & Related Budget Year - 1---------------------------------------------------------------------------- 20

Budget Justification------------------------------------------------------------------------------------------ 23

Research & Related Cumulative Budget-------------------------------------------------------------------------- 24

Research & Related Budget - Consortium Budget (Subaward 1)---------------------------------------------------- 25

Total Direct Costs Less Consortium F&A------------------------------------------------------------------------ 30

SBIR STTR Information----------------------------------------------------------------------------------------- 31

PHS398 Cover Page Supplement---------------------------------------------------------------------------------- 33

PHS 398 Research Plan----------------------------------------------------------------------------------------- 35

 Specific Aims----------------------------------------------------------------------------- 36

 Research Strategy------------------------------------------------------------------------- 37

 Vertebrate Animals------------------------------------------------------------------------ 42

 Select Agent Research--------------------------------------------------------------------- 43

 Bibliography & References Cited----------------------------------------------------------- 44

 Resource Sharing Plan(s)------------------------------------------------------------------ 46

Contact PD/PI: Wagner, David

Table of Contents Page 3
Contact PD/PI: Wagner, David

OMB Number: 4040-0010

Expiration Date: 06/30/2016

Tracking Number: GRANT12240201 Funding Opportunity Number: PA-16-303. Received Date:

2016-09-02T13:28:53.000-04:00

Project/Performance Site Location(s)

Project/Performance Site Primary Location ❍ I am submitting an application as an individual, and not on behalf of

a company, state, local or tribal government, academia, or other type of

organization.

Organization Name: Op-T-Mune,Inc.

Duns Number:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*: 

Zip / Postal Code*: -

Project/Performance Site Congressional District*: CO-001

Project/Performance Site Location 1 ❍ I am submitting an application as an individual, and not on behalf of

a company, state, local or tribal government, academia, or other type of

organization.

Organization Name: University of Colorado Denver, Anschutz

Medical Campus

DUNS Number:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*: 

Zip / Postal Code*: -

Project/Performance Site Congressional District*: CO-006

File Name Mime Type

Additional Location(s)

 Page 4
Tracking Number: GRANT12240201 Funding Opportunity Number: PA-16-303. Received Date:

2016-09-02T13:28:53.000-04:00

OMB Number: 4040-0001

Expiration Date: 06/30/2016

RESEARCH & RELATED Other Project Information

1. Are Human Subjects Involved?* ❍ Yes ● No

1.a. If YES to Human Subjects

 Is the Project Exempt from Federal regulations? ❍ Yes ❍ No

If YES, check appropriate exemption number: 1 2 3 4 5 6

If NO, is the IRB review Pending? ❍ Yes ❍ No

IRB Approval Date:

Human Subject Assurance Number

2. Are Vertebrate Animals Used?* ● Yes ❍ No

2.a. If YES to Vertebrate Animals

Is the IACUC review Pending? ❍ Yes ● No

IACUC Approval Date: 10-04-2015

Animal Welfare Assurance Number A3269-01

3. Is proprietary/privileged information included in the application?* ● Yes ❍ No

4.a. Does this project have an actual or potential impact - positive or negative - on the environment?* ❍ Yes ● No

4.b. If yes, please explain:

4.c. If this project has an actual or potential impact on the environment, has an exemption been authorized or an

environmental assessment (EA) or environmental impact statement (EIS) been performed?

❍ Yes ❍ No

4.d. If yes, please explain:

5. Is the research performance site designated, or eligible to be designated, as a historic place?* ❍ Yes ● No

5.a. If yes, please explain:

6. Does this project involve activities outside the United States or partnership with international

collaborators?*

❍ Yes ● No

6.a. If yes, identify countries:

6.b. Optional Explanation:

Filename

7. Project Summary/Abstract* 1236-Abstract_Diabetes_STTR.pdf

8. Project Narrative* 1237-Narrative_STTR.pdf

9. Bibliography & References Cited 1238-REFS_STTR_2016.pdf

10.Facilities & Other Resources 1239-Facilities_OpTMune.pdf

11.Equipment 1240-Equipment.pdf

12. Other Attachments 1241-SBC_000405651.pdf

Contact PD/PI: Wagner, David

 Page 5
Abstract: Type 1 Diabetes (T1D) affects an ever growing population. While this disease 

typically has been associated with juveniles, the disease in adult populations is rapidly 

increasing. The defining clinical component is insulin loss, which occurs because of sustained 

inflammation in the islets. At present there is no means to prevent or reverse insulin loss. A 

major inflammatory pathway in T1D that contributes to insulin loss is the CD40 – CD154 dyad. 

CD40 is expressed on a wide array of cells and when engaged by CD154 creates localized 

inflammation. This pathway is decisive in T1D; blocking the interaction prevents diabetes onset 

and reverses hyperglycemia in new onset diabetic mice. A major impediment to drug 

development in diabetes has been the failure of therapeutics to translate from mouse to human. 

Mindful of this, we discovered that CD40 provides a link between mouse and human during 

T1D. We discovered that NOD mice, the industry standard model for T1D, increase CD40 

expression, including on a sub population of T cells during diabetes development. Those cells, 

termed Th40, not only expand in number as diabetes develops but Th40 cells are singularly 

capable of transferring T1D to scid recipients. In a translational approach, we discovered that 

Th40 cells become prominent in human T1D patients, regardless of the age, HLA haplotype, 

auto-antibody status, or duration of disease. Like in the mouse model, Th40 cells start at low 

percentages but increase as human subjects progress to T1D and remain at high levels even up 

to 40 years after diagnosis. New onset as well as long – term diabetic patients have highly 

expanded numbers of Th40 cells when compared to non-autoimmune, or type 2 diabetic 

controls. A portion of TrialNet defined Pre-T1D subjects also have expanded Th40 cell numbers, 

suggesting that these cells become pathogenic over time, depending upon CD40 expression. 

Controlling CD40 therefore will be therapeutically advantageous. Methods to control CD40 have 

relied upon monoclonal antibodies or randomly generated, small organic molecules. Both those 

options have failed clinically. To address this, we developed a series of peptides derived from 

the CD154 protein sequence that are designed to target CD40 binding sites. These peptides do 

not function like antibodies and unlike the random generated organic molecule approach, have 

high specificity for CD40. In preliminary work we determined that some of the peptides prevent 

diabetes onset in NOD mice and one of the peptides (thus far) reversed hyperglycemia in new 

onset diabetic mice. The goals of this grant are to establish clinical parameters that will allow 

further development of a lead candidate for therapeutic development. We propose to determine

how candidate peptides impact glucose tolerance testing, serum insulin levels and c-peptide 

levels. 

Contact PD/PI: Wagner, David

Project Summary/Abstract Page 6
Methods to control CD40 CD154 interaction have failed because heretofore they have depended upon 

monoclonal antibody or randomly generated organic molecules. We designed CD40 targeting peptides 

that prevent diabetes onset and reverse hyperglycemia in new onset diabetic mice. 

Contact PD/PI: Wagner, David

Project Narrative Page 7
Facilities and Resources:

Op-T-Mune, Inc., located in Denver, CO is a startup company that originated from the WebbWaring Center, Denver CO, prior to WWC joining the University of Colorado School of Medicine. 

WWC operates as an independent center within the University of Colorado Denver Anschutz 

Medical Campus. WWC has provided guaranteed space to Op-T-Mune for business 

development purposes. This includes wet-lab space, and office space. Op-T-Mune also has 

contracted with the Wagner Lab at the University of Colorado School of Medicine, Dr. Wagner 

serves as Chief Scientific Officer for Op-T-Mune as well as being a senior investigator for WWC 

and Professor in the University of Colorado School of Medicine. Lab space includes 

approximately 1500 sq feet with wet bench space, certified sterile hoods, cell culture incubators, 

dedicated cell culture rooms with sterile hoods and incubators, centrifuges etc. An HPLC for 

antibody and peptide analysis, two flow cytometers, owned by the Wagner lab, are available.

Microscopy core facility including light and fluorescent microscopes, a two photon microscope, 

and confocal microscopes are available. Histology proceeding facilities, microtomes, both 

standard and refrigerated are available. Blood collection and procession stations are available 

with a phlebotomist working in the lab. IRB and IACUC approvals are in place. Use of lab space 

and equipment is established through contractual agreements with the University of Colorado 

Denver. The Wagner Lab currently is housed in the School of Pharmacy building and Dr. 

Wagner has been joined the Pharmaceutical Sciences and Toxicology research group. This 

adds mass spectrometer and ion source mass spectrometer use capabilities. In addition the 

expertise of the Pharmaceutical Sciences faculty are available for consult. In addition Op-TMune has resources and expertise in Boulder Colorado from Nostix, Inc, a diagnostic test 

development firm. Mr. Charlie Henry, CEO of OpTMune is currently the CEO of Nostix (which is

under sale after IPO on the NYSE). 

Contact PD/PI: Wagner, David

Facilities & Other Resources Page 8
Equipment : 

The equipment available for completion of these studies includes: a functional immunology lab, Wagner 

group, and a functional protein chemistry/pharmaceutical sciences lab, Carpenter group. Major 

equipment include 2 flow cytometers, centrifuges, freezers, refrigerators, heating ovens, Fast Protein 

Liquid Chromotography (FPLC), Mass Spectrometer, electrospray triple quadrupole time of flight mass 

spectrometer, InfraRed spectrometer, Microplate readers for optical density studies, Agilent HPLC 

capable of performing reverse phase HPLC and size exclusion HPLC. 

Contact PD/PI: Wagner, David

Equipment Page 9
Contact PD/PI: Wagner, David

SBIR.gov SBC Registration Control ID Form

 SBC CONTROL ID SBC_000405651

 FIRM INFORMATION

 Company Op-T-Mune, Inc

 Address 

 City Denver State CO Zip 80206

 TIN/EIN DUNS 

 Company URL 

 Number of Employees: 5

 Is this SBC majority-owned by multiple venture capital operating companies,

 hedge funds, or private equity firms?

 No

 What percentage (%) of the SBC is majority-owned by multiple venture capital

 operating companies, hedge funds, or private equity firms?

 0%

 SBC CONTROL ID SBC_000405651

Powered by TCPDF (www.tcpdf.org)

Generated June 18, 2013 | U.S. Small Business Association | SBIR.gov

1241-SBC_000405651 Page 10
Contact PD/PI: Wagner, David

OMB Number: 4040-0001

Expiration Date: 06/30/2016

Tracking Number: GRANT12240201 Funding Opportunity Number: PA-16-303 . Received Date:

2016-09-02T13:28:53.000-04:00

RESEARCH & RELATED Senior/Key Person Profile (Expanded)

PROFILE - Project Director/Principal Investigator

Prefix: Dr. First Name*: David Middle Name  Last Name*: Wagner Suffix: 

Position/Title*: Chief Scientific Officer

Organization Name*: Op-T-Mune, Inc.

Department:

Division:

Street1*:

Street2:

City*: Denver

County:

State*: CO: Colorado

Province:

Country*: USA: UNITED STATES

Zip / Postal Code*: 80206-3961

Phone Number*:  Fax Number: 

E-Mail*: 

Credential, e.g., agency login: 

Project Role*:  PD/PI Other Project Role Category: 

Degree Type:  PhD Degree Year:  1995

Attach Biographical Sketch*:             File Name: 1248-Wagner_Bio_2016.pdf

Attach Current & Pending Support:   File Name:

 Page 11
Contact PD/PI: Wagner, David

Tracking Number: GRANT12240201 Funding Opportunity Number: PA-16-303 . Received Date:

2016-09-02T13:28:53.000-04:00

PROFILE - Senior/Key Person

Prefix: Dr. First Name*: Martin Middle Name  Last Name*: Yussman Suffix: 

Position/Title*: Chief Medical Officer

Organization Name*: Op-T-Mune, Inc

Department:

Division:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*: 

Zip / Postal Code*: -

Phone Number*:  Fax Number: 

E-Mail*: 

Credential, e.g., agency login: 

Project Role*:  Other (Specify) Other Project Role Category: Medical Advisor

Degree Type:  MD Degree Year:  1997

Attach Biographical Sketch*:             File Name: 1249-Yussman_Biosketch_2016.pdf

Attach Current & Pending Support:   File Name:

 Page 12
NAME

David H. Wagner, Jr.

POSITION TITLE

Associate Professor, Univ of Colo School of Med

Chief Scientific Officer, Op-T-Mune, Inc

eRA COMMONS USER NAME (credential, e.g., agency login)

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and 

residency training if applicable.)

INSTITUTION AND LOCATION DEGREE

(if applicable) MM/YY FIELD OF STUDY

King College, Bristol, TN

East Tennessee State Univ. Johnson City, TN

Quillen College of Medicine, ETSU

National Jewish Medical Center, Denver, CO

Barbara Davis Childhood Diabetes Center

B.S.

M.S.

PhD

Postdoc

Senior 

Postdoc

06/84

12/87

12/94

12/98

06/01

Chemistry/Biology

Biochemistry

Biomedical Sciences

Immunology

Diabetes/Immunology

A. Personal Statement: Over the last 15 years I have worked to understand the role of CD40 in autoimmune 

inflammation. As a PI my lab defined the Th40 cell subset. These are T cells within the CD4+ helper T cell category that 

express the CD40 co-receptor. We defined these T cells in autoimmune diseases including type 1 diabetes and multiple 

sclerosis. We demonstrated that Th40 cells achieve full effector function and that CD40 acts as a co-stimulus for these 

cells, thus describing an importantly overlooked alternative mechanism of T cell activation. We have explored Th40 cells 

in mouse models of disease and translationally moved these studies to human disease. We have examined over 200 

T1D, 70 T2D and 150 control (non-autoimmune) human subjects to date. Now that my lab has developed a unique 

mechanism to control CD40 induced inflammation through use of a peptide that targets the CD154 binding site on CD40 

expressing cells, we will be able to explore this novel therapeutic approach in appropriate atherosclerotic models. In 

addition this work will define a new means of examining atherosclerosis perhaps defining a new biomarker for this 

pernicious disease. As Chief Scientific Officer and Co-Founder of Op-T-Mune, Inc., I have worked to develop Intellectual 

Properties for improving diabetes diagnosis and treating autoimmune inflammation.

B. Positions and Honors:

Employment

2000 - 2002 Instructor Dept. of Medicine, Univ. of Colorado Denver, School of Medicine

2002 - 2008 Assistant Professor Dept of Medicine and Webb-Waring Center, UCDSOM

2006 - Section Head Immunology Section, Webb-Waring Center

2007- Co-Founder Op-T-Mune, Inc., Denver CO

2008 - Associate Professor Dept of Medicine, Univ. of Colorado Denver, School of Medicine

2009- Chief Scientific Officer Op-T-Mune, Inc., Denver, CO

2014 - Associate Professor Dept of Neurology, Univ. of Colorado Denver, School of Medicine

Professional Activities and Memberships:

2001 - Member, American Association of Immunologists

2004 - Professional Member, American Diabetes Association

Editor, 2011 Type 1 Diabetes: Pathogenesis, Genetics and Immunotherapy\ Publisher: Intech 

Editor, 2011 Type 1 Diabetes: Disease Complications\ Intech Open Access Publisher

Editor, 2012 Conference Papers in Immunology/ Online journal

Senior Editor, 2013 American Journal of Clinical and Experimental Immunology

Editor 2014 Medical Genetics and Biomarkers

Session Chair 2005 Aegean Conferences, Santorini, Greece Mechanisms and Treatment of Autoimmunity,

Session Chair 2009 FASEB, San Francisco, CA T cells and Autoimmunity

Session Chair 2011 (AAI) Mechanisms in Autoimmunity Block Symposium, San Francisco, CA

Session Chair 2014 (AAI) “Genetic, Environmental and Cellular Risk for Disease”, Pittsburgh, PA

NIH Study Section, 2004- HAI, Ad Hoc

NIH Study Section, 2013- ETTN-M, 

ADA Study Section 2009-2014 Regular Member

Contact PD/PI: Wagner, David

Biosketches Page 13
Honors:

2013 AAI Faculty Travel Award

2007 Philip R Lee Outstanding Career Development Award, American Diabetes Association

2002 AAI Outstanding Junior Faculty Award

C. Contributions to Science:

(1) Functional expression of CD40 on CD4+ T cells. CD40 was long considered an Antigen Presenting Cell (APC)

molecule. Its function was defined as inducing B cell antibody class switching. On other APCs CD40s’ function was 

described as inducing pro-inflammatory cytokines. In early work I was involved in exploring CD40 on macrophages. My 

first significant independent discovery was that CD40 is functionally expressed on CD4+ T cells. These cells arise in the 

thymus and occur in the periphery of all mouse strains tested. Importantly CD40 expressing CD4+ cells occur in the 

periphery of all human subjects tested. One of the functions of CD40 on T cells is to subvert cell death by promoting Bcl2, 

Bcl-XL and cFLIP, molecules that promote cell survival. This discovery is related to autoimmune conditions. If fact when 

CD40 expressing T cells are isolated from autoimmune conditions, CD40 signals override Fas-mediated cell death 

signals. This discovery described a new biomarker for T cells; therefore we explored this finding further in disease models.

1. Wagner DH, Jr., Vaitaitis G, Sanderson R, Poulin M, Dobbs C, Haskins K: Expression of CD40 identifies a unique 

pathogenic T cell population in type 1 diabetes. Proc Natl Acad Sci U S A 2002, 99(6):3782-3787.

2. Vaitaitis G, Waid DM, Wagner Jr. D: The Expanding Role of TNF-Receptor Super Family Member CD40 (tnfrsf5) 

in Autoimmune Disease: Focus on Th40 Cells Curr Immunol Rev 2010, 6(2):130-137.

3. Vaitaitis GM, Wagner DH, Jr.: Galectin-9 controls CD40 signaling through a Tim-3 independent mechanism and 

redirects the cytokine profile of pathogenic T cells in autoimmunity. PLoS One 2012, 7(6):e38708.

4. Poe JC, Wagner DH, Jr., Miller RW, Stout RD, Suttles J: IL-4 and IL-10 modulation of CD40-mediated signaling of 

monocyte IL-1beta synthesis and rescue from apoptosis. J Immunol 1997, 159(2):846-852.

5. Wagner DH, Jr., Stout RD, Suttles J: Role of the CD40-CD40 ligand interaction in CD4+ T cell contact-dependent 

activation of monocyte interleukin-1 synthesis. Eur J Immunol 1994, 24(12):3148-3154.

(2) CD40 induces RAG1/RAG2 production: In exploring the potential function of CD40, my lab determined that CD40 

engagement of peripheral CD4+ T cells induced RAG1 and RAG2 proteins, which constitute the machinery for TCR 

generation. This was the first demonstration of a cellular mechanism to induce RAG proteins. Secondarily to this we 

showed that peripheral T cells alter TCR expression in a process called TCR revision. While we not the first to 

demonstrate TCR revision, we were the first to demonstrate a mechanism to induce TCR revision in peripheral T cells. 

TCR revision is an immunologic paradigm shift; our contribution is the discovery of how revision can be induced. By being 

able to control TCR revision, this finding could lead to therapeutics to alter auto-aggressive TCR bearing cells, thus 

serving as an autoimmune treatment; increase vaccine potency by generating antigen specific T cells; and potentially 

promote the development of tumor specific T cells. Each of these applications have direct therapeutic potential.

1. Vaitaitis GM, Poulin M, Sanderson RJ, Haskins K, Wagner DH, Jr.: Cutting edge: CD40-induced expression 

of recombination activating gene (RAG) 1 and RAG2: a mechanism for the generation of autoaggressive T 

cells in the periphery. J Immunol 2003, 170(7):3455-3459.

2. Vaitaitis GM, Wagner DH, Jr.: CD40 interacts directly with RAG1 and RAG2 in autoaggressive T cells and 

Fas prevents CD40-induced RAG expression. Cell Mol Immunol 2013, 10(6):483-489.

3. Wagner DH, Jr.: Re-shaping the T cell repertoire: TCR editing and TCR revision for good and for bad. Clin 

Immunol 2007, 123(1):1-6.

Th40 cells and effector functions: We discovered that CD40 expressing CD4+ cells now termed Th40, achieve effector 

function and that CD40 engagement acts as a T cell co-stimulus. Importantly Th40 cells are capable of producing both 

Th1 (IFN, TNF, and IL-2) but coincidentally produce IL-17 and IL-23 that are associated with Th17 cells. Th40 cells 

express both t-bet and RORt transcription factors. We discovered that Th40 effector cells can express Foxp3, but rather 

than being classic regulatory cells, Foxp3 functions as an effector cell regulator. This is novel in terms of thinking about 

Foxp3 as being a T cell regualtory molecule rather than only defining regulatory T cells. In addition CD40 negatively 

impacts CTLA-4 expression to further ablate tolerance. We created a thymic expressed neo-self antigen to demonstrate 

that thymic expression of antigen drives T cell CD40 expression. The contribution here is first that self-antigen exposure in 

the thymus promotes CD40 expressing T cells, that CD40 constitutes an alternative T cell co-stimulatory molecule and 

that CD40 expression negatively impacts T cell tolerance. This opens entire new approaches to T cell biology. We 

described that infection models cause significant increases in CD40-bearing T cells. Infection further causes systemic 

increases in CD154, the natural ligand for CD40. This means that an entire realm of T cell co-stimulation has been 

overlooked. These findings also are paradigm shifting and require new focus on the biology of T cell responses.

1. Vaitaitis G, Waid DM, Wagner Jr. D: The Expanding Role of TNF-Receptor Super Family Member CD40 

(tnfrsf5) in Autoimmune Disease: Focus on Th40 Cells Curr Immunol Rev 2010, 6(2):130-137.

Contact PD/PI: Wagner, David

Biosketches Page 14
2. Vaitaitis GM, Wagner DH, Jr.: High distribution of CD40 and TRAF2 in Th40 T cell rafts leads to preferential 

survival of this auto-aggressive population in autoimmunity. PLoS One 2008, 3(4):e2076.

3. Vaitaitis GM, Carter JR, Waid DM, Olmstead MH, Wagner DH, Jr.: An alternative role for Foxp3 as an effector 

T cell regulator controlled through CD40. J Immunol 2013, 191(2):717-725.

4. Waid DM, Wagner RJ, Putnam A, Vaitaitis GM, Pennock ND, Calverley DC, Gottlieb P, Wagner DH, Jr.: A 

unique T cell subset described as CD4loCD40+ T cells (TCD40) in human type 1 diabetes. Clin Immunol 

2007, 124(2):138-148.

5. Carter J, Vaitaitis GM, Waid DM, Wagner DH, Jr.: CD40 engagement of CD4+ CD40+ T cells in a neo-self 

antigen disease model ablates CTLA-4 expression and indirectly impacts tolerance. Eur J Immunol 2012, 

42(2):424-435.

(4) Th40 cells and MS and T1D: We discovered that Th40 cells are associated with human disease. Th40 cells occur in 

all humans tested, over 600 thus far, but are significantly expanded in type 1 diabetes and in multiple sclerosis. The major 

risk factors associated with either T1D or MS are HLA haplotype. We discovered that regardless of HLA haplotype if 

autoimmune disease is present Th40 cells are highly expanded in percentage of peripheral lymphocytes as well as in 

actual number. Importantly, if human subjects carry high risk HLA alleles but do not have autoimmunity, Th40 levels are 

“normal”. As in mice, Th40 cells from human autoimmune patients achieve effector function, with CD40 acting as a crucial 

co-stimulus. In T1D and MS Th40 cells achieve a unique antigen signature profile. Each patient tested have Th40 cells 

that display unique responses to associated self-antigens. These studies demonstrate the importance of Th40 cells as 

pathogenic effector cells in two distinct but related autoimmune diseases. This is especially relevant given the growing 

number of patients co-presenting with T1D and MS. 

1. Wagner, DH. 2016. “Of the multiple mechanisms leading to type 1 diabetes, T cell receptor revision may play a 

prominent role (Is type 1 diabetes more than a single disease?). Clin Exp. Immunol. In Press.

2. Waid DM, Schreiner T, Vaitaitis G, Carter JR, Corboy JR, Wagner DH, Jr.: Defining a new biomarker for the 

autoimmune component of Multiple Sclerosis: Th40 cells. J Neuroimmunol 2014, 270(1-2):75-85.

3. Siebert JC, Inokuma M, Waid DM, Pennock ND, Vaitaitis GM, Disis ML, Dunne JF, Wagner DH, Jr., Maecker HT: 

An analytical workflow for investigating cytokine profiles. Cytometry A 2008, 73(4):289-298.

4. Waid DM, Wagner RJ, Putnam A, Vaitaitis GM, Pennock ND, Calverley DC, Gottlieb P, Wagner DH, Jr.: A 

unique T cell subset described as CD4loCD40+ T cells (TCD40) in human type 1 diabetes. Clin Immunol 

2007, 124(2):138-148.

5. Wagner DH, Jr.: The specific antigen approach in multiple sclerosis: can it ever be enough? Clin Immunol 

2012, 144(2):139-141.

6. Wagner J, D.H.: The Role of T cells in type 1 diabetes. In: Type 1 Diabetes: Pathogenesis, Genetics and 

Immuntherapy. Edited by Wagner J, D.H., vol. 1. Croatia: INTECH; 2011: 660.

(5) A novel means of controlling CD40 mediated inflammation: Controlling CD40 mediated inflammation has long 

been a research area of hot pursuit. The only available means thus far has been monoclonal antibody, which has 

created numerous problems. Antibodies against CD40 do not work because they are agonistic. Antibodies against 

CD154 initially were successful but failed ultimately because human platelets express high levels of CD154 and the 

treatment antibodies coagulated platelets leading to thrombosis. We created a 15-amino acid peptide that targets the 

CD154 binding site of CD40. This peptide prevented diabetes onset in NOD mice and reversed hyperglycemia in 50% 

of new onset mice. The peptide restricted T cell infiltrations in to pancreatic islets. This discovery provides a completely 

new way of targeting a major inflammatory pathway without the use of antibodies. In fact this discovery may lead to 

entirely new ways of controlling receptor – ligand interactions by use of small targeting peptides designed to bind 

directly to receptors. 

1. Vaitaitis GM, Olmstead MH, Waid DM, Carter JR, Wagner DH, Jr.: A CD40-targeted peptide controls and 

reverses type 1 diabetes in NOD mice. Diabetologia 2014, 57(11):2366-2373.

D. Research Support:

ACTIVE

Contact PD/PI: Wagner, David

Biosketches Page 15
1R41AI113977-01 (Wagner) 06/15/2014 – 05/31/2015

NIH/NIAID $211,808

Project title: Developing a new treatment for MS: Controlling CD40 in EAE. 

The aims of this project are to test a new treatment module, a small peptide designed to interact with CD40, to impact 

EAE disease development.

Previous Awards: 

Granting agency: NIH/NIAID, R21 AI106011, Start and end dates: 04/01/2013 – 03/31/2015

Project title: Mechanism of action of a CD40 control peptide

Total award: $426,250

Role of applicant: PI

Granting agency: NIH/NIAID, R01 DK075013-01, Start and end dates: 04/01/2007 – 03/31/2013

Project title: CD40 induces RAG1 and RAG2: Mechanism for Breach of Tolerance. 

Total award: $1,732,500

Role of applicant: PI

The purpose of this grant was to examine how T cell receptor changes induced by CD40 engagement led to alteration in 

tolerance. The primary focus was in type 1 diabetes. This grant explored CD40 induction of RAG1 and RAG2 proteins that 

are crucial for T cell receptor changes. The grant provided direct evidence of TCR revision, the ability of T cells to alter 

TCR expression in the periphery. 

Op-T-Mune, Inc.

Granting Agency: NIHCD, R41 (SBIR Phase I), Start/End: 04/01/20013 – 07/01/2014

Project Title: Screening for Type 1 Diabetes: A blood test based on the CD40 biomarker

PI: Peter E. Nelson, Percent Effort: 51%

Consultant: David H. Wagner, Jr., Percent Effort, 5%

The aims of this grant are to determine how Th40 cell levels predict type 1 diabetes onset. Risk levels for 

high/intermediate/low to develop diabetes are the dominant feature. Pre-diabetic subjects are compared to new onset and 

long term diabetic subjects. Differences from non autoimmune controls are noted

Contact PD/PI: Wagner, David

Biosketches Page 16
Contact PD/PI: Wagner, David

OMB No. 0925-0001/0002 (Rev. 08/12 Approved Through 8/31/2015) 

BIOGRAPHICAL SKETCH 

Provide the following information for the Senior/key personnel and other significant contributors. 

Follow this format for each person. DO NOT EXCEED FIVE PAGES. 

NAME: Martin Glenn Yussman

eRA COMMONS USER NAME 

POSITION TITLE: Assistant Professor University of Colorado Medical School 

 Chief Medical Officer, Op-T-Mune, Inc.

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such 

as nursing, include postdoctoral training and residency training if applicable. Add/delete rows as 

necessary.)

INSTITUTION AND LOCATION

DEGREE

(if 

applicable)

Completion 

Date

MM/YYYY

FIELD OF STUDY

University of Wisconsin, Madison, WI

University of Wisconsin, Madison, WI

Universitatsklinik fur Frauenheilkunde, 

Innsbruck, Austria

University of Louisville, Louisville, KY

University of Cincinnati, Cincinnati, OH

University of Cincinnati, Cincinnati, OH

University of Cincinnati, Cincinnati, OH

BA

Associate 

Research 

Specialist

Research 

Scholars 

exchange 

program

MD

Residency

Research 

Fellowship

Clinical 

Fellowship

06/1991

06/1993

8/1994

06/1997

6/2000

6/2002

6/2004

Molecular Biology

Calcium signaling 

and Microscopy

cytokines and 

vitamins influence 

on the expression of 

EGFr and HER-2

Medicine

Internal Medicine

Molecular 

mechanisms of 

cardiomyopathy

Invasive Cardiology

Biosketches Page 17
NOTE: The Biographical Sketch may not exceed five pages. Follow the formats and 

instructions below. 

A. Personal Statement 

Repaying the cost of medical training detoured my research career. Though I enjoyed success 

in clinical medicine, my fervor toward research involvement continued. During my career as a 

clinical cardiologist, I was a site lead investigator for the VIRGO study, IRB member and 

instructor for residency programs. This experience encouraged ongoing research discussions 

with Dr. Wagner, co-investigator for this project. Dr. Wagner is an immunologist whose chief 

interest has been both discovering the etiology of type 1 diabetes as well as developing earlier 

diagnostics. The collaborative effort with Dr. Wagner as well as previous research experience 

with groups at the University of Wisconsin and the University of Cincinnati have provided me 

the expertise necessary to successfully carry out the proposed work. I have become well versed 

in constructing realistic plans, timelines, and budgets as well as creating collaborative networks 

instilled from my administrative positions within hospital and office. Inspiring my network of 

prior community physician partners to collaborate in the research endeavor illustrates these 

capabilities. With my background in molecular biology and genetics and with advanced 

microscopy, immunohistochemistry and echo cardiology skills I have successfully researched 

and published in journals such as Circulation Research and Nature Medicine. The research 

involved pathologic molecular mechanisms characterized by structural derangements and 

rearrangements of myocardial tissue, cardiomyocyte death with gain and loss of function 

design and use of transgenic expression. I have returned to a full-time research career with a 

more mature outlook, expanding my fellowship goals. I am working toward a successful path 

with support from the Webb Waring Center and Cardiopulmonary division of the University of 

Colorado and my prior community cardiology division. I have forfeited my position as senior 

clinician with that division to return to my research passion which has now matured and 

expanded by clinical experience. My record is unique with both clinical and basic science 

expertise and experience preparing me to lead this and future projects. I am become Chief 

Medical Officer with Op-T-Mune to help this startup company expand its clinical/therapeutic 

development opportunities. 

B. Positions and Honors 

List in chronological order previous positions, concluding with the present position. List any 

honors. Include present membership on any Federal Government public advisory committee. 

2014-Present Affiliate Faculty University of Colorado Denver, Webb Waring Center 

2014-Present Chief Medical Officer, Op-T-Mune, Inc, Denver CO 

2011-Oct 2014 General Invasive Cardiologist; SCL Rocky Mountain Cardiovascular 

Assoc. 

Cardiology Instructor St. Joes Internal medicine residency and Family 

practice residency programs. 

 Admin/lead physician EMR transition/coordination 

2009-2011 General Invasive Cardiologist; Rocky Mountain Cardiovascular Assoc. 

Contact PD/PI: Wagner, David

Biosketches Page 18
Cardiology Instructor St. Anthony Family practice residency program. 

Cardiology Preceptor, University of Colorado Denver school of Medicine 

 Admin/lead physician EMR transition/coordination 

2008-2009 Site Principal Investigator VIRGO study; Centura Research Center-Porter 

Adventist Hospital 

Heart Failure Quality Director, Porter Adventist Hospital 

2004-2009 General Invasive Cardiologist; Cardiovascular Associates 

 IRB Cardiology committee member, St. Anthony Hospital 

 IRB Cardiology committee member, Porter Adventist Hospital 

Cardiology Instructor, St. Anthony Family practice residency program. 

Fellow American College of Cardiology 

 2002 First Prize Award, University of Cincinnati 

 Fellowship Research Program 

 2002 Accepted to 8th Annual Astra Zeneca 

 Young Investigators Forum 

 2001 First Prize Award, University of Cincinnati 

 Fellowship Research Program 

 2001 Accepted to 7th Annual Astra Zeneca 

 Young Investigators Forum 

 

C. Contribution to Science 

My publications through the research fellowship addressed the inciting molecular mechanisms 

regulating subcellular protein trafficking in the heart, and the pathological consequences of 

aberrant cardiomyocyte protein transport causing dilated cardiomyopathy. Additionally, by use 

of microarray analysis, a genetic program was identified for myocardial apoptosis in Gqmediated and pressure –overload cardiac hypertrophy. A critical component of this apoptotic 

program was Nix/Bnip3L. Nix localized to mitochondria and caused release of cytochrome c, 

activation of caspase-3 and apoptotic cell death, when expressed in HEK293 fibroblasts. Nix 

overexpression showed the sufficiency of Nix to provoke apoptotic heart failure and identified 

downstream events mediating this response. This article has been cited over 225 times, and 

useful as a model for testing potential therapeutic interventions directed at mitochondrial 

stabilization. I served as the primary or co-investigator in all of these studies. 

1. Hahn, H.S., et al., Protein kinase Calpha negatively regulates systolic and diastolic function in 

pathological hypertrophy. Circ Res, 2003. 93(11): p. 1111-9.

2. Hahn, H.S., et al., Ischemic protection and myofibrillar cardiomyopathy: dose-dependent effects 

of in vivo deltaPKC inhibition. Circ Res, 2002. 91(8): p. 741-8.

3. Yussman, M.G., et al., Mitochondrial death protein Nix is induced in cardiac hypertrophy and 

triggers apoptotic cardiomyopathy. Nat Med, 2002. 8(7): p. 725-30.

4. Wu, G., et al., Increased myocardial Rab GTPase expression: a consequence and cause of 

cardiomyopathy. Circ Res, 2001. 89(12): p. 1130-7.

Contact PD/PI: Wagner, David

Biosketches Page 19
OMB Number: 4040-0001

Expiration Date: 06/30/2016

Tracking Number: GRANT12240201 Funding Opportunity Number: PA-16-303 . Received Date: 2016-09-02T13:28:53.000-04:00

RESEARCH & RELATED BUDGET - SECTION A & B, Budget Period  1

ORGANIZATIONAL DUNS*:   

Budget Type*: ● Project ❍ Subaward/Consortium

Enter name of Organization: Op-T-Mune, Inc.

Start Date*: 04-01-2017 End Date*: 03-31-2018 Budget Period: 1

A. Senior/Key Person

# Prefix First Name* Middle

Name

Last Name* Suffix Project Role* Base

Salary ($)

Calendar

Months

Academic

Months

Summer

Months

Requested

Salary ($)*

Fringe

Benefits ($)*

Funds Requested ($)*

1 . Dr. David Wagner PD/PI 3.60

2 . Dan Waid Research

Coordinator and

Professional

Research

Assistant

3.60

3 . Dr. Martin Yussman MD Chief Medical

Officer, Medical

Advisor

1.08

Total Funds Requested for all Senior Key Persons in the attached file

Additional Senior Key Persons: File Name: Total Senior/Key Person

Mime Type:

B. Other Personnel

Number of

Personnel*

Project Role* Calendar Months Academic Months Summer Months Requested Salary ($)* Fringe Benefits* Funds Requested ($)*

Total Number Other Personnel Total Other Personnel

Total Salary, Wages and Fringe Benefits (A+B)

RESEARCH & RELATED Budget {A-B} (Funds Requested)

Contact PD/PI: Wagner, David

 Page 20
Contact PD/PI: Wagner, David

Tracking Number: GRANT12240201 Funding Opportunity Number: PA-16-303 . Received Date:

2016-09-02T13:28:53.000-04:00

RESEARCH & RELATED BUDGET - SECTION C, D, & E, Budget Period 1

ORGANIZATIONAL DUNS*: 

Budget Type*: ● Project ❍ Subaward/Consortium

Organization: Op-T-Mune, Inc.

Start Date*: 04-01-2017 End Date*: 03-31-2018 Budget Period: 1

C. Equipment Description

List items and dollar amount for each item exceeding $5,000

Equipment Item Funds Requested ($)*

Total funds requested for all equipment listed in the attached file

Total Equipment

Additional Equipment: File Name:

Mime Type:

D. Travel Funds Requested ($)*

1. Domestic Travel Costs ( Incl. Canada, Mexico, and U.S. Possessions)

2. Foreign Travel Costs

Total Travel Cost

E. Participant/Trainee Support Costs Funds Requested ($)*

1. Tuition/Fees/Health Insurance

2. Stipends

3. Travel

4. Subsistence

5. Other: 

 Number of Participants/Trainees Total Participant Trainee Support Costs

RESEARCH & RELATED Budget {C-E} (Funds Requested)

 Page 21
Contact PD/PI: Wagner, David

Tracking Number: GRANT12240201 Funding Opportunity Number: PA-16-303 . Received Date:

2016-09-02T13:28:53.000-04:00

RESEARCH & RELATED BUDGET - SECTIONS F-K, Budget Period 1

ORGANIZATIONAL DUNS*: 

Budget Type*: ● Project ❍ Subaward/Consortium

Organization: Op-T-Mune, Inc.

Start Date*: 04-01-2017 End Date*: 03-31-2018 Budget Period: 1

F. Other Direct Costs Funds Requested ($)*

1. Materials and Supplies

2. Publication Costs

3. Consultant Services

4. ADP/Computer Services

5. Subawards/Consortium/Contractual Costs

6. Equipment or Facility Rental/User Fees

7. Alterations and Renovations

8 . Travel to one national meeting

Total Other Direct Costs

G. Direct Costs Funds Requested ($)*

Total Direct Costs (A thru F)

H. Indirect Costs

Indirect Cost Type Indirect Cost Rate (%) Indirect Cost Base ($) Funds Requested ($)*

1 . Modified Direct Costs 40.00

Total Indirect Costs

Cognizant Federal Agency

(Agency Name, POC Name, and POC Phone Number)

I. Total Direct and Indirect Costs Funds Requested ($)*

Total Direct and Indirect Institutional Costs (G + H)

J. Fee Funds Requested ($)*

K. Budget Justification* File Name: 1234-

Justification_OpT_Diabetes_2016.pdf

Mime Type: application/pdf

(Only attach one file.)

RESEARCH & RELATED Budget {F-K} (Funds Requested)

 Page 22
Budget Justification STTR:

Salaries: Dr. David Wagner PhD. PI, Chief Scientific Officer. Dr. Wagner is Co-Founder and 

Chief Scientific Officer of Op-T-Mune. Dr. Wagner will be r responsible for the biomedical 

research aspect of the grant. The basis for this application has extended from work in the 

Wagner Laboratory. Dr. Wagner will oversee experiments, assist in analyzing data, oversee 

maintenance. Dr. Wagner plays a central role in the operations of OpTMune including writing 

grants, coordinating business relations with the University of Colorado etc. 30% Effort, 3.6 cal 

months is requested in the program section. Dr. Wagner’s effort will be split between Op-TMune and the University of Colorado Denver. UCD request is for 10% effort as described in the 

sub award budget to satisfy the University requirements.

Mr. Dan Waid, BSN, Medical Coordinator, Professional Research Assistant for University of 

Colorado and Chief Financial Officer for Op-T-Mune. Mr. Waid will be involved with animal 

experiments including EAE induction, obtaining tissues, flow cytometry including performing flow 

experiments (the Wagner Lab maintains its own flow cytometer, Mr. Waid oversees all aspects 

of upkeep for that machine) and flow analysis. Mr. Waid also will perform experiments with 

human blood (Physician’s Assistant United States Navy, 1990 – 2000) and has been 

instrumental in establishing the CD4CD40/diabetes database. 30% effort, 3.6 Cal Months is 

requested. 

Dr. Martin Yussman, MD. Dr. Yussman is a board certified Internal Medicine physician. He has 

been recruited as Chief Medical Officer to help Op-T-Mune expand clinical development 

expertise. For this project Dr. Yussman will be involved in data analysis, and chart reviews. We 

will be coordinating medical history and clinical observations to our testing module and Dr. 

Yussman will provide counsel in that respect. We are requesting 9%. 

Materials and supplies: Antibodies for CD4, CD3 and CD40 are produced by Op-T-mune. Inhouse antibodies will be compared to commercial antibodies for titration and standardizations. 

For QC, fluorescenated antibodies will be purchased from commercial vendors to compare to 

our antibodies Antibody budget is $12,000. Calibration beads will be purchased and loaded with 

purchased fluors (Cost, $3100). Supplies including those for cell culture, tissue hood, culture 

hoods, media etc will be purchased. Peptides are made by the GMP accredited, CMO 

AmBioPharm, North Augusta, SC, peptides are at 99.5% purity. These peptides will be 

generated including the 8 different sized peptides for CD40 molecule. Peptides are generated 

with fluorescent tags. (Cost, $15,364) Buffers for suspending and storing peptides, blood 

collection supplies, needles, collection tubes, etc., will be purchased ($910). Total $28,740. 

Subaward: The nature of the STTR mechanism requires University involvement. The Subaward 

budget is to the University of Colorado Denver Anschutz Medical Campus. The budget for the 

subaward is described in that section.

Indirect Costs: After consultation with NIH about small business indirect rates we set indirect at 

40%. As reported to us by Mr. Kyle Kroneberger, the NIH Grants policy statement 18.5.4.3.1 

cover 40% rule. We were told that if the grant is awarded, this rate will be further negotiated 

with NIH. The Indirect costs as allowed by NIH, NIH Grants policy statement, include 

administrative allowances such as IP protection, business allowance, administrative issues etc.

Contact PD/PI: Wagner, David

Budget Justification Attachment Page 23
Contact PD/PI: Wagner, David

Tracking Number: GRANT12240201 Funding Opportunity Number: PA-16-303 . Received Date:

2016-09-02T13:28:53.000-04:00

RESEARCH & RELATED BUDGET - Cumulative Budget

Totals ($)

Section A, Senior/Key Person

Section B, Other Personnel

Total Number Other Personnel

Total Salary, Wages and Fringe Benefits

(A+B)

Section C, Equipment

Section D, Travel

1. Domestic

2. Foreign

Section E, Participant/Trainee Support

Costs

1. Tuition/Fees/Health Insurance

2. Stipends

3. Travel

4. Subsistence

5. Other

6. Number of Participants/Trainees

Section F, Other Direct Costs

1. Materials and Supplies

2. Publication Costs

3. Consultant Services

4. ADP/Computer Services

5. Subawards/Consortium/Contractual

Costs

6. Equipment or Facility Rental/User

Fees

7. Alterations and Renovations

8. Other 1

9. Other 2

10. Other 3

Section G, Direct Costs

(A thru F)

Section H, Indirect Costs

Section I, Total Direct and Indirect Costs

(G + H)

Section J, Fee

 Page 24
OMB Number: 4040-0001

Expiration Date: 06/30/2016

Tracking Number: GRANT12240201 Funding Opportunity Number: PA-16-303 . Received Date: 2016-09-02T13:28:53.000-04:00

RESEARCH & RELATED BUDGET - SECTION A & B, Budget Period  1

ORGANIZATIONAL DUNS*:   

Budget Type*: ❍ Project ● Subaward/Consortium

Enter name of Organization: University of Colorado Anschutz Medical Campus

Start Date*: 04-01-2017 End Date*: 03-31-2018 Budget Period: 1

A. Senior/Key Person

# Prefix First Name* Middle

Name

Last Name* Suffix Project Role* Base

Salary ($)

Calendar

Months

Academic

Months

Summer

Months

Requested

Salary ($)*

Fringe

Benefits ($)*

Funds Requested ($)*

1 . Dr. David H Wagner PD/PI 1.20

2 . Gisela Vaitaitis Professional

Research

Assistant

1.20

Total Funds Requested for all Senior Key Persons in the attached file

Additional Senior Key Persons: File Name: Total Senior/Key Person

Mime Type:

B. Other Personnel

Number of

Personnel*

Project Role* Calendar Months Academic Months Summer Months Requested Salary ($)* Fringe Benefits* Funds Requested ($)*

Total Number Other Personnel Total Other Personnel

Total Salary, Wages and Fringe Benefits (A+B)

RESEARCH & RELATED Budget {A-B} (Funds Requested)

Contact PD/PI: Wagner, David

 Page 25
Contact PD/PI: Wagner, David

Tracking Number: GRANT12240201 Funding Opportunity Number: PA-16-303 . Received Date:

2016-09-02T13:28:53.000-04:00

RESEARCH & RELATED BUDGET - SECTION C, D, & E, Budget Period 1

ORGANIZATIONAL DUNS*: 

Budget Type*: ❍ Project ● Subaward/Consortium

Organization: University of Colorado Anschutz Medical Campus

Start Date*: 04-01-2017 End Date*: 03-31-2018 Budget Period: 1

C. Equipment Description

List items and dollar amount for each item exceeding $5,000

Equipment Item Funds Requested ($)*

Total funds requested for all equipment listed in the attached file

Total Equipment

Additional Equipment: File Name:

Mime Type:

D. Travel Funds Requested ($)*

1. Domestic Travel Costs ( Incl. Canada, Mexico, and U.S. Possessions)

2. Foreign Travel Costs

Total Travel Cost

E. Participant/Trainee Support Costs Funds Requested ($)*

1. Tuition/Fees/Health Insurance

2. Stipends

3. Travel

4. Subsistence

5. Other: 

 Number of Participants/Trainees Total Participant Trainee Support Costs

RESEARCH & RELATED Budget {C-E} (Funds Requested)

 Page 26
Contact PD/PI: Wagner, David

Tracking Number: GRANT12240201 Funding Opportunity Number: PA-16-303 . Received Date:

2016-09-02T13:28:53.000-04:00

RESEARCH & RELATED BUDGET - SECTIONS F-K, Budget Period 1

ORGANIZATIONAL DUNS*: 

Budget Type*: ❍ Project ● Subaward/Consortium

Organization: University of Colorado Anschutz Medical Campus

Start Date*: 04-01-2017 End Date*: 03-31-2018 Budget Period: 1

F. Other Direct Costs Funds Requested ($)*

1. Materials and Supplies

2. Publication Costs

3. Consultant Services

4. ADP/Computer Services

5. Subawards/Consortium/Contractual Costs

6. Equipment or Facility Rental/User Fees

7. Alterations and Renovations

8 . Animal housing

Total Other Direct Costs

G. Direct Costs Funds Requested ($)*

Total Direct Costs (A thru F)

H. Indirect Costs

Indirect Cost Type Indirect Cost Rate (%) Indirect Cost Base ($) Funds Requested ($)*

1 . Modified Indirect Costs

Total Indirect Costs

Cognizant Federal Agency

(Agency Name, POC Name, and POC Phone Number)

DHHS, Wally Chan, (415) 437-7829

I. Total Direct and Indirect Costs Funds Requested ($)*

Total Direct and Indirect Institutional Costs (G + H)

J. Fee Funds Requested ($)*

K. Budget Justification* File Name: 1242-

Justification_Subaward_STTR.pdf

Mime Type: application/pdf

(Only attach one file.)

RESEARCH & RELATED Budget {F-K} (Funds Requested)

 Page 27
Justification: University of Colorado Anschutz MedicalCampus

Salaries

Dr. David Wagner PhD. Professor, Dept. of Medicine and Dept. of Neurology. With STTR grants, the

University of Colorado requires faculty to include a portion of salary in the University budget. Dr.

Wagner is not being paid twice, but his FTE is being divided between Univ. Colorado and Op-T-Mune. Dr.

Wagner will be responsible for the biomedical research aspect of the grant. The basis for thisapplication

has extended from work in the Wagner Laboratory. Dr. Wagner will oversee experiments, be responsible

for analyzing data, oversee maintenance of the diabetes database and coordinate future experiments.

FTE will be 10% which is 1.2 calendarmonths.

Gisela Vaitaitis, MS, Professional Research Associate, 10% FTE year 1 and 8% for the last 6 months in

year 2. Mrs. Vaitaitis will perform molecular biology and protein chemistry experiments. She oversees

the Wagner Lab animal facility. She induces EAE in mice.

Animals: Because animals will be housed at the University of Colorado Anschutz Medical Campus, we are 

requesting a portion of animal housing on the University sub-award. Based on the number of animals requested 

in the vertebrate animal form, and housing for 1 year, we are requesting $12,000.

Supplies: Supply budget includes basic lab supplies, buffers, protein chemistry supplies, suppliesfor

Mass spectrometer, mass spec rental time paid to school of pharmacy, MOG protein, pertussis toxin,

CFA etc.

Indirect Costs: The University of Colorado Anschutz Medical Campus has a negotiated rate of 55.5%.We

are including that as a separate Modified Direct Cost.

Contact PD/PI: Wagner, David

Budget Justification Attachment Page 28
Contact PD/PI: Wagner, David

Tracking Number: GRANT12240201 Funding Opportunity Number: PA-16-303 . Received Date:

2016-09-02T13:28:53.000-04:00

RESEARCH & RELATED BUDGET - Cumulative Budget

Totals ($)

Section A, Senior/Key Person

Section B, Other Personnel

Total Number Other Personnel

Total Salary, Wages and Fringe Benefits

(A+B)

Section C, Equipment

Section D, Travel

1. Domestic

2. Foreign

Section E, Participant/Trainee Support

Costs

1. Tuition/Fees/Health Insurance

2. Stipends

3. Travel

4. Subsistence

5. Other

6. Number of Participants/Trainees

Section F, Other Direct Costs

1. Materials and Supplies

2. Publication Costs

3. Consultant Services

4. ADP/Computer Services

5. Subawards/Consortium/Contractual

Costs

6. Equipment or Facility Rental/User

Fees

7. Alterations and Renovations

8. Other 1

9. Other 2

10. Other 3

Section G, Direct Costs

(A thru F)

Section H, Indirect Costs

Section I, Total Direct and Indirect Costs

(G + H)

Section J, Fee

 Page 29
Contact PD/PI: Wagner, David

Tracking Number: GRANT12240201 Funding Opportunity Number: PA-16-303. Received Date:

2016-09-02T13:28:53.000-04:00

 

Total Direct Costs less Consortium F&A

 

NIH policy (NOT-OD-05-004) allows applicants to exclude consortium/contractual F&A costs when determining if an application falls at or beneath any applicable direct cost limit. When a direct

cost limit is specified in an FOA, the following table can be used to determine if your application falls within that limit. 

Category Budget

Period 1

Budget

Period 2

Budget

Period 3

Budget

Period 4

Budget

Period 5

TOTALS

Total Direct Costs less Consortium F&A 0 0 0 0

 Page 30
Tracking Number: GRANT12240201 Funding Opportunity Number: PA-16-303. Received Date:

2016-09-02T13:28:53.000-04:00

SBIR/STTR Information

OMB Number: 4040-0001

Expiration date: 06/30/2016

Program Type (select only one)*

❍ SBIR ● STTR ❍ Both  (See agency-specific instructions to determine whether a particular agency allows a single submission for

both SBIR and STTR)

SBIR/STTR Type (select only one)*

● Phase I ❍ Phase II ❍ Fast-Track (See agency-specific instructions to determine whether a particular agency participates in FastTrack)

Questions 1-7 must be completed by all SBIR and STTR Applicants:

1a. Do you certify that at the time of award your organization will meet the eligibility criteria for a

small business as defined in the funding opportunity announcement?*

● Yes ❍ No

1b. Anticipated Number of personnel to be employed at your organization at the time of award.* 5

2. Does this application include subcontracts with Federal laboratories or any other Federal

Government agencies?*

❍ Yes ● No

If yes, insert the names of the Federal laboratories/agencies:*

3. Are you located in a HUBZone? To find out if your business is in a HUBZone, use the mapping

utility provided by the Small Business Administration at its web site: http://www.sba.gov *

● Yes ❍ No

4. Will all research and development on the project be performed in its entirety in the United

States?*

● Yes ❍ No

If no, provide an explanation in an attached file. Explanation:*

5. Has the applicant and/or Program Director/Principal Investigator submitted proposals for

essentially equivalent work under other Federal program solicitations or received other Federal

awards for essentially equivalent work?*

❍ Yes ● No

If yes, insert the names of the other Federal agencies:*

6. Disclosure Permission Statement: If this application does not result in an award, is the

Government permitted to disclose the title of your proposed project, and the name, address,

telephone number and e-mail address of the official signing for the applicant organization, to

organizations that may be interested in contacting you for further information (e.g., possible

collaborations, investment)?*

● Yes ❍ No

7. Commercialization Plan: If you are submitting a Phase II or Phase I/Phase II Fast-Track Application, include a Commercialization

Plan in accordance with the agency announcement and/or agency-specific instructions.*

Attach File:*

Contact PD/PI: Wagner, David

 Page 31
Tracking Number: GRANT12240201 Funding Opportunity Number: PA-16-303. Received Date:

2016-09-02T13:28:53.000-04:00

SBIR/STTR Information

OMB Number: 4040-0001

Expiration date: 06/30/2016

SBIR-Specific Questions:

Questions 8 and 9 apply only to SBIR applications. If you are submitting ONLY an STTR application, leave questions 8 and 9 blank and

proceed to question 10.

8. Have you received SBIR Phase II awards from the Federal Government? If yes, provide a

company commercialization history in accordance with agency-specific instructions using this

attachment.*

❍ Yes ❍ No

Attach File:*

9. Will the Project Director/Principal Investigator have his/her primary employment with the small

business at the time of award?*

❍ Yes ❍ No

STTR-Specific Questions:

Questions 10 and 11 apply only to STTR applications. If you are submitting ONLY an SBIR application, leave questions 10 and 11

blank.

10. Please indicate whether the answer to BOTH of the following questions is TRUE:*

(1) Does the Project Director/Principal Investigator have a formal appointment or commitment either with the small business directly

(as an employee or a contractor) OR as an employee of the Research Institution, which in turn has made a commitment to the small

business through the STTR application process; AND

(2) Will the Project Director/Principal Investigator devote at least 10% effort to the proposed project?

● Yes ❍ No

11. In the joint research and development proposed in this project, does the small business

perform at least 40% of the work and the research institution named in the application perform at

least 30% of the work?*

● Yes ❍ No

Contact PD/PI: Wagner, David

 Page 32
PHS 398 Cover Page Supplement

OMB Number: 0925-0001

Expiration Date: 10/31/2018

Tracking Number: GRANT12240201 Funding Opportunity Number: PA-16-303 . Received Date:

2016-09-02T13:28:53.000-04:00

1. Human Subjects Section

Clinical Trial? ❍ Yes ● No

*Agency-Defined Phase III Clinical Trial? ❍ Yes ❍ No

2. Vertebrate Animals Section

Are vertebrate animals euthanized? ● Yes ❍ No

If "Yes" to euthanasia

Is the method consistent with American Veterinary Medical Association (AVMA) guidelines?

● Yes ❍ No

If "No" to AVMA guidelines, describe method and proved scientific justification

3. *Program Income Section

*Is program income anticipated during the periods for which the grant support is requested?

❍ Yes ● No

If you checked "yes" above (indicating that program income is anticipated), then use the format below to reflect the amount and

source(s). Otherwise, leave this section blank.

*Budget Period *Anticipated Amount ($) *Source(s)

Contact PD/PI: Wagner, David

 Page 33
PHS 398 Cover Page Supplement

Tracking Number: GRANT12240201 Funding Opportunity Number: PA-16-303 . Received Date:

2016-09-02T13:28:53.000-04:00

4. Human Embryonic Stem Cells Section

*Does the proposed project involve human embryonic stem cells? ❍ Yes ● No

If the proposed project involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the

following list: http://grants.nih.gov/stem_cells/registry/current.htm. Or, if a specific stem cell line cannot be referenced at this time,

please check the box indicating that one from the registry will be used:

Specific stem cell line cannot be referenced at this time. One from the registry will be used.

Cell Line(s) (Example: 0004):

5. Inventions and Patents Section (RENEWAL)

*Inventions and Patents: ❍ Yes ❍ No

If the answer is "Yes" then please answer the following:

*Previously Reported: ❍ Yes ❍ No

6. Change of Investigator / Change of Institution Section

❏ Change of Project Director / Principal Investigator

Name of former Project Director / Principal Investigator

Prefix:

*First Name:

Middle Name:

*Last Name:

Suffix:

❏ Change of Grantee Institution

*Name of former institution:

Contact PD/PI: Wagner, David

 Page 34
 

PHS 398 Research Plan

OMB Number: 0925-0001

Expiration Date: 10/31/2018

 

Tracking Number: GRANT12240201 Funding Opportunity Number: PA-16-303. Received Date:

2016-09-02T13:28:53.000-04:00

Introduction

1. Introduction to Application

(Resubmission and Revision)

Research Plan Section

2. Specific Aims 1243-Specific_Aims_Diabetes.pdf

3. Research Strategy* 1244-STTR_Diabetes_Peptide_Body.pdf

4. Progress Report Publication List

Human Subjects Section

5. Protection of Human Subjects

6. Data Safety Monitoring Plan

7. Inclusion of Women and Minorities

8. Inclusion of Children

Other Research Plan Section

9. Vertebrate Animals 1245-VertebrateAnimals.pdf

10. Select Agent Research 1246-Select_Agents.pdf

11. Multiple PD/PI Leadership Plan

12. Consortium/Contractual Arrangements  

13. Letters of Support

14. Resource Sharing Plan(s) 1247-Resource_Sharing.pdf

15. Authentication of Key Biological and/or

Chemical Resources

Appendix

16. Appendix

Contact PD/PI: Wagner, David

 Page 35
Significance: In 2012, 9.3% of the population corresponding to over 29.1 million Americans were 

diagnosed with diabetes; this from the American Diabetes Association Statistics Fact Sheet in 2016. There are 

estimated to be an additional 7 million undiagnosed and 86 million “pre-diabetic” people in the US , up from 79 

million in 2010. It is estimated from the most recent statistics than in 2012 1.25 million people had type 1 

diabetes with possibly 350,000 undiagnosed [American Diabetes Association 2016 Fact Sheet]. Both the

American Diabetes Association and the NIH estimate the yearly rate of new T1D diagnoses in the US to be 

approximately 95,000 people with that number inexplicably continuing to increase. T1D certainly affects 

children, but the fastest expansion in disease is occurring in individuals over age 20. Treatment for the disease 

is very limited; at present there are no medications that either prevent insulin loss, or restore insulin production.

Impact: Op-T-Mune, Inc. is a startup company created by Mr. Charlie Henry, entrepreneur, former CEO of 

Nostix, Inc., and Dr. David Wagner, Associate Professor at the University of Colorado School of Medicine. Our 

goals include the prevention and reversal of T1D by controlling the pathogenic cells that target islet beta cells

and lead to loss of insulin production [1-10]. The Wagner Lab identified T cells demarcated by CD40 

expression and reference them as Th40, in the NOD mouse model of T1D [3, 4, 6, 8-16]. An important

translational link followed in that Th40 cells are significantly (p < 10-4) increased in number in human T1D 

subjects, including long term and new onset diabetic subjects, compared to non-autoimmune controls [10]. 

Importantly, Th40 cells are expanded in number in a cohort of diagnosed pre-diabetic, type 1 human subjects 

(preliminary data), suggesting that induction of CD40 expression on T cells is involved early in disease 

development. Multiple studies show that controlling CD40 induced inflammation is both disease preventative 

and therapeutic [2, 9, 17-22]. Because CD40-expressing cells are highly pro-inflammatory and expand in 

number as T1D progresses [1, 5, 6, 9, 23, 24], they are a prime target for therapeutic control. Until now the 

only way to control CD40/CD154 interaction has been by monoclonal antibody or randomly generated, 

synthetic small molecules. Neither of these options has been successful and each creates unique

complications [25-28]. A therapeutic alternative that is effective and can be tolerated for long periods would 

therefore prove paradigm altering. 

We created a unique means to modulate CD40 through peptides derived from the CD154 protein sequence

that encompass known hot spots for receptor – ligand interaction. These peptides avoid the complications 

associated with antibodies, and are physiologically precise, unlike random generated CD40-like small organic 

molecules. We have identified potential candidates one of which has proven effective at preventing diabetes 

onset in 96% of NOD mice, total of 200 tested, and reversing hyperglycemia in 57% of treated new onset 

diabetic mice, totaling 24 mice thus far (preliminary data). 

Goals for Phase I: Establish a lead candidate peptide through:

1. Determining the impact on glucose tolerance testing (GTT) 

2. Determining the duration of protection KGYY15 versus KGYY6

3. Determining the impact on insulin and c-peptide production

Goals for Phase II: 

Determine the cells types that are impacted by the CD40 targeting peptide(s) 

Determine potential immunogenicity of treatment peptide in short-term and long-term studies

Determine how the treatment impacts natural immunity 

 Perform extended pharmaco-kinetic studies 

 Perform toxicity studies 

The end deliverable of the phase II funding will be to submit the IND application with the FDA. 

Innovation: While blocking CD40/CD154 interaction to control autoimmune inflammation is not innovative, the 

method we have developed is. Current immuno-modulatory therapies predominantly rely upon monoclonal 

antibody treatments, which are less than ideal. Antibodies cross-react with unintended targets and cause

nephritic complications. We propose using small peptides that bind to the CD40 receptor interactive site to 

prevent CD154 interaction. The other approach has been randomly generated, small organic molecules to 

theoretically target CD40. The approach, thus far, has proven completely imprecise and does not show clinical 

positive outcome. Our approach constitutes an entirely new way to precisely target known receptor/ligand pairs 

and avoid the complications and shortcomings of other methods. 

Contact PD/PI: Wagner, David

Specific Aims Page 36
Background of the Invention: T1D pathogenesis occurs when auto-aggressive cells infiltrate pancreatic 

islets, creating localized inflammation that leads to loss of insulin production. We determined that CD4+ effector

T cells become capable of expressing the CD40 receptor [3, 4, 6, 8-10, 12-15, 29], which then correlates with 

auto-inflammation. In the NOD model of T1D, CD40 is expressed on diabetogenic T cell clones [6] and as 

NOD mice progressively develop insulitis and eventually diabetes, CD40 expressing T cells (referred by us as

Th40 for simplicity) drastically increase as a percentage of the CD4+ compartment and in total number [3, 6, 8, 

9, 11]. Th40 cells alone from pre-diabetic or diabetic NOD mice are necessary and sufficient to transfer

diabetes to NOD.scid recipients, thus confirming cell type pathogenicity [6, 9]. Mice that receive CD40- T cells, 

even if those T cells were pre-activated, do not develop diabetes [8, 9]. Depletion of CD40 followed by

depletion of Tregs also does not transfer diabetes [8]. These data demonstrate that controlling CD40

expression or function is therapeutic in T1D.

Important translational studies showed that Th40 cells are significantly increased in number in human T1D 

subjects compared to non-autoimmune or type 2 diabetes controls [10] and Figure 1]. This includes newonset

as well as long – term (diagnosed up to 40 years in some cases) T1D subjects. In addition, we examinedblood

samples from recipients of islet transplants. That protocol requires severe immunosuppressive therapy for one 

year that is withdrawn over a 6-month period. Samples were taken 6 months after immunosuppressive therapy

had ceased and Th40 cells already had rebounded to pathogenic levels (Fig. 1). All those subjectseventually 

experienced islet transplant failure suggesting a pathogenic link between Th40 cell levels and islet loss in

human diabetes.

We examined samples from subjects meeting the American Diabetes Association (ADA) and NIDDKcriteria

for “pre-diabetes” determining that a cohort of those subjects has elevated Th40 cells (Fig. 2). Pre-diabetic 

subjects, normal glycemic index but many with abnormal glucose tolerance test (GTT) results, with Th40 cell 

levels at “high risk’ responded to human islets while pre-diabetic subjects with Th40 cell levels at “low risk”did 

not respond to human islets (not shown). These data demonstrate that elevated Th40 levels pre-date 

hyperglycemia onset but more importantly may be causal in diabetes pathogenesis. Th40 cells aresignificantly

increased in subjects carrying the T1D risk factors, HLA-DR4, DR3, DQ2 or DQ8 [10]. Importantly in subjects 

who do not carry the predictive haplotypes but nonetheless are

diabetic, Th40 cells occur at highly expanded numbers [10]. 

Furthermore, in non-autoimmune subjects who carry diabetes

associated haplotypes but do not develop T1D, Th40 percentages

remain at normal levels [10]. These findings suggest that expanded

percentages of Th40 cells are a more reliable risk factor/biomarker

than HLA haplotypes and that disease occurs irrespective of HLA

haplotype. In a double blind study measuring the levels of Th40

cells in individual subject (268 subjects thus far), we were 

successfully able to identify 97% of T1D subjects, differentiating

T1D from controls or T2D subjects.

Th40 cells from T1D but not from control subjects proliferate and

achieve effector function when exposed to insulin peptides, GAD 

peptides, and human islets [10, 13]. Th40 cells from T1D subjects 

but not from non-autoimmune controls produce significantly

elevated levels of IL-6, IFN and TNF, pro-inflammatory cytokines

[10]. CD40 on T cells serves as a functional co-stimulus even

independently of CD28, the classic T cell costimulus, inducing T 

cell activation, this makes CD40 an ideal candidate for

therapeutic control.

Controlling CD40 in Autoimmunity: Injecting an antibody

to block CD40 – CD154 interaction (anti-CD154) prevents 

diabetes onset in NOD mice [9, 17]. We determined that the

mechanism of action was to control the number of Th40 cells

[9]. Importantly, Th40 cells were not eliminated but were

contained at normal levels [9]. Based on these observations, 

CD40 proves to be not only a reliable biomarker in T1D but

serves as a target molecule for controlling destructive T cell

mediated inflammation.

Modulating CD40 – CD154 interactions thus far has relied

Figure 1: Th40 cells in human T1D subjects 

compared to controls. Th40 cell levels were 

determined from peripheral blood of T1D (n=268); 

controls (n=172); T2D (n=82); pT1D (n=32) subjects 

recruited from the Barbara Davis Childhood Diabetes 

Clinic. pT1D are NIH TrialNet subjects, meeting NIH 

and ADA criteria for pre-diabetes. Subjects were all 

healthy. T1D diagnosis met the ADA and NIH criteria. 

Average age of T1D is 26.2, control is 33.7. T1D 

subjects were further categorized as new onset 

(diagnosis < 6 months) and long term (diagnosis > 2 

years, in some cases 40 years). Islet Transplant 

recipients (4 total) were > 2 years’ post-transplant.

80

not significant

60 p < 0.0001

40

20

not significant

%

0

Th40 in peripheral blood

Contact PD/PI: Wagner, David

Research Strategy Page 37
Contact PD/PI: Wagner, David

A.

upon monoclonal antibodies, specifically anti-CD154, or randomly generated small synthetic molecules.Using 

anti-CD154 proved unsuccessful in clinical trials because platelets express CD154 and the treatmentresulted

in platelet aggregation and life-threatening thrombotic events [30] halting the Phase II trials. The block of 

CD40/CD154 was efficacious towards controlling the autoimmune diseases; the problems were that the antiCD154 antibodies coagulated platelets [31-33]. Attempting to control CD40 using anti-CD40 has likewise 

proven non-fruitful, because the anit-CD40 antibodies that have been developed thus far are allagonistic.

While this approach works well in cancer treatment (there currently is a Phase 2 trial anti-CD40 antibody) it is

ineffective in autoimmunity that results from CD40 induced inflammation. Treating with anti-CD40 antibodywill

only exacerbate the autoimmune condition. The organically synthesized, randomly generated small molecule 

option also proved ineffective, lacking any specificity and demonstrating general performance failure as well as

resulting in relatively high toxicity [34]. In view of these studies, we devised an innovative approach toblocking

CD40 – CD154 interactions avoiding use of antibodies or synthetic organic chemical approaches. Wedesigned

peptides derived from the CD154 protein sequence that are capable of binding directly to the CD40 receptorto

interrupt the inflammatory signal pathways. This approach is highly specific while avoiding toxicityproblems.

Design of the peptides: The

interaction motif between

CD154 and CD40 was identified

[35, 36]. Mutational analysis in

the CD154 protein sequence 

suggested that a lysine (K) and

two tyrosine (Y) residues at 

positions 145, 147 and 148,

respectively, are necessary for 

CD40 interaction [36].

Considering this, we designed a 

series of small peptides that

encompass those three amino

acids (Fig. 2). Each peptide

Figure 2: Design of the KGYY15 peptide. Based on the amino acid sequence of the 

murine CD154 sequence (the human sequence is >90% homologous) a series of 

peptides was generated. The known CD40/CD154 interaction amino acids are 

highlighted. The 15-mer peptide is in green. The 15-mer sequence at the bottom shows 

the amino acids that are essential for functionality (orange) of the KGYY15 peptide in 

diabetes prevention assays. The relative peptide stability assessed by ExPaSY analysis 

is shown to the far right.

contains the K-G-Y-Y motif and the additional amino

acids were taken from the actual CD154 protein 

sequence, thus all the peptides represent naturally 

occurring products. The stability of each peptide was

initially assessed by ExPaSY, a software program that

evaluates chemical stability based on numerous 

parameters; those values are indicated (Fig. 2). Stability 

was further measured using mass spec. The most stable 

peptide determined by both measures was the 15-mer 

(green), even though that peptide contains methionine in 

position 12 (Fig. 3). Structure to Activity Relationship 

(SAR) analysis revealed that the methionine residue

surprisingly was critical to diabetes prevention

functionality (Figure 5). The SAR was done under the

Figure 3, KGYY15 prevents diabetes onset in NOD mice. (A) 

NOD mice at 6 weeks of age were injected weekly iv with 25 ug (1

mg/kg) of each peptide through 45 weeks. Each cohort included 10

animals. Blood glucoses were measured and levels at 250 mg/dl or 

higher for 3 consecutive measures were considered diabetic. The 

RGD peptide is a 15 amino acid sequence from the CD154 

sequence that does not include the CD40 binding motif. (B) 

Pancreata were excised and examined by histology for cellular 

infiltrates: 0 = no infiltrate; 1 = one pole infiltrate; 2 = peri-insulitis,bipolar infiltrates; 3 = 75% infiltrate and 4 = full infiltration. (C) 

Representative level 2 islet. (D) CD4 to CD8 ratio in treated versus 

diabetic mice. Mice were gender and age matched. Data represent 

several experiments over a 5-year period.

100

80

60

13-mer

15-mer

Scrambled 

24-mer

6-mer

RGD peptide

40

20

0

Weeks of Age

B. C.

D.

% Diabetic

Research Strategy Page 38
100

80

60

40

Scrambled 

Gly-1

GLY-2

Gly-3 

Gly-4 

Gly-5 

Gly-6 

GLY-7

15-mer KGYY15

Gly-9 

GLY-10

 Gly-11

GLY-12

20

0

Age

guidance of Dr. John Carpenter, PhD, Co-Director of the University of Colorado Center for Pharmaceutical

Biotechnology. Dr. Carpenter has extensive expertise in peptide to drug development and drug formulation and

his expertise is sought out by many Pharmaceutical companies.

Preliminary Data: We established that a monoclonal antibody recognizing CD154 prevents diabetesonset

in NOD mice and that prevention coincides with lower levels of Th40 cells in treated mice only [9].Numerous

studies show that blocking CD40 prevents inflammation and prevents or improves a variety of autoimmune

diseases [9, 17, 18, 37, 38]. To address how our CD40 - targeting peptides perform, we administered each 

peptide to NOD mice and monitored diabetes

onset (Fig. 3). Cohorts of 6 – week old mice, 

10 in each group, were treated with the

different peptides (Fig. 3). The data from six 

different experiments were consolidated.

Based on the size, i.e. number of amino acids

in the peptide, diabetes was increasingly 

controlled in treated animals (Fig. 3). The

peptides at 8 and 10 amino acids were 

ineffective at diabetes prevention (not shown); 

while the 13-mer and 15-mer, were quite 

effective at diabetes prevention. The 15-mer in 

fact was highly effective, preventing diabetes in 

90% of recipients. A larger 24-mer peptide was

completely ineffective at controlling diabetes

(Fig. 3). Interestingly, the 6 amino acid peptide

was reasonably effective at prevention. A 

control peptide, RGD, composed of a 15 amino

acid region upstream from the KGYY section

that does not contain the CD40 binding motif, 

did not impact diabetes prevention (Fig. 3). In 

addition, a scrambled version of the KGYY15 

peptide did not impact diabetes prevention.

Mice were monitored for onset and severity of 

diabetes as defined by hyperglycemia and

Figure 4: Structure to Activity Relationship in the KGYY15 

peptide: Each amino acid of the 15-mer peptide was sequentially 

replaced by Glycine. Solubility and stability of each was assessed by 

mass spec, as described above. 25 ug (1 mg/kg) doses were 

administered weekly for 45 weeks. Each treatment group included 10 

female NOD mice. Diabetes was considered blood glucose levels of > 

250 mg/dl for 3 separate readings in a one-week period. Controls 

included the scrambled peptide (control) and the 15-mer KGYY15 

unaltered (control) peptide. Positions 2, 9, 11 and 12 were determined 

to be critical. Interestingly position 12 methionine was critical for 

diabetes prevention functionality.

glucosuria. We determined that KGYY15 treated mice 

had significantly (p < 0.01) sustained islet insulin

granulation (not shown). Histology of pancreata from 

KGYY15 treated mice demonstrated some islet infiltration 

(Fig. 3 B and C) but significantly (p < 0.001) less than

untreated mice (Fig. 3 B). These data demonstrate 

certain of the peptide promotes islet cyto-protection. The

numbers and proportionality of CD4 and CD8 cells were 

normal in long term (15 weeks) peptide treated mice (Fig.

3 D). B cell numbers were also at normal levels (not

shown). While not conclusive, these data suggest that

the peptide has limited negative effect on the overall 

immune system, but impacts the Th40 pathogenic 

compartment of that system.

Structure to Activity Relationship (SAR). We

addressed the amino acids in the 15-mer peptide that are 

necessary for diabetes prevention activity. A glycine

walk, sequential substitution of Gly for each amino acid in 

the peptide, was conducted. Substitutions at the Tyr 

position number 9, Thr 11 and Met 12 proved critical for

Figure 5: KGYY15 reverses hyperglycemia in new 

onset diabetic NOD mice. NOD mice were monitored for 

blood glucose levels > 250 mg/dl and were then treated 

with 4 mg/kg KGYY15 followed by an additional dose 2 

days later and a half dose weekly after that. Blood 

glucose levels were monitored with ‘normal’ determined to

be 120 mg/dl or less. Untreated NOD mice demonstrate

normal diabetes kinetics (red line). Treated diabetic 

mice demonstrate reversal of hyperglycemia (green 

line). Data represent 24 mice, with 12 reversals.

diabetes prevention activity (Fig. 4). Methionine reportedly undergoes oxidation more easily than other amino

acids and is considered to restrict peptide stability. However, from this SAR data the methionine in KYGG15is

100

80

60

40 No Peptide

KGYY15

20

0

Weeks post treatment

% Diabetic

%Diabetic

Contact PD/PI: Wagner, David

Research Strategy Page 39
critical for function. We determined that the KGYY15 peptide containing methionine at position 12 is stable, 

demonstrating < 1% breakdown (determined by mass spec analysis) over 6 weeks in solution. The 24-mer 

peptide that did not prevent hyperglycemia experienced rapid breakdown, within 60 minutes, as measuredby

mass spec and as predicted by ExPaSY analysis. This suggests that placement of methionine in thepeptide

dictates overall stability as methionine is more centrally located in the 24-mer peptide and occurs nearthe

terminus of the 15-mer peptide.

Reversing Diabetes: We examined how KGYY15 treatment affects established hyperglycemia in newonset

diabetic NOD mice. Mice with blood glucose greater than 250 mg/dl, were treated with KGYY15 at

hyperglycemia onset. KGYY15 at a concentration of 4 mg/kg was administered intravenously (i.v.) in a single

dose and boosted daily for 7 days. Within 2 days 25% of treated mice had normal (< 120 mg/dl) bloodglucose 

levels and at 3 weeks 57% were euglycemic (Fig. 5).

We determined that KGYY15 directly binds to CD40 cells using in vitro assays. PBL from mice werepurified

and stained with an anti-CD40 antibody and co-stained with a fluorescenated peptide. Human cells were 

stained with anti-CD4 and the peptide. The peptide remained stably bound to T cells for 5 days in cell culture.

Importantly we determined that KGYY15 does not induce clotting (determined with the Department of 

Hematology at the University of Colorado). Human blood treated with KGYY15 had the same clot time as

untreated (data not shown). Blood treated with anti-CD154 clots much more rapidly as the antibody bindsand

coagulates.

Approach: Our goal is to prevent and reverse diabetes onset by controlling the inflammatory CD40 pathway. 

We are developing targeted peptides that block the CD40 receptor. We tested 8 different peptides, each based

on the CD40 interaction site of CD154 protein (Fig. 2). Two peptides show high efficacy at prevention and one, 

thus far, has shown high potential for hyperglycemia reversal (Fig. 4). In order to advance this therapeutic, we 

require reliable clinical readouts that eventually can be tested in human clinical trials. The goal of this phase1 

study is to determine a lead candidate peptide based on impact on glucose tolerance testing, sustained insulin 

production, measured in serum and observed in pancreatic islets, and impact on c-peptide levels as ameasure 

of native insulin production.

Research Approach:

Goal # 1: Impact of the lead candidates on glucose tolerance testing. A final diagnosis of diabetes is 

relatively straight forward, high serum glucose and a failed GTT. Determining risk for developing diabetes is 

much less obvious. In the Diabetes Prevention Trial – Type 1 (DPT-1), a 12-hr fasting blood glucose of>126

mg/dl or a sustained oral GTT > 200 mg/dl, constitutes a diagnosis of T1D [39]. As subjects progress towards

diabetes, glucose tolerance becomes less stable. For example, the clinical designation of pre-T1D includesan

abnormal or deviated GTT. NOD mice, like human subjects who eventually develop T1D, undergo a relatively 

extensive pre-diabetes phase [40]. Over time, beta cell mass reduces substantially, finally insulin secretion is 

halted, thus establishing a diabetic state. As mice progress towards diabetes and beta cell mass is lost, the

mice experience abnormal glucose tolerance [40]. To develop a clinical measure for our therapeutic, we will 

determine how each peptide impacts GTT over time. We will perform GTT on NOD mice at different stages of 

diabetes development; 2 - 4 weeks (no insulitis), 6 – 9 weeks (moderate insulitis, early “pre-Diabetes”, 12 – 15

weeks (extensive insulitis, late ‘pre-diabetes”) and after diabetes onset. As controls we will perform GTT on

diabetes resistant, BALB/c mice. We will inject KGYY15 or KGYY6 into all mice using the prevention (1mg/kg 

weekly for 12 weeks) strategy. Routes of administration will include IV, intra-peritoneal (IP), andsubcutaneous

(SC). Tests will be done on 5 of each test animal (NOD mice) compared to 2 controls (BALB/c); this powers 

the study to 95% confidence as determined by GraphPad Prism statistical analysis program. After 1 week, and

every week following for up to 12 weeks, a GTT will be performed. Recovery rates, how quickly each animal

reduces injected glucose will be determined. Changes over time will be recorded.

Outcomes and concerns: The technical issues of this type of analysis are straight forward, the GTT is 

straight forward. We predict that the therapeutics will improve GTT scores over the proposed time periods. Our 

strategy will determine if the 15-mer or 6-mer peptide provide optimal protection, thus establishing a singlelead

candidate. If no differences are detected, we will alter the dosing strategy and re-examine. If successful, this 

analysis will provide important documentation that GTT after therapeutic treatment is an appropriate monitor.

We established both protective and reversal strategies in preliminary data. This aim clarifies a moreclinically 

appropriate monitor that can be developed for eventual human trials. Future considerations: In Phase 2studies

we will address more long-term issues. Can either peptide be used after diabetes onset to 1) improve GTT; 2)

Contact PD/PI: Wagner, David

Research Strategy Page 40
improve natural insulin production and 3) reverse hyperglycemia. Time frame. Because we will befollowing 

mice for long periods, up to 45 weeks of age, 12 months will be required to complete the proposedstudies.

Note: In phase 2 studies, PK will be done on both the 15-mer and the 6-mer. The 15-mer peptide ismore 

stable than other peptides and therefore may prove to have more long-lasting effect. The 6-mer has amuch 

shorter predicted stability profile and may therefore prove to be fast – acting. Thus both options couldprove 

highly valuable for drug development.

Goal #2: Determine the duration of protection using KGYY peptides? KGYY15 [2] and KGYY6 (Fig.

3) efficaciously control hyperglycemia when administered at 1 mg/kg on a weekly basis. It is important to 

determine how long each peptide is effective at sustaining euglycemia. This will be necessary fordeveloping

dosing standards. Experiment #1: Female NOD mice, 11 from each age group compared to 22 controls

(This study is powered at 90% confidence determined by ClinCal.com sample size calculator) at the ages

described above will be treated with 1, 2, 4, 8, 16 and 32 mg/kg of KGYY15 and KGYY6 in a single dose. 

Routes of administration will include: IV, IP, SC and IM. Mice then will be monitored until hyperglycemia

occurs.

Experiment #2: Female NOD mice will be treated with 1, 2, 4 and 8 mg/kg of each peptide at 2 and 3 week

intervals for 18 weeks. Mice will be continually monitored for hyperglycemia. Outcomes and concerns: The 

proposed experiments are straight forward. We have extensive experience with this experimental approach

[2]. By performing dose escalation studies, we will effectively determine the most efficacious dose and route

of administration. While the primary concern is lack of success, we showed that a 1mg/kg dose of KGYY15 

administered weekly, prevents diabetes onset (Fig 3 and [2]); therefore, relative success isassured.

Goal #3: We will determine how each of the peptides maintain insulin and c-peptide. Mice will be 

treated as described in Goal 2, i.e. untreated, versus the various concentrations of KGYY15 and KGYY6.

NOD and control BALB/c mice will be treated. At weekly intervals we will examine serum levels of insulin 

and c- peptide, using commercially available kits. At each time point, some mice will be removed for

pancreatic analysis, including islet mass determination, and histology to determine islet infiltration and 

insulin production. Histologic sections will be stained by H&E to measure cell infiltrates and with anti-insulin. 

Outcomes and concerns: These studies will correlate insulin production in the pancreas to corresponding 

levels that can be detected in serum. This type of analysis cannot be performed in human. However, given 

that the NODT1D model is a reasonable model for human disease; and more importantly, because CD40 

now has beenclearly demonstrated to have strong pathogenic roles in NOD mice [1, 2, 4, 5, 8, 11, 23, 24, 

41] and human disease [7, 10, 13], this approach is valid. The outcome will be to determine that serum 

insulin levels and c-peptide levels will be sustained by the peptide treatments. That is, while the peptides are 

preventing hyperglycemia, insulin production and c-peptide can be detected in serum. This will translate as 

another clinical measurefor eventual human clinical trials.

Milestones and Rationale: Preliminary data establish proof of principle i.e., the 15-mer peptide prevents

and reverses diabetes onset. An interesting aspect is that the 15-mer peptide is extremely stable, even 

though it contains methionine at position 12 and the 6-mer, while relatively effective at diabetes prevention 

has ashort half-life. Either option binds extracellularly as opposed to requiring cellular uptake for 

functionality. Thus our peptides are less subjected to cellular breakdown. When the receptor with bound 

peptide is internalized,the drug can be re-administered; this may explain the functionality of the 6-mer, 

which will be addressed specifically in phase 2. We speculate that the cell surface binding alters cellular 

responses. It is possiblethat the peptide binds to dendritic cells, B cells, or monocytes and alters the 

response of those cells to generate pathogenic effector cells. In either case, the administration of the 

peptide prevents T1Donset.

Time course: Goal 1 will require 12 months. Goals 2 and 3 can be done concurrently. These projects

should be completed in 12 months.

Phase II studies will focus on PK and toxicology studies and will require up to 4 years. An expert in

peptide drug development, Dr. John Carpenter will be recruited as a collaborator for the phase 2 application.

Dr. Carpenter and his team will greatly facilitate development of KGYY15 from a bulk drug substance to a

viable drug option. In addition, Op-T-mune has hired a consulting firm, CliniPace, LLC, for advancing IND

filing. Patent coverage for the peptides is in place PCT Application No. PCT/US2015/022033. Office action:

We received notification that the patent will be awarded in the US and Europe.

Contact PD/PI: Wagner, David

Research Strategy Page 41
1. Animals to be used in this study. Animals used in this study will include NOD 

mice that spontaneously develop type 1 diabetes, NOD.scid for adoptive 

transfers and BALB/c as controls. Mice will be injected with each of the test 

peptides, scrambled control, non-relevant peptide control and vehicle controls.

Based on previous observations where significant differences in disease onset

were achieved sample size powered to 90% require 10 mice per treatment 

group. There will be 5 treatment groups and 5 different treatment concentrations

requiring 250 mice. Glucose tolerance testing will be done on each set of mice.

NOD.scid recipient mice will be used for adoptive transfers requiring 80 mice

based on previous cell transfer studies. 

2. Justification of animal numbers. The number of mice required for statistical 

significance was determined by t-test sample size calculations based on an alpha 

of 0.05, confidence of 0.95 and known differences of the mean between age of 

onset and delayed onset with treatments.

3. Veterinary care for animals. All animals will be housed at the University of 

Colorado Denver, vivarium. Four full time veterinarians and a large Vet Tech staff 

are available. The UCD animal facility is full accredited by PHS and AAALAC. All 

procedures are performed under IACUC approved protocols.

4. Discomfort, distress, pain and injury are always alleviated. Most procedures 

require only simple injections. There is limited discomfort, distress pain or injury

with injections. Discomfort and distress are associated with diabetes

development, but animals are never allowed to proceed to severe categories. At 

hyperglycemia animals typically are euthanized immediately. If animals are 

maintained for longer than 1 week when diabetic, animals are administered 

insulin and blood glucose levels monitored. Analgesics will be provided when 

they don’t alter experimental results. Insulin will be provided when animals 

become hyperglycemic. Glucose tolerance testing will involve injecting mice with 

high dose glucose and monitoring by tail vein clip to obtain blood. Blood glucose 

is analyzed every 30 mins, but additional tail clip is not required, just removing 

the scab.

5. Euthanasia of Mice: Euthanasia of mice will be done by controlled CO2 

exposure followed by cervical dislocation. This method is the more common and 

least painful method available. This method is approved by the Panel on 

Euthanasia of the American Veterinary Medical Association, and by the UCD 

IACUC.

Contact PD/PI: Wagner, David

Vertebrate Animals Page 42
No select agents will be used. 

Contact PD/PI: Wagner, David

Select Agent Research Page 43
REFERENCES:

1. Vaitaitis GM, Carter JR, Waid DM, Olmstead MH, Wagner DH, Jr.: An alternative role for Foxp3 as 

an effector T cell regulator controlled through CD40. J Immunol 2013, 191(2):717-725.

2. Vaitaitis GM, Olmstead MH, Waid DM, Carter JR, Wagner DH, Jr.: A CD40-targeted peptide controls 

and reverses type 1 diabetes in NOD mice. Diabetologia 2014, 57(11):2366-2373.

3. Vaitaitis GM, Poulin M, Sanderson RJ, Haskins K, Wagner DH, Jr.: Cutting edge: CD40-induced 

expression of recombination activating gene (RAG) 1 and RAG2: a mechanism for the 

generation of autoaggressive T cells in the periphery. J Immunol 2003, 170(7):3455-3459.

4. Vaitaitis GM, Wagner DH, Jr.: High distribution of CD40 and TRAF2 in Th40 T cell rafts leads to 

preferential survival of this auto-aggressive population in autoimmunity. PLoS One 2008, 

3(4):e2076.

5. Vaitaitis GM, Wagner DH, Jr.: CD40 glycoforms and TNF-receptors 1 and 2 in the formation of 

CD40 receptor(s) in autoimmunity. Mol Immunol 2010, 47(14):2303-2313.

6. Wagner DH, Jr., Vaitaitis G, Sanderson R, Poulin M, Dobbs C, Haskins K: Expression of CD40 

identifies a unique pathogenic T cell population in type 1 diabetes. Proc Natl Acad Sci U S A 

2002, 99(6):3782-3787.

7. Waid DM, Schreiner T, Vaitaitis G, Carter JR, Corboy JR, Wagner DH, Jr.: Defining a new biomarker 

for the autoimmune component of Multiple Sclerosis: Th40 cells. J Neuroimmunol 2014, 270(1-

2):75-85.

8. Waid DM, Vaitaitis GM, Pennock ND, Wagner DH, Jr.: Disruption of the homeostatic balance 

between autoaggressive (CD4+CD40+) and regulatory (CD4+CD25+FoxP3+) T cells promotes 

diabetes. J Leukoc Biol 2008, 84(2):431-439.

9. Waid DM, Vaitaitis GM, Wagner DH, Jr.: Peripheral CD4loCD40+ auto-aggressive T cell expansion 

during insulin-dependent diabetes mellitus. Eur J Immunol 2004, 34(5):1488-1497.

10. Waid DM, Wagner RJ, Putnam A, Vaitaitis GM, Pennock ND, Calverley DC, Gottlieb P, Wagner DH, 

Jr.: A unique T cell subset described as CD4loCD40+ T cells (TCD40) in human type 1 diabetes. 

Clin Immunol 2007, 124(2):138-148.

11. Vaitaitis G, Waid DM, Wagner Jr. DH: The expanding role of TNF-receptor super family member 

CD40 (tnfrsf5) in autoimmune disease: Focus on Th40 cells. Curr Immunol Rev 2010, 6(2):130-

136.

12. Vaitaitis G, Wagner Jr. DH: CD40 Glycoforms and TNF-Receptors 1 and 2 in the Formation of 

CD40 Receptor(s) in Autoimmunity. Mol Immunol 2010, 47:2303-2313.

13. Siebert JC, Inokuma M, Waid DM, Pennock ND, Vaitaitis GM, Disis ML, Dunne JF, Wagner DH, Jr., 

Maecker HT: An analytical workflow for investigating cytokine profiles. Cytometry A 2008, 

73(4):289-298.

14. Vaitaitis GM, Poulin M, Sanderson RJ, Haskins KJ, Wagner Jr. DH: CD40-Induced Expression of 

Recombination Activating Gene (RAG) 1 and RAG2: A Mechanism for the Generation of

Autoaggressive T Cells in the Periphery. Cutting Edge, J Immunol 2003, 170:3455-3459.

15. Baker RL, Wagner DH, Jr., Haskins K: CD40 on NOD CD4 T cells contributes to their activation 

and pathogenicity. J Autoimmun 2008, 31(4):385-392.

16. Wagner DH, Jr.: The co-evolution of our understanding of CD40 and inflammation. Diabetologia 

2009, 52(6):997-999.

17. Balasa B, Krahl T, Patstone G, Lee J, Tisch R, McDevitt HO, Sarvetnick N: CD40 Ligand-CD40 

interactions are necessary for the initiation of insulitis and diabetes in nonobese diabetic mice.

Journal of Immunology 1997, 159:4620-4627.

18. Girvin AM, Dal Canto MC, Miller SD: CD40/CD40L interaction is essential for the induction of EAE 

in the absence of CD28-mediated co-stimulation. J Autoimmun 2002, 18(2):83-94.

19. Howard LM, Dal Canto MC, Miller SD: Transient anti-CD154-mediated immunotherapy of ongoing 

relapsing experimental autoimmune encephalomyelitis induces long-term inhibition of disease 

relapses. J Neuroimmunol 2002, 129(1-2):58-65.

20. Howard LM, Miga AJ, Vanderlugt CL, Dal Canto MC, Laman JD, Noelle RJ, Miller SD: Mechanisms of 

immunotherapeutic intervention by anti-CD40L (CD154) antibody in an animal model of multiple 

sclerosis. J Clin Invest 1999, 103(2):281-290.

Contact PD/PI: Wagner, David

References Cited Page 44
21. Howard LM, Ostrovidov S, Smith CE, Dal Canto MC, Miller SD: Normal Th1 development following 

long-term therapeutic blockade of CD154-CD40 in experimental autoimmune encephalomyelitis. 

J Clin Invest 2002, 109(2):233-241.

22. Durie FH, Fava RA, Foy TM, Aruffo A, Ledbetter JA, Noelle RJ: Prevention of collagen-induced 

arthritis with an antibody to gp39, the ligand for CD40. Science 1993, 261(5126):1328-1330.

23. Carter J, Vaitaitis GM, Waid DM, Wagner DH, Jr.: CD40 engagement of CD4+ CD40+ T cells in a 

neo-self antigen disease model ablates CTLA-4 expression and indirectly impacts tolerance. Eur 

J Immunol 2012, 42(2):424-435.

24. Vaitaitis GM, Wagner DH, Jr.: Galectin-9 controls CD40 signaling through a Tim-3 independent 

mechanism and redirects the cytokine profile of pathogenic T cells in autoimmunity. PLoS One 

2012, 7(6):e38708.

25. Davis JC, Jr., Totoritis MC, Rosenberg J, Sklenar TA, Wofsy D: Phase I clinical trial of a monoclonal 

antibody against CD40-ligand (IDEC-131) in patients with systemic lupus erythematosus. J 

Rheumatol 2001, 28(1):95-101.

26. Kalunian KC, Davis JC, Jr., Merrill JT, Totoritis MC, Wofsy D, Group I-LS: Treatment of systemic 

lupus erythematosus by inhibition of T cell costimulation with anti-CD154: a randomized, 

double-blind, placebo-controlled trial. Arthritis Rheum 2002, 46(12):3251-3258.

27. Nakayamada S, Saito K, Nakano K, Tanaka Y: Activation signal transduction by beta1 integrin in T 

cells from patients with systemic lupus erythematosus. Arthritis Rheum 2007, 56(5):1559-1568.

28. Koyama I, Kawai T, Andrews D, Boskovic S, Nadazdin O, Wee SL, Sogawa H, Wu DL, Smith RN, 

Colvin RB et al: Thrombophilia associated with anti-CD154 monoclonal antibody treatment and 

its prophylaxis in nonhuman primates. Transplantation 2004, 77(3):460-462.

29. Vaitaitis GM, Wagner DH, Jr.: CD40 glycoforms and TNF-receptors 1 and 2 in the formation of 

CD40 receptor(s) in autoimmunity. Mol Immunol, 47(14):2303-2313.

30. Sidiropoulos PI, Boumpas DT: Lessons learned from anti-CD40L treatment in systemic lupus 

erythematosus patients. Lupus 2004, 13(5):391-397.

31. Henn V, Steinbach S, Buchner K, Presek P, Kroczek RA: The inflammatory action of CD40 ligand 

(CD154) expressed on activated human platelets is temporally limited by coexpressed CD40. 

Blood 2001, 98(4):1047-1054.

32. Danese S, Fiocchi C: Platelet activation and the CD40/CD40 ligand pathway: mechanisms and 

implications for human disease. Crit Rev Immunol 2005, 25(2):103-121.

33. Libby P: Multiple mechanisms of thrombosis complicating atherosclerotic plaques. Clinical 

cardiology 2000, 23 Suppl 6:VI-3-7.

34. Margolles-Clark E, Jacques-Silva MC, Ganesan L, Umland O, Kenyon NS, Ricordi C, Berggren PO, 

Buchwald P: Suramin inhibits the CD40-CD154 costimulatory interaction: a possible mechanism 

for immunosuppressive effects. Biochem Pharmacol 2009, 77(7):1236-1245.

35. Bajorath J, Seyama K, Nonoyama S, Ochs HD, Aruffo A: Classification of mutations in the human 

CD40 ligand, gp39, that are associated with X-linked hyper IgM syndrome. Protein Sci 1996, 

5(3):531-534.

36. Bajorath J, Chalupny NJ, Marken JS, Siadak AW, Skonier J, Gordon M, Hollenbaugh D, Noelle RJ, 

Ochs HD, Aruffo A: Identification of residues on CD40 and its ligand which are critical for the 

receptor-ligand interaction. Biochemistry 1995, 34(6):1833-1844.

37. Daoussis D, Antonopoulos I, Andonopoulos AP, Liossis SN: Increased expression of CD154 (CD40L) 

on stimulated T-cells from patients with psoriatic arthritis. Rheumatology (Oxford) 2007, 

46(2):227-231.

38. Desai-Mehta A, Lu L, Ramsey-Goldman R, Datta S: Hyperexpression of CD40 ligand by B and T 

cells in human lupus and its role in pathogenic autoantibody production. J Clin Invest 1996, 

97:2063-2073.

39. Sosenko JM, Skyler JS, Herold KC, Palmer JP, Type 1 Diabetes T, Diabetes Prevention Trial-Type 1 

Study G: The metabolic progression to type 1 diabetes as indicated by serial oral glucose 

tolerance testing in the Diabetes Prevention Trial-type 1. Diabetes 2012, 61(6):1331-1337.

40. Liang K, Du W, Zhu W, Liu S, Cui Y, Sun H, Luo B, Xue Y, Yang L, Chen L et al: Contribution of 

different mechanisms to pancreatic beta-cell hyper-secretion in non-obese diabetic (NOD) mice 

during pre-diabetes. J Biol Chem 2011, 286(45):39537-39545.

41. Vaitaitis GM, Wagner DH, Jr.: CD40 interacts directly with RAG1 and RAG2 in autoaggressive T 

cells and Fas prevents CD40-induced RAG expression. Cell Mol Immunol 2013, 10(6):483-489.

Contact PD/PI: Wagner, David

References Cited Page 45
Resource Sharing: The resources in this grant include proprietary peptides. Patent coverage for the 

peptides is in place. Otherwise, standard mice that are commercially available will be used.

Contact PD/PI: Wagner, David

Resource Sharing Plan(s) Page 46