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1 CONTRACT NUMBER: W81XWH TITLE: Expliting Inhibitry Siglecs t Cmbat Fd Allergies PARTNERING INVESTIGATOR: Matthew Macauley, Ph.D. CONTRACTING ORGANIZATION: The Scripps Research Institute La Jlla, CA REPORT DATES: Octber 2017 TYPE OF REPORT: Annual PREPARED FOR: U.S. Army Medical Research and Materiel Cmmand Frt Detrick, Maryland DISTRIBUTION STATEMENT: Apprved fr Public Release; Distributin Unlimited The views, pinins and/r findings cntained in this reprt are thse f the authr(s) and shuld nt be cnstrued as an fficial Department f the Army psitin, plicy r decisin unless s designated by ther dcumentatin.

2 REPORT DOCUMENTATION PAGE Frm Apprved OMB N Public reprting burden fr this cllectin f infrmatin is estimated t average 1 hur per respnse, including the time fr reviewing instructins, searching existing data surces, gathering and maintaining the data needed, and cmpleting and reviewing this cllectin f infrmatin. Send cmments regarding this burden estimate r any ther aspect f this cllectin f infrmatin, including suggestins fr reducing this burden t Department f Defense, Washingtn Headquarters Services, Directrate fr Infrmatin Operatins and Reprts ( ), 1215 Jeffersn Davis Highway, Suite 1204, Arlingtn, VA Respndents shuld be aware that ntwithstanding any ther prvisin f law, n persn shall be subject t any penalty fr failing t cmply with a cllectin f infrmatin if it des nt display a currently valid OMB cntrl number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. 1. REPORT DATE 2. REPORT TYPE 3. DATES COVERED Annual Octber TITLE AND SUBTITLE Expliting Inhibitry Siglecs t Cmbat Fd Allergies September 30, September 29, a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Michael Kulis, PhD Principal Investigatr PP Matthew Macauley, PhD Partnering PI mmacaule@scripps.edu and/r macauley@ualberta.ca 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) The Scripps Research Institute N. Trrey Pines Rad La Jlla, CA SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) U.S. Army Medical Research and Materiel Cmmand Frt Detrick, Maryland d. PROJECT NUMBER 5e. TASK NUMBER 5f. WORK UNIT NUMBER 8. PERFORMING ORGANIZATION REPORT NUMBER 10. SPONSOR/MONITOR S ACRONYM(S) 11. SPONSOR/MONITOR S REPORT NUMBER(S) 12. DISTRIBUTION / AVAILABILITY STATEMENT Apprved fr Public Release; Distributin Unlimited 13. SUPPLEMENTARY NOTES Please nte that fr the cming year, Dr. James Paulsn (TSRI) will be taking ver as the Partnering PI, as Dr. Macauley has mved t the University f Alberta (Edmntn, Alberta, Canada). Dr. Macauley will cntinue t participate in the prject as a sub-cntract frm TSRI. An fficial request fr this change in PI was submitted as a separate package. 14. ABSTRACT During this first year f the award, we were able t prduce imprtant data, develp a new tl t track allergen-specific B cells, and generate tw humanized muse mdels. We demnstrated that Ah2 STALs targeting CD22 n B cells can prevent IgE prductin and allergic reactins with nly a single injectin in mice. We als demnstrated that Ah2 STALs targeting CD33 n human mast cells and basphils can prevent degranulatin in cell culture experiments. A nvel tl, Ah2 tetramer, was prduced t quantify numbers f allergenspecific B cells in mice. A mdel t test Ah2 STALs as a therapy was develped in which naïve mice were adptively transferred memry B and T cells frm peanut allergic mice. The resulting mice make rbust levels f Ah2-IgE and experience anaphylaxis upn challenge. This mdel will be used t test STALs as a therapy t deplete memry B cells. Furthermre, tw nvel transgenic muse mdels were generated, ne expresses human CD22 n B cells and the ther expresses human CD33 n mast cells. These mdels are vital t ur prpsed research. 15. SUBJECT TERMS Fd allergy; peanut allergy; Siglec; IgE; Ara h 2; CD22; CD33; mast cell; basphil 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT a. REPORT U b. ABSTRACT U c. THIS PAGE U 18. NUMBER OF PAGES UU 16 19a. NAME OF RESPONSIBLE PERSON USAMRMC 19b. TELEPHONE NUMBER (include area cde) Standard Frm 298 (Rev. 8-98) Prescribed by ANSI Std. Z39.18

3 Table f Cntents Page 1. Intrductin 4 2. Keywrds 4 3. Accmplishments Impact Changes/Prblems Prducts Participants & Other Cllabrating Organizatins Special Reprting Requirements Appendices 16

4 1. INTRODUCTION: In this pre-clinical, translatinal prject, we will utilize muse mdels, human B cells, and human mast cells and basphils t assess the ability f Siglec-engaging Tlerance-inducing Antigenic Lipsmes (STALs) t induce immunlgical tlerance t peanut allergens. STALs are biengineered nanparticles that c-display a selected antigen and high affinity Siglec ligand. STALs targeting the Siglec CD22 n B cells induce antigen-specific B cell tlerance thrugh deletin f the B cells recgnizing the antigen. Applying this apprach t animals with an existing peanut allergy will allw us t deplete memry B cells respnsible fr prducing IgE, and establish a nvel therapeutic strategy fr fd allergies. STALs targeting the human Siglec CD33 will be used t desensitize mast cells. This apprach will be investigated as a therapeutic strategy fr preventing acute allergic reactins, allwing fr tlerizing dses f antigen t be delivered safely. By expliting the inhibitry functins f CD22 n B cells, and CD33 n mast cells and basphils, ur primary bjectives are (1) t develp a nvel prphylactic and therapeutic apprach fr peanut allergy and (2) t develp a targeted apprach t prevent mast cell and basphil degranulatin t peanut allergens. 2. KEYWORDS: Fd allergy; Peanut allergy; Siglec; CD22; CD33; STAL; nanparticle; Ara h 2; mast cell; basphil; B cell 3. ACCOMPLISHMENTS: What were the majr gals f the prject? Specific Aim 1: Establish the therapeutic ptential f Ara h 2 STALs targeting CD22 t abrgate peanut allergies. Majr Task 1: Determine ptimal cnditins t induce B cell tlerance t Ara h 2 and whle peanut extract in a prphylactic muse mdel. Target date: Mnths 1-12; percentage f cmpletin: 100% (cmpleted) Majr Task 2: Use Ara h 2 STALs t induce tlerance by deletin f memry B cells. Target date: Mnths 10-30; percentage f cmpletin: 15% Majr Task 3: Determine translatability f STALs t human CD22 and human B cells. Target date: Mnths 5-24; percentage f cmpletin: 20% Specific Aim 2: Demnstrate the applicability f Ara h 2 STALs targeting CD33 t prevent mast cell- and basphil-mediated allergic respnses t peanut allergen. Majr Task 1: Determine inhibitry effects and lngevity f inhibitin using LAD-2 mast cells. Target date: Mnths 1-7; percentage f cmpletin: 100% (cmpleted) Majr Task 2: Determine inhibitry effects and lngevity f inhibitin using human basphils. Target date: Mnths 7-18 ; percentage f cmpletin: 15% Majr Task 3: Determine preventive effects f STALs targeting CD33 n mast cells in viv in allergic mice. Target date: Mnths 6-30; percentage f cmpletin: 20% Majr Task 4: Determine therapeutic utility f STALs targeting CD33 and CD22 simultaneusly in allergic mice. Target date: 18-36; percentage f cmpletin: 0% What was accmplished under these gals? 4

5 Please nte, this award is fr a Partnering PI prject with the PI, Michael Kulis at the University f Nrth Carlina (UNC) and Matthew Macauley at The Scripps Research Institute (TSRI) and each have cntributed t the prgress f the prject. In the accmplishments listed belw, we have nted which PI (Kulis/UNC r Macauley/TSRI) was invlved in specific experiments cnducted during the reprting perid. Specific Aim 1: Establish the therapeutic ptential f Ara h 2 STALs targeting CD22 t abrgate peanut allergies Majr Task 1 - Determine ptimal cnditins t induce B cell tlerance t Ara h 2 and whle peanut extract in a prphylactic muse mdel. Significant Results and Achievements: IACUC prtcls were written and apprved at UNC, and then submitted and received apprval frm USAMRMC ACURO. The majr peanut allergen, Ara h 2, was prpsed as the mdel antigen t demnstrate key cncepts in preventing and treating peanut allergy. Purified Ara h 2 (Ah2) was prduced and shipped frm UNC t TSRI where Ah2 was cnjugated t lipid and saved as a frzen stck. The frzen stck was validated at TSRI fr making Ah2 STALs with CD22 ligand (CD22L), and will be used fr subsequent experiments thrughut the prject perid. The initial experiment was designed t test the Ah2 STALs with muse CD22L in a prphylactic apprach in mice. The results generated at UNC demnstrated that ne i.v. injectin f Ah2 STALs prevented Ah2- IgE and Ah2-IgG prductin fllwing the peanut plus chlera txin sensitizatin (Figure 1), and ultimately prevented anaphylaxis with Ah2 challenge. This cnfirms Figure 1. Ara h 2-specific IgE and IgG1 levels in serum. Mice were given PBS (Naïve), Ah2 STALs, r Immungenic Ah2, then sensitized with peanut plus chlera txin r sham sensitized with PBS (Naïve). Bld was cllected ne week after sensitizatin fr serum IgE and IgG1 measurements. Significantly lwer levels f Ara h 2-specific IgE and IgG1 were fund in mice treated with Ah2 STALs cmpared t mice treated with immungenic Ah2 lipsmes. These findings indicate that Ah2 STALs deplete naïve B cells thus preventing the prductin f Ah2-specific IgE and anaphylaxis upn allergen challenge. that the STALs wrk apprpriately and the mdel can be used t track Ah2-specific B cell respnses in further experiments. Results frm a similar series f experiments were published in the Jurnal f Allergy and Clinical Immunlgy (Impact Factr: ) using preliminary data that was submitted with the DD prpsal. 5

6 We are very interested in tracking Ah2-specific B cells t understand the mechanism f preventing IgE prductin. T d this, we prpsed wrking with a cllabratr, Justin Taylr at the Fred Hutchinsn Cancer Research Center, wh is an expert in identifying very rare ppulatins f B cells in mice. Dr. Taylr successfully prduced an Ah2 tetramer by linking bitinylated Ah2 t streptavidin that is tagged with an APC flurphre. We have since perfrmed experiments at UNC t demnstrate that we can identify Ah2-specific B cells in mice, where peanutsensitized mice have many mre Ah2-specific B cells than naïve mice (Figure 2). This nvel tl will be critical t further mechanistic studies in Aim 1. Figure 2. Identificatin f Ah2-specific B cells in naïve (uninjected) mice and peanut immunized mice. The tp panels shw naïve muse B cells with very few germinal center (CD38- GL7+) Ah2-specific B cells (0.37% f B220+ cells). The bttm panels shw immunized mice have ~3 times as many B220+CD38-GL7+ cells (2.99%) as the naïve mice. Majr Task 2: Use Ara h 2 STALs t induce tlerance by deletin f memry B cells. Significant Results and Achievements: A majr fcus f Aim 1 is t mve beynd preventin f peanut allergy and int the realm f therapy. We prpsed t test this first in mice with cnferred memry respnses t Ah2. T d this, we needed t develp a muse mdel in which memry T and B cells were adptively transferred int naïve mice. At UNC, BALB/cJ mice were sensitized t peanut using the typical peanut plus chlera txin prtcl, then splencytes (cntaining memry T and B cells) were harvested and transferred int naïve BALB/cJ mice. Mice were then given either PBS (sham treated) r Ah2 injectins t test memry recall respnses. Mice were bled fr Ah2-IgE quantificatin and later challenged with Ah2 t assess anaphylaxis. The Ah2 injectins bsted Ah2-IgE and led t anaphylaxis whereas this did nt happen in mice given PBS nly Figure 3. Ah2-specific IgE in mice with adptively transferred memry B and T cells that were then immunized with Ah2. Mice given nly PBS did nt generate a large recall respnse as evidenced by lw levels f Ah2-IgE, whereas mice given Ah2 lipsmes had large increases in Ah2-IgE demnstrating that memry B cells were activated and able t prduce Ah2-IgE. (Figure 3). This mdel has been repeated twice and will serve as ur initial mdel t test the Ah2 STALs with muse CD22L as a therapeutic t blunt memry B cell respnses. The Ah2 Tetramer will als be used in this mdel t track memry B cell numbers and functinal markers. 6

7 Majr Task 3: Determine translatability f STALs t human CD22 and human B cells Significant Results and Achievements: IACUC prtcls were written and apprved at TSRI and received apprval frm USAMRMC ACURO fr studies cnducted at TSRI. A majr questin is the ability f STALs t be translated int humans. T begin addressing this questin, Dr. Macauley prduced transgenic mice expressing human CD22 n B cells. These mice were prduced and human CD22 expressin cnfirmed n the B-cells (Figure 4A) at levels nly mdestly lwer than genuine human peripheral bld B-cells (Figure 4B). Several chrts f WT and hcd22 transgenic mice were sensitized with crude peanut extract accrding t the prtcl develped and implemented by Dr. Kulis. We find that hcd22 mice munt similar level f anti-ara h 2 antibdies cmpared t WT mice (Figure 4C), and have a similar degree f anaphylactic respnse as WT mice fllwing a challenge with peanut extract (Figure 4D). This wrk was recently published in the Jurnal f Immunlgy (Impact Factr: 4.92) as a part f wrk describing this muse mdel, which cncluded the hcd22 is sufficient t functinally substitute fr mcd22 in mst aspects f B-cell bilgy. This muse mdel sets up future experiments using human CD22L n Ah2 STALs as a therapeutic apprach in mice expressing the human CD22 receptr. Figure 4: Develpment f a hcd22 transgenic muse. (A) Expressin f mcd22 (left) and hcd22 (right) n WT (mcd22 + hcd22 - ; grey), hcd22 (mcd22 -/- hcd22 + ; red), and CD22KO (mcd22 -/- hcd22 - ). (B) Cmparisn f hcd22 expressin n genuine primary human peripheral bld B-cells and murine hcd22 transgenic B-cells. On the right is a histgram shwing the relative expressin levels in multiple patients/mice. (C,D) WT r hcd22 transgenic mice were rally sensitized with crude peanut extract and chlera txin fr fur cnsecutive weeks, then challenged with crude peanut extract a week later intraperitneally. (C) Antibdy respnses t the majr peanut allergen (Ara h 2) in mice fllwing the fur sensitizatins. (D) Anaphylactic respnse in mice fllwing the challenge. N differences in antibdy respnses r temperature decreases were bserved between the tw types f mice. 7

8 Specific Aim 2: Demnstrate the applicability f Ara h 2 STALs targeting CD33 t prevent mast celland basphil-mediated allergic respnses t peanut allergen Majr Task 1: Determine inhibitry effects and lngevity f inhibitin using LAD-2 mast cells. Significant Results and Achievements: The first majr task was t ptimize the rati f Ah2 and human CD33L n STALs t generate fully hypallergenic STALs. At TSRI, several frmulatins f STALs were prepared and ultimately tested n the human mast cell line, LAD-2. An ideal rati was fund that led t essentially n degranulatin frm the LAD-2 cells, whereas Ah2 lipsmes withut the human CD33L led t rbust degranulatin (Figure 5A). Majr Task 2: Determine inhibitry effects and lngevity f inhibitin using human basphils. Figure 5. Ah2 STALs targeting CD33 n human mast cells and basphils. Left panel (A) shws Ah2-STALs (red circles) des nt cause beta-hex release (i.e. degranulatin), whereas Ah2-lipsmes (black circles) cause large amunts f degranulatin. Right panel (B) demnstrates that 10 ng/ml Ah2 causes CD63+ degranulatin f human basphils whereas when basphils are first treated with the Ah2 STAL then 10 ng/ml Ah2 there is a >50% reductin in degranulatin, which is still smewhat higher than the media alne cntrl. Significant Results and Achievements: IRB apprval was btained at UNC and then with USAMRMC HRPO. At UNC, an initial pilt study was cnducted using the whle bld basphil activatin assay. The data lk prmising as STAL treated basphils degranulate less than untreated cells when stimulated with Ah2 (Figure 5B). Further experiments, including additinal cntrls, are planned fr the human basphil assay t better ptimize the STALs treatments. 8

9 Majr Task 3: Determine preventive effects f STALs targeting CD33 n mast cells in viv in allergic mice. Significant Results and Achievements: Apprpriate IACUC apprval was btained at TSRI and then with USAMRMC ACURO. In rder t test the efficacy f Ah2 STALs targeting CD33 in viv, transgenic mice expressing the human CD33 receptr n mast cells needed t be generated. At TSRI, transgenic mice were prduced and expressin f human CD33 n mast cells was verified (Figure 6). These transgenic mice will be a critical tl mving frward with Ah2 STALs, with the aim f silencing mast cells in respnse t peanut challenge. Figure 6. Mast cells in transgenic mice express human CD33. Left panel shws c-kit+ mast cells d nt express human CD33 in wild-type (WT) mice, as expected. The right panel shws c-kit+ mast cells d express human CD33 in the transgenic mice. What pprtunities fr training and prfessinal develpment has the prject prvided? At UNC, Dr. Kulis wrked with Kelly Orgel (an MD/PhD student) t cntinue mentring her n basic science experiments and techniques. Additinally, results frm the CD22 STALs in mice were presented by Kelly Orgel during the UNC Pediatric Day f Schlarship, in which she was awarded with the Best basic science abstract. At TSRI, thrugh ne-n-ne mentring between Dr. Macualey and Shiteng Duan, Shiteng has becme very efficient in all aspects needed fr the prpsed prject including muse handling, lipsme preparatin, and in vitr assays, such as ELISAs. Shiteng and Kevin Wrrell participated in grups meetings, regularly presenting their research updates. Dr. Macauley wrked clsely with Jana Juan t carry ut the muse gentyping and crdinate the muse experiments. Shiteng als rally presented his research at tw departmental meetings at TSRI: nce in the Department f Immunlgy and Micrbial Sciences, and nce in the Department f Mlecular Medicine. Hw were the results disseminated t cmmunities f interest? Nthing t Reprt. What d yu plan t d during the next reprting perid t accmplish the gals? At UNC, we will cntinue t wrk with the Ara h 2 tetramer t examine B cell frequencies in mice treated with Ara h 2 STALs. We will als determine the efficacy f Ah2 STALs targeting memry B cells in the adptive transfer mdel develped during the reprting perid. Furthermre, we will assess the capacity f Ah2 STALs in depletin f human B cells frm peanut allergic subjects. Finally, we will cntinue perfrming studies using human basphil assays t assess CD33 STALs in preventing degranulatin. At TSRI, the hcd33 mice will be used in functinal studies t determine if hcd33l STALs can prevent an anaphylactic respnse (Majr Task 3 in Aim 2). Shiteng Duan will cntinue cllabrating with Dr. Kulis n the basphil assay. At UAlberta, the hcd22 mice will be used in functinal studies t determine if hcd22l STALs can prevent an anaphylactic respnse (Majr Task 3 in Aim 1). 9

10 4. IMPACT: Dr. Macauley will cntinue prvide Dr. Kulis with mcd22l STALs fr studies in mice at UNC. What was the impact n the develpment f the principal discipline(s) f the prject? Tw nvel muse mdels were develped by Dr. Macauley s grup at Scripps. These are the humanized CD22 B cell mice and the humanized CD33 mast cell mice. These mdels prvide a vital tl fr scientists explring the in viv rles f CD22 and CD33, bth in a basic scientific sense as well as applied. What was the impact n ther disciplines? Nthing t Reprt. What was the impact n technlgy transfer? Nthing t Reprt. What was the impact n sciety beynd science and technlgy? Nthing t Reprt. 5. CHANGES/PROBLEMS: Changes in apprach and reasns fr change There were n significant changes t the apprach during the reprting perid. Actual r anticipated prblems r delays and actins r plans t reslve them Nthing t Reprt. Changes that had a significant impact n expenditures At UNC, a pstdc was hired, which did nt happen until apprximately 8 mnths int the funding perid. This likely decreased persnnel csts fr the reprting perid at UNC. At TSRI, since Dr. Macauley was mving institutins he had t hld ff n hiring a pstdc fr the prject. Hwever, by Dr. Macauley pulling sme f the experimental weight, the bjectives culd be achieved. Significant changes in use r care f human subjects, vertebrate animals, bihazards, and/r select agents Nthing t Reprt. Significant changes in use r care f human subjects. Nthing t Reprt. Significant changes in use r care f vertebrate animals. Nthing t Reprt. Significant changes in use f bihazards and/r select agents. Nthing t Reprt. 6. PRODUCTS: Publicatins, cnference papers, and presentatins Jurnal publicatins. Human CD22 Inhibits Murine B Cell Receptr Activatin in a Human CD22 Transgenic Muse Mdel. Bednar KJ, Shanina E, Ballet R, Cnnrs EP, Duan S, Juan J, Arlian BM, Kulis MD, Butcher EC, Fung-Leung WP, Ra TS, Paulsn JC, Macauley MS. 10

11 J Immunl Sep 29. [Epub ahead f print] PMID: Yes, DD Federal Funding was acknwledged. Expliting CD22 n antigen-specific B cells t prevent allergy t the majr peanut allergen Ara h 2. Orgel KA, Duan S, Wright BL, Maleki SJ, Wlf JC, Vickery BP, Burks AW, Paulsn JC, Kulis MD, Macauley MS. J Allergy Clin Immunl Jan;139(1): e2 Epub 2016 Aug 20. PMID: This paper used data submitted as Preliminary Data fr the DD prpsal and went t press prir t receiving DD Federal Funding, s funding frm DD was nt listed. Bks r ther nn-peridical, ne-time publicatins. Nthing t Reprt. Other publicatins, cnference papers, and presentatins. Cmpleted: 1. UNC Pediatric Schlarship Day April 20, 2017, UNC, Chapel Hill, NC Title: Expliting CD22 n Ara h 2-Specific B-Cells t Prevent Allergy t the Majr Peanut Allergen Ara h 2 Authrs: Kelly Orgel, Shiteng Duan, Brian Vickery, A. Wesley Burks, James Paulsn, Matt Macauley, Michael Kulis Accepted Abstracts (will be presented in the cming year): 1. American Academy f Allergy, Asthma, and Immunlgy Annual Cnference March 2-5, 2018, Orland, FL Title: Using Siglec-engaging Tlerance-inducing Antigenic Lipsmes (STALs) t reduce memry B cell respnses t the majr peanut allergen Ara h 2 Authrs: L. Hardy, K. Orgel, S. Duan, Sheila Maleki, A.W. Burks, J. Paulsn, M. Macauley, M.Kulis 2. Grdn Cnference n Fd Allergy Jan 7-12, 2018, Ventura, CA Title: Targeting inhibitry Siglecs t prevent IgE dependent anaphylaxis Authrs: Shiteng Duan, Crwin M. Nychlat, Matthew S. Macauley, Zhu Zhu, Bruce S Bchner, and James C. Paulsn 3. Sciety fr Glycbilgy Annual Meeting Nvember 5-8, 2017, Prtland, OR Title: Develpment f a New Human CD22 Transgenic Muse 11

12 Authrs: Matthew S Macauley, Kyle J Bednar, Elena Shanina, Rmain Ballet, Edward P Cnnrs, Shiteng Duan, Jana Juan, Britni M. Arlian, Mike D Kulis, Eugene C Butcher, Wai-Ping Fung-Leung, Tadimeti S Ra, James C Paulsn 4. American Chemical Sciety Spring Natinal Meeting March 18-22, 2018, New Orleans, LA Title: Expliting the Inhibitry Functin f CD22 n B-cells t Prevent Antibdy Respnses Authrs: Matthew S. Macauley, Britni M. Arlian, Kyle J. Bednar, Shiteng Duan, Wai-Ping Fung-Leung, Mike D. Kulis, Lakeya Hardy, Crwin M. Nychlat, Kelly A. Orgel, Lijuan Pang, James C., Paulsn, Tadimeti S Ra 5. American Chemical Sciety Spring Natinal Meeting March 18-22, 2018, New Orleans, LA Title: Siglecs: Putting the brakes n the immune system Authrs: James C. Paulsn, Britni Arlian, Shiteng Duan, Landn Edgar, Chika Kikuchi, Matthew Macauley, Crwin M. Nychlat, Lijuan Pang, Amrita Srivastiva Website(s) r ther Internet site(s) Nthing t Reprt. Technlgies r techniques Nthing t Reprt. Inventins, patent applicatins, and/r licenses Nthing t Reprt. Other Prducts At TSRI, Dr. Macauley develped tw transgenic muse mdels mice expressing either human CD22 n their B-cells r human CD33 n their mast cells 7. PARTICIPANTS & OTHER COLLABORATING ORGANIZATIONS What individuals have wrked n the prject? Prject Rle: Michael Kulis, PhD Initiating PI (UNC) Researcher Identifier (e.g. ORCID ID): Nearest persn mnth wrked: 4 Cntributin t Prject: Funding Supprt: As PI n the prject, Dr. Kulis led and versaw all f the wrk cnducted at UNC. Specifically, Dr. Kulis was invlved in experimental design, cllabrating with Dr. Macauley at Scripps, data cllectin, data analysis, and trubleshting. Prject Rle: Rishu Gu, PhD Research Scientist (UNC) 12

13 Researcher Identifier (e.g. ORCID ID): Nearest persn mnth wrked: 2 Cntributin t Prject: Funding Supprt: Dr. Gu perfrmed flw cytmetry and cell culture experiments. These include wrk n the muse B cells and human basphil assays. Prject Rle: Researcher Identifier (e.g. ORCID ID): Nearest persn mnth wrked: 3 Cntributin t Prject: Funding Supprt: Lakeya Hardy Graduate Student (UNC) Lakeya develped the adptive transfer mdel. Specifically, she sensitized mice t peanut, harvested splencytes, transferred the cells int naïve animals and then immunized with Ara h 2. She als cntributed by running sme ELISA experiments. Prject Rle: Researcher Identifier (e.g. ORCID ID): Nearest persn mnth wrked: 6 Cntributin t Prject: Funding Supprt: Kelly Orgel Graduate Student (UNC) Kelly wrked primarily n the use f Ah2 STALs targeting B cells t induce tlerance. Kelly perfrmed many hands-n muse prcedures and peanut challenges. She als assisted with cellular studies, flw cytmetry, and ELISA. Kelly perfrmed the human CD33 basphil assays as well. Prject Rle: Researcher Identifier (e.g. ORCID ID): Nearest persn mnth wrked: 2 Cntributin t Prject: Jhanna Smeekens, PhD PstDc Scientist (UNC) Dr. Smeekens assisted with hands-n muse experiments, particularly the allergic challenge experiments. She helped t harvest splencytes, stained cells fr flw cytmetry, and assisted with the human CD33 basphil assay experiments. 13

14 Funding Supprt: Jada Suber Prject Rle: Researcher Identifier (e.g. ORCID ID): Nearest persn mnth wrked: 1 Cntributin t Prject: Funding Supprt: Graduate Student (UNC) Jada helped with flw cytmetry panel design fr the Ah2 B cell tetramer experiments. Jada cllected cells and stained them with these panels fr later cllectin n the flw cytmeter. Xiahng Yue, MS Prject Rle: Researcher Identifier (e.g. ORCID ID): Nearest persn mnth wrked: 3 Cntributin t Prject: Funding Supprt: Research Assciate (UNC) Xiahng helped with rutine technical wrk, including ELISA, cell preparatin and cunting, flw cytmetry after cell culture, and preparatin f reagents needed thrughut the experiments. Prject Rle: Matthew Macauley, PhD Partnering PI (TSRI) Researcher Identifier (e.g. ORCID ID): Nearest persn mnth wrked: 7 Cntributin t Prject: Funding Supprt: Dr. Macauley prvided guidance f prject at TSRI as well as sme experimental prcedures. Dr. Macauley was respnsible fr getting animal prtcls apprved. Experimentally, Dr. Macauley cnjugated Ah2 t lipid, fr making f the STALs., and sensitized mice with the peanut allergen. Jana Juan Prject Rle: Research Assistant (TSRI) Researcher Identifier (e.g. ORCID ID): Nearest persn mnth wrked: 8 14

15 Cntributin t Prject: Funding Supprt: Jana perfrmed all the muse gentyping and helped setup the apprpriate muse breeders. Jana als perfrmed retr-rbital bleeds and analyzed antibdy titers by ELISA Prject Rle: Researcher Identifier (e.g. ORCID ID): Nearest persn mnth wrked: 7 Cntributin t Prject: Funding Supprt: Shiteng Duan Grad Student (TSRI) X Shiteng develped the hcd33 trangenic mice. Shiteng als preparing the Ah2 STALs fr use at TSRI and UNC, and helped challenge the mice with peanut extract and mnitred the anaphylactic respnse. Prject Rle: Researcher Identifier (e.g. ORCID ID): Nearest persn mnth wrked: 6 Cntributin t Prject: Funding Supprt: Kevin Wrrell Research Assistant (TSRI) Kevin was respnsible fr synthesizing the Siglec ligands and cnjugating these glycans t lipids fr incrpratin int STALs. Has there been a change in the active ther supprt f the PD/PI(s) r senir/key persnnel since the last reprting perid? Fr Dr. Kulis at UNC, nthing significant t reprt. Fr Dr. Macauley, NIH grant R01AI and R21AI have been funded. Dr. Macauley will put 3 calendar mnths tward R21AI and 4 calendar mnths tward R01AI in the cming year. Given the situatin with Dr. Macauley mving t University f Alberta and sub-cntracting a prtin f the wrk with Dr. James Paulsn taking ver as the partnering PI at TSRI, Dr. Macauley s effrt n this prject will decrease t apprximately calendar mnths in the cming year. These changes have been submitted as a part f package sent fr changing the partnering PI frm Dr. Macauley t Dr. Paulsn. What ther rganizatins were invlved as partners? Justin Taylr, PhD Organizatin Fred Hutchinsn Cancer Research Center Lcatin f Organizatin: Seattle, WA Partner's cntributin t the prject: Prduced the Ara h 2 tetramers with purified Ara h 2 shipped frm UNC and TSRI. Financial supprt; 15

16 In-kind supprt Facilities Cllabratin Dr. Kulis, Dr. Macauley, and Lakeya Hardy wrked with Dr. Taylr and his staff t develp and ptimize tetramers Persnnel exchanges prject staff used materials prvided by Dr. Taylr Other. Sheila Maleki, PhD Organizatin United States Department f Agriculture Lcatin f Organizatin: New Orleans, LA Partner's cntributin t the prject: Purified and prvided the peanut allergen, Ara h 2. Financial supprt; In-kind supprt Facilities Cllabratin Dr. Kulis, wrked with Dr. Maleki and her staff t prvide the Ara h 2 Persnnel exchanges prject staff (bth UNC and TSRI) used materials prvided by Dr. Maleki Other. 8. SPECIAL REPORTING REQUIREMENTS COLLABORATIVE AWARDS: A duplicative reprt will be submitted as tasks have been clearly marked with the respnsible PI and research site. QUAD CHARTS: 9. APPENDICES: 16