maturation of antigen presenting cells in mice and humans ... · a mixed-leukocyte reaction. data...
TRANSCRIPT
Figure3.ImprimebindstoandlicensestheDCtoprimean9gen-specificCD8Tcells.Ex vivo effect on human Monocyte-derived dendri9c cells (MoDC) -MoDC were prepared byculturing Imprime-boundmonocytesenriched fromwholeblood inXVivo15mediasupplementedwith10%autologousserumand50ng/mLrhGM-CSFfor6daysandthenmaturingthecellswithLPSandTNF-α(50ng/ml)for48hrs. MoDCweresubsequentlyevaluatedfor,(A)phenotype,(B)enhancing CD4/CD8 T cell proliferaSon by CFSE-diluSon assay and IFN-γ producSon in thesupernatant.ShownherearerepresentaSveresultsfrom4differentexperiments.InvivoeffectonmouseDC-(C)NaïveOT-ITCRtransgenicCD8TcellsspecifictoH-2Kb/OVA257-264pepSde were transferred into congenic hosts. The next day, mice were immunized with OVApepSdewithorwithout Imprime.OT-Iexpansionwasanalyzed inthespleen7days later.(D)ForfuncSonalevaluaSon,splenocytesweresSmulated invitrowith1uMOVApepSdefor5hrsinthepresence of brefeldin A. Cells were then stained intracellularly for producSon of cytokines. ToevaluatedegranulaSon,anS-CD107aanSbodywaspresentduringthe5hrsSmulaSon.
CD107a(Lamp1)
IFN-g
nopepSde
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OVAp
sSmulaSon
OVAp+Imprime
Abstract LB-089 AACR Annual Meeting
April 16-20, 2016
Imprime PGG, a β-glucan PAMP (pathogen-associated molecular pattern), effectively elicits in vivo maturation of antigen presenting cells in mice and humans, suggesting potential
synergy with checkpoint inhibitor therapy
Ross B. Fulton, Steven M. Leonardo, Adria B. Jonas, Kathryn A. Fraser, Anissa S.H. Chan, Nadine R. Ottoson, Michael E. Danielson, Nandita Bose, Jeremy R. Graff and Keith Gorden. Biothera Pharmaceuticals, Inc., Eagan MN, 55121
Abstract
Background A general structure of yeast-
derived Imprime PGG.
Summary
Acknowledgements All experiments funded by Biothera, Inc. No external funding was received to support the work. The authors would like to thank Steve Jameson and Kris Hogquist at the Center for Immunology, University of Minnesota for providing OT-I TCR transgenic mice and technical support.
1. In mice and humans, Imprime PGG rapidly binds in vivo to myeloid lineage cells including monocytes and DC subsets. Imprime induces monocyte mobilization and entry into lymphoid tissue.
2. As a PAMP, Imprime PGG provides a “danger” signal to monocytes/DCs that results in increased expression of co-stimulatory molecules. DCs exhibit a type I IFN transcriptional profile and transcription of IFN stimulatory genes (MX1) are increased in the LNs of mice.
3. In the absence of a “danger” signal, MHC class I-restricted peptide immunization results in anergy/poorly functional effector CD8 T cells. Imprime co-administration with peptide increases the magnitude and effector functions of antigen-specific CD8 T cells.
4. These data support a model whereby Imprime drives a coordinated immune response in cancer patients by providing cross-talk between the innate and adaptive immune system that facilitates functional tumor-specific T cell activation.
Recognition of PAMPs via pattern recognition receptors is central to immune recognition of foreign threats and to the generation of a coordinated innate and adaptive immune response. Cancers lack PAMPs and are poorly immunogenic. Consequently, the immune system often fails to mount an effective, coordinated anti-cancer immune attack. Though immunotherapies (e.g. checkpoint inhibitors) are effective in some cancer patients, the majority of patients fail to achieve substantial therapeutic benefit. To fully realize the potential of immune checkpoint inhibitors, there is substantial interest in developing therapeutically-viable PAMPs capable of enabling the maturation and function of professional antigen presenting cells (e.g. dendritic cells, DCs). Bacterial and viral PAMPs (i.e. TLR and STING agonists) can elicit DC maturation but are poorly tolerated systemically and are thereby limited to intra-tumoral delivery. The soluble yeast β-1,3/1,6 glucan, Imprime PGG (Imprime), is a PAMP that has been successfully administered intravenously (IV), is well-tolerated and shows promising efficacy in a series of clinical trials in > 400 total subjects. We sought to determine whether Imprime could drive maturation and enhanced function of antigen presenting cells in vivo. We now show that Imprime binds in vivo to various dendritic cell (DC) subsets in both human and mouse. In mice dosed IV, Imprime also elicits DC maturation as indicated by CD86 upregulation and successfully induces a type I interferon transcriptional profile. In C57Bl/6 mice immunized with the OVA257-264 model antigen, Imprime treatment elicits the specific expansion of adoptively transferred OT-I CD8 T cells (transgenic T cells engineered to recognize the OVA antigen). These OT-I T cells are functional effector cells, showing enhanced degranulation and increased capacity to produce IFN-γ and IL-2 when compared to OT-I cells isolated from mice challenged with OVA peptide alone. In ex vivo human whole blood, Imprime also enables DC maturation (enhanced expression of CD86, CD83, MHC class II), T cell expansion and production of the potent anti-tumor cytokine IFN-γ. Importantly, we now show preliminary data that peripheral blood monocytes and classical DCs from Imprime-treated cancer patients show elevated CD86 expression. Collectively, these data show that Imprime, a novel, systemically-administered, well-tolerated PAMP, can effectively elicit the maturation and function of antigen presenting cells in vivo and may thereby enhance T cell priming and anti-tumor immune response elicited by checkpoint inhibitors.
• Imprime is a soluble, yeast-derived β-1,3/1,6 glucan immunomodulator being developed for cancer treatment in combination with anti-tumor antibodies.
• Imprime is administered intravenously and is well-tolerated (> 400 patients to date).
• In 1st line stage IV NSCLC patients, Imprime, when combined with bevacizumab/carbo/taxel increased ORR (60% vs 44% in control) and yielded median overall survival of 16.1 months (control = 11.6 months).
• Imprime binds to various leukocytes including monocytes, neutrophils, B cells and DCs via a combination of Dectin-1, Fc and complement receptors.
• CD8 T cell differentiation and acquisition of effector functions is shaped by co-stimulation provided by professional antigen-presenting cells and cytokines (e.g. IL-12 or type I IFN).
• Here we evaluate the hypothesis that Imprime matures monocytes/DCs and enhance T cell priming.
Human dendritic cells bind Imprime in vivo
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Figure 4. Circulating DCs in h e a l t h y h u m a n s b i n d Imprime in vivo. Healthy human volunteers were intravenously infused with 4mg/kg Impr ime. Whole b lood was collected prior to infusion and at various times after the end-of-infusion. (A) Representative flow plots showing Imprime binding to c o n v e n t i o n a l ( c D C ) a n d p l a s m a c y t o i d D C s ( p D C ) . Extracellular binding of Imprime to DCs was assessed using an anti-β-glucan antibody. (B) Detection of extracellular Imprime plotted as the fold-MFI relative to pre-infusion DCs. Each set of connected circles represents a unique individual (n=12).
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Figure 6. Imprime enhances the expansion and capacity of CD8 T cells to produce multiple effector cytokines. (A) Naïve CD44lo OT-I CD8 T cel ls were transferred into congenic hosts. The next day, mice were immunized IV with 100µg OVA peptide +/- 1.2mg Imprime. 7 days later, spleens were harvested and the OT-I response was analyzed by flow. (B) The frequency of OT-I in the sp leen . (C ) Sp lenocy tes we re stimulated in vitro with OVA peptide for 5hrs in the presence of brefeldin A and anti-CD107a antibody and then stained for intracellular cytokines and granzyme B. (D) Representative plot of Tbet expression. (E) Frequency of OT-I capable of producing IFN-γ, TNF-α and IL-2. (F) Boolean combinations of cytokine production by OT-I cells. **p<0.01, ***p<0.001 using unpaired Student’s t-test.
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Figure 1. Imprime binds to mouse and human monocytes in vivo and induces mobilization of murine monocytes into the blood and secondary lymphoid organs. C57BL/6 mice were injected IV with 1.2mg DTAF-conjugated Imprime and blood, spleens and peripheral LNs were harvested at various times. (A) Imprime binding to peripheral blood Ly6Clo and Ly6Chi
monocytes in mice. (B) Change in the frequency of murine monocytes 16hrs after Imprime injection. (C) Fold change in total # of murine monocytes in the spleen and LNs. (D) Imprime binds to human monocytes in vivo. Healthy human volunteers were given an IV infusion of Imprime at 4mg/kg. Extracellular Imprime binding to peripheral blood monocytes was assessed using an anti-β-glucan antibody. **p<0.01, ***p<0.001 using unpaired Student’s t-test.
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Figure 2. Imprime increases expression of co-stimulatory molecules on human monocyte-derived DCs and enhances T cell priming. (A) Whole blood was incubated at 37°C for 30min with vehicle or 25µg/mL Imprime. Monocytes were then purified via negative selection using Dynabeads (ThermoFisher) and cultured for 10 days in the presence of IL-4 and GM-CSF. CD11c+ cells were then analyzed by flow. (B) MoDCs were incubated with CFSE-labeled, purified allogeneic CD3 T cells at a 10:1 T cell to DC ratio, and cultured for 5 days. Immunogenicity is measured in a mixed-leukocyte reaction. Data cumulative from 3 separate donors.
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Figure 3. Imprime binds to resident and migratory DC populations within secondary lymphoid organs of mice. C57BL/6 mice were injected IV with 1.2mg DTAF-conjugated Imprime and spleens and peripheral LNs were harvested 30mins post injection. Imprime binding to splenic DC subsets (A), migratory LN DC subsets (B), and plasmacytoid DCs (pDC) in the spleen. Plots representative of 3 individual mice.
Figure 4.
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Figure 7. Imprime induces a type I IFN transcriptional profile. (A) In order to generate a large number of murine DCs, recombinant B16F10 melanoma cells expressing Flt3L were implanted s.c. into C57BL/6 mice. When the tumor was ~1000mm3, spleens were harvested and cDCs were isolated using positive selection. Purified DCs were stimulated for 1hr with 25µg/mL Imprime or 6µg/mL CpG 1826. We then isolated total RNA (Qiagen), generated cDNA, and performed transcriptional analysis using a custom Taqman array (Life Technologies). (B) PBS or Imprime (1.2mg) was injected IV into C57BL/6 mice. Peripheral LNs were harvested 16hrs later and placed into RNALater (ThermoFisher). Taqman primers were used to examine expression of the IFN-stimulated genes MX1, Ifit1, and ISG15.
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16hr in vivo stimulation B Figure 8. Colorectal cancer p a t i e n t s t r e a t e d w i t h I m p r i m e t r e n d t o w a r d increased CD86 expression on monocytes and CD1c+ DCs. Subjects were given 4mg/kg Imprime IV weekly and a cycle constituted 6 weeks of treatment. The change in CD86 expression levels from baseline pre-treatment (cycle 1) to cycle 3 on peripheral blood CD1c+ DCs and CD14+ monocytes was determined by flow cytometry. All samples were stained and run at the same time. Each symbol represents a patient.
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Figure 5. Imprime increases DC expression of co-stimulatory molecules in vivo. C57BL/6 mice were administered 1.2mg DTAF-Imprime and splenic DC populations were analyzed for expression levels of co-stimulatory and activation markers. Representative flow plots are shown in (A) at either 24 or 48hrs post Imprime injection. (B) Time course showing fold MFI (median) change over vehicle. MFI’s in Imprime-treated mice were calculated on DTAF-Imprime+ DCs.
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Abstract # Abstract
Poster #A014
InnateimmunemodulaSon:thenovelimmunotherapeuScImprimePGGtriggerstheanS-cancerimmunitycycleinconcertwithtumor-targeSng,anS-angiogenicandcheckpointinhibitoranSbodies
NanditaBose,KeithGorden,AnissaChan,AdriaBykowskiJonas,NadineCOioson,RichardMWalsh,XiaohongQiu,BenHarrison,TakashiKangas,KathrynFraser,RossFulton,StevenMLeonardo,MarkUhlik,andJeremyGraff.
BiotheraPharmaceuScalsInc.Eagan,MN.
Cancer immunotherapeuScs largely focusonawakeningTcellmediatedrecogniSonanderadicaSonoftumorcells. Indeed,checkpointinhibitoranSbodies(e.g.pembrolizumab)unleash T cells already involved in anS-cancer responses and have shown remarkableclinicalacSvity,thoughonlyin~20-30%ofsolidtumorpaSents.NumerousapproachesarebeingexploredtoenhancethepercentofpaSentswhobenefitfromcheckpointinhibitortherapies.Chiefamongst thesearethe innate immunemodulaSngtherapiescollecSvelydesignated as PAMPs- pathogen- associatedmolecular paierns. PAMPs operate as thecriScal“non-self”signalsthat,inresponsetopathogeninfecSon,ignitethefuncSonoftheinnate immune system to trigger the immunity cycle. TLR and STING agonists act asPAMPs and reflect bacterial and viral danger signals that can drive dendriSc cellmaturaSon, enhancingT cell funcSon. Theseagents are indevelopment in combinaSonwith other immunotherapies, including checkpoint inhibitors, but inspire intolerablecytokinestormsandaretherebylimitedtodirect intra-tumoraldeliveryapproaches.Wetherefore sought to discover and develop a novel, systemically administered PAMP-ImprimePGG(Imprime).Ex vivo studies with whole blood from healthy human donors show that Imprimeconsistently elicits the acSvaSon of innate immune cells. M2 state macrophagesrepolarize, showing increased expression ofM1markers (CD86, PD-L1) with coincidentreducSoninM2markers(CD163,CD206).DendriSccells(DCs)mature,showingenhancedsurfaceexpressionofCD80,CD86andMHCclassII.FuncSonally,theanSgenpresentaSoncapabilityofthesere-polarizedmacrophagesandacSvatedDCsissubstanSallyenhancedand drives the robust expansion of co-cultured CD8 T cells as well as the markedupregulaSon of the potent anS-tumor cytokine interferon gamma. In preclinical tumorstudies, Imprime is administered IV and profoundly enhances the efficacy of numerousanSbodytherapies.UsingtheB16experimentalmetastasismodel,weshowthatImprime(administered IV) synergizes with the anS-TRP1 tumor-targeSng anSbody TA-99, nearlyeradicaSng B16 metastases as measured by visual counts, TRP-1 RT-PCR and in situimmunofluorescence for TRP1. In the H441 and H1299 non-small cell lung cancerxenogrars, Imprime synergizeswith the anS-VEGFR2 anSbodyDC101 to flat-line tumorgrowth. IntheMC-38andCT-26syngeneictumormodels, ImprimesynergizeswithbothanS-PD-1 and PD-L1 checkpoint inhibitor anSbodies to repress tumor growth and/or toeradicate cancer lesions. In situ imaging of these preclinical tumor Sssues shows thatImprime insSgatesa re-orientaSonof the immunemicroenvironment,promoSnganM1state (e.g. increased iNOS2,decreasedArginase1),aswellas the influxofmyeloidcellsand,inthesyngeneicmodels,CD8Tcells.Inclinicaltrialsin>400totalpaSentstodate,Imprime has been safely administered by IV infusion (4mg/kg over 2 hours) and hasrepeatedly shown evidence for efficacy in combinaSon with tumor targeSng or anS-angiogenic anSbodies. Studies with checkpoint inhibitor anSbodies are slated to beginsummer of 2016.We now provide the first evidence in healthy human volunteers thatImprime(IV-4mg/kg,2hours)drivesthesameinnateimmuneacSvaSoneventsevidentinthe preclinical studies (e.g. chemokine and cytokine release, PD-L1 and CD86upregulaSon) verifying that the clinical dose acSvates the innate immune system.Together, these preclinical and clinical studies provide evidence that the novel PAMP,Imprime PGG, can be safely administered systemically and can drive the criScal innateimmuneacSvaSonnecessarytosparktheanS-cancerimmunitycycle.
2016AACRTumorImmunlogyandImmunotherapy
#A89
Background
Figure1.Imprime(PAMP)Ac9vatesAn9-CancerImmunityCycle
• Imprime PGG, a yeast-derived pharmaceuScal-grade soluble 1,3/1,6 β-glucan is beingdeveloped for the treatment of cancer in conjuncSon with tumor targeSng andimmunomodulatoryanSbodies(Abs).
- ImprimehasshownpromisingresultsinmulSplePhase2clinicaltrialsinnon-smallcelllung cancer (NSCLC), and chronic lymphocySc leukemia (CLL) with addiSonal studiesongoing.
• β-glucans are conserved microbial structures found in the cell wall of unicellular andmulScellular pathogens. They are considered pathogen-associated molecular paierns(PAMPs) recognized by the paiern recogniSon receptors including DecSn-1 andComplement Receptor 3 (CR3). Imprime forms an immune complex with endogenousserum immunoglobulin IgG or IgM anS-beta-glucan anSbodies (ABA) before beingrecognizedbyCR3andFcgRIIAoninnateimmunecells.
Results
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C D
• SyngeneicB16melanomamodel-Imprime+An9-Tryp1,TA99
Figure2.Imprimetreatmentenhancesefficacyofan9-tumoran9bodies.Exvivostudies– NeutrophilswereisolatedfromWBbynegaSveselecSonandco-incubatedwithRajiBcelllymphomacellspre-treatedwithvehicleorcoatedwiththeanS-CD20anSbodyRituximab(1μg/ml).ROSgeneraSonwasmeasuredbytheluminolmethodandisshownasrelaSvelightunits(RLU). (B) M2c macrophages were prepared from enriched monocytes isolated from WB, andculturedinX-Vivo10medium(supplementedwith5%autologousserumand50ng/mlrhM-CSF)for6dayswith10ng/mlrhIL-10addiSonforthelast24hrs.M2C’sabilitytophagocytosefluorescently-labeledRituximab-opsonizedRajicellswasmeasuredbyflowcytometry.DataarerepresentaSveof>3independentexperimentsfromdifferentdonors.Invivostudies–C57BL/6micewereinjectedviathetailveinwith100,000B16F10melanomacells.TheanS-Tryp1monoclonalanSbodyTA99andImprimewereadministeredintraperitoneally(50ug/mouse D1,3,5,7,10) and intravenously (1.2 mg/mouse D1,3,7,10,14), respecSvely. Mice wereeuthanized10daysarertumorchallengetocountlungmetastases.(C)Meannumberofmetastasesper treatment group (± SEM). (D) RepresentaSve immunohistochemical staining of tumor SssuesshowingcompleterepressionofoutgrowthofmetastasesintheTA99+Imprimetreatmentgroup.
ImprimeenhancesDCmatura9onandan9genpresenta9ontoTcells
0
5
10
15
20
25
% o
f Pro
lifera
ting
Cells
**p = 0.0038 ***p = 0.0004
Allo-CD4T Allo-CD8T
0
50
100
150
IFNγ
(pg/
ml)
**p = 0.0026
C
B
InVivoStudies
Imprimerepolarizestumormicroenvironment
Figure 7. ABA-dependent Clinical Responses. (A) Immunopharmacodynamic (IPD) responseselicited by IV administraSon of Imprime in healthy human subjects.Whole blood or serumwasdrawnfrom24healthyvolunteersatvariousSmepointsbeforeandarerasingledose(cohort1)ormulSpleweeklydoses(cohort2)ofImprimeinfusion.CellmobilizaSonwasmeasuredbycompleteblood cell counts, plus differenSals. Cytokine/chemokinemeasurement in serumwas performedusingNovexmagneScmulSplex assay (Life Technologies) the LuminexXMAP technology. (B) IgGABAweredeterminedbyELISAforallevaluablepaSentsinthePrimustrial(thirdlineCRCpaSentstreatedwithCetuximaborImprime+Cetuximab).Upperlerpanel-HazardRaSo(HR)vsABAlevelisgraphicallyrepresented.TheABAlevelforeachpaSentisshownonthexaxis.ThesolidlineinthegraphrepresentstheHRwhilethedoiedlinesthatbracketthislinerepresentthe95%confidenceintervals.VerScal lines imposedon thegraph simply represent the cut-points chosen forKaplan-Meieranalyses.Theinsetshowsthe%ofpaSentsinthistrialatthedifferentABAcutpoints.Otherpanels-each representsKaplan-Meieranalyses forOSat the indicatedABAcut-points (20,35,or45μg/ml).HRforeach,andstaSsScalsignificancearenotedintheinset.
CD163 CD86 PD-L1
Coun
t
0 102 103 104 105
Median86
2434759
Median103439790
MeanFluorescenceIntensity
Median12911752108
IsotypectrlStaining
M2-VehicleM2-Imprime
A
Week 1
Pre-Infusio
n
Week 1 15min
Week 1 30min
Week 1
EOI
Week 1 1 Hour
Week 1 4 Hour
Week 1 24 Hour
Week 2
Pre-Infusio
n
Week 2 15min
Week 2 30min
Week 2
EOI
Week 2 1 Hour
Week 2 4 Hour
Week 2 24 Hour
Week 2 48 Hour
Week 3
Pre-Infusio
n0
200
400
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1000
Mon
ocyt
es
(cell
/ul)
IgG Positive Group (>35ug/ml)
4102234ImprimeSaline
Week 1
Pre-Infusio
n
Week 1 15min
Week 1 30min
Week 1
EOI
Week 1 1 Hour
Week 1 4 Hour
Week 1 24 Hour
Week 2
Pre-Infusio
n
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EOI
Week 2 1 Hour
Week 2 4 Hour
Week 2 24 Hour
Week 2 48 Hour
Week 3
Pre-Infusio
n0
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1000
Mon
ocyt
es
(cell
/ul)
ABA Negative Group
ImprimeSaline
473925
Week 1
Pre-Infusio
n
Week 1 15min
Week 1 30min
Week 1
EOI
Week 1 1 Hour
Week 1 4 Hour
Week 1 24 Hour
Week 2
Pre-Infusio
n
Week 2 15min
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Week 2
EOI
Week 2 1 Hour
Week 2 4 Hour
Week 2 24 Hour
Week 2 48 Hour
Week 3
Pre-Infusio
n0
200
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1000
Mon
ocyt
es
(cell
/ul)
IgM Positive Group (>50ug/ml)
ImprimeSaline37162
PRE-IMPRIM
E
POST-IMPRIM
E
1 HOUR
24 HOURS
020406080
100
2000400060008000
10000
IL-8
(pg/
ml)
IgG Positive Group (>35 ug/ml)IL-8
010022004034
PRE-IMPRIM
E
POST-IMPRIM
E
1 HOUR
24 HOURS
020406080
100
2000400060008000
10000
IL-8
(pg/
ml)
IgM Positive Group (>50 ug/ml) IL-8
002016037
PRE-IMPRIM
E
POST-IMPRIM
E
1 HOUR
24 HOURS
020406080
100
2000400060008000
10000
IL-8
(pg/
ml)
ABA Negative Group IL-8
039025
PRE-IMPRIM
E
POST-IMPRIM
E
1 HOUR
24 HOURS
020406080
100
2000400060008000
10000
IL-8
(pg/
ml)
IgG Positive Group (>35 ug/ml)IL-8
010022004034
PRE-IMPRIM
E
POST-IMPRIM
E
1 HOUR
24 HOURS
020406080
100
2000400060008000
10000
IL-8
(pg/
ml)
IgM Positive Group (>50 ug/ml) IL-8
002016037
PRE-IMPRIM
E
POST-IMPRIM
E
1 HOUR
24 HOURS
020406080
100
2000400060008000
10000
IL-8
(pg/
ml)
ABA Negative Group IL-8
039025
ImprimeinducesmonocytemobilizaSoninhighIgGABAsubjects
Imprimeinducescytokine/chemokineproducSoninhighABAsubjects
Vehicl
e
Impri
meTA
99
TA99
+ Im
prime
0
20
40
60
# M
ets
(D10
)
P value 0.0054
Coun
t
CD80 CD86 CD83 HLA-DR
MeanFluorescenceIntensity(MFI)
Median15142867049
Median223640744
Median162168466
Median151871
10759
Isotypectrlstaining Imprime
A
M1polariza9onofmacrophages:Exvivohumanstudies
B
MDSCDifferen9a9on–ExvivohumanstudiesA
B
Figure5.Imprime-treatedMDSChaveAPC-likecharacteris9cs.(A)PhenotypeandfuncSonofhumanMDSC–HumanMDSCwerepreparedfromhumancordbloodperamodifiedversionofthepublishedprotocol (Wu et al., PNAS, 2014). Flow cytometry analyses showed increased CD86 expression onbothmonocyScMDSC (Mo-MDSC) and PMN-MDSC (not shown). FuncSonal assessment in a T-cellproliferaSonassayshowedthatImprime-treatedMDSCwerelesssuppressivetoT-cellproliferaSon.(B) H1299 lung cancer xenograr model: In vivo administraSon of Imprime also enhanced CD86expressiononPMN-MDSCinthetumor.
Significant (p<0.05) tests: 36 out of 86 (41.9%)
2
1
1/2
1/4
1/8
1/16
HR
with
95%
CI
10 20 30 40 50
1.0
0.8
0.2
0.0
0.6
0.4
Sur
viva
l Pro
babi
lity
HR=0.24 (0.1-0.55) p=0.00031
ABA ≥ 45
ABA < 45
0 10 20 30 40 50
0 10 20 30 40 50
ABA < 20
ABA ≥ 20
1.0
0.8
0.2
0.0
0.6
0.4
HR=0.77 (0.49-1.22) p=0.27
Sur
viva
l Pro
babi
lity
% population at ABA cutpoints ≥ 20 µg/ml = 49% ≥ 35 µg/ml = 24% ≥ 45 µg/ml = 16%
57 25 5 1 0 55 28 9 2 1 1
94 40 10 1 0 18 13 4 2 1 1
1.0
0.8
0.2
0.0
0.6
0.4
0 10 20 30 40 50
ABA ≥ 35
ABA < 35
HR=0.40 (0.22-0.73) p=0.0018
Surv
ival
Pro
bab
ility
85 36 8 1 0 0 27 17 6 2 1 1
ABA levels (µg/ml) in evaluable patients Time (months)
Time (months) Time (months)
Conclusions
AcknowledgementsAll experiments funded by Biothera Pharmaceuticals Inc. No external funding was received to support the work.
ABAandOSinImprime-treatedcolorectalcancerpa9ents
PhaseIhealthyhumanvolunteertrialExVivoHumanStudies
ExVivoHumanStudies
A
B
Imprimeenhancesinnateimmunecellkillinginconcertwithtumor-targe9ngan9bodies
DAPITryp1
Veh
Imp
TA99
TA99+Imp
Imprimeenhancestheefficacyofan9-angiogenicsandcheckpointinhibitors
ClinicalbiomarkerresearchInvitrostudies
Invivostudies
Imprime Vehicle
2:1 PBMC:MDSC
Div
isio
n In
dex 1.0
0.5
0.0
1.5 *0.0118
Citrate
Imprim
e PGG
Vehicle Imprime
Mo-MDSC CD86 3500
3000
2500
2000
1500
MFI
**0.0082
Figure6.Imprimeenhancesinvivoan9-tumorefficacyofan9-angiogenicsandcheckpointinhibitors.(A)ImprimeincombinaSonwithDC101(murinesurrogateforramucirumab)inH441andH1229NSCLCxenograrmodels,respecSvely.Oncetumorsreached~100mm3,micewereadministeredDC101(10mg/kgtwiceweeklyIPforuptosixweeks)and/orImprime(1.2mg/mousei.Vtwiceweeklyforuptosixweeks).(B)ImprimeincombinaSonwithanS-PD-1anSbodywastestedinCT26coloncancer-bearingBALB/cmiceorwithanS-PD-L1anSbodyinMC38coloncancer–bearingC57Bl/6mice.3daysarersubcutaneousinjecSonoftumor,themicewereadministeredImprimeand/oranS-PD-1/PD-L1(100μg/mousetwiceweeklyIPforuptofiveweeks).
0.0
0.2
0.4
0.6
0.8
1.0
Div
isio
n In
dex
****p <0.0001 ns
0
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800
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IFNγ
(pg/
ml)
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(pg/
ml)
**p = 0.0015
ns
IL-4 IFN-γ
CD
4 T
cell
expa
nsio
n
(Div
isio
n In
dex)
An9-angiogenics–immunomodulatoryeffectsofan9-VEGFR2
0 5 10 15 20 250
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Study Days
Tum
or V
olum
e (m
m3 )
VehicleImprimeDC101-10mg/kgImprime + DC-101
Vehicle Imprime PD-L1 Imprime+PD-L1
%tu
mor
free
mic
e (d
ay 2
9)
5.6% 11%
33%
83%
Checkpointinhibitors–An9-PD-1/PD-L1an9bodies
A
B
0 10 20 30 400
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Study Days
Tum
or V
olum
e (m
m3)
VehicleImprimePD-1Imprime + PD-1
*
****
H441
MC38CT26
H1299
D
PRE-IMPRIM
E
POST-IMPRIM
E
1 HOUR
24 HOURS
020406080
100
2000400060008000
10000
IL-8
(pg/
ml)
ABA Negative Group IL-8
039025
Donors
BevBev+Imprime
Vehicle
Coun
t
CD86
An9-TumorTcellImmunity-Tcellkillingandimmunememory
Enhancesan9genpresenta9ontoTcells
DrivesDCmaturaSon&acSvaSon
ImprimePGG-Bindstoinnateimmunecells&triggersacoordinatedresponse
Repolarizestumorimmunemicroenvironment
M2toM1polarizaSon
MDSCdifferenSaSon
EnhancedTcellpriming EnhancedTcell
effectorfuncSons
NeutrophilMacrophage
NKCell
Enablesinnateimmunecellkilling
PossibleImmunogenic
celldeath
1 2 3
Imprime PGG acts as a PAMP to enlist the broad funcSonality of the innateimmune system to enhance the anS-tumor efficacy of tumor-targeSng, anS-angiogenic, and immune checkpoint inhibitor monoclonal anSbody therapies.ImprimePGGacSvatesanS-cancerinnateimmuneeffectorfuncSonsby:1)triggeringdirecttumorkillingbyinnateeffectorcells2)acSvaSngthematuraSonofanSgenpresenSngcells3)overridingtumor-inducedimmunosuppressionandThese innate immune funcSons of Imprime PGG are criScal for triggering theimmunitycyclethatulSmatelydrivestheanS-tumorTcell-basedimmunity.
0
1
10
100iNOS
CXCL11CXCL9
IL-12b
CXCL10
IL-6
PD-L1
TNFIFNr
CCR7CD86CCL2
CCL3
Arg1
CCL17
TGFb
Fizz-1
CD206YM-1
MouseBMM-Imprime
Figure4.ImprimetreatmentresultsinM1polariza9onofmacrophages.Exvivostudies-M2macrophageswerepreparedbyculturingImprime-boundmonocytesenrichedfrom human whole blood in the presence of M2-polarizing polarizing condiSon as described inFigure2Bfor6days.Macrophagesweresubsequentlyevaluatedfor,(A)phenotype,(B)CD4TcellproliferaSonbyCFSE-diluSonassay,andIFN-γandIL-4producSoninthesupernatantbyELISA.Invivo studies - (C)Bonemarrow-derivedmacrophages (BMM)waspreparedbyculturingbonemarrowcellsharvestedfromImprime-treatedmiceinRPMImediumsupplementedwith10%fetalcalfserumand20ng/mlrmM-CSFfpr7days,andsubsequentlymeasuredbyqRT-PCR.(D)qRT-PCRassayshowup-regulaSonofM1markersanddown-regulaSonsofM2markers.
M1polariza9onofmacrophages:Invivostudies
C D MouseBMM-Vehicle
Vehicle
0.1
1
10
100CXCL11
CXCL10
IL-12b
TNFa
Arg1
TGFb
IL-10
CD206
Mousetumor-VehicleMousetumor-Imprime
0 10 20 30 400
400
800
1200
Study Days
Tum
or V
olum
e (m
m3)
PBSImprime DC-101DC-101 + Imprime
*