Download - maturation of antigen presenting cells in mice and humans ... · a mixed-leukocyte reaction. Data cumulative from 3 separate donors. CD4 T cells CD8 T cells 0 5 10 15 20 % of proliferation

Transcript

Page 1: maturation of antigen presenting cells in mice and humans ... · a mixed-leukocyte reaction. Data cumulative from 3 separate donors. CD4 T cells CD8 T cells 0 5 10 15 20 % of proliferation

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

OVApepSde

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

[email protected]

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

even

ts (

% o

f max

)

Imprime

CD1c+ cDC CD16+ cDC CD304+ pDC Pre-infusion 1hr post infusion 24hr post infusion

1 4 24 720

hrs post "end of treatment"

Bfd

IV M

FI (n

orm

aliz

)

CD16+ DCs

1 4 24 720

hrs post "end of treatment"

Bfd

IV M

FI (n

orm

aliz

)

pDCs

1 4 24 720

hrs post "end of treatment"

Bfd

IV M

FI (n

orm

aliz

)

CD1c+ DCs

Impr

ime

I (no

rmal

ized

)

hrs after end-of-infusion

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).

OVA peptide +/- Imprime (1.2mg)

Exp 9996

Transfer 1x105 naïve OT-I CD8 T cells specific to OVA257-264 peptide

C57BL/6 Day 7 Harvest spleen/LN Assess OT-I response

(peptide and Imprime are NOT conjugated)

0.0 0.5 1.0 1.5

OVApep + Imp

OVApep2.3x**

day 7 spleen

% OT-I of total CD8

OVApep OVApep + Imprime

CD107a

Gra

nzym

e B

Tbet

IFN

-γ

CD44

C D

even

ts (

% o

f max

)

total CD8 OT-I OVApep OT-I OVApep + Imp F

80 ***

% o

f OT-

*** *** IFN-γ TNF-α IL-2

total CD8 OT-I

E pepti

pepti

de +

Impr

ime

100

% o

f OT

-I p

rodu

cing

toki

ne c

ombi

natio

SPDPTP

No cytokines

Triple producer Double producer Single producer Non-producer

pepti

de +

Impr

ime

100

% o

f OT

-I p

rodu

cing

toki

ne c

ombi

natio

SPDPTP

No cytokines

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.

0 612

100

Peripheral blood monocytes

Ly6Clow

Ly6Chi

time post injection (hrs)

% D

TAF

-Im

prim

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.

A Imprime binding Peripheral blood monocytes

PBS

Impr

ime

PBS

Impr

ime

Blood 16hrs Ly6Clow

Ly6Chi0.9x

3.1x**

% o

f to

tal

PBS

Impr

ime

PBS

Impr

ime

0.0

0.5

1.0

1.5

2.0

LNs 16hrs Ly6Clow

Ly6Chi

1.1x

15.4x***

% o

f to

tal

PBS

Impr

ime

PBS

Impr

ime

0.0

0.5

1.0

1.5

2.0

2.5

Spleen 16hrsLy6Clow

Ly6Chi

2.1x

4.0x

***

% o

f to

tal

Ly6Clow

Ly6ChiMonocyte mobilization

0 612

780

Monocyte mobilizationLy6Clow

Ly6ChiLNs

Ly6Clow

Ly6Chi

time post injection (hrs)

Fold

change in

tota

l #

Spleen

0 1 2 3 4 5 20 300

80CD14+ monocytes

Time (hrs after infusion begins)

% b

indin

g Im

prim

In vivo binding in humans D B

0 612

100

Peripheral blood monocytes

Ly6Clow

Ly6Chi

time post injection (hrs)

% D

TAF

-Im

prim

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.

CD4 T cells

CD8 T cells0

% o

f pro

lifera

tion c

ells

moDC-vehiclemoDC-Imprime

even

ts (

% o

f max

)

HLA-DR

CD80

CD86

Vehicle Imprime

A B day 5 MLR

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.

CD8α"

11b

CD103 (αE integrin)

11b

CD8α+ CD11b+

CD11b+

eve

nts

of m

ax)

ven

ts (

% o

f m

ax)

Splenic resident DC

Lymph node migratory DC

PDCA-1"D

total Spleen pDC

eve

nts

of m

ax)

Imprime

vehicle Imprime vehicle Imprime

CD103+

vehicle Imprime vehicle Imprime

Imprime

vehicle Imprime B

A C

Dendritic cells bind Imprime in vivo in mice

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.

0.1$

10$

100$Ccl3

Ccl4 Ccr7

Cd274

Cd40

Cd80

Cd86

Cxcl1

Cxcl2

Fam26f

Ifna1;Ifna5;Ifna6

Ifna2 Ifnb1

Ifng Il10 Il12a

Il12b

Il1a

Il1b

Il2

Il21

Il23a

Il6

Marco

Tnfa

Tnfsf10 Tnfsf9

Control'

CpG*1hr'

Imprime*1hr'

PBS CpG 1826 Imprime

1hr DC in vitro stimulation A

Ifit1

PBS

Impr

ime

rela

tive e

xpre

ssio

nMX1

PBS

Impr

ime

100

101

102

103

104

rela

tive e

xpre

ssio

PBS

Impr

ime

rela

tive e

xpre

ssio

n ISG15

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.

Cetux

imab

Impr

ime+

Cetux

imab

ΔM

FI f

rom

cle 1

to c

ycle

3 o

f tre

atm

ent

Average delta in CD86 expression on classic DC cycle1 to cycle 3

CD86 on CD1c+ DC

Cetux

imab

Impr

ime+

Cetux

imab

0.0

0.5

1.0

1.5

2.0

2.5

ΔM

FI f

rom

cle 1

to c

ycle

3 o

f tre

atm

ent

CD86 on CD14+ monos A B

Fol

monocyte-derived DCs

Production of IFN-γ/TNF-α/IL-2

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.

0 10 20 30 40 50 160

170

0.5

1.0

1.5

2.0

2.5

3.0

hrs post injection

I fol

d ch

ange

er v

ehic

CD8α+ DC CD86CD40PDL-1MHC class II

A B CD8α+ CD11b+

CD86

MHC class II

Splenic DC

CD40

even

ts (

% o

f max

)

24hrs

48hrs 0 10 20 30 40 50 16

017

00.51.01.52.02.53.03.5

hrs post injection

I fol

d ch

ange

er v

ehic

CD11b+ DC CD86CD40PDL-1MHC class II

0 10 20 30 40 50 160

170

0.0

0.5

1.0

1.5

2.0

2.5

hrs post injection

I fol

d ch

ange

er v

ehic

pDC CD86MHC class II

PD-L1 48hrs

Vehicle Imprime+ Murine splenic DCs A

0 10 20 30 40 50 160

170

0.5

1.0

1.5

2.0

2.5

3.0

hrs post injection

I fol

d ch

ange

er v

ehic

CD8α+ DC CD86CD40PDL-1MHC class II

A B CD8α+ CD11b+

CD86

MHC class II

Splenic DC

CD40

even

ts (

% o

f max

)

24hrs

48hrs 0 10 20 30 40 50 16

017

00.51.01.52.02.53.03.5

hrs post injection

I fol

d ch

ange

er v

ehic

CD11b+ DC CD86CD40PDL-1MHC class II

0 10 20 30 40 50 160

170

0.0

0.5

1.0

1.5

2.0

2.5

hrs post injection

I fol

d ch

ange

er v

ehic

pDC CD86MHC class II

PD-L1 48hrs

0 61

4 CD86 CD8α+

CD11b+

time post injection (hrs)

I fold

change o

ver ve

hic

0 61

3 CD40 CD8α+

CD11b+

time post injection (hrs)

I fold

change o

ver ve

hic

0 61

4 CD86 CD8α+

CD11b+

time post injection (hrs)

I fold

change o

ver ve

hic

0 61

4 CD86 CD8α+

CD11b+

time post injection (hrs)

I fold

change o

ver ve

hic

PBS

OVA pepti

de +

Impri

me0.0

0.1

0.2

0.3

0.4

0.5

% O

T-I o

f tot

al C

Frequency of OT-I

PBS

OVA pepti

de +

Impri

me0

1×104

2×104

3×104

4×104

5×104

tota

l OT-

I per

spl

een

OT-I totals

2.3x**

CD90.1+

~1x105naïveOT-I

OVA257-264pepSde+/-Imprime(1.2mg/mouse)

CD90.2+

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

0 15 30 450

100

Time(Minutes)

RLU

Vehicle NoneImprime PGG NoneVehicle RituximabImprime PGG Rituximab

PMN treatment Tumor treatment

ReacSveOxygen

Species(RO

S)inRLU

% A

80***

Abs: None Rituximab

Raji

Vehicle Imprime

A B

Invivostudies

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

% o

f Pro

lifera

ting

Cells

**p = 0.0038 ***p = 0.0004

Allo-CD4T Allo-CD8T

100

150

IFNγ

(pg/

ml)

**p = 0.0026

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

0 102 103 104 105

Median86

2434759

Median103439790

MeanFluorescenceIntensity

Median12911752108

IsotypectrlStaining

M2-VehicleM2-Imprime

Week 1

Pre-Infusio

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

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

200

400

600

800

1000

Mon

ocyt

(cell

/ul)

IgG Positive Group (>35ug/ml)

4102234ImprimeSaline

Week 1

Pre-Infusio

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

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

200

400

600

800

1000

Mon

ocyt

(cell

/ul)

ABA Negative Group

ImprimeSaline

473925

Week 1

Pre-Infusio

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

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

200

400

600

800

1000

Mon

ocyt

(cell

/ul)

IgM Positive Group (>50ug/ml)

ImprimeSaline37162

PRE-IMPRIM

POST-IMPRIM

1 HOUR

24 HOURS

020406080

100

2000400060008000

10000

IL-8

(pg/

ml)

IgG Positive Group (>35 ug/ml)IL-8

010022004034

PRE-IMPRIM

POST-IMPRIM

1 HOUR

24 HOURS

020406080

100

2000400060008000

10000

IL-8

(pg/

ml)

IgM Positive Group (>50 ug/ml) IL-8

002016037

PRE-IMPRIM

POST-IMPRIM

1 HOUR

24 HOURS

020406080

100

2000400060008000

10000

IL-8

(pg/

ml)

ABA Negative Group IL-8

039025

PRE-IMPRIM

POST-IMPRIM

1 HOUR

24 HOURS

020406080

100

2000400060008000

10000

IL-8

(pg/

ml)

IgG Positive Group (>35 ug/ml)IL-8

010022004034

PRE-IMPRIM

POST-IMPRIM

1 HOUR

24 HOURS

020406080

100

2000400060008000

10000

IL-8

(pg/

ml)

IgM Positive Group (>50 ug/ml) IL-8

002016037

PRE-IMPRIM

POST-IMPRIM

1 HOUR

24 HOURS

020406080

100

2000400060008000

10000

IL-8

(pg/

ml)

ABA Negative Group IL-8

039025

ImprimeinducesmonocytemobilizaSoninhighIgGABAsubjects

Imprimeinducescytokine/chemokineproducSoninhighABAsubjects

Vehicl

Impri

meTA

TA99

+ Im

prime

# M

ets

(D10

)

P value 0.0054

Coun

CD80 CD86 CD83 HLA-DR

MeanFluorescenceIntensity(MFI)

Median15142867049

Median223640744

Median162168466

Median151871

10759

Isotypectrlstaining Imprime

M1polariza9onofmacrophages:Exvivohumanstudies

MDSCDifferen9a9on–ExvivohumanstudiesA

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%)

1/2

1/4

1/8

1/16

with

95%

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

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

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

200

400

600

800

100

200

300

400

500

600

IFNγ

(pg/

ml)

IL-4

(pg/

ml)

**p = 0.0015

IL-4 IFN-γ

4 T

cell

expa

nsio

(Div

isio

n In

dex)

An9-angiogenics–immunomodulatoryeffectsofan9-VEGFR2

0 5 10 15 20 250

400

800

1200

1600

2000

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

5.6% 11%

33%

83%

Checkpointinhibitors–An9-PD-1/PD-L1an9bodies

0 10 20 30 400

400

800

1200

1600

2000

Study Days

Tum

or V

olum

e (m

m3)

VehicleImprimePD-1Imprime + PD-1

****

H441

MC38CT26

H1299

PRE-IMPRIM

POST-IMPRIM

1 HOUR

24 HOURS

020406080

100

2000400060008000

10000

IL-8

(pg/

ml)

ABA Negative Group IL-8

039025

Donors

BevBev+Imprime

Vehicle

Coun

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.

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

100CXCL11

CXCL10

IL-12b

TNFa

Arg1

TGFb

IL-10

CD206