0

1000

2000

3000

4000

Ex vivo Imprime Treatment Induces C5a in Healthy Human Volunteers (HHV) and Modulates C5aR Expression

Hypothesis

Veh

Imp

Imp

+C5aRA(0.01) Im

p

+C5aRA(0.1) Im

p

+C5aRA(1)

0

500

1000

1500

Veh

Imp+IgGA

BA

Imp+IgGA

BA

+C5aRA(0.01)

Imp+IgGA

BA

+C5aRA(0.1)

Imp+IgGA

BA

+C5aRA(1)

0

500

1000

1500

0

2000

4000

6000

8000

0 15 30 45 60 75 900

100

200

300

400

Time(Minutes)

ROS(RLU

)

ImprimeImprime+serumImprime+serum+C5aAb(1)

0

20

40

60

80

% In

crea

se in

Cyt

otox

icity

Vehicle Imprime

Raji +Rituximab

Neutrophil Pre-treatment

**

0 10 20 30 40 50 600

50

100

150

200

250

300

Time(Minutes)

ROS(RLU

)

Veh

Veh+C5aRA(1) Im

p

Imp+C5aRA(0.01)

Imp+C5aRA(0.1)

Imp+C5aRA(1)

0

500

1000

1500

C5a-C5aR pathway is Critical for Imprime-induced Innate Immune Functions (Cytokine Production)

Background

SITC 2018# P527

Imprime PGG, a novel cancer immunotherapeutic, engages the complement system to prime innate immune effector functions

Xiaohong Qiu, Ben Harrison, Adria B. Jonas, Anissa SH Chan, Nadine R. Ottoson, Nandita Bose and Keith B GordenBiothera Pharmaceuticals, Inc., Eagan, MN55121

Background: Imprime PGG (Imprime), an intravenously-administered soluble,

yeast β-1,3/1,6-glucan, is currently in clinical development with tumor-

targeting antibodies, anti-angiogenics, and checkpoint inhibitors. The

fundamental mechanistic rationale for these therapeutic combinations is that

Imprime, being a PAMP, primes innate immune effector functions to

ultimately inspire an adaptive immune response-based anti-cancer immunity

cycle. Imprime forms a tripartite immune complex (IC) comprised of Imprime,

naturally occurring anti-β-glucan antibodies (ABA) and iC3b complement

opsonin in subjects with sufficient ABA levels. Ex vivo human and in vivomouse studies have shown that the innate immune receptor, FcgRIIA, and the

pattern recognition receptors, complement receptor 3 (CR3) and Dectin-1, are

critical for Imprime’s innate immune responses. However, the contributions of

the complement system, a vital component of innate immunity, towards the

functional activity of Imprime have not been thoroughly investigated.

Imprime-ABA IC activates the classical complement pathway and releases

C5a. As C5a is a well-known priming agent, and involved in cross-talk with the

other innate immune receptors, we hypothesized that Imprime-induced C5a

will engage the C5a-C5a receptor (C5aR) signaling pathway to enhance

Imprime binding and innate immune effector functionalities.

Methods: The role of C5a in Imprime-ABA binding to isolated neutrophils was

evaluated by: a) adding exogenous C5a; b) using C5a-depleted serum, and c)

using C5aR antagonist (C5aRA). Cytokine production in healthy subjects with

sufficient ABA levels were measured 24hrs post-Imprime treatment in the

presence or absence of C5aRA by multiplex Luminex assays. The effect of C5a

inhibitors was also evaluated in a chemiluminescence-based oxidative burst

assay measuring reactive oxygen species (ROS) generated by Imprime-treated

isolated neutrophils in response to Rituxan-bound B cell lymphoma cells. In

order to test these endpoints in complement-depleted conditions, the whole

blood was washed extensively to remove the plasma.

Results: Addition of exogenous C5a increased the percentage of neutrophils

binding to Imprime in a dose-dependent manner. Furthermore, Imprime

binding in the presence of C5aRA and C5a-depleted serum was significantly

reduced. Functionally, C5aRA abrogated cytokine production ( IL-8, MCP-1,

MIP-1alpha, and IL-6) in Imprime-treated blood. Likewise, Imprime-ABA

induced ROS in high-ABA blood was greatly inhibited in C5a-depleted serum

and could be rescued by replenishing complement. C5aRA also inhibited

Imprime-induced ROS production. In a non-physiological, complement-

depleted condition, Imprime bound predominantly via FcgRIIA, resulting in

diminished cytokine and ROS responses.

Conclusions: These results collectively demonstrate that Imprime-induced

C5a plays a critical role in enhancing Imprime binding and functional

responses, potentially by lowering the signaling threshold of the other innate

immune receptors.

All experiments funded by Biothera

Pharmaceuticals Inc. No external funding was

received to support the work.

Conclusion

Acknowledgements

Abstract Results

• Imprime PGG, a yeast-derived pharmaceutical-grade soluble 1,3/1,6 β-glucan

is being developed for the treatment of cancer in conjunction with tumor

targeting and immunomodulatory antibodies (Abs).

-Imprime has shown promising results in multiple Phase 2 clinical trials in

non-small cell lung cancer (NSCLC) and chronic lymphocytic leukemia (CLL),

and is currently in Phase 2 clinical trials in combination with anti-PD-1 in

TNBC, Melanoma and HNSCC.

•β-glucans are conserved microbial structures found in the cell wall of

unicellular and multicellular pathogens. They are considered pathogen-

associated molecular patterns (PAMPs) recognized by the pattern

recognition receptors (PRR) including Dectin-1, and Complement Receptor 3

(CR3). Imprime forms an immune complex with endogenous serum

immunoglobulin IgG or IgM anti-beta-glucan antibodies (ABA) before being

recognized by these PRRs.

Effective antigen presentation• Active, mature dendritic cells

• M1-state macrophages

T-Cell based anti-cancer immunity

• CD8, CD4 T cell infiltration• PD-L1 + Tumor, myeloid cells• Adaptive immune signature

Sufficient antigen debris field

• Sufficiently foreign• Presentable

Proposed Mechanism: Imprimetriggers a series of innateimmune activation events thatculminate in enhanced T cellbased anti-cancer immunity

Repolarizes the immune microenvironment

Activates antigen presentation Activates tumor cell killing

Permissive immune microenvironment

• M1>>> M2 polarized macrophages• Reduced/differentiated MDSCs

The Imprime Immune Complex

IgG ABA Complement opsoniniC3b

iC3b

Figure 1. Imprime impacts multiple points of the anti-cancer immunitycycle

0

5

10

15

20

25

Veh Imp

**p=0.0020

C5

a (

ng

/m

L)

Neutrophils Monocytes

Mean Fluorescence Intensity (MFI)

Vehicle Imprime

Co

un

t

1787

799

1642

952

A. B.

In vivo Evidence of C5a Production (SC5b-9 Measured as a Surrogate Marker) in HHV and Cancer Subjects

Figure 3. Ex vivo Imprime treatment of human whole blood results in production of C5a and modulation of C5aR (CD88) expression on human neutrophils and monocytes. Shown here are (A) the average

concentration of C5a in the plasma of HHV treated ex vivo with 10 µg/mL of Imprime (N = 6), and (B)

histogram showing down-modulation of CD88 on human neutrophils and monocytes measured by flow

cytometry after 30 min-1 hr treatment of human whole blood with 10 µg/mL of Imprime (representative of N

= 5)

Figure 4. Systemic administration of Imprime results in C5a production. As a surrogate marker for C5a,

average plasma levels of SC5b-9 was measured 2-3 hrs post-Imprime administration in (A) HHV (N=11) and (B)

triple negative breast cancer subjects (N=12) using commercial enzyme immunoassay kits.

Pre-dose Post-dose

SC

5b

-9 (

ng

/m

L)

Pre-dose Post-dose

**p= 0.0011

SC

5b

-9 (

ng

/m

L)

A. B.

C5a-C5aR Plays a Role in Imprime Binding

100µM 10µM 1µMImprime

Imprime + C5a receptor antagonist

BfD

IVB

fDIV

Imprime 1 µg/mL 100 ng/mL 10 ng/mL

Imprime + C5a

A.

B.

CD16

CD15

Figure 5. Imprime binding is influenced by C5a-C5aR engagement. Binding of Imprime to human neutrophils

was evaluated by incubating 10 µg/mL Imprime to isolated human neutrophils resuspended in media

containing 20% serum and either different concentrations of (A) C5aR antagonist or (B) recombinant C5a, and

then determining the percentage of cells positive for BfD IV (anti-Imprime specific monoclonal antibody) by

flow cytometry.

Veh

Imp

Imp+C5aAb(10)

Imp+C5aAb(50)

0

200

400

600

800

Re

lati

ve

IL-8

mR

NA

Re

lati

ve

IL-8

mR

NA

A.

0

500

1000 Unstim

Stim

Stim + C5aRA

Imp LPS

B.

C5a-C5aR engagement is Critical for Imprime-induced Innate Immune Functions (Reactive Oxygen Species Production-mediated Tumor Killing)

NeutrophilTreatment

RajiTreatment

nonenone

VehicleImprimeVehicleImprime

RituximabRituximab

A.

C.

B.

Proposed Mechanism: Imprime triggers a series of innate immune activation eventsthat culminate in enhanced T cell based anti-cancer immunity

IL-8

(p

g/m

L)

Re

lati

ve

IL-8

mR

NA

IL-8

(p

g/m

L)

AU

C

Serum Complement

Proteins

C3 C3

Imprime-IgG ABA Immune Complex

C5a

iC3b

CD88

(C5aR) FcgRIIA

Complement Receptors

Dectin-1

Opsonized Imprime

Activation of Classical Complement Pathway

C3iC3b

iC3b

Prime

?

Innate Immune Activation

Does Imprime-ABA-induced C5a engage

C5aR signaling pathway to prime Imprime’sbinding and innate

immune effector functionalities?

C5a as priming agent

Nature Reviews Immunology – 2004

Vol4: 133-142

0 15 30 45 60 75 900

100

200

300

400

Time(Minutes)

ROS(RLU

)

Veh

ImpVeh+C5aRA(0.1)

Imp+C5aRA(0.01)Imp+C5aRA(0.1)Imp+C5aRA(1)

Figure 6. C5a-C5aR inhibitors specifically block Imprime-induced cytokines. (A) mRNA levels of IL-8 from human whole blood incubated with 25 µg/mL Imprime for

6 hrs in the presence of anti-C5a Ab (left) or C5aRA (right) were measured by qRT-PCR. The inset in the right panel shows the specificity of C5aRA to Imprime vs LPS-

mediated IL-8 production. (B) Protein levels of IL-8 from human whole blood incubated with 25 µg/mL Imprime for 24 hrs in the presence of C5aRA were measured

by Luminex. The inset panel shows that C5aRA does not inhibit IL-8 produced by Imprime-ABA immune complex in the absence of serum.

• Imprime-ABA immune complex activates classical complement pathway and as a result, releases C5a, a potent complement-

activation product

• C5a, a known priming agent of myeloid cells and PMN, acts via C5aR to enhance Imprime binding

• C5a-C5aR axis is critical for Imprime-ABA immune complex-mediated innate immune effector functions, including

cytokines/chemokine production and anti-tumor cytotoxicity via ROS production.

• Further investigation is on going to, a) confirm whether C5a lowers the threshold of ABA required for Imprime’s functionality, and

b) understand the cross-talk between the C5a-C5aR signaling pathway and the known receptors for Imprime

(CR3/FcgRIIA/Dectin-1)

Figure 7. Imprime-induced neutrophil ROS production against antibody-opsonized tumor cells requires complement. (A) Whole Blood (WB) was treated with

vehicle or Imprime (25µg/mL) at 37°C for 2 hrs. Neutrophils were isolated via negative selection directly from WB (Stemcell technologies) and cells were re-

suspended at between 2-3 x 106 cells per well in a 96 well plate. Neutrophils were stimulated in the presence of luminol (50µM) with Raji B cell lymphoma cells that

were pretreated with or without rituximab (1µg/mL). Luminescence (RLU) was read at 30 second intervals corresponding to reactive oxygen species (ROS)

production. The panel on the right shows that the Imprime-treated neutrophils also showed enhanced cytotoxicity when co-cultured with Raji cells that had been

labeled with Calcein AM dye with Rituximab at a 50:1 effector to target ratio. Cells were incubated for 3hrs and then the co-culture was stained with a live/dead dye

and analyzed by FACS to determine cytotoxicity. Raji cells were identified as Calcein+ and % cytotoxicity was calculated by live/dead dye staining. Data show the %

increase in cytotoxicity over vehicle treated neutrophils co-cultured with Raji alone. Data representative of 2 independent donors. (B) The assay described above was

run in a low-ABA subject by supplementing with different doses of ABA in the presence and absence of serum. For quantitative estimation of ROS produced, the area

under the curve (AUC) was calculated and plotted as a bar graph shown on the right. (C) For evaluation of the role of C5a/C5aR, WB was either pre-incubated with

anti-C5a Ab for 30 mins at room temperature or C5aR antagonist at the indicated concentrations at the time of Imprime treatment and then followed with isolation

of neutrophils for use in the assay. (*p<0.05, **p<0.01,***p<0.001, and ****p<0.0001).

0 15 30 45 60 75 900

1000

2000

3000

Time(Minutes)RO

S(RLU

)

WashedWB+IgGABA(0)WashedWB+IgGABA(20)

WashedWB+IgGABA(100)WashedWB+IgGABA(50)

WB+IgGABA(100)WB+IgGABA(200)WashedWB+IgGABA(200)

WB+IgGABA(0)WB+IgGABA(20)WB+IgGABA(50)

IgGABA(0)

IgGABA(20)

gGABA(50)

IgGABA(100)

IgGABA(200)

IgGABA(0)

IgGABA(20)

gGABA(50)

IgGABA(100)

IgGABA(200)

0

50000

100000

150000

200000

Washed WB WB

*p= 0.0455