IFNgIDO1STAT1GZMAGZMKCCR5

CXCL10CXCR3CXCR6CD2

CD3dIL2RA

HLA-DRAPRF1

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SITC 2018

#10876

Abstract

Background

Background: Though efficacious, checkpoint inhibitor (CPI) monotherapy

fails to elicit response in the majority of patients. TNBC is one such cancer

type where CPI antibodies (pembrolizumab, avelumab, atezolizumab),

have demonstrated only a ~5-10% response rate, irrespective of PD-L1

expression. We are developing Imprime PGG (Imprime), a novel yeast

derived β-glucan PAMP in combination with pembrolizumab, to enhance

the benefit that TNBC patients derive from CPI-based therapy.

Methods: In this analysis, we present the serum and cellular IPD responses

elicited by Imprime and pembrolizumab in the peripheral blood of 12

TNBC subjects who previously failed front-line chemotherapy, enrolled as

part of a Phase 2 study (NCT02981303). Subjects received Imprime (4

mg/kg qw) + pembro IV (200 mg q3w) in 3 week cycles. Anti-beta glucan

antibodies (ABA), circulating immune complexes (CIC), complement

activation, cytokine production, gene expression changes, and phenotypic

changes on immune cells were evaluated.

Results: As Imprime is known to complex with serum IgG ABA, a drop in

the free ABA levels and a concomitant increase in the CIC was observed at

the end of infusion (EOI) of every Imprime dose. Interestingly, 11 of 12

subjects showed increased ABA levels between cycles 1 and 2, with peak

levels increasing ~1.5 to 35-fold over baseline. In line with this ABA

increase, peak levels in serum CIC levels (range ~3 to 22-fold) and

complement protein SC5b-9 (~1.4 to 41-fold) were also observed at cycle 2

EOI. In a subset of patients, a maximum increase of ~10-1000-fold in

several chemokines was detected at cycle 2 EOI. Gene expression analyses

of whole blood indicated peak activation of several genes at cycle 2

associated with activation of innate immune cells and T-cells. In 8 of 12

subjects, an increased frequency up to 11-fold in the CD16+ monocytes,

cells known for their enhanced cytotoxicity as well as M1-polarizing

functions, was observed between cycles 1 and 2. We also observed an

increase, up to 2-fold, in CD16+ inflammatory DC in 8 of 12 subjects. The

maximal increase (~4 to 20-fold) in newly proliferating (Ki67+), activated

CD8 T cells (PD-1+ CD38+ HLADR+) was observed at cycle 2 in 4 subjects.

Of all these immunological responses, robust cytokine production together

with an increased frequency of activated CD8 T cells, correspond with

objective tumor responses.

Conclusions: These data provide the first evidence in cancer patients that

Imprime can drive the critical IPD changes known to be associated with

efficacy in preclinical cancer models.

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

• 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) with additional studies ongoing.

•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 including Dectin-1 and Complement Receptor 3

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

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

recognized by CR3 and FcgRIIA on innate immune cells.

All experiments funded by Biothera

Pharmaceuticals Inc. No external funding was

received to support the work.

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

Hypothesis: Imprime in combination with pembrolizumab will enhance sensitivityto checkpoint inhibitors (CPI) by stimulation of the patient’s innate andadaptive immune systems in those patients who have failed prior therapy (CPI-naive)

Repolarizes the immune microenvironment

Activates antigen presentationActivates tumor cell killing

Permissive immune microenvironment• M1>>> M2 polarized macrophages• Reduced/differentiated MDSCs

The Imprime Immune Complex

IgG ABA Complement opsoniniC3b

iC3b

Figure 3. Imprime administration resulted in drop of free IgG ABA concentrations and increase in CIC levels. Shown here

are serological changes in (A) IgG ABA and, (B) CIC levels tested at pre-dose, and ~EOI of Imprime and pembro for 3 cycles

using an ELISA method.

Figure 4. Imprime and pembrolizumab dosing resulted in several innate and adaptive immune activatingpharmacodynamic changes that generally peaked between cycle 1 and cycle 2. We explored peripheral blood changes at

pre-dose and EOI of Cycles 1, 2, 3, 4, 5, 6, and beyond. The peak IPD responses are observed between cycles 1 and 2.

(A) Maximum fold-increase in serum levels of free ABA, CIC, and complement-activation product SC5b-9 measured at cycle

2 EOI by ELISA, (B) Fold increase over C1 in cytokine levels measured at cycles 1-3 EOI by Luminex in a representative

subject (103102), and (C) Representative plot from a patient showing the appearance of CD16+ intermediate/ non-classical

monocytes (107107) and inflammatory DC (104104) in the peripheral whole blood at cycle 2 (pre-dose) as measured by flow

cytometry. Changes in the frequencies of CD16+ monocytes and inflammatory DC from cycle 1 to cycle 2 (pre-dose) in all 12

subjects are also shown, (D) A representative plot from a patient (109128) showing the appearance of of Ki67+ PD-1+

responding CD8 T cells in whole blood as measured by flow cytometry. The responding Ki67+ PD-1+ population is activated

(CD38+ HLA-DR+) and is largely contained within the memory CD8 T cells. Changes in the frequency of Ki67+ PD-1+ CD8 T cells

from cycle 1 to cycle 2 (pre-dose) in all 12 subjects are also shown.

Acknowledgements

Immunopharmacodynamic (IPD) responses of Imprime PGG combined with pembrolizumab in chemotherapy-resistant, metastatic triple negative breast cancer (TNBC) subjects in a Phase 2 trial: Analyses of stage 1 patients

Nadine R. Ottoson1, Adria B. Jonas1, Anissa SH Chan1, Xiaohong Qiu1, Blaine T. Rathmann1, Richard M. Walsh1, Ben Harrison1, Mike Danielson1, Kyle Michel1, Michaela Finley1, Mark Uhlik1, Jamie Lowe1, Paulette Mattson1, Michele Gargano1, Michael Chisamore2, Joanna Cox1, Bruno Osterwalder3, Jeremy R. Graff1, and Nandita Bose1

1Biothera Pharmaceuticals, Inc., Eagan MN, 55121. 2Merck & Co. Inc., Kenilworth, NJ. 3B.O. Consulting GmbH, Riehen, Switzerland

Fold

In

cre

ase

Ove

r P

re-d

ose

Cyc

le 1

Evidence of Imprime-ABA Immune Complex Formation In Vivo

Pre-dose EOI

Cycle 1 Cycle 2 Cycle 3

**p=0.004 **p=0.003 **p=0.0051

IgG

AB

A (µg

/mL)

Pre-dose EOI Pre-dose EOI

A. B.Cycle 1 Cycle 2 Cycle 3

*p=0.0218 ***p=0.0001 ***p=0.0008

Pre-dose EOI Pre-dose EOI Pre-dose EOI

CIC

(µg

Eq

/mL)

Peak Immunopharmacodynamic Responses Measured in Serum/Plasma and at Cellular Levels (cycle 2)

Fold

in

cre

ase

ove

r b

ase

lin

e

Figure 2. Study Schema

Peripheral Immunological Responses Associated with Clinical Response

B.

Fre

qu

en

cy o

f C

D1

6+

mo

no

Ki67+ PD-1+

A.

C.

Figure 5. Association between peripheral immunological responses and objective clinical response. Shown here are trends

in association of some of the select immunological changes to the objective clinical responses, including (A) more robust

cytokine/chemokine profile regulating both innate and adaptive immunity observed in all the responders (B) T-cell

activation gene expression profile using RNA from whole blood, and (C) higher magnitude of increase in the frequency of

Ki67+ PD-1+ CD8 T cells in all the responders. Fold maximal change between C2 and C6 over pre-dose C1 is shown.

Results

Responder (confirmed)Responder (unconfirmed)

Summary•For the first time, this study provides evidence for Imprime-ABA immune complex formation and the

downstream peripheral innate and adaptive immune activation responses in cancer subjects.

•For the first time in TNBC patients, treatment with Imprime (in combination with Pembrolizumab)

elicits peripheral innate immune-activating immunopharmacodynamic changes including complement

activity, select chemokine production, and phenotypic activation of monocytes and DC. These

activities have been previously evident in pre-clinical efficacy models as well as healthy volunteers.

•The strong association between the clinical responses and the innate/adaptive immune responses are

suggestive of interplay between the therapeutic mechanisms of Imprime and pembrolizumab.

•The correlative work between the peripheral immunological responses and the tumor

microenvironmental changes are ongoing.

118102

103102

109128

107107

111101

104104

115107

109116

110130

112103

107113

Stable DiseaseProgressive Disease

*OR=overall response

Stage 1 = 12 pts

Imprime PGG 4 mg/kg QW

Pembro 200 mg Q3W

NO

YES

Repeat Stage 1 with

Imprime PGG 2

mg/kg QW

NO STOP

N = 41 TNBC patients

≤4 Gr 3/4 toxicities

in each tumor type

≥1 OR in TNBC*

Success: ≥5 OR

Stage 2 = 29 pts

Note: Subject 109112 was excluded from exploratory analyses due to discontinuation of treatment prior to C2

ABA CIC

SC5b-9

0

10

20

30

40

50

A.

C.

Cycle1 Cycle20

20

40

60

8017.8%

CD16+ DC14.9%

3.2%

Nonclassical

IntermediateCycle 1

Pre-dose

Cycle 2

Pre-dose

23.4%

5.5%

CD

16

29.3%

Monocytes Dendritic Cells

CD1cCD14

Monocytes Dendritic Cells

Cycle1 Cycle20

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Ki67+ PD-1+

CD

38

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