imprime pgg, a novel cancer immunotherapeutic, …...0 1000 2000 3000 4000 ex vivo imprimetreatment...
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Ex vivo Imprime Treatment Induces C5a in Healthy Human Volunteers (HHV) and Modulates C5aR Expression
Hypothesis
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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
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C5
a (
ng
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Neutrophils Monocytes
Mean Fluorescence Intensity (MFI)
Vehicle Imprime
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un
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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
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ng
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Pre-dose Post-dose
**p= 0.0011
SC
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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
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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.
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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
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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
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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).
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*p= 0.0455