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Utilizing Tri-specific Killer Engagers Against B7H3 to Overcome Dexamethasone-Mediated Immunosuppression of NK Cells

8/16/2023

Virginia Hsiao

Miller Lab

Medical Student Summer Research Program in Infection and Immunity

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Glioblastoma Multiforme

Fast growing, aggressive brain tumor
Incidence of 4 in 100,000 people
14% of all primary brain tumors
Median survival is 14 months
Dexamethasone (steroid) treatment for cerebral edema poses a challenge to efficacy of immunotherapies due to its immunosuppressive nature
Critical need for new efficacious treatment

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NK Cells

NK Cells as a potential cellular therapy
Benefits of NK Cells

Not MHC restricted
No priming needed
No GVHD
~5-15% of circulating lymphocytes

Prior studies have shown efficacy of NK cells (e.g., AML)
Prior studies have also shown that NK cells can lyse GSCs and mature GBM cells

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NK Cell Mechanisms

Figure from Rasa Islam et al

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Strategies to enhance NK cell therapy

Tumor

cell

NK

cell

CD16

IL-15

B7H3

Created with BioRender.com by Melissa Khaw

Tri-specific Killer Engager (TriKE)

Different Sources for Adoptive Transfer

NK cell

Expansion with K562** feeder cells

Expansion with K562 feeder cells

Expanded PBNK

Expanded Adaptive NK

From CMV+ seropositive Donors*

(NKG2C+CD57+)

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Expanded Cell Phenotype

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Phenotypic markers identify distinct populations in Expanded PBNK and Adaptive NKs post-expansion

Expanded PBNK

Expanded Adaptive NK

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Specific Aims

Specific Aim: Evaluate the suppressive effects of the steroid Dexamethasone on NK cells and the ability of various activation signals to overcome immune suppression.

TriKE
Different sources of adoptive transfer
IL-15 levels

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To Explore this

Dexamethasone titration

Impact on phenotype and activation markers

Functional Assessment

Impact on degranulation and cytokine production

Live Imaging Assay

Killing kinetics

Cell proliferation
Pre-complexing

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Dexamethasone Titration

Impact on phenotypic and activation markers

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Dexamethasone does not robustly impact NK phenotype and activation markers

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Dexamethasone does not robustly impact NK activation and phenotypic markers

CD16

NKG2D

CD69

Dex

No Dex

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Functional Assessment

Impact on degranulation and cytokine production

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Dexamethasone decreases cytokine production in expanded Adaptive cells but not degranulation. Addition of TriKE improves function of Dexamethasone treated cells

IL-15 Treatment

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Dexamethasone decreases cytokine production in expanded PBNK cells but not degranulation. Addition of TriKE improves function of Dexamethasone treated cells

IL-15 Treatment

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Dexamethasone impacts cytokine production and degranulation in T cells

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Real-time Imaging Assays

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Dexamethasone slows the killing kinetics of expanded Adaptive NK cells and is restored by the addition of TriKE

Dex: 1µM

TriKE: 3nM

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Cell Proliferation

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Dexamethasone impairs cell proliferation of expanded PBNK cells, which is abrogated by TriKE

1 µM Dex, 10 ng/mL IL-15

1 µM Dex, 1 ng/mL IL-15

1 ng/mL IL-15

10 ng/mL IL-15

1 µM Dex, 30 nM TriKE

30 nM TriKE

1 µM Dex, 3 nM TriKE

3 nM TriKE

Expanded PBNK

Expanded Adaptive NK

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Pre-Complexing

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NK

cell

CD16

IL-15

B7H3

Pre-complexed (B7H3 TriKE to NK Cell)

NK

cell

TriKE

Initial Components

Challenge: how do we address dosing for a potential phase 1 trial?

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TriKE can be precomplexed to Expanded PBNK and Adaptive NK

90 nM TriKE

60 nM TriKE

30 nM TriKE

0 nM TriKE

99.8%

99.5%

99.0%

0.94%

68.9%

66.0%

55.7%

0.99%

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Conclusions

Understanding the immunosuppressive effects of Dexamethasone is critical for increasing treatment efficacy for GBM

Dexamethasone is immunosuppressive but does not notably affect activation or phenotype of NK cells

Functionally, Dexamethasone decreases cytokine production of expanded PBNK and Adaptive NK cells, but does not impact degranulation via CD107a, while T cells are suppressed for both

Increasing IL-15 concentrations does seem to have a subtle impact on functionality
TriKE does seem to improve function of NK cells when exposed to dexamethasone

Dexamethasone does impact the killing kinetics of expanded Adaptive NKs and TriKE restores this

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Discussion

Overall, this demonstrates the potential of NK cells as a therapy in the context of dexamethasone treatment and immunosuppression

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Future Directions

Future experiments will seek to repeat these experiments and explore the following:

The impact of Dexamethasone with the complex tumor microenvironment (e.g., hypoxia, TGF-β)
Specific testing using tumors isolated from GBM patient samples

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Acknowledgements

Miller Lab

Hongbo Wang
Ross Cromarty
Zachary Davis
Bin Zhang
Gwen Phung
Melissa Khaw
Rhett Waller
Jacob Myers
Pippa Kennedy
Jeffrey Miller

NIH Summer T35 Program

Dr. Daniel Mueller
Drew Keup
The incredible seminar speakers this summer!

Thank you everyone for supporting my learning this summer!

Flow Cytometry Seminar Leads

Maude Ashby
Andrew Soerens
Matous Voboril

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Supplemental

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Dexamethasone does not robustly impact T cell activation and phenotypic markers

NKG2D

CD28

CD69

NKG2D

Dex

No Dex

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PBNK CD107a

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PBNK IFNg

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Adapt NK CD107a

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Adapt NK IFNg

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PBNK TNFa

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Adaptive NK TNFa

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PBNK MFI

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PBNK IFNg MFI

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PBNK TNFa (MFI)

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AdaptNK, CD107a (MFI)

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AdaptNK, IFNg (MFI)

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AdaptNK, TNFa, (MFI)

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Dex Titration

CD4+ T cells

+ 1 µM Dex, 48 hours

0 µM Dex

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Dexamethasone impairs cell proliferation of expanded PBNK cells, which is abrogated by TriKE

1 µM Dex, 10 ng/mL IL-15

1 µM Dex, 1 ng/mL IL-15

1 ng/mL IL-15

10 ng/mL IL-15

1 µM Dex, 30 nM TriKE

30 nM TriKE

1 µM Dex, 3 nM TriKE

3 nM TriKE

Expanded PBNK

Expanded Adaptive NK