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Evaluation of NXI-101 in non-small cell lung cancer (NSCLC) using patient-derived
organotypic tumor spheroids (PDOTS): Targeting a novel tumorigenic pathway..
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Julia Vail1, Hyung Gun Maeng2, Ji Woong Jang2, Kyungjin Boo2, Ji-In Youn2, Danielle Hagee1, Emma Cui1, Alyssa Martin1, Aaron Goldman3, Kyoung Wan Yoon2, Michael A. Perricone1
1Xsphera Biosciences, Inc., Boston, MA, 2NEX-I, Seoul, Korea, Republic of, 3Harvard Medical School, Boston, MA

BackgroundResults

About Xsphera PDOTS Platform

40

Atezolizumab 300 µg/ml
NXI-101 1 mg/ml
Atezolizumab 300 µg/ml + NXI-101 1 mg/ml

30

Δ AW % DeadONCOKINE-1 (OK-1), a soluble protein released by tumor cells, is thought tocontribute significantly to immunotherapy resistance by suppressing tumor
apoptosis and dendritic cell (DC) activation while elevating TGF-β levels. To
counteract this, NEX-I (Seoul, South Korea) has developed NXI-101, amonoclonal antibody aimed at neutralizing OK-1. Expected outcomes of NXI101 treatment include increased tumor cell death, enhanced DC activationand M1 polarization, and suppression of resistance mechanisms, offering apromising avenue for improving immunotherapy outcomes in cancer patients.

Treatment

Cytotoxicity and Target Expression

Patient-derived organotypic tumor spheroids (PDOTS), developed at the DanaFarber Cancer Institute (Boston, MA) and commercialized by Xsphera
Biosciences, Inc. (Boston, MA), offer a powerful preclinical model forevaluating efficacy, mechanism of action, and patient response variability totest articles (1,2). PDOTS encompass key components of the tumor
microenvironment (TME), in particular immune cells, making them an ideal
model for assessing the performance of immunotherapeutics andinvestigating related drug resistance and immune evasion mechanisms.

About NXI-101

Study Design*20-10Cytotoxicity
Proteomics
Transcriptomics

Cell Profile

P7P6P10P5P4P5

P3 P2

P7 P4 P2

P9P11

P3P9P6P8P8P10ECM

Live tumor samples collected from patients diagnosed with Non-Small Cell
Lung Cancer (NSCLC) are dissociated into spheroids using a mechanical andenzymatic digestion process. Patient derived organotypic tumor spheroids(PDOTS) 40 µm - 100 µm diameter are loaded into microfluidic devices
containing an extracellular matrix to support the tumor microenvironmentof the PDOTS. Following treatment with therapeutics, PDOTS cytotoxicity,growth, cytokine expression and RNA transcription are determined to study
the post-treatment effects on tumor growth and immune responses. Allgraphs were produced in the Xsphera Cloud.

Δ Norm RNA Count

PDOTs (S2)

P3

P6

P8

FIGURE 2. The cytotoxicity observed with NXI-101 was directly
correlated with baseline target expression level. Simple linear
regression of the cytotoxic response induced by NXI-101 with thebaseline OK-1 H-Score (p = 0.003). The cytotoxicity values for NXI101 were expressed as the difference in area-weighted % deadvalues from the control.

100 µg/ml
1 mg/ml• Gene Expression(Nanostring)

NXI-101

NXI-101/atezo

Media

Atezolizumab

NXI-101

AB

FIGURE 3. Cytotoxicity is associated with decreased tumor
cell survival in the cytotoxic responsive patient tumors.
Tumor cell associated RNA counts were decreased posttreatment relative to media in patients P1, P3, P6, P8. This
indicates a decrease in tumor cell abundance. Both NXI-101
and NXI-101/atezo treatment groups had marked decreases.
There were no differences in tumor associated RNA counts inthe non-responsive patient tumors (data not shown).

Atezolizumab

NXI-101

Conclusions• Potent Cytotoxic Effects: NXI-101/atezo combination enhanced the efficacy
of each monotherapy and elicited potent cytotoxic effects in 40% of thepatient tumors.
• Correlation with Target Expression: The magnitude of cytotoxic response
induced by NXI-101 correlated significantly with expression levels of thetarget, OK-1, suggesting a targeted therapeutic effect.

Atezolizumab + NXI-101
MediaFIGURE 5. Fluorescence images of Hoechst/PI stained NSCLCPDOTS within the microfluidic channels 3 days post-treatment.
Each channel contains hundreds of spheroids. NXI-101 andcombination NXI-101/atezo treatment groups have a higher
level of PI stain than the media control. Data from responsive
sample P1 are shown.

• Xsphera Ex Vivo Platform: Proven as a reliable tool for assessing the efficacy
and mechanism of action of immunotherapeutics, while also serving as amodel for investigating factors contributing to patient variability intreatment responses.

Molecular Pathways Associated with Response
Tumor Cell Survival

Media channel

P1

FIGURE 1. NXI-101 and NXI-101 + atezo elicited cytotoxic effects on select patient tumors. Changein area-weighted % dead (Δ AW % Dead) of NSCLC PDOTS expressed as difference from the media
control. 3 days post-treatment with atezolizumab (atezo; anti-PD-L1), NXI-101 or NXI-101/atezo
combination. The combination treatment elicited significant (p<0.05) cytotoxicity in 4 of 10 patienttumors. These tumors (P1, P3, P6, P8) were designated as “Responsive” tumors. While OK-1 HScores were correlated with NXI-101 cytotoxic response (Figure 5), PD-L1 H-Scores did not correlatewith atezolizumab cytotoxic response. While atezo had 1 of 10 response (P7) by AW % Dead, atezo
significantly reduced the live spheroid areas in 5 of 10 (data not shown).

THE MICROFLUIDIC PLATFORM
Drugs

Atezo
NXI-101

3 Days

P1P1600Tumor Cells
Immune Cells
Targets• Hundreds of spheroids in eachtreatment channel• Each spheroid contains tumor,stromal and immune cells and ECMcomponents• The platform preserves eachpatient’s tumor microenvironment(TME) containing tumor cells andthe immune contexture

1 mg/ml

Hoechst
PIΔ Norm RNA Count

Patientsample

Drug Treatment

Mechanical&enzymatic
digestion

NXI-101

0Δ Norm RNA Count

H&E
OK-1 IHC
PD-L1 IHC

100 µg/ml• Cytotoxicity(HPI Imaging)

10

PD-L1 H-Score

Histopathology

Atezolizumab

Endpoints**

OK-1 H-Score

The Xsphera Platform-Time***MediaMedia

*

Dose

Atezolizumab + NXI-101FIGURE 4. NXI-101 and NXI-101 + atezolizumab altered anti-tumor immune pathways in responsive
tumors, with variable effects across patients. (A) Combination treatment notably boosted immune effector
activity gene sets. (B) TGF-β suppression and c-Myc reduction with NXI-101 and NXI-101/atezo are consistent
with anti-tumor responses. Pathway analysis utilized Nanostring, MSigDB curated gene sets, and Gene Set
Enrichment Analysis.

• Activation of Immune Pathways: NXI-101 and NXI-101/atezo consistently
modulated immune pathways in cytotoxic responsive patient tumors
consistent with anti-tumor activities.
• De-Risking Drug Development: Xsphera’s functional platform provides
valuable data for exploring mechanisms of drug resistance and the potential
for patient stratification as drugs progress towards clinical application.
References:(1) Jenkins et al. (2018) Cancer Discovery 8 (2):196.
(2) Aref et al. (2018) Lab Chip, 2018, 18, 3129.