LB218/13

Advanced Ex Vivo Platform for Target Expression and Drug Response Analysis:

Evaluating the Potent Effects of NMC-521 mAb on Fresh Mouse- and Patient-Derived Organotypic Tumor Spheroids..
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Amir Aref1, Hamidreza Aboulkheyr2, Alyssa Martin1, Patryk K Krzesaj2, Victor Adler2, Houman Mahjour2, Anthony Attardo1, Sinal Patel1, Ketki Bhise1, Ehsan Sarafraz-Yazdi2, Michael A. Perricone1
1Xsphera Biosciences, Inc., Boston, MA, 2NomoCan Pharmaceuticals, Inc., New York, NYResults

Background
About Xsphera PDOTS Platform

Murine CT26 Tumors
EX VIVO CT26 TUMOR SPHEROID PLATFORM (MDOTS)
A

In tumor cells, NMC-1, typically a nuclear protein, undergoes alternativesplicing, leading to a membrane-bound variant on the cell surface. NMC-521,a monoclonal antibody (mAb), was developed by NomoCan Pharmaceuticals(New York, NY) to target NMC-1 as a first-in-class therapeutic selectively
targeting cancers that express NMC-1. NMC-521 is an IgG1 mAb with antitumor activities that include antibody dependent cell cytotoxicity (ADCC) andcomplement-dependent cytotoxicity (CDC) as well as robust activation of an
NK-mediated tumor killing. On this poster, NMC-521 refers to the α-hu mAb
as well as the murine surrogate mAb against the NMC-1 target.

NMC-521 (µg/ml)

FIGURE 1. NMC-521 elicited cytotoxicity on CT26
MDOTS ex vivo in a dose-dependent manner. A-B.
NMC-1 target expression on CT-26 cell line assessedby flow cytometry and IF. C. NMC-521 suppresses
tumor spheroid outgrowth and enhances tumor
spheroid killing ex-vivo in the CT-26 MDOTS model. D.
NMC-521 elicited cytotoxicity (Live Cell Area) in dosedependent manner.

- H&E
- IHC
- Genomics

Drug Treatment- Cytotoxicity- Proteomics- Transcriptomics

Cell Profile

FIGURE 2. Mechanism of NMC-521 involved NK
cell-mediated killing. CT26 MDOTS treated withNMC-521 elicited significant cytotoxicity relativeto IgG (p<0.0001). Efficacy was partially reducedby neutralizing NK cell activity
by coadministration with ⍺NKG2D mAb (p<0.05),
whereas ⍺CD8 neutralizing mAb did not reduce
activity of NMC-521. Mean ± sd shown.

FIGURE 3. NMC-521 suppressed the growth of tumors in theCT26 BALB/c murine syngeneic tumor model. A. Tumor growth
was significantly suppressed in the NMC-521 alone or in
combination with 5-FU treated mice. The combination treatment
had greater efficacy than either of the monotherapies as wassignificantly reduced from the isotype control, B. Synergistic effectof CTLA4 with NMC-521 in tumor growth inhibition. p<0.0001.The
results with the ex vivo CT26 MDOTS platform corroborate the invivo CT26 tumor model and provide a potential MOA.

Patient-Derived Organotypic Tumor Spheroids (PDOTS)
Treatment

THE MICROFLUIDIC PLATFORM
Media channel

IgG1

100 µg/ml

Nivolumab

40 µg/ml

NMC-52150 µg/ml

Nivolumab +NMC-521

50 µg/ml
40 µg/ml

Time3 Days

% change in live area

ECM

DoseEndpoints• Gene Expression(Nanostring)

P02

P06

P03

• Potent Cytotoxic Effects: NMC-521 was highly effective in the ex vivo
platform using murine CT26 MDOTS and mirrored the effects in thesyngeneic in vivo CT26 mouse tumor model.

Immune activating
pathways** p< 0.05

• Xsphera Ex Vivo Platform: The Xsphera ex vivo platform is a proven tool forassessing the efficacy and mechanism of action of immunotherapeutics
and serving as a model to investigate factors contributing to variability intreatment responses.

Cell survival andProliferation
pathways**P01

B• Cytotoxicity(HPI Imaging)

*

P04

A

4 03 0Control
2 5 1 6 3 1 05 06 04 02
P0 IgGP P P0 P0 P0 P0 P0NMC-521
P P P P

* *P05FIGURE 4. NMC-521 induced cytotoxicity in 3 of 6 patient
tumors using the PDOTS ex vivo platform. The live
spheroid areas were decreased relative to IgG1 control inpatient tumors (P06, P03, P05).
Combination withnivolumab did not improve the cytotoxic response of NMC521.

FIGURE 5. Fluorescence images of PDOTS demonstrating thecytotoxic effects of NMC-521. Hoechst-PI stained endometrial (A)and non-small cell lung cancer (B) PDOTS from shown from aportion of the microfluidic channel. The PDOTS had were viable(limited PI stain) in the IgG and nivolumab treatment groups, while
the NMC-521 treatment arm had smaller spheroids and a highproportion of PI-stained spheroids. (Figure-5B is a high projected
image of Hoechst-PI staining).

Conclusions

Norm RNA count- Tumor Cells- Immune Cells- Targets

Live tumor specimens collected from murine CT-26 tumors or humanendometrial cancer patients were dissociated into spheroids using amechanical and enzymatic digestion process. Murine- or patient-derived
organotypic tumor spheroids (MDOTS or PDOTS) of 40 µm - 100 µm
diameter were size fractionated and loaded into microfluidic devices with an
extracellular matrix to support the tumor microenvironment of the PDOTS.
Following treatment with therapeutics, cytotoxicity is measured by imaging
and RNA transcription profiles are measured to study the post-treatment
effects on tumor growth and immune responses.

Bb 2D 21 2D D8 D82gG KG C-5 NKG + ⍺C ⍺C
IN M ⍺++⍺
1
D1N + 52
2
G
1 CGIg2
K* p<0.05
5 M ⍺NCN+** p<0.01
M
1N2
5*** p<0.001
CM**** p<0.0001
NLive Spheroid Area
Dead Spheroid Area

PIHoechst

NSCLC

Live Cell Area (µm2)

BHistopathology• Hundreds of spheroids in eachDrugs
treatment channelPDOTs (S2)
• Each spheroid contains tumor,
stromal and immune cells and ECMcomponents• The platform preserves the tumor
microenvironment (TME) containing
tumor cells and the immunecontexture

No Spheroid Overgrowth

Dead MDOTSThe Xsphera Platform

Patientsample

A

Spheroids Overgrow Channel

Viable MDOTSDA

EpCAM NMC-1 DAPI

488-B-A :: NMC-1

C

IN VIVO CT26 TUMOR MODELB

NMC-1

About NMC-521

Mechanical&enzymatic
digestion

PDOTS Imaging
Endometrial

Patient-derived organotypic tumor spheroids (PDOTS), developed at theDana-Farber 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.

P05
P03
P06

FIGURE 5. The drug treatments modulated the molecular immune profile in cytotoxicity responsive patientsusing Nanostring IO360 panel. A. Post-treatment pathway enrichment analysis (log 2 norm RNA count) of all
patient tumors treated with NMC-521 (right side) compared to IgG control (left side). The heatmap clearly
illustrates the robust activation of immune-activity related pathways (high NES score) and reduction of cell
survival pathways in response to NMC-521 compared to IgG control. B. Nivolumab, NMC-521, and their
combination consistently elevated the RNA counts of CD8+ T cell and NK cell associated RNAs in the cytotoxic
responsive patients identified in Figure 4. Both NMC-521 and the combination treatment showed greater
potency in enhancing these cell signatures compared to nivolumab alone.

• Mechanism of Action (MOA): A potential MOA for NMC-521 was identified
using neutralizing antibodies: neutralizing NK cell antibodies reduced
cytotoxicity, while CD8+ T cell neutralization had no effect.
• De-Risking Drug Development: NMC-521 elicited cytotoxic effects in 3 of 6
patient tumors tested and immune pathways were activated in theresponsive patient tumors. NMC-521 has clinical potential as a naked mAbor as an ADC-based therapeutic.
References:(1) Jenkins et al. (2018) Cancer Discovery 8 (2):196.
(2) Aref et al. (2018) Lab Chip, 2018, 18, 3129.