# Stanford Project 2

> **NIH NIH U54** · UNIVERSITY OF CALIFORNIA, SAN FRANCISCO · 2021 · $516,569

## Abstract

SUMMARY
Non-small cell lung cancer (NSCLC) is an aggressive malignancy in which limited treatment options are further
compromised by treatment resistance. Immunotherapy, particularly against the PD-1/PD-L1 immune checkpoint,
has transformed NSCLC treatment with durable responses and comparatively minimal side effects in both
second-line treatment of metastatic disease and, recently, first line therapy. Despite these impressive responses
there are equally impressive but poorly defined intrinsic and/or acquired resistance mechanisms. Across solid
tumor types, the response rate targeting the PD-1 axis in unselected patients is only ~20-30%. In previously
untreated NSCLC, the overall response rate (ORR) to the anti-PD-1 antibody pembrolizumab is only ~45% even
with patient pre-selection for >50% IHC PD-L1 tumor positivity, PD-L1 negative patients also exhibit anti-tumor
response and the active search for alternative biomarkers of response in NSCLC has been unfulfilled. Thus, the
significant resistance mechanisms impairing response to PD-1-targeted agents in NSCLC and other diverse
solid tumors have remained intractable to both biomarker discovery and accompanying mechanistic definition.
 Project 2 of this U54 application thus directly addresses the pressing issue of intrinsic and acquired
resistance to PD-1-targeted immunotherapy in NSCLC through analysis of an invaluable cohort of on-treatment
longitudinal biopsies. Aim 1 pursues deep single-cell RNA-seq profiling of the immune component of NSCLC
anti-PD-1 on-treatment biopsies using a highly efficient, microfluidic bead-based protocol allowing unsupervised
discovery of cell clusters, T cell activation or exhaustion states and transcriptomic insights into immunotherapy
resistance. Aim 2 exploits our 3D Patient-Derived Tumor Organoid (PDO) cultures that represent the first in
vitro functional recapitulation of the PD-1-dependent immune checkpoint and tumor infiltrating lymphocytes
(TILs) within clinical NSCLC biopsies. Here, we create functional organoid culture models of NSCLC
immunotherapy resistance from longitudinal biopsies, measuring TIL activation upon in vitro anti-PD-1 organoid
treatment and correlating against patient response. Lastly, Aim 3 performs prospective liquid biopsy and
exome sequencing to determine mutational signatures of anti-PD-1 resistance that functionally regulate
immune checkpoints. These Aims utilize synergistic expertise from the Stanford site of the U54 with Calvin Kuo
(Project 2 PI; organoid culture, single cell RNA-seq), Ron Levy (tumor immunotherapy) and Heather Wakelee
and Suki Padda (NSCLC immunotherapy trials), all in close coordination with Project 1 clinical biopsies, liquid
biopsies and whole exome sequencing from Trever Bivona and Sourav Bandyopadhyay of the UCSF site.
Overall, we present a comprehensive approach to intrinsic and acquired resistance to PD-1 inhibition in NSCLC
via complementary single cell, organoid and sequencing analysis of longitudinal on-t...

## Key facts

- **NIH application ID:** 10241310
- **Project number:** 5U54CA224081-04
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
- **Principal Investigator:** CALVIN J KUO
- **Activity code:** U54 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $516,569
- **Award type:** 5
- **Project period:** 2017-09-30 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10241310

## Citation

> US National Institutes of Health, RePORTER application 10241310, Stanford Project 2 (5U54CA224081-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10241310. Licensed CC0.

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