RON is a member of the c-MET receptor tyrosine kinase family. Like c-MET, RON is expressed by a variety of epithelial-derived tumors and cancer cell lines and it is thought to play a functional role in tumorigenesis. To date, antagonists of RON activity have not been tested in vivo to validate RON as a potential cancer target. In this report, we used an antibody phage display library to generate IMC-41A10, a human immunoglobulin G1 (IgG1) antibody that binds with high affinity (ED 50 = 0.15 nmol/L) to RON and effectively blocks interaction with its ligand, macrophage-stimulating protein (MSP; IC 50 = 2 nmol/L). We found IMC-41A10 to be a potent inhibitor of receptor and downstream signaling, cell migration, and tumorigenesis. It antagonized MSP-induced phosphorylation of RON, mitogen-activated protein kinase (MAPK), and AKT in several cancer cell lines. In HT-29 colon, NCI-H292 lung, and BXPC-3 pancreatic cancer xenograft tumor models, IMC-41A10 inhibited tumor growth by 50% to 60% as a single agent, and in BXPC-3 xenografts, it led to tumor regressions when combined with Erbitux. Western blot analyses of HT-29 and NCI-H292 xenograft tumors treated with IMC-41A10 revealed a decrease in MAPK phosphorylation compared with control IgG-treated tumors, suggesting that inhibition of MAPK activity may be required for the antitumor activity of IMC-41A10. To our knowledge, this is the first demonstration that a RON antagonist and specifically an inhibitory antibody of RON negatively affects tumorigenesis. Another major contribution of this report is an extensive analysis of RON expression in f100 cancer cell lines and f300 patient tumor samples representing 10 major cancer types. Taken together, our results highlight the potential therapeutic usefulness of RON activity inhibition in human cancers. (Cancer Res 2006; 66(18): 9162-70)

Exceptional clinical responses produced by the first chimeric antigen receptor T [CAR‐T] cell therapies, and their entry into commercial markets prompted a logarithmic increase in the number of next generation CAR‐T clinical trials. As a result, there is a growing interest in understanding the analytical approaches utilized for reliable monitoring of these “living” drugs, and the challenges encountered during their clinical development. Multiparametric flow cytometry (MFC) assays have played a crucial role in understanding the phenotype and function of first approved CAR‐T therapies. Herein, three main areas for monitoring CAR‐T therapies in clinical trials are discussed: (1) analytical considerations critical for development of MFC assays for the reliable enumeration of CAR‐T levels, (2) operational challenges associated with clinical trial sampling and transportation, and (3) differential cellular kinetics observed by MFC and qPCR analyses and their relationship with efficacy (measurable residual disease levels). Initial experiences described here may enable design of fit‐for‐purpose tools and help to more rapidly advance the development of next generation CAR‐T therapies.

Introduction: Rapidly advancing single cell genomics technology enables uncovering cell heterogeneity missed by bulk sequencing/analysis. Multi-omic single cell sequencing has ability to assess up to a hundred cell surface proteins, hundreds of transcripts to whole transcriptome, or TCR/BCR gene rearrangement in thousands of cells. Here we report deep cell immune profiling of PBMC by assessing selected cell surface proteins by AbSeq and targeted immune response genes using two single cell sequencing platforms. Experimental procedures: For targeted RNASeq on 10xGenomics Chromium platform, droplets containing single PBMC cell/bead were generated. After cDNA synthesis, libraries with genes of interest were generated using BD Rhapsody Human Immune Response Targeted Panel. For BD Rhapsody multi-omic single cell analysis, PBMC were stained using tagged AbSeq antibodies and sample specific cell tag antibodies. Then viable cells after incubation with cell viability dyes were counted on BD Rhapsody scanner. Some samples were processed for removal of apoptotic cells using EasySep Dead Cell Removal (Annexin V) Kit (Stem Cell Tech). Labeled PBMC from multiple subjects were pooled and loaded to a BD Rhapsody chip. Followed by cell capture beads loading and lysis, mRNA and antibody tags were captured. cDNA was synthesized and libraries containing genes of interest were generated using Human immune response panel along with AbSeq and sample tag libraries. Sequencing was performed on NextSeq 500 (Illumina). Data analysis from 10xGenomics were conduct using its Cell Ranger software. Data analysis from BD Rhapsody workflow were conduct using Seven Bridges Genomics Pipeline and SeqGeq (FlowJo). For comparison, PBMC were also profiled for general major cell populations by flow cytometry. Summary and Conclusions: Targeted RNASeq using both single cell sequencing platforms enables deep cell profiling with benefits of lower cost and higher sensitivity compared with whole transcriptome RNASeq. The results for general cell populations generated by single cell sequencing are comparable to those generated by flow cytometry, however, single cell sequencing classifies sub-populations in greater details based on gene expression markers. Including tagged AbSeq antibodies to assess cell surface proteins in the workflow further helps classification in the case of post-transcriptional regulation, while identifying alternatively spliced isoforms (CD45RO and CD45RA) elusive by RNASeq. One of the challenges is the requirement for viable cells. Although some apoptotic cells could be removed, less viable cells leftover could interfere single cell sequencing and thus careful data QC bioinformatics is warranted. Overall, multi-omic single cell sequencing could enable deep cell profiling and assessing patient samples has great potential to provide biological insights and identify predictive biomarkers. Citation Format: Wenge Shi, Christian Laing, Jane Gao, Kerri Burns, Shyam Sarikonda, Reinhold Pollner, Hua Gong. Multi-omic single cell sequencing for deep cell immune profiling and identification of potential biomarkers for cell therapy and immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4290.

Supplementary Figure 1 from Therapeutic Implications of a Human Neutralizing Antibody to the Macrophage-Stimulating Protein Receptor Tyrosine Kinase (RON), a c-MET Family Member

Supplementary Figure 2 from Therapeutic Implications of a Human Neutralizing Antibody to the Macrophage-Stimulating Protein Receptor Tyrosine Kinase (RON), a c-MET Family Member