Supplementary Data from Immuno-PET Monitoring of Lymphocytes Using the CD8-Specific Antibody REGN5054

Abstract Assessment of immune-cell subsets within the tumor immune microenvironment is a powerful approach to better understand cancer immunotherapy responses. However, the use of biopsies to assess the tumor immune microenvironment poses challenges, including the potential for sampling error, restricted sampling over time, and inaccessibility of some tissues/organs, as well as the fact that single biopsy analyses do not reflect discordance across multiple intrapatient tumor lesions. Immuno-positron emission tomography (PET) presents a promising translational imaging approach to address the limitations and assess changes in the tumor microenvironment. We have developed 89Zr-DFO-REGN5054, a fully human CD8A-specific antibody conjugate, to assess CD8+ tumor-infiltrating lymphocytes (TIL) pre- and posttherapy. We used multiple assays, including in vitro T-cell activation, proliferation, and cytokine production, and in vivo viral clearance and CD8 receptor occupancy, to demonstrate that REGN5054 has minimal impact on T-cell activity. Preclinical immuno-PET studies demonstrated that 89Zr-DFO-REGN5054 specifically detected CD8+ T cells in lymphoid tissues of CD8-genetically humanized immunocompetent mice (VelociT mice) and discerned therapy-induced changes in CD8+ TILs in two models of response to a CD20xCD3 T-cell activating bispecific antibody (REGN1979, odronextamab). Toxicology studies in cynomolgus monkeys showed no overt toxicity, and immuno-PET imaging in cynomolgus monkeys demonstrated dose-dependent clearance and specific targeting to lymphoid tissues. This work supports the clinical investigation of 89Zr-DFO-REGN5054 to monitor T-cell responses in patients undergoing cancer immunotherapy.

Despite the enormous promise of T cell therapies, the isolation and study of human T cell receptors (TCRs) of dedicated specificity remains a major challenge. To overcome this limitation, we generated mice with a genetically humanized system of T cell immunity. We used VelociGene technology to replace the murine TCRαβ variable regions, along with regions encoding the extracellular domains of co-receptors CD4 and CD8, and major histocompatibility complex (MHC) class I and II, with corresponding human sequences. The resulting “VelociT” mice have normal myeloid and lymphoid immune cell populations, including thymic and peripheral αβ T cell subsets comparable with wild-type mice. VelociT mice expressed a diverse TCR repertoire, mounted functional T cell responses to lymphocytic choriomeningitis virus infection, and could develop experimental autoimmune encephalomyelitis. Immunization of VelociT mice with human tumor-associated peptide antigens generated robust, antigen-specific responses and led to identification of a TCR against tumor antigen New York esophageal squamous cell carcinoma-1 with potent antitumor activity. These studies demonstrate that VelociT mice mount clinically relevant T cell responses to both MHC-I– and MHC-II–restricted antigens, providing a powerful new model for analyzing T cell function in human disease. Moreover, VelociT mice are a new platform for de novo discovery of therapeutic human TCRs.

Profiling T cell receptor (TCR) repertoire via short read transcriptome sequencing (RNA-Seq) has a unique advantage of probing simultaneously TCRs and the genome-wide RNA expression of other genes. However, compared to targeted amplicon approaches, the shorter read length is more prone to mapping error. In addition, only a small percentage of the genome-wide reads may cover the TCR loci and thus the repertoire could be significantly under-sampled. Although this approach has been applied in a few studies, the utility of transcriptome sequencing in probing TCR repertoires has not been evaluated extensively. Here we present a systematic assessment of RNA-Seq in TCR profiling. We evaluate the power of both Fluidigm C1 full-length single cell RNA-Seq and bulk RNA-Seq in characterizing the repertoires of different diversities under either naïve conditions or after immunogenic challenges. Standard read length and sequencing coverage were employed so that the evaluation was conducted in accord with the current RNA-Seq practices. Despite high sequencing depth in bulk RNA-Seq, we encountered difficulty quantifying TCRs with low transcript abundance (<1%). Nevertheless, top enriched TCRs with an abundance of 1-3% or higher can be faithfully detected and quantified. When top TCR sequences are of interest and transcriptome sequencing is available, it is worthwhile to conduct a TCR profiling using the RNA-Seq data.

The characteristics of the data traffic generated by the use of micro-browser-enabled PCS phones to gain access to the Web is of particular interest to cellular network operators. Questions such as the frequency and length of browser sessions, and the specific characteristic of the traffic generated, need to be answer by researchers. These answers are valuable in network capacity planning as more subscribers use their cellular phones to interact with the Web.In this paper, we analyse network traces generated by a Mobile Browser application. We observe daily and weekly cycles and, some evidence of self-similarity in the network traffic produced by the application. We show that the activity factor for data generated by this application is lower than voice activity factor. This fact is significant for the design of wireless networks because it is possible to multiplex a large number of data sources onto existing channels with the appropriate protocols. We also compare and contrast the Mobile Browser traffic characteristics with results for WWW traffic published in the literature.