Takara (United States)

Abstract Kirsten rat sarcoma (KRAS) is the most frequently altered oncogene, making mutant KRAS (mKRAS) an attractive target for neoantigen-specific T cell-based therapies. Identification of T cell receptors (TCRs) with strong and specific binding capabilities enables T cell therapies for cancer patients who harbor the respective mKRAS/HLA allele pair. Here, we generated the so far largest TCR warehouse library covering approximately one quarter of pancreatic ductal adenocarcinoma patients and 10% of colorectal cancer patients. There are eight TCRs in this library, targeting two KRAS G12D and six G12V neoantigens. The TCRs compare favorably based on expression, functional avidity, and cytotoxicity to existing clinical and literature benchmark TCRs. Warehouse TCRs are strong binders, exhibit robust cytotoxicity, and efficacy in in vivo models. Additionally, to evaluate potential safety risks, we performed cross-reactivity and alloreactivity analyses on two TCRs and did not observe any cross- or alloreactivities. Furthermore, detailed characterization of each TCR revealed unique biology for specific KRAS neoantigens. We show that TCRs recognize KRAS G12V on HLA-C*03:03 and HLA-C*03:04 interchangeably, thereby resulting in a single neoantigen with one of the highest frequencies. We observed a unique and striking enrichment in the gene segment, TRAV4*01, amongst 100 TCRs targeting G12V/C*03:03 and /C*03:04. Deeper analysis and understanding of these gene segments could be advantageous for future TCR discovery or optimization. Additionally, in the case of KRAS G12V/HLA-C*01:02, TCRs do not distinguish the mutant epitope from the wild-type epitope when exogenously loaded onto target cells, a phenomenon which has not been reported thus far. Our data suggests that in the natural processing and presentation setting, the level of wild-type epitope abundance appears to be significantly lower, creating a therapeutic window. In summary, an mKRAS-targeted TCR warehouse is introduced, comprising candidates for further preclinical and clinical development. Citation Format: Kendra Foley, Margaret Hallisey, Christian Yee, Tejashri Jadhav, Jared Dietze, Hannah Miller, Maria Massaro, Sebastian Newrzela, Marit van Buuren, Gavin Palowitch, Charles Dulberger, John Srouji, Richard Gaynor, Vikram Juneja. A LIBRARY OF POTENT TCRS DERIVED FROM HEALTHY DONORS TARGETING KRAS G12D AND G12V NEOANTIGENS [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr B002.

Abstract Knee osteoarthritis (KOA) is a common degenerative joint disease impairing mobility and quality of life, especially in the elderly. Conventional treatments often fail to address its multifactorial nature. This study explores invasive laser acupuncture integrated with electroacupuncture, delivering precise and minimally invasive multimodal stimulation. A randomized, sham-controlled, assessor-blinded, pilot sized clinical trial will evaluate the safety and efficacy of laser acupuncture combined with electroacupuncture in 45 KOA patients. Participants will be assigned to one of three groups: (1) 650 nm laser with electroacupuncture, (2) 830 nm laser with electroacupuncture, or (3) sham laser with sham electroacupuncture. Treatments occur twice weekly for four weeks. Primary outcome: pain reduction (VAS). Secondary outcomes: functional improvement (KOOS), quality of life (EQ-5D-5L), and safety. Of 45 randomized patients, 43 completed the trial. At week 5, VAS at rest decreased from 62.5 ± 11.3 to 32.7 ± 15.6 in the 650-nm group, 59.7 ± 8.6 to 30.5 ± 23.0 in the 830-nm group, and 66.9 ± 10.3 to 41.9 ± 20.5 in the control group. VAS during activity decreased from 68.7 ± 8.7 to 32.8 ± 14.6, 67.8 ± 7.6 to 31.4 ± 22.0, and 69.3 ± 10.2 to 43.8 ± 20.6, respectively. KOOS improved to 84.7 ± 19.5 (650 nm) and 83.3 ± 25.5 (830 nm) but not in controls (91.1 ± 21.1). EQ-5D scores improved slightly but without significance. PGA rose to 6.7 ± 1.1 (650 nm), 7.1 ± 1.5 (830 nm), and 6.8 ± 1.0 (control). No serious adverse events occurred; mild bruising was the most common event. This pilot-sized trial proposes an innovative, patient-centered approach to KOA management, potentially reducing reliance on medications and invasive procedures. And is a cornerstone to larger clinical trials. Trial Registration: CRIS PRE20231022-003.

Abstract Adoptive cell transfer (ACT) with T cells targeting KRAS neoantigens can drive anti-tumor immunity. However, due to the broad range of immunogenicity, the majority of ACT therapies have been focused on a small number of the over twenty different known KRAS neoantigens. Here, we develop a single process starting from peripheral blood that can prime and expand T cell responses to any KRAS neoantigen based on each individual’s HLA profile. We started with an ex vivo induction process, NEO-STIM, previously developed to induce T cell responses to a broad range of neoantigens. To account for the low fraction of antigen-specific T cells anticipated against individual KRAS neoantigens with low immunogenicity, we used a simple peptide pulse to upregulate 4-1BB (CD137) on cells specific for mutant KRAS and enriched using magnetic beads. We optimized both the initial stimulation and expansion steps such that nine of ten runs reached our target dose. Between the initial and optimized process runs, we conducted the process in 21 different healthy donors and generated T cell responses to 48 of 49 evaluated neoantigens with a range of antigen-specific frequency commensurate with the immunogenicity of the neoantigen. G12V neoantigens appeared more immunogenic on average than G12D neoantigens, with several HLA alleles (A*11:01, C*03:03, and C*03:04) presenting these epitopes in a particularly immunogenic fashion. Using tumor lines expressing the target KRAS mutation and HLA restriction elements, we demonstrated that the T cells generated through this process were able to slow tumor growth in vitro using a kinetic cytotoxicity assay and in vivo using a mouse xenograft model. We additionally cloned more than 150 KRAS T cell receptors (TCRs) in total, with multiple to each neoantigen. The strongest TCRs specific for KRAS G12D presented on HLA-A*11:01 and HLA-C*08:02 were similar or better than clinically active benchmark TCRs against these same targets, demonstrating that this approach can also be used for the discovery of clinically relevant TCRs from healthy donors. Together, this process can now be used as the basis for the development of a therapeutic or to discover a library of TCRs against each KRAS neoantigen regardless of immunogenicity. Citation Format: Brandon P Conn, Jared L Dietze, Christian J Yee, Margaret M Hallisey, Irais Ortiz-Caraveo, Marit M van Buuren, Richard B Gaynor, Kendra C Foley, Jaewon Choi, Vikram R Juneja. Generation of T cell responses against broad KRAS hotspot neoantigens using a single large-scale process as the basis for an adoptive cell therapy or for TCR discovery [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr PR009.

Abstract Single-cell RNA sequencing (scRNA-seq) has become a very powerful technology for biomedical research and is becoming much more affordable as methods continue to evolve, but it is unknown how reproducible different platforms are using different bioinformatics pipelines, particularly the recently developed scRNA-seq batch correction algorithms. We carried out a comprehensive multi-center cross-platform comparison on different scRNA-seq platforms using standard reference samples. We compared six pre-processing pipelines, seven bioinformatics normalization procedures, and seven batch effect correction methods including CCA, MNN, Scanorama, BBKNN, Harmony, limma and ComBat to evaluate the performance and reproducibility of 20 scRNA-seq data sets derived from four different platforms and centers. We benchmarked scRNA-seq performance across different platforms and testing sites using global gene expression profiles as well as some cell-type specific marker genes. We showed that there were large batch effects; and the reproducibility of scRNA-seq across platforms was dictated both by the expression level of genes selected and the batch correction methods used. We found that CCA, MNN, and BBKNN all corrected the batch variations fairly well for the scRNA-seq data derived from biologically similar samples across platforms/sites. However, for the scRNA-seq data derived from or consisting of biologically distinct samples, limma and ComBat failed to correct batch effects, whereas CCA over-corrected the batch effect and misclassified the cell types and samples. In contrast, MNN, Harmony and BBKNN separated biologically different samples/cell types into correspondingly distinct dimensional subspaces; however, consistent with this algorithm’s logic, MNN required that the samples evaluated each contain a shared portion of highly similar cells. In summary, we found a great cross-platform consistency in separating two distinct samples when an appropriate batch correction method was used. We hope this large cross-platform/site scRNA-seq data set will provide a valuable resource, and that our findings will offer useful advice for the single-cell sequencing community.

Continued interest in protein therapeutics has motivated the development of improved bioanalytical tools to support development programs. LC-MS offers specificity, sensitivity, and multiplexing capabilities without the need for target-specific reagents, making it a valuable alternative to ligand binding assays. Immunoaffinity purification (IP) and enzymatic digestion are critical, yet extensive and time-consuming components of the "gold standard" bottom-up approach to LC-MS-based protein quantitation. In the present work, commercially available technology, based on membrane-immobilized reagents in spin column and plate format, is applied to reduce IP and digestion times from hours to minutes. For a standard monoclonal antibody, the lower limit of quantitation was 0.1 ng μL-1 compared to 0.05 ng μL-1 for the standard method. A pharmacokinetics (PK) study dosing Herceptin in rat was analyzed by both the membrane and the standard method with a total sample processing time of 4 h and 20 h, respectively. The calculated concentrations at each time point agreed within 8% between both methods, and PK values including area under the curve (AUC), half-life (T1/2), mean residence time (MRT), clearance (CL), and volume of distribution (Vdss) agreed within 6% underscoring the utility of the membrane methodology for quantitative bioanalysis workflows.

IntroductionSingle-cell T-cell receptor (scTCR) clonotype analysis permits the determination of the specific TCR alpha-beta (α/β) chain pairing expressed on each cell. This pairing information allows researchers to gain insight into Tcell heterogeneity and plasticity, determine the contribution of the pairing to antigen specificity of the individual TCR, and design therapeutic antibodies. Here we employ a 5’-RACE-like approach and SMART technology, in conjunction with two novel next-generation sequencing (NGS) library preparation kits, using the same primer pairs, to capture full-length variable regions of TCR-α and -β chains.Material and methodsMethod 1, using the SMARTer Human scTCRa/b Profiling Kit, permits NGS library preparation of FACS-sorted cells in 96-well plates. Method 2 is an adaptation of the process above that scales to ~1200 single cells using the SMARTer ICELL8 Single-Cell System, which enables single-cell isolation and nanoliter PCR in a nanowell chip.Results and discussionsWe present data showing α/β pairing from Jurkat, CCRF, PBMCs, and CD4 +T cells. In addition to the sensitivity of this method, the ability to pool the cDNA from 96wells into 12 sequencing libraries adds to the ease of use. Consistent with immunology reports, unstimulated CCRF-CEM cells examined with this kit expressed a TCR-β but not a TCR-α chain.scTCR pofiling with SMARTer ICELL8 Single-Cell System as a proof of principle of scTCRα/β was performed on Jurkat cells and CCRF-CEM cells.Jurkat cells and CCRF-CEM cells were processed using an ICELL8 chip preprinted with barcoded oligos. Paired TCRα/β Jurkat clonotypes were detected in 77% and 87% cells in mixed and single cell populations, respectively.ConclusionThe ability of the core biochemistry and PCR components of these kits to be used with either FACS-sorted cells in 96-well plates or >1,000 cells in novel ICELL8 chips (in development) points to the general utility and scalability of this approach in understanding paired scTCR clonotype diversity.

We have developed a high-throughput single-cell ATAC-seq (assay for transposition of accessible chromatin) method to measure physical access to DNA in whole cells. Our approach integrates fluorescence imaging and addressable reagent deposition across a massively parallel (5184) nano-well array, yielding a nearly 20-fold improvement in throughput (up to ~1800 cells/chip, 4-5 hour on-chip processing time) and cost (~98¢ per cell) compared to prior microfluidic implementations. We applied this method to measure regulatory variation in Peripheral Blood Mononuclear Cells (PBMCs) and show robust,de-novoclustering of single cells by hematopoietic cell type.

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.