Single Cell Line Research Articles

Establishment of a novel experimental system using single cell-derived pleomorphic rhabdomyosarcoma cell lines expressing K-RasG12V and deficient in p53.

Pleomorphic rhabdomyosarcoma (PRMS) predominantly arises in adult skeletal musculature and is usually associated with poor prognosis. Thus, effective treatments must be developed. PRMS is a rare tumor; therefore, it is critical to develop an experimental system to understand the cellular and molecular mechanisms of PRMS. We previously demonstrated that PRMS develops after p53 gene deletion and oncogenic K-Ras expression in the skeletal muscle tissue. In that study, oncogenic K-Ras-expressing cells were diverse and the period until disease onset was difficult to control. In this study, we developed an experimental system to address this problem. Single cell-derived murine cell lines, designated as RMS310 and RMSg2, were established by limiting the dilution of cells from a lung metastatic tumor colony that were positive for various cancer stem cells and activated skeletal muscle-resident stem/progenitor cell marker genes by RT-PCR. All cell lines stably recapitulated the histological characteristics of human PRMS as bizarre giant cells, desmin-positive cells, and lung metastases in C57BL/6 mice. All subclones of the RMSg2 cells by the limiting dilution in vitro could seed PRMS subcutaneously, and as few as 500 RMSg2 cells were sufficient to form tumors. These results suggest that the RMSg2 cells are multipotent cancer cells that partially combine the properties of skeletal muscle-resident stem/progenitor cells and high tumorigenicity. Thus, our model system's capacity to regenerate tumor tissue in vivo and maintain stable cells in vitro makes it useful for developing therapeutics to treat PRMS.

Synthesis and antiproliferative activity of (5R)-cleistenolide and analogues

(5R)-Cleistenolide and a few related analogues have been synthesized starting from d-glucose. The key steps of the synthesis included a Z-selective Wittig olefination and an intramolecular Mitsunobu reaction with an inversion of configuration at the C-5 position. In vitro antiproliferative activity of synthesized compounds was tested on a panel of eight human tumour cells and against a single normal cell line (MRC-5). The majority of tested compounds showed strong antiproliferative effects on certain human tumour cells and all of them showed negligible toxicity to normal foetal lung fibroblasts (MRC-5). The most active compound obtained in this work is lactone 5, which in MDA-MB 231 cell culture showed the same activity as doxorubicin (IC50 0.09 ?M). Strong antiproliferative activities of analogues 2, 5 and 6 were recorded in the K562 cell line (IC50 0.21, 0.34 and 0.33 ?M, respectively), in which they showed very similar activities to doxorubicin (IC50 0.25 ?M). A performed SAR study revealed that a change in the stereochemistry at the C-5 position may increase the activity of resulting stereoisomers.

Divergent synthesis and antitumour activity of novel conformationally constrained (−)-muricatacin analogues

Four novel conformationally restricted (?)-muricatacin analogues, bearing a methoxy group at the C-5 position and with an alkoxymethyl group ?s the C-7 side chain, have been synthesised and their in vitro antiproliferative activity was evaluated against a panel of seven human tumour cell lines, as well as a single normal cell line. All analogues (9?12) showed diverse antiproliferative effects against all tested human malignant cell lines, but were devoid of any significant cytotoxicity towards the normal foetal lung fibroblasts (MRC-5). A structure?activity relationship study reveals that the introduction of tetrahydrofuran ring, the replacement of C-8 methylene group in the side chain of muricatacin analogues with the O-8 ether functionality, as well as the length of side chain may be beneficial for the antiproliferative effects of these lactones. All novel analogues were more potent than lead compound, (?)-muricatacin, against HL-60 cell line.

Patient-by-Patient Deep Transfer Learning for Drug-Response Profiling Using Confocal Fluorescence Microscopy of Pediatric Patient-Derived Tumor-Cell Spheroids.

Image-based phenotypic drug profiling is receiving increasing attention in drug discovery and precision medicine. Compared to classical end-point measurements quantifying drug response, image-based profiling enables both the quantification of drug response and characterization of disease entities and drug-induced cell-death phenotypes. Here, we aim to quantify image-based drug responses in patient-derived 3D spheroid tumor cell cultures, tackling the challenges of a lack of single-cell-segmentation methods and limited patient-derived material. Therefore, we investigate deep transfer learning with patient-by-patient fine-tuning for cell-viability quantification. We fine-tune a convolutional neural network (pre-trained on ImageNet) with 210 control images specific to a single training cell line and 54 additional screen -specific assay control images. This method of image-based drug profiling is validated on 6 cell lines with known drug sensitivities, and further tested with primary patient-derived samples in a medium-throughput setting. Network outputs at different drug concentrations are used for drug-sensitivity scoring, and dense-layer activations are used in t-distributed stochastic neighbor embeddings of drugs to visualize groups of drugs with similar cell-death phenotypes. Image-based cell-line experiments show strong correlation to metabolic results ( R ≈ 0.7 ) and confirm expected hits, indicating the predictive power of deep learning to identify drug-hit candidates for individual patients. In patient-derived samples, combining drug sensitivity scoring with phenotypic analysis may provide opportunities for complementary combination treatments. Deep transfer learning with patient-by-patient fine-tuning is a promising, segmentation-free image-analysis approach for precision medicine and drug discovery.

Tumors derived from lung cancer cells respond differently to treatment with sodium valproate (a HDAC inhibitor) in a chicken embryo chorioallantoic membrane model.

Lung cancer is the most frequent cause of cancer death. Some human lung malignant tumors have a combined small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) histology, with tumor cell phenotype changing during tumor progression. Valproic acid is used as an anti-seizure medication to treat migraine, and bipolar mood disorders. Recently, its efficacy as an adjuvant therapy was shown in cancer due to its histone deacetylase (HDAC) inhibitory property. HDACs are upregulated in lung tumors, and HDAC inhibitors, including valproic acid, inhibit endothelial cell proliferation in vitro and in vivo and have antiproliferative and antimigratory properties. We tested valproic acid for possible antiangiogenic and antimigratory effects on experimental lung tumors grafted onto the chicken embryo chorioallantoic membrane (CAM). Tumors were formed from two NSCLC cell lines and a single SCLC cell line. To investigate tumor and CAM interactions, in vivo biomicroscopy, visualization of blood vessels with injected fluorescent dextran, histological, immunohistochemical and histomorphometric methods were applied. Our results showed that a sodium valproate (NaVP) treatment-induced a dose-dependent decrease of experimental tumor invasion into the CAM mesenchyme and a reduction in angiogenesis. Both the invasion and the angiogenic response were dependent on the type of cell line used: invasion and angiogenesis of tumors derived from A549 and NCI-H146 cell lines responded to increasing doses of NaVP from 4 to 8 mM, whereas Sk_Lu_1 cells response were antimigratory and antiangiogenic when NaVP was used up to 6 mM. When 8mM NaVP was used, stimulated invasion and angiogenesis in tumors from Sk_Lu_1 cells were observed.

Organ-Specific Endothelial Dysfunction Following Total Body Irradiation Exposure.

As the single cell lining of the heart and all blood vessels, the vascular endothelium serves a critical role in maintaining homeostasis via control of vascular tone, immune cell recruitment, and macromolecular transit. For victims of acute high-dose radiation exposure, damage to the vascular endothelium may exacerbate the pathogenesis of acute and delayed multi-organ radiation toxicities. While commonalities exist between radiation-induced endothelial dysfunction in radiosensitive organs, the vascular endothelium is known to be highly heterogeneous as it is required to serve tissue and organ specific roles. In keeping with its organ and tissue specific functionality, the molecular and cellular response of the endothelium to radiation injury varies by organ. Therefore, in the development of medical countermeasures for multi-organ injury, it is necessary to consider organ and tissue-specific endothelial responses to both injury and candidate mitigators. The purpose of this review is to summarize the pathogenesis of endothelial dysfunction following total or near total body irradiation exposure at the level of individual radiosensitive organs.

Feeder cell-dependent primary culture of single blastula-derived embryonic cell lines from marine medaka (Oryzias dancena).

Fish embryonic stem cells (ESCs) are derived from blastomeres that have been cultured from blastula embryos. The most widely used method for derivation of fish ESCs is the culture of blastomeres that have been isolated from approximately 10 blastula embryos under feeder-free conditions. However, this method leads to intercellular genetic heterogeneity among the cultured cells, which is a major obstacle to the development of stable ESC culture conditions. In this study, to establish ESC lines with intercellular genetic homogeneity at the early stage of culture, we attempted to derive embryonic cell lines from single blastula-derived blastomeres of marine medaka (Oryzias dancena) in a feeder cell culture system. Using basic fibroblast growth factor-expressing feeder cells during primary culture, we successfully established 22 single blastula-derived embryonic cell lines that could be subcultured more than 20 times. In contrast, we were unable to efficiently derive cell lines using wild-type feeder cells and under feeder-free conditions. The established cell lines exhibited ESC-like cell characteristics in terms of alkaline phosphatase activity, pluripotency-related gene expression, and embryoid body formation. The results of this study will contribute to the development of methods for derivation of fish ESCs.

A Systematic Study of Size Correlation and Young's Modulus Sensitivity for Cellular Mechanical Phenotyping by Microfluidic Approaches.

Cellular mechanical properties are a class of intrinsic biophysical markers for cell state and health. Microfluidic mechanical phenotyping methods have emerged as promising tools to overcome the challenges of low throughput and high demand for manual skills in conventional approaches. In this work, two types of microfluidic cellular mechanical phenotyping methods, contactless hydro-stretching deformability cytometry (lh-DC) and contact constriction deformability cytometry (cc-DC) are comprehensively studied and compared. Polymerized hydrogel beads with defined sizes are used to characterize a strong negative correlation between size and deformability in cc-DC (r=-0.95), while lh-DC presents a weak positive correlation (r=0.13). Young's modulus sensitivity in cc-DC is size-dependent while it is a constant in lh-DC. Moreover, the deformability assessment for human breast cell line mixture suggests the lh-DC exhibits better differentiation capability of cells with different size distributions, while cc-DC provides higher sensitivity to identify cellular mechanical changes within a single cell line. This work is the first to present a quantitative study and comparison of size correlation and Young's modulus sensitivity of contactless and contact microfluidic mechanical phenotyping methods, which provides guidance to choose the most suitable cellular mechanical phenotyping platform for specific cell analysis applications.

Lab resource: Single cell line generation and characterization of a human-derived induced pluripotent stem cell line (IGIBi005-A) from a patient with spastic paraplegia/ataxia/ALS phenotype due to the mutation of the gene Kinesin Family Member 5A (KIF5A)

Human Kinesin Family Member 5A (KIF5A) gene mutations have been identified as a putative genetic cause of amyotrophic lateral sclerosis (ALS). Disease modelling using human-induced pluripotent stem cells (HiPSCs) is the next-generation approach to studying numerous human diseases. For the current investigation, we report the generation of patient-specific KIF5A iPSC lines with a mutation at the splice site mutation (c.3020 + 3 A > T) in the intronic region. The resulting line displayed markers for pluripotency, a healthy karyotype, the ability to differentiate into three germ layers in vitro, vector clearance, the KIF5A mutation, STR-based genomic identity, and contamination-free culture.

NKp44-based chimeric antigen receptor effectively redirects primary T cells against synovial sarcoma

BackgroundT-cell receptor-engineered T-cell therapies have achieved promising response rates against synovial sarcoma in clinical trials, but their applicability is limited owing to the HLA matching requirement. Chimeric antigen receptor (CAR) can redirect primary T cells to tumor-associated antigens without requiring HLA matching. However, various obstacles, including the paucity of targetable antigens, must be addressed for synovial sarcoma. Ligands for natural killer (NK) cell-activating receptors are highly expressed by tumor cells. MethodsThe surface expression of ligands for NK cell-activating receptors in synovial sarcoma cell lines was analyzed. We analyzed RNA sequencing data deposited in a public database to evaluate NKp44-ligand expression. Primary T cells retrovirally transduced with CAR targeting NKp44 ligands were evaluated for their functions in synovial sarcoma cells. Alterations induced by various stimuli, including a histone deacetylase inhibitor, a hypomethylating agent, inflammatory cytokines, and ionizing radiation, in the expression levels of NKp44 ligands were investigated. Results: Ligands for NKp44 and NKp30 were expressed in all cell lines. NKG2D ligands were barely expressed in a single cell line. None of the cell lines expressed NKp46 ligand. Primary synovial sarcoma cells expressed the mRNA of the truncated isoform of MLL5, a known cellular ligand for NKp44. NKp44-based CAR T cells specifically recognize synovial sarcoma cells, secrete interferon-γ, and exert suppressive effects on tumor cell growth. No stimulus altered the expression of NKp44 ligands. ConclusionNKp44-based CAR T cells can redirect primary human T cells to synovial sarcoma cells. CAR-based cell therapies may be an option for treating synovial sarcomas.

Case Studies Exemplifying the Transition to Animal Component-free Cell Culture.

Cell culture techniques are strongly connected with modern scientific laboratories and production facilities. Thus, choosing the most suitable medium for the cells involved is vital, not only directly to optimise cell viability but also indirectly to maximise the reliability of the experiments performed with the cells. Fetal bovine or calf serum (FBS or FCS, respectively) is the most commonly used cell culture medium supplement, providing various nutritional factors and macromolecules essential for cell growth. Yet, the use of FBS encompasses a number of disadvantages. Scientifically, one of the most severe disadvantages is the lot-to-lot variability of animal sera that hampers reproducibility. Therefore, transitioning from the use of these ill-defined, component-variable, inconsistent, xenogenic, ethically questionable and even potentially infectious media supplements, is key to achieving better data reproducibility and thus better science. To demonstrate that the transition to animal component-free cell culture is possible and achievable, we highlight three different scenarios and provide some case studies of each, namely: i) the adaptation of single cell lines to animal component-free culture conditions by the replacement of FBS and trypsin; ii) the adaptation of multicellular models to FBS-free conditions; and (iii) the replacement of FBS with human platelet lysate (hPL) for the generation of primary stem/stromal cell cultures for clinical purposes. By highlighting these examples, we aim to foster and support the global movement towards more consistent science and provide evidence that it is indeed possible to step out of the currently smouldering scientific reproducibility crisis.

Quality and Purity of L929 Cell Cultures: Isoenzyme Profiles As a Sensitive Marker of Cross-Contamination.

Objective: Inappropriate handling of cells can generate modifications in the genomic DNA. The additional risk is cross-contamination. Isoenzyme analysis with gel agarose electrophoresis is a known, fast, and cheap technique for detection of species-specific isoforms of intracellular enzymes. The aim of the experimental work was to check if variations in the cell growth conditions can affect morphology and/or nuclear anomalies including micronuclei (MN) in the L929 cells; and to define how sensitive and selective is the classic gel agarose electrophoresis for analysis of isoforms of the selected enzymes to detect cross-contamination of L929 cultures with HeLa cells or with the related species, such as CHO-K1 cells, in the case of unavailability of the commercial kits. Methods: The experiments were done with use of the National Collection of Type Cultures clone 929 (L929)-mouse fibroblasts from subcutaneous connective tissue; HeLa-human cervix adenocarcinoma; and CHO-K1-epithelial-like hamster ovary cells. Cell morphology was evaluated with a light/fluorescence microscope. MN were determined with the cytokinesis-block micronucleus assay, and the isoenzyme analysis was performed using gel agarose electrophoresis. Results: As shown, the overgrown cultures result in a significant increase of the MN in the L929 cells. The band patterns for lactate dehydrogenate, glucose-6-phosphate dehydrogenase, or malate dehydrogenase allow identification of the single L929, HeLa, or CHO-K1 cell line and to detect the cross-contamination, even up to 0.4%. Conclusions: There can be no exceptions from the recommended cell culture conditions in the passage scheme. The sensitivity of the gel agarose separation depends on the cells and on the type of enzyme tested and seems to be sufficient in a quick screening of the CHO-K1, L929, or HeLa cell cultures through the possible mutual contamination.

Abstract CT520: The safety summary of the phase 1a/b trial of QL1706, a novel dual immune checkpoint blockade containing a mixture of anti-PD1 IgG4 and anti-CTLA4 IgG1 antibodies, for advanced malignant tumors

Abstract Background: Anti-cytotoxic T lymphocyte antigen 4 (CTLA-4) antibody may enhance the efficacy of programmed cell death 1 (PD-1) inhibitor. QL1706 is a novel dual immune checkpoint blockade containing a mixture of anti-PD-1 IgG4 and anti-CTLA4 IgG1 antibodies produced by a single cell line. Here we reported the pooled data of the phase 1a/b trial. Methods: In the phase 1a dose escalation and expansion study (NCT04296994), dose escalation involved six levels (0.3, 1.0, 3.0, 5.0, 7.5, and 10.0 mg/kg, intravenously q3w) using an accelerated 3+3 design. The dose expansion cohorts received selected doses. In phase 1b (NCT05171790), patients with advanced solid tumors were given intravenous QL1706 5.0 mg/kg q3w according to the data in phase 1a. The primary objectives were to define the safety, tolerability, and recommended phase 2 dose (RP2D) of QL1706 in phase 1a, and to evaluate the preliminary efficacy in phase 1b. Results: As of Sept 30, 2021, totally 518 patients with multiple types of advanced cancer (99 in phase 1a and 419 in phase 1b) were enrolled, including lung cancer (175 [33.8%]), nasopharyngeal cancer (134 [25.9%]), cervical cancer (58 [11.2%]), liver cancer (34 [6.6%]), colorectal cancer (27 [5.2%]), and kidney cancer (18 [3.5%]), etc. 494 [95.4%] were previously treated, and 182 (35.1%) received previous immunotherapy. Two patients at 10 mg/kg suffered dose-limiting toxicity. The RP2D of QL1706 was defined as 5.0 mg/kg. After a median follow-up of 6.7 months, treatment-related adverse events (TRAEs) were observed in 374 patients (72.2%). 70 (13.5%) experienced grade ≥3 TRAEs. The most common TRAEs were rash (91 [17.6%]), pruritus (65 [12.5%]), hypothyroidism (60 [11.6%]), AST increased (48 [9.3%]), and hyperthyroidism (46 [8.9%]). The TRAEs that led to drug interruption and drug discontinuation were observed in 67 (12.9%) and 31 patients (6.0%), respectively. The immune-related TRAEs and treatment-related serious adverse events were observed in 223 (43.1%) and 59 patients (11.4%), respectively. 79 (16.9%) patients had a confirmed objective response, 237 (50.6%) patients had a confirmed disease control. Conclusion: QL1706 had a manageable safety profile. QL1706 still showed promising anti-tumor activity although over one-third of the patients received prior immunotherapy. These results supported further investigation of QL1706 for advanced solid tumors. Citation Format: Li Zhang, Hongyun Zhao, Yan Huang, Wenfeng Fang, Yuxiang Ma, Yang Zhang, Xiaoli Wei, Yanhua Yang, Wei Yang, Furong Liu, Zuan Lin, Jianing Li, Qianwen Liu, Benyan Zou, Kunlun Liao, qun Liu. The safety summary of the phase 1a/b trial of QL1706, a novel dual immune checkpoint blockade containing a mixture of anti-PD1 IgG4 and anti-CTLA4 IgG1 antibodies, for advanced malignant tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT520.

Abstract 2947: Surveying the tumor suppressive genetic network underlying chr4p deletion in TNBC

Abstract Triple negative breast cancer (TNBC) is a breast cancer subtype lacking targetable biomarkers, resulting in the worst prognosis compared to other breast cancer subtypes. TNBC is characterized by many large copy number variants that result in the deletion and amplifications of many genes, with TP53 being the only common oncogenic driver. Using TCGA data and in-depth functional genomic analysis of TNBC patient-derived xenografts (PDX), our group showed that chr4p is a recurrently deleted region in basal breast cancer, which TNBC is an enriched subtype. This correlated with poor prognosis and a highly proliferative state. Here, we set out to survey the tumor suppressive genetic network underlying the TNBC-specific chr4pdeletion. Using an arrayed CRISPR-enCas12 screening approach, I will generate a panel of mutant cell lines deleted for all protein-coding genes residing within chr4p. MCF10A series of cell lines will be used for mutant cell line construction, because it is an established normal human breast epithelial model system with a normal karyotype to ensure the diploid state ofchr4p and includes other derivatives (MCF10A(-E7-Bcl2)) that show basal anchorage independent growth in 3D to assess cell transformation. The resulting panel of single gene deletion mutant cell lines will be characterized for their effects on proliferation, apoptosis, cell transformation and senescence. Additionally, the tumor suppressive genetic interaction network of chr4p will be mapped using a multiplexed CRISPR-enCas12 screening methodology. A dual guide-RNA library will be generated for all protein-coding genes to test all pairwise combinations for tumor suppressive genetic interactions. The proliferation due to double gene deletions will be monitored and compared to single gene deletions to identify tumor suppressive interactions. This study will be the first to systematically identify tumor suppressor genetic network underlying chr4p. Ultimately, it will provide an in-depth understanding of the genetic network of large copy number variants in TNBC and insight into new avenues for precision oncology. Citation Format: Joseph Del Corpo, Rohan Dandage, Lea Harrington, Elena Kuzmin. Surveying the tumor suppressive genetic network underlying chr4p deletion in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2947.

Abstract 2902: Development of PSB202, a bifunctional antibody pair that target CD20 and CD37, for the treatment of B-cell malignancies

Abstract CD20 and CD37 are co-expressed on most B-NHL and CLL tumors. Systemic depletion of normal and malignant B-cells by anti-CD20 antibodies (e.g. rituximab and obinutuzumab) and anti-CD37 antibodies (e.g. otlertuzumab and BI 836826) has been well tolerated in clinical studies. Depletion of B-cells is reversible, with the ability to repopulate B-cells from hematopoietic stem cells. Dual targeting CD20 and CD37 may improve the clinical efficacy and decrease drug resistance. PSB202 is a novel biological entity composed of two engineered monoclonal antibodies which are produced from a single stable CHO cell line - a humanized anti-CD20 antibody (PSB102) and a humanized anti-CD37 antibody (PSB107) at a 1:1 molecular ratio. In vitro binding studies demonstrated that PSB202 specifically binds to human CD20 and CD37. The antibodies’ binding to FcγRIIIa was enhanced by reducing the fucose level on each antibody component. PSB202 directly induced potent autonomous killing via target engagement. PSB202 mediated depletion of human and monkey blood B-cells and malignant human B-cell lines through ADCC activity at EC50 of double digit pM and CDC activity at EC50 of low single digit nM. Importantly, PSB202 effectively depleted B-cells in ex vivo cultures of blood cells from CLL and NHL patients who were refractive to rituximab treatment. The antitumor activity of PSB202 was demonstrated in human xenograft models in mice. PSB202 was able to reduce different human lymphoma tumors by up to ~97.9% in a dose-dependent manner, suggesting that PSB202 might be potentially efficacious against multiple types of B-cell malignancies. Synergy was demonstrated for PSB202 in combination with lenalidomide. Toxicokinetic assessment in cynomolgus monkeys showed a dose proportional exposure for both antibody components, with mean terminal elimination half-life (T1/2) of ~60 and ~68 hours for the anti-CD20 and anti-CD37 component, respectively. PBS202 was well tolerated, with observed toxicities mostly related to its activity as a B-cell depletion agent. As expected, PSB202 when administered at ≥ 10 mg/kg resulted in nearly complete depletion of B lymphocytes 24 hours following the 1st dose. B-cells returned to near baseline within 2 months after the last dose at 10 mg/kg. PSB202 can deplete normal and malignant B-cells by targeting two clinically validated targets through multiple distinct mechanisms including direct B-cell killing, enhanced ADCC activity, and CDC activity. PSB202 offers the potential to improve clinical efficacy, decrease drug resistance, provide administration convenience especially when combining with additional therapeutics, lower the cost for treatment, and reduce the unwanted side effects from chemotherapy. PSB202 is currently being evaluated in patients with previously treated, relapsed, indolent B-cell malignancies (NCT05003141). Citation Format: Paul Algate, Zhi Liu, Saying Yuan, Hua Liu, Cristina Domeier, Haiming Jiang, Yuanzhi Lao, Yanling Xu, William C. Fanslow, Ron Schoner, Wei Yan. Development of PSB202, a bifunctional antibody pair that target CD20 and CD37, for the treatment of B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2902.

Abstract 3578: Optimization of shared neoantigen vaccine design to increase vaccine potency: From bench to bedside and back

Abstract Vaccines targeting neoantigens identified from common tumor driver mutations are of increasing interest as evidence of clinical benefit builds, and opportunities to combine such vaccines with immune modulators are growing. Our individualized neoantigen vaccine (GRANITE) has shown clinical benefit and strong, consistent CD8 T cell induction in patients. We have developed an analogous off-the-shelf product (SLATE) targeting shared neoantigens that offers manufacturing simplicity and faster administration to patients with shared driver mutations. A Phase 1/2 trial of a heterologous prime/boost vaccine regimen using a chimpanzee adenovirus (ChAd) prime and self amplifying mRNA (SAM) boosts (SLATE, NCT03953235) was initiated to assess safety, tolerability, and immunogenicity in patients with advanced cancers. SLATE version 1 encodes 20 unique neoantigens to various shared driver mutations (KRAS, TP53, etc.). Patients were selected if their tumors harbored one of the 20 neoantigens encoded by the vaccine cassette and an HLA Class I allele that presents that neoantigen. Administration of ChAd prime and repeated administration of 30, 100, or 300µg SAM doses were safe and well tolerated in all subjects dosed (n=26), with no evidence of increasing reactogenicity with sequential dosing. Early efficacy signals (molecular responses; one unconfirmed RECIST response) were observed in NSCLC subjects all treated with and progressed on prior anti-PD(L)1. Analysis of T cell responses pre and post immunizations by ex vivo IFNγ ELISpot did not show robust responses to KRAS neoantigens across all patients. However, objective CD8 T cell responses to KRAS antigens post vaccination were detectable after in vitro stimulation, suggesting the induction of low-level KRAS specific T cell responses in vivo. In contrast, HLA-matched responses to TP53 neoantigens encoded by the vaccine were consistently detected via ex vivo ELISpot in these same patients. These data suggests that an immunodominant T cell response to the TP53 mutations may have outcompeted the response to the less immunogenic KRAS mutations restricted and presented by the same HLA in vivo. Differential surface peptide-HLA (pHLA) density may explain these discordant findings, and subsequent targeted mass spectrometry analyses revealed detection of TP53 pHLA complexes at a higher frequency compared to KRAS mutations in single HLA-allele cell lines. Redesigned vaccine cassettes excluding the TP53 epitopes and repeating KRAS epitopes demonstrated increased immune responses (ex vivo IFNγ ELISpot) compared to cassette version 1 in HLA transgenic mice, further supporting the tumor neoantigen immunodominance hierarchy observed in humans dosed with SLATE version 1. A re-designed product (SLATE v2) focusing exclusively on KRAS mutations (G12C, G12D, G12V and Q61H) is currently being assessed in phase 2 in patients with advanced KRAS-driven tumors. Citation Format: Amy R. Rappaport, Christine D. Palmer, Annie Shen, Claudia X. Dominguez, Meghan G. Hart, Lauren D. Kraemer, Sonia Koulavouth, Martina Marrali, Jason R. Jaroslavsky, Charmaine N. Nganje, Ciaran D. Scallan, Sue-Jean Hong, Leonid Gitlin, Monica Lane, Daniel V. Catenacci, Chrisann Kyi, David P. Carbone, Hossein Borghaei, Raphael Rousseau, Andrew Ferguson, Karin Jooss. Optimization of shared neoantigen vaccine design to increase vaccine potency: From bench to bedside and back [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3578.

Implementing microwell slides for detection and isolation of single circulating tumor cells from complex cell suspensions.

Cell loss during detection and isolation of circulating tumor cells (CTCs) is a challenge especially when label-free pre-enrichment technologies are used without the aid of magnetic particles. Although microfluidic systems can remove the majority of "contaminating" white blood cells (WBCs), their remaining numbers are still impeding single CTC isolation, thus making additional separation steps needed. This study aimed to develop a workflow from blood-to-single CTC for complex cell suspensions by testing two microwell formats. In the first step, different cell lines were used to compare the performances of Sievewell™ 370 K (TOK, Japan) and CellCelector™ Nanowell U25 (ALS Automated Lab Solutions, Germany) slides for cell labelling and single-cell micromanipulation. Confounding levels of auto-fluorescence inherent to different plastic materials used to cast the microwells, staining recovery rates, and cell isolation rates were determined. In the second step, three different blood preservation tubes were tested for RNA analysis. Lastly, the established workflow was applied to isolate CTCs from peripheral blood samples obtained from metastasized breast cancer (mBC) patients for single-cell DNA and RNA analysis. The detection of CTCs in Sievewell slides profit from better signal-to-noise ratios in the fluorescence channels mainly used for CTC detection. In addition, due to its design, Sievewell supports direct in situ CTC labelling, which minimizes cell loss and leads to single-cell recovery rates after staining of approx. 94%. Detection of PIK3CA mutations in single CTCs verified the applicability of the workflow for the analysis of genomic DNA of CTCs. Furthermore, combined with blood preservation up to 48 h at room temperature in LBguard tubes, panel RT-PCR transcript analysis was successful for single cell line cells and CTCs, respectively. The combined use of Sievewell microwell slides and CellCelector™ automated micromanipulation system improves single CTC detection, labelling and isolation from complex cell suspensions. This approach is especially valuable when samples of high cellular content are processed.

Efficient Detection of the Alternative Spliced Human Proteome Using Translatome Sequencing.

Alternative splicing (AS) isoforms create numerous proteoforms, expanding the complexity of the genome. Highly similar sequences, incomplete reference databases and the insufficient sequence coverage of mass spectrometry limit the identification of AS proteoforms. Here, we demonstrated full-length translating mRNAs (ribosome nascent-chain complex-bound mRNAs, RNC-mRNAs) sequencing (RNC-seq) strategy to sequence the entire translating mRNA using next-generation sequencing, including short-read and long-read technologies, to construct a protein database containing all translating AS isoforms. Taking the advantage of read length, short-read RNC-seq identified up to 15,289 genes and 15,906 AS isoforms in a single human cell line, much more than the Ribo-seq. The single-molecule long-read RNC-seq supplemented 4,429 annotated AS isoforms that were not identified by short-read datasets, and 4,525 novel AS isoforms that were not included in the public databases. Using such RNC-seq-guided database, we identified 6,766 annotated protein isoforms and 50 novel protein isoforms in mass spectrometry datasets. These results demonstrated the potential of full-length RNC-seq in investigating the proteome of AS isoforms.

Efficacy and safety of QL1706, a novel dual immune checkpoint blockade containing a mixture of anti-PD1 IgG4 and anti-CTLA4 IgG1 antibodies, for advanced cervical cancer: Cohort data from a phase 1b trial.

5535 Background: Anti-cytotoxic T lymphocyte antigen 4 (CTLA-4) and anti-programmed cell death 1 (PD-1) antibodies have synergistic effect. QL1706 is a novel dual immune checkpoint blockade containing a mixture of anti-PD-1 IgG4 and anti-CTLA4 IgG1 antibodies produced by a single cell line. QL1706 showed promising anti-tumor activity in multiple solid tumors. Here we reported the cervical cancer cohort data from the phase 1b trial of QL1706 (NCT05171790). Methods: Eligible patients had a pathologically confirmed diagnosis of squamous cell carcinoma, adenosquamous carcinoma, or adenocarcinoma of the cervix, with recurrent or metastatic, immunotherapy-naive disease at the time of enrollment. Patients were required to have ≥one lesion measurable by RECIST v1.1 and disease that had relapsed after a first-line, platinum-based regimen. Patients were included regardless of PD-L1 expression status at baseline. Patients received intravenous QL1706 5.0 mg/kg q3w (i.e., recommended phase 2 dose, defined in a phase 1a trial), for up to 24 months. The primary endpoint was confirmed overall response rate (ORR) per RECIST v1.1 by investigator. The secondary endpoints included disease control rate, duration of response (DoR), progression-free survival (PFS), overall survival (OS), safety, etc. Results: As of Dec 31, 2021, totally 53 patients with cervical cancer were enrolled (median age, 52 years [range 33-72]). Median follow-up was 5.6 months. Most patients (45 [85%]) had ECOG performance status of 1. Histological breakdown was squamous cell carcinoma (44 [83%]) and adenocarcinoma (nine [17%]). 33 (62%) patients had received one prior chemotherapy, 20(38%) patients had received ≥two prior chemotherapy. The confirmed ORR was 28% (95% CI 17%-42%; 15 patients), including one (2%) complete response and 14 (26%) partial response, with median duration of response not reached (95% CI 2.8 months-not estimable). Disease control was observed in 29 (55%; 95% CI 40%-68%) patients. Median PFS was 4.2 months (95% CI 1.7-6.9), and 6-month PFS rate was estimated as 37% (95% CI 24%-51%). Treatment-related adverse events (TRAE) were observed in 40 (75%) patients. Nine (17%) experienced grade ≥3 TRAE. The most common TRAE were rash (eight [15%]), hyperthyroidism (eight [15%]), and pyrexia (seven [13%]). Three (6%) patients suffered TRAE leading to dose discontinuation. The immune-related TRAE and serious TRAE were observed in 28 (53%) and eight (15%) patients, respectively. Conclusions: QL1706 demonstrated promising and durable clinical activity, with favorable tolerability in patients with recurrent and/or metastatic cervical cancer. Further investigations in this setting are continuing. Clinical trial information: NCT05171790.

Two for one: targeting BCMA and CD19 in B-cell malignancies with off-the-shelf dual-CAR NK-92 cells

BackgroundChimeric antigen receptor (CAR) T-cell therapy has proven to be a valuable new treatment option for patients with B-cell malignancies. However, by applying selective pressure, outgrowth of antigen-negative tumor cells can occur, eventually resulting in relapse. Subsequent rescue by administration of CAR-T cells with different antigen-specificity indicates that those tumor cells are still sensitive to CAR-T treatment and points towards a multi-target strategy. Due to their natural tumor sensitivity and highly cytotoxic nature, natural killer (NK) cells are a compelling alternative to T cells, especially considering the availability of an off-the-shelf unlimited supply in the form of the clinically validated NK-92 cell line.MethodsGiven our goal to develop a flexible system whereby the CAR expression repertoire of the effector cells can be rapidly adapted to the changing antigen expression profile of the target cells, electrotransfection with CD19-/BCMA-CAR mRNA was chosen as CAR loading method in this study. We evaluated the functionality of mRNA-engineered dual-CAR NK-92 against tumor B-cell lines and primary patient samples. In order to test the clinical applicability of the proposed cell therapy product, the effect of irradiation on the proliferative rate and functionality of dual-CAR NK-92 cells was investigated.ResultsCo-electroporation of CD19 and BMCA CAR mRNA was highly efficient, resulting in 88.1% dual-CAR NK-92 cells. In terms of CD107a degranulation, and secretion of interferon (IFN)-γ and granzyme B, dual-CAR NK-92 significantly outperformed single-CAR NK-92. More importantly, the killing capacity of dual-CAR NK-92 exceeded 60% of single and dual antigen-expressing cell lines, as well as primary tumor cells, in a 4h co-culture assay at low effector to target ratios, matching that of single-CAR counterparts. Furthermore, our results confirm that dual-CAR NK-92 irradiated with 10 Gy cease to proliferate and are gradually cleared while maintaining their killing capacity.ConclusionsHere, using the clinically validated NK-92 cell line as a therapeutic cell source, we established a readily accessible and flexible platform for the generation of highly functional dual-targeted CAR-NK cells.