Acute myeloid leukemia (AML) is characterized by antigen heterogeneity and poor prognosis. Here, to tackle the heterogeneity of AML, we combined FLT3 with CD33 in a combinatorial OR-gate approach using our split, universal, programmable (SUPRA) chimeric antigen receptor (CAR) platform. The split platform affords tunability over activation levels and multiplexed targeting. We characterized the specificity and sensitivity of different SUPRA CAR adapters for each target across a panel of target cell lines. Our results demonstrate that this CAR system can effectively target two antigens with equivalent efficacy to conventional CARs while reducing the engineering burden of designing CAR T cells against multiple antigens. Furthermore, we can characterize an effective dose range where off-target cytotoxicity against hematopoietic stem and progenitor cells is minimized. Our SUPRA OR gate has the potential to provide an effective and safer solution to treating AML.

Background: Patients with CD33 and/or FLT3 malignancies, which encompass most myeloid malignancies such as acute myeloid leukemia (AML), have a grim prognosis and a high unmet need. There are no approved cell therapies for patients with AML due to paucity of targets and an immunosuppressive bone marrow milieu. SENTI-202, a preclinical CAR-NK cell is engineered with a logic-gated gene circuit to overcome these challenges. Methods: SENTI-202 is engineered with a FLT3 OR CD33 NOT EMCN logic-gated gene circuit and a proprietary calibrated release IL-15 (crIL-15). These 3 chimeric proteins are engineered onto a natural killer (NK) cell (healthy adult peripheral blood-derived) to create an off-the-shelf chimeric antigen receptor (CAR) NK cell therapy. The bivalent CD33 OR FLT33 (OR GATE) activating CAR (aCAR) enables concurrent targeting of CD33+ and/or FLT3+ AML cells which kills both AML blasts and leukemic stem cells (LSCs) in vitro and in vivo. This concurrent targeting of two tumor antigens could potentially provide longer remission and less chance of relapse. The NOT EMCN (NOT GATE) inhibitory CAR (iCAR) protects healthy EMCN+ hematopoietic stem cells (HSCs) and early hematopoietic progenitor cells (HPCs) from off-tumor toxicity to potentially aid in post-treatment reconstitution of a healthy hematopoietic system. The crIL-15 provides both autocrine and paracrine IL-15 stimulation to the CAR-NK cells (and surrounding immune cells) to promote cell expansion, persistence, and tumor killing. While previous presentations (Garrison et al., 2021) focused on the performance of individual SENTI-202 components in a cell, this presentation focuses on functional data from the complete logic-gated gene circuit and the crIL-15 together in a cell (i.e., the final CAR-NK with all 3 genetic elements). Results: We demonstrated delivery of all circuit components using a single retroviral vector. We then investigated activity of the resulting CAR-NK cells using a traditional in vitro cytotoxicity assay (E:T=1:2; 20hrs) in which SENTI-202 showed >90% killing of leukemia cells (e.g. SEM; P≤0.001), maintained significant serial killing potential (3 rounds; P≤0.0001; P≤0.001; P≤0.001), as well as robust killing against primary AML blasts and LSC-enriched target cell populations. Importantly, SENTI-202 also exhibited significant tumor cell killing and improved survival (P≤0.001) in an MV4-11 xenograft AML mouse model. We investigated the ability of SENTI-202 to reduce off-tumor toxicity toward EMCN+ healthy cells, including primary HSCs. Within in vitro assays, SENTI-202 provided >50% protection to FLT3+ CD33+ model healthy cells that also expressed the EMCN safety antigen, and similar significant protection was maintained through serial protection assays (3 rounds; P≤0.0001; P≤0.001; P≤0.0001). As further evidence of the selectivity imparted using the OR/NOT logic gating approach, in vitro assays with SENTI-202 resulted in killing of leukemia cells but not primary healthy human HSCs and HPCs (~42% protection) (P≤0.01). Finally, in the presence of SENTI-202 in vivo, the NOT GATE protected and enabled significant (P≤0.0001) expansion of EMCN+ FLT3+ CD33+ model healthy cells. Conclusions: In summary, preclinical evaluation of SENTI-202 demonstrated selective killing of CD33 and/or FLT3 expressing AML cell lines while protecting healthy HSCs and HPCs. This selectivity is imparted by the novel logic-gated gene circuit engineered into healthy adult NK cells. Clinical evaluation of SENTI-202 is planned to evaluate the safety and efficacy in patients with hematologic malignancies with high unmet need, including AML.

Background: While chimeric antigen receptor (CAR) cell therapies have provided extraordinary clinical responses in some hematological malignancies, developing effective CAR cell therapies for acute myeloid leukemia (AML) has been challenging due to: (a) the lack of a single target antigen robustly expressed across both AML leukemic stem cell (LSC) and immature leukemic blast cell subpopulations, and (b) the lack of truly AML-specific target antigens, since current targets are also expressed on healthy tissues and may result in off-tumor toxicity. Using logic gated gene circuits, we are engineering our SENTI-202 CAR-NK cell therapy to overcome these long-standing challenges to treating AML patients.

Abstract Background: Liquid biopsies represent a noninvasive alternative to traditional tissue biopsies, enabling the detection and tracking of cancer driver mutations from a simple blood draw. Biocept’s Target SelectorTM test platform offers the unique ability to analyze biomarkers from both circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA). Here we performed a clinical concordance study, comparing the ABI 7900 to the newer ABI QuantStudio 5 (QS5) for integration of the QS5 into Biocept’s CAP/CLIA certified laboratory. TargetSelectorTM ctDNA mutation tests were validated for five targets, including EGFR (Del19, L858R, or T790M), BRAF, and KRAS, all markers integral to devising personalized therapies for non-small cell lung cancer (NSCLC) patients. Methods: Prior to assessing clinical samples, extensive analytical validation was performed with DNA extracted from cancer cell lines containing the relevant EGFR, BRAF or KRAS mutations. TargetSelectorTM ctDNA mutant assays procedurally incorporate real-time PCR and DNA sequencing. The analytical validation was conducted to compare the performance of the ABI 7900 vs QS5 instruments within Biocept’s ctDNA testing platform. Evaluation of 3000 samples across the five TargetSelectorTM assays demonstrated single mutant copy detection sensitivity on the QS5 platform, with >99% sensitivity and >99% specificity for each of the ctDNA mutant assays. Following analytical evaluation, synchronized aliquots of 13 patient ctDNA samples, extracted from whole blood collected in Biocept CEE-SureTM Blood Collection tubes, were used to test the performance of both the ABI 7900 and QS5 instruments in the TargetSelectorTM ctDNA assays. Results: EGFR, BRAF and KRAS TargetSelectorTM assays that incorporate the ABI QS5 vs the ABI 7900 enable more sensitive ctDNA testing, as demonstrated by analytical validation and subsequent analyses of clinical samples. In patient samples, TargetSelectorTM tests using the QS5 identified all of the mutations detected by same assays on the ABI 7900 platform. Utilization of the QS5 instrument within the TargetSelectorTM also enabled identification of additional mutations not detected in the assays where the ABI 7900 was used. Conclusions: Implementation of the QuantStudio 5 real-time PCR instrument into Biocept’s TargetSelectorTM ctDNA assays has improved performance over the older TargetSelectorTM platform that utilized the ABI 7900. The more sensitive QS5-based TargetSelectorTM assays increase the likelihood of identifying molecular drivers linked to a patient’s cancer. Liquid biopsy detection of EGFR, BRAF and KRAS mutant ctDNA provides a minimally invasive means to gain valuable information towards developing personalized treatment strategies, monitoring therapeutic response, and identifying potential resistance mechanisms, all of which are vital for disease management and NSCLC patient care. Citation Format: Shan Fu Wu, Jason C. Poole, Tim T. Lu, Lyle J. Arnold, Jeffrey Chen, Anh Pham, Veena M. Singh. Validation of the QuantStudio5 instrument for use in Biocept’s TargetSelectorTM ctDNA lung cancer assays [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A20.

Prediction of human physical traits and demographic information from genomic data challenges privacy and data deidentification in personalized medicine. To explore the current capabilities of phenotype-based genomic identification, we applied whole-genome sequencing, detailed phenotyping, and statistical modeling to predict biometric traits in a cohort of 1,061 participants of diverse ancestry. Individually, for a large fraction of the traits, their predictive accuracy beyond ancestry and demographic information is limited. However, we have developed a maximum entropy algorithm that integrates multiple predictions to determine which genomic samples and phenotype measurements originate from the same person. Using this algorithm, we have reidentified an average of >8 of 10 held-out individuals in an ethnically mixed cohort and an average of 5 of either 10 African Americans or 10 Europeans. This work challenges current conceptions of personal privacy and may have far-reaching ethical and legal implications.

Background: Circulating cell-free fetal DNA has enabled non-invasive prenatal fetal aneuploidy testing without direct discrimination of the maternal and fetal DNA. Testing may be improved by specifically enriching the sample material for fetal DNA. DNA methylation may allow for such a separation of DNA; however, this depends on knowledge of the methylomes of circulating cell-free DNA and its cellular contributors.Results: We perform whole genome bisulfite sequencing on a set of unmatched samples including circulating cell-free DNA from non-pregnant and pregnant female donors and genomic DNA from maternal buffy coat and placenta samples. We find CpG cytosines within longer fragments are more likely to be methylated. Comparison of the methylomes of placenta and non-pregnant circulating cell-free DNA reveal many of the 51,259 identified differentially methylated regions are located in domains exhibiting consistent placenta hypomethylation across millions of consecutive bases. We find these placenta hypomethylated domains are consistently located within regions exhibiting low CpG and gene density. Differentially methylated regions identified when comparing placenta to non-pregnant circulating cell-free DNA are recapitulated in pregnant circulating cell-free DNA, confirming the ability to detect differential methylation in circulating cell-free DNA mixtures.Conclusions: We generate methylome maps for four sample types at single-base resolution, identify a link between DNA methylation and fragment length in circulating cell-free DNA, identify differentially methylated regions between sample groups, and uncover the presence of megabase-size placenta hypomethylated domains.

Small noncoding antisense RNAs (sasRNAs) guide epigenetic silencing complexes to target loci in human cells and modulate gene transcription. When these targeted loci are situated within a promoter, long-term, stable epigenetic silencing of transcription can occur. Recent studies suggest that there exists an endogenous form of such epigenetic regulation in human cells involving long noncoding RNAs. In this article, we present and validate an algorithm for the generation of highly effective sasRNAs that can mimic the endogenous noncoding RNAs involved in the epigenetic regulation of gene expression. We validate this algorithm by targeting several oncogenes including AKT-1, c-MYC, K-RAS, and H-RAS. We also target a long antisense RNA that mediates the epigenetic repression of the tumor suppressor gene DUSP6, silenced in pancreatic cancer. An algorithm that can efficiently design small noncoding RNAs for the epigenetic transcriptional silencing or activation of specific genes has potential therapeutic and experimental applications.

Background: Synthetic lethal (SL) interactions are used to develop targeted cancer therapy. However, novel SL interactions discovered in mammalian cell cultures are often cell type specific and are therefore only relevant to a small, or difficult to define, subset of patients. We developed a strategy in which we prioritize potential SL drug targets using the genetically tractable model system Saccharomyces cerevisiae. Material and Methods: Weperformed a SL screen by expressing a constitutively active RAS allele, RAS2(V19), in ~4800 S. cerevisiae strains in which each individual gene is deleted. Next we tested if SL interactions were conserved in human cancer cell lines. Results: The yeast screen yielded a hit list highly enriched for mutants with a defect in ‘endoplasmic reticulum (ER)-to-Golgi-to-vacuole’ transport. Moreover, we found that this list had a significant overlap with strains sensitive to b-mercaptoethanol, DTT and tunicamycin. We hypothesized that ER homeostasis was disturbed in these cells. The two gene deletion mutants most sensitive to ER stress are IRE1 and HAC1. These genes make up the unfolded protein response (UPR) in yeast; the signaling pathway that restores ER homeostasis. Both UPR genes were SL with RAS2(V19). Next we asked if we could detect a SL interaction between oncogenic RAS and the UPR in human cells. We find that a SL interaction between oncogenic RAS and the UPR is dependent on specific RAS effector pathways in human cell cultures. Conclusions: The UPR is conserved in evolution. However, signaling pathways downstream of RAS have diverged over time. We will present how the interaction between oncogenic RAS and the UPR has evolved in human cells and how this interaction can be exploited for therapeutic intervention.

OBJECTIVES: Several well-designed clinical trials, including two trials conducted in China, have demonstrated the feasibility and accuracy of sequencing based noninvasive prenatal test (NIPT). However, the failure rate, sensitivity and specificity of NIPT for a large population in the real life clinical setting have not been reported. METHOD: Within a period of 15 months, under a provincially approved program, we applied NIPT to more than 50,000 pregnant women of various demographic and maternal background, using the published methodology developed for the clinical trials that we had conducted (Prenatal Diagnosis, 2013, Vol. 32: 1-7). RESULTS: We found that the detection sensitivity and specificity for trisomy 21, trisomy 18, and trisomy 13 were almost the same as we had observed in the clinical trials, each at >99%. The failure rate was at ~0.6%, better than the rate that we had encountered during the clinical trials. We also observed that NIPT could accurately detect aneuploidies of other chromosomes, with slightly higher “false positive” rates mainly caused by confined placenta mosaicism (CPM) and maternal mosaicism (MM). CONCLUSIONS: Our results indicate that NIPT can be applied to real life clinical setting, with low failure rate and high accuracy. The results suggested that CPM and MM should be factored into the interpretation of the data, particularly for aneuploidies other than trisomy 21. 1-2

Background: Circulating cell-free (ccf) fetal DNA comprises 3-20% of all the cell-free DNA present in maternal plasma. Numerous research and clinical studies have described the analysis of ccf DNA using next generation sequencing for the detection of fetal aneuploidies with high sensitivity and specificity. We sought to extend the utility of this approach by assessing semi-automated library preparation, higher sample multiplexing during sequencing, and improved bioinformatic tools to enable a higher throughput, more efficient assay while maintaining or improving clinical performance.Methods: Whole blood (10mL) was collected from pregnant female donors and plasma separated using centrifugation. Ccf DNA was extracted using column-based methods. Libraries were prepared using an optimized semi-automated library preparation method and sequenced on an Illumina HiSeq2000 sequencer in a 12-plex format. Z-scores were calculated for affected chromosomes using a robust method after normalization and genomic segment filtering. Classification was based upon a standard normal transformed cutoff value of z = 3 for chromosome 21 and z = 3.95 for chromosomes 18 and 13.Results: Two parallel assay development studies using a total of more than 1900 ccf DNA samples were performed to evaluate the technical feasibility of automating library preparation and increasing the sample multiplexing level. These processes were subsequently combined and a study of 1587 samples was completed to verify the stability of the process-optimized assay. Finally, an unblinded clinical evaluation of 1269 euploid and aneuploid samples utilizing this high-throughput assay coupled to improved bioinformatic procedures was performed. We were able to correctly detect all aneuploid cases with extremely low false positive rates of 0.09%, <0.01%, and 0.08% for trisomies 21, 18, and 13, respectively.Conclusions: These data suggest that the developed laboratory methods in concert with improved bioinformatic approaches enable higher sample throughput while maintaining high classification accuracy.

OBJECTIVE: We sought to evaluate a multiplexed massively parallel shotgun sequencing assay for noninvasive trisomy 21 detection using circulating cell-free fetal DNA.STUDY DESIGN: Sample multiplexing and cost-optimized reagents were evaluated as improvements to a noninvasive fetal trisomy 21 detection assay. A total of 480 plasma samples from high-risk pregnant women were employed.RESULTS: In all, 480 prospectively collected samples were obtained from our third-party storage site; 13 of these were removed due to insufficient quantity or quality. Eighteen samples failed prespecified assay quality control parameters. In all, 449 samples remained: 39 trisomy 21 samples were correctly classified; 1 sample was misclassified as trisomy 21. The overall classification showed 100% sensitivity (95% confidence interval, 89-100%) and 99.7% specificity (95% confidence interval, 98.5-99.9%).CONCLUSION: Extending the scope of previous reports, this study demonstrates that plasma DNA sequencing is a viable method for noninvasive detection of fetal trisomy 21 and warrants clinical validation in a larger multicenter study.