Introduction: Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment landscape for B-cell lymphomas and leukemias. However, most patients do not achieve durable response. Mechanisms that facilitate resistance and impact survival outcomes include antigen escape, loss of CD58 co-stimulation, and CAR T exhaustion. Novel CAR T-cell designs are critical to address these challenges. Construct design and product characterizations: CRG-023 is a tri-specific CAR T that targets the B-cell lineage antigens CD19, CD20, and CD22 via tri-cistronic expression of 3 distinct second-generation CARs from a single lentiviral vector. The CD19- and CD20-targeting CARs employ novel, human single-chain variable fragment (scFv) binders selected using methods to assess CAR-mediated T-cell functionality. The CD22-targeting CAR employs the human scFv m971 (Frank M et al. The Lancet 2024). Each CAR incorporates a CD3ζ signaling domain and a distinct costimulatory domain derived from 4-1BB (CD22-targeting CAR), CD28 (CD19-targeting CAR), or CD2 (CD20-targeting CAR). CD2 is the costimulatory receptor required for CD58 engagement. Each CAR sequence and arrangement within the tri-cistronic vector were engineered to achieve optimal CAR T-cell activity. Further codon optimization and removal of splice sites were performed to limit potential recombination and to ensure stable CAR expression. For the tri-specific construct, flow cytometry analysis demonstrated that each CAR expressed well and the aggregate surface expression of all 3 receptors was comparable to levels measured on monospecific CAR T cells. Importantly, the tri-cistronic lentiviral vector was evaluated for manufacturability to ensure that sufficient titers of transducing virus were generated. In vitro results: The tri-specific CAR T cells sustained durable anti-tumor control when repeatedly challenged with new tumor cells expressing all three antigens. Moreover, durable functionality was demonstrated with repeat challenges by tumor cells expressing a single cognate antigen, indicating that the activity of CRG-023 is not dependent on any single CAR specificity. Superior tumor control was also observed against target cells expressing low CD19 antigen levels (~1000-6000 molecules per cell) as compared to FMC63 scFv CD19-targeting CAR T-cell benchmarks. In these assays, the tri-specific CAR T cells were less differentiated and sustained proliferation of both CD4 and CD8 T-cell subsets. Importantly, the expression of three distinct CARs did not result in excessive cytokine secretion or activation marker expression relative to the monospecific CAR T-cell benchmark controls. In vivo results: CRG-023 CAR T cells generated from healthy donor material in a full-scale manufacturing process demonstrated superior anti-tumor activity in mouse models of disseminated lymphoma compared to monospecific CAR T cells. Additional dose-response studies showed complete tumor clearance was achieved in Raji lymphoma models with the lowest dose tested (105 CRG-023 CAR T cells per mouse). We also modeled in vivo CD19 antigen escape using a mixture of wild-type Raji cells and CD19 knockout Raji (CD19ko) cells to address the potential for CD19 antigen loss to limit durable response as seen in CD19-targeting CAR T cells (Zurko J el al. Blood Advances 2023). Using this model, the tri-specific CAR T cells effectively eradicated all tumors, in contrast to FMC63-containing CD19 CAR T cells, for which tumor escape was observed. Collectively the data show that CRG-023 exhibits enhanced, durable clearance of lymphoma tumors in vivo when using low doses and in antigen loss models relative to benchmark controls. Conclusions: Significant engineering and screening were undertaken to develop CRG-023, a potent tri-specific CAR T-cell product candidate with differentiated pre-clinical activity. CRG-023 demonstrated durable potency across a range of antigen levels in stringent in vitro and in vivo models, with each of the three CARs contributing to its functionality. By addressing factors known to be associated with inferior CAR T-cell response, CRG-023 may improve outcomes for patients afflicted with B-cell malignancies, paving the way for further clinical development and translation into the clinic.

A gynaecological ultrasound on an adolescent patient can involve a transabdominal (TAUS), transvaginal (TVUS), trans‐perineal (TPUS) or transrectal (TRUS) approach. Following TAUS a TVUS or TRUS may be suitable on an adolescent patient if they are determined to be a ‘mature minor’/ Gillick competent and provide informed consent. Legal information on adolescent consent to medical treatment is difficult for sonographers to obtain as current professional guidelines are not sufficiently detailed on the laws of consent to medical treatment and workplace policies are often not specific to adolescent patients. This manuscript provides information on informed consent to medical treatment, ‘mature minor’/ Gillick competence determination, Australian and New Zealand legislation for ‘mature minors’ to consent to medical treatment, adolescent psychology and avoiding coercion when obtaining consent.

Cleft lip and/or palate (CL/CP) is the most common congenital craniofacial anomaly and parents often ask, “how did this happen?” Patients and families may benefit from access to a multidisciplinary team (MDT) from prenatal diagnosis into early adulthood. Multiple factors can contribute to the development of a cleft. We discuss the epidemiology and risk factors that increase the likelihood of having a newborn with a cleft. The purpose of this article is to review the prenatal investigations involved in the diagnosis and workup of these patients in addition to postpartum treatment, prognostic factors, and counseling families regarding future recurrence risk.

Neoantigens are peptides derived from non-synonymous mutations presented by human leukocyte antigens (HLAs), which are recognized by antitumour T cells1-14. The large HLA allele diversity and limiting clinical samples have restricted the study of the landscape of neoantigen-targeted T cell responses in patients over their treatment course. Here we applied recently developed technologies15-17 to capture neoantigen-specific T cells from blood and tumours from patients with metastatic melanoma with or without response to anti-programmed death receptor 1 (PD-1) immunotherapy. We generated personalized libraries of neoantigen-HLA capture reagents to single-cell isolate the T cells and clone their T cell receptors (neoTCRs). Multiple T cells with different neoTCR sequences (T cell clonotypes) recognized a limited number of mutations in samples from seven patients with long-lasting clinical responses. These neoTCR clonotypes were recurrently detected over time in the blood and tumour. Samples from four patients with no response to anti-PD-1 also demonstrated neoantigen-specific T cell responses in the blood and tumour to a restricted number of mutations with lower TCR polyclonality and were not recurrently detected in sequential samples. Reconstitution of the neoTCRs in donor T cells using non-viral CRISPR-Cas9 gene editing demonstrated specific recognition and cytotoxicity to patient-matched melanoma cell lines. Thus, effective anti-PD-1 immunotherapy is associated with the presence of polyclonal CD8+ T cells in the tumour and blood specific for a limited number of immunodominant mutations, which are recurrently recognized over time.

Background NeoTCR-P1 is a personalized autologous T cell therapy for treatment of patients with solid tumors. Neoantigen-specific T cell receptors (neoTCRs) were isolated from the patients' own circulating CD8 T cells using the imPACT Isolation Technology®, followed by non-viral precision genome engineering into an autologous apheresis product for infusion back into the patient. Methods This phase 1 trial is a first-in-human, multi-center, dose-escalation study to evaluate the safety, tolerability, and manufacturing feasibility of NeoTCR-P1 alone or in combination with IL-2 in solid tumors. Patients with TCRs identified at screening and meeting eligibility criteria underwent apheresis to manufacture personalized NeoTCR-P1 cell product. Lymphodepleted patients received a single dose of up-to-three distinct NeoTCR cell products at dose levels of 0.4, 1.2, or 4×109 NeoTCR-edited T cells. Pre- and post-treatment blood and biopsy samples were collected to evaluate NeoTCR-P1 pharmacokinetics, tumor trafficking, signs of T cell engagement or potential mechanisms of resistance. Results Sixteen patients were infused with NeoTCR-P1 T cells including patients with MSS-colorectal cancer (11), breast cancer (2), ovarian cancer (1), melanoma (1), or non-small cell lung cancer (1). Four of the sixteen patients were treated with NeoTCR-P1 + IL-2. Two patients experienced toxicities associated with NeoTCR-P1 cell infusions: a grade 1 CRS and a grade 2 ICANS. Five patients had stable disease as their best response at their first tumor assessment (day 28). NeoTCR+ T cells detected in the peripheral blood had an average peak of 3.6% (range 0.9-7.3%) for DL1, 11.7% (7.7-20.8%) for DL2, and 19.8% (12.0-37.3%) for DL3. Increases in NeoTCR T cells were observed at higher dose levels, stronger lymphodepletion, or higher gene editing rates of the infused product. Eight post-infusion biopsies were available for sequencing and imaging analysis; 17 of 22 neoTCR-T cells were detected in post-infusion biopsies with 12 neoTCRs among the top 4% of CDR3 sequences detected. The targeted neoantigens were detected in 7 of 8 post-treatment biopsies (15 of 22 targets), and personalized ctDNA confirmed targeting of a predicted sub-clonal mutation. An APOBEC signature and HLA-LOH were identified as potential mechanisms of resistance. By single-cell, spatial molecular imaging, neoTCR-T cells were visualized in post-treatment biopsies and found to differentially express potential markers of engagement. Conclusions This study demonstrates the feasibility of isolating and manufacturing NeoTCR-T cells using non-viral precision genome engineering, the safety of infusing up-to-three gene edited NeoTCR-T cell products, and T cell persistence and trafficking to a variety of solid tumors. Trial Registration NCT03970382 Ethics Approval Ethics approvals have been obtained from each clinical site enrolling patients: City of Hope, Duarte California; University of California Los Angeles, Los Angeles California; University of California, Irvine Medical Center, Orange, California; University of California, Davis, Sacramento California; University of California, San Francisco, San Francisco California; Northwestern University Medical Center, Chicago Illinois; Memorial Sloan Kettering Cancer Center, New York, New York; Tennessee Oncology, Nashville, Tennessee; and Fred Hutchinson Cancer Research Center, Seattle, Washington.

T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells 1 – 3 . Here we developed a clinical-grade approach based on CRISPR–Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRβ). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen–HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial ( NCT03970382 ). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.

cells are functional and show prolonged persistence and improved tumor control in an in vivo model of T cell-mediated

Background The primary target of T-cell responses to cancer cells are peptides derived from non-synonymous mutations presented by HLA. However, the large diversity of HLA alleles and restricted availability of clinical samples has limited the study of the antigenic determinants recognized by T cells (termed neoepitopes) at the scale needed for a landscape analysis of antitumor immune responses in patients. Methods We applied a newly developed technology to perform a longitudinal landscape analysis of the neoepitope-specific T cells in peripheral blood and tumor from 11 patients with metastatic melanoma, 7 with response (R) or 4 with no response (NR) to immune checkpoint blockade (ICB) immunotherapy. Briefly, based on the computational prediction of patient-specific putative neoepitopes, hundreds of capture reagents were made consisting of the patient HLA class I subtypes loaded with the corresponding predicted neoepitope; neoepitope-specific T cells were then isolated, and the TCR alpha and beta sequenced. The tumor reactivity of the isolated neoepitope-specific TCRs (neoTCR) was assessed upon co-culture of autologous melanoma cell lines from each patient with primary human T cells expressing the neoTCRs generated using a CRISPR-based non-viral precision genome engineering to replace the endogenous TCRs. Results The tumor mutation burden ranged between 2562 and 54 and 297 to 31 for patients with R and NR, respectively. We screened an average of 157 (range 243 to 17) predicted neoepitope-HLA per patient across their 6 HLA molecules, and isolated neoTCRs in all 11 patients. The number of mutations targeted ranged between 13 and 1. We assessed tumor reactivity in samples from 3 R and 3 NR; 39 of the 64 neoTCRs demonstrated specific recognition and cytotoxicity to patient-matched melanoma cell lines. Multiple T cells with different neoTCRs (T cell clonotypes) recognized a limited number of mutations in 7 patients with R (average of 31 different neoTCR clonotypes per patient). These T cell specificities were recurrently detected at different time points in blood and tumors. Samples from 4 patients with NR also demonstrated neoepitope-specific T cell responses in blood and tumor to a similarly restricted number of mutations but lacked TCR polyclonality (average 3 neoTCR clonotypes per patient) and were not recurrently detected in sequential samples. Conclusions Effective ICB therapy is associated with polyclonal neoepitope-specific T cell responses in the tumor and blood that recognize a limited number of immunodominant mutations and are recurrently recognized over time. Ethics Approval Patients with metastatic melanoma were selected as they signed an informed consent to collect PBMC and tumour biopsies while receiving therapy with anti-PD-1 therapy alone or in combination with other drugs. Biopsies and blood samples were collected under the University of California, Los Angeles (UCLA) Institutional Review Board approvals 11–003254.

BACKGROUND: The American College of Obstetricians and Gynecologists and the Society for Maternal-Fetal Medicine recently recommended offering genetic counseling and diagnostic testing for enlarged nuchal translucency at >= 3.0 mm, regardless of previous negative screening with noninvasive prenatal testing. OBJECTIVE: This study aimed to perform a population-based, individual record linkage study to determine the optimal definition of an enlarged nuchal translucency for the detection of atypical chromosome abnormalities. STUDY DESIGN: This was a retrospective study of women resident in Victoria, Australia, undergoing combined first-trimester screening during the 24-month period from January 2015 to December 2016. Linkages between statewide results for combined first-trimester screening, prenatal diagnostic procedures, and postnatal cytogenetic results from products of conception and infants up to 12 months of age were used to ascertain the frequency and type of chromosome abnormality by gestation and nuchal translucency measurement. An atypical chromosome abnormality was defined as any major chromosome abnormality other than whole chromosome aneuploidy involving chromosomes 21, 18, 13, X, and Y. RESULTS: Of the 81,244 singleton pregnancies undergoing combined first-trimester screening, 491 (0.60%) had a nuchal translucency of >= 3.5 mm, 534 (0.66%) had a nuchal translucency of 3.0 to 3.4 mm, and 80,219 (98.74%) had a nuchal translucency of < 3.0 mm. When grouped by nuchal translucency multiples of the median (MoM), 192 (0.24%) had a nuchal translucency of >= 3.0 MoM, 513 (0.63%) had a nuchal translucency of 1.9 to 2.9 MoM, and 80,539 (99.13%) had a nuchal translucency of <1.9 MoM. A total of 1779 pregnancies underwent prenatal or postnatal diagnostic testing, of which 89.60% were performed by whole-genome single-nucleotide polymorphism chromosomal microarray. The frequency of total major chromosome abnormalities was significantly higher in the group with a nuchal translucency of >= 3.5 mm (147 of 491, 29.94%) than the group with a nuchal translucency of 3.0 to 3.4 mm(21 of 534, 3.93%) or a nuchal translucency of<3.0 mm (71 of 80,219, 0.09%) (P<.001). There were 93 atypical chromosome abnormalities in the total screened cohort. The frequency of an atypical chromosome abnormality was 4.07% (95% confidence interval, 2.51-6.22), 0.37% (95% confidence interval, 0.05-1.35), and 0.09% (95% confidence interval, 0.07-0.11) in the groups with a nuchal translucency of >= 3.5 mm, 3.0 to 3.4 mm, and <3.0 mm, respectively. The frequency of atypical chromosome abnormalities was 4.69% (95% confidence interval, 2.17-8.71), 2.53% (95% confidence interval, 1.36-4.29), and 0.09% (95% confidence interval, 0.07-0.11) in the groups with a nuchal translucency of >= 3.0 MoM, 1.9 to 2.9 MoM, and<1.9 MoM, respectively. When defining thresholds for offering diagnosis with chromosomal microarray at 11 to 13 weeks, both a nuchal translucency threshold of 1.9 MoM and a fixed threshold of 3.0 mm captured 22 of 93 fetuses (23.7%) with an atypical chromosome abnormality. Of these, 50.0% had a coexisting fetal abnormality on ultrasound. However, the gestation-specific threshold of 1.9 MoM had a better specificity than 3.0 mm. The positive predictive value of an enlarged nuchal translucency for any atypical chromosome abnormality was 1 in 47 for nuchal translucency of>3.0 mm and 1 in 32 for nuchal translucency of >1.9 MoM. Our nuchal translucency threshold of 1.9 MoM captured 0.87% of fetuses, thus approximating the 99th centile. CONCLUSION: A gestational age-adjusted nuchal translucency threshold of 1.9 MoM or 99th centile is superior to the fixed cutoff of 3.0 mm for the identification of atypical chromosome abnormalities. The risk of an atypical chromosome abnormality in a fetus with an enlarged nuchal translucency is more than tripled in the presence of an additional ultrasound abnormality.

STUDY QUESTION What is the frequency of major chromosome abnormalities in a population-based diagnostic data set of genomic tests performed on miscarriage, fetal and infant samples in a state with >73 000 annual births? SUMMARY ANSWER The overall frequency of major chromosome abnormalities in the entire cohort was 28.2% (2493/8826), with a significant decrease in the detection of major chromosome abnormalities with later developmental stage, from 50.9% to 21.3% to 15.6% of tests in the miscarriage, prenatal and postnatal cohorts, respectively. WHAT IS KNOWN ALREADY Over the past decade, technological advances have revolutionized genomic testing at every stage of reproduction. Chromosomal microarrays (CMAs) are now the gold standard of chromosome assessment in prenatal diagnosis and pediatrics. STUDY DESIGN, SIZE, DURATION A population-based cohort study including all chromosome analysis was performed in the Australian state of Victoria during a 24-month period from January 2015 to December 2016. All samples obtained via invasive prenatal diagnosis and postnatal samples from pregnancy tissue and infants ≤12 months of age were included. PARTICIPANTS/MATERIALS, SETTING, METHODS A research collaboration of screening and diagnostic units in the Australian state of Victoria was formed (the Perinatal Record Linkage collaboration), capturing all instances of prenatal and postnatal chromosome testing performed in the state. Victoria has over 73 000 births per annum and a median maternal age of 31.5 years. We analyzed our population-based diagnostic data set for (i) chromosome assessment of miscarriage, prenatal diagnosis and postnatal samples; (ii) testing indications and diagnostic yields for each of these cohorts; (iii) and the combined prenatal/infant prevalence of 22q11.2 deletion syndrome (DS) as a proportion of all births ≥20 weeks gestation. MAIN RESULTS AND THE ROLE OF CHANCE During the 24-month study period, a total of 8826 chromosomal analyses were performed on prenatal and postnatal specimens in Victoria. The vast majority (91.2%) of all chromosome analyses were performed with CMA.The overall frequency of major chromosome abnormalities in the entire cohort was 28.2% (2493/8826). There was a significant decreasing trend in the percentage of chromosome abnormalities with later developmental stage from 50.9% to 21.3% to 15.6% in the miscarriage, prenatal and postnatal cohorts, respectively (χ2 trend = 790.0, P < 0.0001). The total frequency of abnormalities in the live infant subgroup was 13.4% (244/1816). The frequencies of pathogenic copy number variants (CNVs) detected via CMA for the miscarriage, prenatal and postnatal cohorts were 1.9% (50/2573), 2.2% (82/3661) and 4.9% (127/2592), respectively. There was a significant increasing trend in the frequency of pathogenic CNVs with later developmental stage (χ2 trend = 39.72, P < 0.0001). For the subgroup of live infants, the pathogenic CNV frequency on CMA analysis was 6.0% (109/1816). There were 38 diagnoses of 22q11.2 DS, including 1 miscarriage, 15 prenatal and 22 postnatal cases. After excluding the miscarriage case and accounting for duplicate testing, the estimated prevalence of 22q11 DS was 1 in 4558 Victorian births. LIMITATIONS, REASONS FOR CAUTION Clinical information was missing on 11.6% of postnatal samples, and gestational age was rarely provided on the miscarriage specimens. We were unable to obtain rates of termination of pregnancy and stillbirth in our cohort due to incomplete data provided by clinical referrers. We therefore cannot make conclusions on pregnancy or infant outcome following diagnostic testing. Childhood and adult diagnoses of 22q11 DS were not collected. WIDER IMPLICATIONS OF THE FINDINGS Our study marks a complete transition in genomic testing from the G-banded karyotype era, with CMA now established as the first line investigation for pregnancy losses, fetal diagnosis and newborn/infant assessment in a high-income setting. Integration of prenatal and postnatal diagnostic data sets provides important opportunities for estimating the prevalence of clinically important congenital syndromes, such as 22q11 DS. STUDY FUNDING/COMPETING INTEREST(S) L.H. is funded by a National Health and Medical Research Council Early Career Fellowship (1105603); A.L. was funded by a Mercy Perinatal Research Fellowship; J.H. was funded by a National Health and Medical Research Council Senior Research Fellowship (10121252). The funding bodies had no role in the conduct of the research or the manuscript. Discretionary funding from the Murdoch Children's Research Institute has supported the prenatal diagnosis data collection and reporting over the years.Dr Ricardo Palma-Dias reports a commercial relationship with Roche Diagnostics, personal fees from Philips Ultrasound, outside the submitted work. Debbie Nisbet reports a commercial relationship with Roche Diagnostics, outside the submitted work. TRIAL REGISTRATION NUMBER NA.

The Australasian Sonographers Association (ASA) is the peak body and leading voice for sonographers in Australasia and leads the profession in delivering excellence in sonography to the community. A vital part of the ASA's dedication to promoting best practice in medical sonography involves producing and providing resources to guide sonographers in their work. This guideline has been developed by the authors and reviewed by the ASA Special Interest Group Obstetric and Gynaecology (SIG O&G) committee and the Royal Australian and New Zealand College of Obstetricians and Gynaecologists (RANZCOG). The guideline has been approved for release by the ASA Sonographer Policy and Advisory Committee (SPAC). Vasa praevia is an obstetric condition where fetal vessels (unsupported by either the Wharton's jelly in the umbilical cord or placental tissue) run through the amniotic membranes in close proximity to (<20mm), or are overlying the cervical internal os (IOS).1-4 These unsupported vessels are at risk of shearing at the time of membrane rupture causing rapid fetal exsanguination. The estimated incidence of vasa praevia is 1:1250 - 1:4667 pregnancies.1-4 Current evidence demonstrates that antenatal diagnosis of vasa praevia is associated with improved perinatal outcomes.3 If vasa praevia is undiagnosed and appropriate care is not instituted the perinatal survival rate is 56%1 verses 97% if diagnosed prenatally.1, 3 Vasa praevia has three main risk factors; velamentous cord insertion, succenturiate lobe and a low lying placenta.2, 3 Universal screening of the lower uterine segment with colour Doppler should be performed transabdominally at every mid trimester ultrasound as 17% of vasa praevia cases will have no identifiable risk factors on ultrasound.3, 5 This is achieved by performing a dynamic assessment sweeping the transducer longitudinally and transversely with a colour Doppler box (on appropriate settings) over the lower uterine segment to identify vessels within 20mm of the cervical IOS.2, 3, 6 The information in this publication is current when published and is general in nature; it does not constitute professional advice. Any views expressed are those of the author and may not reflect ASA's views. ASA does not endorse any product or service identified in this publication. You use this information at your sole risk and ASA is not responsible for any errors or for any consequences arising from that use.

Clinical benefit observed with immuno-oncology trials often depends on the unleashing of a pre-existing intrinsic T cell immune response in each cancer patient. The targets of these intrinsic T cells are commonly ascribed to recognition of patient-specific neoantigens that arose from cancer mutations. PACT Pharma has developed the ability to selectively capture neoantigen-specific CD8+ T cells from peripheral blood of the patient. Leveraging this technology, PACT Pharma is developing personalized, autologous neo-epitope specific TCR-engineered T cell therapies for the eradication of solid tumors. Briefly, using PACT's proprietary TCR isolation technology neoepitope-specific TCRs are cloned and autologous CD8+ and CD4+ T cells from the same patient with cancer are precision genome engineered (using a DNA-mediated (non-viral) method) to express the neoTCR. NeoTCR expressing T cells are then expanded in a manner that preserves a “younger” T cell phenotypes, resulting in a NeoTCR-P1 product in which the majority of the T cells exhibit T memory stem cell and T central memory phenotypes. Upon cognate antigen encounter, NeoTCR-P1 rapidly differentiate into potent effector T cells. Engineered NeoTCR-P1 cells rapidly expand, secrete effector molecules such as perforin and granzyme B, and cytokines such as interferon-gamma (IFN-γ), IL-2 and TNF-alpha (TNF-α). Single cell secretome analysis demonstrates that NeoTCR-P1 cells are highly polyfunctional (secretion of two or more cytokines or effector proteins). These results demonstrate that PACT’s autologous ex vivo engineered NeoTCR-P1 T cells represent a highly personalized adoptive T cell therapy with potential to provide significant clinical benefit to subjects with solid tumors. Citation Format: Barbara Sennino, Andrew Conroy, Bhamini Purandare, Adam Litterman, Kyle Jacoby, Robert Moot, William Lu, Diana Nguyen, Fabrizia Urbinati, Susan Foy, Theresa Hunter, Olivier Dalmas, Michael Bethune, Tim Park, Songming Peng, Alex Franzusoff, Stefanie Mandl. NeoTCR-P1, a novel neoepitope-specific adoptive cell therapy, consists of T cells with ‘younger’ phenotypes that rapidly proliferate and kill target cells upon recognition of cognate antigen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1433.

To ascertain Australian obstetric sonographers knowledge and current practice when assessing for vasa praevia in the mid‐trimester ultrasound with the view to improve prenatal diagnosis.

T cell receptor (TCR) ligand discovery is essential for understanding and manipulating immune responses to tumors. We developed a cell-based selection platform for TCR ligand discovery that exploits a membrane transfer phenomenon called trogocytosis. We discovered that T cell membrane proteins are transferred specifically to target cells that present cognate peptide-major histocompatibility complex (MHC) molecules. Co-incubation of T cells expressing an orphan TCR with target cells collectively presenting a library of peptide-MHCs led to specific labeling of cognate target cells, enabling isolation of these target cells and sequencing of the cognate TCR ligand. We validated this method for two clinically employed TCRs and further used the platform to identify the cognate neoepitope for a subject-derived neoantigen-specific TCR. Thus, target cell trogocytosis is a robust tool for TCR ligand discovery that will be useful for studying basic tumor immunology and identifying new targets for immunotherapy.

T cells targeting neoepitopes derived from mutations exclusive to the tumor are one of the main drivers of cancer immunotherapy efficacy. Tracking these neoepitope-specific T cells during cancer immunotherapy has been hampered by the impracticality of repeated sampling from the tumor, and by the low frequency of neoepitope-specific T cells in peripheral blood. An ultra-sensitive and high-throughput technology (imPACT) has been developed for the identification and isolation of neoepitope-specific T cells from peripheral blood. Subjects with colorectal cancer, endometrial adenocarcinoma, nasopharyngeal carcinoma and other solid tumors were treated with AB122 (anti-PD-1 antibody) as part of an ongoing dose-escalation clinical trial. Pre-treatment blood samples were analyzed to identify the basal repertoire of neoepitope-specific T cells. Evolution of this repertoire during AB122 treatment was monitored to enable immune phenotyping and correlation with clinical outcomes. In addition, transcriptional profile changes were monitored at the single-cell level for each neoepitope-specific T cell. These data will enable us to analyze T cells targeting neoepitopes and identify driver mutations that correlate with and may be responsible for therapeutic benefit. More broadly, this platform technology promises to significantly advance our understanding of T cell-mediated mechanisms of cancer immunotherapy. Citation Format: Songming Peng, Benjamin Yuen, Joanne Tan, Fangfang Yin, Robert Bao, Zheng Pan, Olivier Dalmas, Duo An, Boi Quach, Michael Yi, Michael Bethune, Stefanie Mandl, Matt Walters, Juan Jaen, Alex Franzusoff. Longitudinal monitoring of neoepitope-specific T cell repertoires in patient blood following cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4042.

TCR ligand discovery is essential for elucidating targets of anti-tumor immunity and designing targeted immunotherapies.Here, we describe a cell-based selection platform for TCR ligand discovery that exploits a membrane transfer phenomenon called trogocytosis.

CD8+ T cells recognize and eliminate tumors in an antigen-specific manner. Despite progress in characterizing the antitumor T cell repertoire and function, the identification of target antigens remains a challenge. Here we describe the use of chimeric receptors called signaling and antigen-presenting bifunctional receptors (SABRs) in a cell-based platform for T cell receptor (TCR) antigen discovery. SABRs present an extracellular complex comprising a peptide and major histocompatibility complex (MHC), and induce intracellular signaling via a TCR-like signal after binding with a cognate TCR. We devised a strategy for antigen discovery using SABR libraries to screen thousands of antigenic epitopes. We validated this platform by identifying the targets recognized by public TCRs of known specificities. Moreover, we extended this approach for personalized neoantigen discovery.

Methods used to engineer cells for adoptive cell therapies (ACT) utilizing receptors that are constant across many patients (CAR or shared Ag TCRs) typically rely on Lenti-, retro-, or adeno-associated virus to deliver specificity-altering sequences to T cells. However, for personalized therapies such as the generation of neoepitope-specific TCR T cell therapies, use of viral vectors is not feasible due to long manufacturing timelines and prohibitive per-patient costs. PACT Pharma has developed a highly efficient, DNA-mediated (non-viral) proprietary precision genome engineering approach to engineer neoepitope-specific primary human T cells. This method can be widely utilized to generate T cells at research scale, as well as for ex vivo manufacturing. Briefly, genomes of individual primary human CD8 and CD4 T cells are engineered with site-specific nucleases in a single-step transfection process to yield efficient, targeted replacement of the endogenous TCR with the therapeutic neoTCR sequences. In this way, the expression of the endogenous TCR is abolished ensuring natural expression and regulation of the inserted neoTCR. The precision of neoTCR-T cell genome engineering was evaluated by Targeted Locus Amplification (TLA) for off-target integration hot spots or translocations, and by next generation sequencing based off-target cleavage assays and found to lack evidence of unintended outcomes. Engineered neoepitope-specific T cells are highly functional as demonstrated by antigen-specific proliferation, killing and cytokine production. Phenotype and detailed functional characterization of PACTs neoTCR-P1 T cells were performed and are described in a separate abstract. PACT’s precision genome engineering approach enables highly efficient generation of bespoke NeoTCR T cells for personalized adoptive cell therapy for patients with solid tumors. Furthermore, PACT precision genome engineering method is not restricted to the use in T cells and has also been applied successfully to other primary cell types, including natural killer and hematopoietic stem cells. Citation Format: Kyle Jacoby, Robert Moot, William Lu, Diana Nguyen, Barbara Sennino, Andrew Conroy, Bhamini Purandare, Adam J. Litterman, Fabrizia Urbinati, Susan P. Foy, Theresa Hunter, Albert Tai, Michael T. Bethune, Songming Peng, Olivier Dalmas, Alex Franzusoff, Stefanie J. Mandl. Highly efficient, non-viral precision genome engineering for the generation of personalized neoepitope-specific adoptive T cell therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4783.