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MANA-SRM parameters for peptide targets.

Structure of plasmid construct used for transfection.

Supplementary Figure and Table Legends

Many immunotherapeutic approaches under development rely on T-cell recognition of cancer-derived peptides bound to human leukocyte antigen molecules on the cell surface. Direct experimental demonstration that such peptides are processed and bound is currently challenging. Here, we describe a method that meets this challenge. The method entailed an optimized immunoprecipitation protocol coupled with two-dimensional chromatography and mass spectrometry. The ability to detect and quantify minute amounts of predefined antigens should be useful both for basic research in tumor immunology and for the development of rationally designed cancer vaccines.

Mutation-associated neoantigens (MANAs) are exquisitely cancer-specific therapeutic targets. However, MANAs are present at ultra-low densities on the cancer cell surface (as few as 1-2 copies per cell), leading to the challenge of eliciting a sufficiently robust therapeutic effect. We combined components of both T cell receptors (TCRs) and chimeric antigen receptors (CARs) to create a new receptor with improved potency against an ultra-low-density MANA. From CARs, we utilized the antibody-based antigen recognition domain (i.e. the single chain variable fragment, scFv) and the integrated co-stimulation that amplifies T cell activation. From TCRs, we utilized the multi-chain signaling platform that facilitates high antigen sensitivity. This new receptor, termed a TCR Embedded ScFv for Long-term Activation (TESLA), showed promising characteristics when tested with the H2-scFv which targets the p53 R175H mutation presented on HLA-A*02:01 (R175H/A2). Using CRISPR-based homology directed repair in primary human T cells, we tested 15 configurations of appending the H2-scFv to subunits of the TCR complex to identify a design that maximized T cell cytotoxicity and interferon gamma release in co-cultures with cancer cells expressing endogenous levels of the R175H/A2 antigen. In this system, we showed that the optimal TCR-embedded configuration of the H2-scFv produced similar levels of cytotoxicity and interferon gamma secretion as patient-derived TCRs targeting the same R175H/A2 MANA, while conventional H2-CARs were unable to produce any T-cell activation. We then used a multiple stimulation co-culture system to identify a co-stimulation domain combination (MyD88 and CD40) that improved serial cytotoxicity and proliferation of H2-TESLAs when incorporated on the intracellular side of the TCRbeta chain. Finally, we compared the H2-TESLA receptor to patient-derived TCRs modified with the same MyD88 and CD40 co-stimulation domains. In vivo, H2-TESLAs cured all mice in a tumor model, while co-stimulation-modified TCRs produced only temporary tumor control. Moreover, in vivo, H2-TESLAs elicited 100-fold greater T cell expansion than co-stimulation-modified TCRs. In conclusion, we demonstrated that by combining aspects of both CARs and TCRs, the TESLA receptor improved T cell reactivity against an ultra-low-density neoantigen compared to conventional CARs and patient-derived TCRs. Citation Format: Brian J. Mog, Sarah R. DiNapoli, Michael S. Hwang, Tushar D. Nichakawade, Jacqueline Douglass, Emily Han-Chung Hsiue, Katharine M. Wright, Alexander H. Pearlman, Maximilian F. Konig, Suman Paul, Nicolas Wyhs, Nikita Marcou, Stephanie Glavaris, Jiaxin Ge, Michelle S. Miller, P. Aitana Azurmendi, Evangeline Watson, Drew M. Pardoll, Sandra B. Gabelli, Chetan Bettegowda, Nickolas Papadopoulos, Kenneth W. Kinzler, Bert Vogelstein, Shibin Zhou. Hybrid TCR-CAR design surpasses conventional CARs and patient-derived TCRs in targeting an ultra-low-density neoantigen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB095.

HIV-1 infection is incurable due to the persistence of the virus in a latent reservoir of resting memory CD4+ T cells. “Shock-and-kill” approaches that seek to induce HIV-1 gene expression, protein production, and subsequent targeting by the host immune system have been unsuccessful due to a lack of effective latency-reversing agents (LRAs) and kill strategies. In an effort to develop reagents that could be used to promote killing of infected cells, we constructed T cell receptor (TCR)-mimic antibodies to HIV-1 peptide-major histocompatibility complexes (pMHC). Using phage display, we panned for phages expressing antibody-like variable sequences that bound HIV-1 pMHC generated using the common HLA-A*02:01 allele. We targeted three epitopes in Gag and reverse transcriptase identified and quantified via Poisson detection mass spectrometry from cells infected in vitro with a pseudotyped HIV-1 reporter virus (NL4.3 dEnv). Sequences isolated from phages that bound these pMHC were cloned into a single-chain diabody backbone (scDb) sequence, such that one fragment is specific for an HIV-1 pMHC and the other fragment binds to CD3ε, an essential signal transduction subunit of the TCR. Thus, these antibodies utilize the sensitivity of T cell signaling as readouts for antigen processing and as agents to promote killing of infected cells. Notably, these scDbs are exquisitely sensitive and specific for the peptide portion of the pMHC. Most importantly, one scDb caused killing of infected cells presenting a naturally processed target pMHC. This work lays the foundation for a novel therapeutic killing strategy toward elimination of the HIV-1 reservoir.

The presentation of neoantigens on the cell membrane is the foundation for most cancer immunotherapies. Due to their extremely low abundance, analyzing neoantigens in clinical samples is technically difficult, hindering the development of neoantigen-based therapeutics for more general use in the treatment of diverse cancers worldwide. Here, we describe an integrated system, “Valid-NEO”, which reveals patient-specific cancer neoantigen therapeutic targets from minute amounts of clinical samples through direct observation, without computer-based prediction, in a sensitive, rapid, and reproducible manner. The overall four-hour procedure involves mass spectrometry analysis of neoantigens purified from tumor samples through recovery of HLA molecules with HLA antibodies. Valid-NEO could be applicable to the identification and quantification of presented neoantigens in cancer patients, particularly when only limited amounts of sample are available.

Several current immunotherapy approaches target private neoantigens derived from mutations that are unique to individual patients’ tumors. However, immunotherapeutic agents can also be developed against public neoantigens derived from recurrent mutations in cancer driver genes. The latter approaches target proteins that are indispensable for tumor growth, and each therapeutic agent can be applied to numerous patients. Here we review the opportunities and challenges involved in the identification of suitable public neoantigen targets and the development of therapeutic agents targeting them.

Background: Pivotal clinical trials provide critical evidence to regulators regarding a product’s suitability for marketing approval. The objectives of this study are (1) to characterize select features of trials for oncology products approved by the U.S. Food and Drug Administration between 2015 and 2017; and (2) to quantify the costs of these trials and how such costs varied based on trial characteristics. Methods: We identified novel oncology therapeutic drugs, and their respective pivotal trials, approved between 2015 and 2017 using annual summary reports from the Food and Drug Administration. Cost estimates for each pivotal trial were calculated using IQVIA’s CostPro, a clinical trial cost estimating tool based on executed contracts between pharmaceutical manufacturers and contract research organizations. Measures of drug and trial characteristics included trial design, end point, patient enrollment, and regulatory pathway. We also performed sensitivity analyses that varied assumptions regarding how efficiently each trial was conducted. Results: A total of 39 pivotal clinical trials provided the basis for Food and Drug Administration approval of 30 new oncology drugs from 2015 to 2017. Among these trials, primary end points were objective response rate in 20 (51.3%), progression-free survival in 13 (33.3%), and overall survival in 6 (15.4%). Twenty trials (51.3%) were single-arm studies. The median estimated cost of oncology pivotal trials was $31.7 million (interquartile range = $17.0–$60.4 million). Trials with objective response rate as primary end point had a median estimate of $17.7 million (interquartile range = $11.9–$27.1 million), compared with trials examining progression-free survival ($42.3 million, interquartile range = $34.6–$101.2 million) or overall survival ($79.4 million, interquartile range = $56.9–$97.7 million) (p Conclusions: Relative to the estimated costs of drug development, the costs of these oncology pivotal trials were modest, with trials that produced more valuable scientific information costing more than their counterparts.

Several current immunotherapy approaches target private neoantigens derived from mutations that are unique to individual patients’ tumors. However, immunotherapeutic agents can also be developed against public neoantigens derived from recurrent mutations in cancer driver genes. The latter approaches target proteins that are indispensable for tumor growth, and each therapeutic agent can be applied to numerous patients. Here we review the opportunities and challenges involved in the identification of suitable public neoantigen targets and the development of therapeutic agents targeting them. Zhou and colleagues discuss the opportunities and challenges in targeting public neoantigens for cancer immunotherapy.

BackgroundCurrent therapy for osteosarcoma pulmonary metastases (PMs) is ineffective. The mechanisms that prevent successful immunotherapy in osteosarcoma are incompletely understood. We investigated the tumor microenvironment of metastatic osteosarcoma with the goal of harnessing the immune system as a therapeutic strategy.Methods66 osteosarcoma tissue specimens were analyzed by immunohistochemistry (IHC) and immune markers were digitally quantified. Tumor-infiltrating lymphocytes (TILs) from 25 specimens were profiled by functional cytometry. Comparative transcriptomic studies of distinct tumor-normal lung ‘PM interface’ and ‘PM interior’ regions from 16 PMs were performed. Clinical follow-up (median 24 months) was available from resection.ResultsIHC revealed a statistically significantly higher concentration of TILs expressing immune checkpoint and immunoregulatory molecules in PMs compared with primary bone tumors (including programmed cell death 1 (PD-1), programmed death ligand 1 (PD-L1), lymphocyte-activation gene 3 (LAG-3), T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), and indoleamine 2,3-dioxygenase (IDO1). Remarkably, these lymphocytes are excluded at the PM interface compared with PM interior. TILs from PMs exhibited significantly higher amounts of PD-1 and LAG-3 and functional cytokines including interferon-γ (IFNγ) by flow cytometry. Gene expression profiling further confirmed the presence of CD8 and CD4 lymphocytes concentrated at the PM interface, along with upregulation of immunoregulatory molecules and IFNγ-driven genes in the same region. We further discovered a strong alternatively activated macrophage signature throughout the entire PMs along with a polymorphonuclear myeloid-derived suppressor cell signature focused at the PM interface. Expression of PD-L1, LAG-3, and colony-stimulating factor 1 receptor (CSF1R) at the PM interface was associated with significantly worse progression-free survival (PFS), while gene sets indicative of productive T cell immune responses (CD8 T cells, T cell survival, and major histocompatibility complex class 1 expression) were associated with significantly improved PFS.ConclusionsOsteosarcoma PMs exhibit immune exclusion characterized by the accumulation of TILs at the PM interface. These TILs produce effector cytokines, suggesting their capability of activation and recognition of tumor antigens. Our findings suggest cooperative immunosuppressive mechanisms in osteosarcoma PMs including immune checkpoint molecule expression and the presence of immunosuppressive myeloid cells. We identify cellular and molecular signatures that are associated with patient outcomes, which could be exploited for successful immunotherapy.

Background: Whether the weaning outcome of solid cancer patients receiving mechanical ventilation (MV) in the intensive care unit (ICU) is comparable to that in non-cancer patients is unknown. The aim of this study was to compare the weaning outcomes between non-cancer patients and patients with different types of cancer. Methods: We studied patients requiring MV during ICU stay for medical reasons between 2012 and 2014. Cancer patients were grouped into those with lung cancer (LC), head and neck cancer (HNC), hepatocellular carcinoma (HCC), and other cancers (OC). The primary endpoint was successful weaning at day 90 after the initiation of MV, and the main secondary endpoints were 28-day and 90-day mortality after ICU admission. Results: Five hundred and eighteen patients with solid cancers and 1362 non-cancer patients were recruited. The rate of successful weaning at day 90 was 57.9% in cancer patients, which was lower than 68.9% in non-cancer patients (p

BACKGROUND: Recent studies have reported T cell immunity to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in unexposed donors, possibly due to crossrecognition by T cells specific for common cold coronaviruses (CCCs). True T cell crossreactivity, defined as the recognition by a single TCR of more than one distinct peptide-MHC ligand, has never been shown in the context of SARS-CoV-2. METHODS: We used the viral functional expansion of specific T cells (ViraFEST) platform to identify T cell responses crossreactive for the spike (S) glycoproteins of SARS-CoV-2 and CCCs at the T cell receptor (TCR) clonotype level in convalescent COVID-19 patients (CCPs) and SARS-CoV-2–unexposed donors. Confirmation of SARS-CoV-2/CCC crossreactivity and assessments of functional avidity were performed using a TCR cloning and transfection system. RESULTS: Memory CD4(+) T cell clonotypes that crossrecognized the S proteins of SARS-CoV-2 and at least one other CCC were detected in 65% of CCPs and unexposed donors. Several of these TCRs were shared among multiple donors. Crossreactive T cells demonstrated significantly impaired SARS-CoV-2–specific proliferation in vitro relative to monospecific CD4(+) T cells, which was consistent with lower functional avidity of their TCRs for SARS-CoV-2 relative to CCC. CONCLUSIONS: Our data confirm, for what we believe is the first time, the existence of unique memory CD4(+) T cell clonotypes crossrecognizing SARS-CoV-2 and CCCs. The lower avidity of crossreactive TCRs for SARS-CoV-2 may be the result of antigenic imprinting, such that preexisting CCC-specific memory T cells have reduced expansive capacity upon SARS-CoV-2 infection. Further studies are needed to determine how these crossreactive T cell responses affect clinical outcomes in COVID-19 patients. FUNDING: NIH funding (U54CA260492, P30CA006973, P41EB028239, R01AI153349, R01AI145435-A1, R21AI149760, and U19A1088791) was provided by the National Institute of Allergy and Infectious Diseases, the National Cancer Institute, and the National Institute of Biomedical Imaging and Bioengineering. The Bloomberg~Kimmel Institute for Cancer Immunotherapy, The Johns Hopkins University Provost, and The Bill and Melinda Gates Foundation provided funding for this study.

Significance The lack of viable tumor-specific targets continues to thwart efforts to implement selective anticancer drugs in the clinic. Clonal loss of heterozygosity (LOH) occurs in the great majority of human tumors and represents an irreversible genetic alteration present in cancer cells that unequivocally distinguishes them from normal cells. Here, we report the development of NASCAR (Neoplasm-targeting Allele-Sensing CAR), a platform comprising pairwise chimeric receptors for detecting and targeting LOH events in cancer. As proof-of-concept, we demonstrate specific NASCAR T cell responses in models of HLA LOH in vitro and in vivo. This work lays the foundation for future exploration and exploitation of LOH-mediated vulnerabilities for precision cellular immunotherapy., Developing therapeutic agents with potent antitumor activity that spare normal tissues remains a significant challenge. Clonal loss of heterozygosity (LOH) is a widespread and irreversible genetic alteration that is exquisitely specific to cancer cells. We hypothesized that LOH events can be therapeutically targeted by “inverting” the loss of an allele in cancer cells into an activating signal. Here we describe a proof-of-concept approach utilizing engineered T cells approximating NOT-gate Boolean logic to target counterexpressed antigens resulting from LOH events in cancer. The NOT gate comprises a chimeric antigen receptor (CAR) targeting the allele of human leukocyte antigen (HLA) that is retained in the cancer cells and an inhibitory CAR (iCAR) targeting the HLA allele that is lost in the cancer cells. We demonstrate that engineered T cells incorporating such NOT-gate logic can be activated in a genetically predictable manner in vitro and in mice to kill relevant cancer cells. This therapeutic approach, termed NASCAR (Neoplasm-targeting Allele-Sensing CAR), could, in theory, be extended to LOH of other polymorphic genes that result in altered cell surface antigens in cancers.

Turning a tumor suppressor into a target Tumor-suppressor genes such as TP53 (tumor protein P53) play key roles in the pathogenesis of cancer but, unfortunately, they are difficult to target because they do not create an overactive protein that can be inhibited with a drug. Hsiue et al. discovered a way to target a cancer-associated mutant form of the p53 protein using the body's own immune system (see the Perspective by Weidanz). The authors identified a distinct fragment of this mutant protein, characterized the structural basis for its presentation to T cells, and designed a bispecific antibody to stimulate T cell killing of p53-mutant cancer cells. Science , this issue p. eabc8697 ; see also p. 996

Mutations in the RAS oncogenes occur in multiple cancers, and ways to target these mutations has been the subject of intense research for decades. Most of these efforts are focused on conventional small-molecule drugs rather than antibody-based therapies because the RAS proteins are intracellular. Peptides derived from recurrent RAS mutations, G12V and Q61H/L/R, are presented on cancer cells in the context of two common human leukocyte antigen (HLA) alleles, HLA-A3 and HLA-A1, respectively. Using phage display, we isolated single-chain variable fragments (scFvs) specific for each of these mutant peptide-HLA complexes. The scFvs did not recognize the peptides derived from the wild-type form of RAS proteins or other related peptides. We then sought to develop an immunotherapeutic agent that was capable of killing cells presenting very low levels of these RAS-derived peptide-HLA complexes. Among many variations of bispecific antibodies tested, one particular format, the single-chain diabody (scDb), exhibited superior reactivity to cells expressing low levels of neoantigens. We converted the scFvs to this scDb format and demonstrated that they were capable of inducing T cell activation and killing of target cancer cells expressing endogenous levels of the mutant RAS proteins and cognate HLA alleles. CRISPR-mediated alterations of the HLA and RAS genes provided strong genetic evidence for the specificity of the scDbs. Thus, this approach could be applied to other common oncogenic mutations that are difficult to target by conventional means, allowing for more specific anticancer therapeutics.

PD-1 blockade unleashes CD8 T cells1, including those specific for mutation-associated neoantigens (MANA), but factors in the tumour microenvironment can inhibit these T cell responses. Single-cell transcriptomics have revealed global T cell dysfunction programs in tumour-infiltrating lymphocytes (TIL). However, the majority of TIL do not recognize tumour antigens2, and little is known about transcriptional programs of MANA-specific TIL. Here, we identify MANA-specific T cell clones using the MANA functional expansion of specific T cells assay3 in neoadjuvant anti-PD-1-treated non-small cell lung cancers (NSCLC). We use their T cell receptors as a ‘barcode’ to track and analyse their transcriptional programs in the tumour microenvironment using coupled single-cell RNA sequencing and T cell receptor sequencing. We find both MANA- and virus-specific clones in TIL, regardless of response, and MANA-, influenza- and Epstein–Barr virus-specific TIL each have unique transcriptional programs. Despite exposure to cognate antigen, MANA-specific TIL express an incompletely activated cytolytic program. MANA-specific CD8 T cells have hallmark transcriptional programs of tissue-resident memory (TRM) cells, but low levels of interleukin-7 receptor (IL-7R) and are functionally less responsive to interleukin-7 (IL-7) compared with influenza-specific TRM cells. Compared with those from responding tumours, MANA-specific clones from non-responding tumours express T cell receptors with markedly lower ligand-dependent signalling, are largely confined to HOBIThigh TRM subsets, and coordinately upregulate checkpoints, killer inhibitory receptors and inhibitors of T cell activation. These findings provide important insights for overcoming resistance to PD-1 blockade., Single-cell RNA sequencing and T cell receptor sequencing are combined to identify transcriptional programs specific to mutation-associated neoantigen-specific T cells in non-small cell lung cancers treated with anti-PD-1, providing insights into resistance to PD-1 blockade.

Chimeric antigen receptor (CAR) T cells have emerged as a promising class of therapeutic agents, generating remarkable responses in the clinic for a subset of human cancers. One major challenge precluding the wider implementation of CAR therapy is the paucity of tumor-specific antigens. Here, we describe the development of a CAR targeting the tumor-specific isocitrate dehydrogenase 2 (IDH2) with R140Q mutation presented on the cell surface in complex with a common human leukocyte antigen allele, HLA-B*07:02. Engineering of the hinge domain of the CAR, as well as crystal structure-guided optimization of the IDH2R140Q-HLA-B*07:02-targeting moiety, enhances the sensitivity and specificity of CARs to enable targeting of this HLA-restricted neoantigen. This approach thus holds promise for the development and optimization of immunotherapies specific to other cancer driver mutations that are difficult to target by conventional means., Chimeric antigen receptor T cells in the clinic currently target cell-type-specific extracellular antigens on malignant cells. Here, authors engineer tumor-specific chimeric antigen receptor T cells that target human leukocyte antigen-presented neoantigens derived from mutant intracellular proteins.

Background Patients with relapsed metastatic osteosarcoma have no effective treatments available to them,1 and immunotherapy thus far has not succeeded in improving outcomes.2–5 We aim to understand the immune architecture of the tumor microenvironment (TME) of osteosarcoma, with the goal of harnessing the immune system as a major therapeutic strategy for the treatment of patients with osteosarcoma. Methods 66 osteosarcoma tissue specimens were stained and analyzed by immunohistochemistry. Tumor-infiltrating lymphocytes (TILs) from 25 specimens were profiled by functional multiparameter flow cytometry (MFC). Distinct regions from 16 pulmonary metastases (PMs) were microdissected, and RNA was extracted to perform comparative transcriptomic studies. Clinical follow-up (median 24 months) was available from resection. Results Digital image analysis of immunohistochemistry demonstrated significantly higher infiltrating immune cells in the PMs compared to primary bone tumors, concentrated at the tumor-normal lung ‘PM interface’ region, and elevated expression of multiple immune checkpoint molecules at the PM interface (figure 1). MFC confirmed the increased expression of the immune checkpoint molecules programmed cell death 1 (PD-1, p Conclusions In contrast to primary bone osteosarcoma ‘immune deserts,’ osteosarcoma PMs represent an ‘immune-excluded’ TME where immune cells are present but are halted at the PM interface. TILs can produce effector cytokines, suggesting their capability of activation and recognition of tumor antigens. Our findings suggest cooperative immunosuppressive mechanisms in osteosarcoma PMs that prevent TILs from penetrating into the PM interior, including immune checkpoint molecule expression and the presence of immunosuppressive myeloid cells. We identify cellular and molecular signatures that are associated with PFS of patients, which could be potentially manipulated for successful immunotherapy. Ethics Approval This study was approved by Johns Hopkins University’s Ethics Board, approval number FWA00005752. References Mirabello L, Troisi RJ, Savage SA. Osteosarcoma incidence and survival rates from 1973 to 2004: Data from the surveillance, epidemiology, and end results program. Cancer 2009;115(7):1531–43. Tawbi HA, Burgess M, Bolejack V, Van Tine BA, Schuetze SM, Hu J, et al. Pembrolizumab in advanced soft-tissue sarcoma and bone sarcoma (SARC028): A multicentre, two-cohort, single-arm, open-label, phase 2 trial. Lancet Oncol 2017;18(11):1493–501. Davis KL, Fox E, Merchant MS, Reid JM, Kudgus RA, Liu X, et al. Nivolumab in children and young adults with relapsed or refractory solid tumours or lymphoma (ADVL1412): A multicentre, open-label, single-arm, phase 1–2 trial. Lancet Oncol 2020;21(4):541–50. D’Angelo SP, Mahoney MR, Van Tine BA, Atkins J, Milhem MM, Jahagirdar BN, et al. Nivolumab with or without ipilimumab treatment for metastatic sarcoma (alliance A091401): Two open-label, non-comparative, randomised, phase 2 trials. Lancet Oncol 2018;19(3):416–26. Paoluzzi L, Cacavio A, Ghesani M, Karambelkar A, Rapkiewicz A, Weber J, et al. Response to anti-PD1 therapy with nivolumab in metastatic sarcomas. Clin Sarcoma Res 2016;6:24.

Background TP53 is the most commonly mutated cancer driver gene but drugs that target TP53 are not yet available. A peptide derived from the most common p53 mutation R175H (HMTEVVRHC) can be presented by a common human leukocyte antigen (HLA-A*02:01) after proteasomal degradation.1 We aimed to develop T cell receptor (TCR)-mimic antibody targeting this shared neoantigen. Methods We constructed a single-chain variable fragment (scFv) phage display library that presents scFvs at an estimated diversity of 3.6e10. Mutant peptide-HLA (pHLA)-specific scFvs were enriched through five rounds of positive and negative selections. Mutant pHLA-specific scFv clones were converted into bispecific T cell retargeting antibodies in the single-chain diabody (scDb) format by linking with the anti-CD3 scFv UCHT1.2 These scDbs were tested in T cell co-culturs in the presence of target pulsed cells or target cells that either overexpress the p53 neoantigen or present the p53 neoantigen at endogenous levels. In vivo efficacy was assessed by administering scDb in NSG xenograft mouse model. The structural basis of the binding specificity was evaluated by X-ray crystallography. Results We identified an scFv, termed clone H2, that specifically binds p53 R175H/HLA-A*02:01 pHLA and not its wild-type counterpart at a Kd of 86 nM (figure 1A). H2-scDb induced T cell cytokine release and cytotoxicity in the presence of 1) HLA-A*02:01-expressing cells pulsed with the p53R175H peptide, 2) cells transfected with HLA-A*02:01 and p53 R175H, and 3) cancer cell lines KMS26, KLE, and TYK-nu that express endogenous HLA-A*02:01/p53 R175H (figure 1B-E). T cell activation was diminished when TP53 was knocked out from these cancer cell lines using CRISPR (figure 1E). When administered to NOD scid gamma (NSG) mice systemically engrafted with the KMS-26 cell line, H2-scDb significantly suppressed tumor growth (figure 1 F, G). The structure of p53 R175H/HLA-A2 bound to the H2-Fab fragment shows that four complementarity-determining region loops of the H2 antibody formed a cage-like configuration around the C-terminus of the p53 R175H peptide, trapping the mutant histidine (His175) and the adjacent arginine (Arg174) residues in a stable interaction, which provides the structural basis for the specificity (figure 2). Conclusions We have developed a TCR-mimic bispecific T cell engager H2-scDb that recognized the shared neoantigen HLA-A*02:01/p53 R175H pHLA complex with exquisite specificity. It effectively activated T cells and lysed tumor cells both in vitro and in vivo. This approach could in theory be used to target cancers containing mutations that are difficult to target in conventional ways. Acknowledgements We thank Surojit Sur, Nicolas Wyhs, Ashley Cook, Marco Dal Molin, Richard L. Blosser, Lilian Dasko-Vincent, Christopher Thoburn, Jianhong Cao, and Jose Rodriguez Molina for assistance with this study. Trial Registration NA Ethics Approval NA Consent NA References Lo W, Parkhurst M, Robbins PF, Tran E, Lu YC, Jia L, Gartner JJ, Pasetto A, Deniger D, Malekzadeh P, Shelton TE, Prickett T, Ray S, Kivitz S, Paria BC, Kriley I, Schrump DS, Rosenberg SA. Immunologic recognition of a shared p53 mutated neoantigen in a patient with metastatic colorectal cancer. Cancer Immunol. Res 2019;7:534–543. Stork R, Campigna E, Robert B, Muller D, Kontermann RE. Biodistribution of a bispecific single-chain diabody and its half-life extended derivatives. J Biol Chem 2009;284:25612–25619

Immunotherapies such as chimeric antigen receptor (CAR) T cells and bispecific antibodies redirect healthy T cells to kill cancer cells expressing the target antigen. The pan-B cell antigen-targeting immunotherapies have been remarkably successful in treating B cell malignancies. Such therapies also result in the near-complete loss of healthy B cells, but this depletion is well tolerated by patients. Although analogous targeting of pan-T cell markers could, in theory, help control T cell cancers, the concomitant healthy T cell depletion would result in severe and unacceptable immunosuppression. Thus, therapies directed against T cell cancers require more selective targeting. Here, we describe an approach to target T cell cancers through T cell receptor (TCR) antigens. Each T cell, normal or malignant, expresses a unique TCR β chain generated from 1 of 30 TCR β chain variable gene families (TRBV1 to TRBV30). We hypothesized that bispecific antibodies targeting a single TRBV family member expressed in malignant T cells could promote killing of these cancer cells, while preserving healthy T cells that express any of the other 29 possible TRBV family members. We addressed this hypothesis by demonstrating that bispecific antibodies targeting TRBV5-5 (α-V5) or TRBV12 (α-V12) specifically lyse relevant malignant T cell lines and patient-derived T cell leukemias in vitro. Treatment with these antibodies also resulted in major tumor regressions in mouse models of human T cell cancers. This approach provides an off-the-shelf, T cell cancer selective targeting approach that preserves enough healthy T cells to maintain cellular immunity.

The association between the response to first-line epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) and survival in EGFR mutation-positive non-small-cell lung cancer (NSCLC) remains unclear. We studied the association between the response to first-line EGFR-TKIs and survival using Response Evaluation Criteria In Solid Tumors (RECIST) and maximal tumor shrinkage.We analyzed data from patients with advanced EGFR mutation-positive NSCLC enrolled in first-line gefitinib and afatinib trials. A total of 98 patients who achieved a response or stable disease and had ≥ 1 measurable target lesion were included. The association between the best response by RECIST or maximal tumor shrinkage and survival was analyzed in Kaplan-Meier and Cox regression models with the landmark method. The specified landmark time points were 8 weeks, the median time to maximal tumor shrinkage (16.5 weeks), and median progression-free survival (PFS; 56 weeks).A total of 76 patients (77%) responded to gefitinib or afatinib. Of these 76 patients, 49 (64%) and 75 (99%) had achieved a response at 8 and 16.5 weeks, respectively. All responders had achieved a response by 56 weeks. The responders had a significantly longer PFS and overall survival (OS) compared with those with stable disease at 16.5 weeks (PFS, P = .003; OS, P .001) and 56 weeks (PFS, P = .026; OS, P = .016) but not at 8 weeks (PFS, P = .104; OS, P = .313). Among the responders, greater tumor shrinkage was not associated with longer PFS or OS.Those with a response to first-line gefitinib or afatinib had more favorable PFS and OS compared with those with stable disease. A sufficient observation period was required for the response to occur and predict outcomes. Greater maximal tumor shrinkage in the responders was not predictive of survival.

Non-small cell lung cancer patients harboring uncommon epidermal growth factor receptor (EGFR) mutations together account for approximately 10% of all EGFR mutations. The most common of which being G719X, S768I, L861Q, and exon 20 insertions. The clinical significance, particularly their response to EGFR tyrosine kinase inhibitors (TKIs) is largely unclear. Previous data is limited to a small fraction of patients in prospective studies and retrospective series. Recently, a combined analysis of patients with uncommon EGFR mutations in the Lux-Lung 2, Lux-Lung 3, Lux-Lung 6 trials provide new perspectives of uncommon EGFR mutations. Areas covered: This review reports the existing evidence from major prospective and retrospective studies, along with new data that focus on the clinical significance of uncommon EGFR mutations. Expert commentary: The clinical data of uncommon EGFR mutations should be interpreted carefully as data from prospective and retrospective studies are not considered at the same level of evidence.

Current classification and treatment of lung cancer rely increasingly on molecular and genetic testing. Obtaining tumor tissue is not always feasible and multiple biopsies are undesirable. In response to the demand for non-invasive molecular and genetic testing in cancer care, several liquid biopsy technologies, including circulating DNA (ctDNA), have been developed. ctDNA analysis is now technically feasible to be carried out in large scales and integrated into clinical practice owing to the advances in technology. Despite the challenges in improving test accuracy and cost-effectiveness, there are huge potentials for ctDNA analysis in lung cancer management. This review focuses on the clinical utility of ctDNA analysis in lung cancer, including early detection, monitoring treatment response and detecting residual disease, identification of genetic determinants for targeted therapy, and predicting efficacy of immune checkpoint blockade.

Many immunotherapeutic approaches under development rely on T-cell recognition of cancer-derived peptides bound to human leukocyte antigen molecules on the cell surface. Direct experimental demonstration that such peptides are processed and bound is currently challenging. Here, we describe a method that meets this challenge. The method entailed an optimized immunoprecipitation protocol coupled with two-dimensional chromatography and mass spectrometry. The ability to detect and quantify minute amounts of predefined antigens should be useful both for basic research in tumor immunology and for the development of rationally designed cancer vaccines.

HIV-1 infects CD4+ T cells and macrophages and is currently incurable due to a latent reservoir. Targeting infected cells using TCR-mimic antibodies to peptide-MHC (pMHC) would provide a specific, high-affinity platform for immune system activation and monitoring antigen processing. However, antibodies to pMHC are notoriously difficult to isolate. Using a phage display platform, we panned for phage expressing variable fragments against immunodominant HIV pMHC complexes. These Fab fragments were cloned into a bispecific antibody (bsAb) backbone, such that one Fab fragment is specific for an HIV pMHC and the other fragment binds to CD3. These antibodies utilize the sensitivity of T cell signaling as readouts for antigen processing and potentially as therapeutic formats. Using this approach, we have shown that bsAbs specific for the most conserved MHC-II HIV capsid epitope (Gag293) can recognize the processed epitope on human monocyte-derived dendritic cells (moDCs) fed with the whole Gag protein antigen. Recognition is exquisitely epitope-specific. Additionally, human monocyte-derived macrophages infected with HIV were able to present Gag293 using the endogenous MHC-II pathway, as read out by a Gag-specific bsAb. To our knowledge, this is the first description of endogenous class II presentation of HIV in a major cell type that supports viral replication. By developing bsAbs to detect HIV peptide presentation, we can begin to address the kinetics, pathways, and cell types contributing to the priming events in early HIV infection, which will inform improved vaccine therapies. Additionally, given the potential for therapeutic application, these bsAbs may be used to redirect cytolytic effector cells towards infected targets.

Background In 2011, two national policies aiming to foster hospice services for terminal cancer patients took effect in Taiwan. The single-payer National Health Insurance of Taiwan started to reimburse full hospice services. The national hospital accreditation program, which graded all hospitals, incorporated hospice utilization in its evaluation. We assessed the impact of these national policies. Methods A cohort of 249,394 patients aged ≥18 years who died of cancer between 2008 and 2013 were identified from the National Death Registry. We retrieved utilization data of medical services and compared the health care utilization in the final month of life before and after the implementation of the new policies. Results After the policy changes, hospice utilization increased from 20.8% to 36.2%. In a multivariate analysis adjusting for patient demographics, cancer features, and hospital characteristics, hospice utilization significantly increased after 2011 (adjusted odds ratio [AOR] 2.35, p Conclusion The national policy changes fostering hospice care significantly increased hospice utilization, decreased invasive end-of-life care, and reduced the medical costs of terminal cancer patients.

Epithelioid trophoblastic tumor (ETT) is a rare chemoresistant gestational trophoblastic neoplasm that typically presents as an intrauterine lesion. To our knowledge, no isolated abdominal wall ETT around a Cesarean scar has been reported. Here we describe a 54-yr-old woman with a complex obstetric history who presented with a solitary abdominal wall tumor adjacent to the abdominal Cesarean section scar. The tumor demonstrated typical morphologic and immunophenotypic features of ETT. The gestational origin of the tumor was confirmed by microsatellite genotyping. The tumor enlarged despite the patient undergoing multiagent chemotherapy. Whole-exome sequencing was performed to explore the mechanisms underlying chemoresistance. The ATP-binding cassette subfamily B member 1 (ABCB1) 3435CC genotype, and a putative deleterious x-ray cross-complementing group 4 (XRCC4) Ala73Pro mutations were found. In conclusion, ETT may present as a solitary abdominal wall lesion and microsatellite genotyping could facilitate the determination of its gestational origin. More studies are required to provide mechanistic insights into the chemoresistance of ETT.

2524 Background: Increased tumor suppressor protein p53 expression is observed in a wide range of human cancers. As a result there is intense interest in targeting p53 for cancer therapy. Intracellular p53 is inaccessible to therapeutic antibodies that bind cell surface proteins. However, intracellular proteins including p53 are degraded into peptides that are presented on cell surface in association with HLA class I molecules. Thus p53 peptide-HLA (p53-HLA) complexes can be antibody targets. Methods: Using phage display we identified a novel anti-p53-HLA single chain variable fragment (scFv) clone-43 that recognizes a wild-type p53 10-mer epitope bound to HLA-A*2402. By coupling our clone-43 scFv with an anti-CD3 scFv, we generated a single chain diabody (scDb) designed to activate T-cells against p53-expressing target cells. Results: In-vitro co-culture of clone-43 scDb with donor human T-cells and p53 expressing SIG-M5 cancer cells results in SIG-M5 cell killing and concomitant T-cell interferon gamma (IFNγ) release. In contrast, similar co-culture with SIG-M5 p53-knock out (KO) cells showed no cell killing and minimal IFNγ release demonstrating specificity of clone-43 to p53 expressing cells. Additionally, in-vivo growth of p53 expressing SW480 cancer cell xenografts in NSG mice was completely terminated by clone-43 scDb injections. A major concern for wild-type p53 epitope targeting is potential on-target off-tumor effect on non-cancerous tissue. We observed significant in-vitro clone-43 scDb mediated killing of human HLA-A*24:02 peripheral blood mononuclear cells. To better evaluate effect of clone-43 scDb on non-neoplastic human cells, we engrafted HLA-A*24:02 human CD34+ hematopoietic stem cells into NSG mice to generate a humanized mouse model with circulating mature human CD45+ cells. Clone-43 scDb treatment resulted in selective depletion of circulating human cells while the same cells persisted in mice treated with unrelated control scDb. Conclusions: Our observation that immune targeting of wild-type p53 epitope results in significant off-tumor hematopoietic cell death is contrary to previously published reports and carries important implications for future anti-p53 antibody and vaccine design for cancer immunotherapy.

Dear Editor, Mitochondrial DNA 3243A[G is the most common pathogenic mutation of the tRNA gene, accounting for 80 % of mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) [1]. As a result of multi-organ involvement, patients harboring m.3243A[G may develop complications requiring intensive care unit (ICU) admission. However, relevant literature remains scarce [2– 4]. We retrospectively reviewed 33 patients from six medical centers diagnosed with m.3243A[G mutation between January 1997 and August 2014 at the National Taiwan University Hospital, which receives country-wide genetic referrals. Participants were followed until 15 August 2014 by medical records or phone contact if there were no clinic visits within 3 months. Excluding two asymptomatic carriers and one lost to follow-up, 30 symptomatic patients were followed for a median of 5.6 years (range, 9 months to 34 years). Prognostic factors were analyzed by the Kaplan–Meier method and Cox regression model. The hospital ethics committee approved the protocol. Fourteen patients (47 %) were admitted to ICU at a median of 5.0 years after disease onset. Only 8 patients (57 %) had m.3243A[G diagnosed before admission. The diagnosis was made during ICU and more than 1 year after discharge in 4 (29 %) and 2 (14 %) patients, respectively. The reasons for admission included seizure (n = 9), lactic

Gefitinib is recently approved by the US Food and Drug Administration as a first-line treatment for non-small cell lung cancer (NSCLC) patients harboring EGFR mutations. The therascreen® EGFR RGQ PCR Kit is approved as a companion diagnostic to select patients with EGFR exon 19 deletions and L858R mutation for treatment with gefitinib. Areas covered: This article reviews the methods for detecting EGFR mutations, the technology and indication of the therascreen® kit, and the clinical utility of the assay in phase 3 and phase 4 clinical trials. Studies that compared the performance of the therascreen® kit with other assays and assessed the kit's application in non-tissue samples are also discussed. Expert commentary: The therascreen® kit is a highly sensitive real-time polymerase chain reaction test that provides standardised testing and automated interpretation of EGFR mutation status in formalin-fixed, paraffin-embedded (FFPE) tissue samples. Although not indicated for these applications, the test has also shown utility in detecting uncommon sensitizing EGFR mutations as well as mutations in non-tissue samples.

Here we report the clinical features and treatment outcomes of three patients with Candida haemulonii infection. Candida haemulonii was confirmed by sequence analysis of the internal transcribed spacer (ITS) regions of the rRNA genes and the 18S rRNA genes. Two of the three isolates were associated with fungaemia and reduced susceptibility to fluconazole [minimum inhibitory concentrations (MICs) of 16 mg/L] and amphotericin B (MICs of 2 mg/L). However, one of these two patients responded to fluconazole therapy. Echinocandins, voriconazole and posaconazole demonstrated excellent in vitro potency against the isolates.

We describe a 69-year-old female farmer with diabetes mellitus who developed subcutaneous infection due to a plant pathogen, Corynespora cassiicola. The organism was identified based on characteristic morphotypes and confirmed by sequence analysis of the internal transcribed spacer (ITS) regions. The patient was treated successfully with amphotericin B therapy.

e18113Background: Admitting cancer patients to the intensive care unit (ICU) remains controversial due to perceived poor outcome. This study aimed to (1) assess the predictive value of Acute Physio...

10008Background: In 2011, two significant policy changes to foster hospice service for terminal cancer patients became effective in Taiwan. The single-payer National Health Insurance (NHI) of Taiwa...

e20721 Background: Opioids are crucial for cancer pain management, but not all opioids are recommended by guidelines. We examined the use of opioids not recommended by the National Comprehensive Ca...

The association between sarcoidosis and lymphoproliferative diseases is well-recognized. As fluorine-18 fluorodeoxyglucose (FDG)-positron emission tomography (PET) is increasingly utilized in lymphoma staging, it is important for clinicians to be aware of lymphoma mimickers on FDG-PET. Here we describe two patients with diffuse large B-cell lymphoma who presented with high muscular FDG-uptake. The first patient had histologically proven concomitant muscular sarcoidosis, while the second patient had primary skeletal muscle lymphoma. FDG-PET is of limited utility in discerning sarcoidosis and lymphoma, as both lesions are FDG-avid. Magnetic resonance imaging may provide clues to differentiate these two entities non-invasively. The “3 stripes” and “dark star” signs are characteristic for muscular sarcoidosis, whereas muscular lymphoma usually demonstrates homogenous gadolinium enhancement. Nevertheless, histological examination remains the gold standard in distinguishing hese two diseases. In conclusion, while FDG-PET may be useful in monitoring disease extent and activity of both sarcoidosis and lymphoma, and MRI may provide additional diagnostic information, biopsy is still recommended when clinical decision-making dictates confirmatory diagnosis.