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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.

One of the most intriguing characteristics of cell-free DNA (cfDNA) from plasma is the sequence at the ends of the fragments. Previous studies have shown that these end-sequences are somewhat different in cancer patients than in healthy individuals. While investigating this characteristic, we noticed that the bases at the 5’-ends of a double-stranded fragment were highly correlated with the GC content of that particular fragment. This led us to develop a method, called MendSeqS (Modified End-based sequencing System), that incorporates the correlation between end-motifs and GC content into the analysis of shallow (0.5x) whole genome sequencing (WGS). When applied to plasma samples, MendSeqS was able to classify patients with a sensitivity of 96% at 98% specificity in a cohort comprised of 107 individuals evaluated in our laboratory (43 with cancer and 64 without). In cohorts evaluated in three other laboratories, comprising a total of 401 individuals (193 with cancer and 208 without), MendSeqS achieved a sensitivity of 87% at 98% specificity. MendSeqS could in principle be combined with other methods of cfDNA analysis to enhance cancer detection.

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.

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.

The therapeutic applications of antibodies are manifold and the emergence of SARS-CoV-2 provides a cogent example of the value of rapidly identifying biologically active antibodies. We describe an approach called SLISY (Sequencing-Linked ImmunoSorbent assaY) that in a single experiment can assess the binding specificity of millions of clones, be applied to any screen that links DNA sequence to a potential binding moiety, and requires only a single round of biopanning. We demonstrate this approach using an scFv library applied to cellular and protein targets to identify specific or broadly reacting antibodies. For a cellular target, we use paired HLA knockout cell lines to identify a panel of antibodies specific to HLA-A3. For a protein target, SLISY identifies 1279 clones that bound to the Receptor Binding Domain of the SARS-CoV-2 spike protein, with >40% of tested clones also neutralizing its interaction with ACE2 in in vitro assays. Using a multi-comparison SLISY against the Beta, Gamma, and Delta variants, we recovered clones that exhibited broad-spectrum neutralizing potential in vitro. By evaluating millions of scFvs simultaneously against multiple targets, SLISY allows the rapid identification of candidate scFvs with defined binding profiles facilitating the identification of antibodies with the desired biological activity.

Mutations in CTNNB1, the gene encoding β-catenin, are common in colon and liver cancers, the most frequent mutation affecting Ser-45 in β-catenin. Peptides derived from WT β-catenin have previously been shown to be presented on the cell surface as part of major histocompatibility complex (MHC) class I, suggesting an opportunity for targeting this common driver gene mutation with antibody-based therapies. Here, crystal structures of both the WT and S45F mutant peptide bound to HLA-A*03:01 at 2.20 and 2.45 Å resolutions, respectively, confirmed the accessibility of the phenylalanine residue for antibody recognition. Phage display was then used to identify single-chain variable fragment clones that selectively bind the S45F mutant peptide presented in HLA-A*03:01 and have minimal WT or other off-target binding. Following the initial characterization of five clones, we selected a single clone, E10, for further investigation. We developed a computational model of the binding of E10 to the mutant peptide–bound HLA-A3, incorporating data from affinity maturation as initial validation. In the future, our model may be used to design clones with maintained specificity and higher affinity. Such derivatives could be adapted into either cell-based (CAR-T) or protein-based (bispecific T-cell engagers) therapies to target cancer cells harboring the S45F mutation in CTNNB1.

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.

Metabolic reprogramming is a hallmark of Pancreatic Ductal Adenocarcinomas (PDAC) and offers unique opportunities for therapeutic development. TCGA datasets and pancreatic tissue microarrays reveal that nearly 30% of PDACs exhibit a marked overexpression of Monocarboxylate Transporter 1 (MCT-1), a known lactate/pyruvate transporter. In addition, several clinical studies have correlated MCT-1 expression to poor patient prognosis in other cancers and potential resistance to therapies in PDAC, making MCT-1 an intriguing therapeutic target for pancreatic cancer. In this study, we leverage 3-Bromopyruvate (3BP), a cytotoxic pyruvate analog, to target MCT-1 overexpressing PDACs and optimize it for systemic delivery. First, we establish that the sensitivity of PDAC cells to 3BP correlates to MCT-1 expression. In vitro studies with a panel of pancreatic cancer cell lines highlight that the cytotoxicity of 3BP is limited to PDAC cells with elevated MCT-1 and the mode of action of 3BP is distinctly different from AZD-3965, an MCT-1 inhibitor under clinical investigation. We demonstrate that 3BP exerts its effect on PDAC cells using MCT-1 as gate to enter a cell and then induce cytotoxicity. While 3BP is considered promising, poor serum stability, pharmacokinetics, and excessive in vivo toxicity have hampered its clinical development. We show here, a novel formulation where 3BP is entrapped inside a cyclodextrin cage (sCD-3BP) stabilizes 3BP in vivo, retains its MCT-1 specific activity, and delivers significantly more drug to the tumor with a single dose. Additionally, we show that even short exposures of sCD-3BP are adequate for induction of cell death when compared to current standard-of-care therapeutics such as gemcitabine and components of FOLFIRINOX. Finally, we test our MCT-1 gated approach in multiple in vivo models of pancreatic cancer: an orthotopic pancreatic cancer cell line model and two patient-derived orthotopic xenografts (PDoX) models originating from a primary pancreatic and a metastatic site. Arguably more clinically relevant, the PDoX models used in our study closely mimic clinical features of donor disease, causing significant metastasis to lung and liver, marked invasion into the pancreas, and development of duct-like structures within the tumor. In these models, sCD-3BP significantly reduces pancreatic cancer growth and metastatic spread. In summary, we show that intravenous administration of sCD-3BP is safe, causes no damage to normal tissue and effectively suppresses PDAC tumor growth and reduces metastatic burden. In addition, elucidating the mechanism of action of 3BP allows us to identify potential candidates for this therapy. Our study demonstrates that sCD-3BP can be a powerful therapeutic tool against MCT-1 overexpressed PDACs and present a strong rationale for future human clinical trials. Citation Format: Surojit Sur, Jordina Rincon-Toroella, Marco Dal Molin, Kathleen Gabrielson, Chetan Bettegowda, Shibin Zhou, Ken Kinzler, Bert Vogelstein. sCD-3BP: An MCT-1-“gated” therapeutic for pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B004.

We describe the creation of an isogenic cell line panel representing common cancer pathways, with features optimized for high-throughput screening. More than 1,800 cell lines from three normal human cell lines were generated using CRISPR technologies. Surprisingly, most of these lines did not result in complete gene inactivation despite integration of sgRNA at the desired genomic site. A subset of the lines harbored biallelic disruptions of the targeted tumor suppressor gene, yielding a final panel of 100 well-characterized lines covering 19 frequently lost cancer pathways. This panel included genetic markers optimized for sequence-based ratiometric assays for drug-based screening assays. To illustrate the potential utility of this panel, we developed a high-throughput screen that identified Wee1 inhibitor MK-1775 as a selective growth inhibitor of cells with inactivation of

SummaryWe describe the creation and characterization of an isogenic cell line panel representing common cancer pathways, with multiple features optimized for high-throughput screening. More than 1,800 cell lines from three normal human cells were generated using CRISPR-technologies. Surprisingly, we discovered most of these lines did not result in complete gene inactivation, despite integration of sgRNA at the desired genomic site. However, a subset of the lines harbored true, biallelic disruptions of the targeted tumor suppressor gene, yielding a final panel of 100 well-characterize lines covering 19 pathways frequently subject to loss of function in cancers. This panel included genetic markers optimized for sequence-based ratiometric assays for drug-based screening assays. To illustrate the potential utility of this panel, we developed a multiplexed high-throughput screen that identified Wee1 inhibitor MK-1775 as a selective growth inhibitor of cells with inactivation ofTP53. These cell lines and screening approach should prove useful for researchers studying a variety of cellular and biochemical phenomena.

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.

PURPOSE: Vascular disrupting therapy of cancer has become a promising approach not only to regress tumor growth directly but also to boost the delivery of chemotherapeutics in the tumor. An imaging approach to monitor the changes in tumor vascular permeability, therefore, has important applications for monitoring of vascular disrupting therapies. METHODS: Mice bearing CT26 subcutaneous colon tumors were injected intravenously with 150 kD dextran (Dex150, diameter, d~ 20 nm, 375 mg/kg), tumor necrosis factor-alpha (TNF-α; 1 μg per mouse), or both (n = 3 in each group). The Z-spectra were acquired before and 2 h after the injection, and the chemical exchange saturation transfer (CEST) signals in the tumors as quantified by asymmetric magnetization transfer ratio (MTR(asym)) at 1 ppm were compared. RESULTS: The results showed a significantly stronger CEST contrast enhancement at 1 ppm (ΔMTR(asym) = 0.042 ± 0.002) in the TNF-α-treated tumors than those by Dex150 alone (ΔMTR(asym) = 0.000 ± 0.005, P = 0.0229) or TNF-α alone (ΔMTR(asym) = 0.002 ± 0.004, P = 0.0264), indicating that the TNF-α treatment strongly augmented the tumor uptake of 150 kD dextran. The MRI findings were verified by fluorescence imaging and immunofluorescence microscopy. CONCLUSIONS: High molecular weight dextrans can be used as safe and sensitive CEST MRI contrast agents for monitoring tumor response to vascular disrupting therapy and, potentially, for developing dextran-based theranostic drug delivery systems.

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 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.

Medications that target catecholamine-associated inflammation may prevent cytokine storm syndrome associated with COVID-19 and other diseases

A real-time trial of a cancer blood test Cancers diagnosed early are often more responsive to treatment. Blood tests that detect molecular markers of cancer have successfully identified individuals already known to have the disease. Lennon et al. conducted an exploratory study that more closely reflects the way in which such blood tests would be used in the future. They evaluated the feasibility and safety of incorporating a multicancer blood test into the routine clinical care of 10,000 women with no history of cancer. Over a 12-month period, the blood test detected 26 cancers of different types. A combination of the blood test and positron emission tomography–computed tomography (PET-CT) imaging led to surgical removal of nine of these cancers. Use of the blood test did not result in a large number of futile follow-up procedures. Science , this issue p. eabb9601

The mortality of Coronavirus disease 2019 (COVID-19) appears to be driven by acute respiratory distress syndrome (ARDS) and a dysregulated immune response to SARS-CoV-2. Emerging evidence suggests that a subset of COVID-19 is characterized by the development of a cytokine storm syndrome (CSS), and interleukin (IL)-6 levels are predictors of COVID-19 severity and in-hospital mortality. Targeting hyper-inflammation in COVID-19 may be critical for reducing mortality. Catecholamines enhance inflammatory injury by augmenting the production of IL-6 and other cytokines through a self-amplifying feed-forward loop in immune cells that requires alpha-1 adrenergic receptor (α1-AR) signaling. Prophylactic inhibition of catecholamine synthesis with the α1-AR antagonist prazosin reduced catecholamines and cytokine responses in mice, and resulted in markedly increased survival following various hyper-inflammatory stimuli. These findings offer a rationale for studying α1-AR antagonists in the prophylaxis of patients with COVID-19-CSS and ARDS. As high infection rates threaten to overwhelm hospital capacity during this pandemic, preventative approaches that ameliorate COVID-19 severity and reduce excessive mortality are desperately needed. We hypothesize that treatment with prazosin of individuals who test positive for SARS-CoV-2 could reduce catecholamine surges, secondary cytokine dysregulation, and mortality. To investigate a potential role for α1-AR antagonists in preventing poor outcomes in ARDS, we conducted a retrospective analysis of hospitalized patients diagnosed with ARDS. Using data from the Truven Health MarketScan Research Database (2010-2017), we identified 13,125 men (age 45-64) with ARDS, of whom 655 patients (5.0%) were prescribed α1-AR antagonists in the previous year. Applying logistic regression models, we found that patients with prior use of α1-AR antagonists had lower odds of invasive mechanical ventilation compared to non-users (adjusted OR=0.75, 95% CI 0.59-0.95, p=0.019). Perhaps more importantly, those patients had a ~36% lower incidence of both being ventilated and dying in the hospital (adjusted OR=0.59, 95% CI 0.34-0.95, p=0.042). By contrast, prior use of beta-adrenergic receptor (β-AR) antagonists was not correlated with either outcome. We extended these analyses to patients admitted with pneumonia. Of 108,956 subjects in this cohort, 5,498 patients (5.0%) were taking α1-AR antagonist. Similar to ARDS, patients with pneumonia on α1-AR antagonists (but no β-AR antagonists) had a lower odds of mechanical ventilation (adjusted OR=0.83, 95% CI 0.75-0.92, p

In this White Paper, we discuss the current state of microbial cancer therapy. This paper resulted from a meeting (‘Microbial Based Cancer Therapy’) at the US National Cancer Institute in the summer of 2017. Here, we define ‘Microbial Therapy’ to include both oncolytic viral therapy and bacterial anticancer therapy. Both of these fields exploit tumor-specific infectious microbes to treat cancer, have similar mechanisms of action, and are facing similar challenges to commercialization. We designed this paper to nucleate this growing field of microbial therapeutics and increase interactions between researchers in it and related fields. The authors of this paper include many primary researchers in this field. In this paper, we discuss the potential, status and opportunities for microbial therapy as well as strategies attempted to date and important questions that need to be addressed. The main areas that we think will have the greatest impact are immune stimulation, control of efficacy, control of delivery, and safety. There is much excitement about the potential of this field to treat currently intractable cancer. Much of the potential exists because these therapies utilize unique mechanisms of action, difficult to achieve with other biological or small molecule drugs. By better understanding and controlling these mechanisms, we will create new therapies that will become integral components of cancer care.

We here describe a selected reaction monitoring (SRM)-based approach for the discovery and validation of peptide biomarkers for cancer. The first stage of this approach is the direct identification of candidate peptides through comparison of proteolytic peptides derived from the plasma of cancer patients or healthy individuals. Several hundred candidate peptides were identified through this method, providing challenges for choosing and validating the small number of peptides that might prove diagnostically useful. To accomplish this validation, we used 2D chromatography coupled with SRM of candidate peptides. We applied this approach, called sequential analysis of fractionated eluates by SRM (SAFE-SRM), to plasma from cancer patients and discovered two peptides encoded by the peptidyl-prolyl cis-trans isomerase A (PPIA) gene whose abundance was increased in the plasma of ovarian cancer patients. At optimal thresholds, elevated levels of at least one of these two peptides was detected in 43 (68.3%) of 63 women with ovarian cancer but in none of 50 healthy controls. In addition to providing a potential biomarker for ovarian cancer, this approach is generally applicable to the discovery of peptides characteristic of various disease states.

Purpose To investigate the use of natural dextrans as nano-sized chemical exchange saturation transfer (CEST) MRI probes for characterizing size-dependent tumor vascular permeability. Methods Dextrans of different molecular weight (10, 70, 150, and 2000 kD) were characterized for their CEST contrast. Mice (N = 5) bearing CT26 subcutaneous colon tumors were injected intravenously with 10 kD (D10, 6 nm) and 70 kD (D70, 12 nm) dextran at a dose of 375 mg/kg. The CEST-MRI signal in the tumors was assessed before and approximately 40 min after each injection using a dynamic CEST imaging scheme. Results All dextrans of different molecular weights have a strong CEST signal with an apparent maximum of approximately 0.9 ppm. The detectability and effects of pH and saturation conditions (B1 and Tsat ) were investigated. When applied to CT26 tumors, the injection of D10 could produce a significant "dexCEST" enhancement in the majority of the tumor area, whereas the injection of D70 only resulted in an increase in the tumor periphery. Quantitative analysis revealed the differential permeability of CT26 tumors to different size particles, which was validated by fluorescence imaging and immunohistochemistry. Conclusions As a first application, we used 10- and 70-kD dextrans to visualize the spatially variable, size-dependent permeability in the tumor, indicating that nano-sized dextrans can be used for characterizing tumor vascular permeability with dexCEST MRI and, potentially, for developing dextran-based theranostic drug delivery systems. Magn Reson Med 79:1001-1009, 2018. © 2017 International Society for Magnetic Resonance in Medicine.