Mutations derived from horseshoe bat ACE2 orthologs enhance ACE2-Fc neutralization of SARS-CoV-2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates infection of cells expressing angiotensin-converting enzyme 2 (ACE2). ACE2 is also the viral receptor of SARS-CoV (SARS-CoV-1), a related coronavirus that emerged in 2002-2003. Horseshoe bats (genus Rhinolophus) are presumed to be the original reservoir of both viruses, and a SARS-like coronavirus, RaTG13, closely related to SARS-CoV-2, has been identified in one horseshoe-bat species. Here we characterize the ability of the S-protein receptor-binding domains (RBDs) of SARS-CoV-1, SARS-CoV-2, pangolin coronavirus (PgCoV), RaTG13, and LyRa11, a bat virus similar to SARS-CoV-1, to bind a range of ACE2 orthologs. We observed that the PgCoV RBD bound human ACE2 at least as efficiently as the SARS-CoV-2 RBD, and that both RBDs bound pangolin ACE2 efficiently. We also observed a high level of variability in binding to closely related horseshoe-bat ACE2 orthologs consistent with the heterogeneity of their RBD-binding regions. However five consensus horseshoe-bat ACE2 residues enhanced ACE2 binding to the SARS-CoV-2 RBD and neutralization of SARS-CoV-2 pseudoviruses by an enzymatically inactive immunoadhesin form of human ACE2 (hACE2-NN-Fc). Two of these mutations impaired neutralization of SARS-CoV-1 pseudoviruses. An hACE2-NN-Fc variant bearing all five mutations neutralized both SARS-CoV-2 pseudovirus and infectious virus more efficiently than wild-type hACE2-NN-Fc. These data suggest that SARS-CoV-1 and -2 originate from distinct bat species, and identify a more potently neutralizing form of soluble ACE2.

Polo-like kinase 1 promotes pulmonary hypertension.

BACKGROUND: Pulmonary hypertension (PH) is a lethal vascular disease with limited therapeutic options. The mechanistic connections between alveolar hypoxia and PH are not well understood. The aim of this study was to investigate the role of mitotic regulator Polo-like kinase 1 (PLK1) in PH development. METHODS: Mouse lungs along with human pulmonary arterial smooth muscle cells and endothelial cells were used to investigate the effects of hypoxia on PLK1. Hypoxia- or Sugen5416/hypoxia was applied to induce PH in mice. Plk1 heterozygous knockout mice and PLK1 inhibitors (BI 2536 and BI 6727)-treated mice were checked for the significance of PLK1 in the development of PH. RESULTS: Hypoxia stimulated PLK1 expression through induction of HIF1α and RELA. Mice with heterozygous deletion of Plk1 were partially resistant to hypoxia-induced PH. PLK1 inhibitors ameliorated PH in mice. CONCLUSIONS: Augmented PLK1 is essential for the development of PH and is a druggable target for PH.

Prediction model based on radiomics and clinical features for preoperative lymphovascular invasion in gastric cancer patients.

Background: We explored whether a model based on contrast-enhanced computed tomography radiomics features and clinicopathological factors can evaluate preoperative lymphovascular invasion (LVI) in patients with gastric cancer (GC) with Lauren classification. Methods: Based on clinical and radiomic characteristics, we established three models: Clinical + Arterial phase_Radcore, Clinical + Venous phase_Radcore and a combined model. The relationship between Lauren classification and LVI was analyzed using a histogram. Results: We retrospectively analyzed 495 patients with GC. The areas under the curve of the combined model were 0.8629 and 0.8343 in the training and testing datasets, respectively. The combined model showed a superior performance to the other models. Conclusion: CECT-based radiomics models can effectively predict preoperative LVI in GC patients with Lauren classification.

Computer-aided classification of MRI for pathological complete response to neoadjuvant chemotherapy in breast cancer.

Background: To determine suitable optimal classifiers and examine the general applicability of computer-aided classification to compare the differences between a computer-aided system and radiologists in predicting pathological complete response (pCR) from patients with breast cancer receiving neoadjuvant chemotherapy. Methods: We analyzed a total of 455 masses and used the U-Net network and ResNet to execute MRI segmentation and pCR classification. The diagnostic performance of radiologists, the computer-aided system and a combination of radiologists and computer-aided system were compared using receiver operating characteristic curve analysis. Results: The combination of radiologists and computer-aided system had the best performance for predicting pCR with an area under the curve (AUC) value of 0.899, significantly higher than that of radiologists alone (AUC: 0.700) and computer-aided system alone (AUC: 0.835). Conclusion: An automated classification system is feasible to predict the pCR to neoadjuvant chemotherapy in patients with breast cancer and can complement MRI.

Affinity maturation, humanization, and co-crystallization of a rabbit anti-human ROR2 monoclonal antibody for therapeutic applications.

Antibodies are widely used as cancer therapeutics, but their current use is limited by the low number of antigens restricted to cancer cells. A receptor tyrosine kinase, receptor tyrosine kinase-like orphan receptor 2 (ROR2), is normally expressed only during embryogenesis and is tightly down-regulated in postnatal healthy tissues. However, it is up-regulated in a diverse set of hematologic and solid malignancies, thus ROR2 represents a candidate antigen for antibody-based cancer therapy. Here we describe the affinity maturation and humanization of a rabbit mAb that binds human and mouse ROR2 but not human ROR1 or other human cell-surface antigens. Co-crystallization of the parental rabbit mAb in complex with the human ROR2 kringle domain (hROR2-Kr) guided affinity maturation by heavy-chain complementarity-determining region 3 (HCDR3)-focused mutagenesis and selection. The affinity-matured rabbit mAb was then humanized by complementarity-determining region (CDR) grafting and framework fine tuning and again co-crystallized with hROR2-Kr. We show that the affinity-matured and humanized mAb retains strong affinity and specificity to ROR2 and, following conversion to a T cell-engaging bispecific antibody, has potent cytotoxicity toward ROR2-expressing cells. We anticipate that this humanized affinity-matured mAb will find application for antibody-based cancer therapy of ROR2-expressing neoplasms.

Potent and selective antitumor activity of a T cell-engaging bispecific antibody targeting a membrane-proximal epitope of ROR1.

T cell-engaging bispecific antibodies (biAbs) present a promising strategy for cancer immunotherapy, and numerous bispecific formats have been developed for retargeting cytolytic T cells toward tumor cells. To explore the therapeutic utility of T cell-engaging biAbs targeting the receptor tyrosine kinase ROR1, which is expressed by tumor cells of various hematologic and solid malignancies, we used a bispecific ROR1 × CD3 scFv-Fc format based on a heterodimeric and aglycosylated Fc domain designed for extended circulatory t1/2 and diminished systemic T cell activation. A diverse panel of ROR1-targeting scFv derived from immune and naïve rabbit antibody repertoires was compared in this bispecific format for target-dependent T cell recruitment and activation. An ROR1-targeting scFv with a membrane-proximal epitope, R11, revealed potent and selective antitumor activity in vitro, in vivo, and ex vivo and emerged as a prime candidate for further preclinical and clinical studies. To elucidate the precise location and engagement of this membrane-proximal epitope, which is conserved between human and mouse ROR1, the 3D structure of scFv R11 in complex with the kringle domain of ROR1 was determined by X-ray crystallography at 1.6-Å resolution.

Chimeric Antigen Receptor Library Screening Using a Novel NF-κB/NFAT Reporter Cell Platform.

Chimeric antigen receptor (CAR)-T cell immunotherapy is under intense preclinical and clinical investigation, and it involves a rapidly increasing portfolio of novel target antigens and CAR designs. We established a platform that enables rapid and high-throughput CAR-screening campaigns with reporter cells derived from the T cell lymphoma line Jurkat. Reporter cells were equipped with nuclear factor κB (NF-κB) and nuclear factor of activated T cells (NFAT) reporter genes that generate a duplex output of enhanced CFP (ECFP) and EGFP, respectively. As a proof of concept, we modified reporter cells with CD19-specific and ROR1-specific CARs, and we detected high-level reporter signals that allowed distinguishing functional from non-functional CAR constructs. The reporter data were highly reproducible, and the time required for completing each testing campaign was substantially shorter with reporter cells (6 days) compared to primary CAR-T cells (21 days). We challenged the reporter platform to a large-scale screening campaign on a ROR1-CAR library, and we showed that reporter cells retrieved a functional CAR variant that was present with a frequency of only 6 in 1.05 × 106. The data illustrate the potential to implement this reporter platform into the preclinical development path of novel CAR-T cell products and to inform and accelerate the selection of lead CAR candidates for clinical translation.

Chemically Programmable and Switchable CAR-T Therapy.

Although macromolecules on cell surfaces are predominantly targeted and drugged with antibodies, they harbor pockets that are only accessible to small molecules and constitutes a rich subset of binding sites with immense potential diagnostic and therapeutic utility. Compared to antibodies, however, small molecules are disadvantaged by a less confined biodistribution, shorter circulatory half-life, and inability to communicate with the immune system. Presented herein is a method that endows small molecules with the ability to recruit and activate chimeric antigen receptor T cells (CAR-Ts). It is based on a CAR-T platform that uses a chemically programmed antibody fragment (cp-Fab) as on/off switch. In proof-of-concept studies, this cp-Fab/CAR-T system targeting folate binding proteins on the cell surface mediated potent and specific eradication of folate-receptor-expressing cancer cells in vitro and in vivo.

Targeting Stereotyped B Cell Receptors from Chronic Lymphocytic Leukemia Patients with Synthetic Antigen Surrogates.

Chronic lymphocytic leukemia (CLL) is a disease in which a single B-cell clone proliferates relentlessly in peripheral lymphoid organs, bone marrow, and blood. DNA sequencing experiments have shown that about 30% of CLL patients have stereotyped antigen-specific B-cell receptors (BCRs) with a high level of sequence homology in the variable domains of the heavy and light chains. These include many of the most aggressive cases that haveIGHV-unmutated BCRs whose sequences have not diverged significantly from the germ line. This suggests a personalized therapy strategy in which a toxin or immune effector function is delivered selectively to the pathogenic B-cells but not to healthy B-cells. To execute this strategy, serum-stable, drug-like compounds able to target the antigen-binding sites of most or all patients in a stereotyped subset are required. We demonstrate here the feasibility of this approach with the discovery of selective, high affinity ligands for CLL BCRs of the aggressive, stereotyped subset 7P that cross-react with the BCRs of several CLL patients in subset 7p, but not with BCRs from patients outside this subset.

Tsc1 deficiency-mediated mTOR hyperactivation in vascular endothelial cells causes angiogenesis defects and embryonic lethality.

This is a study on the role of tuberous sclerosis complex1 (TSC1) mutation and mTOR activation in endothelial cells during angiogenic and embryonic development. Past studies had shown that Tsc1/Tsc2 mutant genes lead to overactivation of mTOR in the regulating pathways in developing fetus. We used conditional Cre-loxp gene knockout approach to delete Tsc1 in mice's endothelial cells in our experimental models. Similarly, activation of mTOR signaling in endothelial cells of these embryos (Tie2-Cre/Tsc1(-/-)) was found. Majority of Tie2-Cre/Tsc1(-/-) embryos died at embryonic day 14.5 in utero. Cardiovascular defects, subcutaneous edema and hemorrhage were present among them. Whole-mount immunostaining in these embryos revealed a disorganized vascular network, defective sprouting of vessels in yolk sac and thickening of the labyrinth layer in the placenta. A thinner ventricular wall with disorganized trabeculae was present in the hearts of Tie2-Cre/Tsc1(-/-) embryos. Endothelial cells in Tsc1-deficient mice showed defective mitochondrial and endoplasmic reticular morphology, but no significant change was observed in cell junctions. The mutant embryos displayed significantly reduced cell proliferation, increased apoptosis and disturbed expression of angiogenic factors. A cohort of mice was treated prenatally with mTOR inhibitor rapamycin. The offspring of these mutant mice survived up to 22 days after birth. It was concluded that physiological TSC1-mTOR signaling in endothelial cells is crucial for vascular development and embryogenesis. We postulated that disruption of normal angiogenic pathways through hyperactive mTOR signaling maybe the mechanism that lead to deranged vascular pathogenesis in the tuberous sclerosis complex.

mTORC1 Up-Regulates GP73 to Promote Proliferation and Migration of Hepatocellular Carcinoma Cells and Growth of Xenograft Tumors in Mice.

BACKGROUND & AIMS: Levels of the Golgi protein 73 (GP73) increase during development of hepatocellular carcinoma (HCC); GP73 is a serum marker for HCC. However, little is known about the mechanisms or effects of GP73 during hepatic carcinogenesis. METHODS: GP73 was overexpressed from a retroviral vector in HepG2 cells, which were analyzed in proliferation and migration assays. Xenograft tumors were grown from these cells in nude mice. The effects of monoclonal antibodies against GP73 were studied in mice and cell lines. GP73(-/-), GP73(+/-), and GP73(+/+) mice were given injections of diethylnitrosamine to induce liver injury. Levels of GP73 were reduced in MHCC97H, HCCLM3, and HepG2.215 cell lines using small hairpin RNAs; xenograft tumors were grown in mice from MHCC97H-small hairpin GP73 or MHCC97H-vector cells. We used microarray analysis to compare expression patterns between GP73-knockdown and control MHCC97H cells. We studied the effects of the mechanistic target of rapamycin (mTOR) inhibitor rapamycin on GP73 expression in different cancer cell lines and on growth of tumors in mice. Levels of GP73 and activated mTOR were quantified in human HCC tissues. RESULTS: Xenograft tumors grown from HepG2 cells that expressed GP73 formed more rapidly and more metastases than control HepG2 cells in mice. A monoclonal antibody against GP73 reduced proliferation of HepG2 cells and growth of xenograft tumors in mice. GP73(-/-) mice had less liver damage after administration of diethylnitrosamine than GP73(+/-) or GP73(+/+) mice. In phosphatase and tensin homolog-null mouse embryonic fibroblasts with constitutively activated mTOR, GP73 was up-regulated compared with control mouse embryonic fibroblasts; this increase was reversed after incubation with rapamycin. Expression of GP73 also was reduced in HCC and other cancer cell lines incubated with rapamycin. mTORC1 appeared to regulate expression of GP73 in cell lines. Activated mTOR correlated with the level of GP73 in human HCC tissues. Injection of rapamycin slowed the growth of xenograft tumors from MHCC97H-vector cells, compared with MHCC97H-short hairpin GP73 cells. CONCLUSIONS: Increased expression of GP73 promotes proliferation and migration of HCC cell lines and growth of xenograft tumors in mice. mTORC1 regulates the expression of GP73, so GP73 up-regulation can be blocked with rapamycin. mTOR inhibitors or other reagents that reduce the level or activity of GP73 might be developed for the treatment of HCC.

PDK4 protein promotes tumorigenesis through activation of cAMP-response element-binding protein (CREB)-Ras homolog enriched in brain (RHEB)-mTORC1 signaling cascade.

Mechanistic target of rapamycin (mTOR) integrates multiple extracellular and intracellular signals to regulate cell growth and survival. Hyperactivation of mTOR has been observed in various cancers. Regulation of mTOR activity is thus of importance in physiological processes and tumor development. Here, we present pyruvate dehydrogenase kinase 4 (PDK4) as a novel regulator of mTORC1 signaling. mTORC1 activity was augmented with PDK4 overexpression and reduced by PDK4 suppression in various cell lines. Furthermore, PDK4 bound to cAMP-response element-binding protein (CREB) and prevented its degradation. The enhanced CREB consequently transactivated the expression of Ras homolog enriched in brain (RHEB), a direct key activator of mTORC1, independent of AMP-activated protein kinase or tuberous sclerosis complex protein 2. PDK4 potentiated the mTORC1 effectors hypoxia-inducible factor 1α and pyruvate kinase isozymes M2 and promoted aerobic glycolysis (Warburg effect). Knockdown of PDK4 suppressed the tumor development of cancer cells with activated mTORC1. The abundance of PDK4 dictated the responsiveness of cells to the mTOR inhibitor, rapamycin. Combinatory suppression of mTOR and PDK4 exerted synergistic inhibition on cancer cell proliferation. Therefore, PDK4 promotes tumorigenesis through activation of the CREB-RHEB-mTORC1 signaling cascade.

Mammalian target of rapamycin up-regulation of pyruvate kinase isoenzyme type M2 is critical for aerobic glycolysis and tumor growth.

Although aerobic glycolysis (the Warburg effect) is a hallmark of cancer, key questions, including when, how, and why cancer cells become highly glycolytic, remain less clear. For a largely unknown regulatory mechanism, a rate-limiting glycolytic enzyme pyruvate kinase M2 (PKM2) isoform is exclusively expressed in embryonic, proliferating, and tumor cells, and plays an essential role in tumor metabolism and growth. Because the receptor tyrosine kinase/PI3K/AKT/mammalian target of rapamycin (RTK/PI3K/AKT/mTOR) signaling cascade is a frequently altered pathway in cancer, we explored its potential role in cancer metabolism. We identified mTOR as a central activator of the Warburg effect by inducing PKM2 and other glycolytic enzymes under normoxic conditions. PKM2 level was augmented in mouse kidney tumors due to deficiency of tuberous sclerosis complex 2 and consequent mTOR activation, and was reduced in human cancer cells by mTOR suppression. mTOR up-regulation of PKM2 expression was through hypoxia-inducible factor 1α (HIF1α)-mediated transcription activation, and c-Myc-heterogeneous nuclear ribonucleoproteins (hnRNPs)-dependent regulation of PKM2 gene splicing. Disruption of PKM2 suppressed oncogenic mTOR-mediated tumorigenesis. Unlike normal cells, mTOR hyperactive cells were more sensitive to inhibition of mTOR or glycolysis. Dual suppression of mTOR and glycolysis synergistically blunted the proliferation and tumor development of mTOR hyperactive cells. Even though aerobic glycolysis is not required for breach of senescence for immortalization and transformation, the frequently deregulated mTOR signaling during multistep oncogenic processes could contribute to the development of the Warburg effect in many cancers. Components of the mTOR/HIF1α/Myc-hnRNPs/PKM2 glycolysis signaling network could be targeted for the treatment of cancer caused by an aberrant RTK/PI3K/AKT/mTOR signaling pathway.

In vivo affinity maturation of mouse B cells reprogrammed to express human antibodies.

Mice adoptively transferred with mouse B cells edited via CRISPR to express human antibody variable chains could help evaluate candidate vaccines and develop better antibody therapies. However, current editing strategies disrupt the heavy-chain locus, resulting in inefficient somatic hypermutation without functional affinity maturation. Here we show that these key B-cell functions can be preserved by directly and simultaneously replacing recombined mouse heavy and kappa chains with those of human antibodies, using a single Cas12a-mediated cut at each locus and 5' homology arms complementary to distal V segments. Cells edited in this way to express the human immunodeficiency virus type 1 (HIV-1) broadly neutralizing antibody 10-1074 or VRC26.25-y robustly hypermutated and generated potent neutralizing plasma in vaccinated mice. The 10-1074 variants isolated from the mice neutralized a global panel of HIV-1 isolates more efficiently than wild-type 10-1074 while maintaining its low polyreactivity and long half-life. We also used the approach to improve the potency of anti-SARS-CoV-2 antibodies against recent Omicron strains. In vivo affinity maturation of B cells edited at their native loci may facilitate the development of broad, potent and bioavailable antibodies.

Reciprocal activation between PLK1 and Stat3 contributes to survival and proliferation of esophageal cancer cells.

BACKGROUND & AIMS: Aberrant activation of the signal transducer and activator of transcription (Stat)3 and overexpression of polo-like kinase (PLK)1 each have been associated with cancer pathogenesis. The mechanisms and significance of dysregulation of Stat3 and PLK1 in carcinogenesis and cancer progression are unclear. We investigated the relationship between Stat3 and PLK1 and the effects of their dysregulation in esophageal squamous cell carcinoma (ESCC) cells. METHODS: We used immunoblot, quantitative reverse-transcription polymerase chain reaction, immunochemistry, chromatin immunoprecipitation, mobility shift, and reporter assays to investigate the relationship between Stat3 and PLK1. We used colony formation, fluorescence-activated cell sorting, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, and xenograft tumor assays to determine the effects of increased activation of Stat3 and PLK1 in proliferation and survival of ESCC cells. RESULTS: Stat3 directly activated transcription of PLK1 in esophageal cancer cells and mouse embryonic fibroblast cell NIH3T3. PLK1 then potentiated the expression of Stat3; ß-catenin was involved in PLK1-dependent transcriptional activation of Stat3. This mutual regulation between Stat3 and PLK1 was required for proliferation of esophageal cancer cells and resistance to apoptosis in culture and as tumor xenografts in mice. Furthermore, phosphorylation of Stat3 and overexpression of PLK1 were correlated in a subset of ESCC. CONCLUSIONS: Stat3 and PLK1 control each other's transcription in a positive feedback loop that contributes to the development of ESCC. Increased activity of Stat3 and overexpression of PLK1 promote survival and proliferation of ESCC cells in culture and in mice.

A mammalian cell display platform based on scFab transposition.

In vitro display technologies have been successfully utilized for the discovery and evolution of monoclonal antibodies (mAbs) for diagnostic and therapeutic applications, with phage display and yeast display being the most commonly used platforms due to their simplicity and high efficiency. As their prokaryotic or lower eukaryotic host organisms typically have no or different post-translational modifications, several mammalian cell-based display and screening technologies for isolation and optimization of mAbs have emerged and are being developed. We report here a novel and useful mammalian cell display platform based on the PiggyBac transposon system to display mAbs in a single-chain Fab (scFab) format on the surface of HEK293F cells. Immune rabbit antibody libraries encompassing ~7 × 107 independent clones were generated in an all-in-one transposon vector, stably delivered into HEK293F cells and displayed as an scFab with rabbit variable and human constant domains. After one round of magnetic activated cell sorting and two rounds of fluorescence activated cell sorting, mAbs with high affinity in the subnanomolar range and cross-reactivity to the corresponding human and mouse antigens were identified, demonstrating the power of this platform for antibody discovery. We developed a highly efficient mammalian cell display platform based on the PiggyBac transposon system for antibody discovery, which could be further utilized for humanization as well as affinity and specificity maturation.

In vivo affinity maturation of murine B cells reprogrammed to express human antibodies.

CRISPR-edited murine B cells engineered to express human antibody variable chains proliferate, class switch, and secrete these antibodies in vaccinated mice. However, current strategies disrupt the heavy-chain locus, resulting in inefficient somatic hypermutation without functional affinity maturation. Here we show that recombined murine heavy- and kappa-variable genes can be directly and simultaneously overwritten, using Cas12a-mediated cuts at their 3'-most J segments and 5' homology arms complementary to distal V segments. Cells edited in this way to express the HIV-1 broadly neutralizing antibodies 10-1074 or VRC26.25-y robustly hypermutated and generated potent neutralizing plasma in vaccinated recipient mice. 10-1074 variants isolated from these mice bound and neutralized HIV-1 envelope glycoprotein more efficiently than wild-type 10-1074 while maintaining or improving its already low polyreactivity and long in vivo half-life. We further validated this approach by generating substantially broader and more potent variants of the anti-SARS-CoV-2 antibodies ZCB11 and S309. Thus, B cells edited at their native loci affinity mature, facilitating development of broad, potent, and bioavailable antibodies and expanding the potential applications of engineered B cells.

Concerted Antibody and Antigen Discovery by Differential Whole-cell Phage Display Selections and Multi-omic Target Deconvolution.

Monoclonal antibody (mAb)-based biologics are well established treatments of cancer. Antibody discovery campaigns are typically directed at a single target of interest, which inherently limits the possibility of uncovering novel antibody specificities or functionalities. Here, we present a target-unbiased approach for antibody discovery that relies on generating mAbs against native target cell surfaces via phage display. This method combines a previously reported method for improved whole-cell phage display selections with next-generation sequencing analysis to efficiently identify mAbs with the desired target cell reactivity. Applying this method to multiple myeloma cells yielded a panel of >50 mAbs with unique sequences and diverse reactivities. To uncover the identities of the cognate antigens recognized by this panel, representative mAbs from each unique reactivity cluster were used in a multi-omic target deconvolution approach. From this, we identified and validated three cell surface antigens: PTPRG, ICAM1, and CADM1. PTPRG and CADM1 remain largely unstudied in the context of multiple myeloma, which could warrant further investigation into their potential as therapeutic targets. These results highlight the utility of optimized whole-cell phage display selection methods and could motivate further interest in target-unbiased antibody discovery workflows.

ROR1-targeting switchable CAR-T cells for cancer therapy.

The success of chimeric antigen receptor T cell (CAR-T) therapy in the treatment of hematologic malignancies has prompted the development of numerous CAR-T technologies, including switchable CAR-T (sCAR-T) systems that combine a universal CAR-T with bispecific adapter proteins. Owing to their controllability and versatility, sCAR-Ts have received considerable attention. To explore the therapeutic utility of sCAR-Ts targeting the receptor tyrosine kinase ROR1, which is expressed in hematologic and solid malignancies, and to identify bispecific adaptor proteins that efficiently mediate universal CAR-T engagement, a panel of switches based on ROR1-targeting Fabs with different epitopes and affinities was compared in in vitro and in vivo models of ROR1-expressing cancers. For switches targeting overlapping or identical epitopes, potency correlated with affinity. Surprisingly, however, we identified a switch targeting a unique epitope with low affinity but mediating potent and selective antitumor activity in vitro and in vivo. Converted to a conventional CAR-T, the same anti-ROR1 mAb (324) outperformed a clinically investigated conventional CAR-T that is based on an anti-ROR1 mAb (R12) with ~200-fold higher affinity. Thus, demonstrating therapeutic utility on their own, sCAR-Ts also facilitate higher throughput screening for the identification of conventional CAR-T candidates for preclinical and clinical studies.

A Clinical Assessment of a Magnetic Resonance Computer-Aided Diagnosis System in the Detection of Pathological Complete Response After Neoadjuvant Chemotherapy in Breast Cancer.

Purpose: This study aimed to assess the diagnostic performance and the added value to radiologists of different levels of a computer-aided diagnosis (CAD) system for the detection of pathological complete response (pCR) after neoadjuvant chemotherapy (NAC) in patients with breast cancer. Besides, to investigate whether tumor molecular typing is associated with the efficiency of diagnosis of the CAD systems. Methods: 470 patients were identified with breast cancers who underwent NAC and post MR imaging between January 2016 and March 2019. The diagnostic performance of radiologists of different levels and the CAD system were compared. The added value of the CAD system was assessed and subgroup analyses were performed according to the tumor molecular typing. Results: Among 470 patients, 123 (26%) underwent pCR. The CAD system showed a comparable specificity as the senior radiologist (83.29% vs. 84.15%, p=0.488) and comparable area under the curve (AUC) (0.839 vs. 0.835, p =0.452). The performance of all radiologists significantly improved when aided by the CAD system (P<0.05), And there were no statistical differences in terms of sensitivity, specificity and accuracy between the two groups with CAD assistance(p>0.05).The AUC values for identifying pCR in TN patients were significant (0.883, 95%CI: 0.801-0.964, p < 0.001). Conclusion: The CAD system assessed in this study improves the performance of all radiologists, regardless of experience. The molecular typing of breast cancer is potential influencer of CAD diagnostic performance.