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The compatibility of elastomers used in engine fuel system with Fischer–Tropsch (FT) fuel is essential, but there is very limited research available on it. In this work, the compatibility between four types of commonly used elastomers in fuel system and FT fuel was experimentally investigated. The assessed elastomers including nitrile butadiene rubber (NBR), ethylene propylene diene-monomer rubber (EPDM), fluoroelastomer (FKM), and chlorinated polyethylene (CPE) were exposed to FT fuel which derived from natural gas for 21 days. The properties of the elastomers such as mass, volume, hardness, tensile strength, elongation at break, and tear strength were measured before and after immersion, at the same time the change rates of these properties were calculated. The results demonstrated that all properties of EPDM deteriorated significantly after soak, and its compatibility with FT fuel was the worst, making it unsuitable for FT engines. NBR, FKM, and CPE all showed low rates of property change, with FKM exhibiting the greatest compatibility performance with FT fuel. Additionally, the O-rings made of FKM were submerged in FT fuel and diesel for 7 days, 14 days, and 21 days, respectively. It was found that the mass and volume variations of FKM O-rings in FT fuel were negligible and comparable with those of diesel fuel.

Background 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. Methods We report here a novel and useful mammalian cell display platform based on the PiggyBac transposon system to display mAbs in single chain Fab (scFab) format on the surface of HEK293F cells. Results 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 scFab with rabbit variable and human constant domains. After one round of magnetic activated cell sorting (MACS) and two rounds of fluorescence activated cell sorting (FACS), 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. Conclusions 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.

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.

Antibody–drug conjugates (ADCs) are targeted therapeutics generated by conjugation of cytotoxic small molecules to monoclonal antibodies (mAbs) via chemical linkers. Due to their selective delivery of toxic payloads to antigen-positive cancer cells, ADCs demonstrate wider therapeutic indexes compared with conventional chemotherapy. After decades of intensive research and development, significant advances have been made in the field, leading to a total of 10 U.S. food and drug administration (FDA)-approved ADCs to treat cancer patients. Currently, ~80 ADCs targeting different antigens are under clinical evaluation for treatment of either hematological or solid malignancies. Notably, three ADCs targeting the same oncofetal protein, receptor tyrosine kinase like orphan receptor 1 (ROR1), have attracted considerable attention when they were acquired or licensed successively in the fourth quarter of 2020 by three major pharmaceutical companies. Apparently, ROR1 has emerged as an attractive target for cancer therapy. Since all the components of ADCs, including the antibody, linker and payload, as well as the conjugation method, play critical roles in ADC’s efficacy and performance, their choice and combination will determine how far they can be advanced. This review summarizes the design and development of current anti-ROR1 ADCs and highlights an emerging trend to target ROR1 for cancer therapy.

BackgroundDespite numerous therapeutic options, safe and curative therapy is unavailable for most patients with chronic lymphocytic leukemia (CLL). A drawback of current therapies such as the anti-CD20 monoclonal antibody (mAb) rituximab is the elimination of all healthy B cells, resulting in impaired humoral immunity. We previously reported the identification of a patient-derived, CLL-binding mAb, JML-1, and identified sialic acid-binding immunoglobulin-like lectin-6 (Siglec-6) as the target of JML-1. Although little is known about Siglec-6, it appears to be an attractive target for cancer immunotherapy due to its absence on most healthy cells and tissues.MethodsWe used a target-specific approach to mine for additional patient-derived anti-Siglec-6 mAbs. To assess the therapeutic utility of targeting Siglec-6 in the context of CLL, T cell-recruiting bispecific antibodies (T-biAbs) that bind to Siglec-6 and CD3 were engineered into single-chain variable fragment–Fc and dual-affinity retargeting (DART)–Fc constructs. T-biAbs were evaluated for their activity in vitro, ex vivo, and in vivo.ResultsWe discovered the anti-Siglec-6 mAbs RC-1 and RC-2, which bind with higher affinity than JML-1 yet maintain similar specificity. Both JML-1 and RC-1 T-biAbs were effective at activating T cells and killing Siglec-6+target cells. The RC-1 clone in the DART–Fc format was the most potent T-biAb tested and was the only anti-Siglec-6 T-biAb that eliminated Siglec-6+primary CLL cells via autologous T cells at pathological T-to-CLL cell ratios. Tested at healthy T-to-B cell ratios, it also eliminated a Siglec-6+fraction of primary B cells from healthy donors. The subpicomolar potency of the DART–Fc format was attributed to the reduction in the length and flexibility of the cytolytic synapse. Furthermore, the RC-1 T-biAb was effective at clearing MEC1 CLL cells in vivo and demonstrated a circulatory half-life of over 7 days.ConclusionSiglec-6-targeting T-biAbs are highly potent and specific for eliminating Siglec-6+leukemic and healthy B cells while sparing Siglec-6−healthy B cells, suggesting a unique treatment strategy for CLL with diminished suppression of humoral immunity. Our data corroborate reports that T-biAb efficacy is dependent on synapse geometry and reveal that synapse architecture can be tuned via antibody engineering. Our fully human anti-Siglec-6 antibodies and T-biAbs have potential for cancer immunotherapy.Trial registration numberNCT00923507.

Objective Lymphovascular invasion (LVI) is an independent risk factor of gastric cancer (GC) prognosis; however, LVI cannot be determined preoperatively. We explored whether a model based on contrast-enhanced computed tomography (CECT) radiomics features combined with clinicopathological factors can evaluate preoperative LVI in patients with GC with a clear Lauren classification. Methods We retrospectively analyzed 495 patients with GC, including 288 with LVI. The extracted CECT features were standardized, followed by consistency testing, correlation analysis, univariate analysis, and multivariate least absolute shrinkage and selection operator (LASSO) regression analysis. The radiomics score (Radcore) was calculated for each patient. Univariate (p

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.

The mechanism of the hump formation during the laser wire feeding process has been investigated in this paper. A numerical model was established to analyze the heat transfer and melt flow of molten pool during process with or without hump formation. And effects of laser power, traverse speed, and wire feeding speed on hump formation were also investigated through such a numerical model. The results showed that, under the condition with high wire feeding speed, the molten metal flowed rapidly to the tail of the molten pool due to the impact force of droplet and the metal vapor recoil pressure. Simultaneously, due to the high speed of laser movement, the thin channel of the trough solidified prematurely under the effect of surface tension, which isolated the heat transfer of the molten pool, and then formed periodic humps. By comparative analysis, the numerical simulation results were in good agreement with the experimental results.

In recent years, dedicated hardware accelerators for the acceleration of the convolutional neural network (CNN) have been extensively studied. Although many studies have presented efficient designs on FPGAs for image classification neural network models such as AlexNet and VGG, there are still little implementations for CNN-based object detection applications. This paper presents an OpenCL-based high-throughput FPGA accelerator for the YOLOv2 object detection algorithm on Arria-10 GX1150 FPGA. The proposed hardware architecture adopts a scalable pipeline design to support multi-resolution input image and full 8-bit fixed-point datapath to improve hardware resource utilization. Layer fusion technology that merges the convolution, batch normalization and Leaky-ReLU is also developed to avoid transmission of intermediate data between FPGA and external memory. Experimental results show that the final design achieves a peak throughput of 566 GOP/s under the working frequency of 190 MHz. The accelerator can execute YOLOv2 inference computation ( $$288\times 288$$ resolution) and tiny YOLOv2 ( $$416\times 416$$ resolution) at the speed of 35 and 71 FPS, respectively.

BackgroundThere is a demand for additional alternative methods that can allow the differentiation of the breast tumor into molecular subtypes precisely and conveniently.PurposeThe present study aimed to determine suitable optimal classifiers and investigate the general applicability of computer-aided diagnosis (CAD) to associate between the breast cancer molecular subtype and the extracted MR imaging features.MethodsWe analyzed a total of 264 patients (mean age: 47.9 ± 9.7 years; range: 19–81 years) with 264 masses (mean size: 28.6 ± 15.86 mm; range: 5–91 mm) using a Unet model and Gradient Tree Boosting for segmentation and classification.ResultsThe tumors were segmented clearly by the Unet model automatically. All the extracted features which including the shape features,the texture features of the tumors and the clinical features were input into the classifiers for classification, and the results showed that the GTB classifier is superior to other classifiers, which achieved F1-Score 0.72, AUC 0.81 and score 0.71. Analyzed the different features combinations, we founded that the texture features associated with the clinical features are the optimal features to different the breast cancer subtypes.ConclusionCAD is feasible to differentiate the breast cancer subtypes, automatical segmentation were feasible by Unet model and the extracted texture features from breast MR imaging with the clinical features can be used to help differentiating the molecular subtype. Moreover, in the clinical features, BPE and age characteristics have the best potential for subtype.

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., Author summary The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), like the closely related virus SARS-CoV (SARS-CoV-1), infects cells by interacting with the cellular receptor angiotensin-converting enzyme 2 (ACE2). This interaction is mediated by the viral spike (S) protein through an independently folded subdomain, described as its receptor-binding domain (RBD). The susceptibility of a species to SARS-CoV-1 or -2 infection correlates with the binding affinity of their respective RBD for the ACE2 orthologs of that species. We therefore investigated the binding affinity of the RBD regions of multiple SARS-like coronaviruses with a range of ACE2 orthologs. Our results are consistent with the hypothesis that pangolins serve as an intermediate between humans and horseshoe bats. We further observed a high level of variability in the ability of the SARS-CoV-2 RBD to bind horseshoe bat ACE2 orthologs, suggesting ongoing selection pressure on their receptor ACE2 proteins from SARS-like viruses. Indeed, mutations derived from different horseshoe bat orthologs introduced into a soluble form of human ACE2 differentially impacted SARS-CoV-1 and SARS-CoV-2 S-protein-mediated infection. A combination of five residues present in multiple horseshoe bats increased the ability of a soluble form of ACE2 to neutralize SARS-CoV-2 S-protein-mediated infection. Thus horseshoe bats ACE2 orthologs can provide insight useful to improving the potency of ACE2-based therapeutics.

Phage display of combinatorial antibody libraries is a versatile tool in the field of antibody engineering, with diverse applications including monoclonal antibody (mAb) discovery, affinity maturation, and humanization. To improve the selection efficiency of antibody libraries, we developed a new phagemid display system that addresses the complication of bald phage propagation. The phagemid facilitates the biotinylation of fragment of antigen binding (Fab) antibody fragments displayed on phage via Sortase A catalysis and the subsequent enrichment of Fab-displaying phage during selections. In multiple contexts, this selection approach improved the enrichment of target-reactive mAbs by depleting background phage. Panels of cancer cell line-reactive mAbs with high diversity and specificity were isolated from a naïve chimeric rabbit/human Fab library using this approach, highlighting its potential to accelerate antibody engineering efforts and to empower concerted antibody drug and target discovery.

Although the 5-year survival rate of chronic lymphocytic leukemia (CLL) patients has risen to >80%, the only potentially curative treatment is allogeneic hematopoietic stem cell transplantation (alloHSCT). To identify possible new monoclonal antibody (mAb) drugs and targets for CLL, we previously developed a phage display–based human mAb platform to mine the antibody repertoire of patients who responded to alloHSCT. We had selected a group of highly homologous post-alloHSCT mAbs that bound to an unknown CLL cell surface antigen. Here, we show through next-generation sequencing of cDNAs encoding variable heavy-chain domains that these mAbs had a relative abundance of ∼0.1% in the post-alloHSCT antibody repertoire and were enriched ∼1,000-fold after three rounds of selection on primary CLL cells. Based on differential RNA-seq and a cell microarray screening technology for discovering human cell surface antigens, we now identify their antigen as Siglec-6. We verified this finding by flow cytometry, ELISA, siRNA knockdown, and surface plasmon resonance. Siglec-6 was broadly expressed in CLL and could be a potential target for antibody-based therapeutic interventions. Our study reaffirms the utility of post-alloHSCT antibody drug and target discovery. Cancer Immunol Res; 6(9); 1008–13. ©2018 AACR.

SARS-CoV-2 variants with spike (S)-protein D614G mutations now predominate globally. We therefore compare the properties of the mutated S protein (SG614) with the original (SD614). We report here pseudoviruses carrying SG614 enter ACE2-expressing cells more efficiently than those with SD614. This increased entry correlates with less S1-domain shedding and higher S-protein incorporation into the virion. Similar results are obtained with virus-like particles produced with SARS-CoV-2 M, N, E, and S proteins. However, D614G does not alter S-protein binding to ACE2 or neutralization sensitivity of pseudoviruses. Thus, D614G may increase infectivity by assembling more functional S protein into the virion., SARS-CoV-2 variants with spike (S)-protein D614G mutations currently predominate globally. Here, Zhang et al. hypothesize that D614G variant may increase infectivity by increasing S protein abundance on the virion since pseudoviruses carrying S-G614 incorporate higher amounts of S protein and enter cells more efficiently than those carrying S-D614.

Antibody-drug conjugates directed against tumor-specific targets have allowed targeted delivery of highly potent chemotherapy to malignant cells while sparing normal cells. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal protein with limited expression on normal adult tissues and is overexpressed on the surface of malignant cells in mantle cell lymphoma, acute lymphocytic leukemia with t(1;19)(q23;p13) translocation, and chronic lymphocytic leukemia. This differential expression makes ROR1 an attractive target for antibody-drug conjugate therapy, especially in malignancies such as mantle cell lymphoma and acute lymphocytic leukemia, in which systemic chemotherapy remains the gold standard. Several preclinical and phase 1 clinical studies have established the safety and effectiveness of anti-ROR1 monoclonal antibody–based therapies. Herein we describe a humanized, first-in-class anti-ROR1 antibody-drug conjugate, huXBR1-402-G5-PNU, which links a novel anti-ROR1 antibody (huXBR1-402) to a highly potent anthracycline derivative (PNU). We found that huXBR1-402-G5-PNU is cytotoxic to proliferating ROR1+ malignant cells in vitro and suppressed leukemia proliferation and extended survival in multiple models of mice engrafted with human ROR1+ leukemia. Lastly, we show that the B-cell lymphoma 2 (BCL2)-dependent cytotoxicity of huXBR1-402-G5-PNU can be leveraged by combined treatment strategies with the BCL2 inhibitor venetoclax. Together, our data present compelling preclinical evidence for the efficacy of huXBR1-402-G5-PNU in treating ROR1+ hematologic malignancies.

SUMMARYThe 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 SARS-CoV-2, has been isolated from one horseshoe-bat species. Here we characterize the ability of S-protein receptor-binding domains (RBDs) of SARS-CoV-1, SARS-CoV-2, and RaTG13 to bind a range of ACE2 orthologs. We observed that the SARS-CoV-2 RBD bound human, pangolin, and horseshoe bat (R. macrotis) ACE2 more efficiently than the SARS-CoV-1 or RaTG13 RBD. Only the RaTG13 RBD bound rodent ACE2 orthologs efficiently. Five mutations drawn from ACE2 orthologs of nine Rhinolophus species enhanced human ACE2 binding to the SARS-CoV-2 RBD and neutralization of SARS-CoV-2 by an immunoadhesin form of human ACE2 (ACE2-Fc). Two of these mutations impaired neutralization of SARS-CoV-1. An ACE2-Fc variant bearing all five mutations neutralized SARS-CoV-2 five-fold more efficiently than human ACE2-Fc. These data narrow the potential SARS-CoV-2 reservoir, suggest that SARS-CoV-1 and -2 originate from distinct bat species, and identify a more potently neutralizing form of ACE2-Fc.

Although the cell surfaceome is predominantly targeted and drugged with antibodies, its pocketome fraction is 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 their 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 sites in the cell surfaceome mediated potent and specific eradication of folate receptor-expressing cancer cells in vitro and in vivo. The method harnesses an increasing quantity and quality of cell surfaceome-targeting small molecules derived from virtual and actual small molecule libraries.

Metastatic medullary thyroid cancer (MTC) is a rare but often aggressive thyroid malignancy with a 5-year survival rate of less than 40% and few effective therapeutic options. Adoptive T cell immunotherapy using chimeric antigen receptor (CAR)-modified T cells (CAR Ts) is showing encouraging results in the treatment of cancer, but development is challenged by the availability of suitable target antigens. We identified glial-derived neurotrophic factor (GDNF) family receptor alpha 4 (GFRα4) as a putative antigen target for CAR-based therapy of MTC. We show that GFRα4 is highly expressed in MTC, in parafollicular cells within the thyroid from which MTC originates, and in normal thymus. We isolated two single-chain variable fragments (scFvs) targeting GFRα4 isoforms a and b by antibody phage display. CARs bearing the CD3ζ and the CD137 costimulatory domains were constructed using these GFRα4-specific scFvs. GFRα4-specific CAR Ts trigger antigen-dependent cytotoxicity and cytokine production in vitro, and they are able to eliminate tumors derived from the MTC TT cell line in an immunodeficient mouse xenograft model of MTC. These data demonstrate the feasibility of targeting GFRα4 by CAR T and support this antigen as a promising target for adoptive T cell immunotherapy and other antibody-based therapies for MTC., Graphical Abstract, Metastatic medullary thyroid carcinoma (MTC) is a rare but aggressive malignancy with no curative options. By sequencing mRNA from a patient’s tumor, Siegel and colleagues identified and characterized GFRα4 as a promising tumor target antigen and demonstrated that CAR T immunotherapy targeting this antigen controlled MTC in tumor xenograft models.

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.

Owing to their high affinities and specificities, rabbit monoclonal antibodies (mAbs) have demonstrated value and potential primarily as basic research and diagnostic reagents, but, in some cases, also as therapeutics. To accelerate access to rabbit mAbs bypassing immunization, we generated a large naive rabbit antibody repertoire represented by a phage display library encompassing > 10 billion independent antibodies in chimeric rabbit/human Fab format and validated it by next-generation sequencing. Panels of rabbit mAbs selected from this library against two emerging cancer targets, ROR1 and ROR2, revealed high diversity, affinity, and specificity. Moreover, ROR1- and ROR2-targeting rabbit mAbs demonstrated therapeutic utility as components of chimeric antigen receptor-engineered T cells, further corroborating the value of the naive rabbit antibody library as a rich and virtually unlimited source of rabbit mAbs.

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 kappa B (NF kappa B) 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 x 10(6). 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.

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 have IGHV-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.

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 naive 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-A resolution.

T-cell engaging bispecific antibodies 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 bispecific antibodies 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 half-life 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. A ROR1-targeting scFv with a membrane-proximal epitope, R11, revealed potent and selective antitumor activityin vitroandin vivoand 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 three-dimensional structure of scFv R11 in complex with the kringle domain of ROR1 was determined by X-ray crystallography at 1.6-Å resolution.

The exhaust gases contain large amounts of unburned hydrocarbons during cranking without combustion. The effects of unburned hydrocarbon recirculation on ignition and combustion during diesel engine cold starts were investigated using both experiments and simulations. Experiments were conducted on a single-cylinder DI (direct injection) diesel engine equipped with a common rail injection system. The amount of unburned hydrocarbon recirculation was jointly controlled by an EGR (exhaust gas recirculation) valve and a back pressure valve. The investigation showed that optimal opening of recirculation control valves allowed the first firing cycle to be advanced from 19 to 6 and reduced the duration of heavy smoke emission (opacity > 50%) by 77%. However, the enhancement to the in-cylinder LTR (low temperature reaction) decreased gradually as the amount of unburned hydrocarbon recirculation increased. An analysis of the chemical kinetics showed that the reaction intermediates present in unburned hydrocarbons, such as ketohydroperoxides, were the most significant factor in enhancing the LTR during non-firing cycles. At the same time, the substantial heat capacity of unburned hydrocarbons suppressed the LTR for higher recirculation rates.

Engine start/stop strategy is one of the most important methods for hybrid electric vehicles (HEVs) to achieve fuel economy and low emission. The engine of HEV would experience frequent start/stop during a normal drive. In this paper, an independent fuel injection strategy (IFIS) for port fuel injection (PFI) engine was proposed to realize a quick HEV hot start with low-emission. The purpose of the IFIS is to supply fuel injection independently for each cylinder according to their initial piston positions (IPPs) at the beginning of the cranking start. With independent injection timing and optimized fuel pulse width (FPW), appropriate mixtures can be produced for all cylinders shortly after the start of cranking. Thus, robust and stable combustion could be obtained. By introduction of the IFIS, not only HEVs could experience better startability and lower hydrocarbon (HC) emissions during hot re-starts, but also vehicles with conventional drivetrain would have start–stop capabilities. In this work, the mixture preparation characteristics based on four typical IPPs were studied at first. Then the IFIS was proposed and experimentally tested. The results of the IFIS tests are compared with the original fuel injection strategy (OFIS) of the engine control unit (ECU) in terms of the starting time, indicated mean effective pressure (IMEP) and engine-out hydrocarbon (EOHC). The results of the IFIS tests suggest that the IFIS is a very effective method to improve the HEV hot start. With IFIS, shorter starting time and lower HC emissions were both achieved.

In this review, we explain why and how rabbit monoclonal antibodies have become outstanding reagents for laboratory research and increasingly for diagnostic and therapeutic applications. Starting with the unique ontogeny of rabbit B cells that affords highly distinctive antibody repertoires rich in in vivo pruned binders of high diversity, affinity and specificity, we describe the generation of rabbit monoclonal antibodies by hybridoma technology, phage display and alternative methods, along with an account of successful humanization strategies.

Introduction: Receptor tyrosine kinase like orphan receptor 1 (ROR1) is expressed on the surface of multiple blood and solid tumors. There have been increased interest in ROR1 as a potential therapeutic target in ROR1 positive leukemia and lymphomas. In this study, we present a novel first-in-class anti-ROR1 monoclonal antibody drug conjugate (ADC) based on chimeric rabbit/human mAb “XBR1-402” or its humanized version “huXBR1-402”. Conjugated to a highly potent anthracycline derived toxin, PNU-159682 (PNU) via a non-cleavable peptide/amide linker, it shows both in vitro and in vivo efficacy at eliminating ROR1 positive chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and pre B cell acute lymphocytic leukemia (pre B cell ALL) cells. Results: We saw a decrease in viability for all ADC treated ROR1 positive leukemic cell lines. We saw a significant decrease in viability of the ROR1 positive pre B cell ALL cell lines 697 and Kasumi-2 when treated with XBR1-402-PNU (EC50 = 77 and 1 ng/mL) when compared to treatment with isotype-matched control Trastuzumab-PNU (Tras-PNU) (EC50 = 2987 and 5330 ng/mL). In in vitro models of MCL, we saw decreased viability for huXBR1-402-PNU treated ROR1 positive Jeko and Mino cell lines but not ROR1 negative Mec-1 cell line when compared to Tras-PNU. While we were unable to observe significant direct cytotoxicity in primary CLL cells, we show that PNU ADCs mediates antibody dependent cellular cytotoxicity and phagocytosis in vitro. We also show that PNU ADCs mediates a G2/M cell cycle arrest in affected cell lines and hypothesize that this is necessary for direct cytotoxicity. As ex vivo primary CLL cells are non-proliferative, we postulate that PNU would target actively dividing cells in proliferation centers (e.g. secondary lymphoid organs). To test this hypothesis, we set up in vivo studies to test with XBR1-402-PNU and huXBR1-402-PNU in disseminated and aggressive ROR1+ ALL and CLL models, respectively. In vivo studies on a murine 697 ALL model suggested XBR1-402-PNU treatment increased overall survival when compared to treatment with Tras-PNU control (Median survival of 32 and 23 days post implantation, p=0.021, n=6). In vivo CLL studies with engrafted murine human-ROR1 expressing TCL1 leukemic cells showed that huXBR1-402-PNU treatment (3 times/week for 1 week) both suppressed leukemia burden and increased overall survival when compared with Tras-PNU control (Median survival of 62 and 41 days post implantation, p=0.0028, n=4). Ongoing proliferation and cell cycling studies will confirm the mechanism of the in vivo cytotoxicity of huXBR1-402-PNU. Conclusion: Our results suggest that anti-ROR1 ADC huXBR1-402-PNU is an effective and promising targeted cytotoxic therapy for ROR1 positive leukemic cells of CLL, MCL, and pre B ALL and warrants further evaluation for clinical consideration as either a single agent or combination therapy in patients with ROR1 positive leukemia and lymphomas. Citation Format: Eileen Hu, Priscilla Do, Rajeswaran Mani, Frank Frissora, Rebecca Pearson, Gerard Lozanski, Haiyong Peng, Lorenz Waldmeier, Roger Beerli, Christoph Rader, Ulf Grawunder, John Byrd, Natarajan Muthusamy. Evaluation of ROR1 targeted antibody drug conjugate in ROR1 positive leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1541.

Hyperactivation of mechanistic target of rapamycin (MTOR) is a common feature of human cancers, and MTOR inhibitors, such as rapamycin, are thus becoming therapeutics in targeting certain cancers. However, rapamycin has also been found to compromise the efficacy of chemotherapeutics to cells with hyperactive MTOR. Here, we show that loss of TSC2 or PTEN enhanced etoposide-induced DNA damage and apoptosis, which was blunted by suppression of MTOR with either rapamycin or RNA interference. cAMP response element-binding protein 1 (CREB1), a nuclear transcription factor that regulates genes involved in survival and death, was positively regulated by MTOR in mouse embryonic fibroblasts (MEFs) and cancer cell lines. Silencing Creb1 expression with siRNA protected MTOR-hyperactive cells from DNA damage-induced apoptosis. Furthermore, loss of TSC2 or PTEN impaired either etoposide or nutrient starvation-induced autophagy, which in turn, leads to CREB1 hyperactivation. We further elucidated an inverse correlation between autophagy activity and CREB1 activity in the kidney tumor tissue obtained from a TSC patient and the mouse livers with hepatocyte-specific knockout of PTEN. CREB1 induced DNA damage and subsequent apoptosis in response to etoposide in autophagy-defective cells. Reactivation of CREB1 or inhibition of autophagy not only improved the efficacy of rapamycin but also alleviated MTOR inhibition-mediated chemoresistance. Therefore, autophagy suppression of CREB1 may underlie the MTOR inhibition-mediated chemoresistance. We suggest that inhibition of MTOR in combination with CREB1 activation may be used in the treatment of cancer caused by an abnormal PI3K-PTEN-AKT-TSC1/2-MTOR signaling pathway. CREB1 activators should potentiate the efficacy of chemotherapeutics in treatment of these cancers.

Tuberous sclerosis complex 1 (TSC1) and TSC2 are suppressors of mechanistic target of rapamycin (mTOR). mTOR is the major component of two protein complexes: mTOR complex 1 (mTORC1) and mTORC2. Inactive mutation of either TSC1 or TSC2 unleashes mTOR signaling and consequently causes TSC, a benign tumor syndrome affecting multiple organs. We report here that expression of αB-crystallin was upregulated in Tsc1-/- or Tsc2-/- mouse embryonic fibroblasts, Eker rat uterine leiomyoma-derived Tsc2-deficient ELT3 cells, mutant Tsc2-associated mouse kidney tumors, and human lung lymphangioleiomyomatosis nodules. αB-crystallin was transcriptionally activated by mTOR complex 2 (mTORC2): nuclear factor-kappa B (NFκB) signaling cascade. The augmented αB-crystallin was critical for the migration, invasion and apoptotic resistance of Tsc2-defective cells. Disruption of αB-crystallin suppressed Tsc2-null cell proliferation and tumorigenesis. Therefore, enhanced αB-crystallin has an essential role in TSC1/2 complex deficiency-mediated tumorigenesis, and inhibition of αB-crystallin may complement the current therapy for TSC.

For Port Fuel Injection (PFI) engines, excessive fuel is needed for the first few cycles during the cold-start because of the unfavorable evaporation environment. The excessive fuel usually forms a fuel film which causes non-uniform mixture and becomes a major source of the Engine-Out Hydrocarbons (EOHC). In this paper, a partial-heating method was proposed to improve the evaporation of the deposited fuel film at the beginning of the cold-start of PFI engine. The partial-heating method focuses on increasing the surface temperature of the intake port end before the cranking start. In this study, the effect of the partial-heating was researched based on the first cranking cycle. The effect was experimentally investigated and discussed in terms of the mixture preparation, combustion and EOHC. The results showed that within the capacity of the on-board battery, the partial-heating led to efficient and effective heating of the surface of the intake port end. The fuel delivery efficiency was increased by over 10%. Fuel enrichment was reduced from 40 ms FPW to 30 ms FPW within 15 s of the partial-heating. Moreover, the mixture was found to burn faster in the partial-heating cases. Higher Indicated Mean Effective Pressure (IMEP) could be obtained with optimized spark timing in the partial-heating cases.

Loss of either TSC1 or TSC2 causes tuberous sclerosis complex (TSC) via activation of mTOR signaling pathway. The two prominent features of TSC are skin lesions including hypomelanic macules and benign tumors in multiple organs, whose molecular alterations are largely unknown. We report here that X(c)(−) cystine/glutamate antiporter (xCT) was elevated in Tsc2(−/−) or Pten(−/−) cells, Tsc1 knockout mouse tissues and TSC2-deficient human kidney tumor. xCT was transcriptionally boosted by mTOR-mediated Oct1 signaling cascade. Augmented xCT led to reduction of eumelanin and elevation of pheomelanin in Tsc1 skin knockout mice through mTOR signaling pathway. Disruption of xCT suppressed the proliferation and tumorigenesis of Pten-null cells and Tsc2-null cells. mTOR hyperactive cells were more sensitive to inhibitors of mTOR or xCT. Combined inhibition of mTOR and xCT synergistically blocked the propagation and oncogenesis of mTOR hyperactive cells. Therefore, oncogenic mTOR activation of xCT is a key connection between aberrant melanin synthesis and tumorigenesis. We suggest that xCT is a novel therapeutic target for TSC and other aberrant mTOR-related diseases.

As a hallmark of cancer, the Warburg effect (aerobic glycolysis) confers a selective advantage for the survival and proliferation of cancer cells. Due to frequent aberration of upstream proto-oncogenes and tumor suppressors, hyperactive mammalian/mechanistic target of rapamycin (mTOR) is a potent inducer of the Warburg effect. Here, we report that overexpression of a glycolytic enzyme, phosphoglyceric acid mutase-1 (PGAM1), is critical to oncogenic mTOR-mediated Warburg effect. mTOR stimulated PGAM1 expression through hypoxia-inducible factor 1α-mediated transcriptional activation. Blockage of PGAM1 suppressed mTOR-dependent glycolysis, cell proliferation, and tumorigenesis. PGAM1 expression and mTOR activity were positively correlated in non-small cell lung cancer (NSCLC) tissues and PGAM1 abundance was an adverse predictor for patient survival. PGAM1 is thus a downstream effector of mTOR signaling pathway and mTOR-PGAM1 signaling cascade may contribute to the development of Warburg effect observed in cancer. We consider PGAM1 as a novel prognostic biomarker for NSCLC and a therapeutic target for cancer.

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.