Potentiating CD20 monoclonal antibody therapy by targeting complement C3 fragments covalently deposited on lymphoma cells.

Monoclonal antibodies (mAbs) improve survival of patients with mature B-cell malignancies. Fcγ receptor-dependent effector mechanisms kill tumor cells but can promote antigen loss through trogocytosis, contributing to treatment failures. Cell-bound mAbs trigger the complement cascade to deposit C3 activation fragments and lyse cells.

Phage Display Selection of Antibody Libraries: Panning Procedures.

The mining of naive, immune, and synthetic antibody repertoires by phage display has been widely applied to the de novo generation and in vitro evolution of monoclonal antibodies from multiple species. Once built, phage display antibody libraries can be selected by a variety of different strategies tailored toward the desired antigen binding properties. Here, we describe the selection of antibody libraries generated in a phage display vector of the pComb3 phagemid family.

Cloning, Expression, and Purification of Phage Display-Selected Fab for Biophysical and Biological Studies.

The antigen-binding fragment (Fab) is the ∼50-kDa monovalent arm of an antibody molecule. In the laboratory, the Fab can be produced via either enzymatic digestion or recombinant expression, and its use facilitates the accurate assessment of affinity and specificity of monoclonal antibodies. The high melting temperature of the Fab, together with its low tendency to aggregate and ready conversion to natural and nonnatural immunoglobulin (Ig) formats (without affecting antigen binding properties), have made it a preferred format for phage display, as well as a tool for accurate assessment of affinity, specificity, and developability of monoclonal antibodies.

Generation of Antibody Libraries for Phage Display: Chimeric Rabbit/Human Fab Format.

Rabbit monoclonal antibodies are attractive reagents for research, and have also found use in diagnostic and therapeutic applications. This is owed to their high affinity and specificity, along with their ability to recognize epitopes conserved between mouse and human antigens. Phage display is a powerful method for the de novo generation, affinity maturation, and humanization of rabbit monoclonal antibodies from naive, immune, and synthetic antibody repertoires.

Generation of Antibody Libraries for Phage Display: Human Fab Format.

Phage display is a powerful method for the de novo generation and affinity maturation of human monoclonal antibodies from naive, immune, and synthetic antibody repertoires. The pComb3 phagemid family of phage display vectors facilitates the selection of human monoclonal antibody libraries in the monovalent Fab format, which consists of human variable domains V and V (V or V), fused to the human constant domains C1 of IgG1 and C (C or C), respectively. Here, we describe the use of a pComb3 derivative, phagemid pC3C, for the generation of human Fab libraries with randomly combined human variable domains (V, V, and V) of high sequence diversity, starting from the preparation of mononuclear cells from blood and bone marrow.

Generation of Antibody Libraries for Phage Display: Preparation of Helper Phage.

The generation and selection of antibody libraries by phagemid-based phage display requires three components; namely, phagemid library, host bacterial cells, and helper phage. The use of helper phage is necessary for the selection of phagemid libraries by phage display because it provides all genes needed for production of infectious phage particles. Here, we describe the generation of high-titer helper phage preparations suitable for phagemid-based phage display.

Phage Display Selection of Antibody Libraries: Screening of Selected Binders.

Phage display selection of antibody libraries is a powerful method for generating and evolving monoclonal antibodies. The pComb3 phagemid family of phage display vectors facilitates the mining of antibody libraries in Fab format from human and nonhuman antibody repertoires. Here, we describe the screening for monoclonal Fab binders after selection of a polyclonal pool of Fab binders to an antigen of interest, with the goal of identifying and sequencing monoclonal antibodies that bind the antigen with high affinity and specificity.

Generation of Antibody Libraries for Phage Display: Library Reamplification.

Phage display of Fab libraries enables the de novo discovery and in vitro evolution of monoclonal antibodies. Fab libraries are collections of millions to billions of different antibodies that collectively cover a large antigen or epitope binding space. To preserve the diversity of the Fab library for repeated selection campaigns, it is recommended to use the original phage from the Fab library generation rather than reamplified phage, if practically possible.

Generation of Antibody Libraries for Phage Display: Preparation of Electrocompetent .

The size of an antibody library, that is, the phage display-selectable diversity, is restricted mainly by its transformation into the host bacterial cells. Electroporation is the most efficient method for transforming with plasmids, including phagemids. Here, we describe the preparation of electrocompetent for the generation of phagemid-encoded antibody libraries encompassing 10-10 independent transformants.

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.

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.

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.

A mammalian cell display platform based on scFab transposition.

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.

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

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. 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.

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.

Patient-derived Siglec-6-targeting antibodies engineered for T-cell recruitment have potential therapeutic utility in chronic lymphocytic leukemia.

Background: Despite 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.

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.

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.

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

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. 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.

Perspectives on the development of antibody-drug conjugates targeting ROR1 for hematological and solid cancers.

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.

The ROR1 antibody-drug conjugate huXBR1-402-G5-PNU effectively targets ROR1+ leukemia.

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.

Computer-Aided Diagnosis Evaluation of the Correlation Between Magnetic Resonance Imaging With Molecular Subtypes in Breast Cancer.

Background: There is a demand for additional alternative methods that can allow the differentiation of the breast tumor into molecular subtypes precisely and conveniently.

Purpose: The 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.

Methods: We analyzed a total of 264 patients (mean age: 47.

Adoptive T cell immunotherapy for medullary thyroid carcinoma targeting GDNF family receptor alpha 4.

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.