Affinity For Antigen Research Articles

Retrospective SARS-CoV-2 human antibody development trajectories are largely sparse and permissive

Immunological interventions, like vaccinations, are enabled by the predictive control of humoral responses to novel antigens. While the development trajectories for many broadly neutralizing antibodies (bnAbs) have been measured, it is less established how human subtype-specific antibodies develop from their precursors. In this work, we evaluated the retrospective development trajectories for eight anti-SARS-CoV-2 Spike human antibodies (Abs). To mimic the immunological process of BCR selection during affinity maturation in germinal centers (GCs), we performed deep mutational scanning on anti-S1 molecular Fabs using yeast display coupled to fluorescence-activated cell sorting. Focusing only on changes in affinity upon mutation, we found that human Ab development pathways have few mutations which impart changes in monovalent binding dissociation constants and that these mutations can occur in nearly any order. Maturation pathways of two bnAbs showed that while they are only slightly less permissible than subtype-specific Abs, more development steps on average are needed to reach the same level of affinity. Many of the subtype-specific Abs had inherent affinity for antigen, and these results were robust against different potential inferred precursor sequences. To evaluate the effect of differential affinity for precursors on GC outcomes, we adapted a coarse-grained affinity maturation model. This model showed that antibody precursors with minimal affinity advantages rapidly outcompete competitors to become the dominant clonotype.

Antigen affinity and site of immunization dictate B cell recall responses.

Protective antibodies against HIV-1 require unusually high levels of somatic mutations introduced in germinal centers (GCs). To achieve this, a sequential vaccination approach was proposed. Using HIV-1 antibody knockin mice with fate-mapping genes, we examined if antigen affinity affects the outcome of B cell recall responses. Compared to a high-affinity boost, a low-affinity boost resulted in decreased numbers of memory-derived B cells in secondary GCs but with higher average levels of somatic mutations, indicating an affinity threshold for memory B cells to enter GCs. Furthermore, upon boosting local lymph nodes (LNs), the composition of primary GCs was modified in an antigen-affinity-dependent manner to constitute less somatically mutated B cells. Our results demonstrate that antigen affinity and location of the boost affect the outcome of the B cell recall response. These results can help guide the design of vaccine immunogens aiming to selectively engage specific B cell clones for further diversification.

Phage-ELISA for ultrasensitive detection of Salmonella enteritidis.

The M13 phage carries approximately 5 copies of the pIII protein, each of which is capable of displaying a single-chain variable fragment (scFv) that targets a specific antigen. This feature enables the M13 phage to be widely employed in the construction of scFv libraries, thereby facilitating the identification of antibodies with high specificity and affinity for target antigens. In this study, mice were immunized three times with Salmonella enteritidis (strain C50041) to induce diverse antibodies. The variable region sequences were subsequently amplified by PCR using genome extracted from the mice's splenic cells and fused to the pIII protein to construct the scFv phage display library (C50041-M13-scFv). Through biopanning with the C50041-M13-scFv library, a phage clone (C50041-scFv-4) exhibiting high affinity for the target bacteria was successfully obtained. Moreover, the scFv antibody (scFv-4) derived from C50041-scFv-4 was expressed in a prokaryotic expression system and validated to possess high specificity and affinity for C50041 through in vitro adsorption assays. Additionally, a phage-ELISA method was established: initially, bacteria were immobilized on the bottom surface of a 96-well plate. Next, the positive clone C50041-scFv-4 was introduced to specifically bind to the host cells. Finally, horseradish peroxidase (HRP)-conjugated anti-pVIII antibodies were used to detect the pVIII proteins of the bound phage clones. Owing to the capacity of multiple C50041-scFv-4 probes to simultaneously bind to a single target Salmonella and each phage clone's ability to accommodate hundreds of HRP-labeled antibodies, the proposed phage-ELISA demonstrated remarkable sensitivity (104 CFU mL-1) for detecting Salmonella enteritidis samples. This sensitivity surpasses that of traditional ELISA by one order of magnitude in this study. Our phage-ELISA technology exhibits broad applicability across various biological species and provides an improved and robust platform for pathogen detection including bacteria and viruses.

Biophysical and Structural Features of αβT-Cell Receptor Mechanosensing: A Paradigmatic Shift in Understanding T-Cell Activation.

αβT cells protect vertebrates against many diseases, optimizing surveillance using mechanical force to distinguish between pathophysiologic cellular alterations and normal self-constituents. The multi-subunit αβT-cell receptor (TCR) operates outside of thermal equilibrium, harvesting energy via physical forces generated by T-cell motility and actin-myosin machinery. When a peptide-bound major histocompatibility complex molecule (pMHC) on an antigen presenting cell is ligated, the αβTCR on the T cell leverages force to form a catch bond, prolonging bond lifetime, and enhancing antigen discrimination. Under load, the αβTCR undergoes reversible structural transitions involving partial unfolding of its clonotypic immunoglobulin-like (Ig) domains and coupled rearrangements of associated CD3 subunits and structural elements. We postulate that transitions provide critical energy to initiate the signaling cascade via induction of αβTCR quaternary structural rearrangements, associated membrane perturbations, exposure of CD3 ITAMs to phosphorylation by non-receptor tyrosine kinases, and phase separation of signaling molecules. Understanding force-mediated signaling by the αβTCR clarifies long-standing questions regarding αβTCR antigen recognition, specificity and affinity, providing a basis for continued investigation. Future directions include examining atomistic mechanisms of αβTCR signal initiation, performance quality, tissue compliance adaptability, and T-cell memory fate. The mechanotransduction paradigm will foster improved rational design of T-cell based vaccines, CAR-Ts, and adoptive therapies.

BIOTECHNOLOGICAL SYSTEM FOR THE SEARCH OF SUBSTANCES WITH POTENTIAL ACTIVITY AGAINST CORONAVIRUS

Aim. Development of a biotechnological system based on a non-pathogenic coronavirus strain for humans and a sensitive cell line to the selected strain aimed at identifying compounds with potential antiviral activity. Methods. The study was conducted on the cell lines CEF, CEFs, and ВНК-21, which are sensitive to the avian infectious bronchitis virus (IBV). Cell cultivation was performed in flasks and microplates with adhesive surfaces at 37 °C in a 5% CO2 atmosphere. To detach the cells from the growth surface, a Versene solution (0.02%) was used, and for trypsinization of CEF, a trypsin solution (0.25%) was applied. Growth media for all cell cultures were prepared based on a mixture of RPMI-1640 and DMEM in a 1:1 ratio, supplemented with 5% fetal serum. Results. The adaptation of the model virus IBV strain H120 to cultivation in ВНК-21 cell cultures was carried out using intermediate CEF and CEF cultures. In ВНК-21 cells, IBV induced a pronounced cytopathic effect and demonstrated high infectious titers, reaching 5.5 lg TCID50/mL. The use of intermediate CEF and CEF cell cultures facilitated the gradual adaptation of the virus to the new cultivation conditions due to the antigenic affinity between chicken embryo fibroblast cells and avian embryos. Conclusions: As a result of the conducted research, the vaccine virus IBV H-120 was successfully adapted to cultivation conditions in ВНК-21 cell cultures, using primary trypsinized chicken embryo fibroblast cells as an intermediate system. The obtained system "ВНК-21 cell culture + IBV H-120," cultivated at 37 °C in a 5% CO2 atmosphere, can be recommended for use in further biotechnological and virological studies, particularly for evaluating the antiviral activity of potential drugs against coronaviruses.

Machine learning application to predict binding affinity between peptide containing non-canonical amino acids and HLA0201.

Class 1 major histocompatibility complexes (MHC-I), encoded by the highly polymorphic HLA-A, HLA-B, and HLA-C genes in humans, are expressed on all nucleated cells. Both self and foreign proteins are processed to peptides of 8 to 10 amino acids, loaded into MCH-1 within the endoplasmic reticulum and then presented on the cell surface. Foreign peptides presented in this fashion activate CD8+ T cells and their immunogenicity correlates with their affinity for the MHC-1 binding groove. Thus, predicting antigen binding affinity for MHC-I is a valuable tool for identifying potentially immunogenic antigens. While quite a few predictors for MHC-I binding exist, there are no currently available tools that can predict antigen/MHC-I binding affinity for antigens with explicitly labeled post-translational modifications or unusual/non-canonical amino acids (NCAAs). However, such modifications are increasingly recognized as critical mediators of peptide immunogenicity. In this work, we propose a machine learning application that quantifies the binding affinity of epitopes containing NCAAs to MHC-I and compares its performance with other commonly used regressors. Our model demonstrates robust performance, with 5-fold cross-validation yielding an R2 value of 0.477 and a root-mean-square error (RMSE) of 0.735, indicating strong predictive capability for peptides with NCAAs. This work provides a valuable tool for the computational design and optimization of peptides incorporating NCAAs, potentially accelerating the development of novel peptide-based therapeutics with enhanced properties and efficacy.

High-affinity chimeric antigen receptor signaling induces an inflammatory program in human regulatory T cells

Regulatory Tcells (Tregs) are promising cellular therapies to induce immune tolerance in organ transplantation and autoimmune disease. The success of chimeric antigen receptor (CAR) Tcell therapy for cancer has sparked interest in using CARs to generate antigen-specific Tregs. Here, we compared CAR with endogenous Tcell receptor (TCR)/CD28 activation in human Tregs. Strikingly, CAR Tregs displayed increased cytotoxicity and diminished suppression of antigen-presenting cells and effector T (Teff) cells compared with TCR/CD28-activated Tregs. RNA sequencing revealed that CAR Tregs activate Teff cell gene programs. Indeed, CAR Tregs secreted high levels of inflammatory cytokines, with a subset of FOXP3+ CAR Tregs uniquely acquiring CD40L surface expression and producing IFN-γ. Interestingly, decreasing CAR antigen affinity reduced Teff cell gene expression and inflammatory cytokine production by CAR Tregs. Our findings showcase the impact of engineered receptor activation on Treg biology and support tailoring CAR constructs to Tregs for maximal therapeutic efficacy.

Endemicity factors and risks of false-positive serological reactions in brucellosis testing of farm animals

The article presents generalized data on the endemicity and epizootology of animal brucellosis. The work was carried out to analyze and summarize the information from the Centralized Research Institute of Veterinary Medicine and Epidemiology of Kharkiv region on research on brucellosis of farm animals. Material samples were examined at the Laboratory of Brucellosis of the National Scientific Center «Institute of Experimental and Clinical Veterinary Medicine» to clarify the diagnosis. The study revealed that brucellosis continues circulating among wildlife populations, especially pigs, despite control efforts. It has been shown that the enzooticity of detecting false-positive reactions in traditional serological tests, namely the dipstick test (DST), agglutination reaction (RA), complement binding reaction (CBR), long-term complement binding reaction (LTCR), and milk ring reaction (MR). The presence of antigenic affinity of the lipopolysaccharide antigen of pathogenic Brucella and other gram-negative bacteria, in particular, Enterobacteriacea, has been confirmed, which causes false positive results that lead to diagnostic errors and unjustified culling of animals. To increase the specificity and reduce the likelihood of false positive results, it is proposed to conduct additional studies of sera at a reduced dose of antigen, which can significantly increase the specificity of RBP as a screening method while maintaining sensitivity. The main risks of occurrence and the presence of pathogen sources, pathways, and mechanisms of transmission of pathogenic Brucella sp. (B. abortus, B. suis, B. melitensis) are identified. Special attention is paid to the aspects of effective diagnostics, which are currently crucial for controlling the spread of this infection. It is shown that the endemic threshold for brucellosis in some regions is a problem related to the complication of serological tests, particularly the risk of false-positive reactions, which are significant obstacles to the diagnosis and prevention of the disease. The authors argue that the results highlight the need to continue research to develop more specific methods and diagnostic tools to control this zoonotic disease effectively

Genetically modified extracellular vesicles loaded with activated gasdermin D potentially inhibit prostate-specific membrane antigen-positive prostate carcinoma growth and enhance immunotherapy

Prostate cancer (PCa) is associated with poor immunogenicity and lymphocytic infiltration, and immunotherapy effective against PCa remains unavailable. Pyroptosis, a novel immunotherapeutic modality for cancer, promotes systemic immune responses leading to immunogenic cell death in solid tumors. This paper describes the preparation and analysis of PSMAscFv-EVN-GSDMD; this genetically engineered recombinant extracellular vesicle (EV) expresses a single-chain variable antibody fragment (scFv) with high affinity for prostate-specific membrane antigen (PSMA) on their surfaces and is loaded with the N-terminal domain of gasdermin D (GSDMD). Both in vitro and in vivo, PSMAscFv-EVN-GSDMD effectively targeted PSMA-positive PCa cells and induced pyroptosis through the carrier properties of EVs and the specificity of PSMAscFv. In the 22RV1 and PSMA-transfected RM-1-inoculated PCa mouse models, PSMAscFv-EVN-GSDMD efficiently inhibited tumor growth and promoted tumor immune responses. In conclusion, PSMAscFv-EVN-GSDMD can convert the immunosuppressive “cold” tumor microenvironment of PCa into an immunogenic “hot” tumor microenvironment.

T-cell engagers: model interrogation as a tool to quantify the interplay of relative affinity and target expression on trimer formation.

T-cell engagers (TCEs) represent a promising therapeutic strategy for various cancers and autoimmune disorders. These bispecific antibodies act as bridges, connecting T-cell receptors (TCRs) to target cells (either malignant or autoreactive) via interactions with specific tumour-associated antigens (TAAs) or autoantigens to form trimeric synapses, or trimers, that co-localise T-cells with target cells and stimulate their cytotoxic function. Bispecific TCEs are expected to exhibit a bell-shaped dose-response curve, with a defined optimal TCE exposure for maximizing trimer formation. The shape of the dose-response is determined by a non-trivial interplay of binding affinities, exposure and antigens expression levels. Furthermore, excessively low binding to the TCR may reduce efficacy, but mitigate risk of over-stimulating cytokine secretion or induce effector cell exhaustion. These inevitable trade-off highlights the importance of quantitatively understanding the relationship between TCE concentration, target expression, binding affinities, and trimer formation. We utilized a mechanistic target engagement model to show that, if the TCE design parameters are close to the recommended ranges found in the literature, relative affinities for TCR, TAA and target expression levels have qualitatively different, but predictable, effects on the resulting dose-response curve: higher expression levels shift the curve upwards, higher antigen affinity shifts the curve to the left, and higher TCR affinity shifts the curve upwards and to the left.

Natural TCRs targeting KRASG12V display fine specificity and sensitivity to human solid tumors.

BACKGROUNDNeoantigens derived from KRASMUT have been described, but the fine antigen specificity of T cell responses directed against these epitopes is poorly understood. Here, we explore KRASMUT immunogenicity and the properties of 4 T cell receptors (TCRs) specific for KRASG12V restricted to the HLA-A3 superfamily of class I alleles.METHODSA phase 1 clinical vaccine trial targeting KRASMUT was conducted. TCRs targeting KRASG12V restricted to HLA-A*03:01 or HLA-A*11:01 were isolated from vaccinated patients or healthy individuals. A comprehensive analysis of TCR antigen specificity, affinity, crossreactivity, and CD8 coreceptor dependence was performed. TCR lytic activity was evaluated, and target antigen density was determined by quantitative immunopeptidomics.RESULTSVaccination against KRASMUT resulted in the priming of CD8+ and CD4+ T cell responses. KRASG12V -specific natural (not affinity enhanced) TCRs exhibited exquisite specificity to mutated protein with no discernible reactivity against KRASWT. TCR-recognition motifs were determined and used to identify and exclude crossreactivity to noncognate peptides derived from the human proteome. Both HLA-A*03:01 and HLA-A*11:01-restricted TCR-redirected CD8+ T cells exhibited potent lytic activity against KRASG12V cancers, while only HLA-A*11:01-restricted TCR-T CD4+ T cells exhibited antitumor effector functions consistent with partial coreceptor dependence. All KRASG12V-specific TCRs displayed high sensitivity for antigen as demonstrated by their ability to eliminate tumor cell lines expressing low levels of peptide/HLA (4.4 to 242) complexes per cell.CONCLUSIONThis study identifies KRASG12V-specific TCRs with high therapeutic potential for the development of TCR-T cell therapies.TRIAL REGISTRATIONClinicalTrials.gov NCT03592888.FUNDINGAACR SU2C/Lustgarten Foundation, Parker Institute for Cancer Immunotherapy, and NIH.

Abstract B047: Antigenic properties determine the outcome of B cell driven anti-tumor immunity

Abstract B cells have the capacity to elicit both anti- and pro-tumorigenic effects; yet the determinants of productive B cell responses in cancer remain elusive. Using a system where a B cell neoantigen is either soluble or tethered to the surface of pancreatic cancer cells, we demonstrate that membrane-tethered antigen is sufficient to elicit activation of anti-tumor immunity and reduce tumor growth in an NK cell-dependent manner. We also show that decreased antigen affinity to the BCR promotes regulatory B cell function which can be overcome by increased avidity of membrane-tethered antigen. Furthermore, soluble and membrane-tethered antigens drive qualitatively distinct germinal center, plasma cell, memory B cell, and Treg responses. Finally, we describe two classes of membrane-associated antigens that correlate with GC B cell signature and improved survival in pancreatic cancer patients. Thus, antigen localization impacts basic mechanisms governing B cell differentiation and the resulting immune response to solid malignancy with implications for vaccine engineering. Citation Format: Colleen Sturdevant, Nancy Kren, Harrison Silva, Ismael Gomez, Austin Hepperla, Yuliya Pylayeva-Gupta. Antigenic properties determine the outcome of B cell driven anti-tumor immunity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B047.

A Novel Tetravalent Bispecific Immune Cell Engager Activates Natural Killer Cells to Kill Cancer Cells without Mediating Fratricide.

We previously reported the structure, affinity, and anticancer activity of a bivalent bispecific natural killer cell engager (BiKE) composed of one anti-CD16a VHH and one anti-HER2 VHH fused via a linker. In this study, we explored the engineering of a tetravalent BiKE by fusing two anti-CD16a and two anti-HER2 VHHs in tandem, using bivalent BiKE as a template. The tetravalent BiKE was genetically engineered, and its tertiary structure was predicted using in silico modeling. The antigen binding and affinity of the tetravalent BiKE were assessed using ELISA, flow cytometry, and biolayer interferometry. The ability of the BiKEs to kill cancer cells was evaluated through classical and residual antibody-dependent cellular cytotoxicity (ADCC) assays. Additionally, we investigated the potential for NK cell fratricide via CD16a-CD16a crosslinking. Our results revealed that the tetravalent BiKE exhibited at least 100-fold higher affinity toward its target antigens compared to its bivalent counterpart. The residual ADCC assay indicated that the tetravalent BiKE was more effective in killing cancer cells than the bivalent BiKE, attributable to its lower Koff value, which prolonged its binding to NK cell surfaces. Fratricide assays demonstrated that neither the bivalent nor the tetravalent BiKE mediated fratricide. Notably, our findings showed that daratumumab-induced NK fratricide was restricted to CD38-CD38 crosslinking and was not related to ADCC via CD16a-CD38 crosslinking. This study is the first in the literature to show the successful engineering of a tetravalent immune cell engager composed of tandem VHH units, which achieves high affinity and anticancer activity without mediating fratricide.

Engineered Antibodies as Cancer Radiotheranostics.

Radiotheranostics is a rapidly growing approach in personalized medicine, merging diagnostic imaging and targeted radiotherapy to allow for the precise detection and treatment of diseases, notably cancer. Radiolabeled antibodies have become indispensable tools in the field of cancer theranostics due to their high specificity and affinity for cancer-associated antigens, which allows for accurate targeting with minimal impact on surrounding healthy tissues, enhancing therapeutic efficacy while reducing side effects, immune-modulating ability, and versatility and flexibility in engineering and conjugation. However, there are inherent limitations in using antibodies as a platform for radiopharmaceuticals due to their natural activities within the immune system, large size preventing effective tumor penetration, and relatively long half-life with concerns for prolonged radioactivity exposure. Antibody engineering can solve these challenges while preserving the many advantages of the immunoglobulin framework. In this review, the goal is to give a general overview of antibody engineering and design for tumor radiotheranostics. Particularly, the four ways that antibody engineering is applied to enhance radioimmunoconjugates: pharmacokinetics optimization, site-specific bioconjugation, modulation of Fc interactions, and bispecific construct creation are discussed. The radionuclide choices for designed antibody radionuclide conjugates and conjugation techniques and future directions for antibody radionuclide conjugate innovation and advancement are also discussed.

Enhancement of CAR-T cell-targeted immune responses against solid tumors: role of lowered CARs affinity and addition of PD-1 chimeric antigen complexes

Cancers rising prevalence demands novel treatments. Chimeric Antigen Receptor T-cell (CAR-T) therapy emerges as a transformative option. Conventional cancer treatments of-ten lack efficacy and carry severe side effects. This study focuses on enhancing CAR-T therapy against solid tumors. Strategies include lowering CAR affinity for antigens and in-tegrating PD-1&CD-28 chimeric antigen complexes. While CAR-T therapy excels against hematological malignancies, challenges remain, such as immune checkpoint upregulation and potential toxicity. The research aims to optimize CAR-T therapys efficacy through innovative modifications. Alternative strategies like multi-targeted CARs and combina-tions with immune checkpoint inhibitors are explored. Overall, this study advances can-cer therapy by addressing CAR-T therapys limitations and proposing complementary strategies for improved personalized treatments.

Screening and anti-angiogenesis activity of Chiloscyllium plagiosum anti-human VEGFR2 single-domain antibody.

Recently, the incidence of malignant tumors is on the rise and searching for new treatments on it has become the research priority. Blocking the vascular endothelial growth factor (VEGF) and its receptor (VEGFR) is one of the treatment strategies that used in the development of specific anti-angiogenic drugs. The deficiencies in tissue penetration and affinity maturation become the weakness of these drugs in anti-tumors applications. The single heavy chain antibody found in Chiloscyllium plagiosum, which has a low molecular weight and superior tissue penetration of variable region (variable new antigen receptor, VNARs), was considered to have the high antigen-binding activity and stability. This type of antibody has a simple structure that can be prokaryoticaly expressed, which makes it easily to produce new antiangiogenic target drugs. Specific anti-IgNAR rabbit multiple antibodies have been used to assess the level of VNARs in sharks and have shown a significant enrichment of IgNAR after triple immunization. An anti-VEGFR2 phage library was used for the targeted VNARs screening, and five candidate VNARs sequences were subsequently obtained by phage screening, followed by combined screening with the transcriptome library, and analysis of conserved regions along with 3D modelling matched the VNAR profile. ELISA and cell-based assays showed that two of the VNARs, VNAR-A6, and VNAR-E3, had a superior antigen affinity and anti-angiogenic activity thereby being able to inhibit human Umbilical Vein Endothelial Cells proliferation and migration. The anti-VEGFR2 VNARs derived from the immunized C. plagiosum and screened by phage library, which provide the new research ideas and specific approaches for the development of new drugs. The anti-VEGFR2 VNARs are capable for blocking the VEGF-VEGFR pathway, which of these may contribute to expanding the use of anti-angiogenic drugs.

Hu8F4-CAR T cells with mutated Fc spacer segment improve target specificity and mediate anti-leukemia activity in vivo

Background aimsHu8F4 is a T-cell receptor–like antibody with high affinity for the leukemia-associated antigen PR1/HLA-A2 epitope. Adapted into a chimeric antigen receptor (CAR) format, Hu8F4-CAR is composed of the Hu8F4 single-chain variable fragment, the human IgG1 CH2CH3 extracellular spacer domain, a human CD28 costimulatory domain and the human CD3ζ signaling domain. We have demonstrated high efficacy of Hu8F4-CAR-T cells against PR1/HLA-A2-expressing cell lines and leukemic blasts from patients with acute myeloid leukemia in vitro. Previous studies have shown that modification of the Fc domains of IgG4 CH2CH3 spacer regions can eliminate activation-induced cell death and off-target killing mediated by mouse Fc gamma receptor–expressing cells. MethodsWe generated Hu8F4-CAR(PQ) with mutated Fc receptor binding sites on the CH2 domain of Hu8F4-CAR to prevent unwanted interactions with Fc gamma receptor–expressing cells in vivo. ResultsThe primary human T cells transduced with Hu8F4-CAR(PQ) can specifically lyse HLA-A2+ PR1-expressing leukemia cell lines in vitro. Furthermore, both adult donor-derived and cord blood-derived Hu8F4-CAR(PQ)-T cells are active and can eliminate U937 leukemia cells in NSG mice. ConclusionsHerein, we demonstrate that modification of the IgG1-based spacer can eliminate Fc receptor binding–induced adverse effects and Hu8F4-CAR(PQ)-T cells can kill leukemia in vivo.

Preparation of interleukin-1β-targeted nanobodies and their effects on apoptosis in hypoxic cardiomyocytes of mice

Objective: To prepare interleukin-1β-targeted nanoantibodies and observe their effects on apoptosis in hypoxic cardiomyocyte of mice. Methods: Using DNA recombination technology, the pET-16b and pHEN1 expression vectors were used to construct the prokaryotic expression plasmids of interleukin-1β-targeted nanobodies (pET-16b-4G6M-VHH, pET-16b-5BVP-VHH, pET-16b-5MVZ-VHH, pHEN1-4G6M-VHH, pHEN1-5BVP-VHH and pHEN1-5MVZ-VHH, where VHH is a variable domain of heavy chain antibody, 4G6M-VHH, 5BVP-VHH, 5MVZ-VHH were three interleukin-1β-targeted nanoantibodies respectively). The constructed plasmids were transferred into Escherichia coli Rosetta2 (DE3) for induction of expression and nickel column purification, respectively. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were employed to identify the expression product and purified product, and the enzyme-linked immune sorbent assay (ELISA) was performed to determine their affinity. The cardiomyocyte hypoxia model was used with the highest affinity IL-1β-targeted nanobody (pHEN1-5MVZ-VHH), and cell survival and apoptosis rates were detected (the experiment was divided into normal control group, hypoxia model group, blank plasmid group and 12.5, 25.0, 50.0 μg/ml pHEN1-5MVZ-VHH treatment groups). Results: SDS-PAGE and Western blotting results showed that the anti-interleukin-1β (IL-1β) nanobodies with a relative molecular mass of about 15 000 were successfully obtained. Likewise, ELISA results found that the nanobodies expressed in pHEN1 vector group had higher affinity for IL-1β antigen compared with pET-16b vector group (4G6M-VHH group: 3.20±0.03 vs 1.20±0.03, P<0.001; 5BVP-VHH group: 3.18±0.06 vs 1.21±0.02, P<0.001; 5MVZ-VHH group: 3.38±0.05 vs 1.62±0.04, P<0.001). Additionally, the results of cell survival assay and apoptosis assay detected that compared with the hypoxia model group, HL-1 cell activity was significantly increased in the 25.0 μg/ml and 50.0 μg/ml pHEN1-5MVZ-VHH treatment groups [(75.55±2.23)% vs (46.90±2.51)%, P<0.001; (74.36±1.96)% vs (46.90±2.51)%, P<0.001], and apoptosis rate was significantly reduced [(6.83±0.27)% vs (10.24±0.76)%, P<0.001; (6.68±0.38)% vs (10.24±0.76)%, P<0.001]. Conclusions: 4G6M-VHH, 5BVP-VHH, and 5MVZ-VHH are expressed by both pET-16b and pHEN1 expression vectors and the nanobodies produced by the pHEN1 vector display enhanced antigen affinity. Furthermore, in hypoxic cardiomyocytes, pHEN1-5MVZ-VHH treatment reduces cell apoptosis.

A guide to adaptive immune memory.

Immune memory - comprising T cells, B cells and plasma cells and their secreted antibodies - is crucial for human survival. It enables the rapid and effective clearance of a pathogen after re-exposure, to minimize damage to the host. When antigen-experienced, memory T cells become activated, they proliferate and produce effector molecules at faster rates and in greater magnitudes than antigen-inexperienced, naive cells. Similarly, memory B cells become activated and differentiate into antibody-secreting cells more rapidly than naive B cells, and they undergo processes that increase their affinity for antigen. The ability of T cells and B cells to form memory cells after antigen exposure is the rationale behind vaccination. Understanding immune memory not only is crucial for the design of more-efficacious vaccines but also has important implications for immunotherapies in infectious disease and cancer. This 'guide to' article provides an overview of the current understanding of the phenotype, function, location, and pathways for the generation, maintenance and protective capacity of memory T cells and memory B cells.

Comparative study of optical fiber immunosensors based on traditional antibody or nanobody for detecting HER2

In this study, we present a novel biomarker detection platform employing a modified S-tapered fiber coated with gold nanoparticle/graphene oxide (GNP/GO) for quantifying human epidermal growth factor receptor-2 (HER2) concentrations, using antibodies as sensing elements. The fabrication of this device involves implementing an in-situ layer-by-layer technique coupled with a chemical adsorption step to achieve the self-assembly of GNP, GO, and antibodies on the STF surface. The detection mechanism relies on monitoring the refractive index changes induced by the adsorption of HER2 onto the immobilized antibodies. For comparative analysis, both monoclonal antibody (mAb) and the novel nanobody (Nb) were employed in constructing the STF immunosensor, referred to as the mAb immunosensor and Nb immunosensor, respectively. Spectral analysis results highlight that the Nb immunosensor exhibits twice the sensitivity of the mAb immunosensor. This enhanced sensitivity is attributed to the small size, high antigen affinity, strong specificity, and structural stability of Nb. The Nb immunosensor demonstrated an impressive detection limit of 0.001 nM for HER2, surpassing the detection limit of the mAb immunosensor. These findings underscore the potential of the proposed Nb immunosensor as a promising and sensitive tool for HER2 detection, contributing to the diagnosis and prognosis of breast cancer. Furthermore, the simplicity of production and excellent optical performance position the Nb immunosensor as a prospective real-time biosensor with minimal cytotoxicity.