CAR T-cell bioengineering: Single variable domain of heavy chain antibody targeted CARs.

Redirecting the recognition specificity of T lymphocytes to designated tumour cell surface antigens by transferring chimeric antigen receptor (CAR) genes is becoming an effective strategy to combat cancer. Today, CAR T-cell therapy has proven successful in the treatment of haematological malignancies and the first CD19 CAR T-cell products has already entered the market. This success is expanding CAR design for broader malignancies including solid tumours. Nevertheless, CARs such as those built on antigen-specific single chain antibody variable fragment (scFv) may induce some adverse effects. Here, we briefly review CAR T-cell bioengineering and discuss selected important initiatives for improved T-cell reprogramming, function and safety. In this respect, we further elaborate on unconventional CARs structured on single variable domain of heavy chain (VHH) antibodies (single-domain antibodies) as an alternative to scFv, because of their interesting immunological and physicochemical characteristics and unique structure, which shows a high degree of homology with human VH3 gene family.

HIV-1 Gag p24-Nef fusion peptide induces cellular and humoral immune response in a mouse model.

UNLABELLED: Many Human immunodeficiency virus (HIV) candidate vaccines have been tested in clinical trials, but none was sufficiently effective in the prevention of HIV infection. A HIV vaccine should induce humoral as well as cell-mediated response, the latter including the cytotoxic CD8+ T lymphocyte (CTL) response. In this study, we immunized BALB/c mice with a purified fusion peptide Gag p24-Nef and evaluated immune responses. As for the cellular responses, the adjuvanted fusion peptide induced lymphocyte proliferation, CTL response, and cytokines IFN-gamma and IL-4 in the Th1 pattern. Humoral immune response to the adjuvanted fusion peptide included an increase in IgG antibodies of more IgG2a than IgG1 subtype. These results indicate that the employed HIV-1 peptide construct can elicit both cellular and humoral immune responses in mice. Further studies aimed at memory T cells and other aspects of immune responses are needed before a comprehensive assessment of this candidate vaccine could be provided. KEYWORDS: epitopes; fusion peptide; HIV-1 p24-Nef; immune response.

Production of chimeric recombinant single domain antibody-green fluorescent fusion protein in Chinese hamster ovary cells.

There is an increasing interest in the application of nanobodies such as VHH in the field of therapy and imaging. In the present study a stable genetically engineered cell line of Chinese hamster ovary (CHO) origin transfected using two sets of expression vectors was constructed in order to permit the cytoplasmic and extracellular expression of single domain antibody along with green fluorescent protein (GFP) as reporter gene. The quality of the constructs were examined both by the restriction map as well as sequence analysis. The gene transfection and protein expression was further examined by reverse transcription-polymerase chain reaction (RT-PCR). The transfected cells were grown in 200 microg/mL hygromycin containing media and the stable cell line obtained showed fluorescent activity for more than a period of 180 days. The production of fusion protein was also detected by fluorescent microscopy, fluorescent spectroscopy as well as by enzyme-linked immunosorbent assay (ELISA) analysis. This strategy allows a rapid production of recombinant fluobodies involving VHH, which can be used in various experiments such as imaging and detection in which a primary labeled antibody is required.

Elevated expression of human alpha-1 antitrypsin mediated by yeast intron in Pichia pastoris.

Intron-mediated enhancement has been documented in many cases to involve large positive effect on gene expression. To address this, human Alpha-1 antitrypsin (hAAT) gene was integrated into Pichia pastoris with and without a yeast intron generated from the final plasmid pBlu-exII-int-exIII and ligated into the EcoRI/BamHI multiple cloning site of the yeast shuttle vector pHIL-S1. The chimeric exon-intron complex in the middle of the naturally occurring hAAT exons II and III caused a 23-fold enhancement of hAAT expression in P. pastoris, measured through SDS-PAGE and immunoblot analyses.

Expression and characterization of a recombinant single-domain monoclonal antibody against MUC1 mucin in tobacco plants.

A promising alternative to conventional antibodies is the single-domain antibody fragment of the Camelidae (V(HH)), which (because of features such as small length, high expression, solubility, and stability) is preferred to other antibody derivatives. In this report, a recombinant single-domain antibody (V(HH)) against MUC1 mucin in the tobacco plant, which may be considered as a suitable and economical alternative expression system, was produced. This antibody was expressed under the control of a strong constitutive promoter, CaMV35S, and NOS terminator. A plant high-expression sequence (Kozak sequence) was linked at the 5' end for overexpression of the V(HH) gene. The constructed cassette (pBIV(HH)) was transferred to agrobacterium, and the VHH gene was inserted into the plant genome by agrobacterium-mediated transformation. Transgenic lines were selected on kanamycin (100 mg/L) and maintained in soil, and subsequent generations were obtained. The presence and expression of the transgene was confirmed in the transformants by polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and Western blot. Tobacco transgenic lines leave expressed V(HH) at levels varying from 1.12% to 1.63% of the total soluble protein. This report examines the transformation and expression of recombinant single-domain antibody (V(HH)) against antigen-associated tumor in tobacco plants.

A new competitive enzyme linked immunosorbent assay (MRP83-CA15-3) for MUC1 measurement in breast cancer.

A new competitive enzyme linked immunosorbent assay was developed in this study. Monoclonal antibody (PR81) against the tandem repeat of the core protein was prepared, characterized, purified, and conjugated to HRP. This antibody exhibited no cross reactions with proteins such as bovine serum albumin, keyhole limpet homocyanin, human serum albumin, casein, human milk fat globin (HMFG), and peptone. The native cancerous MUC1 protein was purified from ascites fluid of a patient suffering from small cell lung carcinoma by immunoaffinity chromatography and used as a standard preparation in the assay buffer. The standard curve was constructed following a competitive procedure in the range of 0-200 U/mL. The level of MUC1 in normal and cancerous samples was compared following this procedure and using available CA15-3 EIA (Can Ag), as well as LIAISON CA15-3 commercial kits. The correlation coefficient between the procedure reported in this work (MRP83-CA15-3) and CA15-3 EIA (Can Ag) was 0.68 and was 0.95 with the LIAISON CA15-3 kit. We concluded that the present assay can detect MUC1 in breast cancer patients with great sensitivity and accuracy.

Modified salting-out method: high-yield, high-quality genomic DNA extraction from whole blood using laundry detergent.

Different approaches have been used to extract DNA from whole blood. In most of these methods enzymes (such as proteinase K and RNAse A) or toxic organic solvents (such as phenol or guanidine isothiocyanate) are used. Since these enzymes are expensive, and most of the materials that are used routinely are toxic, it is desirable to apply an efficient DNA extraction procedure that does not require the use of such materials. In this study, genomic DNA was extracted by the salting-out method, but instead of using an analytical-grade enzyme and chemical detergents, as normally used for DNA isolation, a common laundry powder was used. Different concentrations of the powder were tested, and proteins were precipitated by NaCl-saturated distilled water. Finally, DNA precipitation was performed with the use of 96% ethanol. From the results, we conclude that the optimum concentration of laundry powder for the highest yield and purity of isolated DNA is 30 mg/mL. The procedure was optimized, and a final protocol is suggested. Following the same protocol, DNA was extracted from 100 blood samples, and their amounts were found to be >50 microg/mL of whole blood. The integrity of the DNA fragments was confirmed by agarose gel electrophoresis. Furthermore, the extracted DNA was used as a template for PCR reaction. The results obtained from PCR showed that the final solutions of extracted DNA did not contain any inhibitory material for the enzyme used in the PCR reaction, and indicated that the isolated DNA was of good quality. These results show that this method is simple, fast, safe, and cost-effective, and can be used in medical laboratories and research centers.

Production of novel recombinant single-domain antibodies against tandem repeat region of MUC1 mucin.

Recently, the existence of "heavy-chain" antibody in Camelidae has been described. However, as yet there is no data on the binding of this type of antibody to peptides. In addition, there was not any report of production of single-domain antibodies in two-humped camels (Camelus bactrianus). In the present study, these questions are addressed. We showed the feasibility of immunizing old world camels, cloning the repertoire of the variable domain of their heavy-chain antibodies, panning and selection, leading to the successful identification of minimum-sized antigen binders. Antigen-specific fragments of the heavy-chain IgGs (V(HH)) are of great interest in biotechnology because they are very stable, highly soluble, and react specifically and with high affinity to the antigens. In this study, we immunized two camels (Camelus dromedarius and Camelus bactrianus) with homogenized cancerous tissues, synthetic peptide, and human milk fat globule membrane (HMFG), and generated two V(HH) libraries displayed on phage particles. Some single-domain antibody fragments have been isolated that specifically recognize the tandem repeat region of MUC1. The camels' single-domain V(HH) harbor the original, intact antigen binding site and reacted specifically and with high affinity to the tandem repeat region of MUC1. Indeed soluble, specific antigen binders and good affinities (in the range of 0.2 x 10(9) M(-1) to 0.6 x 10(9) M(-1)) were identified from these libraries. This is the first example of the isolation of camel anti-peptide V(HH) domains.

Stabilization of penicillinase-hapten conjugate for enzyme immunoassay.

The influence of various additives, such as organic solvents, polyhydric alcohols, salts, polymers, and cross-linker, on the stability and storage ability of penicillinase-morphine conjugate was studied in liquid and solid (freeze dried) states. The results of these experiments showed that using low concentrations of CaCl2 (0.1-0.2%) could stabilize enzyme activity in both states for more than seven months. The immunoreactivity of antigen toward the antibody did not change significantly. However, a cross-linker such as glutaraldehyde and various additives such as dimethylsulfoxide, glycerol, polyethylene glycol, gelatin, dextran, ammonium sulfate, lactose, and sucrose did not have any effect on stability. In addition, it was found that the presence of lactose and sucrose in the lyophilization procedure gives a significant amount of protection to the enzyme, which could last for a period of seven months and preserve almost 95% of the enzyme activity, as well as immunoreactivity of the tracer molecule.

Characterization of a monoclonal antibody against neopterin using an enzyme-linked immunosorbent assay with penicillinase as label.

An active ester derivative of neopterin was prepared using 4-(N-maleimidomethyl) cyclohexan 4-carboxilic acid N-hydroxy succinimide ester (MCH-NHS), conjugated to bovine serum albumin (BSA) and injected for antibody production (for both monoclonal and polyclonal antibodies). High titer antibody producing spleen cells were removed and fused with myeloma cells of Sp2/0 origin. Neopterin was conjugated to the enzyme penicillinase by a one-step glutaraldehyde method, which was used as tracer. A novel enzyme immunoassay was developed using this conjugate to screen and characterize the monoclonal antibody (MAb) produced in these experiments. After limiting dilutions, it was found that antibody produced by one clone with a Ka value of 7.6 x 10-7 mol/L was specific for a number of structurally related molecules. This clone was found to be of IgG class and IgG2a subclass. The standard curvewas constructed with a sensitivity of 10 pg/well (100 pg/mL) covering up to 1 ng/mL.

Preparation and characterization of specific and high-affinity monoclonal antibodies against morphine.

A C6-hemisuccinate derivative of morphine was prepared and conjugated to bovine serum albumin. High titer antibody producing spleen cells were removed and fused with myeloma cells of Sp2/0 origin. A C3-hemisuccinate derivative of morphine was prepared and conjugated to enzyme penicillinase used as a tracer molecule. A novel enzyme-linked immunoadsorbent assay was developed using this conjugate to screen and characterize the monoclonal antibody produced in these experiments. After two successive limiting dilutions, antibodies produced by 5 clones with good affinities ranging from 10(8) to 10(12) M(-1) and less cross-reaction (least for codeine and other structurally related molecules) were selected. These clones were found to be of IgG class with kappa light chain. Subclass determination showed that two of the clones produced IgG2b and three of them produced IgG1 type of antibody. Affinity purifications were performed for the selected clone (MOR-I). Purified antibody was coated onto the wells of microtiter plate. The standard curve was constructed with a sensitivity of 100 pg/mL covering up to 10 ng/mL in buffer and urine. The slope of the standard curve for selected clone in buffer and urine was calculated to be -0.7 and -0.64, respectively.