Cytotoxic activity of T- and NK-cells can be efficiently retargeted against cancer cells using chimeric antigen receptors (CARs) and rTCRs. In the context of solid cancers, use of armored CAR T- and ...NK cells secreting additional anti-cancer molecules such as cytokines, chemokines, antibodies, BiTEs, inverted cytokine receptors, and checkpoint inhibitors, appears particularly promising, as this may help overcome immunosuppressive tumor microenvironment, attract bystander immune cells, and boost CAR T/NK-cell persistence. Placing the expression of such molecules under the transcriptional control downstream of CAR-mediated T/NK-cell activation offers the advantage of targeted delivery, high local concentration, and reduced toxicity. Several canonic DNA sequences that are known to function as activation-inducible promoters in human T and B cells have been described to date and typically encompass the multimers of NFkB and NFAT binding sites. However, relatively little is known about the DNA sequences that may function as activation-driven switches in the context of NK cells. We set out to compare the functionality of several activation-inducible promoters in primary human T cells, as well as in NK cell lines NK-92 and YT.
Lentiviral constructs were engineered to express two fluorescent reporters: mCherry under 4xNFAT, 2xNFkB, 5xNFkB, 10xNFkB, 30xNFkB promoters, as well as two variants of the CD69 promoter, and copGFP under the strong constitutive promoter of the human EF1a gene. Pseudotyped lentiviral particles obtained using these constructs were transduced into primary human T cells and NK-92 and YT cell lines expressing a CAR specific for PSMA. The transgenic cells obtained were activated by CD3/CD28 beads (T cells) or via a CAR (CAR-NK cell lines). Promoter activity before and after activation was assayed using FACS analysis.
In T cells, the CD69 promoter encompassing CNS1 and CNS2 regions displayed the highest signal/noise ratio. Intriguingly, in the context of CAR-YT cell line neither of the seven promoters tested displayed acceptable activation profile. In CAR-NK-92 cells, the largest fold activation (which was modest) was achieved with the 10xNFkB and 30xNFkB promoters, however its expression was clearly leaky in "resting" non-activated cells.
Unlike in T cells, the robust activation-driven inducible expression of genetic cassettes in NK cells requires unbiased genome-wide identification of promoter sequences.
In the absence of virus-targeting small-molecule drugs approved for the treatment and prevention of COVID-19, broadening the repertoire of potent SARS-CoV-2-neutralizing antibodies represents an ...important area of research in response to the ongoing pandemic. Systematic analysis of such antibodies and their combinations can be particularly instrumental for identification of candidates that may prove resistant to the emerging viral escape variants. Here, we isolated a panel of 23 RBD-specific human monoclonal antibodies from the B cells of convalescent patients. A surprisingly large proportion of such antibodies displayed potent virus-neutralizing activity both in vitro and in vivo. Four of the isolated nAbs can be categorized as ultrapotent with an apparent IC
below 16 ng/mL. We show that individual nAbs as well as dual combinations thereof retain activity against currently circulating SARS-CoV-2 variants of concern (such as B.1.1.7, B.1.351, B.1.617, and C.37), as well as against other viral variants. When used as a prophylactics or therapeutics, these nAbs could potently suppress viral replication and prevent lung pathology in SARS-CoV-2-infected hamsters. Our data contribute to the rational development of oligoclonal therapeutic nAb cocktails mitigating the risk of SARS-CoV-2 escape.
Introduction. The SARS-CoV-2 coronavirus pandemic has been a major challenge for all areas of medical science, causing a surge of new developments in various fields ranging from diagnostic techniques ...to therapeutic and preventive approaches. Intranasal vaccination is an innovative approach to immunization against SARS-CoV-2, which has attracted the attention of many drug developers. Dynamics of blood virus-neutralizing antibodies (VNAs) in recovered COVID-19 patients or vaccinated healthy volunteers is one of the objective parameters for assessing vaccine immunological efficacy, which requires high standards of bioanalytical techniques within the framework of clinical trials. Immunogenicity data on the two-component Salnavak®(intranasal) and Gam-COVID-Vac®(intramuscular) vaccination obtained in randomized double-blind multicenter phase 3 clinical trial interim analysis are presented. The objective of the study was to assess immunogenicity of intranasal and intramuscular vaccination against COVID-19 using a neutralization reaction with pseudoviral particles and HEK293T-hACE2 cell culture.Materials and methods.A total of 137 healthy volunteers with a baseline anti-RBD IgG level not exceeding 100 BAU/ml received immunization by a two-component (Ad26 and Ad5 based) intranasal or intramuscular vaccine administered on day 1 and day 21. Immunogenicity level based on VNA quantitative analysis using a neutralization reaction with pseudoviral particles and a HEK293T-hACE2 cell culture as well as to SARS-CoV-2 S-protein receptor-binding domain (anti-RBD) IgG antibodies on days 21 and 42 after administration of component I was assessed.Results.The geometric mean VNA titer against SARS-CoV-2 on day 42 was 238.34±3.93 and 616.94±3.73 in the Salnavac®and Gam-COVID-Vac®groups, respectively. Trial data shows sufficient immunological efficacy of both intramuscular and intranasal vaccines based on a high protection level at VNA titer of more than 100 while using the pseudoviral neutralization method. The geometric mean of the anti-RBD IgG level by day 42 was 131.22±3.91 and 782.03±3.04 in the Salnavac®and Gam-COVID-Vak®groups, respectively. A direct moderate correlation was shown between VHA and anti-RBD IgG.Conclusion. Neutralization reaction using pseudoviral particles was successfully validated and used to determine the VNA titer during clinical trial. Trial interim data revealed that intranasal vaccine Salnavac®vs intramuscular vaccine Gam-COVID-Vak®resulted in lower but sufficient stringency of humoral immunity.
The emergence of SARS-CoV-2 mutant variants has posed a significant challenge to both the prevention and treatment of COVID-19 with anti-coronaviral neutralizing antibodies. The latest viral variants ...demonstrate pronounced resistance to the vast majority of human monoclonal antibodies raised against the ancestral Wuhan variant. Less is known about the susceptibility of the evolved virus to camelid nanobodies developed at the start of the pandemic. In this study, we compared nanobody repertoires raised in the same llama after immunization with Wuhan's RBD variant and after subsequent serial immunization with a variety of RBD variants, including that of SARS-CoV-1. We show that initial immunization induced highly potent nanobodies, which efficiently protected Syrian hamsters from infection with the ancestral Wuhan virus. These nanobodies, however, mostly lacked the activity against SARS-CoV-2 omicron-pseudotyped viruses. In contrast, serial immunization with different RBD variants resulted in the generation of nanobodies demonstrating a higher degree of somatic mutagenesis and a broad range of neutralization. Four nanobodies recognizing distinct epitopes were shown to potently neutralize a spectrum of omicron variants, including those of the XBB sublineage. Our data show that nanobodies broadly neutralizing SARS-CoV-2 variants may be readily induced by a serial variant RBD immunization.
SARS-CoV-2 has a relatively high mutation rate, with the frequent emergence of new variants of concern (VOCs). Each subsequent variant is more difficult to neutralize by the sera of vaccinated ...individuals and convalescents. Some decrease in neutralizing activity against new SARS-CoV-2 variants has also been observed in patients vaccinated with Gam-COVID-Vac. In the present study, we analyzed the interplay between the history of a patient's repeated exposure to SARS-CoV-2 antigens and the breadth of neutralization activity. Our study includes four cohorts of patients: Gam-COVID-Vac booster vaccinated individuals (revaccinated, RV), twice-infected unvaccinated individuals (reinfected, RI), breakthrough infected (BI), and vaccinated convalescents (VC). We assessed S-protein-specific antibody levels and the ability of sera to neutralize lentiviral particles pseudotyped with Spike protein from the original Wuhan variant, as well as the Omicron variants BA.1 and BA.4/5. Individuals with hybrid immunity (BI and VC cohorts) exhibited significantly higher levels of virus-binding IgG and enhanced breadth of virus-neutralizing activity compared to individuals from either the revaccination or reinfection (RV and RI) cohorts. These findings suggest that a combination of infection and vaccination, regardless of the sequence, results in significantly higher levels of S-protein-specific IgG antibodies and the enhanced neutralization of SARS-CoV-2 variants, thereby underscoring the importance of hybrid immunity in the context of emerging viral variants.
The continued emergence of SARS-CoV-2 variants with immune evasion properties of concern, such as Delta (B.1.617.2) and Omicron (B.1.1.529), calls into question the extent of the antibody-mediated ...immune response from the virus. The presence of virus-neutralizing antibodies against SARS-CoV-2 in the blood serum of recovered and immunized volunteers is the most accurate indicator of the level of protective activity. Methods for reliable, sensitive and rapid detection of anti-SARS-CoV-2 nAbs are needed for preclinical and clinical vaccine research. In addition, quantification of virus-neutralizing antibodies in recovered COVID-19 subjects may be useful in identifying potential donors for passive immunization and therapeutic use of class G immunoglobulins. Pseudoviruses are actively used as an alternative to infectious viral isolates of pathogenicity groups III in serological studies to determine the titers of neutralizing antibodies formed in vaccinated or infected volunteers. In addition, using several pseudotypes with different reporter genes, it is possible to simultaneously detect antibodies to different types of viruses in one biological sample. Currently, the pseudolentiviral system is widely used, in which pseudoviral particles are obtained by transfection of producer cells with vectors of a multiplasmid system of 45 plasmids: one for the vector genome, the second for Gag-Pol, the third for Rev, and one or two for protein(s) envelope, or for the co-expression of a labeled viral protein such as GAG-GFP or VPR-GFP, the main advantage of which is safety due to the minimal risk of generating a replication-competent virus. The article discusses the development of a technique that allows to determine the presence of virus-specific neutralizing antibodies to the SARS-CoV-2 antigen in the blood serum of volunteers who have had a new coronavirus infection COVID-19 and/or immunized with specific prophylaxis drugs, healthy volunteers in a neutralization reaction on a HEK 293-cell culture. T-hAce2 using pseudotyped viral constructs based on human immunodeficiency virus. The results of the development and validation of the method, as well as its subsequent prospects for use, are shown.
The adoptive transfer of allogeneic CAR NK cells holds great promise as an anticancer modality due to the relative ease of manufacturing and genetic modification of NK cells, which translates into ...affordable pricing. Compared to the pronounced efficacy of CAR T cell therapy in the treatment of B cell malignancies, rigorous clinical and preclinical assessment of the antitumor properties of CAR NK cells has been lagging behind. In this brief review, we summarize the biological features of NK cells that may help define the therapeutic niche of CAR NK cells as well as create more potent NK cell-based anticancer products. In addition, we compare T cells and NK cells as the carriers of CARs using the data of single-cell transcriptomic analysis.
Fc receptor-like A (FCRLA) is an unusual member of the extended Fc receptor family. FCRLA has homology to receptors for the Fc portion of Ig (FCR) and to other FCRL proteins. However, unlike these ...other family representatives, which are typically transmembrane receptors with extracellular ligand-binding domains, FCRLA has no predicted transmembrane domain or N-linked glycosylation sites and is an intracellular protein. We show by confocal microscopy and biochemical assays that FCRLA is a soluble resident endoplasmic reticulum (ER) protein, but it does not possess the amino acid sequence KDEL as an ER retention motif in its C-terminus. Using a series of deletion mutants, we found that its ER retention is most likely mediated by the amino terminal partial Ig-like domain. We have identified ER-localized Ig as the FCRLA ligand. FCRLA is unique among the large family of Fc receptors, in that it is capable of associating with multiple Ig isotypes, IgM, IgG and IgA. Among hemopoietic cells, FCRLA expression is restricted to the B lineage and is most abundant in germinal center B lymphocytes. The studies reported here demonstrate that FCRLA is more broadly expressed among human B lineage cells than originally reported; it is found at significant levels in resting blood B cells and at varying levels in all B-cell subsets in tonsil.
The aim of this study was to fill important gaps in the evolutionary history of immunoglobulins by examining the structure and diversity of IgL genes in non-teleost ray-finned fish. First, based on ...the bioinformatic analysis of recent transcriptomic and genomic resources, we experimentally characterized the IgL genes in the chondrostean fish,
(sterlet). We show that this species has three loci encoding IgL kappa-like chains with a translocon-type gene organization and a single VJC cluster, encoding homogeneous lambda-like light chain. In addition, sterlet possesses sigma-like VL and J-CL genes, which are transcribed separately and both encode protein products with cleavable leader peptides. The Acipenseriformes IgL dataset was extended by the sequences mined in the databases of species belonging to other non-teleost lineages of ray-finned fish: Holostei and Polypteriformes. Inclusion of these new data into phylogenetic analysis showed a clear subdivision of IgL chains into five groups. The isotype described previously as the teleostean IgL lambda turned out to be a kappa and lambda chain paralog that emerged before the radiation of ray-finned fish. We designate this isotype as lambda-2. The phylogeny also showed that sigma-2 IgL chains initially regarded as specific for cartilaginous fish are present in holosteans, polypterids, and even in turtles. We conclude that there were five ancient IgL isotypes, which evolved differentially in various lineages of jawed vertebrates.
T and NK cells armed with chimeric antigen receptors (CAR) are promising tools for the specific elimination of cancer cells. In most CAR designs implemented to date, the recognition of target cells ...is mediated by single-chain variable fragments (scFvs) derived from murine monoclonal antibodies. This format, however, has a number of limitations, including its relatively large size and potential immunogenicity in humans. In this study, we explored the feasibility of using human fibronectin type III domains (Fn3) as the antigen recognition domain in CARs. Human Fn3 domains have lower predicted immunogenicity compared to mouse-derived sequences, and a reduced molecular weight compared to scFvs. We created a functional CAR using a VEGFR2-specific Fn3 module replacing the conventional scFv. The resulting FnCAR specifically potentiates the cytotoxic activity of human T cells and YT NK cells in the presence of VEGFR2-positive targets. These findings demonstrate that Fn3 domains can be used in CARs for antigen recognition.
PDF
