CAR-T Cell Therapy for Classical Hodgkin Lymphoma.

Classical Hodgkin lymphoma (cHL) is a malignancy characterized by the presence of Hodgkin and Reed-Sternberg (HRS) cells within a complex tumor microenvironment (TME). Despite advances in conventional therapies, a subset of cHL patients experience relapse or refractory disease, necessitating the exploration of novel treatment strategies. Chimeric antigen receptor T cell (CAR-T cell) therapy has emerged as a promising approach for the management of cHL, harnessing the power of genetically modified T cells to recognize and eliminate tumor cells. In this article, we provide an overview of the pathogenesis of cHL, highlighting the key molecular and cellular mechanisms involved. Additionally, we discuss the rationale for the development of CAR-T cell therapy in cHL, focusing on the identification of suitable targets on HRS cells (such as CD30, CD123, LMP1, and LMP2A), clonotypic lymphoma initiating B cells (CD19, CD20), and cells within the TME (CD123, CD19, CD20) for CAR-T cell design. Furthermore, we explore various strategies employed to enhance the efficacy and safety of CAR-T cell therapies in the treatment of cHL. Finally, we present an overview of the results obtained from clinical trials evaluating the efficacy of CAR-T cell therapies in cHL, highlighting their potential as a promising therapeutic option. Collectively, this article provides a comprehensive review of the current understanding of cHL pathogenesis and the rationale for CAR-T cell therapy development, offering insights into the future directions of this rapidly evolving field.

Design and Immunogenicity of SARS-CoV-2 DNA Vaccine Encoding RBD-PVXCP Fusion Protein.

The potential of immune-evasive mutation accumulation in the SARS-CoV-2 virus has led to its rapid spread, causing over 600 million confirmed cases and more than 6.5 million confirmed deaths. The huge demand for the rapid development and deployment of low-cost and effective vaccines against emerging variants has renewed interest in DNA vaccine technology. Here, we report the rapid generation and immunological evaluation of novel DNA vaccine candidates against the Wuhan-Hu-1 and Omicron variants based on the RBD protein fused with the Potato virus X coat protein (PVXCP). The delivery of DNA vaccines using electroporation in a two-dose regimen induced high-antibody titers and profound cellular responses in mice. The antibody titers induced against the Omicron variant of the vaccine were sufficient for effective protection against both Omicron and Wuhan-Hu-1 virus infections. The PVXCP protein in the vaccine construct shifted the immune response to the favorable Th1-like type and provided the oligomerization of RBD-PVXCP protein. Naked DNA delivery by needle-free injection allowed us to achieve antibody titers comparable with mRNA-LNP delivery in rabbits. These data identify the RBD-PVXCP DNA vaccine platform as a promising solution for robust and effective SARS-CoV-2 protection, supporting further translational study.

Isolation of an escape-resistant SARS-CoV-2 neutralizing nanobody from a novel synthetic nanobody library.

The COVID-19 pandemic not only resulted in a global crisis, but also accelerated vaccine development and antibody discovery. Herein we report a synthetic humanized VHH library development pipeline for nanomolar-range affinity VHH binders to SARS-CoV-2 variants of concern (VoC) receptor binding domains (RBD) isolation. Trinucleotide-based randomization of CDRs by Kunkel mutagenesis with the subsequent rolling-cycle amplification resulted in more than 1011 diverse phage display library in a manageable for a single person number of electroporation reactions. We identified a number of nanomolar-range affinity VHH binders to SARS-CoV-2 variants of concern (VoC) receptor binding domains (RBD) by screening a novel synthetic humanized antibody library. In order to explore the most robust and fast method for affinity improvement, we performed affinity maturation by CDR1 and CDR2 shuffling and avidity engineering by multivalent trimeric VHH fusion protein construction. As a result, H7-Fc and G12x3-Fc binders were developed with the affinities in nM and pM range respectively. Importantly, these affinities are weakly influenced by most of SARS-CoV-2 VoC mutations and they retain moderate binding to BA.4\5. The plaque reduction neutralization test (PRNT) resulted in IC50 = 100 ng\ml and 9.6 ng\ml for H7-Fc and G12x3-Fc antibodies, respectively, for the emerging Omicron BA.1 variant. Therefore, these VHH could expand the present landscape of SARS-CoV-2 neutralization binders with the therapeutic potential for present and future SARS-CoV-2 variants.

Safety and Immunogenicity of Combined DNA-Polyethylenimine and Oral Bacterial Idiotypic Vaccine for Patients with B-Cell Non-Hodgkin Lymphoma: A Pilot Study.

We report, in brief, the results of a phase I, non-randomized study of idiotypic DNA vaccination in patients with B-cell non-Hodgkin's lymphoma (ISRCTN31090206). The DNA sequence of lymphoma-derived immunoglobulin variable regions was used as a tumor-specific antigen fused to the potato virus X coat protein. A conjugate of plasmid DNA with polyethylenimine was used for the intramuscular injections, followed by a boost with an oral live-attenuated Salmonella vaccine carrying the same plasmid. The patients with a complete or partial response to previous chemotherapy received one or two courses of vaccination, including four injections at monthly intervals. The vaccine was well tolerated, with low-grade adverse events. The T-cell immune responses were assessed by ELISpot, at last vaccine, one week and one month post-vaccination, and were detected in 11/14 (78.6%) of the patients. In cases of progression requiring chemotherapy, or the presence of a positive MRD after the first course of vaccination, the patients underwent a second course of vaccination. At the end point, 6/19 vaccinated patients had disease stabilization, while 13/19 were in complete remission. The overall survival was 100% at follow-up, of a median of 2.3 years.

Combining of synthetic VHH and immune scFv libraries for pregnancy-associated glycoproteins ELISA development.

Nanobodies (VHH) from camelid antibody libraries hold great promise as therapeutic agents and components of immunoassay systems. Synthetic antibody libraries that could be designed and generated once and for various applications could yield binders to virtually any targets, even for non-immunogenic or toxic ones, in a short term. One of the most difficult tasks is to obtain antibodies with a high affinity and specificity to polyglycosylated proteins. It requires antibody libraries with extremely high functional diversity and the use of sophisticated selection techniques. Here we report a development of a novel sandwich immunoassay involving a combination of the synthetic library-derived VHH-Fc fusion protein as a capture antibody and the immune single-chain fragment variable (scFv) as a tracer for the detection of pregnancy-associated glycoprotein (PAG) of cattle (Bos taurus). We succeeded in the generation of a number of specific scFv antibodies against PAG from the mouse immune library. Subsequent selection using the immobilized scFv-Fc capture antibody allowed to isolate 1.9 nM VHH binder from the diverse synthetic library without any overlapping with the capture antibody binding site. The prototype sandwich ELISA based on the synthetic VHH and the immune scFv was established. This is the first successful example of the combination of synthetic and immune antibody libraries in a single sandwich immunoassay. Thus, our approach could be used for the express isolation of antibody pairs and the development of sandwich immunoassays for challenging antigens. KEY POINTS: • Heavily glycosylated PAG Bos Taurus were used for immune library construction and specific scFv isolation by phage display. • Nanomolar affinity VHH for PAG was selected from the original synthetic nanobodies library. • A novel VHH/scFv-based immunoassay for Bos Taurus pregnancy determination was developed.

Development of CYB5-fusion monitoring system for efficient periplasmic expression of multimeric proteins in Escherichia coli.

Despite all advances of heterologous expression of recombinant proteins in Escherichia coli, expression of multidomain disulphide-rich proteins faces some problems due to the absence of the possibility to monitor the process in real-time. Here we provide a CYB5-fusion system - cytochrome b5 fusion system for periplasmic expression of multimeric proteins with the possibility of process monitoring. We validated this system by Fab and scFv antibody fragments expression in order to improve antibody-derived molecules characterization and to simplify their usage. The combination of redox dependent absorbance spectrum of red-colored cytochrome b5 with its high value molar extinction coefficient allows us to monitor antibody fusion proteins localization, redox state and quantify them in reliable way in turbid solutions. Moreover, it was revealed that due to outstanding stability and solubility, cytochrome b5 significantly enhances expression level of Fab/scFv antibody fragments in Escherichia coli periplasm.