Payload diversification: a key step in the development of antibody-drug conjugates.

Antibody-drug conjugates (ADCs) is a fast moving class of targeted biotherapeutics that currently combines the selectivity of monoclonal antibodies with the potency of a payload consisting of cytotoxic agents. For many years microtubule targeting and DNA-intercalating agents were at the forefront of ADC development. The recent approval and clinical success of trastuzumab deruxtecan (Enhertu®) and sacituzumab govitecan (Trodelvy®), two topoisomerase 1 inhibitor-based ADCs, has shown the potential of conjugating unconventional payloads with differentiated mechanisms of action. Among future developments in the ADC field, payload diversification is expected to play a key role as illustrated by a growing number of preclinical and clinical stage unconventional payload-conjugated ADCs. This review presents a comprehensive overview of validated, forgotten and newly developed payloads with different mechanisms of action.

Radiotherapy in Combination With Cytokine Treatment.

Radiotherapy (RT) plays an important role in the management of cancer patients. RT is used in more than 50% of patients during the course of their disease in a curative or palliative setting. In the past decades it became apparent that the abscopal effect induced by RT might be dependent on the activation of immune system, and that the induction of immunogenic cancer cell death and production of danger-associated molecular patterns from dying cells play a major role in the radiotherapy-mediated anti-tumor efficacy. Therefore, the combination of RT and immunotherapy is of a particular interest that is reflected in designing clinical trials to treat patients with various malignancies. The use of cytokines as immunoadjuvants in combination with RT has been explored over the last decades as one of the immunotherapeutic combinations to enhance the clinical response to anti-cancer treatment. Here we review mainly the data on the efficacy of IFN-α, IL-2, IL-2-based immunocytokines, GM-CSF, and TNF-α used in combinations with various radiotherapeutic techniques in clinical trials. Moreover, we discuss the potential of IL-15 and its analogs and IL-12 cytokines in combination with RT based on the efficacy in preclinical mouse tumor models.

High hydrostatic pressure in cancer immunotherapy and biomedicine.

High hydrostatic pressure (HHP) has been known to affect biological systems for >100 years. In this review, we describe the technology of HHP and its effect macromolecules and physiology of eukaryotic cells. We discuss the use of HHP in cancer immunotherapy to kill tumor cells for generation of whole cell and dendritic cell-based vaccines. We further summarize the current use and perspectives of HHP application in biomedicine, specifically in orthopedic surgery and for the viral, microbial and protozoan inactivation to develop vaccines against infectious diseases.

Severe, but not mild heat-shock treatment induces immunogenic cell death in cancer cells.

The mechanisms of immunogenicity underlying mild heat-shock (mHS) treatment < 42°C of tumor cells are largely attributed to the action of heat-shock proteins; however, little is known about the immunogenicity of tumor cells undergoing severe cytotoxic heat-shock treatment (sHS > 43°C). Here, we found that sHS, but not mHS (42°C), induces immunogenic cell death in human cancer cell lines as defined by the induction of ER stress response and ROS generation, cell surface exposure of calreticulin, HSP70 and HSP90, decrease of cell surface CD47, release of ATP and HMGB1. Only sHS-treated tumor cells were efficiently killed and phagocytosed by dendritic cells (DCs), which was partially dependent on cell surface calreticulin. DCs loaded with mHS or sHS-treated tumor cells displayed similar level of maturation and stimulated IFNγ-producing CD8+ T cells without any additional adjuvants in vitro. However, only DCs loaded with sHS-treated tumor cells stimulated antigen-specific CD4+ T cells and induced higher CD8+ T-cell activation and proliferation. sHS-treated murine cells also exposed calreticulin, HSP70 and HSP90 and activated higher DC maturation than mHS treated cells. Vaccination with sHS-treated tumor cells elicited protective immunity in mice. In this study, we defined specific conditions for the sHS treatment of human lung and ovarian tumor cells to arrive at optimal ratio between effective cell death, immunogenicity and content of tumor antigens for immunotherapeutic vaccine generation.

High hydrostatic pressure affects antigenic pool in tumor cells: Implication for dendritic cell-based cancer immunotherapy.

High hydrostatic pressure (HHP) can be used to generate dendritic cell (DC)-based active immunotherapy for prostate, lung and ovarian cancer. We showed here that HHP treatment of selected human cancer cell lines leads to a degradation of tumor antigens which depends on the magnitude of HHP applied and on the cancer cell line origin. Whereas prostate or ovarian cell lines displayed little protein antigen degradation with HHP treatment up to 300MPa after 2h, tumor antigens are hardly detected in lung cancer cell line after treatment with HHP 250MPa at the same time. On the other hand, quick reduction of tumor antigen-coding mRNA was observed at HHP 200MPa immediately after treatment in all cell lines tested. To optimize the DC-based active cellular therapy protocol for HHP-sensitive cell lines the immunogenicity of HHP-treated lung cancer cells at 150, 200 and 250MPa was compared. Lung cancer cells treated with HHP 150MPa display characteristics of immunogenic cell death, however cells are not efficiently phagocytosed by DC. Despite induction of the highest number of antigen-specific CD8+ T cells, 150 MPa-treated lung cancer cells survive in high numbers. This excludes their use in DC vaccine manufacturing. HHP of 200MPa treatment of lung cancer cells ensures the optimal ratio of efficient immunogenic killing and delivery of protein antigens in DC. These results represent an important pre-clinical data for generation of immunogenic killed lung cancer cells in ongoing NSCLC Phase I/II clinical trial using DC-based active cellular immunotherapy (DCVAC/LuCa).

Generation of dendritic cell-based vaccine using high hydrostatic pressure for non-small cell lung cancer immunotherapy.

High hydrostatic pressure (HHP) induces immunogenic death of tumor cells which confer protective anti-tumor immunity in vivo. Moreover, DC pulsed with HHP-treated tumor cells induced therapeutic effect in mouse cancer model. In this study, we tested the immunogenicity, stability and T cell stimulatory activity of human monocyte-derived dendritic cell (DC)-based HHP lung cancer vaccine generated in GMP compliant serum free medium using HHP 250 MPa. DC pulsed with HHP-killed lung cancer cells and poly(I:C) enhanced DC maturation, chemotactic migration and production of pro-inflammatory cytokines after 24h. Moreover, DC-based HHP lung cancer vaccine showed functional plasticity after transfer into serum-containing media and stimulation with LPS or CD40L after additional 24h. LPS and CD40L stimulation further differentially enhanced the expression of costimulatory molecules and production of IL-12p70. DC-based HHP lung cancer vaccine decreased the number of CD4+CD25+Foxp3+ T regulatory cells and stimulated IFN-γ-producing tumor antigen-specific CD4+ and CD8+ T cells from non-small cell lung cancer (NSCLC) patients. Tumor antigen specific CD8+ and CD4+ T cell responses were detected in NSCLC patient's against a selected tumor antigens expressed by lung cancer cell lines used for the vaccine generation. We also showed for the first time that protein antigen from HHP-killed lung cancer cells is processed and presented by DC to CD8+ T cells. Our results represent important preclinical data for ongoing NSCLC Phase I/II clinical trial using DC-based active cellular immunotherapy (DCVAC/LuCa) in combination with chemotherapy and immune enhancers.

Suppressive effect of citalopram on plasma concentrations of thromboxane B2.

BACKGROUND: Citalopram, a selective serotonin reuptake inhibitor (SSRi), is widely used to treat major depression. Patients treated with SSRIs suffer more frequently from bleeding disorders caused by the antiplatelet effect of SSRIs. METHODS: To investigate the potential suppressive effect of citalopram treatment on plasma thromboxane B2 levels and its possible correlation with actual plasma concentration of citalopram. Plasma concentrations of thromboxane B2 and citalopram were examined in a cohort of 77 aspirin-treated geriatric patients before and in the third week of citalopram therapy. RESULTS: Citalopram therapy led to a significant decrease of plasma concentrations of thromboxane B2 compared to its levels before initiation of the therapy. Furthermore, we have shown negative correlation in thromboxane B2 levels and actual plasma concentration of citalopram. Actual plasma concentrations of citalopram were significantly higher compared to younger adult patients treated with similar dose. CONCLUSIONS: In this study we have shown that even short-term citalopram therapy led to a suppression of thromboxane B2 production in aspirin-treated patients. This suppressive effect correlates with actual plasma concentration of citalopram.

Calcium influx rescues adenylate cyclase-hemolysin from rapid cell membrane removal and enables phagocyte permeabilization by toxin pores.

Bordetella adenylate cyclase toxin-hemolysin (CyaA) penetrates the cytoplasmic membrane of phagocytes and employs two distinct conformers to exert its multiple activities. One conformer forms cation-selective pores that permeabilize phagocyte membrane for efflux of cytosolic potassium. The other conformer conducts extracellular calcium ions across cytoplasmic membrane of cells, relocates into lipid rafts, translocates the adenylate cyclase enzyme (AC) domain into cells and converts cytosolic ATP to cAMP. We show that the calcium-conducting activity of CyaA controls the path and kinetics of endocytic removal of toxin pores from phagocyte membrane. The enzymatically inactive but calcium-conducting CyaA-AC⁻ toxoid was endocytosed via a clathrin-dependent pathway. In contrast, a doubly mutated (E570K+E581P) toxoid, unable to conduct Ca²âº into cells, was rapidly internalized by membrane macropinocytosis, unless rescued by Ca²âº influx promoted in trans by ionomycin or intact toxoid. Moreover, a fully pore-forming CyaA-ΔAC hemolysin failed to permeabilize phagocytes, unless endocytic removal of its pores from cell membrane was decelerated through Ca²âº influx promoted by molecules locked in a Ca²âº-conducting conformation by the 3D1 antibody. Inhibition of endocytosis also enabled the native B. pertussis-produced CyaA to induce lysis of J774A.1 macrophages at concentrations starting from 100 ng/ml. Hence, by mediating calcium influx into cells, the translocating conformer of CyaA controls the removal of bystander toxin pores from phagocyte membrane. This triggers a positive feedback loop of exacerbated cell permeabilization, where the efflux of cellular potassium yields further decreased toxin pore removal from cell membrane and this further enhances cell permeabilization and potassium efflux.

The purification step is not crucial in EIA measurements of thromboxane B2 and 11-dehydrothromboxane B2 in human plasma.

BACKGROUND: Thromboxane B2 (TxB2) and particularly 11-dehydrothromboxane B2 (11-dTxB2) are widely used as prognostic risk markers of platelet activation in cardiovascular diseases. The main errors in TxB2 and 11-dTxB2 determination include either low concentrations of circulating TxB2 (1 - 2 pg/mL) and 11-dTxB2 (0.9 - 4.3 pg/mL) or rather high transiency (mean TxB2 half-life is approximately 5 minutes) as well as an incorrect pre-analytical phase set up. The aim of this study was to investigate the impact of a widely used purification step on the results of enzyme immunosorbent assay (EIA)--based measurement of the two selected thromboxanes. METHODS: For the purpose of this study, 20 plasma samples (10 healthy donors, 10 patients under treatment with acetylsalicylic acid) were screened for TxB2 and 11-dTxB2 concentrations using commercial competitive EIA kits (Cayman Chemicals, Tallinn, Estonia; Neogen, Lexington, KY, USA) with or without the introduction of the purification procedure. RESULTS: The purification step does not significantly affect the results of EIA measurements of the two of TxA2 metabolites (TxB2, 11-dTxB2) in human plasma. The levels of TxB2 and 11-dTxB2 determined in the plasma samples were not significantly changed (p < 0.05) when the purification step was omitted compared to the purified samples. CONCLUSIONS: This study establishes a protocol allowing for reliable and reproducible plasma TxB2 and 11-dTxB2 EIA measurement for routine basic screening of platelet function.

Bacteria and their toxins tamed for immunotherapy.

Bacterial toxins share the ability to enter host cells to target various intracellular proteins and to modulate host immune responses. Over the last 20 years, toxins and their mutated variants, as well as live attenuated bacteria, have been exploited for vaccination and immunotherapy of various infectious, malignant and autoimmune diseases. The ability of Bordetella pertussis adenylate cyclase toxin to translocate its adenylate cyclase domain across the host cell membrane, as well as the pathways of intracellular trafficking of Bacillus anthracis lethal and edema toxins, Shigella dysenteriae shiga toxin or Escherichia coli shiga-like toxin, have been repeatedly exploited for the delivery of antigenic epitopes into host cells and for stimulation of antigen-specific T cell responses. Similarly, E. coli α-hemolysin, or effector proteins of Yersinia and Salmonella secreted by the type III secretion systems, were used to facilitate the delivery of fused heterologous proteins or peptides for antigenic presentation. Vibrio cholerae cholera toxin, E. coli heat-labile enterotoxin, B. pertussis pertussis toxin or the Cry1A protein of Bacillus thuringiensis have shown a great potential to act as adjuvants and to stimulate mucosal as well as systemic immune responses. The immunotherapeutic potential of some toxins, like Clostridium perfringens perfringolysin O, Streptococcus intermedius intermedilysin, or Streptococcus pneumoniae pneumolysin needs to be evaluated further. The Bordetella adenylate cyclase toxoid used as a vaccine delivery tool, or Corynebacterium diphtheriae diphtheria toxin and Pseudomonas aeruginosa exotoxin A-based immunotoxins, are currently in various phases of clinical trials for cancer immunotherapy, as are some antigen-delivering Salmonella and Listeria monocytogenes strains.

Evaluation of aspirin's effect on platelet function early after coronary artery bypass grafting.

OBJECTIVE: Aspirin therapy decreases mortality and ischemic complication rates after coronary artery bypass grafting (CABG). However, platelet inhibition after oral aspirin seems to be insufficient in the early postoperative period. There are incomplete data reporting aspirin efficacy early after CABG. The aim of this study was to assess the pharmacologic effect of aspirin on platelets in the first postoperative days using the most specific laboratory tests for the evaluation of aspirin efficacy. DESIGN: A prospective study. SETTING: A clinical study in one cardiac surgery center and measurements in two pharmacologic institutions. PARTICIPANTS: Thirty patients. INTERVENTIONS: Postoperative aspirin efficacy (200 mg/d) was assessed by the suppression of serum thromboxane B(2) (TxB(2)) and by arachidonic acid-induced aggregometry using the MULTIPLATE analyzer. Samples were collected before surgery and on postoperative days 1-5. METHODS AND MAIN RESULTS: The median baseline value (range) of serum TxB(2) was 1.6 ng/mL (1.4-1.9). The median TxB(2) inhibition >90% (the value required for full platelet inhibition) was not achieved until day 5 (-91%, 0.13 ng/mL [0.08-0.22], p < 0.001) and in only 55% of patients. The median baseline ASPI value was 805 (640-975) aggregation units (AU)*min. A significant decrease in aspirin insufficiency was not seen before postoperative day 5 (390 [243-621], p < 0.003) and only 34% of patients reached an effective platelet inhibition on day 5 (cutoff < 300 AU*min). CONCLUSIONS: The effect of aspirin on inhibition of TxB(2) production and arachidonic acid-induced platelet aggregation is impaired during the first postoperative days after CABG. A more effective antiplatelet strategy presumably could increase early graft patency and improve clinical outcomes after CABG.

Type IV fimbrial subunit protein ApfA contributes to protection against porcine pleuropneumonia.

Porcine pleuropneumonia caused by Actinobacillus pleuropneumoniae accounts for serious economic losses in the pig farming industry worldwide. We examined here the immunogenicity and protective efficacy of the recombinant type IV fimbrial subunit protein ApfA as a single antigen vaccine against pleuropneumonia, or as a component of a multi-antigen preparation comprising five other recombinant antigens derived from key virulence factors of A. pleuropneumoniae (ApxIA, ApxIIA, ApxIIIA, ApxIVA and TbpB). Immunization of pigs with recombinant ApfA alone induced high levels of specific serum antibodies and provided partial protection against challenge with the heterologous A. pleuropneumoniae serotype 9 strain. This protection was higher than that engendered by vaccination with rApxIVA or rTbpB alone and similar to that observed after immunization with the tri-antigen combination of rApxIA, rApxIIA and rApxIIIA. In addition, rApfA improved the vaccination potential of the penta-antigen mixture of rApxIA, rApxIIA, rApxIIIA, rApxIVA and rTbpB proteins, where the hexa-antigen vaccine containing rApfA conferred a high level of protection on pigs against the disease. Moreover, when rApfA was used for vaccination alone or in combination with other antigens, such immunization reduced the number of pigs colonized with the challenge strain. These results indicate that ApfA could be a valuable component of an efficient subunit vaccine for the prevention of porcine pleuropneumonia.

RTX proteins: a highly diverse family secreted by a common mechanism.

Repeats-in-toxin (RTX) exoproteins of Gram-negative bacteria form a steadily growing family of proteins with diverse biological functions. Their common feature is the unique mode of export across the bacterial envelope via the type I secretion system and the characteristic, typically nonapeptide, glycine- and aspartate-rich repeats binding Ca(2+) ions. In this review, we summarize the current state of knowledge on the organization of rtx loci and on the biological and biochemical activities of therein encoded proteins. Applying several types of bioinformatic screens on the steadily growing set of sequenced bacterial genomes, over 1000 RTX family members were detected, with the biological functions of most of them remaining to be characterized. Activities of the so far characterized RTX family members are then discussed and classified according to functional categories, ranging from the historically first characterized pore-forming RTX leukotoxins, through the large multifunctional enzymatic toxins, bacteriocins, nodulation proteins, surface layer proteins, up to secreted hydrolytic enzymes exhibiting metalloprotease or lipase activities of industrial interest.

Single-step affinity purification of recombinant proteins using a self-excising module from Neisseria meningitidis FrpC.

Purification of recombinant proteins is often a challenging process involving several chromatographic steps that must be optimized for each target protein. Here, we developed a self-excising module allowing single-step affinity chromatography purification of untagged recombinant proteins. It consists of a 250-residue-long self-processing module of the Neisseria meningitidis FrpC protein with a C-terminal affinity tag. The N terminus of the module is fused to the C terminus of a target protein of interest. Upon binding of the fusion protein to an affinity matrix from cell lysate and washing out contaminating proteins, site-specific cleavage of the Asp-Pro bond linking the target protein to the self-excising module is induced by calcium ions. This results in the release of the target protein with only a single aspartic acid residue added at the C terminus, while the self-excising affinity module remains trapped on the affinity matrix. The system was successfully tested with several target proteins, including glutathione-S-transferase, maltose-binding protein, beta-galactosidase, chloramphenicol acetyltransferase, and adenylate cyclase, and two different affinity tags, chitin-binding domain or poly-His. Moreover, it was demonstrated that it can be applied as an alternative to two currently existing systems, based on the self-splicing intein of Saccharomyces cerevisiae and sortase A of Staphylococcus aureus.