Recombinant Molecules Research Articles

Mitochondrial DNA duplication, recombination, and introgression during interspecific hybridization

mtDNA recombination events in yeasts are known, but altered mitochondrial genomes were not completed. Therefore, we analyzed recombined mtDNAs in six Saccharomyces cerevisiae × Saccharomyces paradoxus hybrids in detail. Assembled molecules contain mostly segments with variable length introgressed to other mtDNA. All recombination sites are in the vicinity of the mobile elements, introns in cox1, cob genes and free standing ORF1, ORF4. The transplaced regions involve co-converted proximal exon regions. Thus, these selfish elements are beneficial to the host if the mother molecule is challenged with another molecule for transmission to the progeny. They trigger mtDNA recombination ensuring the transfer of adjacent regions, into the progeny of recombinant molecules. The recombination of the large segments may result in mitotically stable duplication of several genes.

Comparative Analysis of the Clinical Use of 99mTechnetium-Labeled Recombinant Target Molecules in Different Dosages for the Radionuclide Diagnosis of Her2-Positive Breast Cancer

Background. The chief goal in assessing the Her2/neu status in clinical practice is to identify indications for targeted therapy. The main methods for determining the Her2/neu status are an immunohistochemical method and fluorescence in situ hybridization (FISH); however, despite their widespread use, they have a number of significant disadvantages. Over the past few years, radionuclide diagnosis using a new class of alternative scaffold proteins that meet all the requirements for optimal radionuclide delivery to tumor cells has become widespread. Objective: comparative analysis of the effectiveness of radionuclide imaging of Her2-positive breast cancer using 99m technetium-labeled recombinant molecules in different dosages. Material and methods. The investigation enrolled 22 patients with breast cancer (T1-4N0-2M0) before systemic therapy: 11 had Her2/neu overexpression; 11 had no marker expression. The patients’ mean age was 50.7 ± 2.3 years. Morphological and immunohistochemical studies were performed in all cases. FISHanalysis was carried out in the presence of the Her2/neu 2+ value. The agent was prepared immediately before using in dosages of 500 and 1000 µg, after which it was slowly administered intravenously to the patient. Whole-body scintigraphy and chest single-photon emission computed tomography were conducted at 2, 4, 6 and 24 hours after administration. Results. The radiochemical yield and radiochemical purity values were 77 ± 9% and 99 ± 1%, respectively. The activity of the agent immediately before administration was 416 ± 135 MBq for the 500 µg group and 349 ± 133 MBq for 1000 µg group. Analysis of the findings indicated that the higher uptake of the agent by organs without tumor lesion was observed in the kidneys, liver, and lungs. The highest renal absorption of the agent was observed in both study groups (0.135 ± 0.042 and 0.191 ± 0.047 mGy, respectively). The effective dose was 0.009 ± 0.002 mGy for the 500 µg group and 0.010 ± 0.003 mGy for the 1000 µg group. The better distribution between the tumors with Her2/neu positive and negative statuses was observed 2 hours after administration in the 500 µg group with the mean tumor/background value of 37 ± 19 for Her2-positive tumors, and 5 ± 2 for Her2-negative tumors (p < 0.001). Conclusion. The findings suggest that the test radiopharmaceutical agent at a dose of 500 µg can be considered as a new additional method to diagnose Her2-positive breast tumors.

Bioengineered microRNAs in the control of folate cycle related one‐carbon metabolism in NSCLC metabolism

Lung cancer remains as the leading cause of cancer deaths in the US and worldwide, of which non-small cell lung cancer (NSCLC) accounts for 80-85%. Very recently, we have established a novel technology to achieve in vivo fermentation production of bioengineered miRNA agents for the study of cancer biology and new therapies. Using recombinant miRNA molecules produced and folded in living cells, a number of the top most effective miRNAs against NSCLC cell variability were identified, among which miR-22-3p, miR-9-5p and miR-218-5p were all predicted to interfere with folate cycle of one-carbon metabolism. The serine hydroxymethyltransferase-1 (SHMT-1), a tumor biomarker and important enzyme in folate biotransformation, was verified as a new target for both miR-9-5p and miR-218-5p. The methylenetetrahydrofolate dehydrogenase 1 like (MTHFD1L) and methylenetetrahydrofolate dehydrogenase-2 (MTHFD2) were proven to be regulated by miR-9-5p directly. Furthermore, both methylenetetrahydrofolate reductase (MTHFR) and MTHFD2 were significantly suppressed by miR-22-3p in NSCLC cells. LC-MS/MS methods revealed that folate metabolites and amino acid metabolome were altered remarkably by individual miRNAs in human NSCLC cells. Further isotope labeled glucose feeding experiments showed that miR-22-3p affected intracellular supply of serine through the inhibition of glucose transporter GLUT1, and miR-9-5p and miR-218-5p decreased serine levels via downregulation of serine biosynthesis enzymes. Consequently, glycolytic rate and mitochondrial function were significantly inhibited in NSCLC cells by these miRNAs, accompanied by cell cycle arrest, ROS accumulation and NADP+/NADPH imbalance. Finally, miR-22-3p-loaded lipopolyplex was shown to significantly inhibit tumor growth in NSCLC patient-derived xenograft (PDX) mouse models in vivo, attributable to on-target actions. In conclusion, the study demonstrates that bioengineered miR-22-3p, miR-9-5p, and miR-218-5p act on folate cycle to suppress NSCLC cell metabolism, among which miR-22-3p is effective to control NSCLC tumor growth in PDX mice. These findings provide new insights into mechanistic actions of tumor suppressive miRNAs and development of new therapeutic strategies for NSCLC.

Dendritic Cell Tumor Vaccination via Fc Gamma Receptor Targeting: Lessons Learned from Pre-Clinical and Translational Studies.

Despite significant recent improvements in the field of immunotherapy, cancer remains a heavy burden on patients and healthcare systems. In recent years, immunotherapies have led to remarkable strides in treating certain cancers. However, despite the success of checkpoint inhibitors and the advent of cellular therapies, novel strategies need to be explored to (1) improve treatment in patients where these approaches fail and (2) make such treatments widely and financially accessible. Vaccines based on tumor antigens (Ag) have emerged as an innovative strategy with the potential to address these areas. Here, we review the fundamental aspects relevant for the development of cancer vaccines and the critical role of dendritic cells (DCs) in this process. We first offer a general overview of DC biology and routes of Ag presentation eliciting effective T cell-mediated immune responses. We then present new therapeutic avenues specifically targeting Fc gamma receptors (FcγR) as a means to deliver antigen selectively to DCs and its effects on T-cell activation. We present an overview of the mechanistic aspects of FcγR-mediated DC targeting, as well as potential tumor vaccination strategies based on preclinical and translational studies. In particular, we highlight recent developments in the field of recombinant immune complex-like large molecules and their potential for DC-mediated tumor vaccination in the clinic. These findings go beyond cancer research and may be of relevance for other disease areas that could benefit from FcγR-targeted antigen delivery, such as autoimmunity and infectious diseases.

Microarray-Based Allergy Diagnosis: Quo Vadis?

More than 30% of the world population suffers from allergy. Allergic individuals are characterized by the production of immunoglobulin E (IgE) antibodies against innocuous environmental allergens. Upon allergen recognition IgE mediates allergen-specific immediate and late-phase allergic inflammation in different organs. The identification of the disease-causing allergens by demonstrating the presence of allergen-specific IgE is the key to precision medicine in allergy because it allows tailoring different forms of prevention and treatment according to the sensitization profiles of individual allergic patients. More than 30 years ago molecular cloning started to accelerate the identification of the disease-causing allergen molecules and enabled their production as recombinant molecules. Based on recombinant allergen molecules, molecular allergy diagnosis was introduced into clinical practice and allowed dissecting the molecular sensitization profiles of allergic patients. In 2002 it was demonstrated that microarray technology allows assembling large numbers of allergen molecules on chips for the rapid serological testing of IgE sensitizations with small volumes of serum. Since then microarrayed allergens have revolutionized research and diagnosis in allergy, but several unmet needs remain. Here we show that detection of IgE- and IgG-reactivity to a panel of respiratory allergens microarrayed onto silicon elements is more sensitive than glass-based chips. We discuss the advantages of silicon-based allergen microarrays and how this technology will allow addressing hitherto unmet needs in microarray-based allergy diagnosis. Importantly, it described how the assembly of silicon microarray elements may create different microarray formats for suiting different diagnostic applications such as quick testing of single patients, medium scale testing and fully automated large scale testing.

A hepatitis B virus transgenic mouse model with a conditional, recombinant, episomal genome

Background & AimsDevelopment of new and more effective therapies against hepatitis B virus (HBV) is limited by the lack of suitable small animal models. The HBV transgenic mouse model containing an integrated overlength 1.3-mer construct has yielded crucial insights, but this model unfortunately lacks covalently closed circular DNA (cccDNA), the episomal HBV transcriptional template, and cannot be cured given that HBV is integrated in every cell.MethodsTo solve these 2 problems, we generated a novel transgenic mouse (HBV1.1X), which generates an excisable circular HBV genome using Cre/LoxP technology. This model possesses a HBV1.1-mer cassette knocked into the ROSA26 locus and is designed for stable expression of viral proteins from birth, like the current HBV transgenic mouse model, before genomic excision with the introduction of Cre recombinase.ResultsWe demonstrated induction of recombinant cccDNA (rcccDNA) formation via viral or transgenic Cre expression in HBV1.1X mice, and the ability to regulate HBsAg and HBc expression with Cre in mice. Tamoxifen-inducible Cre could markedly downregulate baseline HBsAg levels from the integrated HBV genome. To demonstrate clearance of HBV from HBV1.1X mice, we administered adenovirus expressing Cre, which permanently and significantly reduced HBsAg and core antigen levels in the murine liver via rcccDNA excision and a subsequent immune response.ConclusionsThe HBV1.1X model is the first Cre-regulatable HBV transgenic mouse model and should be of value to mimic chronic HBV infection, with neonatal expression and tolerance of HBV antigens, and on-demand modulation of HBV expression.Lay summaryHepatitis B virus (HBV) can only naturally infect humans and chimpanzees. Mouse models have been developed with the HBV genome integrated into mouse chromosomes, but this prevents mice from being cured. We developed a new transgenic mouse model that allows for HBV to be excised from mouse chromosomes to form a recombinant circular DNA molecule resembling the natural circular HBV genome. HBV expression could be reduced in these mice, enabling curative therapies to be tested in this new mouse model.

SP-A and SP-D: Dual Functioning Immune Molecules With Antiviral and Immunomodulatory Properties.

Surfactant proteins A (SP-A) and D (SP-D) are soluble innate immune molecules which maintain lung homeostasis through their dual roles as anti-infectious and immunomodulatory agents. SP-A and SP-D bind numerous viruses including influenza A virus, respiratory syncytial virus (RSV) and human immunodeficiency virus (HIV), enhancing their clearance from mucosal points of entry and modulating the inflammatory response. They also have diverse roles in mediating innate and adaptive cell functions and in clearing apoptotic cells, allergens and other noxious particles. Here, we review how the properties of these first line defense molecules modulate inflammatory responses, as well as host-mediated immunopathology in response to viral infections. Since SP-A and SP-D are known to offer protection from viral and other infections, if their levels are decreased in some disease states as they are in severe asthma and chronic obstructive pulmonary disease (COPD), this may confer an increased risk of viral infection and exacerbations of disease. Recombinant molecules of SP-A and SP-D could be useful in both blocking respiratory viral infection while also modulating the immune system to prevent excessive inflammatory responses seen in, for example, RSV or coronavirus disease 2019 (COVID-19). Recombinant SP-A and SP-D could have therapeutic potential in neutralizing both current and future strains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus as well as modulating the inflammation-mediated pathology associated with COVID-19. A recombinant fragment of human (rfh)SP-D has recently been shown to neutralize SARS-CoV-2. Further work investigating the potential therapeutic role of SP-A and SP-D in COVID-19 and other infectious and inflammatory diseases is indicated.

Development of Antibody-Based PROTACs for the Degradation of the Cell-Surface Immune Checkpoint Protein PD-L1.

Targeted protein degradation has emerged as a new paradigm to manipulate cellular proteostasis. Proteolysis-targeting chimeras (PROTACs) are bifunctional small molecules that recruit an E3 ligase to a target protein of interest, promoting its ubiquitination and subsequent degradation. Here, we report the development of antibody-based PROTACs (AbTACs), fully recombinant bispecific antibodies that recruit membrane-bound E3 ligases for the degradation of cell-surface proteins. We show that an AbTAC can induce the lysosomal degradation of programmed death-ligand 1 by recruitment of the membrane-bound E3 ligase RNF43. AbTACs represent a new archetype within the PROTAC field to target cell-surface proteins with fully recombinant biological molecules.

Phase I clinical study of a new radiopharmaceutical based on recombinant target molecules DARPin9_29 labeled with technetium-99m for radionuclide diagnosis of the Her2/neu-positive breast cancer

Phase I clinical study of a new radiopharmaceutical based on recombinant target molecules DARPin9_29 labeled with technetium-99m for radionuclide diagnosis of the Her2/neu-positive breast cancer

Expression and Purification of tRNA/ pre-miRNA-Based Recombinant Noncoding RNAs.

Research on RNA function and therapeutic potential is dominated by the use of chemoengineered RNA mimics. Recent efforts have led to the establishment of novel technologies for the production of recombinant or bioengineered RNA molecules, which should better recapitulate the structures, functions and safety profiles of natural RNAs because both are produced and folded in living cells. Herein, we describe a robust approach for reproducible fermentation production of bioengineered RNA agents (BERAs) carrying warhead miRNAs, siRNAs, aptamers, or other forms of small RNAs, based upon an optimal hybrid tRNA/pre-miRNA carrier. Target BERA/sRNAs are readily purified by fast protein liquid chromatography (FPLC) to a high degree of homogeneity (>97%). This approach offers a consistent high-level expression (>30% of total bacterial RNAs) and large-scale production of ready-to-use BERAs (multiple to tens milligrams from 1L bacterial culture).

In vivo fermentation production of humanized noncoding RNAs carrying payload miRNAs for targeted anticancer therapy.

Rationale: Noncoding RNAs (ncRNAs) such as microRNAs (miRs or miRNAs) play important roles in the control of cellular processes through posttranscriptional gene regulation. However, ncRNA research is limited to utilizing RNA agents synthesized in vitro. Recombinant RNAs produced and folded in living cells shall better recapitulate biologic RNAs.Methods: Herein, we developed a novel platform for in vivo fermentation production of humanized recombinant ncRNA molecules, namely hBERAs, carrying payload miRNAs or siRNAs. Target hBERAs were purified by anion exchange FPLC method. Functions of hBERA/miRNAs were investigated in human carcinoma cells and antitumor activities were determined in orthotopic osteosarcoma xenograft spontaneous lung metastasis mouse models.Results: Proper human tRNAs were identified to couple with optimal hsa-pre-miR-34a as new fully-humanized ncRNA carriers to accommodate warhead miRNAs or siRNAs. A group of 30 target hBERAs were all heterogeneously overexpressed (each accounting for >40% of total bacterial RNA), which facilitated large-scale production (8-31 mg of individual hBERAs from 1L bacterial culture). Model hBERA/miR-34a-5p and miR-124-3p were selectively processed to warhead miRNAs in human carcinoma cells to modulate target gene expression, enhance apoptosis and inhibit invasiveness. In addition, bioengineered miR-34a-5p and miR-124-3p agents both reduced orthotopic osteosarcoma xenograft tumor growth and spontaneous pulmonary metastases significantly.Conclusion: This novel ncRNA bioengineering technology and resulting recombinant ncRNAs are unique additions to conventional technologies and tools for basic research and drug development.

The Abtamer: A Novel Antibody-Aptamer Conjugate for Electrochemical Biosensing Applications

Introduction: We propose an innovated semisynthetic biosensing molecule, the Abtamer, an antibody-aptamer one-to-one complex. An important consideration in the design of electrochemical biosensors is the molecular recognition element (MRE) from which the signal to be transduced is generated. Among MREs utilized for affinity principle based electrochemical biosensors, antibodies are commonly used due to their high sensitivity and selectivity towards specific targets. However, antibodies’ structural rigidity proves limiting in biosensing applications.In contrast, DNA/RNA aptamers are easily-synthesized, versatile biomolecules, which are able to exhibit a high affinity and sensitivity towards their selected target [1]. Owing to their structure, aptamers are inherently flexible which allows for pronounced conformational changes upon binding. Furthermore, their small size allows for the immobilization of a larger density of molecules on the sensor surface, aiding in their sensitivity [2]. However, these same characteristics put aptamers at a disadvantage in terms of their selectivity towards targets. Without various post-synthesis modifications, DNA/RNA oligonucleotides can exhibit nonspecific electrostatic interactions with other analytes in solution.To this end, we propose a new biosensing molecule for biomedical sensor applications based on antibody-aptamer conjugates, designated as “Abtamers,” which includes advantages of both antibodies and aptamers; e.g. high sensitivity, selectivity with molecular dynamics upon target binding, facilitating signals for sensing. We propose to use insulin as a model target for an electrochemical sensor employing this novel biorecognition element. The electrochemical detection of insulin levels in persons with diabetes would provide another useful metric in diabetes technology, as insulin measurements are typically done via ELISA. Methods: The antibody-aptamer conjugate constructed utilized an insulin single chain variable fragment (scFv) and an insulin aptamer. To circumvent the non-optimal binding of polypeptides to oligonucleotides, we utilized the SpyCatcher/SpyTag complex as the bioconjugation system to couple the scFv to the aptamer in a one-to-one manner [3-4]. We created a recombinant fusion molecule of the insulin scFv and SpyCatcher, and created a SpyTag-insulin aptamer fusion molecule. The combination of these two fusion molecules results in an insulin antibody-insulin aptamer conjugate. We aim to employ this new MRE to detect insulin via square wave voltammetry. Results and Discussion: Through the binding of the two fusion molecules, we have created an irreversibly bound conjugate, conserving the structures of both the antibody and aptamer. The modification of a redox probe to the insulin aptamer domain in the Abtamer conjugate allows for a simple and facile method to create an electroactive biomolecule which can be immobilized to an electrode surface. We expect the binding affinity of this molecule to be higher towards specified targets due to both an antibody and aptamer binding event. Furthermore, the flexibility of the aptamer allows for conformational changes upon binding which can be exploited in electrochemical sensing applications. The developed insulin antibody-insulin aptamer conjugate can be applied as a biorecognition element for an electrochemical sensing platform, thereby realizing a unique biosensing principle, the “Abta-sensor”

Turoctocog alfa is safe for the treatment of Indian patients with hemophilia A: Guardian 10 trial results

BackgroundHemophilia A is an X chromosome–linked bleeding disorder caused by the deficiency of coagulation factor VIII (FVIII). The majority of the Indian population with hemophilia A use plasma‐derived clotting factors and, in some instances, fresh frozen plasma and cryoprecipitate. Safer and more efficient treatment options are needed for this group of patients. ObjectivesTo assess the safety of turoctocog alfa, a third‐generation recombinant FVIII molecule, for the treatment and prophylaxis of bleeding episodes in previously treated Indian patients with moderate or severe hemophilia A. Patients/MethodsThis single‐country, multicenter, open‐label, nonrandomized trial enrolled 60 patients who received prophylactic treatment with turoctocog alfa for 8 weeks, which corresponded to a minimum of 20 exposure days. Confirmed development of FVIII inhibitors during the 8‐week treatment period was evaluated. Other assessments included frequencies of adverse drug reactions (ARs), serious adverse reactions, drug‐related allergic reactions, and infusion reactions during the 12‐week period after the first treatment; hemostatic effect of turoctocog alfa for the treatment of bleeding episodes; and total annualized dose of turoctocog alfa administered during the 8‐week treatment period. ResultsNo incidence of FVIII inhibitors was detected. No safety concerns such as ARs, serious ARs, or drug‐related allergic reactions were noted. The hemostatic success rate for the treatment of bleeding episodes with turoctocog alfa was 81.6%. ConclusionsThe trial results demonstrated that turoctocog alfa is a safe treatment option for the prophylaxis and treatment of bleeding episodes in previously treated adolescent and adult patients with hemophilia A in the Indian population.

Combination of epidural electrical stimulation with ex vivo triple gene therapy for spinal cord injury: a proof of principle study.

Despite emerging contemporary biotechnological methods such as gene- and stem cell-based therapy, there are no clinically established therapeutic strategies for neural regeneration after spinal cord injury. Our previous studies have demonstrated that transplantation of genetically engineered human umbilical cord blood mononuclear cells producing three recombinant therapeutic molecules, including vascular endothelial growth factor (VEGF), glial cell-line derived neurotrophic factor (GDNF), and neural cell adhesion molecule (NCAM) can improve morpho-functional recovery of injured spinal cord in rats and mini-pigs. To investigate the efficacy of human umbilical cord blood mononuclear cells-mediated triple-gene therapy combined with epidural electrical stimulation in the treatment of spinal cord injury, in this study, rats with moderate spinal cord contusion injury were intrathecally infused with human umbilical cord blood mononuclear cells expressing recombinant genes VEGF165, GDNF, NCAM1 at 4 hours after spinal cord injury. Three days after injury, epidural stimulations were given simultaneously above the lesion site at C5 (to stimulate the cervical network related to forelimb functions) and below the lesion site at L2 (to activate the central pattern generators) every other day for 4 weeks. Rats subjected to the combined treatment showed a limited functional improvement of the knee joint, high preservation of muscle fiber area in tibialis anterior muscle and increased H/M ratio in gastrocnemius muscle 30 days after spinal cord injury. However, beneficial cellular outcomes such as reduced apoptosis and increased sparing of the gray and white matters, and enhanced expression of heat shock and synaptic proteins were found in rats with spinal cord injury subjected to the combined epidural electrical stimulation with gene therapy. This study presents the first proof of principle study of combination of the multisite epidural electrical stimulation with ex vivo triple gene therapy (VEGF, GDNF and NCAM) for treatment of spinal cord injury in rat models. The animal protocols were approved by the Kazan State Medical University Animal Care and Use Committee (approval No. 2.20.02.18) on February 20, 2018.

Targeted Injection of a Truncated Form of Tissue Inhibitor of Metalloproteinase 3 Alters Post-Myocardial Infarction Remodeling.

Infarct expansion can occur after myocardial infarction (MI), which leads to adverse left ventricular (LV) remodeling and failure. An imbalance between matrix metalloproteinase (MMP) induction and tissue inhibitors of MMPs (TIMPs) can accelerate this process. Past studies have shown different biologic effects of TIMP-3, which may depend upon specific domains within the TIMP-3 molecule. This study tested the hypothesis that differential effects of direct myocardial injections of either a full-length recombinant TIMP-3 (F-TIMP-3) or a truncated form encompassing the N-terminal region (N-TIMP-3) could be identified post-MI. MI was induced in pigs that were randomized for MI injections (30 mg) and received targeted injections within the MI region of F-TIMP-3 (n = 8), N-TIMP-3 (n = 9), or saline injection (MI-only, n = 11). At 14 days post-MI, LV ejection fraction fell post-MI but remained higher in both TIMP-3 groups. Tumor necrosis factor and interleukin-10 mRNA increased by over 10-fold in the MI-only and N-TIMP-3 groups but were reduced with F-TIMP-3 at this post-MI time point. Direct MI injection of either a full-length or truncated form of TIMP-3 is sufficient to favorably alter the course of post-MI remodeling. The functional and differential relevance of TIMP-3 domains has been established in vivo since the TIMP-3 constructs demonstrated different MMP/cytokine expression profiles. These translational studies identify a unique and more specific therapeutic strategy to alter the course of LV remodeling and dysfunction after MI. SIGNIFICANCE STATEMENT: Using different formulations of tissue inhibitor of matrix metalloproteinase-3 (TIMP-3), when injected into the myocardial infarction (MI) region, slowed the progression of indices of left ventricular (LV) failure, suggesting that the N terminus of TIMP-3 is sufficient to attenuate early adverse functional events post-MI. Injections of full-length recombinant TIMP-3, but not of the N-terminal region of TIMP-3, reduced relative indices of inflammation at the mRNA level, suggesting that the C-terminal region affects other biological pathways. These unique proof-of-concept studies demonstrate the feasibility of using recombinant small molecules to selectively interrupt adverse LV remodeling post-MI.

Retro-inverso D-peptides as a novel targeted immunotherapy for Type 1 diabetes

Over the past four decades, the number of people with Type 1 Diabetes (T1D) has increased by 4% per year, making it an important public health challenge. Currently, no curative therapy exists for T1D and the only available treatment is insulin replacement. HLA-DQ8 has been shown to present antigenic islet peptides driving the activation of CD4+ T-cells in T1D patients. Specifically, the insulin peptide InsB:9-23 activates self-reactive CD4+ T-cells, causing pancreatic beta cell destruction. The aim of the current study was to identify retro-inverso-d-amino acid based peptides (RI-D-peptides) that can suppress T-cell activation by blocking the presentation of InsB:9-23 peptide within HLA-DQ8 pocket. We identified a RI-D-peptide (RI-EXT) that inhibited InsB:9-23 binding to recombinant HLA-DQ8 molecule, as well as its binding to DQ8 expressed on human B-cells. RI-EXT prevented T-cell activation in a cellular antigen presentation assay containing human DQ8 cells loaded with InsB:9-23 peptide and murine T-cells expressing a human T-cell receptor specific for the InsB:9-23–DQ8 complex. Moreover, RI-EXT blocked T-cell activation by InsB:9-23 in a humanized DQ8 mice both ex vivo and in vivo, as shown by decreased production of IL-2 and IFN-γ and reduced lymphocyte proliferation. Interestingly, RI-EXT also blocked lymphocyte activation and proliferation by InsB:9-23 in PBMCs isolated from recent onset DQ8-T1D patients. In summary, we discovered a RI-D-peptide that blocks InsB:9-23 binding to HLA-DQ8 and its presentation to T-cells in T1D. These findings set the stage for using our approach as a novel therapy for patients with T1D and potentially other autoimmune diseases.

The effect of cell-mediated delivery of combination VEGF165, GDNF, and NCAM1 genes on molecular and cellular reactions in the spinal cord of pigs with contusion trauma

Currently, the treatments for spinal cord injury are limited. Gene therapy is one of the most promising approaches aimed at overcoming negative post-traumatic consequences in the spinal cord. Numerous studies performed in rodents indicate a positive effect of the delivery of therapeutic genes to the spinal cord to stimulate neuroregeneration. However, to bring the developed protocols of gene therapy to the stage of clinical trials, it is necessary to verify the results obtained in experiments on large laboratory animals. Objective: Immunofluorescence analysis of the response of markers of cell stress and apoptosis, synaptic proteins and neuroglia in the spinal cord of female vietnamese pot-bellied pigs after intrathecal delivery of genes encoding vascular endothelial growth factor (VEGF165), glial-derived neurotrophic factor and neuronal cell adhesion molecule (NCAM1), using human umbilical cord blood mononuclear cells (UCBMC). In experimental pigs (n = 2), 4 hours after modeling a dosed contusion injury of the spinal cord at the Th8-Th9 level, 2х106 genetically modified UCBMCs overexpressing recombinant VEGF, GDNF, and NCAM molecules in 200 |jl of saline were intrathecally injected. Control animals (n = 2) were injected with 200 jl of saline into the cerebrospinal fluid. Intact pigs (n = 2) were used to obtain baseline values for immunofluorescence analysis of post-traumatic molecular and cellular responses. After 60 days, immunofluorescence analysis in the rostral and caudal parts of the spinal cord relative to the epicenter of injury revealed positive changes in experimental pigs against the background of cell-mediated delivery of the VeGf165, GDNF, and NCAM1 genes. In the anterior horns of the rostral and caudal spinal cord of animals from the therapeutic group, a higher level of fluorescence of the synaptic protein synaptophysin, a lower number of astrocytes and microglial cells were found, which may indicate functional recovery of neurons and suppression of the development of astrogliosis. In the rostral section, in the area of the corticospinal tract, gene therapy maintained the number of oligodendrocytes, which ensure myelination of regenerating axons. The results obtained suggest that genetically modified UCBMCs, overexpressing recombinant molecules VEGF and GDNF (as therapeutic molecules) and NCAM (as a molecule providing survival and targeted targeting of cell carriers), contribute to post-traumatic regeneration of the spinal cord.

Antithrombin p.Thr147Ala: The First Founder Mutation in People of African Origin Responsible for Inherited Antithrombin Deficiency.

Hereditary antithrombin deficiency is a rare autosomal-dominant disorder predisposing to recurrent venous thromboembolism (VTE). To date, only two founder mutations have been described. We investigated the antithrombin p.Thr147Ala variant, found in 12 patients of African origin. This variant is known as rs2227606 with minor allele frequency of 0.5% in Africans and absent in Europeans. A possible founder effect was investigated. Phenotypical characterization was established through immunological and functional methods, both under basal and stress conditions. Recombinant antithrombin molecules were constructed by site-directed mutagenesis and expressed in HEK-293T cells. Secreted antithrombin was purified and functionally characterized. Structural modeling was performed to predict the impact of the mutation on protein structure. A novel nanopore sequencing approach was used for haplotype investigation. Ten patients experienced VTE, stroke, or obstetric complications. Antithrombin antigen levels and anti-IIa activity were normal or slightly reduced while anti-Xa activity was reduced with only one commercial assay. On crossed immunoelectrophoresis, an increase of antithrombin fractions with reduced heparin affinity was observed under high ionic strength conditions but not under physiological conditions. The recombinant p.Thr147Ala protein displayed a reduced anti-Xa activity. Structural modeling revealed that residue Thr147 forms three hydrogen bonds that are abolished when mutated to alanine. The investigated patients shared a common haplotype involving 13 SERPINC1 intragenic single nucleotide polymorphisms. Antithrombin p.Thr147Ala, responsible for antithrombin type II heparin binding site deficiency, is the first founder mutation reported in people of African ancestry. This study further emphasizes the limitations of commercial methods to diagnose this specific subtype.

Abstract 5597: Tumor-derived GDF-15 suppresses T-lymphocyte recruitment to the tumor microenvironment

Abstract Background: Growth and differentiation factor 15 (GDF-15) is a remote member of the TGF-β protein family with low to absent expression in healthy tissue. During pregnancy GDF-15 is secreted in high amounts by placenta contributing to feto-maternal tolerance. Similarly, GDF-15 expression is upregulated following tissue injury serving as a key inhibitor of excessive and disruptive immune infiltration. Importantly, various major tumor types secrete high levels of GDF-15 correlating with poor prognosis and reduced overall survival. Methods: In a flow-adhesion assay different immune cell subsets pre-treated +/- GDF-15 were perfused over an activated layer of endothelial cells or recombinant adhesion molecules. Adhesion and transmigration processes were monitored by live imaging microscopy. Impact of a proprietary GDF-15 neutralizing antibody (CTL-002) regarding T cell trafficking were analyzed. In a humanized (CD34+-HPSC engrafted) PDX mouse model inoculated with the PD-L1hi and GDF-15 secreting human melanoma tumor cell line HV18-MK T cell infiltration was analysed +/- CTL-002 by FACS. Results: Adhesion of T cells to the endothelial cell layer was significantly impaired by addition of GDF-15. Among T-cell subsets CD8+ T-cells were most affected while adhesion of other immune cells was not reduced. Inhibitory effects of GDF-15 on CD8+ T-cell adhesion were comparable to potent blockade of LFA-1 by TS1/18 antibody and could be rescued by the anti-GDF-15 antibody CTL-002. In vivo, neutralization of GDF-15 in HV18-MK melanoma-bearing humanized mice by CTL-002 resulted in a strong increase of tumor infiltrating leukocyte numbers. Subset analysis revealed an overproportional enrichment of T-cells, especially CD8+-T cells. Conclusion: Our in vitro and in vivo data show that elevated GDF-15 levels block T-cell infiltration into the tumor tissue. Neutralizing GDF-15 with a proprietary antibody (CTL-002) restored the ability of T cells (especially CD8+-T-cells) to extravasate blood vessels and enter tumor tissue both in vitro and in vivo. As it is known that presence of tumor-infiltrating lymphocytes correlates with better patient outcomes in a multitude of cancers and is a predictor of response to checkpoint inhibitors, high levels of GDF-15 in the tumor may contribute to failure of immunotherapies and poor overall survival. Neutralization of GDF-15 could be a new and promising approach to increase response rates of immunotherapies and increase overall survival of cancer patients. Citation Format: Markus Haake, Neha Vashist, Sabrina Genssler, Kristin H. Eichler, Birgitt Fischer, Jessica Kammer, Paula S. Romer, Manfred Rudiger, Eugen Leo, Falk Nimmerjahn, Christine Schuberth-Wagner, Jorg Wischhusen. Tumor-derived GDF-15 suppresses T-lymphocyte recruitment to the tumor microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5597.

Red blood cell adhesion to ICAM-1 is mediated by fibrinogen and is associated with right-to-left shunts in sickle cell disease

AbstractSickle cell disease (SCD), which afflicts 100 000 Americans, as well as millions worldwide, is associated with anemia, lifelong morbidity, and early mortality. Abnormal adhesion of sickle red blood cells (RBCs) to activated vascular endothelium may contribute acutely to the initiation of painful vaso-occlusive crises and chronically to endothelial damage in SCD. Sickle RBCs adhere to activated endothelium through several adhesion mechanisms. In this study, using whole blood from 17 people with heterozygous SCD (HbS variant) and 55 people with homozygous SCD (HbSS) analyzed in an in vitro microfluidic assay, we present evidence for the adhesion of sickle RBCs to immobilized recombinant intercellular adhesion molecule 1 (ICAM-1). We show that sickle RBC adhesion to ICAM-1 in vitro is associated with evidence of hemolysis in vivo, marked by elevated lactate dehydrogenase levels, reticulocytosis, and lower fetal hemoglobin levels. Further, RBC adhesion to ICAM-1 correlates with a history of intracardiac or intrapulmonary right-to-left shunts. Studies of potential ICAM-1 ligands on RBC membranes revealed that RBC–ICAM-1 interactions were mediated by fibrinogen bound to the RBC membrane. We describe, for the first time, RBC rolling behavior on ICAM-1 under high shear rates. Our results suggest that firm adhesion of sickle RBCs to ICAM-1 most likely occurs in postcapillary venules at low physiological shear rates, which is facilitated by initial rolling in high shear regions (eg, capillaries). Inhibition of RBC and ICAM-1 interactions may constitute a novel therapeutic target in SCD.