Recombinant Chimeric Protein Research Articles

Infusion Reactions to Infliximab in Pediatric Patients with Inflammatory Bowel Disease

Infliximab (IFX) is a recombinant DNA-derived chimeric IgG monoclonal antibody protein that inhibits tumor necrosis factor alpha (TNF-α). IFX, like other agents derived from foreign proteins, can cause infusion reactions both during and after the infusion. The incidence of infusion reactions ranges between 0% and 15% in pediatric patients. The potential underlying mechanisms for these reactions may include anaphylaxis and anaphylactoid reactions, cytokine release syndrome, serum sickness-like reactions, and the development of antibodies against IFX. Several precautions can help reduce the risk of a new infusion reaction, such as a gradual increase in the infusion rate, scheduled infusions, and administering premedication or immunomodulators alongside IFX. Acute mild to moderate reactions often resolve spontaneously after a temporary cessation of the infusion or reduction in the infusion rate. Strategies like graded dose challenges and premedication can be utilized to prevent recurrence. In cases of severe reactions, desensitization or switching to an alternative biologic may be considered. This article aims to review the most recent guidelines for managing IFX-related infusion reactions in pediatric patients with inflammatory bowel disease (IBD), relying on the best available evidence.

Development and evaluation of a recombinant multi-epitopic protein (TsMEP) as an antigen candidate for Tityus serrulatus antivenom production

Scorpionism is Brazil's most prevalent envenomation. Treatment typically involves the use of heterologous antivenoms derived from the immunization of horses with crude T. serrulatus venom (TsV). Due to the high toxicity of this immunogen, as well as the particular challenges associated with the use of venoms as antigens, there is interest in exploring new alternatives for reducing their use in antivenom production. In this study, ten linear B-cell epitopes from hyaluronidase and voltage-gated sodium channel toxins, previously identified using the SPOT-synthesis method, were selected to construct a recombinant chimeric MultiEpitopic Protein from T. serrulatus scorpion venom (TsMEP). We demonstrated that TsMEP is non-toxic, and antibodies elicited in rabbits against this antigen exhibited reactivity in ELISA with Brazilian T. serrulatus, T. bahiensis, T. obscurus, and T. stigmurus venoms, as well as with Peruvian Hadruroides lunatus and North African Androctonus australis hector scorpion venoms. In vivo and in vitro neutralization assays revealed that rabbit anti-TsMEP antibodies partially neutralize the hyaluronidase activity of T. serrulatus venom and its lethality. Data presented here suggests that this multiepitopic protein could be a promising candidate in experimental immunization approaches for antivenom production for clinical use against Tityus spp. venoms.

Immunogenicity analysis based on VP1 and VP2 proteins of bovine enterovirus

Bovine enterovirus (BEV) infection manifests as a spectrum of clinical signs affecting the respiratory, gastrointestinal, and reproductive systems in cattle. Outbreaks of this disease results in large economic losses to the bovine industry worldwide. Currently there are no efficacious vaccines and medicines to prevent BEV infection. In the present study, reverse transcription-polymerase chain reaction was used to amplify the VP1 and VP2 genes of BEV, enabling the synthesis of a chimeric recombinant protein which contained partial sequences from both proteins. Subsequently, the emulsified purified proteins with Freund’s adjuvant were used for triple-fold immunization of 4-week-old Institute of Cancer Research (ICR) mice. The mice were subsequently subjected to a challenge assay which elicited an immune response that was characterized by elevated titers of BEV-specific neutralizing antibodies. Notably, the results showed that the purification of pET32a-VP1 and pET32a-VP2 proteins markedly curtailed virus excretion and mitigated the histopathological damage usually associated with BEV infections. These were observed in the small intestines, kidneys and brain in infected animals. It also alleviated clinical symptoms such as hypothermia and weight loss. In summary, this study offers a theoretical and practical basis for BEV vaccine development.

Immunogenicity and Neutralization Potential of Recombinant Chimeric Protein Comprising the Catalytic Region of Gp63 of Leishmania and LTB against Leishmania donovani

Aim: To study the inhibition potential of antibody against a recombinant chimera comprising of the catalytic epitope of gp63 of Leishmania donovani and B subunit of heat-labile enterotoxin (LTB) in the functional activity of L. donovani. Background: Visceral leishmaniasis, caused by the protozoan parasite Leishmania donavani, is a major health problem and causes mortality in tropical regions. Protozoan proteases play a crucial role in the pathogenesis of the disease and in establishing infection by countering the host's innate immune responses, namely complement-mediated lysis and phagocytosis. A surface-bound metalloprotease (gp63) has been reported to be a major virulence factor resulting in the evasion of complement- mediated lysis, cleaving host extracellular and intracellular substrates, resulting in intra- phagolysosomal survival. Method: The epitope corresponding to the catalytic motif of gp63 of Leishmania donovani was fused with the B subunit of heat-labile enterotoxin, which is known to be immunogenic. The chimera was cloned to a prokaryotic expression vector and purified using Ni NTA affinity chromatography. Antibodies were generated against the purified fusion protein and analyzed for its ability to bind to the gp63 catalytic motif peptide by ELISA. The effect of fusion protein antibody on the functional activity of gp63 was evaluated by assessing the effect of purified IgGs on the protease activity and complement-mediated lysis of L. donovani promastigotes in vitro. Results: The present study reports that a recombinant chimera of the catalytic epitope of gp63 and B subunit of heat-labile enterotoxin (LTB) of E. coli, a potent adjuvant of humoral response can mount significant immune response towards the catalytic epitope. ELISA and Western blot analysis showed that the anti-fusion protein antiserum could recognize the native gp63. Also, it significantly inhibited the protease activity of promastigotes and subsequently increased complement-mediated lysis of the promastigotes in vitro. Conclusion: It could be concluded that the hybrid protein containing catalytic motif L. donovani gp63 protein and carrier protein (LTB) could elicit antibodies that could neutralise the functional activity of gp63 and thus could be a potential candidate for subunit leishmaniasis vaccine.

Enhancing the cytotoxicity of immunotoxins by facilitating their dissociation from target receptors under the reducing conditions of the endocytic pathway

Immunotoxins (ITs) are recombinant chimeric proteins that combine a protein toxin with a targeting moiety to facilitate the selective delivery of the toxin to cancer cells. Here, we present a novel strategy to enhance the cytosolic access of ITs by promoting their dissociation from target receptors under the reducing conditions of the endocytic pathway. We engineered monobodySS, a human fibronectin type III domain-based monobody with disulfide bond (SS)-containing paratopes, targeting receptors such as EGFR, EpCAM, Her2, and FAP. MonobodySS exhibited SS-dependent target receptor binding with a significant reduction in binding under reducing conditions. We then created monobodySS-based ITs carrying a 25 kDa fragment of Pseudomonas exotoxin A (PE25), termed monobodySS-PE25. These ITs showed dose-dependent cytotoxicity against target receptor-expressing cancer cells and a wider therapeutic window due to higher efficacy at lower doses compared to controls with SS reduction inhibited. ERSS/28-PE25, with a KD of 28 nM for EGFR, demonstrated superior tumor-killing potency compared to ER/21-PE25, which lacks an SS bond, at equivalent and lower doses. In vivo, ERSS/28-PE25 outperformed ER/21-PE25 in suppressing tumor growth in EGFR-overexpressing xenograft mouse models. This study presents a strategy for developing solid tumor-targeting ITs using SS-containing paratopes to enhance cytosolic delivery and antitumor efficacy.

Impact of vaccination with the Anaplasma phagocytophilum MSP4 chimeric antigen on gene expression in the rabbit host

There are currently no vaccines available to prevent and control of Anaplasma phagocytophilum, an intracellular bacterial pathogen transmitted by ticks that occurs in many regions of the world and causes disease in a wide range of domestic and wild hosts, including humans. Vaccines induce long-lasting immunity and could prevent or reduce transmission of this pathogen.Understanding how vaccines induce a protective response can be difficult due to the complexity of the immune system, which operates at many levels throughout the organism. New perspectives in vaccinology, based on systems biology approaches, integrate many scientific disciplines to fully understand the biological responses to vaccination, where a transcriptomic approach could reveal relevant information of the host immune system, allowing profiling for rational design of vaccine formulations, administration, and potential protection.In the present study we report the gene expression profiles by RNA-seq followed by functional analysis using whole blood samples from rabbits immunized with a recombinant chimeric protein containing peptides from the MSP4 protein of A. phagocytophilum, which showed satisfactory results in terms of potential protection. Transcriptomic analysis revealed differential expression of 720 genes, with 346 genes upregulated and 374 genes downregulated. Overrepresentation of biological and metabolic pathways correlated with immune response, protein signaling, cytoskeleton organization and protein synthesis were found. These changes in gene expression could provide a complete and unique picture of the biological response to the epitope candidate vaccine against A. phagocytophilum in the host.

Antiviral therapy in cats progressively infected with feline leukaemia virus: lessons from a series of 18 consecutive cases from Australia.

It is doubtful that any of the treatments proposed for feline leukaemia virus (FeLV) infection are effective, despite the entity being described 60 years ago. Eighteen pet cats with progressive FeLV infections were recruited in Australia. One or more antiviral drugs were trialled in 16 cats, while two FeLV-infected cats were not handleable and served as untreated controls. Six cats were administered RetroMAD1™ only (0.5 mg/kg orally twice daily), a commercially available recombinant chimeric protein with proposed antiretroviral activity. Three cats were administered the integrase inhibitor raltegravir only (10-15 mg/kg orally twice daily), a drug used as a component of highly effective antiretroviral therapy for human immunodeficiency virus (HIV-1) infection. Three cats were administered RetroMAD1™ and raltegravir concurrently, and four cats were administered raltegravir and the reverse transcriptase inhibitor zidovudine (AZT, 5 mg/kg orally twice daily) concurrently. FeLV RNA and p27 antigen loads were measured at two timepoints (T1-2 months and T3-5 months) during therapy and compared to baseline (pretreatment) levels, to assess the response to therapy using linear modelling. The median survival time (MST) of the cats from commencement of FeLV treatment to death was also determined and compared between treatments. The MST for the 16 FeLV-positive cats which received antiviral therapy was 634 days, while the MST from FeLV diagnosis to death for the two untreated control cats was 780 days. In cats treated with RetroMAD1™, FeLV viral load decreased from T0 to T1-2 months (median viral load reduced from 1339 × 106 to 705 × 106 copies/mL plasma; P = 0.012), but MST was reduced compared to cats not given RetroMAD1™ (426 days vs 1006 days; P = 0.049). Cats treated with raltegravir and AZT had no significant changes in FeLV viral load over time, but p27 antigen load was decreased from T0 to T3-5 months in cats treated with raltegravir (median p27 antigen level reduced from 50.2% to 42.7%; P = 0.005). All other results were not significantly affected by the treatment provided. Importantly, statistically significant and substantial associations were found between age at FeLV diagnosis and survival time (P = 0.046, R2 = 18.6) and between FeLV viral load at T0 and survival time (P = 0.004, R2 = 44.4). Younger cats, and cats with higher levels of pretreatment FeLV RNA, had reduced survival times. Cats treated with RetroMAD1™ were typically younger (median age 2.0 vs 8.0 years), likely explaining the observed reduction in MST. A significant association was found between FeLV viral load and p27 antigen load at T0 (P = 0.015, R2 = 32.9). Results from this small case series do not provide convincing support for the use of RetroMAD1™, raltegravir or AZT, alone or in combination, for the treatment of cats progressively infected with FeLV. The changes observed were biologically insignificant. Age and FeLV viral load at diagnosis are useful prognostic markers, and p27 antigen concentration can be used to predict viral load. Larger field trials should be performed examining antiretroviral therapy in FeLV-positive cats with progressive infections, preferably using three or more drugs from at least two classes, as is standard with human antiretroviral therapy. Future studies would be easier in countries with a higher prevalence of FeLV infections than Australia.

Immunoinformatic approaches for ErpY-LemA chimeric protein design for use in leptospirosis control.

Currently, immunoinformatic approaches have shown promise in rapidly and cost-effectively identifying new antigens from the Leptospira proteome. Chimeric multiepitope proteins offer a strategy with significant potential for implementation in diagnosis and vaccines development. In this study, we detail the immunoinformatic analyses and design of a new recombinant chimeric protein constructed with epitopes identified from the sequences of ErpY-like and LemA proteins, previously identified as potential antigens for controlling leptospirosis. We expressed the chimeric protein using Escherichia coli heterologous systems, evaluated its antigenicity using serum from naturally infected patients, and its immunogenicity in mice as an animal model, with Freund as an adjuvant. The resulting recombinant chimeric protein, named rErpY-LemA, was successfully expressed and purified using a prokaryotic system, with an expected mass of 35kDa. Serologic assays using serum samples from naturally infected patients demonstrated recognition of the chimera protein by antibodies present in sera. Animals immunized with the chimera exhibited a significant IgG antibody response from the 7th day (P<0.001), persisting until day 49 of experimentation, with a titer of 1:12,800 (P<0.05). Notably, significant production of IgA, IgM, and IgG subclasses was observed in animals immunized with the chimera. These results highlight the promising role of immunoinformatics in rapidly identifying antigens and the potential of chimeric multiepitope proteins in developing effective strategies for leptospirosis control.

Treating late-onset Tay Sachs disease: Brain delivery with a dual trojan horse protein

Tay-Sachs (TS) disease is a neurodegenerative disease resulting from mutations in the gene encoding the α-subunit (HEXA) of lysosomal β –hexosaminidase A (HexA). We report that 1) recombinant HEXA alone increased HexA activity and reduced GM2 content in human TS glial cells and peripheral mononuclear blood cells; 2) a recombinant chimeric protein comprised of HEXA linked to two blood brain barrier (BBB) entry elements, a transferrin receptor binding sequence and G-CSF, associates with HEXB in vitro; reaches human cultured TS cells lysosomes and mouse brain cells, especially neurons, in vivo; lowers GM2 in cultured human TS cells; lowers whole brain GM2 concentration by ∼40% within 6 weeks , when injected intravenously to adult TS mutant mice mimicking the slow course of late onset TS; and increases forelimbs grip strength. Hence, a chimeric protein equipped with dual BBB entry elements can transport a large protein such as HEXA to the brain, reduce the accumulation of GM2 and improve muscle strength, thereby providing potential treatment for late-onset TS.

IgY-mediated protection against Vibrio cholerae infection: Efficacy of avian antibodies targeting a chimeric recombinant protein

Introduction: Vibrio cholerae, the etiologic pathogen of diarrheal disease, prevails mainly in developing countries, transmitted through contaminated water or food. The unique genetic makeup and remarkable competency has prompted intensive research to unravel the bacterium virulence properties. Egg yolk immunoglobulins (IgY) have emerged as innovative biotherapeutics for both passive immunotherapy and prophylactic strategies. Methods: In the present study, we generated avian antibodies against a chimeric recombinant protein comprising OmpW-TcpA-CtxB (OTC) antigens from V. cholerae, and examined its efficacy against bacterial toxins and infection. The chimeric protein was expressed in E. coli BL21 (DE3) and purified using Ni-NTA affinity chromatography. Leghorn chickens were intramuscularly immunized with the recombinant protein and the purity of extracted IgYs was assessed through SDS-PAGE analysis. The immunoreactivity and specificity of anti-OTC-IgYs were evaluated through protein and whole-cell ELISA, and their ability to neutralize cholera toxin (CT) of V. cholerae was evaluated in Y1 cell line. Finally, the protective efficacy of orally administered anti-OTC-IgY was investigated in V. cholerae-infected infant mice. Results: Anti-OTC-IgY successfully neutralized the cytotoxic effects of CT at a concentration of 250 µg/mL. Oral administration of two 100 µg doses of anti-OTC-IgY and resulted in 60% and 20% survival rates in suckling mice infected with LD and 10 LD of V. cholerae, respectively. Conclusion: The anti-OTC-IgY antibodies exhibited significant immunoreactivity, toxin-neutralizing potency, and protective effects, establishing their potential as promising antimicrobials against the bacterial pathogenicity through passive immunotherapy.

Immunogenicity of PvCyRPA, PvCelTOS and Pvs25 chimeric recombinant protein of Plasmodium vivax in murine model.

In the Americas, P. vivax is the predominant causative species of malaria, a debilitating and economically significant disease. Due to the complexity of the malaria parasite life cycle, a vaccine formulation with multiple antigens expressed in various parasite stages may represent an effective approach. Based on this, we previously designed and constructed a chimeric recombinant protein, PvRMC-1, composed by PvCyRPA, PvCelTOS, and Pvs25 epitopes. This chimeric protein was strongly recognized by naturally acquired antibodies from exposed population in the Brazilian Amazon. However, there was no investigation about the induced immune response of PvRMC-1. Therefore, in this work, we evaluated the immunogenicity of this chimeric antigen formulated in three distinct adjuvants: Stimune, AddaVax or Aluminum hydroxide (Al(OH)3) in BALB/c mice. Our results suggested that the chimeric protein PvRMC-1 were capable to generate humoral and cellular responses across all three formulations. Antibodies recognized full-length PvRMC-1 and linear B-cell epitopes from PvCyRPA, PvCelTOS, and Pvs25 individually. Moreover, mice's splenocytes were activated, producing IFN-γ in response to PvCelTOS and PvCyRPA peptide epitopes, affirming T-cell epitopes in the antigen. While aluminum hydroxide showed notable cellular response, Stimune and Addavax induced a more comprehensive immune response, encompassing both cellular and humoral components. Thus, our findings indicate that PvRMC-1 would be a promising multistage vaccine candidate that could advance to further preclinical studies.

A recombinant chimeric antigen constructed with B-cell epitopes from Mycobacterium leprae hypothetical proteins is effective for the diagnosis of leprosy

The diagnosis if leprosy is difficult, as it requires clinical expertise and sensitive laboratory tests. In this study, we develop a serological test for leprosy by using bioinformatics tools to identify specific B-cell epitopes from Mycobacterium leprae hypothetical proteins, which were used to construct a recombinant chimeric protein, M1. The synthetic peptides were obtained and showed good reactivity to detect leprosy patients, although the M1 chimera have showed sensitivity (Se) and specificity (Sp) values higher than 90.0% to diagnose both paucibacillary (PB) and multibacillary (MB) leprosy patients, but not those developing tegumentary or visceral leishmaniasis, tuberculosis, Chagas disease, malaria, histoplasmosis and aspergillosis, in ELISA experiments. Using sera from household contacts, values for Se and Sp were 100% and 65.3%, respectively. In conclusion, our proof-of-concept study has generated data that suggest that a new recombinant protein could be developed into a diagnostic antigen for leprosy.

The design of an RGD in situ sustained delivery system utilizing scallop byssal protein through genetic engineering

Although bioactive peptides enhancing bone healing have demonstrated effectiveness in treating bone defects, in vivo instability poses a challenge to their clinical application. Currently reported peptide delivery systems do not meet the demands of bone tissue repair regarding stability and peptide release efficacy. Herein, the self-assembling recombinant chimeric protein (Sbp5–2RGD) is developed by genetic engineering with cell adhesion peptide RGD as the targeted peptide and a newly discovered scallop byssal-derived protein Sbp5–2 that can assemble into wet stable films as the structural domain. In vitro studies show that the Sbp5–2RGD film exhibits excellent extensibility and biocompatibility. In vitro and in vivo degradation experiments demonstrate that the film remains stable due to the layer-by-layer degradation mode, resulting in sustained delivery of RGD in situ for up to 4 weeks. Consequently, the film can effectively promote osteogenesis, which accelerates bone defect healing and the implants osseointegration. Cell-level studies further show that the film up-regulates the expression of genes and proteins (ALP, OCN, OSX, OPN, RUNX2, VEGF) associated with osteogenesis and angiogenesis. Overall, this novel protein film represents an intelligent platform for peptide immobilization, protection, and release through its self-assembly, dense structure, and degradation mode, providing a therapeutic strategy for bone repair.

The Development of Toxoplasma gondii Recombinant Trivalent Chimeric Proteins as an Alternative to Toxoplasma Lysate Antigen (TLA) in Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of Immunoglobulin G (IgG) in Small Ruminants.

This study presents an evaluation of seventeen newly produced recombinant trivalent chimeric proteins (containing the same immunodominant fragment of SAG1 and SAG2 of Toxoplasma gondii antigens, and an additional immunodominant fragment of one of the parasite antigens, such as AMA1, GRA1, GRA2, GRA5, GRA6, GRA7, GRA9, LDH2, MAG1, MIC1, MIC3, P35, and ROP1) as a potential alternative to the whole-cell tachyzoite lysate (TLA) used in the detection of infection in small ruminants. These recombinant proteins, obtained by genetic engineering and molecular biology methods, were tested for their reactivity with specific anti-Toxoplasma IgG antibodies contained in serum samples of small ruminants (192 samples of sheep serum and 95 samples of goat serum) using an enzyme-linked immunosorbent assay (ELISA). The reactivity of six recombinant trivalent chimeric proteins (SAG1-SAG2-GRA5, SAG1-SAG2-GRA9, SAG1-SAG2-MIC1, SAG1-SAG2-MIC3, SAG1-SAG2-P35, and SAG1-SAG2-ROP1) with IgG antibodies generated during T. gondii invasion was comparable to the sensitivity of TLA-based IgG ELISA (100%). The obtained results show a strong correlation with the results obtained for TLA. This suggests that these protein preparations may be a potential alternative to TLA used in commercial tests and could be used to develop a cheaper test for the detection of parasite infection in small ruminants.

Serodiagnosis of paucibacillary and multibacillary leprosy using a recombinant chimeric protein composed of specific B-cell epitopes derived from Mycobacterium leprae proteins

Leprosy diagnosis is difficult due to the clinical similarity with other infectious diseases, and laboratory tests presents problems related to sensitivity and/or specificity. In this study, we used bioinformatics to assess Mycobacterium leprae proteins and formulated a chimeric protein that was tested as a diagnostic marker for the disease. The amino acid sequences from ML0008, ML0126, ML0308, ML1057, ML2028, ML2038, ML2498 proteins were evaluated, and the B-cell epitopes QASVAYPATSYADFRAHNHWWNGP, SLQRSISPNSYNTARVDP and QLLGQTADVAGAAKSGPVQPMGDRGSVSPVGQ were considered M. leprae-specific and used to construct the gene encoding the recombinant antigen. The gene was constructed, the recombinant protein was expressed, purified and tested in ELISA using 252 sera, which contained samples from multibacillary (MB) or paucibacillary (PB) leprosy patients, from their household contacts and healthy individuals, as well as from patients with Chagas disease, visceral and tegumentary leishmaniases (VL/TL), malaria, tuberculosis, and HIV. Sensitivity (Se) and specificity (Sp) for MB and PB samples compared to sera from both healthy subjects and individuals with cross-reactive diseases were 100%. The Se value for MB and PB samples compared to sera from household contacts was 100%, but Sp was 64%. In conclusion, data suggest that this protein could be considered in future studies for leprosy diagnosis.

Enhanced Immunogenicity and Protective Effects against SARS-CoV-2 Following Immunization with a Recombinant RBD-IgG Chimeric Protein.

The unprecedented global impact caused by SARS-CoV-2 imposed huge health and economic challenges, highlighting the urgent need for safe and effective vaccines. The receptor-binding domain (RBD) of SARS-CoV-2 is the major target for neutralizing antibodies and for vaccine formulations. Nonetheless, the low immunogenicity of the RBD requires the use of alternative strategies to enhance its immunological properties. Here, we evaluated the use of a subunit vaccine antigen generated after the genetic fusing of the RBD with a mouse IgG antibody. Subcutaneous administration of RBD-IgG led to the extended presence of the protein in the blood of immunized animals and enhanced RBD-specific IgG titers. Furthermore, RBD-IgG immunized mice elicited increased virus neutralizing antibody titers, measured both with pseudoviruses and with live original (Wuhan) SARS-CoV-2. Immunized K18-hACE2 mice were fully resistant to the lethal challenge of the Wuhan SARS-CoV-2, demonstrated by the control of body-weight loss and virus loads in their lungs and brains. Thus, we conclude that the genetic fusion of the RBD with an IgG molecule enhanced the immunogenicity of the antigen and the generation of virus-neutralizing antibodies, supporting the use of IgG chimeric antigens as an approach to improve the performance of SARS-CoV-2 subunit vaccines.

Abstract 7259: Effectiveness and comparison of CAR-T cell therapies for synovial sarcoma; HER2-targeted CAR-T and NKG2D-based CAR-T

Abstract Introduction: In Japan since 2019, CAR-T cell therapy has been used to treat patients with relapsed or refractory B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphoma and has shown excellent clinical results. Clinical studies of CAR-T cell therapies targeting various antigens, such as HER2, have been conducted, but the therapeutic outcome in sarcoma is still limited. We have previously reported that HER2-targeted CAR-T cells show antitumor effects for synovial sarcoma in vitro. The purpose of this study is to compare HER2-targeted CAR-T cells and NKG2D-based CAR-T cells in antitumor effect and specific responses for synovial sarcoma in vitro. Materials and Methods: Surface expressions of HER2 and NKG2D ligands on three synovial sarcoma cell lines were detected by using recombinant human HER2 Fc chimeric protein and that of NKG2D, followed by staining with PE-conjugated anti-human IgG secondary antibody. We generated 4-1BB-type second generation CAR-T cells targeting NKG2D ligands and examined their antitumor effects and specific responses against synovial sarcoma cells. In addition, we compared the differences between HER2-targeted CAR-T cells and NKG2D-based CAR-T cells by co-culturing with synovial sarcoma cells using short-term assay (WST-8 assay), long-term assay (Real-time Cell Analysis) and CD107a assay. Results: HER2 and NKG2D ligands were found to be expressed by flow cytometry in three synovial sarcoma cell lines. In vitro, NKG2D-based CAR-T cells showed antitumor effects against synovial sarcoma cells, including degranulation and cytokine production. Furthermore, NKG2D-based CAR-T cells showed stronger antitumor effects than HER2-targeted CAR-T cells in both short- and long-term assays and expressed higher levels of CD107a. Conclusions: In this study, NKG2D-based CAR-T cells showed more antitumor effects than HER2-targeted CAR-T cells in vitro. Although the reason for this is not clear, we hypothesize that NKG2D ligands may be induced in synovial sarcoma cells by co-culture with CAR-T cells because HER2 is a cancer-specific antigen, whereas NKG2D ligands are ligands for activating receptors of natural killer cells. Further studies, including in vivo studies, will be conducted to verify the difference in anti-tumor effects between these CAR-T cells. Citation Format: Tomohiro Miyazaki, Chihaya Imai, Naoki Oike, Yudai Murayama, Takashi Ariizumi, Minori Baba, Yasushi Kasahara, Hiroyuki Kawashima. Effectiveness and comparison of CAR-T cell therapies for synovial sarcoma; HER2-targeted CAR-T and NKG2D-based CAR-T [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7259.

Post-therapeutic cure criterion in chronic Chagas disease using Trypanosoma cruzi chimeric proteins.

Chagas disease (CD) is a neglected disease caused by Trypanosoma cruzi Chagas, 1909. Causative treatment can be achieved with two drugs: benznidazole or Nifurtimox. There are some gaps that hinder progress in eradicating the disease. There is no test that can efficiently assess cure control after treatment. Currently, the decline in anti-T. cruzi antibody titres is assessed with conventional serological tests, which can take years. However, the search for new markers of cure must continue to fill this gap. The present study aimed to evaluate the decline in serological titres using chimeric proteins after treatment with benznidazole in chronic patients diagnosed with CD. It was a prospective cross-sectional cohort study between 2000 and 2004 of T. cruzi-positive participants from the Añatuya region (Argentina) treated with benznidazole. Serum samples from ten patients were collected before treatment (day zero) and after the end of treatment (2, 3, 6, 12, 24 and 36 months). For the detection of anti-T. cruzi antibodies, an indirect ELISA was performed using two chimeric recombinant proteins (IBMP-8.1 and IBMP-8.4) as antigens. The changes in reactivity index within the groups before and after treatment were evaluated using the Friedman test. All participants experienced a decrease in serological titres after treatment with benznidazole, especially IBMP-8.1. However, due to the small number of samples and the short follow-up period, it is premature to conclude that this molecule serves as a criterion for sustained cure. Further studies are needed to validate tests based on these or other biomarkers to demonstrate parasitological cure.

Non-clinical toxicity and immunogenicity evaluation of a Plasmodium vivax malaria vaccine using Poly-ICLC (Hiltonol®) as adjuvant

Malaria caused byPlasmodium vivaxis a pressing public health problem in tropical and subtropical areas.However, little progress has been made toward developing a P. vivaxvaccine, with only three candidates being tested in clinical studies. We previously reported that one chimeric recombinant protein (PvCSP-All epitopes) containing the conserved C-terminus of the P. vivax Circumsporozoite Protein (PvCSP), the three variant repeat domains, and aToll-like receptor-3 agonist,Poly(I:C), as an adjuvant (polyinosinic-polycytidylic acid, a dsRNA analog mimicking viral RNA), elicits strong antibody-mediated immune responses in mice to each of the three allelic forms of PvCSP. In the present study, a pre-clinical safety evaluation was performed to identify potential local and systemic toxic effects of the PvCSP-All epitopes combined with the Poly-ICLC (Poly I:C plus poly-L-lysine, Hiltonol®) or Poly-ICLC when subcutaneously injected into C57BL/6 mice and New Zealand White Rabbits followed by a 21-day recovery period. Overall, all observations were considered non-adverse and were consistent with the expected inflammatory response and immune stimulation following vaccine administration. High levels of vaccine-induced specific antibodies were detected both in mice and rabbits. Furthermore, mice that received the vaccine formulation were protected after the challenge with Plasmodium berghei sporozoites expressing CSP repeats from P. vivax sporozoites (Pb/Pv-VK210). In conclusion, in these non-clinical models, repeated dose administrations of the PvCSP-All epitopes vaccine adjuvanted with a Poly-ICLC were immunogenic, safe, and well tolerated.

Targeted Delivery of HSP70 to Tumor Cells via Supramolecular Complex Based on HER2-Specific DARPin9_29 and the Barnase:Barstar Pair.

(1) Background: We have previously shown that the use of an artificial supramolecular two-component system based on chimeric recombinant proteins 4D5scFv-barnase and barstar-heat shock protein 70 KDa (HSP70) allows targeted delivery of HSP70 to the surface of tumor cells bearing HER2/neu antigen. In this work, we studied the possibility to using DARPin9_29-barnase as the first targeting module recognizing HER2/neu-antigen in the HSP70 delivery system. (2) Methods: The effect of the developed systems for HSP70 delivery to human carcinomas SK-BR-3 and BT474 cells hyperexpressing HER2/neu on the activation of cytotoxic effectors of the immune cells was studied in vitro. (3) Results: The results obtained by confocal microscopy and cytofluorimetric analysis confirmed the binding of HSP70 or its fragment HSP70-16 on the surface of the treated cells. In response to the delivery of HSP70 to tumor cells, we observed an increase in the cytolytic activity of different cytotoxic effector immune cells from human peripheral blood. (4) Conclusions: Targeted modification of the tumor cell surface with molecular structures recognized by cytotoxic effectors of the immune system is among new promising approaches to antitumor immunotherapy.