Anti-glycosylphosphatidylinositol (GPI) single-chain recombinant antibody fragment (SCFV) and its use for the treatment of cerebral malaria.

Description

/ 12 Recombinant single-chain antibody fragment (scFv) against glycosylphosphatidylinositol (GPI) and its use for the treatment of cerebral malaria. Field of Invention

[001] The present invention relates to the selection and characterization of a recombinant antibody fragment, the single-chain variable (scFv) anti-glycosylphosphatidylinositol (GPI) moiety of Plasmodium falciparum, as well as its use in the treatment of cerebral malaria. State of the Art

[002] Malaria is a disease caused by protozoa of the genus Plasmodium and transmitted by the Anopheles mosquito. Its distribution occurs mainly in countries with tropical and subtropical climates, with the most affected regions being parts of sub-Saharan Africa, parts of Asia, Central America, and South America. Although there are five species in the genus capable of affecting humans, P. falciparum, P. vivax, and P. malariae stand out epidemiologically, with P. falciparum being predominant in Africa and Asia, while P. vivax is more common in the Americas (AMIR, A. et al. Plasmodium knowlesi malaria: current research perspectives. Infection and drug resistance, p. 1145-1155, 2018.).

[003] In 2022 alone, 249 million cases and 608,000 deaths from malaria were recorded, with the African region accounting for 94% of cases and 95% of deaths. The most vulnerable groups are children under 5 years of age, pregnant women, and immunocompromised individuals, with symptoms ranging from mild to potentially fatal. Mild symptoms include fever, headache, and chills, while severe symptoms include fatigue, cerebral malaria, and respiratory distress (WORLD HEALTH ORGANIZATION. World malaria report 2023).

[004] The most severe form of the disease is cerebral malaria, which is more prevalent in children under five years of age living in endemic environments, with a mortality rate of 10% to 20%. Clinical manifestations may begin with a typical malaria presentation and lead to seizures. Petition 870250000708, dated 06 / 01 / 2025, page 16 / 40 / 12 generalized, which are observed in 80% of children and 15% of adults, and may progress to epileptic stages and degenerate into a coma (Potchen MJ, Kampondeni SD, Seydel KB, et al. 1.5 Tesla Magnetic Resonance Imaging to Investigate Potential Etiologies of Brain Swelling in Pediatric Cerebral Malaria. Am J Trop Med Hyg. 2018;98(2):497-504. doi:10.4269 / ajtmh.17-0309). In addition to possible complications such as acute respiratory, renal and hepatic failure (HORA, R, KAPOOR, P, THIND, KK, MISHRA, PC. Cerebral malaria - clinical manifestations and pathogenesis. Metab Brain Dis, 2016. https: / / doi.org / 10.1007 / s11011-015-97875).

[005] Current treatments for malaria are based on antiparasitic drugs, such as chloroquine, artemisinin, and artesunate, while supportive treatment is tailored to the patient's needs. However, these treatments have limitations, especially in the context of severe malaria, since their mechanisms of action are associated with parasitism and not directly with the condition of cerebral malaria. The growing resistance to antiparasitic drugs, notably artemisinin and combination therapies, is a concern, especially in Southeast Asia, with P. falciparum showing resistance in several regions of Africa, Asia, and South America, and P. vivax showing resistance to chloroquine in places like Papua New Guinea and Indonesia (RASMUSSEN, C.; ALONSO, P.; RINGWALD, P. Current and emerging strategies to combat antimalarial resistance. Expert Review of Antiinfective Therapy. v.20:3, p. 353- 372. 2022).

[006] Glycosylphosphatidylinositol (GPI) is a glycolipid complex that anchors various proteins and glycoproteins to the cell membrane via its C-terminal portion. These so-called GPI anchors are ubiquitous among eukaryotes, having been described in the genus Plasmodium (DELORENZI, M., SEXTON, A., SHAMS-ELDIN, H., SCHWARZ, RT, SPEED, T., SCHOFIELD, L. Genes for glycosylphosphatidylinositol toxin biosynthesis in Plasmodium falciparum. Infect Immun., v. 70, p. 4510-4522, 2002. https: / / doi.org / 10.1128 / IAI.70.8.4510- Petition 870250000708, dated 06 / 01 / 2025, page 17 / 40 / 12 4522.2002), Toxoplasma (TOMAVO, S., SCHWARZ, RT, DUBREMETZ, JF Evidence for glycosyl-phosphatidylinositol anchoring of Toxoplasma gondii major surface antigens. Mol Cell Biol., v. 9, p. 4576-4580, 1989. https: / / doi.org / 10.1128 / MCB.9.10.4576), Trypanosoma (FERGUSON, MA, BRIMACOMBE JS, BROWN JR, et al. The GPI biosynthetic pathway as a therapeutic target for African sleeping sickness. Biochim Biophys Acta, v. 1455, p. 327-340, 1999. https: / / doi.org / 10.1016 / S0925-4439(99)00058-7) e Leishmania (ILGOUTZ, S.C., ZAWADZKI, J.L., RALTON, J.E., MCCONVILLE, M.J. Evidence that free GPI glycolipids are essential for growth of Leishmania mexicana. EMBO J., v. 18, p. 2746-2755, 1999. https: / / doi.org / 10.1093 / emboj / 18.10.2746), bem como em leveduras e mamíferos (PITTET, M., CONZELMANN, A. Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae. Biochim Biophys Acta., v. 1771, p. 405-420,2007).

[007] Despite being ubiquitous, GPIs are found in extremely low concentrations in the plasma membranes of most organisms, except in parasitic protozoa, in which they are very abundant (IKEZAWA, H. Glycosylphosphatidylinositol (GPI)-anchored proteins. Biol Pharm Bull, v. 25, p. 409-417, 2002. https: / / doi.org / 10.1248 / bpb.25.409).

[008] It has been seen that GPIs are proven pathogenicity factors in Plasmodium falciparum (NAIK et al., 2000). And it has been demonstrated that people living in malaria-endemic areas exhibit a specific IgG response to Plasmodium falciparum GPIs. Although adults and older children have high antibody levels, susceptible children have no or very low antibody levels. Furthermore, the absence of anti-GPI antibodies has been verified in patients with fever and anemia, suggesting that antibodies provide protection against clinical forms of malaria (NAIK, RS; DAVIDSON, EA; GOWDA, DC). Developmental stage-specific biosynthesis of glycosylphosphatidylinositol anchors in intraerythrocytic cells. Petition 870250000708, dated 06 / 01 / 2025, page 18 / 40 / 12 Plasmodium falciparum and its inhibition in a novel manner by mannosamine. J. Biol. Chem., v. 275, p. 24506-24511, 2000. (https: / / doi.org / 10.1074 / jbc.M002151200). In this way, GPI becomes an important candidate target for the production of molecules to be used in therapeutic alternatives against parasitic diseases.

[009] Phage display is an efficient technique for identifying peptides or proteins that bind to other molecules with diverse applications, such as the development of immunotherapies and vaccines for different diseases (WANG, Y.; LIN, Y; LV, J. Phage Display technology and its applications in cancer immunotherapy. Anticancer Agents Med. Chem.,2019). The technology is based on the principle that polypeptides can be expressed on the surface of filamentous bacteriophages by inserting a coding DNA segment into their genome, so that the expressed peptide or protein is exposed on the surface of the viral particle fused to a viral protein naturally expressed in the bacteriophage (RUIZ, A., PÉREZ, D., MUNOZ, MC, et al. Targeting essential Eimeria ninakohlyakimovae sporozoite ligands for caprine host endothelial cell invasion with a phage display peptide library. Parasitol Res., v. 114, p. 4327-4331, 2015).

[0010] A therapeutic alternative in combating various diseases is the construction of recombinant artificial antibody molecules that are normally produced in the form of polypeptide fragments, with the ability to behave identically to the original antibody (ZHOU, SJ; WEI, J.; SU, S.; CHEN, FJ; QIU, YD; LIU, BR Strategies for Bispecific Single Chain Antibody in Cancer Immunotherapy. J. Cancer, v. 17, p. 3689-3696, 2017). The most commonly used form in this context is the single-chain variable fragment (scFv), which contains the variable domains of the light and heavy chains linked by a linker peptide, and has a molecular weight of approximately 30 kDa (AHMAD, S. ZUHAIDA, AA; SWEE, KY ABDUL, MA; WAN, YH; NOORJAHAN, BMA; MUHAJIR, H. scFv antibody: Principles and clinical application. Clin. Dev. Petition 870250000708, dated 06 / 01 / 2025, page 19 / 40 / 12 Immun., v. 2012, 2012.).

[0011] scFvs have been used in the diagnosis and immunotherapy of various types of cancer, viral and parasitic diseases, and have shown advantages over monoclonal and conventional antibodies (WANG, Y.; LIN, Y; LV, J. Phage Display technology and its applications in cancer immunotherapy. Anticancer Agents Med. Chem., 2019.).

[0012] The use of these molecules has presented certain advantages over the use of a whole antibody molecule in therapeutic applications: a) the smaller fragments allow for faster and more uniform penetration into tumors and other tissues compared to whole antibodies; b) they exhibit rapid blood circulation. Previous studies have shown that patients treated with scFv showed good localization of the molecules in just one hour after injection; c) good tissue penetration; d) low retention in the kidneys and other non-target organs; e) greater ease of gene manipulation, as it is a monocistronic construct; f) low commercial cost in large-scale production; g) they can be coupled with drugs and radionuclides in order to result in low exposure of healthy tissue (WANG, Y.; LIN, Y; LV, J. Phage Display technology and its applications in cancer immunotherapy. Anticancer Agents Med. Chem., 2019.).

[0013] The production of recombinant artificial antibody molecules, often in the form of polypeptide fragments that mimic the properties of natural antibodies, has emerged as an innovative therapeutic approach against various diseases, such as cancer, viral and parasitic diseases (ZHOU, SJ; WEI, J.; SU, S.; CHEN, FJ; QIU, YD; LIU, BR Strategies for Bispecific Single Chain Antibody in Cancer Immunotherapy. J. Cancer, v. 17, p. 3689-3696, 2017.). A notable example of these molecules is scFv, which consists of variable light and heavy chain domains connected by a linker peptide, with an approximate molecular weight of 30 kDa (AHMAD, S. ZUHAIDA, AA; SWEE, KY ABDUL, M. Petition 870250000708, dated 06 / 01 / 2025, page 20 / 40 / 12 A.; WAN, YH; NOORJAHAN, BMA; MUHAJIR, H. scFv antibody: Principles and clinical application. Clin. Dev. Immun., v. 2012, 2012). The ability to artificially combine functional antibody subunits, based on their domain structure, enables the creation of highly specific molecules targeted at predetermined targets. Furthermore, these antibody fragments offer several advantages over the use of whole antibody molecules: (1) their smaller size allows for faster and more uniform distribution in tissues compared to larger molecules; (2) they exhibit low retention in non-target tissues; (3) they have a reduced commercial cost when produced on a large scale; (4) can be conjugated to drugs and radionucleotides, aiming to minimize exposure to healthy tissues (WANG, A., HUEN, SC, LUAN, HH, et al. Glucose metabolism mediates disease tolerance in cerebral malaria. Proc Natl Acad Sci US A., v. 115, p. 11042-11047, 2018.).

[0014] Currently, molecules have been used for the treatment of diseases and have been patented, such as WO03016354A2, which claims antibodies, their method of production and use of such antibodies. The antibodies are against the MSP-3 antigen, and are capable of passively inducing natural immunity against malaria, i.e., for prevention, which differs from the present invention.

[0015] WO2013035107A1 refers to a cysteine-restricted cyclic peptide against the recombinant B subunit of P. falciparum VH+ ATPase. These peptides inhibit the growth of P. falciparum in vitro and may be explored for their therapeutic value in malaria. It shows promising results in vitro, but has not been tested in vitro.

[0016] WO2024042112A1 refers to the field of malaria drugs, in particular antibodies that bind to Plasmodium falciparum sporozoites, in particular to Plasmodium falciparum circumsporozoite protein for the prevention of malaria.

[0017] WO2024010373A1 refers to antibodies that bind to the factor of Petition 870250000708, dated 06 / 01 / 2025, page 21 / 40 / 12, concerning IgE-dependent histamine release and its uses. The inventors attempted to develop antibodies that specifically bind to HRF, which is associated with various diseases. The anti-HRF antibody according to the invention has been confirmed to inhibit the cytokine-like activity of HRF by specifically binding to HRF with high binding affinity, and is effective in treating allergic diseases, chronic inflammatory diseases, autoimmune diseases, cancer, hypertension, and can be usefully used to prevent and develop treatments for HRF-related diseases such as malaria and osteoporosis. This demonstrates the ability of recombinant antibodies to be employed in the treatment of various diseases.

[0018] CN116888150A also claims an antibody for a vaccine to prevent malaria. The disclosure includes monoclonal antibodies or antibody fragments that bind to Plasmodium-derived antigens. The inventors obtained a variety of monoclonal antibodies using Ripr, which is a Plasmodium-derived protein, as an antigen, and discovered from these monoclonal antibodies some antibodies that show inhibitory activity against Plasmodium growth, and concluded the invention.

[0019] BR 10 2018 072643 9 A2 also claims selection, characterization of an scFv fragment (scFv-D09) by Phage Display) as in the present invention, but in this case against total salivary proteins. Showing the diverse application of antibody fragments for various health conditions. This scFv recognizes an epitope of chain 4 of the alpha-tropomyosin protein as a target antigen for binding, and can be used in the diagnosis, prognosis and therapeutics in oral squamous cell carcinoma.

[0020] BR 112022022503-2 A2 claims antigen-binding proteins, such as fully human antibodies, that specifically bind to the spike (S) protein of the SARS-CoV-2 coronavirus and their uses. Methods for using anti-spike protein antibodies include methods for treating or preventing infection with a coronavirus, such as the SARS-CoV-2 coronavirus, by administering an antibody or antibody fragment, as disclosed in Petition 870250000708, dated 06 / 01 / 2025, p. 22 / 40 / 12 present invention. The inventors show that antibodies can be used for the treatment and prevention of viral diseases, as well as the importance of prospecting for these recombinant molecules for application against diseases of global importance.

[0021] Thus, addressing the shortcomings of current technologies, the present invention proposes the use of the scFV antibody fragment, antiglycosylphosphatidylinositol (GPI) from Plasmodium falciparum, named scFVG7, for the treatment of cerebral malaria. The present invention rescued 71.4% of the animals, promoted the recovery of the initial weight loss caused by the infection, inhibited neurological signs of cerebral malaria (CM), as well as systemic signs of the disease, reduced parasitemia, improved blood parameters, mobilized pro-inflammatory cytokines, and decreased cerebral hemorrhages in the animals, showing a significant therapeutic effect in the pathology of experimental cerebral malaria (ECM). List of Figures

[0022] The present invention may be better understood based on the attached figures, where: FIGURE 1: shows the predicted nucleotide and amino acid sequence of the light (VL) and heavy (VH) chains of the variable region of the scFv-G7 clone. FIGURE 2 (A and B): elucidate the 3D structure of the scFv-G7 clone, highlighting the composition of the secondary structure and showing the surface conformation. The variable light chain (VL) (in red) and variable heavy chain (VH) (in lilac) domains are highlighted, being exposed on the protein surface. FIGURE 3: shows the graphs resulting from the ELISA assay, highlighting the greater reactivity of the scFv-G7 clone with the Plasmodium GPI protein. FIGURE 4: (A) shows the decrease in parasitemia after treatment of animals with scFv-G7; (B) shows the restoration of body weight in treated animals. FIGURE 5: shows the decrease in parasitemia in animals infected with and Petition 870250000708, dated 06 / 01 / 2025, page 23 / 40 / 12 without treatment with scFv-G7. FIGURE 6: (A) shows the increase in survival; (B) shows the restoration of body weight in mice infected with P. berghei-ANKA. FIGURE 7: shows the improvement in the clinical signs of the animals after treatment with scFv-G7, through the analysis of scores for each parameter. (A) Murine rapid coma and behavior scale; (B) Gait; (C) Balance; (D) Motor Performance; (E) Body Position; (F) Limb Strength; (G) Escape to touch; (H) Pinna reflex; (I) Foot reflex; (J) Grooming; (K) Eyes. FIGURE 8: (A) illustrates the decrease in thrombocytopenia; (B) illustrates the decrease in monocytopenia in infected animals treated and not treated with scFv-G7. FIGURE 9: shows the decrease in microhemorrhages after treatment of animals with scFv-G7. Description of the Invention

[0023] The present invention provides the selection of the Plasmodium falciparum anti-glycosylphosphatidylinositol (GPI) scFV (single-chain variable fragment) antibody fragment named scFV-G7 described in the methodology.

[0024] Based on the DNA sequencing of the scFv-G7 antiglycosylphosphatidylinositol (GPI) clone, the subject of the present invention, the sequences of said antibody were designated. SEQ. ID No. 1 corresponds to the sequence of the light chain of the antibody fragment. SEQ. ID No. 2 corresponds to the heavy chain of the antibody fragment. And finally, SEQ. ID No. 3 corresponds to the combination of the light and heavy chains of the antibody fragment, thus being named scFv-G7.

[0025] The complete nucleotide sequence and the predicted amino acid sequence of the variable region of the heavy and light chains of the scFv-G7 clone are shown in FIGURE 1. FIGURE 2 shows the successful prediction by ITASSER of the three-dimensional model, as well as the location of its CDRs. In FIGURE 2A the model is presented showing the composition of the secondary structure and in FIGURE 2B showing the surface conformation. The Petition 870250000708, dated 06 / 01 / 2025, page 24 / 40 / 12 variable light chain (VL) domains (in red) and variable heavy chain (VH) domains (in lilac) are highlighted, being exposed on the protein surface.

[0026] Assays were performed to demonstrate the specificity and reactivity of the scFv anti-glycosylphosphatidylinositol (GPI) antibody fragment for the treatment of cerebral malaria.

[0027] The ELISA assay demonstrated that the scFv-G7 antibody fragment has affinity for Plasmodium GPI, showing 80.5% accuracy (FIGURE 3).

[0028] From the treatment of C57BL / 6 mice infected with P. berghei-ANKA-GFP+, it was possible to observe that the selected antibody fraction decreases the parasitemia of the animals (FIGURE 4A), keeping them alive and without symptoms of cerebral malaria for up to 30 days after infection (FIGURE 4B). This is not observed in untreated animals, which die of cerebral malaria after 6 days of infection. In FIGURE 5, an optical microscopy image can be seen showing the progression of parasitemia between the 4th and 6th days post-infection (dpi).

[0029] scFv-G7 increases survival and restores body weight in mice infected with P. berghei-ANKA. During survival monitoring, it was observed that all infected animals not treated with scFV succumbed between the 7th and 9th day post-infection, showing clinical signs of cerebral malaria (FIGURE 6A). In contrast, unlike the previous group, infected animals treated with scFv-G7 remained alive and free of clinical manifestations of cerebral malaria until the 30th day post-infection. FIGURE 6B illustrates body weight monitoring, where all animals showed an initial weight reduction. From the 3rd dpi, there was a progressive weight reduction in the PbA + PBS group until the 6th dpi. In contrast, infected animals treated with scFv-G7 showed body weight gain on days 2, 3, 5, and 6 post-infection.

[0030] Treatment with scFv-G7 reduced the clinical signs of cerebral malaria and improved the overall clinical condition of infected mice (FIGURE 7). Na. Petition 870250000708, dated 06 / 01 / 2025, page 25 / 40 / 12. In the sum of all scores, the animals in the PbA + scFv-G7 group obtained scores equal to or greater than 17 points in all evaluations. On the other hand, the animals in the PbA + PBS group presented scores of 19.33 and 18.67 on the 4th and 5th dpi, respectively. However, there was a reduction to 13.83 on the 6th dpi and to 6 points 5 minutes before euthanasia, also 290 on the 6th dpi. In the evaluations of each score, the PbA + scFv group showed better performance than the PbA + PBS group, except for Grooming, where there was no statistical difference between the groups (FIGURES 7A to 7K).

[0031] Treatment with scFv-G7 improves thrombocytopenia and monocytopenia in infected mice. Animals in the PbA + PBS group presented polycythemia, evidenced by an increase in hematocrit. Although anemia is common in cases of malaria, no evident anemia was observed due to severe dehydration on the day of collection, resulting in a reduction in circulating fluid volume and, consequently, an increase in red blood cell concentration (FIGURE 8A and 8B).

[0032] Treatment with scFv-G7 reduces micro-hemorrhages in the brains of infected mice. Analysis of cerebral micro-hemorrhages revealed a statistically significant difference (p < 0.0001) between groups treated and not treated with scFv-G7, demonstrating that scFv-G7 offers cerebral protection to infected animals (FIGURE 9).

[0033] Finally, the present invention led to increased survival, reduced parasitemia, recovery of body weight, prevention of neurological signs, improvement in hematological parameters, changes in the immune response, and a reduction in cerebral micro-hemorrhages. The Plasmodium anti-GPI scFv-G7 acts in the treatment of complicated malaria.

[0034] The antibody fragment is synthetically produced and characterized by its use in the treatment and prophylaxis of malaria and its complications, and by neutralizing the progression of complications of cerebral malaria. It can be used in pharmaceutical compositions, including liposomal formulations, and can be associated with biocompatible nanoparticles and conjugated. Petition 870250000708, dated 06 / 01 / 2025, page 26 / 40 / 12 directly with drugs. Petition 870250000708, dated 06 / 01 / 2025, page 27 / 40

Claims

CLAIMS 1- Recombinant single-chain antibody fragment characterized by comprising the scFV-G7 fragment. 2- Antibody fragment, according to claim 1, characterized in that the scFv-G7 fragment is composed of SEQ. ID No. 01 and SEQ. ID No.

02. 3- Antibody fragment, according to claim 2, characterized by the individual use of SEQ. ID No. 01, being the light chain of the antibody fragment. 4- Antibody fragment, according to claim 2, characterized by individual use SEQ. ID No. 02, being the heavy chain of the antibody fragment. 5- Antibody fragment, according to claims 3 and 4, characterized by the combination of the light and heavy chains of the variable antibody fragment, defining SEQ. ID No.

03. 6- Antibody fragment, according to any of the preceding claims, characterized by being produced synthetically or recombinantly. 7- Antibody fragment, according to claims 1 and 5, characterized by being used for the treatment or prophylaxis of malaria and its complications. 8- Antibody fragment, according to claims 1 and 5, characterized by being an integral part of a pharmaceutical composition including liposomal formulations, biocompatible nanoparticles, and direct conjugation with drugs. 9- Antibody fragment, according to claims 1 and 5, characterized by being used as a neutralizer of the progression of complications of cerebral malaria. Petition 870250000708, dated 06 / 01 / 2025, pp. 28 / 40

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