BR-102023019240-B1 - Variable regions of monoclonal antibody fragments with potential in treatment and diagnosis detect toxins present in the venom of spiders of the genus Loxosceles.

BR102023019240B1BR 102023019240 B1BR102023019240 B1BR 102023019240B1BR-102023019240-B1

Abstract

Variable Regions of Monoclonal Antibody Fragments with Potential in Treatment and Diagnosis Detect Toxins Present in the Venom of Spiders of the Genus Loxosceles. Accidental envenomation caused by spiders of the genus Loxosceles represents a public health problem. According to the Health Ministry's Information System for Notifiable Diseases (SINAM), they account for 24% of reported cases of arachnidism between 2016 and 2022. During this period, there were 53,316 cases of Loxoscelism, most of which were registered in the Southern region (80%), especially in Paraná (n= 5,499). Treatment of loxoscelism is carried out through the administration of equine hyperimmune serums, used for over a century, which, despite being effective, are heterologous and can cause adverse immune reactions. The diagnosis, in turn, is subjective and based on ambiguous clinical signs, ideally requiring the victim to bring the spider with them for identification and/or a highly qualified healthcare professional. This condition reinforces the need to propose improvements for the diagnosis and treatment of loxoscelism. The present invention relates to variable light and heavy chain sequences of a monoclonal antibody, LmAb12, which recognizes the low molecular weight toxins of the venom of three species of spiders of the genus Loxosceles. These sequences were used to construct recombinant fragments (scFv (...).

Inventors

Larissa Magalhães Alvarenga
ALESSANDRA BECKER FINCO
Juliana Ferreira De Moura
CAMILA ALVES MONDINI
SABRINA KARIM SILVA
MARTINA BELTRAMINO
MARIANA FERNANDES FONSECA
ISABELLA GIZZI JIACOMINI

Assignees

UNIVERSIDADE FEDERAL DO PARANA

Dates

Publication Date: 20260317
Application Date: 20230921

Claims (8)

1. Monoclonal antibody fragment against venom toxins of spiders of the genus Loxosceles characterized by consisting of: A) a variable heavy chain domain including complementarity-determining regions or CDRs 1, 2 and 3 (CDR1, CDR2 and CDR3) consisting of the following amino acid sequence: SEQ ID No. 5. B) a variable light chain domain including complementarity-determining regions CDR1, CDR2 and CDR3 consisting of the following amino acid sequence: SEQ ID No. 6. C) a 15-amino acid junctional peptide between the variable heavy chain domain and the variable light chain domain generating the single chain fragment variable (scFv) consisting of the following amino acid sequence: SEQ ID No. 10. D) a 5-amino acid junctional peptide between the variable heavy chain domain and the variable light chain domain generating the variable fragment dimer. single-chain (diabody) constituting the following amino acid sequence: SEQ ID No. 11.
2. Amino acid sequences of the variable regions of a monoclonal antibody fragment that participate in the interaction with the venom toxins of spiders of the genus Loxosceles, according to claim 1, characterized by having the following sequences as CDRs of the variable heavy chain fragment: CDR1: SEQ ID No. 7, CDR2: SEQ ID No. 8 and CDR3: SEQ ID No. 9. For the CDRs of the variable light chain fragment comprising: CDR1: SEQ ID No. 12, CDR2: SEQ ID No. 13 and CDR3: SEQ ID No. 14.
3. Amino acid sequences of variable regions of monoclonal antibody fragments that participate in the interaction with the venom toxins of spiders of the genus Loxosceles, according to claims 1 and 2, characterized by having a 15-amino acid junctional peptide SEQ ID No. 10 between the variable heavy chain domain SEQ ID No. 5 and the variable light chain domain SEQ ID No. 6 constituting the single-chain variable fragment scFv12P.
4. Amino acid sequences of variable regions of monoclonal antibody fragments that participate in the interaction with the venom toxins of spiders of the genus Loxosceles, according to claims 1, 2 and 3, characterized by having a 5-amino acid junctional peptide SEQ ID No. 11 between the variable heavy chain domain SEQ ID No. 5 and the variable light chain domain SEQ ID No. 6 constituting the diabody12P variable fragment.
5. Use of recombinant murine monoclonal antibody fragment scFv12P and diabody 12P, according to claims 1, 2, 3 and 4, characterized by being for the detection of venom toxins from spiders of the genus Loxosceles in immunoassays.
6. Use of recombinant murine monoclonal antibody fragment scFv12P and diabody 12P, according to claims 1, 2, 3 and 4, characterized by being for in vitro hemolysis neutralization of venom from spiders of the genus Loxosceles.
7. Use of recombinant murine monoclonal antibody fragment scFv12P and diabody 12P, according to claims 5 and 6, characterized by being an input for use in a diagnostic method for accidents caused by venomous animals such as arachnids of the genus Loxosceles.
8. Use of a recombinant murine monoclonal antibody fragment, scFv12P and diabody 12P, according to claim 6, characterized by being a component of a medicament used as an antivenom.

Description

Field of Invention [001]. The present invention relates to the field of immunochemistry and molecular biology applied to medicine and public health. This document highlights the biochemical and immunological characterization, as well as the amino acid and nucleotide sequence of the variable light and heavy chain regions of a murine monoclonal antibody linked by a junctional peptide, characterizing the recombinant variable fragments, which are capable of detecting and neutralizing the hemolytic effect of toxins present in the venom of spiders of the genus Loxosceles. These recombinant monoclonal antibody fragments can be particularly useful as a tool in the diagnosis and treatment of patients who have suffered envenomation accidents (loxoscelism) caused by these spiders. Fundamentals of the Invention and State of the Art [002]. Loxoscelism, necrotic or gangrenous arachnidism are some of the terms used to describe accidents with spiders of the genus Loxosceles, which is currently the most important form of arachnidism in some countries in the world. The initial records of accidents involving Loxosceles in Brazil were reported in 1954 by BÜCHERL & ROSENFELD (RIBEIRO, 2007). According to data reported in Sinan (2022), of the 1,298,429 accidents involving venomous animals reported between 2017 and 2021, 168,420 (12.97%) were caused by spiders, being the third type of venomous animal with the highest number of notifications. During this period, the year in which the most accidents were reported was 2019, with 38,961 accidents, exceeding, at that time, the number of records of snakebite accidents. In the subsequent period (2020 and 2021), amidst the COVID-19 pandemic, there was a reduction in the number of reported spider bite accidents, perhaps due to increased concern about household cleanliness and fear of exposure to SARS-CoV-2 when seeking hospital care in case of an accident. In all years evaluated, among the spider species identified, loxosceles spider bites were the most common, with 39,409 (23.40%) reported accidents. [003]. Spiders of the genus Loxosceles, popularly known as brown recluse spiders, have a worldwide distribution, and in Brazil, 12 species have already been described, of which the most medically important are: Loxosceles hirsuta (Lh) Mello-Leitão, 1931 (PR and RS), Loxosceles intermedia (Li) Mello-Leitão, 1934 (DF, RJ, SP and RS), Loxosceles gaucho (Lg) Gerstch, 1967 (SP and RS) and Loxosceles laeta (Ll) (RS, SP, RJ, MG and PR) (MARQUES-DA-SILVA, FISCHER, 2005; CABRERIZO et al., 2009; CHAGAS et al., 2010; HASS et al., 2013). They have nocturnal habits and are not aggressive unless they feel threatened. Loxosceles spider bites occur most frequently between October and March, with a seasonality similar to that of snake and scorpion stings. The Southern Region is responsible for approximately 80% of reported Loxosceles spider bites in Brazil. Between 2017 and 2021, the Southern Region was responsible for the largest number of reported spider bites (53.54% of the total), with the state of Paraná registering the most accidents (45,024, 26.73%), followed by Santa Catarina (26,342, 15.64%), São Paulo (26,264, 15.59%), Minas Gerais (22,832, 13.56%), and Rio Grande do Sul (18,811, 11.17%) (SINAM, 2022). [004]. Loxosceles envenomation is divided into cutaneous/dermonecrotic and cutaneous-visceral/systemic (hemolytic) clinical presentations. The cutaneous presentation, which accounts for 87-98% of cases, is initially characterized between 2 and 8 hours by pain, burning, and the formation of an edematous region associated with erythema. These symptoms intensify in the first 24 to 72 hours after the accident, and can lead to 3 types of lesions: uncharacteristic (serous blister, edema, heat and redness, with or without pain and burning); suggestive (induration, blister, ecchymosis and burning pain); characteristic (burning pain, focal hemorrhagic lesions, mixed with pale areas of ischemia, called marbled plaque, and evolving to necrosis). The ulcer caused by loxoscelism requires months to heal (SINAM, 2022). [005]. Cutaneous-visceral or systemic clinical manifestations occur less frequently (1-13%) in cases of Loxosceles envenomation, with L. laeta being the most commonly involved species. In the first 24 hours after the bite, symptoms such as asthenia, fever, vomiting, headache, sensory alterations, insomnia, and coma may appear. More severe manifestations include hematological alterations characterized by anemia, jaundice, hemoglobinuria, neutropenia, platelet aggregation and, consequently, thrombocytopenia, as well as disseminated intravascular coagulation. Systemic manifestations involving renal alterations can lead to Acute Renal Failure (ARF) (hemoglobinuria and hematuria) and even tubular lumen obstruction (oliguric or non-oliguric ARF). Symptoms such as fever, nausea, vomiting, and seizures have been reported, suggesting that the venom acts on the central nervous system (BRASIL, 2014 PINTO, 2009;