Application of bispecific chimeric lectin from Musa acuminata and Xerocomus chrysenteron as an antitumor agent for pancreatic cancer.

Description

/ 17 Application of bispecific chimeric lectin from Musa acuminata and Xerocomus chrysenteron as an antitumor agent for pancreatic cancer. Field of invention

[001] The present invention relates in the international patent classification to the areas of chemistry and human needs listed in sections A61K; A61K 8 / 64; A61K 8 / 72; A61K 31 / 74; A61P 35 / 04; A61K 36 / 07; A61P 43 / 00; A61K 49 / 14; A61K 131 / 00; A61P 17 / 02; C12N 15 / 09; C12N 15 / 29. It relates to the use of a bispecific chimeric lectin, formed by two lectins with different specificities, from the species Musa acuminata (herein referred to as banana lectin, Banlec) and Xerocomus chysenteron (herein referred to as XCL). The chimeric lectin was produced using recombinant DNA technology and possesses antiproliferative activity against pancreatic cancer cells. The innovation refers to the recombinant production from an expression vector and the use of the chimeric lectin in a pancreatic cancer cell line as an antitumor agent. Fundamentals of the invention

[002] Pancreatic cancer has the highest mortality / incidence ratio among all malignant diseases. In general, antitumor drugs are mainly based on key mutations that alter the functioning of proteins that contribute to the tumorigenicity of malignant cells. The most commonly used drugs are tyrosine kinase inhibitors (TKIs), which bind specifically to those mutated proteins to prevent their activity, such as the epidermal growth factor receptor (EGFR) and the mesenchymal-epithelial transition gene (MET). An undesirable result of the use of TKIs is acquired resistance, and new alternatives are still being discovered at a slow pace (WU, Q. et al. Small-molecule inhibitors, immune checkpoint inhibitors, and more: FDA-approved novel therapeutic drugs for solid tumors from 1991 to 2021. Journal of Hematology & Oncology, v. 15, p. 143, Oct. 8, 2022).Specifically in the case of pancreatic cancer, only four mutations characteristic of cancers were detected (namely KRAS, CDKN2A, TP53 and...). Petition 870250004579, dated 01 / 21 / 2025, page 12 / 36 / 17 SMAD4), however, none of these are targets of commonly used therapies, since they depend on mutations affecting membrane receptors related to cell cycle maintenance. The target genes of these drugs are weakly expressed, which impacts the therapeutic potential and, consequently, the success of antitumor treatment (THE CANCER GENOME ATLAS RESEARCH NETWORK. Integrated Genomic Characterization of Pancreatic Ductal Adenocarcinoma. Cancer cell, v. 32, n. 2, p. 185-203.e13, Aug. 14, 2017).

[003] Tumor cells exhibit modified glycans on their surface, a target that is widely present in various types of cancer. The aberrant glycans that occur in tumor cells are generally associated with glycosylated proteins and membrane receptors, such as the Epidermal Growth Factor Receptor (EGFR). More interestingly, these modified glycans are not found in healthy cells, since they are processed by specific post-translational modification enzymes whose activity is not altered by cancer-promoted mutations (PINHO, SS; REIS, CA Glycosylation in cancer: mechanisms and clinical implications. Nature Reviews Cancer, v. 15, n. 9, p. 540-555, set. 2015). In the case of pancreatic cancer, there is a large amount of aberrant sugars exposed on the surface, mainly N-glycans (sugars linked to asparagine) and O-glycans (sugars linked to the hydroxyl group of a serine or threonine).These glycans have not undergone proper processing and are widely present in various types of pancreatic cancer, making them extremely relevant targets. (MUNKLEY, J. The glycosylation landscape of pancreatic cancer. Oncology Letters, v. 17, n. 3, p. 2569-2575, Mar. 2019.).

[004] Lectins are proteins capable of binding to carbohydrates in a specific and selective manner. The therapeutic potential of lectins is mainly determined by their ability to recognize carbohydrates and their binding specificity. Lectins are very diverse, especially in terms of fine specificity: the carbohydrate binding site can accommodate both simple and complex sugars. The efficiency of lectins as an antitumor agent is demonstrated by the inhibition of proliferation in various types of cancer, as these proteins are able to selectively recognize glycans modified by the tumor cell, but do not interact with glycans. Petition 870250004579, dated 01 / 21 / 2025, page 13 / 36 / 17 of healthy cells exposed on the cell surface. Associated with specific recognition, lectins are capable of inducing cell death in tumor cells through apoptosis and autophagy mechanisms (BARRE, A. et al. Mannose-specific lectins from marine algae: Diverse structural scaffolds associated with common virucidal and anti-cancer properties. Marine Drugs, v. 17, n. 8, 26 Jul. 2019. POIROUX, G. et al. Plant Lectins Targeting O-Glycans at the Cell Surface as Tools for Cancer Diagnosis, Prognosis and Therapy. International Journal of Molecular Sciences, v. 18, n. 6, p. 1232, 9 Jun. 2017).

[005] An innovative strategy in the field of lectinology is the combination of lectins with different specificities, forming chimeras or cyborg lectins. This type of study was previously carried out with the insertion of a nonapeptide from the Lens culinaris lectin into the Bauhinia purpurea lectin sequence, making the chimeric protein bispecific (or bispecific) for mannose and galactose, unlike the wild-type protein, which exhibited affinity only for galactose. This has been done in order to explore the potential of already identified molecules more effectively, aiming for enhanced antiproliferative activity in various types of cancer, in addition to using reduced concentrations and without the risk of an exacerbated immune response. (YAMAMOTO, K.; KONAMI, Y.; OSAWA, T. A chimeric lectin formed from Bauhinia purpurea lectin and Lens culinaris lectin recognizes a unique carbohydrate structure. Journal of Biochemistry, v. 127, n. 1, p. 129-135, Jan. 2000).

[006] Recent lectin engineering strategies have shown great promise regarding their therapeutic potential. The binding of lectins to important immune system effectors has been extensively studied. First, a construct targeting high-mannose glycans is the lectibody, which consists of part antibody and part lectin. Specifically, lectins are found in the Fab portion, while the human Fc portion is retained. Promising results have been observed using the Avaren lectin lectibody (AvFc) in non-small cell lung cancer (NSCLC), where AvFc was observed binding to various extracellular matrix glycoproteins, such as EGFR and insulin-like growth factor receptor 1 (IGF1R) (OH, YJ et al. Antitumor activity of a lectibody targeting cancer-associated high-mannose glycans. Molecular Therapy, v. Petition 870250004579, dated 01 / 21 / 2025, p. 14 / 36 / 17 (p. 1523-1535, Apr. 6, 2022). Secondly, the development of modified CAR T cells containing a banana lectin stands out. In this construct, the lectin is anchored to a chimeric antigen receptor (CAR) and expressed on T cells. The mutant Banlec CAR T cells were able to selectively recognize and specifically bind to high mannose from various pancreatic cancer cell lines, as well as disrupt the architecture of stromal cells in 3D culture, another obstacle presented by solid tumors. Furthermore, they were also effective in in vivo studies, specifically penetrating the tumor and, finally, were not toxic to healthy cells (MCKENNA, MK et al. Novel banana lectin CAR-T cells to target pancreatic tumors and tumor-associated stroma. Journal for Immunotherapy of Cancer, v. 11, n. 1, p. e005891, Jan. 18, 2023).

[007] The potential of lectins as broad-spectrum recognition molecules that recognize different aberrant glycans exposed on the cell surface is clear from these studies. Targeting these glycans allows for a broad-spectrum therapeutic approach and emerges as a highly relevant therapeutic alternative, since the most commonly used chemotherapeutic agents against pancreatic cancer are dependent on these mutations in membrane receptors related to cell cycle maintenance. Brief description of the drawings

[008] Figure 1 represents the schematic design of the Banlec mutant-XCL chimera construction. The relevant DNA sequences for Banlec mutant (blue) and XCL (green), retrieved from the Uniprot database, will be connected via a linker (red) and inserted into the pET-24a vector (+) and the histidine marker (His6x) located at the C-terminal.

[009] Figure 2 shows the recombinant chimera expression test. The colony test was performed with the RIL (A) and Origami (B) strains, and the expected band size is 32 kDa. In A, a prominent band can be observed in well 7. Well 1: Uninduced colony 1; 2: Induced colony 1; 3: Uninduced colony 2; 4: Induced colony 2; 5: Uninduced colony 3; 6: Induced colony 3. In B, the same band is also visible at the expected height in colonies that were induced with the Petition 870250004579, dated 01 / 21 / 2025, page 15 / 36 / 17 analogous to lactose. Well 1: Cell extract of the untransformed bacteria; 2: Colony 1 induced with 1 mM IPTG; 3: Colony 1 not induced; 4: Colony 2 not induced; 5: Colony 2 induced; 6: Colony 3 not induced; 7: Colony 3 induced; 8: Colony 4 not induced; 9: Colony 4 induced; 10: Colony 5 not induced; 11: Colony 5 induced; 12: BioRad All Blue 1 KB molecular weight marker.

[0010] Figure 3 shows the expression test of the isolated XCL protein. The colony assay was performed with the RIL and Origami strains, detecting the expected band size (15 kDa) in both. In A, SDS-PAGE gel and WB of the expression in the RIL strain. Well 1: Untransformed RIL; 2: Induced colony 1; 3: Induced colony 2; 4: Induced colony 3; 5: Induced colony 4; 6: Induced colony 5; 7: BioRad 1Kb plus molecular weight marker. In B, SDS-PAGE gel and WB of the expression in the Origami strain. Well 1: Molecular weight marker; 2: Untransformed Origami; 3: Uninduced colony 1; 4: Induced colony 1; 5: Induced colony 2; 6: Induced colony 3; 7: Induced colony 4; 8: Uninduced colony 5.

[0011] Figure 4 presents the structural arrangement analysis of the chimera, XCL, and mutant Banlec in D-PBS buffer. In A, the SDS-PAGE gel of the chimera and its isolated components (mutant Banlec and XCL) are shown in comparison with BSA (1 mg / ml), and in B, the Western blot of the same gel is shown. This analysis confirms that after all production and processing steps, the molecule continues to form its active structures. Well 1: BioRad Dual Color Plus molecular weight marker; 2 and 3: mutant Banlec under non-denaturing and denaturing conditions (presence of beta-mercaptoethanol in the sample buffer); 4 and 5: chimera under non-denaturing and denaturing conditions, respectively; 6 and 7: XCL under non-denaturing and denaturing conditions. The primary antibody used was anti-histidine (1:10000) and the secondary antibody was anti-mouse (1:10000), with the membrane being revealed using the chemiluminescent compound Pierce ECL Western Blotting Substrate™.

[0012] Figure 5 shows cell proliferation assays in CFPAC-1 pancreatic cancer cells. The proteins were incubated with the cells for up to 6 days, when images were captured at 4x magnification (EVOS Microscope, Petition 870250004579, dated 01 / 21 / 2025, page 16 / 36 / 17 (Invitrogen). A marked reduction in tumor cell growth can be observed, which is also confirmed by the MTT assay. In A, the control treatment with D-PBS only. In B, the Banlec mutant, in C the chimera, in D, the XCL protein.

[0013] Figure 6 shows the percentage of normalized cell viability relative to day 0. Average of 3 independent experiments using triplicate treatments. Data were generated using GraphPad software applying 2-way ANOVA and Tukey's post-hoc test. Asterisks indicate a statistically significant difference between groups (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001); error bars were generated from the standard error of the mean (SEM).

[0014] Figure 7 shows the Western blot of proteins associated with cell death signaling pathways by autophagy or apoptosis. CFPAC1 pancreatic cancer cells were treated with culture medium alone, with 10 μM of the chimera, or with their respective isolated portions (Banlec mutant and XCL). Extracts were collected at 24, 72, and 120 h after treatment with the lectins, and 30 μg of total protein were transferred to the membrane. It is possible that the lectins exhibit antiproliferative activity against both apoptosis and autophagy. Description of the invention

[0015] The present invention aims to demonstrate the production capacity and antitumor activity of two lectins with different specificities composing a chimera. For this purpose, the Banlec mutant with affinity for the modified high-mannose N-glycan and the XCL mushroom lectin, with affinity for the T and Tn antigens, truncated O-glycans, were used. It is expected, therefore, to increase the antiproliferative activity and specificity of the lectins when in contact with tumor cells, and, preferably, to decrease the concentration required for effectiveness.

[0016] It is interesting to note that despite the great potential of lectins as therapeutic agents, no lectin has actually reached the clinical phase. This may be due to some characteristics that hinder their use, such as the cytotoxicity and mitogenicity of some lectins, and exacerbated activation of the immune system. Petition 870250004579, dated 01 / 21 / 2025, p. 17 / 36 / 17 resulting in deleterious responses - and difficulty in large-scale production (NABIAFJADI, M. et al. Lectins and lectibodies: potential promising antiviral agents. Cellular & Molecular Biology Letters, v. 27, p. 37, May 13, 2022).

[0017] Banana lectin (Banlec) is a 15 kDa jacalin (JRL)-like lectin that recognizes complex glycans such as high mannose and its antitumor activity has been demonstrated for several types of cancer. Unfortunately, the potent mitogenic response hinders its therapeutic use (SINGH, SS; DEVI, SK; NG, TB Banana Lectin: A Brief Review. Molecules, v. 19, n. 11, p. 18817-18827, 17 nov. 2014.). An engineered Banlec lectin, derived from in silico analyses of the sequences of nine other lectins related to the JRL family, maintained its antiproliferative activity against a melanoma cell line, demonstrating that the carbohydrate binding site was preserved and that it is possible to use it as a new antitumor agent (DE CAMARGO, LJ et al. Characterization of a Molecularly Engineered Banlec-Type Lectin (rBTL). Molecular Biotechnology, v. 66, n. 2, p. 288-299, 1 Feb. 2024).

[0018] Xerocomus chrysenteron lectin (XCL) has great biotechnological potential and antitumor activity. XCL has a molecular weight of 14 kDa and specifically recognizes N-acetylgalactosamine (GalNAc). Its interaction with tumor cells occurs mainly through affinity for T and Tn antigens, stimulating the loss of cell adhesion. The main characteristic of XCL is that it does not trigger mitogenic events or toxicity to normal cells. Small size, lack of mitogenic activity, and binding to modified O-glycan are desirable characteristics studied and implemented in conjunction with new therapeutic technologies, including those targeting antiproliferative activity (DAMIAN, L. et al. Determination of thermodynamic parameters of Xerocomus chrysenteron lectin interactions with N-acetylgalactosamine and Thomsen-Friedenreich antigen by isothermal titration calorimetry. BMC Biochemistry, v. 6, n. 1, p. 11, 1 jun. 2005; MARTY-DETRAVES, C. et al.Inhibitory action of a new lectin from Xerocomus chrysenteron on cell substrate adhesion. Molecular and Cellular Biochemistry, vol. 258, no. 1 / 2, p. 49-55, Mar. 2004). Petition 870250004579, dated 01 / 21 / 2025, page 18 / 36 / 17

[0019] Due to the antiproliferative potential shown by Banlec and XCL, it was hypothesized that it would be possible to increase the affinity for aberrant glycans and inhibit tumor growth by combining two lectins with different specificities.

[0020] Experiments conducted during the course of developing embodiments of the present invention have demonstrated the activity of the chimeric lectin as an antitumor agent capable of inhibiting the proliferation of cancer cells, particularly pancreatic cancer cells.

[0021] Chimeric lectin possesses a variety of properties that make it attractive for development as an antitumor therapeutic component, including its small size, stability, inhibition of cell proliferation, ability to bind to different aberrant glycans, and it can be produced in bacteria, plants, and yeast.

[0022] Therefore, in some embodiments, the present invention provides the possibility of using the product derived from the nucleotide sequence of the Banlec mutant-XCL chimera optimized for heterologous expression, defined in SEQ ID No. 1; or its active fragments.

[0023] The present invention provides for the use of any recombinant DNA expression systems, such as bacteria, yeast, insect cells, animal or plant cells, characterized by having been transfected with the DNA sequence of claims 1 and 2, as well as a method of lectin expression characterized by the fact that it is carried out within the organism from cDNA or genomic DNA or synthetic sequence in native form.

[0024] In some embodiments, the present invention provides for the construction of DNA, characterized by comprising a nucleotide sequence that expresses wholly or partially the Banlec mutant-XCL chimera, operationally linked to a heterologous nucleotide sequence of interest.

[0025] In some embodiment, the present invention provides the possibility of using the product of recombinant expression of a nucleotide sequence comprising a fragment of at least 50% of the optimized sequence, defined in SEQ ID No. 1. In addition Petition 870250004579, dated 01 / 21 / 2025, page 19 / 36 / 17. Furthermore, the present invention provides for the use of a nucleotide sequence comprising a fragment of the sequence defined in SEQ ID No. 1, in which said sequence initiates transcription in a recombinant DNA expression system and in eukaryotic or prokaryotic cells.

[0026] In some embodiments, the present invention provides variant polypeptides wherein a nucleotide sequence comprising a sequence having at least 90% identity with the sequence defined in SEQ ID No. 1, in which said sequence initiates transcription in a recombinant DNA expression system.

[0027] In some embodiments, the present invention provides for the use of any cell characterized by having been transfected with the DNA sequence of claims 1 and 2; and the use of any transgenic organism characterized by having stably incorporated into its genome the DNA sequence of claims 1 and 2;

[0028] Therefore, the present invention provides for the development of a method for expressing the Banlec mutant-XCL chimera characterized in that it is carried out within the organism from cDNA or genomic DNA or a synthetic sequence fused with another protein or peptide.

[0029] Furthermore, the present invention provides for the use of a method of lectin expression characterized by the fact that it is carried out within the organism from cDNA or genomic DNA or synthetic sequence fused with part of the original protein.

[0030] In some embodiment, the present invention provides for the use of the heterologous protein of interest characterized by having been produced using the DNA sequence of claims 1 and 2, not limited to these. Furthermore, this claim contemplates the use of the sequence comprising a variant polypeptide of the Banlec mutant-XCL chimera wherein said variant demonstrates antitumor activity.

[0031] In some embodiments, the expression product of the mutant Banlec chimeric lectin gene (SEQ ID No. 1) is used as a component of chemotherapeutic compositions, for example, to control the growth and spread of cancerous and malignant cells, thereby preventing their proliferation. Petition 870250004579, dated 01 / 21 / 2025, page 20 / 36 / 17

[0032] In some embodiments, the gene expression product of the mutant Banlec-XCL chimeric lectin (e.g., SEQ ID NOs: 1) is used as a component of chemotherapeutic compositions (e.g., to reduce or destroy cancer cells, thus contributing to cancer treatment). For example, in some embodiments, the chimeric lectin and mutants obtained from its genetic engineering are included in formulations for use as an antitumor compound (e.g., alone or in combination with different antitumor treatments). In some embodiments, formulations comprising the recombinant chimeric protein are used in the chemotherapeutic treatment of various types of solid cancers that exhibit specific aberrant glycans, such as, but not limited to, liver, breast, ovarian, prostate, colorectal, and lung cancer.

[0033] In some embodiments, compositions and formulations containing the mutant Banlec-XCL chimeric lectin, for surface application, may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives, such as, but not limited to, penetration enhancers, carrier compounds and other acceptable carriers or excipients.

[0034] Possible compositions of the present invention include, but are not limited to, solutions, emulsions, and formulations containing liposomes. These compositions can be generated from a variety of components, including, but not limited to, pre-formed liquids, self-emulsifying solids, and self-emulsifying semi-solids.

[0035] Possible compositions of the present invention include, but are not limited to, solutions, emulsions, and formulations containing nanoparticles or integrating the present invention with nanoparticles. These compositions may be generated from, or contain, a variety of components including, but not limited to, exosomes, colloidal nanoparticles, polymeric nanoparticles, dendrimers, micelles, protein and cell membrane nanoparticles, mesoporous silica nanoparticles, gold nanoparticles, iron oxide nanoparticles, quantum dots, carbon nanotubes. Petition 870250004579, dated 01 / 21 / 2025, page 21 / 36 / 17

[0036] The formulations of the present invention, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the biotechnology industry. Such techniques include the step of associating the active ingredients with the pharmaceutical vehicle(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately associating the active ingredients with liquid vehicles or finely divided solid vehicles or both, and then, if necessary, molding the product.

[0037] The compositions of the present invention may additionally contain other adjunct components conventionally found in chemical and biotechnological compositions. Thus, for example, they may contain additional materials useful in the physical formulation of various dosage forms of the compositions of the present invention, such as colorants, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents, and stabilizers. On the other hand, these materials, when added, should not interfere with the biological activities of the components of the compositions of the present invention. The formulations may be sterilized by methods that maintain their properties, including chimeric lectin. Examples of embodiments of the invention EXAMPLE 1 - CONSTRUCTION AND PRODUCTION OF CHEMICAL LECTIN AND XCL Bioinformatic analyses

[0038] Prior to constructing the synthetic gene, the configuration and stability of the Banlec mutant-XCL chimera were evaluated using bioinformatics tools. Using the sequences of Banlec mutant (Uniprot ID: O22321) and Xerocomus chrysenteron lectin (XCL) (Uniprot ID: Q8WZC9), different arrangements were tested through 3D modeling in the Colabfold software. The positioning of the histidine tail at the C or N-terminal and different linkers and their quantity were evaluated, these being rigid or flexible.

[0039] The stability of the chimeric protein was evaluated in a 100 ns simulation at the expression induction temperature (16 °C) using molecular dynamics in the GROMACS program. The ProtParam program was used to obtain information Petition 870250004579, dated 01 / 21 / 2025, page 22 / 36 / 17 related to biochemical nature, such as isoelectric point, GRAVY index, molar extinction coefficient and absorbance at 280 nm of 1 mg / ml. The solubility of the chimera was verified using the Protein-sol software.

[0040] Molecular docking studies using the Vina-Carb program and the carbohydrates with which the chimera could interact were performed with the T antigen, Tn, and high-mannose complex glycans. For the evaluation of the interaction between the protomers of the chimera, the ClusPro, UDock, and AlphaFold3 software were used.

[0041] Bioinformatics analyses using 3D models confirmed that the best linker to ensure structural flexibility is flexible. The histidine tail positioned at the C-terminal is suitable for the plasmid, and this configuration proved stable in molecular dynamics simulation, showing low variation in the distance between atoms measured by RMSD, RMSF, and radius of gyration. This was the best arrangement for the present objective, presenting the following configuration: HindIII restriction site - Banlec mutant lectin - GGGS (2x) linker - HindIII restriction site - XCL lectin - histidine tail. Biochemical parameter analyses indicated a molecular weight of 32 kDa, isoelectric point 7.84, aliphatic index 74.01, GRAVY index -0.197, molar extinction coefficient 47330, and absorbance at 280 nm (1 mg / ml) 1.467. The predicted solubility was 0.47, only marginally higher than the cutoff point for soluble proteins (0.45).Molecular docking with carbohydrates demonstrated high binding affinity of the chimeric lectin with the T antigen (-6.4 kcal / mol) and high mannose (-5.6 kcal / mol), while analysis of protomer interaction showed that the isolated portions, both from the mutant Banlec and XCL, interact and form dimers, which has already been detected experimentally. Construction, expression, and purification of chimeric and XCL proteins in E. coli.

[0042] The construction of the synthetic gene encompassed the best arrangement derived from bioinformatics analyses, which consisted of lectins connected by two flexible GGGS linkers, a histidine tail (His6x-tag) in the C-terminal portion, and restriction sites for the HindIII enzyme. This restriction site was added in the N-terminal portion and immediately after the linker, aiming to separate the mutant Banlec portion from the chimeric protein and produce XCL in isolation. The sequences of the chimera and XCL were expressed using a Petition 870250004579, dated 01 / 21 / 2025, page 23 / 36 / 17 synthetic construct obtained from the company Epoch Life Sciences®, inserted into the pET24(+) vector and transformed into Escherichia coli.

[0043] The DH5a cloning strain was used for the propagation of the synthetic plasmid containing the chimera gene in its entirety as well as only the XCL gene after separation with the restriction enzyme HindIII HF (NEB). The plasmid was incubated for 30 min with the restriction enzyme at 37 °C, followed by inactivation for 20 min at 80 °C. For end ligation, the T4 DNA ligase enzyme (Invitrogen™, Catalog #15224017) was used for 10 min at room temperature followed by inactivation at 65 °C. Samples were analyzed using 1% agarose gel in TAE buffer. For recombinant expression, the following modified E. coli BL21 (DE3) strains were tested: STAR, DE(3), PlysS, SOLU, Rosetta (unpublished data), RIL (Agilent Technologies®) and Origami™ 2 (Sigma Aldrich®). The bacteria were cultured at 37 °C until they reached an optical density (OD) of 0.6 to 0.8, at which point they were induced at a final concentration of 1 mM IPTG (Isopropyl ed-1-thiogalactopyranoside).Induction was performed overnight at 16 °C, followed by centrifugation of the cultures at 20,000 x g for 20 min.

[0044] For purification, standard affinity chromatography buffers (IMAC) (46 mM Na2HPO4, 3 mM NaH2PO4, 290 mM NaCl, 3.07 mM Sodium Azide (NaN3), pH 8) were used. Pellet processing was performed with IMAC without imidazole supplemented with 0.04 g of lysozyme (Sigma Aldrich) and 80 pL of DNase I (Thermo Fisher Scientific), with agitation for 30 min at room temperature. Then, IMAC50 buffer (same composition, supplemented with 50 mM imidazole) was added and the samples were incubated on ice for 10 min. Sonication was performed with the samples on ice four times, with 30 seconds at 50% power and 60-second intervals. The sonicated samples were centrifuged at 38,000 x g for 40 min at 4 °C. The resulting pellet was resuspended in IMAC-25 supplemented with 8M urea in order to obtain the proteins from the inclusion bodies.Twelve to fourteen buffer changes were performed via dialysis in D-PBS buffer (Gibco, Catalog #21600069, 2.66 mM Potassium Chloride (KCl), 1.47 mM Monobasic Potassium Phosphate (KH2PO4), 137.93 mM Sodium Chloride (NaCl), 8 mM Anhydrous Dibasic Sodium Phosphate (Na2HPO4)). Confirmation of recombinant expression and quaternary arrangement of the active forms was determined by SDS electrophoresis. Petition 870250004579, dated 01 / 21 / 2025, page 24 / 36 / 17 PAGE under denaturing and non-denaturing conditions (MiniProtean Gel 4-20%, BioRad) and Western blotting with anti-histidine antibody (Qiagen, 34460).

[0045] The Escherichia coli RIL and Origami strains were tested for both chimerism and XCL. Regarding chimerism, the Origami strain proved to express it in greater quantities, which is seen by the more prominent band at the expected height (32 kDa) (Figure 2). In the case of XCL, the RIL strain was more suitable, as seen by the more prominent 15 kDa band (Figure 3). After dialysis in D-PBS, it was observed that the structure and quaternary arrangements were maintained (Figure 4), which was evaluated through the presence or absence of the denaturing agent β-mercaptoethanol. Thus, it is clear that after purification and removal of the purification buffer, the arrangements formed by both proteins are mostly dimers, with tetramers also present.

[0046] After characterizing the best production method for the Banlec mutant XCL chimera, it was concluded that the chimera is expressed in greater quantities in the Origami strain, with overnight induction at 16 °C and forms dimers that are detected in the Western Blot at a height of 64 kDa. In the case of XCL, a 16 kDa protein, in this study it is undergoing dimerization, represented by a 30 kDa band also recognized in the Western Blot. Both proteins are found in inclusion bodies, therefore, in the insoluble fraction, in agreement with the in silico analysis. EXAMPLE 2 - EFFECT OF CHIMERIC LECTIN ON THE PROLIFERATION OF PANCREATIC CANCER CELLS

[0047] The procedures described below are used to evaluate the antiproliferative potential of chimeric lectin. Antiproliferative activity test in cell culture

[0048] For the antiproliferative activity test, we used CFPAC-1 pancreatic cancer cells. The cell line was maintained in 175 cm2 (T-125) flasks with IMDM culture medium (25 mM HEPES + L-glutamine) supplemented with 10% fetal bovine serum until confluence was reached, in a controlled atmosphere of 5% CO2 and a temperature of 37 °C. 3 x 10⁴ cells per well were seeded in 48-well plates, and data were obtained in triplicate. As a control, cells supplemented with D were used. Petition 870250004579, dated 01 / 21 / 2025, page 25 / 36 / 17 Sterile PBS was used to equalize the amount of this buffer present in the protein treatments. The proteins were added to the culture medium at a final concentration of 10 μM and incubated with the cells for 6 days. The antiproliferative activity of the proteins was indirectly measured through cellular metabolic activity using the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). On day 0 and from day 3 onwards, the MTT reagent (12 mM) was added for 3 hours. After careful removal, the cells were incubated for 10 minutes with 100 μL of DMSO to solubilize the formazan crystals for absorbance reading at 540 nm. To calculate cell viability, the DMSO background value was subtracted, and the percentage of viable cells normalized from each triplicate was calculated relative to the average of the triplicates on day 0. The values ​​from each triplicate were entered into GraphPad Prism 10 software (Version 10.1).2) and a two-way ANOVA analysis of variance was performed to identify differences between groups, with Tukey's post-hoc test used to identify which groups differ from each other.

[0049] Tests were performed to evaluate the antiproliferative action in a pancreatic cancer cell line, CFPAC-1. We evaluated the antiproliferative activity of the chimera and its isolated constituents (mutant Banlec or XCL) in vitro. A pronounced cell death effect was observed from the third day of the assay, as already observed for mutant Banlec, but the maximum cell death effect was obtained on the fifth and sixth days after treatment (Figure 5). The percentage of cell viability was obtained through the MTT assay and calculation of the absorbances of the triplicates of each assay, in 3 different assays (Figure 6). The effect of 10 μM of the chimera demonstrated greater antiproliferative activity on day 6 compared to the proteins tested in isolation, however, it is statistically equal to the effect of mutant Banlec alone. In these assays, isolated XCL showed moderate antiproliferative activity compared to the other two proteins. Determination of the mechanism of action of chimera and XCL in pancreatic cancer cells.

[0050] In order to determine by which pathway the expressed lectins were acting to cause cell death, a series of Western blots were performed using CFPAC-1 cell extract. The cells were maintained as previously mentioned until they reached confluence, and then 2x10⁶, 5x10⁵ and 1.25 x 10⁵ cells were seeded per plate. Petition 870250004579, dated 01 / 21 / 2025, pp. 26 / 36 / 17 respectively, in 100 mM diameter culture plates (Thermo Fisher Scientific) and treated after 24h with the proteins. The extracts were recovered by scraping with sterile 1x PBS at timepoints 24h, 72h and 120h after the start of treatment. The total extract was centrifuged at 1500 rpm at 4 °C for 10 min. The supernatant was discarded and the extraction of total proteins proceeded. The cells were lysed with CHAPS buffer (150 mM KCl, 50 mM HEPES, 0.1% CHAPS) supplemented with a 1% phosphatase inhibitor cocktail and a protease inhibitor cocktail with 1X EDTA for 30 min at 4 °C under gentle agitation and then centrifuged at 13.3 rpm for 10 min at 4 °C. The cell extract was quantified using the Bradford method.Specific antibodies for various proteins related to both apoptosis and autophagy were used to characterize the pathways of action: T-EGFR (4267), CASP3 (9662), T-BECLIN-2 (3738S), LC3B (3868), PARP1 (9542), T-AKT (9272), and GAPDH (mouse - 2118). These antibodies were purchased from Cell Signaling® and produced in rabbits. The secondary antibodies used were anti-rabbit (goat - 31462) and anti-mouse (horse 7076; goat - A2554). For detection, a concentration of 30 µg of total protein per well was used, calculated from the Bradford assay (BRADFORD, MM A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analytical Biochemistry, n. 72, p. 248-254, 1976).

[0051] The analyzed proteins that revealed distinct results between treatments were total EGFR (T-EGFR), total Beclin (T-Beclin), LC3B, and uncleaved Caspase-3, but not between T-AKT and PARP-1. These proteins are involved in known cell death pathways caused by lectins. Treatment with each of the lectins shows different levels of these proteins in the cell extracts (Figure 7). In the case of the mutant Banlec, there is a drastic reduction in T-EGFR and T-Beclin levels compared to the control at 24, 72, and 120h; LC3B and caspase-3 are only subtly decreased. Treatment with the chimera revealed a decrease in T-Beclin levels from 72h onwards, and LC3B is practically undetectable up to 120h. T-EGFR levels decrease until 72h, but increase at the last timepoint. Casp-3 appears to be slightly increased after 120 hours.XCL presents a distinct profile from the other two lectins, with no relevant interference in TEGFR and Casp-3, while T-Beclin increases at 120h and LC3B remains practically undetectable until the same timepoint. This analysis is extremely important because there is... Petition 870250004579, dated 01 / 21 / 2025, page 27 / 36 / 17 indicates that the chimera acts differently from the isolated proteins, possibly through either apoptosis or autophagy. However, the cell death mechanism involved in the antiproliferative activity of the chimera cannot yet be determined.

[0052] In summary, the chimera affected cell growth as well as or better than the isolated mutant Banlec, while the isolated XCL did not show equivalent antiproliferative activity. It was characterized that the antiproliferative activity of the chimera may be occurring through both apoptosis and autophagy due to the treatments presenting distinct protein profiles. A potential therapeutic agent that acts on both cell death pathways represents a new possibility for destroying cancer cells. Petition 870250004579, dated 01 / 21 / 2025, pages 28 / 36

Claims

1 / 2 CLAIMS 1. DNA SEQUENCE FOR PRODUCTION OF THE RECOMBINANT CHIMERIC LECTIN “BANLEC MUTANT-XCL” AND USE AS AN ANTITUMORAL AGENT characterized by a synthetic nucleotide sequence (SEQ ID No. 1) encoding the recombinant protein “Banlec mutant-XCL”; 2. DNA SEQUENCE FOR PRODUCTION OF RECOMBINANT LECTIN “BANLEC MUTANT-XCL” AND USE AS AN ANTITUMORAL AGENT, as claimed in claim 1, characterized by a nucleotide sequence that may comprise from 50% to 100% of the sequence defined as SEQ ID No. 1; 3. DNA SEQUENCE FOR PRODUCTION OF RECOMBINANT LECTIN “MUTANT-XCL BANLEC” AND USE AS AN ANTITUMOR AGENT, according to claims 1 and 2, characterized by using a vector for expression of the “mutant-XCL BANLEC” protein in bacterial cells, but not limited to, including yeast, insects, mammals or plants; 4. DNA SEQUENCE FOR PRODUCTION OF RECOMBINANT LECTIN “BANLEC MUTANT-XCL” AND USE AS AN ANTITUMORAL AGENT, according to claims 1, 2 and 3, characterized by comprising a variant polypeptide of the “Banlec mutant-XCL” protein, with 50% to 100% identity, demonstrating biological activity; 5. DNA SEQUENCE FOR PRODUCTION OF RECOMBINANT LECTIN “BANLEC MUTANT-XCL” AND USE AS AN ANTITUMOR AGENT, according to claims 1, 2, 3 and 4, characterized by exhibiting antitumor activity in CFPAC-1 pancreatic cancer cells at a concentration of 0.01 to 10 μM; 6. DNA SEQUENCE FOR THE PRODUCTION OF RECOMBINANT LECTIN “BANLEC MUTANT-XCL” AND USE AS AN ANTITUMOR AGENT, as per Petition 870250061380, dated 07 / 17 / 2025, page 4 / 5 2 / 2 claims 1, 2, 3 and 4, characterized by exhibiting antitumor activity in tumor cell lines at a concentration of 0.01 to 10 μM; 7. DNA SEQUENCE FOR THE PRODUCTION OF RECOMBINANT LECTIN “BANLEC MUTANT-XCL” AND USE AS AN ANTITUMOR AGENT, according to claims 1, 2, 3 and 4, characterized by comprising an antitumor formulation in the form of a solution or emulsion, but not limited to these, containing recombinant “Banlec mutant-XCL” lectin or parts thereof, these compositions being able to be generated from a variety of components including, but not limited to, pre-formed liquids, self-emulsifying solids and self-emulsifying semi-solids. Petition 870250061380, dated 07 / 17 / 2025, page 5 / 5

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