PROTEÍNAS DE LIGAÇÃO AO ANTÍGENO, PROTEÍNA DE FUSÃO, COMPOSIÇÃO FARMACÊUTICA, USO DE UMA PROTEÍNA DE LIGAÇÃO AO ANTÍGENO E USO DA COMPOSIÇÃO FARMACÊUTICA

Abstract

The present invention provides an antigen-binding protein that specifically binds to a conformational epitope formed by domains iii and iv of the human epidermal growth factor receptor 3 (her3) and antigen-binding proteins that compete with it for binding, as well as a fusion protein or conjugate comprising the same. The present invention also provides a nucleic acid molecule comprising a sequence encoding said antigen-binding proteins, vectors comprising the nucleic acid, and cells and drugs comprising the antigen-binding protein, the fusion protein, the nucleic acid, or the vector. The present invention also provides the antigen-binding protein, the fusion protein or conjugate, the nucleic acid, the vector, the cell, or the drug for use as a medicament.The present invention further provides a method of inhibiting tumor growth or treating cancer, comprising administering a therapeutically effective amount of antigen-binding protein, fusion protein or conjugate, nucleic acid, vector, cell or drug.

Description

1 / 148 “ANTIGEN-BINDING PROTEINS, FUSION PROTEIN, PHARMACEUTICAL COMPOSITION, USE OF AN ANTIGEN-BINDING PROTEIN AND USE OF THE PHARMACEUTICAL COMPOSITION”

[0001] The present invention provides an antigen-binding protein that specifically binds to a conformational epitope formed by domains III and IV of the human epidermal growth factor receptor 3 (HER3) and antigen-binding proteins that compete with it for binding, as well as a fusion protein or conjugate comprising the same. The present invention also provides a nucleic acid molecule comprising a sequence encoding said antigen-binding proteins, vectors comprising the nucleic acid, and cells and drugs comprising the antigen-binding protein, the fusion protein, the nucleic acid, or the vector. The present invention also provides the antigen-binding protein, the fusion protein or conjugate, the nucleic acid, the vector, the cell, or the drug for use as a medicament.The present invention further provides a method of inhibiting tumor growth or treating cancer, comprising administering a therapeutically effective amount of antigen-binding protein, fusion protein or conjugate, nucleic acid, vector, cell or drug. Background of the Invention

[0002] The complex signaling network of ErbB family members is tightly regulated in normal human tissue. However, dysregulation of ErbB family members by receptor overexpression, altered receptor function by mutations, or aberrant ligand stimulation is frequently associated with cancer development and spread. EGFR is frequently overexpressed in colorectal cancer, ovarian cancer, squamous cell carcinoma of the head and neck, and other cancer types, and EGFR overexpression has been associated with a Petition 870260062263, dated 06 / 25 / 2026, page 12 / 317 2 / 148 poor prognosis. HER2 is particularly associated with human breast cancer, where it is amplified and / or overexpressed by up to 30%. HER3 has also been previously shown to be mutated in ~11% of colon and gastric cancers, promoting oncogenic signaling in the presence of HER2 (Jaiswal et al., 2013, Oncogenic ErbB3 mutations in human cancers. Cancer Cell 23, 603-617). Furthermore, HER3 has gained special interest due to its potent activation of the PI3K / Akt pathway, which has been reported to be responsible for resistance mechanisms against ErbB-targeted therapies (Holbro et al., 2003, The functions of ErbB2 / ErbB3 heterodimer as an oncogenic unit: ErbB2 requires ErbB3 to drive tumor cell proliferation. Proc. Natl. Acad. Sci. USA 100:8933-8938). The role of HER4 in cancer development has been controversially discussed, but increasingly, studies have revealed that HER4 is associated with tumorigenesis, especially in relation to acquired resistance (Canfield et al.)., 2014, Receptor tyrosine kinase ErbB4 mediates acquired resistance to ErbB2 inhibitors in breast cancer cells. Cell Cycle 14: 648-655).

[0003] Oncogenic mutations in HER3 have been identified, for example, in about 11% of colon and gastric cancers (Jaiswal et al., 2013). Such mutations have been shown to transform colon and breast epithelial cells in a ligand-independent manner (Jaiswal et al., 2013, Oncogenic ErbB3 mutations in human cancers. Cancer Cell 23, 603-617). Mutations in the extracellular region were located in domains I, II, and III, with many hot spots in domain II (A232V, P262H / S, G284R, D297Y, G325R), one in domain I (V104M), and one in domain III (T355A / I) (Gaborit et al. 2015, Emerging anti-cancer antibodies and combination therapies targeting HER3 / ErbB3. Hum. Vaccin. Immunother. 12:576-592).

[0004] Members of the ErbB family can be targeted by antibodies. They can inhibit ligand binding and / or dimerization of Petition 870260062263, dated 06 / 25 / 2026, p. 13 / 317 3 / 148 receptor. Furthermore, antibodies can induce receptor internalization and degradation by cross-linking to the receptor (Friedman et al., 2005, Synthetic downregulation of receptor tyrosine kinases by mAb combinations: implications for cancer therapy. Proc. Natl. Acad. Sci. USA 102:1915-1920; Roepstorff et al., 2008, Endocytic downregulation of ErbB receptors: mechanisms and relevance in cancer. Moody et al., 2015, Receptor crosslinking: a general method to trigger internalization and lysosomal targeting of therapeutic receptor:ligand complexes. Mol. Therapy 23:1888-1898). Additionally, antibodies containing an Fc portion can mediate cancer cell death through effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Antibodies can also be used as a delivery system for cytotoxic agents to cancer cells. Due to its emerging role as a heterodimerization partner involved in the propagation of tumorigenesis and the development of therapy resistance, HER3 has become a target for antibody therapy. Several antibodies targeting HER3 have been developed (Gaborit et al. 2015, Emerging anti-cancer antibodies and combination therapies targeting HER3 / ErbB3. Hum. Vaccin. Immunother. 12: 576-592; Dey et al.2015, A critical role of HER3 in HER2amplified and non-amplified breast cancers: function of a kinase-dead RTK. Am. J. Transl. Res. 7: 733-750; Aurisicchio et al. 2012, The promise of antiErbB3 monoclonals as new cancer therapeutics. Oncotarget 3, 744-758;. Baselga & Swain 2009, Novel anticancer targets: revisiting ErbB2 and discovering ErbB3. Nat. Rev. Cancer 9: 463-475; Gala & Chandariapaty 2014, Molecular pathways: HER3 targeted therapy. Clin. Cancer Res . 20: 1410-1416; Kol et al. 2014, HER3, serious parnter in crime: therapeutic approaches and potential bomarkers for effect of HER3-targeting. Petição 870260062263, de 25 / 06 / 2026, pág. 14 / 317 4 / 148 Pharmacol. Ther. 143: 1-11; Zhang et al. 2016, HER3 / ErbB3, an emerging therapeutic target for cancer. Acta Biochim. Biophys. Syn. 48: 39-48) with several of them directed against domain I or III involving ligand binding, others directed against domain II and / or IV are involved in receptor dimerization. It has been described that an antibody, KTN3379, binds between domains II and III blocking the receptor in an inactive conformation (Lee et al., 2015, Inhibition of ErbB3 by a monoclonal antibody that locks the extracellular domain in an inactive configuration. Proc. Natl. Acad. Sci. USA 112: 13225-13230).

[0005] However, since the domains targeted by these antibodies may comprise one or more oncogenic mutations, they may not be reactive against wild-type HER3 or against mutated HER3, or against an oncogenic mutated HER3 that is mutated at a position other than that targeted by the respective antibody. Thus, there is a need in the state of the art for an antagonist molecule that is reactive to both wild-type and mutated HER3. Furthermore, in order to inhibit HER3 activation in both ligand-independent and ligand-dependent ways, there is a need for an antagonist molecule that binds to HER3 in order to inhibit heterodimerization as well as inhibit ligand binding.

[0006] To solve the above problem, we identified a human anti-HER3 antibody (ErbB3), 3-43, that recognizes a unique epitope in HER3 formed by domains III and IV, which is conserved between human and mouse HER3. This antibody binds as an IgG molecule with EC50 values ​​below 0.1 nM to HER3-expressing tumor cells, efficiently inhibits ligand-independent and ligand-dependent receptor activation and downstream signaling, and leads to rapid and efficient receptor internalization and degradation. Petition 870260062263, dated 06 / 25 / 2026, p. 15 / 317 5 / 148 Brief Description of the Invention

[0007] In a first aspect, the present invention provides an antigen-binding protein that specifically binds to a conformational epitope formed by domains III and IV of the human epidermal growth factor receptor 3 (HER3).

[0008] In a second aspect, the present invention provides an antigen-binding protein that competes with the antigen-binding protein of the first aspect.

[0009] In a third aspect, the present invention provides a protein or fusion conjugate comprising the antigen-binding protein of the first or second aspect.

[0010] In a fourth aspect, the present invention provides a nucleic acid molecule comprising a sequence encoding the antigen-binding protein of the first or second aspect or the fusion protein of the third aspect.

[0011] In a fifth aspect, the present invention provides a vector comprising the nucleic acid of the fourth aspect.

[0012] In a sixth aspect, the present invention provides a cell comprising the antigen-binding protein of the first or second aspect, the fusion protein of the third aspect, the nucleic acid of the fourth aspect or the vector of the fifth aspect.

[0013] In a seventh aspect, the present invention provides a pharmaceutical composition comprising the antigen-binding protein of the first or second aspect, the fusion protein of the third aspect, the nucleic acid of the fourth aspect, or the vector of the fifth aspect.

[0014] In an eighth aspect, the present invention provides the antigen-binding protein of the first or second aspect, the fusion protein or conjugate of the third aspect, the nucleic acid of the fourth aspect or Petition 870260062263, dated 06 / 25 / 2026, p. 16 / 317 6 / 148 the vector of the fifth aspect, the cell of the sixth aspect, or the pharmaceutical composition of the seventh aspect for use as a medicine.

[0015] In a ninth aspect, the present invention provides a method of inhibiting tumor growth or treating cancer, comprising administering a therapeutically effective amount of the antigen-binding protein of the first or second aspect, the fusion protein or conjugate of the third aspect, the nucleic acid of the fourth aspect, or the vector of the fifth aspect, the cell of the sixth aspect, or the pharmaceutical composition of the seventh aspect. Brief Description of the Figures

[0016] Fig. 1: Biochemical characterization and binding studies of IgG 3-43. A) SDS-PAGE analysis (stained with Coomassie) under reducing (R) and non-reducing (NR) conditions. B) HPLC and size exclusion chromatography of IgG 3-43. C) HER3 binding was analyzed by ELISA. An Fc fusion protein of the extracellular domain of HER3 was used as an antigen. Data are represented as mean ± standard deviation (SD) of three independent experiments. D) The quartz crystal microbalance experiment was performed using the Attana system. IgG 3-43 was immobilized on a carboxyl chip and frequency changes representing weight gain or loss were measured through binding of the His-labeled extracellular domain of HER3.

[0017] Fig. 2: Epitope mapping and cross-reactivity with mouse HER3; A) Epitope mapping: Sequences encoding truncated forms of the extracellular domain of HER3 were fused with the sequence of the Fc portion of IgG1. The resulting constructs were transfected and expressed in HEK293 cells, and the proteins were purified from the supernatant via protein A-affinity chromatography. The Fc fusion proteins were used as antigens in an assay. Petition 870260062263, dated 06 / 25 / 2026, page 17 / 317 7 / 148 ELISA and IgG 3-43 binding was detected with an HRP-conjugated anti-Fab antibody. B) Cross-reactivity of 3-43 with human and mouse HER3 using the extracellular region of human and mouse HER3 fused to a human Fc region. Binding of scFv 3-43 to immobilized HER3 fusion protein was detected with an anti-Histag antibody.

[0018] Fig. 3: Binding of IgG 3-43 to HER3-expressing tumor cell lines. Several tumor cell lines (as indicated) were incubated with varying concentrations of IgG 3-43 and bound antibody was detected with a PE-labeled secondary antibody. Cells were analyzed using a Miltenyi MACSquant. EC50 values ​​were calculated from n = 1 to 3 experiments.

[0019] Fig. 4: IgG 3-43 competes with HRG for binding to HER3-expressing cells. Binding of recombinant human his-labeled heregulin-βΐ was measured by flow cytometry via PE-conjugated anti-His antibody. Pre-incubation with excess IgG 3-43 potentially reduced the signal by more than 60%, while the anti-EGFR antibody Cetuximab did not show the same effect.

[0020] Fig. 5: IgG 3-43 inhibits HER3 phosphorylation and downstream targets induced by HRG. The indicated cells were seeded in 6-well plates until they were semi-confluent on the day of the experiment. After ligation, the cells were serum-deprived overnight and incubated for one hour with 100 nM IgG 3-43 or control IgG (Rituximab) (A, B, C) or with different concentrations of IgG 3-43 or IgG 3M6 (D, E). IgG-treated and untreated cells were stimulated with human heregulin-βΐ at 50 ng / mL. Subsequently, the cells were lysed with RIPA buffer containing protease inhibitors and the cell lysates were analyzed by Western blot using the indicated antibodies. Petition 870260062263, dated 06 / 25 / 2026, page 18 / 317 8 / 148

[0021] Fig. 6: IgG 3-43 is internalized into cancer cells and leads to a reduction in cellular HER3 levels. A) MCF-7 cells were incubated with 100 nM IgG 3-43 for the indicated time points, and HER3 levels were analyzed by Western blot. The HER32 signal decreased rapidly, with a reduction already observed after 5 minutes of incubation time. B) Cy5-labeled IgG 3-43 was incubated with MCF-7 cells at 37°C for the indicated time points. Cell membranes were stained with Concanavalin-A, and cells were fixed with 4% paraformaldehyde. Images of treated and control cells were acquired on a spinning disk microscope. Blue: Dapi nuclei staining; green: Con A membrane staining; purple: Cy5-labeled IgG 3-43.

[0022] Fig. 7: IgG 3-43 reduces HRG-mediated cancer cell proliferation in vitro. NCI-N87 (A), BT-474 (B), and MCF-7 (C) cells were seeded at low densities in 96-well plates, allowed to adhere overnight, and incubated for one week under low serum concentrations (0.2%) and in the presence of 10 ng / mL heregulin with 10 pg of IgG 3-43 or Rituximab as a control. D) FaDu cells are known to produce heregulin in an autocrine manner and were subjected to the same proliferation assay, but in the absence of heregulin in the medium. Titration of IgG 3-43 revealed a potent inhibitory effect on growth even at nanomolar concentrations.

[0023] Fig. 8: Fig. 8: IgG 3-43 inhibits tumor growth in the sc xenograft model of FaDu tumor in SCID mice. Mice were treated when tumors reached a size of approximately 100 mm3 (2 weekly injections for 3 weeks, see lines) at the indicated doses. A) Kaplan-Mayer survival curve. B) Tumor volumes on day 42. C) - F) Increases in individual tumors in mice treated with PBS (C), 30 pg of IgG 3-43 (D), 100 pg of IgG 3-43 (E) or 300 pg of IgG 3-43 Petition 870260062263, dated 06 / 25 / 2026, p. 19 / 317 9 / 148 (F).

[0024] Fig. 9: Biochemical characterization of scDb hu225x3-43-Fc. A) Schematic arrangement of variable and constant domains in an scDb-Fc fusion protein. B) SDS-PAGE analysis (10% PAA, stained with Coomassie) of cetuximab (lanes 1, 4), IgG 3-43 (lanes 2, 5) and scDb hu225x3-43-Fc (lanes 3, 6) under reducing (lanes 1-3) and non-reducing (lanes 4-6) conditions. C) Schematic structure of a dimeric scDb-Fc fusion protein. D) Size exclusion chromatography of cetuximab, IgG 3-43 and scDb hu225x3-43-Fc.

[0025] Fig. 10: Binding studies of scDb hu225x3-43-Fc. A) The binding of scDb hu225x3-43-Fc compared to cetuximab and IgG 3-43 to immobilized receptor ECD proteins (0.2 μg / well) was analyzed by ELISA. Antibodies were detected with HRP-conjugated anti-human IgG antibody (specific for the Fc portion). Optical density was measured at 450 nm. Data are represented as mean ± standard deviation (SD) of three independent experiments. B) Binding to FaDu cells was analyzed by flow cytometry. All antibodies were detected with PE-conjugated anti-human Fc antibody. Data are represented as mean ± standard deviation (SD) of three independent experiments.

[0026] Fig. 11: Inhibition of HER3 signaling in MCF7 cells. Cells (cultured in RPMI 1640, 0.2% serum) were treated with 75 nM of the parental IgG molecules or scDb-Fc molecules and 37.5 nM of each parental antibody for the combined treatment for 1 ha at 37°C. An irrelevant IgG1 was used as a control. Cells were stimulated with heregulin (50 ng / mL) for 15 min. at 37°C, before being lysed using RIPA buffer (Tris 50 mM pH 7.5, NaCl 150 mM, NaF 10 mM, β-Glycerophosphate 20 mM, EDTA 1 mM, 1% NP-40, NaaVO4 1 mM, PMSF 0.5 mM, 0.25% DOC, 0.1% SDS) containing a cocktail of inhibitors of Petition 870260062263, dated 06 / 25 / 2026, page 20 / 317 10 / 148 proteinase at 4°C. Cell lysates were analyzed by immunoblotting using antibodies against HER3, phospho-HER3 (Tyr1289), Akt, phospho-Akt (Thr308), Erk1 / 2, phospho-Erk1 / 2 (Thr202 / 204), and α-Tubulin. The data presented are representative of two independent experiments.

[0027] Fig. 12: Inhibition of receptor phosphorylation in different cell lines overexpressing ErbB. Different cell lines (A; MCF-7; B, A-431; C, NCI-N87; D, SK-BR-3; E, FaDu; F, A549) were treated with 50 nM cetuximab, IgG 3-43 or scDb hu225x3-43-Fc for 1 ha at 37°C before stimulation with heregulin (50 ng / mL) or EGF (50 ng / mL) for 15 min. Cells were lysed using RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 10 mM NaF, 20 mM β-Glycerophosphate, 1 mM EDTA, 1% NP40, 1 mM Na3VO4, 0.5 mM PMSF, 0.25% DOC, 0.1% SDS) containing a protease inhibitor cocktail, and the cell lysates were analyzed by immunoblotting using antibodies against EGFR, phospho-EGFR (Tyr1068), HER3, phospho-HER3 (Tyr1289), and α-Tubulin.

[0028] Fig. 13: Inhibition of receptor phosphorylation in FaDu cells. Cells were treated with serial dilutions of scDb hu225x3-43Fc and IgG 3-43 combined with cetuximab for 1 ha at 37°C before stimulation with heregulin (50 ng / mL). Cells were lysed using RIPA buffer (Tris 50 mM pH 7.5, NaCl 150 mM, NaF 10 mM, β-Glycerophosphate 20 mM, EDTA 1 mM, NP-40 at 1%, Na3VO4 1 mM, PMSF 0.5 mM, 0.25% DOC, 0.1% SDS) containing a protease inhibitor cocktail, and the lysates were analyzed by immunoblotting using antibodies against HER3, phospho-HER3 (Tyr1289), and α-Tubulin. Phospho-HER3 levels were quantified relative to the α-tubulin loading control and normalized to the control without antibody. The data presented are representative of at least two independent experiments, with error bars representing mean ± SD values. A, quantified data. B, representative images. Petition 870260062263, dated 06 / 25 / 2026, page 21 / 317 11 / 148

[0029] Fig. 14: Biochemical characterization of scDb 2-35x3-43-Fc. A) SDS-PAGE analysis (10% PAA, stained with Coomassie) of scDb 2-35x3-43-Fc under reducing (R) and non-reducing (NR) conditions. B) Size exclusion chromatography of scDb 2-35x3-43-Fc.

[0030] Fig. 15: Binding studies with scDb 2-35x3-43-Fc. A) The binding of scDb 2-35x3-43-Fc compared to cetuximab and IgG 3-43 to immobilized receptor ECD proteins (0.2 μg / well) was analyzed by ELISA. Antibodies were detected with HRP-conjugated anti-human IgG antibody (specific for the Fc portion). Optical density was measured at 450 nm. Data are represented as mean ± standard deviation (SD) of three independent experiments.

[0031] Fig. 16: Biochemical characterization of scFv-3-43-Fc-scTRAIL. A) SDS-PAGE analysis (10% PAA, stained with Coomassie) under reducing (R) and non-reducing (NR) conditions. B) Size exclusion chromatography of scFv-3-43-Fc-scTRAIL.

[0032] Fig. 17: Binding studies with scFv-3-43-Fc-scTRAIL. Binding to human HER3 (A) and TRAIL-R2 (B) was analyzed by ELISA. Fc fusion proteins of the extracellular domains of human HER3 or TRAIL-R2 were used as antigens. Optical density was measured at 450 nm. Binding to Colo205 (C) and HCT-116 (D) cells was analyzed by flow cytometry. Data are represented as mean ± SD from at least three independent experiments.

[0033] Fig. 18: Induction of cell death compared to an untargeted construct. The induction of cell death by scFv3-43-FcscTRAIL was analyzed in comparison with the corresponding untargeted (non-target) Fc-scTRAIL fusion proteins. The effects on Colo205 were investigated after pre-incubation with medium or bortezomib (650 nM) to sensitize the cells to TRAIL-induced apoptosis. For Petition 870260062263, dated 06 / 25 / 2026, page 22 / 317 12 / 148 To confirm the targeting effects of scFv3-43-Fc-scTRAIL, experiments were additionally performed in the presence of a 200-fold molar excess of scFv3-43-Fc. Data are represented as mean ± standard deviation (SD) of three independent experiments.

[0034] Fig. 19: Biochemical characterization, binding, and IL2 assay of scDb 3-43xCD3. A) Schematic arrangement of variable and constant domains in an scDb construct. B) Schematic structure of an scDb construct. C) SDS-PAGE analysis (12% PAA, stained with Coomassie) of scDb 3-43xCD3 under reducing (R) and non-reducing (NR) conditions. D) Size exclusion chromatography of scDb 3-43xCD3. E) The binding of scDb 3-43xCD3 was analyzed by ELISA using an Fc fusion protein of the extracellular domain of HER3 as an antigen. The protein was detected with HRP-conjugated anti-His antibody. Optical density was measured at 450 nm. F and G) Binding to MCF-7 cells expressing HER3 (F) and Jurkat cells expressing CD3 (G) was analyzed by flow cytometry. The bound protein was detected with anti-His antibody conjugated with PE. H) IL-2 release from PBMCs activated by scDb 3-43xCD3 bound to Colo205 cells expressing HER3.The IL-2 concentration in the supernatant was determined by ELISA according to the manufacturer's instructions (human IL-2 kit, R&D). Data are presented as mean ± SD.

[0035] Fig. 20: Biochemical characterization and binding of a trivalent, bispecific scDb3-43xCD3-scFv3-43 fusion protein. A) Schematic arrangement and structure of variable domains in an scDb-scFv construct. B) SDS-PAGE analysis (10% PAA, stained with Coomassie) of scDb343xCD3-scFv3-43 under reducing (1) and non-reducing (2) conditions. C) Size exclusion chromatography of scDb3-43xCD3-scFv3-43. D) The binding of scDb3-43xCD3-scFv3-43 was analyzed by ELISA using an Fc fusion protein of the extracellular domain of HER3 as an antigen. A Petition 870260062263, dated 06 / 25 / 2026, page 23 / 317 13 / 148 protein was detected with HRP-conjugated anti-His antibody. scDb3-43xCD3 binding was used as a monovalent control (for HER3). Optical density was measured at 450 nm. E) and F) Binding to MCF-7 cells (E) expressing HER3 and Jurkat cells expressing CD3 (F) was analyzed by flow cytometry. Bound protein was detected with PE-conjugated anti-His antibody. Data are represented as mean ± SD.

[0036] Fig. 21: Characterization of a bispecific 4D5x3-43Fc scDb targeting HER2 and HER3. A) SDS-PAGE analysis (10% PAA, Coomassie stained) of 4D5x3-43-Fc scDb under reducing (1) and non-reducing (2) conditions. B) Size exclusion chromatography of 4D5x3-43Fc scDb. C) Binding of 4D5x3-43-Fc scDb was analyzed by ELISA using His-labeled proteins from the extracellular domains of HER2 or HER3 as antigens. Bound protein was detected with HRP-conjugated human anti-Fc antibody. Optical density was measured at 450 nm. D) Binding to FaDu cells expressing HER2 and HER3 was analyzed by flow cytometry. The bound protein was detected with anti-human Fc antibody conjugated with PE. Data are represented as mean ± SD.

[0037] Fig. 22: IgG 3-43 inhibits ligand-independent colony formation of SKBR3 and BT474. A) Colony formation assay with SKBR3 and BT474 incubated for 12 days with IgG 3-43 (50 nM). Untreated cells (con) and trastuzumab-treated cells (Tras., directed against HER2) were included as additional controls. Triplicates are shown. B) Quantification of colonies formed from SKBR3 and BT474 cells incubated as described in (A).

[0038] Fig. 23: Biochemical characterization and binding studies of Db3-43xhu225-Ig. A) Schematic illustration of the light and heavy chains of the Db3-43xhu225-Ig fusion protein. B) Schematic structure of the domains in the Db3-43xhu225-Ig fusion protein. C) SDS-PAGE analysis (PAA at 10%; Petition 870260062263, dated 06 / 25 / 2026, page 24 / 317 14 / 148 stained with Coomassie) of the Db3-43xhu225-Ig fusion protein under reducing (1) and non-reducing (2) conditions (marker: M). D) Size exclusion chromatography of the Db3-43xhu225-Ig fusion protein. E) The binding of the tetravalent, bispecific Db343xhu225-Ig was analyzed by ELISA using His-labeled fusion proteins from the extracellular domain of EGFR or HER3 as antigen. The bound protein was detected with an HRP-conjugated anti-human Fc antibody. Parenteral antibodies (cetuximab and 3-43-IgG) were used as controls. Optical density was measured at 450 nm. F) The simultaneous binding of the bispecific fusion protein Db3-43xhu25-Ig was analyzed by ELISA using an Fc fusion protein from the extracellular domain of EGFR as the first antigen. Db3-43xhu225-Ig in serial dilutions was added to the wells. Finally, the second antigen, HER3-His, was added to the wells.HER3-His bound was detected using an HRP-conjugated anti-His antibody. Optical density was measured at 450 nm. G) Db3-43xhu225-Ig binding to cells was analyzed by flow cytometry. Different tumor cell lines (MCF-7, SKBR-3, and FaDu) were incubated with a serial dilution of bispecific Db3-43xhu225-Ig or the parental monoclonal antibodies (cetuximab and 3-43-IgG). Bound antibody was detected via PE-labeled human anti-Fc secondary antibody. Cells were analyzed using a Miltenyi MACSquant.

[0039] Fig. 24: Pharmacokinetics of Db3-43xhu225-Ig in SWISS mice. The pharmacokinetic profile of Db3-43xhu225-Ig was determined in female SWISS mice (3 mice). 25 μg of protein were injected intravenously into the tail vein. Concentrations of serum samples collected after the indicated time intervals were determined by ELISA using EGFR-Fc or HER3-Fc fusion protein as coated antigen. Bound Db3-43xhu225-Ig molecules were Petition 870260062263, dated 06 / 25 / 2026, page 25 / 317 15 / 148 detected using an HRP-conjugated anti-human Fab antibody.

[0040] Fig. 25: A HER3-targeting scFv3-43-Fc-scTRAIL fusion protein. A) HER3 expression by melanoma cells was analyzed by flow cytometry and quantified via QIFIKIT. B) Schematic composition of the scFv3-43-Fc-scTRAIL polypeptide. C) Schematic composition of the dimeric scFv-Fc-scTRAIL fusion protein. D) Binding of the scFv3-43-Fc-scTRAIL fusion protein to the HER3-positive A375 cell line was assessed by flow cytometry. The cell-bound protein was detected by anti-human IgG (specific for γ chain) R-PE. EC50 values ​​are indicated as dashed lines. EC50 value meanings were calculated in comparison to Fc-scTRAIL in the respective cell line. E) Competitive inhibition with scFv3-43-Fc (inhibitor) was performed in the A375 cell line. Cells were treated with a 200x molar excess of inhibitor before being treated with the protein (10 nM).The cell-bound protein was detected via anti-human TRAIL-PE.

[0041] Fig. 26: Cell death induction of scFv3-43-Fc-scTRAIL targeting HER3 and quantitative analysis of HER3 antigen density on the cell surface in the presence or absence of bortezomib (BZB). For cell death induction assays, cells were pre-incubated with medium or bortezomib for 30 min before being treated with the serial dilution of scFv3-43-Fc-scTRAIL for 16 h. Cell viability was analyzed by crystal violet staining. For statistical analysis, EC50 values ​​of scFv3-43-Fc-scTRAIL were compared with Fc-scTRAIL. EC50 values ​​are indicated as dashed lines if the target effect is significant. HER3 antigen density was determined using QIFIKIT. Therefore, the cells were treated with the same concentration of bortezomib that was used for the cell death induction assays. Statistical analysis was performed using the unpaired t-test (two-tailed, p < 0.05 *, p < 0.01 **, p < Petition 870260062263, dated 06 / 25 / 2026, p. 26 / 317 16 / 148 0.001 ***, p > 0.05 ns).

[0042] Fig. 27: In vivo activity, tolerability, and PK of scFv3-43Fc-scTRAIL and Fc-scTRAIL. A) Nude NMRI mice (6 mice per group) with established Colo205 tumors were treated with 0.2 nmol protein (corresponding to 0.4 nmol scTRAIL units; iv) or PBS twice weekly for three weeks (days 14, 18, 21, 25, 28, 32). Treatments are indicated with dashed lines. B) Statistical analysis of tumor volumes of the different treated groups on day 47 was performed using one-way ANOVA followed by Tukey's post-hoc test (* P <0.05; ** P <0.01; *** P <0.001; ns, P> 0.05). C) ALT activity and D) serum concentration of the molecules was determined 4 hours and 24 hours after the last treatment (day 32).

[0043] Fig. 28: Biochemical characterization and binding of scDbhu225x3-43-Fc. A) Schematic illustration of the scDbhu225x3-43-Fc fusion protein. B) Schematic structure of the domains in the scDbhu225x3-43-Fc fusion protein. C) SDS-PAGE analysis (10% PAA; stained with Coomassie) of the scDbhu225x3-43-Fc fusion protein under reducing (1) and non-reducing (2) conditions (marker:M). D) Size exclusion chromatography of the scDbhu225x3-43-Fc fusion protein. E) The binding of the bispecific, tetravalent scDbhu225x3-43-Fc fusion protein was analyzed by ELISA using a recombinant His-labeled protein from the extracellular domain of EGFR or HER3 as antigen. The bound protein was detected with an HRP-conjugated anti-human Fc antibody. Parental antibodies (hu225-IgG and 3-43-IgG) were used as controls. Optical density was measured at 450 nm.

[0044] Fig. 29: Inhibition of receptor signaling in FaDu cells. Cells were treated with 50 nM of IgG hu225, IgG 3-43, a combination of IgG hu225 and IgG 3-43, scDbhu225x3-43-Fc (GGGGS), or Db3 Petition 870260062263, dated 06 / 25 / 2026, page 27 / 317 17 / 148 43xhu225-Ig for 1 hour before stimulation with heregulin (50 ng / ml) for 15 min at 37°C. Cells were lysed using RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 10 mM NaF, 20 mM β-Glycerophosphate, 1 mM EDTA, 1% NP-40, 1 mM NaaVO4, 0.5 mM PMSF, 0.25% DOC, 0.1% SDS) containing a protease inhibitor cocktail, and the cell lysates were analyzed by immunoblotting using antibodies against EGFR, phosphoEGFR (Tyr1068), phospho-HER2 (Tyr1221 / 1222), HER3, phospho-HER3 (Tyr1289), Akt, phospho-Akt (Thr308), Erk, phospho-Erk (Thr202 / Tyr204), and α-Tubulin. αTubulin, pHER3, EGFR, Akt and Erk were on membrane 1, aTubulin2, HER3, pEGFR, pHER2, pAkt and pErk were on membrane 2.

[0045] Fig. 30: Proliferation assay using scDbhu225x3-43Fc or Db3-43xhu225-Ig. SW620, HCT116 and LoVo cells were used for the 2D (A) and 3D (B) proliferation assays. 2000 cells / well in a 96-well plate format were cultured for 24 hours in RPMI medium containing 10% SBF (for 3D culture: 1:2 mixture of Matrigel:Kollagen, RPMI or DMEM + 10% SBF + 2% Matrigel). Next, the medium was replaced with deprivation medium (RPMI medium containing 0.2% SBF and 1% P / S) and after 24 hours of culture, the cells were treated with different antibodies (Cetuximab, 3-43-IgG: 50 nM alone or 50 nM each in combination; scDbhu225x3-43-Fc, Db3-43xhu225-Ig: 50 nM) in the presence or absence of MEK inhibitor (AZD6244, Selumetinib; unstimulated with HRG: 5 nM for SW620, 45 nM for HCT116, 35 nM for LoVo; stimulated with HRG: 10 nM for SW620, 300 nM for HCT116, 250 nM for LoVo).After 1 hour of incubation, the cells were stimulated with heregulin (6 ng / well) or kept unstimulated. On day 8 after seeding the cells, the plates were analyzed using CelltiterGlo 2.0 kit (A) (25 pl of deprivation medium mixed with 25 pl of reagent from the CelltiterGlo 2.0 kit per well) or CelltiterGlo 3D kit (B) (25 pl of deprivation medium mixed with 25 pl of reagent). Petition 870260062263, dated 06 / 25 / 2026, page 28 / 317 18 / 148 CelltiterGlo 3D per well) measuring luminescence. The luminescence of untreated cells (no antibody, no AZD62244, no HRG) was set to 100%; Mean ± SD, n = 2.

[0046] Fig. 31: Biochemical characterization and bioactivity of scDb4D5x3-43-LL. A) Schematic illustration of the scDb4D5x343-LL fusion protein. B) Schematic structure of the domains in the scDb4D5x3-43-LL fusion protein. C) SDS-PAGE analysis (12% PAA; stained with Coomassie) of the scDb4D5x3-43-LL fusion protein under reducing (1) and non-reducing (2) conditions (marker:M). D) Size exclusion chromatography of the scDb4D5x3-43-LL fusion protein. E) The bispecific, bivalent binding of the scDb4D5x3-43-LL fusion protein was analyzed by ELISA using an extracellular domain fusion protein from HER2 or HER3 as an antigen. The bound protein was detected with HRP-conjugated anti-His antibody. Parenteral antibodies (Trastuzumab and 3,43-IgG) were used as controls. Optical density was measured at 450 nm.

[0047] Fig. 32: Inhibition of receptor signaling in MCF-7 cells. Cells were treated with 50 nM trastuzumab, IgG 3-43, a combination of trastuzumab and IgG 3-43, scDb 4D5x3-43-LL-Fc or scDb 4D5x3-43-LL for 1 h before stimulation with heregulin (50 ng / ml) for 15 min at 37°C. Cells were lysed using RIPA buffer (Tris 50 mM pH 7.5, NaCl 150 mM, NaF 10 mM, β-Glycerophosphate 20 mM, EDTA 1 mM, NP-40 at 1%, NaaVO4 1 mM, PMSF 0.5 mM, 0.25% DOC, 0.1% SDS) containing a protease inhibitor cocktail, and the cell lysates were analyzed by immunoblotting using antibodies against EGFR, phosphoEGFR (Tyr1068), phospho-HER2 (Tyr1221 / 1222), HER3, phospho-HER3 (Tyr1289), Akt, phospho-Akt (Thr308), Erk, phospho-Erk (Thr202 / Tyr204), and α-Tubulin. αTubulinal, pHER3, HER2, Akt, and Erk were in membrane 1, while αTubulinal2, HER3, pHER2, pAkt, and pErk were in membrane 2. Petition 870260062263, dated 06 / 25 / 2026, page 29 / 317 19 / 148

[0048] Fig. 33: Biochemical characterization and bioactivity of multivalent bispecific antibodies directed against HER3 and CD3. (A+B) Schematic illustration (A) and structure (B) of the bivalent (scDb3-43xhuU3), trivalent (scDb3-43xhuU3-scFv3-43) or tetravalent (scFv3-43-scDb3-43xhuU3-scFv3-43) bispecific fusion protein. (C+D) The binding of the different bispecific fusion proteins to the CD3-positive Jurkat cell line (C) and the HER3-positive MCF-7 cell line (D) was analyzed by flow cytometry. A serial dilution of the bispecific antibodies was incubated with the cells for 1 hour at 4°C. The bound antibody was detected via PE-labeled human anti-Fc secondary antibody. Cells were analyzed using a Miltenyi MACSquant. E) IL-2 release from PBMCs activated by multivalent bispecific antibodies bound to HER3-expressing MCF-7 cells.After 24 hours, the IL-2 concentration in the supernatant was determined by ELISA according to the manufacturer's instructions (human IL-2 kit, R&D). F) Bispecific multivalent antibodies were titrated and incubated with MCF7 as target cells for one hour before human PBMCs were added. Cell viability was determined by MTT assay after 48 hours of incubation. Additionally, bispecific multivalent antibodies were titrated and incubated in MCF7 as target cells without the addition of PBMCs. Cell viability was determined via MTT assay after 48 hours of incubation and indicated with gray symbols and dashed lines for each protein.

[0049] Fig. 34: Analysis of mutated HER3-Fc fusion proteins and analysis of binding to 3-43-IgG. A) SDS-PAGE analysis of purified HER3Fc mutants (1, T335A; 2, T389I; 3, M406K; 4, R453H; 5, Y464C; 6, D492H; 7, K498I) under reducing conditions. The gel was stained with Coomassie Blue. B) Binding of IgG 3-43 to immobilized HER3-Fc and wild-type HER3-Fc mutants using 100 nM of IgG 3-43 detected with an anti-IgG antibody. Petition 870260062263, dated 06 / 25 / 2026, page 30 / 317 20 / 148 Human Fab conjugated with horseradish peroxidase and with the signal obtained normalized by the wild-type HER3-Fc fusion protein. The Y464C mutant was not included due to aggregate formation, as revealed by SEC analysis. 3M6-IgG directed against human HER3 domain I was included as a positive control. List of Sequences - Free Text Information

[0050] SEQ ID NO: 1 Amino acid sequence of Her3 (Expasy entry no: P21860).

[0051] SEQ ID NO: 2 IgG heavy chain variable domain amino acid sequence 3-43.

[0052] SEQ ID NO: 3 IgG light chain variable domain amino acid sequence 3-43.

[0053] SEQ ID NO: 4 IgG heavy chain amino acid sequence 3-43.

[0054] SEQ ID NO: 5 IgG light chain amino acid sequence 3-43.

[0055] SEQ ID NO: 6 scFv amino acid sequence 3-43.

[0056] SEQ ID NO: 7 PelB leader amino acid sequence - scFv 3-43 - c-myc - his.

[0057] SEQ ID NO: 8 IgG leader amino acid sequence - scDb hu225x3-43-Fc.

[0058] SEQ ID NO: 9 IgG leader amino acid sequence - 2-35 x 3-43 scDb-Fc.

[0059] SEQ ID NO: 10 IgG leader amino acid sequence - scDb 4D5x3-43-LL-Fc.

[0060] SEQ ID NO: 11 Amino acid sequence of IgK leader - FLAG - ligand - scFv3-43-Fc-scTRAIL. Petition 870260062263, dated 06 / 25 / 2026, page 31 / 317 21 / 148

[0061] SEQ ID NO: 12 - scDb 3-43xCD3 - His.

[0062] SEQ ID NO: 13 IgK leader amino acid sequence IgK leader amino acid sequence - scDb 3-43xCD3-scFv 3-43 - His.

[0063] SEQ ID NO: 14 peptide 1: GGGGS. Ligand amino acid sequence

[0064] SEQ ID NO: 15 peptide 2: GGGGSGGGGS. Ligand amino acid sequence

[0065] SEQ ID NO: 16 peptide 3: GGGGSGGGGSGGGGS. Ligand amino acid sequence

[0066] SEQ ID NO: 17 peptide 4: GSLGGSGG. Ligand amino acid sequence

[0067] SEQ ID NO: 18 peptide 5: GGGSGGGT. Amino acid sequence of ligand

[0068] SEQ ID NO: 19 peptide 6: GGGSGGGTGS. Amino acid sequence of ligand

[0069] SEQ ID NO: 20 peptide 7: GGGSGGGTGSGG. Amino acid sequence of ligand

[0070] SEQ ID NO: 21 Amino acid sequence of peptide ligand 8: GGGGSGGRASGGGGS GGGGS.

[0071] SEQ ID NO: 22 peptide 9: GGGSGGGS. Amino acid sequence of ligand

[0072] SEQ ID NO: 23 peptide 10: EFTRG.Amino acid sequence of ligand

[0073] SEQ ID NO: 24 peptide 11: AAA. Amino acid sequence of ligand

[0074] SEQ ID NO: 25 tag. Amino acid sequence of FLAG-. Petition 870260062263, dated 06 / 25 / 2026, p. 32 / 317 22 / 148

[0075] SEQ ID NO: 26

[0076] SEQ ID NO: 27

[0077] SEQ ID NO: 28 PelB leader sequence.

[0078] SEQ ID NO: 29 IgK leader sequence.

[0079] SEQ ID NO: 30 IL-2 sequence leader.

[0080] SEQ ID NO: 31 43xVLhu225-CL.

[0081] SEQ ID NO: 32 VHhu225xVLhu3-43-CH1-CH2-CH3.

[0082] SEQ ID NO: 33 scDbhu225x3-43-Fc (GGGGS).

[0083] SEQ ID NO: 34 scDb4D5x3-43-LL.

[0084] SEQ ID NO: 35 43-scDb3-43xhuU3-scFv3-43. Amino acid sequence of His-tag. Myc-tag amino acid sequence. Amino acid sequence of Amino acid sequence of Amino acid sequence of Amino acid sequence of VH3- Amino acid sequence of Amino acid sequence of Amino acid sequence of Amino acid sequence of scFv3 Detailed Description of the Invention

[0085] Before the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methodology, protocols, and reagents described herein, as these may vary. It should also be understood that the terminology used herein is used only for the purpose of describing specific embodiments and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is commonly understood by a Petition 870260062263, dated 06 / 25 / 2026, p. 33 / 317 23 / 148 skilled technician in the subject.

[0086] Preferably, the terms used in the present invention are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC recommendations)”, Leuenberger HGW, Nagel B. and Kolbl H., Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland (1995).

[0087] Several documents are cited throughout this descriptive report. Each of the documents cited in the present invention (including all patents, patent applications, scientific publications, manufacturers' specifications, instructions, GenBank accession number sequence submissions, etc.), whether cited above or below, is incorporated herein by reference. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of a prior invention. Definitions

[0088] The word “comprising”, or variations such as “comprises” or “containing”, will be understood as implying the inclusion of a whole or stage, or group of wholes or stages, but not the exclusion of any other whole or stage, or group of wholes or stages.

[0089] As used in the descriptive report and attached claims, the singular forms “a” and “the” cover plural referents, unless the context clearly states otherwise.

[0090] Concentrations, quantities, and other numerical data may be expressed or presented in “range” format. It should be understood that such range format is used merely for convenience and brevity and, therefore, should be interpreted flexibly to include not only the explicitly stated numerical values ​​but also the Petition 870260062263, dated 06 / 25 / 2026, page 34 / 317 24 / 148 range limits, but also include all individual numerical values ​​or sub-intervals covered within that range, as if each numerical value and sub-interval were explicitly quoted. As an example, a numerical range of “150 mg to 600 mg” should be interpreted to include not only the explicitly quoted values ​​from 150 mg to 600 mg, but also individual values ​​and sub-intervals within that stated range. Thus, included in that range are individual values ​​such as 150, 160, 170, 180, 190,... 580, 590, 600 mg and sub-intervals such as 150 to 200, 150 to 250, 250 to 300, 350 to 600, etc. This same principle applies to ranges that quote only a numerical value. Furthermore, such interpretation should be applied regardless of the range width or the characteristics described.

[0091] The term “about” when used in connection with a numerical value shall encompass numerical values ​​within a range with a lower limit that is 5% less than the indicated numerical value and an upper limit that is 5% greater than the indicated numerical value.

[0092] The terms “nucleic acid” and “nucleic acid molecule” are used synonymously and are understood to mean single- or double-stranded oligo- or polymers of deoxyribonucleotide or ribonucleotide bases or both. Nucleotide monomers are composed of a nucleobase, a five-carbon sugar (such as, among others, ribose or 2'-deoxyribose), and one to three phosphate groups. Typically, a nucleic acid is formed through phosphodiester bonds between individual nucleotide monomers. In the context of the present invention, the term nucleic acid includes, but is not limited to, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) molecules, and also includes synthetic molecules. (Science 254:1497-1500, 1991). Typically, nucleic acids are single- or double-stranded molecules and are composed of nucleotides that... Petition 870260062263, dated 06 / 25 / 2026, page 35 / 317 25 / 148 occur naturally. The representation of a single-stranded nucleic acid also defines (at least partially) the complementary strand sequence. Nucleic acid can be single-stranded or double-stranded, or it can contain portions of both double-stranded and single-stranded sequences. Exemplary double-stranded nucleic acid molecules may have 3' or 5' ends and, as such, are not necessarily or assumed to be completely double-stranded along their entire length. Nucleic acid can be obtained by biological, biochemical, or chemical synthesis methods or any of the methods known in the state of the art, including, but not limited to, RNA amplification and reverse transcription methods.The term nucleic acid encompasses chromosomes or chromosome segments, vectors (e.g., expression vectors), expression cassettes, naked DNA or polymeric RNA, primers, probes, cDNA, genomic DNA, recombinant DNA, cRNA, mRNA, tRNA, microRNA (miRNA), or small interfering RNA (siRNA). A nucleic acid may be, for example, single-stranded, double-stranded, or triple-stranded and is not limited to any particular length. Unless otherwise indicated, a particular nucleic acid sequence comprises or encodes complementary sequences in addition to any explicitly indicated sequence.

[0093] Nucleic acids can be degraded by endonucleases or exonucleases, particularly by DNases and RNases that can be found in the cell. For this reason, it can be advantageous to modify nucleic acids in order to stabilize them against degradation, thus ensuring that a high concentration of the nucleic acid is maintained in the cell for a long period of time. Typically, such stabilization can be achieved by introducing one or more internucleotide phosphorus groups or by introducing one or more non-phosphorus internucleotides. Consequently, nucleic acids can be composed of Petition 870260062263, dated 06 / 25 / 2026, page 36 / 317 26 / 148 Non-naturally occurring nucleotides and / or modifications to naturally occurring nucleotides and / or alterations to the molecule's structure. Modified internucleotide phosphate radicals and / or non-phosphorus bridges in a nucleic acid include, but are not limited to, methyl phosphonate, phosphorothioate, phosphoramidate, phosphorodithioate and / or phosphate esters, while analogous non-phosphorus internucleotides include, but are not limited to, siloxane bridges, carbonate bridges, carboxymethyl esters, acetamidate bridges and / or thioether bridges.Additional examples of nucleotide modifications include, but are not limited to: phosphorylation of 5' or 3' nucleotides to allow binding or prevent degradation by exonuclease / polymerase extension, respectively; amino, thiol, alkyne, or biotinyl modifications to covalent and quasi-covalent linkages; fluorophores and quenchers; and modified bases, such as deoxyInosine (dI), 5Bromo-deoxyuridine (5-Bromo-dU), deoxyUridine, 2-Aminopurine, 2,6Diaminopurine, inverted dT, inverted Dideoxy-T, dideoxycytidine (ddC 5-methyl deoxyCitidine (5-methyl dC), locked nucleic acids (LNA's), 5-nitroindole, Iso-dC and -dG bases, RNA bases 2'-O-methyl, hydroxymethyl dC, 5-hydroxybutyn2'-deoxyuridine, 8-aza-7-deazaguanosine and Fluorine Modified Bases.Thus, nucleic acid can also be an artificial nucleic acid, which includes, but is not limited to, polyamide or peptide nucleic acid (PNA), morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA).

[0094] A nucleic acid is “operationally linked” when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operationally linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operationally linked to a coding sequence if it is Petition 870260062263, dated 06 / 25 / 2026, page 37 / 317 27 / 148 positioned to facilitate translation.

[0095] In the context of the present invention, the term “oligonucleotide” refers to a nucleic acid sequence of up to about 50 nucleotides, for example, from 2 to about 50 nucleotides in length.

[0096] The term “polynucleotide” when used in the context of the present invention, refers to a nucleic acid with more than 50 nucleotides in length, for example, 51 or more nucleotides in length.

[0097] Oligonucleotides and polypeptides are prepared by any suitable method, including, but not limited to, isolation from an existing or natural sequence, DNA replication or amplification, reverse transcription, cloning and restriction digestion of appropriate sequences, or direct chemical synthesis by a method such as the phosphotriester method of Narang et al. (Meth. Enzymol. 68:90-99, 1979); the phosphodiester method of Brown et al. (Meth. Enzymol. 68:109-151, 1979); the diethylphosphoramidite method of Beaucage et al. (Tetrahedron Lett. 22:1859-1862, 1981); the triester method of Matteucci et al. (J. Am. Chem. Soc. 103:3185-3191, 1981); automated synthesis methods; or the solid support method of U.S. Patent 4,458,066, or other methods known to those skilled in the art.

[0098] As used in the present invention, the term “vector” refers to a protein or a polynucleotide or a mixture thereof that is capable of being introduced or introducing proteins and / or nucleic acids comprised therein into a cell. Examples of vectors include, but are not limited to, plasmids, cosmids, phages, viruses, or artificial chromosomes. In particular, a vector is used to transport a gene product of interest, such as, for example, foreign or heterologous DNA into a suitable host cell. Vectors may contain sequences Petition 870260062263, dated 06 / 25 / 2026, p. 38 / 317 28 / 148 polynucleotide “replicons” that facilitate autonomous vector replication in a host cell. The foreign DNA is defined as heterologous DNA, which is DNA not naturally found in the host cell that, for example, replicates the vector molecule, encodes a selectable or traceable marker, or encodes a transgene. Once in the host cell, the vector can replicate independently or coincidentally with the host's chromosomal DNA, and multiple copies of the vector and its inserted DNA can be generated. Additionally, the vector may also contain necessary elements that allow transcription of the inserted DNA into an mRNA molecule or otherwise cause replication of the inserted DNA into multiple RNA copies. Vectors may also include “expression control sequences” that regulate the expression of the gene of interest.Typically, expression control sequences are polypeptides or polynucleotides, such as, but not limited to, promoters, enhancers, silencers, isolators, or repressors. In a vector comprising more than one polynucleotide encoding one or more gene products of interest, expression may be controlled jointly or separately by one or more expression control sequences. More specifically, each polynucleotide comprised in the vector may be controlled by a separate expression control sequence, or all polynucleotides comprised in the vector may be controlled by a single expression control sequence. The polynucleotides comprised in a single vector controlled by a single expression control sequence may form an open reading frame.Some expression vectors additionally contain sequence elements adjacent to the inserted DNA that increase the half-life of the expressed mRNA and / or allow the translation of the mRNA into a protein molecule. Many mRNA molecules and polypeptides encoded by the inserted DNA can thus be rapidly... Petition 870260062263, dated 06 / 25 / 2026, p. 39 / 317 29 / 148 synthesized.

[0099] The term “amino acid” generally refers to any monomeric unit comprising a substituted or unsubstituted amino group, a substituted or unsubstituted carboxyl group and one or more side chains or groups, or analogues of any of these groups. Exemplary side chains include, for example, thiol, selene, sulfonyl, alkyl, aryl, acyl, keto, azide, hydroxyl, hydrazine, cyano, halo, hydrazide, alkenyl, alkyne, ether, borate, boronate, phospho, phosphone, phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine or any combination of these groups.Other representative amino acids include, but are not limited to, photoactivatable crosslinking amino acids, metal-binding amino acids, labeled amino acids, fluorescent amino acids, metal-containing amino acids, amino acids with novel functional groups, amino acids that covalently or non-covalently interact with other molecules, photoconditioned and / or photoisomerizable amino acids, radioactive amino acids, amino acids comprising biotin or a biotin analogue, glycosylated amino acids, other carbohydrate-modified amino acids, amino acids comprising polyethylene glycol or polyether, heavy-atom substituted amino acids, chemically cleavable and / or photocleavable amino acids, carbon-linked sugar-containing amino acids, redox-active amino acids, amino acids containing amino thioacids, and amino acids comprising one or more toxic moieties.As used in this invention, the term "amino acid" includes the following twenty naturally occurring amino acids or genetically encoded alpha-amino acids: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys / Cis or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly / Gli or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys / Lis or K), methionine (Met or M), phenylalanine (Phe / Fen or F), proline. Petition 870260062263, dated 06 / 25 / 2026, p. 40 / 317 30 / 148 (Pro or P), serine (Ser or S), threonine (Thr / Tre or T), tryptophan (Trp or W), tyrosine (Tyr / Tir or Y), and valine (Val or V). In cases where the “X” residues are undefined, they should be defined as “any amino acid”. The structures of these twenty naturally occurring amino acids are shown, for example, in Stryer et al., Biochemistry, 5th ed., Freeman and Company (2002). Additional amino acids, such as selenocysteine ​​and pyrrolysine, can also be genetically encoded by (Stadtman (1996) “Selenocysteine”, Annu Rev Biochem. 65:83-100 and Ibba et al. (2002) “Genetic code: introducing pyrrolysine,” Curr Biol. 12(13):R464-R466). The term “amino acid” also includes non-natural amino acids, modified amino acids (e.g., having modified side chains and / or backbones), and amino acid analogs. See, for example, Zhang et al. (2004) “Selective incorporation of 5-hydroxytryptophan into proteins in mammalian cells,” Proc. Natl. Acad. Sci. USA101(24):8882-8887, Anderson et al. (2004) “An expanded genetic code with a functional quadruplet codon” Proc. Natl. Acad. Sci. U.S.A. 101(20):75667571, Ikeda et al. (2003) “Synthesis of a novel histidine analogue and its efficient incorporation into a protein in vivo,” Protein Eng. Des. Sel. 16(9):699706, Chin et al. (2003) “An Expanded Eukaryotic Genetic Code,” Science 301(5635):964-967, James et al. (2001) “ Kinetic characterization of ribonuclease S mutants containing photoisomerizable phenylazophenylalanine residues,” Protein Eng. Des. Sel. 14(12):983-991, Kohrer et al. (2001) “Import of amber and ochre suppressor tRNAs into mammalian cells: A general approach to site-specific insertion of amino acid analogues into proteins,” Proc. Natl. Acad. Sci. U.S.A. 98(25):14310-14315, Bacher et al. (2001) “Selection and Characterization of Escherichia coli Variants Capable of Growth on an Otherwise Toxic Tryptophan Analogue,” J. Bacteriol. 183(18):5414-5425, Hamano-Takaku et al.(2000) “A Mutant Escherichia coli Tyrosyl-tRNA Synthetase Utilizes the Unnatural Amino Acid Azatyrosine More Efficiently than. Petition 870260062263, dated 06 / 25 / 2026, page 41 / 317 31 / 148 Tyrosine,” J. Biol. Chem. 275(51):40324-40328, and Budisa et al. (2001) “Proteins with {beta}-(thienopyrrolyl) alanines as alternative chromophores and pharmaceutically active amino acids,” Protein Sci. 10(7):1281-1292. Amino acids can be incorporated into peptides, polypeptides, or proteins.

[00100] In the context of the present invention, the term “peptide” refers to a short polymer of amino acids linked by peptide bonds. It has the same chemical (peptide) bonds as proteins, but is generally shorter in length. The shortest peptide is a dipeptide, consisting of two amino acids linked by a single peptide bond. There may also be a tripeptide, tetrapeptide, pentapeptide, etc. Typically, the peptide has a length of up to 8, 10, 12, 15, 18 or 20 amino acids. A peptide has an amino end (amino-terminal) and a carboxyl end (carboxy-terminal), unless it is a cyclic peptide.

[00101] In the context of the different aspects of the present invention, the term “polypeptide” refers to a single linear chain of amino acids linked by peptide bonds and typically comprises at least about 21 amino acids. A polypeptide may be a chain of a protein that is composed of more than one chain or may be the protein itself if the protein is composed of a single chain.

[00102] In the context of the present invention, the “primary structure” of a protein or polypeptide is the amino acid sequence in the polypeptide chain. The “secondary structure” in a protein is the overall three-dimensional shape of local segments of the protein. However, it does not describe specific atomic positions in three-dimensional space, which are considered tertiary structure. In proteins, secondary structure is defined by patterns of hydrogen bonding between amide structures and Petition 870260062263, dated 06 / 25 / 2026, page 42 / 317 32 / 148 carboxyl groups. The “tertiary structure” of a protein is the three-dimensional structure of the protein determined by its atomic coordinates. The “quaternary structure” is the arrangement of multiple protein or polypeptide molecules folded and coiled into a multi-subunit complex.

[00103] The terms “folding”, “protein folding” or “folding” refer to the process by which a protein assumes its three-dimensional shape or conformation, that is, the protein is directed to form a specific three-dimensional shape through non-covalent interactions, such as, but not limited to, hydrogen bonding, metal coordination, hydrophobic forces, van der Waals forces, pi-pi interactions and / or electrostatic effects. The term “folded protein”, therefore, refers to a protein in its three-dimensional form, as its secondary, tertiary or quaternary structure.

[00104] The term “fragment” used in the present invention refers to naturally occurring fragments (e.g., processing variants) as well as artificially constructed fragments, in particular those obtained by genetic technological means. Typically, a fragment has a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 amino acids at its N-terminal and / or C-terminal end and / or internally compared to the parent polypeptide, preferably at its N-terminal end, N and C-terminal ends, or C-terminal end.

[00105] An “epitope,” also known as an antigenic determinant, is the segment of a macromolecule recognized by the immune system, specifically by antibodies, B cells, or T cells. Such an epitope is the part or segment of a macromolecule capable of binding to a Petition 870260062263, dated 06 / 25 / 2026, page 43 / 317 33 / 148 antibody or antigen-binding fragment of the antibody. In this context, the term “connection” preferably refers to a specific connection. In the context of the present invention, it is preferred that the term “epitope” refers to the protein or polyprotein segment that is recognized by the immune system. Epitopes generally consist of chemically active surface clusters of molecules, such as amino acids or sugar side chains, and generally possess specific three-dimensional structural features as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished by the fact that binding to the former, but not the latter, is lost in the presence of denaturing solvents.

[00106] As used in the present invention, a “conformational epitope” refers to an epitope of a linear macromolecule (e.g., a polypeptide) that is formed from the three-dimensional structure of said macromolecule. In the context of the present application, a “conformational epitope” is a “discontinuous epitope,” that is, the conformational epitope on the macromolecule (e.g., a polypeptide) that is formed from at least two separate regions in the primary sequence of the macromolecule (e.g., the amino acid sequence of a polypeptide).In other words, an epitope is considered a "conformational epitope" in the context of the present invention if the epitope consists of at least two separate regions in the primary sequence to which a binding portion of the invention (e.g., an antibody or its antigen-binding fragment) binds simultaneously, such that these at least two separate regions are interrupted by one more region in the primary sequence to which the binding portion of the invention does not bind. In particular, such a "conformational epitope" is present in a polypeptide and the two separate regions in the primary sequence are two separate amino acid sequences to which a binding portion of the invention (e.g., an antibody or its antigen-binding fragment) binds. Petition 870260062263, dated 06 / 25 / 2026, p. 44 / 317 34 / 148 antigen binding) binds, such that these at least two separate amino acid sequences are interrupted by one more amino acid sequence in the primary sequence to which the binding portion of the invention does not bind. In particular, the interrupting amino acid sequence is a contiguous amino acid sequence comprising two or more amino acids to which the binding portion does not bind. The (at least) two separate amino acid sequences to which a binding portion of the invention binds are not particularly limited with respect to their length. Such a separate amino acid sequence may consist of only one amino acid provided that the total number of amino acids within said at least two separate amino acid sequences is sufficiently large to effect the specific binding between the binding portion and the conformational epitope.

[00107] A “paratope” is the part of an antibody that recognizes the epitope. In the context of the present invention, a “paratope” is the part of a binding portion (e.g., an antibody or its antigen-binding fragment), as described in the present invention, that recognizes the epitope.

[00108] A “peptide linker” in the context of the present invention refers to an amino acid sequence that statically separates two parts or portions of a complex, for example, two peptides or proteins. Typically, such a linker consists of between 1 and 100 amino acids with a minimum length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids and a maximum length of at least 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16 or 15 amino acids or less. The preferred minimum and maximum lengths of the peptide linker according to the present invention may be combined if such a combination makes mathematical sense, for example, Petition 870260062263, dated 06 / 25 / 2026, p. 45 / 317 35 / 148 Such a linker may consist of 1-15, or 12-40, or 25-75, or 1-100 amino acids. Peptide linkers may also provide flexibility between the two parts that are connected to each other. This flexibility is generally increased if the amino acids are small. Consequently, flexible peptide linkers comprise an increased content of small amino acids, in particular glycines and / or alanines, and / or hydrophilic amino acids such as serines, threonines, asparagines, and glutamines. Preferably, more than 20%, 30%, 40%, 50%, 60% or more of the amino acids in the peptide linker are small amino acids.

[00109] As used in the present invention, the term “variant” should be understood as a polypeptide or polynucleotide that differs from the polypeptide or polynucleotide from which it is derived by one or more alterations in its length or sequence. The polypeptide or polynucleotide from which a polypeptide or polynucleotide is derived is also known as the parent polypeptide or polynucleotide. The term “variant” includes “fragments” or “derivatives” of the parent molecule. Typically, “fragments” are smaller in length or size than the parent molecule, while “derivatives” exhibit one or more differences in their sequences compared to the parent molecule.Modified molecules such as, but not limited to, post-translational modified proteins (e.g., glycosylated, biotinylated, phosphorylated, ubiquitinated, palmitoylated, or proteolytically cleaved proteins) and modified nucleic acids, such as methylated DNA, are also included. Mixtures of different molecules, such as, but not limited to, RNA-DNA hybrids, are also encompassed by the term "variant." Typically, a variant is artificially constructed, preferably by gene-technological means, while the parental protein or polynucleotide is a type protein or polynucleotide. Petition 870260062263, dated 06 / 25 / 2026, page 46 / 317 36 / 148 wild type, or a consensus sequence thereof. However, naturally occurring variants should also be understood as encompassed by the term “variant,” as used in this invention. Furthermore, variants usable in the present invention may also be derived from homologs, orthologs, or paralogs of the parent molecule or from artificially constructed variants, provided that the variant exhibits at least one biological activity of the parent molecule, i.e., it is functionally active.

[00110] In particular, the term “peptide variant”, “polypeptide variant”, “protein variant” should be understood as a peptide, polypeptide or protein that differs from the peptide, polypeptide or protein from which it is derived by one or more changes in the amino acid sequence. The peptide, polypeptide or protein from which a variant peptide, polypeptide or protein is derived is also known as the parent peptide, polypeptide or protein. Furthermore, variants usable in the present invention may also be derived from homologs, orthologs or paralogs of the parent peptide, polypeptide or protein or artificially constructed variant, provided that the variant exhibits at least one biological activity of the parent peptide, polypeptide or protein.Changes in the amino acid sequence can be substitutions, insertions, deletions, N-terminal truncations, or C-terminal truncations of amino acids, or any combination of these changes, which can occur in one or more locations. A variant peptide, polypeptide, or protein may exhibit a total of up to 200 (up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200) amino acid sequence alterations (i.e., exchanges, insertions, deletions, N- or C-terminal truncations). Amino acid exchanges may be conservative and / or non-conservative. Petition 870260062263, dated 06 / 25 / 2026, page 47 / 317 37 / 148 Alternatively or additionally, a “variant,” as used in the present invention, may be characterized by a certain degree of sequence identity to the parent peptide, polypeptide, or protein from which it is derived. More precisely, a variant peptide, polypeptide, or protein in the context of the present invention exhibits at least 80% sequence identity with its parent peptide, polypeptide, or protein. The sequence identity of the variant peptide, polypeptide, or protein is greater than a continuous stretch of 20, 30, 40, 45, 50, 60, 70, 80, 90, 100, or more amino acids.

[00111] The “percentage of sequence identity” is determined by comparing two optimally aligned sequences along a comparison window, where the portion of the sequence in the comparison window may contain additions or deletions (i.e., gaps) when compared to the reference sequence (which does not have the additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to yield the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to give the percentage of sequence identity.

[00112] The term “identical” in the context of two or more nucleic acids or polypeptide sequences refers to two or more sequences or subsequences that are the same, that is, that comprise the same sequence of nucleotides or amino acids. Sequences are “substantially identical” to each other if they have a specific percentage of identical nucleotides or amino acid residues (e.g., at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least Petition 870260062263, dated 06 / 25 / 2026, p. 48 / 317 38 / 148 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity over a specified region), when compared and aligned for maximum match within a comparison window, or designated region, measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. These definitions also apply to the complement of a test sequence. Consequently, the term "at least 80% sequence identity" is used throughout the descriptive report to refer to comparisons of polypeptide and polynucleotide sequences.This expression preferably refers to a sequence identity of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% for the respective reference polypeptide or for the respective reference polynucleotide.

[00113] The term “sequence comparison” refers to the process in which a sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, if necessary the subsequence coordinates are assigned, and the sequence algorithm program parameters are assigned. Preloaded or default program parameters are commonly used, or parameters can be assigned. Petition 870260062263, dated 06 / 25 / 2026, page 49 / 317 39 / 148 alternatives. The sequence comparison algorithm then calculates the percentages of sequence identities or similarities for the test sequences relative to the reference sequence, based on the program parameters. In the case where two sequences are compared and the reference sequence is not specified against which the sequence identity percentage should be calculated, the sequence identity should be calculated with reference to the longer sequence among the two sequences to be compared, unless specifically indicated otherwise. If the reference sequence is indicated, the sequence identity is determined based on the total length of the reference sequence indicated by the SEQ ID, unless specifically indicated otherwise.

[00114] In a sequence alignment, the term “comparison window” refers to those contiguous position spans of a sequence that are compared with a reference span of contiguous positions of a sequence that has the same number of positions. The number of contiguous positions selected can range from 10 to 1000, i.e., it can comprise 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 contiguous positions. Typically, the number of contiguous positions varies from about 20 to 800 contiguous positions, from about 20 to 600 contiguous positions, from about 50 to 400 contiguous positions, from about 50 to about 200 contiguous positions, and from about 100 to about 150 contiguous positions.

[00115] Sequence alignment methods for comparison are well known in the state of the art. Optimal sequence alignment for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2:482, 1970), or by the homology alignment algorithm of Needleman and Wunsch. Petition 870260062263, dated 06 / 25 / 2026, page 50 / 317 40 / 148 (J. Mol. Biol. 48:443, 1970), by the similarity search method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis.), or by manual alignment and visual inspection (see, for example, Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)). Suitable algorithms for determining sequence identity percentage and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (Nuc. Acids Res. 25:3389-402, 1977) and Altschul et al. (J. Mol. Biol. 215:403-10, 1990), respectively. The computer program for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).This algorithm first involves identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy certain T-score thresholds with positive values ​​when aligned with a word of the same length in a sequence from the database. T is referred to as the neighboring word score threshold (Altschul, et al, supra). These initial neighboring matching words (word hits) act as seeds to initiate searches to find larger HSPs that contain them. The matching words (word hits) are extended in both directions along each sequence as the cumulative alignment score can be increased.Cumulative scores are calculated using nucleotide sequences, parameters M (reward score for a matching residue pair, always > 0) and N (penalty score for non-matching residues, always < 0). For amino acid sequences, a scoring matrix is ​​used. Petition 870260062263, dated 06 / 25 / 2026, page 51 / 317 41 / 148 calculate the cumulative score. The extension of matching words (word hits) in each direction is interrupted when the cumulative alignment score falls by an amount X from its maximum value reached, the cumulative score goes to zero or below due to the accumulation of one or more alignments with negative score residues, or when the end of a sequence is reached. The W, T, and X parameters of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a default word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of the two strands. For amino acid sequences, the BLASTP program uses a default word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.).USA 89:10915, 1989) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90: 5873-87, 1993). One similarity measure provided by the BLAST algorithm is the probability of the smallest sum (P(N)), which gives an indication of the probability that a match between two nucleotide or amino acid sequences occurs by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the probability of the lowest sum in a comparison of the test nucleic acid sequence with the reference nucleic acid sequence is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[00116] Semi-conservative and especially conservative amino acid substitutions are preferred, in which one amino acid is Petition 870260062263, dated 06 / 25 / 2026, page 52 / 317 42 / 148 replaced by a chemically related amino acid. Typical substitutions are between aliphatic amino acids, between amino acids with aliphatic hydroxyl side chains, between amino acids with acidic residues, between amide derivatives, between amino acids with basic residues, or between amino acids with aromatic residues. Typical semi-conservative and conservative substitutions are as shown in Table 1 below. Table 1 Amino Acid Conservative Substitution Semiconservative Substitution AG; St; TN; V; CCA; V; LM; I; F; GDE; N; QA; St; T; K; R; HED; Q; NA; St; T; K; R; HFW; Y; L; M; HI; V; AGAS; N; T; D; AND; N; QHY; F; K; RL; M; AIV; L; M; AF; Y; W; GKR; HD; AND; N; Q; St; T; ALM; I; V; AF; Y; W; H; CML; I; V; AF; Y; W; W; NQD; AND; St; T; THE; G; K; RPV; IL; THE; M; W; Y; St; T; W; FQND; AND; THE; St; T; L; M; K; RRK; HN; Q; St; T; D; AND; WING; T; G; ND; AND; R; KTA; St; G; N; VD; AND; R; K; ICMS; L; IM; T; W; NWF; Y; HL; M; I; V; CYF; W; HL; M; I; V; W

[00117] Changing from A, F, H, I, L, M, P, V, W, or Y to C is a semi-conservative substitution if the new cysteine ​​remains as a free thiol. Furthermore, a person skilled in the art will understand that glycines in steric demand positions should not be substituted and that P should not be substituted. Petition 870260062263, dated 06 / 25 / 2026, p. 53 / 317 43 / 148 can be introduced into parts of the protein that have an alpha-helical structure or beta-sheet.

[00118] The EGF receptor family comprises four members, EGFR (erbB1, HER1), ErbB2 (HER2), ErbB3 (HER3), and ErbB4 (HER4). The receptors consist of an extracellular region composed of four domains (I-IV), a transmembrane region, and an intracellular region composed of a tyrosine kinase domain and a carboxy-terminal tail containing tyrosine residues (Baselga & Swain 2009, Novel anticancer targets: revisiting ErbB2 and discovering ErbB3. Nat. Rev. Cancer 9: 463-475). Extracellular domains I and III are involved in ligand binding, domains II and IV in receptor dimerization. Domain II mediates receptor-receptor contacts via a dimerization loop, the so-called dimerization arm (Garrett et al., 2002, Combination of antibody that inhibits ligand-independent HER3 dimerization and a p110 alpha inhibitor potently blocks PI3K signaling and growth of HER2+ breast cancers. Cancer Res. 73: 6013-6023).Several ligands belonging to the EGF ligand family can bind to the receptor. EGF, Transforming Growth Factor-α (TGF-alpha), and amphiregulin bind specifically to EGFR / ErbB1. Betacellulin (BTC), heparin-binding EGF (HB-EGF), and epiregulin (EPR) show dual specificity, binding to both EGFR / ErbB1 and ErbB4. Neuregulins (NRGs) form two subgroups based on their ability to bind to ErbB3 and ErbB4 (NRG-1 and NRG-2) or only to ErbB4 (NRG-3 and NRG-4). None of the ligands bind to ErbB2, but ErbB2 is the preferred dimerization partner for all other ErbB receptors. ErbB3 has impaired kinase activity and only acquires signaling potential when dimerized with another member of the ErbB receptor family. Ligand binding to ErbB receptors induces a large conformational change leading to the formation of receptor homodimers and heterodimers and activation of the receptor. Petition 870260062263, dated 06 / 25 / 2026, p. 54 / 317 44 / 148 intrinsic kinase domain, resulting in the phosphorylation of specific tyrosine residues within the cytoplasmic tail. These phosphorylated residues serve as anchoring sites for intracellular signaling molecules. The ligand determines which tyrosine residues are phosphorylated and, therefore, which signaling molecules are recruited. Three main pathways can be stimulated by ErbB activation: mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)-AKT, and the Janus Kinase (JAKSTAT) pathway, all responsible for regulating cellular metabolism, growth, and survival (Hervent & De Keulenaer, 2012, Molecular mechanisms of cardiotoxicity induced by ErbB receptor inhibitor cancer therapeutics. Int. J. Mol. Sci. 13: 12268-12286).

[00119] A tag is any type of substance capable of indicating the presence of another substance or complex of substances. The tag can be a substance bound to or introduced into the substance to be detected. Detectable tags are used in molecular biology and biotechnology to detect, for example, a protein, a product of an enzymatic reaction, a second messenger, DNA, molecular interactions, etc. Examples of suitable tags include fluorophores, chromophores, radiolabels, metallic colloids, enzymes, or chemiluminescent or bioluminescent molecules. In the context of the present invention, suitable tags are preferably protein tags whose peptide sequences are genetically grafted onto or inscribed on a recombinant protein.Protein tags can include, for example, affinity tags, solubilization tags, chromatography tags, epitope tags, or fluorescence tags.

[00120] “Affinity markers” are attached to proteins so that the protein can be purified from its crude biological source using an affinity technique. These include protein binding to Petition 870260062263, dated 06 / 25 / 2026, p. 55 / 317 45 / 148 chitin (CBP), maltose-binding protein (MBP), and glutathione-S-transferase (GST). Poly(His) tag is a widely used protein marker that binds to metal matrices.

[00121] “Solubilization markers” are used especially for recombinant proteins expressed in chaperone-deficient species, to aid in proper protein folding and prevent precipitation. These include thioredoxin (TRX) and poly(NANP). Some affinity markers have a dual role as solubilizing agents, such as MBP and GST.

[00122] “Chromatography markers” (or chromatography tags) are used to alter the chromatographic properties of a protein and provide different resolution through a specific separation technique. Frequently, these consist of polyanionic amino acids, such as the FLAG-tag.

[00123] “Epitope markers” (or epitope tags) are short peptide sequences that are chosen because high-affinity antibodies can be reliably produced from them in different species. These are usually derived from viral genes, which explains their high immunoreactivity. Epitope tags include V5-tag, Myc-tag, and HA-tag. These markers are particularly useful for Western blotting, immunofluorescence, and immunoprecipitation experiments, although they can also be used in antibody purification.

[00124] “Fluorescence markers” are used to provide a visual reading of a protein. GFP and its variants are the most widely used fluorescence markers. More advanced applications of GFP include using it as a foldable reporter (fluorescent when folded, colorless when not). Other examples of fluorophores include fluorescein, rhodamine, and sulfoindocyanine Cy5 dyes. Petition 870260062263, dated 06 / 25 / 2026, page 56 / 317 46 / 148

[00125] The term “antigen-binding protein,” as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules containing an antigen-binding site that immunospecifically binds an antigen. Also included are immunoglobulin-like proteins that are selected through techniques that include, for example, display on phages to specifically bind to a target molecule or target epitope.In assessing the binding and / or specificity of an antigen-binding protein, for example, an antibody or its immunologically functional fragment, an antibody or fragment may substantially inhibit the binding of a ligand to its binding partner when an excess of antibody reduces the amount of binding partner bound to the ligand by at least about 1-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-98%, 98-99% or more (e.g., measured in an in vitro competitive binding assay). Neutralizing ability can be described in terms of an IC50 or EC50 value.

[00126] The “IC50” value refers to half the maximum inhibitory concentration of a substance and is therefore a measure of a substance’s effectiveness in inhibiting a specific biological or biochemical function. Values ​​are typically expressed as molar concentration. The IC50 of a drug can be determined in functional antagonist assays by constructing a dose-response curve and examining the inhibitory effect of the substance under examination at different concentrations. Alternatively, competitive binding assays can be performed to determine the IC50 value. Typically, inhibitory antibodies exhibit an IC50 value between 50 nM-1 pM, i.e., 50 nM, 10 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 1 pM.

[00127] The value “EC50” refers to half the concentration Petition 870260062263, dated 06 / 25 / 2026, page 57 / 317 The maximum effective concentration (EC50) of a substance is therefore a measure of the concentration of that substance that induces a response halfway between the baseline and the maximum after a specified exposure time. It is commonly used as a measure of drug potency. The EC50 value of a graded dose-response curve therefore represents the concentration of a substance at which 50% of its maximum effect is observed. The EC50 value of a quantal dose-response curve represents the concentration of a compound at which 50% of the population exhibits a response after a specified exposure period. Typically, inhibitory antibodies exhibit an EC50 value between 50 nM-1 pM, that is, 50 nM, 10 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, or 1 pM.

[00128] The term “binding” according to the invention preferably refers to a specific binding. The term “binding affinity” generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., a target or antigen). Unless otherwise indicated, “binding affinity” refers to intrinsic binding affinity reflecting a 1:1 interaction between the members of the binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (Kd). “Specific binding” means that a binding moiety (e.g., an antibody) binds more strongly to a target, such as an epitope for which it is specific, compared to binding to another target.A binding moiety binds more strongly to a first target compared to a second target if it binds to the first target with a dissociation constant (Kd) smaller than the dissociation constant for the second target. The dissociation constant (Kd) for the target to which the binding moiety specifically binds is more than 10 times, preferably more than 20 times, most preferably more than. Petition 870260062263, dated 06 / 25 / 2026, p. 58 / 317 48 / 148 times, or even more preferably more than 100 times, 200 times, 500 times or 1000 times smaller than the dissociation constant (Kd) for the target to which the binding moiety does not specifically bind.

[00129] Thus, the term “Kd” (measured in “mol / L”, sometimes abbreviated as “M”) refers to the dissociation equilibrium constant of the particular interaction between a binding moiety (e.g., an antibody or fragment thereof) and a target molecule (e.g., an antigen or epitope thereof). Affinity can be measured by common methods known in the art, including, but not limited to, surface plasmonic resonance-based assays (such as the BIAcore assay); quartz crystal microbalance assays (such as the Attana assay); enzyme-linked immunosorbent assays (ELISA); and competitive assays (e.g., RIAs). Low-affinity antibodies bind weakly to the antigen and tend to dissociate rapidly, while high-affinity antibodies bind the antigen more strongly and remain bound for longer.A variety of methods for measuring binding affinity are known in the state of the art, any of which can be used for the purposes of the present invention.

[00130] Typically, antibodies bind with sufficient binding affinity to their target, for example, with a Kd value between 500 nM and 1 pM, i.e., 500 nM, 450 nM, 400 nM, 350 nM, 300 nM, 250 nM, 200 nM, 150 nM, 100 nM, 50 nM, 10 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM or 1 pM.

[00131] The term “compete” when used in the context of antigen-binding proteins (e.g., neutralizing antigen-binding proteins or neutralizing antibodies) competing for the same epitope means competition between antigen-binding proteins, as determined by an assay in which the antigen-binding protein Petition 870260062263, dated 06 / 25 / 2026, p. 59 / 317 49 / 148 (e.g., antibody or its immunologically functional fragment) to be tested prevents or inhibits (e.g., reduces) the specific binding of a reference antigen-binding protein (e.g., a ligand, or reference antibody) to a common antigen. Numerous types of competitive binding assays can be used to determine whether an antigen-binding protein competes with another, for example: direct or indirect solid-phase radioimmunoassay (RIA), direct or indirect enzyme immunoassay (EIA), (see, for example, Stahli et al., 1983, Methods in Enzymology. 2:242-253); direct solid-phase biotin-avidin EIA (see, for example, Kirkland et al., 1986, J. Immunol.137:3614-3619) direct solid-phase labeling assay, solid-phase labeled sandwich assay (see, for example, Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); RIA with direct solid-phase labeling using label 125I (see, for example, Morel et al., 1988, Molec. Jmmunol. 25:7-15); direct solid-phase biotin-avidin EIA (see, for example, Cheung, et al., 1990, Virology. 176:546-552); and RIA with direct labeling (Moldenhauer et al., 1990, Scand. J Immunol. 32:77-82). Typically, such an assay involves the use of purified antigen bound to a solid surface or cells containing either an unlabeled test antigen-binding protein and a labeled reference antigen-binding protein. Competitive inhibition is mediated by determining the amount of marker bound to the solid surface or cells in the presence of the test antigen-binding protein. Usually, the test antigen-binding protein is present in excess. Antigen-binding proteins identified by the competition assay (antigen-binding protein competition) include antigen-binding proteins that bind to the same epitope as the reference antigen-binding proteins and antigen-binding proteins that bind to an adjacent epitope sufficiently close to the epitope bound by the reference antigen-binding protein. Petition 870260062263, dated 06 / 25 / 2026, page 60 / 317 50 / 148 reference antigen-binding protein so that steric hindrance occurs. Further details regarding methods for determining competitive binding are provided in the examples presented in the present invention. Typically, when a competing antigen-binding protein is present in excess, it will inhibit (e.g., reduce) the specific binding of a reference antigen-binding protein to a common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[00132] The term “immunoglobulin” (Ig), as used in the present invention, refers to glycoproteins that confer immunity from the immunoglobulin superfamily. “Surface immunoglobulins” are bound to the membrane of effector cells by their transmembrane region and include molecules such as, but not limited to, B cell receptors, T cell receptors, major histocompatibility complex (MHC) class I and II proteins, beta-2 microglobulin (β2M), CD3, CD4 and CD8.

[00133] Typically, the term “antibody” as used in the present invention refers to secreted immunoglobulins that lack the transmembrane region and can thus be released into the blood and body cavities. Human antibodies are grouped into different isotypes based on the heavy chain they possess. There are five types of human Ig heavy chains denoted by the Greek letters: α, γ, δ, ε, and μ. The type of heavy chain present defines the antibody class, i.e., these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively, each playing different roles and directing the appropriate immune response against different types of antigens. Distinct heavy chains differ in size and composition; and may comprise approximately 450 amino acids (Janeway et al. (2001) Immunobiology, Garland Science). A Petition 870260062263, dated 06 / 25 / 2026, page 61 / 317 51 / 148 IgA is found in mucosal areas, such as the intestine, respiratory tract, and urogenital tract, as well as in saliva, tears, and breast milk, and prevents colonization by pathogens (Underdown & Schiff (1986) Annu. Rev. Immunol. 4:389-417). IgD functions primarily as an antigen receptor on B cells that have not been exposed to antigens and is involved in the activation of basophils and mast cells to produce antimicrobial factors (Geisberger et al. (2006) Immunology 118: 429-437; Chen et al. (2009) Nat. Immunol. 10: 889-898). IgE is involved in allergic reactions through its binding to allergens, triggering the release of histamine from mast cells and basophils. IgE is also involved in protection against parasitic worms (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press).IgG provides the majority of antibody-based immunity against invading pathogens and is the only antibody isotype capable of crossing the placenta to provide passive immunity to the fetus (Pier et al. (2004) Immunology, Infection, and Immunity, ASM Press). In humans, there are four different IgG subclasses (IgG1, 2, 3, and 4), named according to their serum abundances, with IgG1 being the most abundant (~66%), followed by IgG2 (~23%), IgG3 (~7%), and IgG4 (~4%). The biological profile of the different IgG classes is determined by the structure of their respective hinge region. IgM is expressed on the surface of B cells in a monomeric form and secreted in a pentameric form with very high avidity. IgM is involved in the elimination of pathogens in the early stages of B cell-mediated (humoral) immunity before sufficient IgG production (Geisberger et al. (2006) Immunology 118:429-437).Antibodies are not only found as monomers, but are also known to form dimers of two Ig units (e.g., IgA), tetramers of four Ig units (e.g., IgM from teleost fish), or pentamers of five Ig units (e.g., IgM from mammals). Antibodies are typically made of four chains. Petition 870260062263, dated 06 / 25 / 2026, page 62 / 317 52 / 148 polypeptides comprising two identical heavy chains and two identical light chains that are linked by disulfide bonds and resemble a Y-shaped macromolecule. Each chain comprises a number of immunoglobulin domains, some of which are constant domains and others are variable domains. The immunoglobulin domains consist of a 2-layer sandwich between 7 and 9 antiparallel strands arranged in two sheets. Typically, the heavy chain of an antibody comprises four Ig domains, with three of them being constant (CH domains: CH1, CH2, CH3) and one being a variable domain (VH). The light chain typically comprises one constant Ig domain (CL) and one variable Ig domain (VL).The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework or structural regions (FR framework). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino-terminal end to the carboxy-terminal end in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable heavy and light chain regions contain a binding domain that interacts with an antigen. The constant antibody regions can mediate the binding of immunoglobulin to tissues or host factors, including via immune system cells (e.g., effector cells) and the first component (C1q) of the classical complement system.

[00134] The term “antigen-binding fragment” of an antibody (or simply “binding portion”), as used in the present invention, refers to one or more fragments of an antibody that retain the ability to bind specifically to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a whole antibody. Petition 870260062263, dated 06 / 25 / 2026, p. 63 / 317 53 / 148

[00135] As used in the present invention, “human antibodies” include antibodies possessing variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). The human antibodies of the invention include antibodies isolated from human immunoglobulin libraries or from transgenic animals for one or more human immunoglobulins and that do not express endogenous immunoglobulins, as described, for example, in U.S. Patent 5,939,598 by Kucherlapati & Jakobovits.

[00136] The term “monoclonal antibody,” as used herein, refers to a preparation of antibody molecules of unique molecular composition. A monoclonal antibody exhibits unique specificity and binding affinity for a particular epitope. In one embodiment, monoclonal antibodies are produced by a hybridoma that includes a B cell obtained from a non-human animal, for example, a mouse, fused to an immortalized cell.

[00137] The term “recombinant antibody,” as used in the present invention, includes all antibodies that are prepared, expressed, created, or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal with respect to immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, for example, from a transfectome, (c) antibodies isolated from a recombinant combinatorial antibody library, and (d) antibodies prepared, expressed, created Petition 870260062263, dated 06 / 25 / 2026, p. 64 / 317 54 / 148 or isolated by any other means involving the processing of immunoglobulin gene sequences into other DNA sequences.

[00138] The term “chimeric antibody” refers to those antibodies in which a portion of each of the amino acid sequences of the heavy and light chains is homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular class, while the remaining segment of the chain is homologous to the corresponding sequences of another species or class. Typically, the variable region of both the light and heavy chains mimics the variable regions of antibodies derived from one mammalian species, while the constant portions are homologous to antibody sequences derived from another. A clear advantage for such chimeric forms is that the variable region can be conveniently derived from currently known sources using readily available B cells or hybridomas of non-human host organisms in combination with constant regions derived from, for example, human cell preparations.While the variable region has the advantage of ease of preparation and specificity unaffected by the source, the constant region, being human, has a lower probability of inducing an immune response from a human subject when antibodies are injected than the constant region from a non-human source. However, the definition is not limited to this specific example.

[00139] The term “humanized antibody” refers to a molecule possessing an antigen-binding site that is substantially derived from an immunoglobulin of a non-human species, wherein the remaining immunoglobulin structure of the molecule is based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may comprise complete variable domains fused into constant domains or only the complementarity-determining regions (CDRs). Petition 870260062263, dated 06 / 25 / 2026, page 65 / 317 55 / 148 grafted into appropriate structural regions in the variable domains. Antigen-binding sites may be wild-type or modified by one or more amino acid substitutions, for example, modified to more closely resemble human immunoglobulins. Some forms of humanized antibodies preserve all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs of the mouse antibody). Other forms have one or more CDRs that are altered relative to the original antibody.

[00140] Different methods for humanizing antibodies are known to experts, as reviewed by Almagro & Fransson, 2008, whose content is incorporated herein by reference in its entirety. The review article by Almagro & Fransson is briefly summarized below. Almagro & Fransson distinguish between rational and empirical approaches. Rational approaches are characterized by generating a few variants of the manipulated antibody and evaluating their binding or any other property of interest. If the designed variants do not produce the expected results, a new cycle of design and binding evaluation is initiated. Rational approaches include CDR grafting, resurfacing, superhumanization, and Human String Content Optimization. In contrast, empirical approaches are based on generating large libraries of humanized variants and selecting the best clones using high-throughput enrichment or screening technologies.Consequently, empirical approaches depend on a reliable selection and / or screening system capable of searching through a vast range of antibody variants. In vitro delivery technologies, such as phage presentation and ribosome presentation, meet these requirements and are well known to those skilled in the art. Empirical approaches include FR libraries, guided selection, etc. Petition 870260062263, dated 06 / 25 / 2026, page 66 / 317 56 / 148 shuffling of FR and humanization.

[00141] A “bivalent antibody” comprises two antigen-binding sites. Such bivalent antibodies can be monospecific or bispecific. In the case of a monospecific bivalent antibody, the two binding sites of the antibody have the same antigen specificities. A “bispecific” or “bifunctional” antigen-binding protein or antibody is a hybrid antigen-binding protein or antibody, respectively, possessing two different antigen-binding sites. The two antigen-binding sites of a bispecific protein or antibody bind to two different epitopes residing on the same or different antigens. Bispecific antigen-binding proteins and antibodies are a type of multispecific antigen-binding protein antibody and can be produced by a variety of methods including, but not limited to, hybridoma fusion, chemical ligation of IgG fragments or IgG as Fab', or by genetic means.See, for example, Songsivilai and Lachmann, 1990, Clin. Immunological exp. 79:315-321; Kostelny et al. 1992, J. Immunol. 148:1547-1553; Kontermann, 2014, MAbs 4: 182-197.

[00142] A “trifunctional antibody” is a type of bispecific antibody comprising two binding sites targeting different antigens, as well as an intact Fc portion that can bind to an Fc receptor on accessory cells (e.g., monocytes / macrophages, natural killer cells, dendritic cells, or others). For example, a trifunctional antibody might comprise one binding site targeting an epitope on the surface of a cancer cell, the second binding site might target an epitope on the surface of a T cell (e.g., CD3), and the Fc portion might bind to the Fc receptor on the surface of a macrophage. This trifunctional antibody is therefore capable of binding to both T cells and macrophages, leading to their destruction. Petition 870260062263, dated 06 / 25 / 2026, p. 67 / 317 57 / 148

[00143] Digestion of antibodies with papain produces two identical antigen-binding fragments, called “Fab fragments” (also called “Fab portion” or “Fab region”), each with a single antigen-binding site, and a residual “Fc fragment” (also referred to as “Fc portion” or “Fc region”) whose name reflects its ability to readily crystallize. The crystal structure of the Fc region of human IgG has been determined (Deisenhofer (1981) Biochemistry 20: 2361-2370). In IgG, IgA, and IgD isotypes, the Fc region is composed of two identical protein fragments, derived from the CH2 and CH3 domains of the two antibody heavy chains; in IgM and IgE isotypes, the Fc regions contain three constant heavy chain domains (CH2-4) in each polypeptide chain. In addition, smaller immunoglobulin molecules exist naturally or have been artificially constructed.The term “Fab' fragment” refers to a Fab fragment that additionally comprises the hinge region of an Ig molecule, while “F(ab')2 fragments” comprise two Fab' fragments that are chemically linked or are linked via a disulfide bond. Although “single-domain antibodies (sdAb)” (Desmyter et al. (1996) Nat. Structure Biol. 3:803-811) and “Nanobodies” comprise only a single VH domain, “single-chain Fv fragments (scFv) comprise the variable heavy chain domain linked via a short peptide linker to the variable light chain domain” (Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85, 5879-5883). Bivalent single-chain variable fragments (di-scFvs) can be designed by linking two scFvs (scFvA-scFvB). This can be done by producing a single peptide chain with two VH and two VL regions, producing “tandem scFvs” (VHA-VLA-VHB-VLB).Another possibility is the creation of scFvs with linkers that are too short for the two variable regions to fold together, forcing the scFvs to dimerize. Generally, the linkers have a length... Petition 870260062263, dated 06 / 25 / 2026, page 68 / 317 58 / 148 of 5 residues are used to generate these dimers. This type is known as “diabodies”. Even shorter linkers (one or two amino acids) between a VH and VL domain lead to the formation of monospecific trimers, the so-called “triabodies” or “tribodies”. Bispecific diabody antibodies are formed by expression in chains with the arrangement VHA-VLB and VHB-VLA or VLA-VHB and VLB-VHA, respectively. Single-chain diabodies (scDb) comprise a VHAVLB and a VHB-VLA fragment that are linked by a 12-20 amino acid linking peptide (P), preferably 14 amino acids (VHA-VLB-P-VHBVLA). “Bispecific occupation antibodies against T cells (BiTEs)” are fusion proteins consisting of two different antibody scFvs in which one of the scFvs binds to T cells via the CD3 receptor and the other to a tumor cell via a tumor-specific molecule (Kufer et al. (2004) Trends Biotechnol. 22: 238-244).Dual-affinity redirection molecules (DART molecules) are diaphragms additionally stabilized via a C-terminal disulfide bond. Single-chain divalent variable fragments can be linked to one or more homo- or heterodimerization domains to create tetravalent, hexavalent, octavalent, or even higher valence molecules. Depending on the respective specificities of the single-chain variable fragments linked through one or more homo- or heterodimerization domains, the resulting dimeric or multimeric proteins will have two, three, four, or more specificities.

[00144] The antibodies described in the present invention are preferably isolated. An “isolated antibody,” as used herein, is intended to refer to an antibody that is substantially free of other antibodies with different antigenic specificities. In addition, an isolated antibody may be substantially free of other material. Petition 870260062263, dated 06 / 25 / 2026, page 69 / 317 59 / 148 cellular and / or chemical products. In one embodiment, a combination of “isolated” monoclonal antibodies refers to antibodies possessing different specificities and being combined into a well-defined composition.

[00145] As used in the present invention, the term “antibody-like protein” refers to a protein that has been modified (e.g., by loop mutagenesis) to bind specifically to a target molecule. Typically, such an antibody-like protein comprises at least one variable peptide loop attached at both ends to a scaffold protein. This double structural restriction greatly increases the binding affinity of the antibody-like protein to levels comparable to those of an antibody. The length of the variable peptide loop typically consists of 10 to 20 amino acids. The scaffold protein can be any protein with good solubility properties. Preferably, the scaffold protein is a small globular protein. Antibody-like proteins include, without limitation, afibodies, anticalins, and ankyrin repeat proteins (for review see: Binz HK et al.(2005) Engineering novel binding proteins from nonimmunoglobulin domains. Nat. Biotechnol. 23 (10): 1257-1268). Antibody-like proteins can be derived from large mutant libraries, for example, they can be searched from large display libraries in phages and can be isolated in analogy with regular antibodies. In addition, antibody-like binding proteins can be obtained by combinatorial mutagenesis of surface-exposed residues in globular proteins. Sometimes, antibody-like proteins are called “peptide aptamers”.

[00146] As used in the present invention, a “mimetic peptide” is a small protein-like chain designed to Petition 870260062263, dated 06 / 25 / 2026, page 70 / 317 60 / 148 mimic a peptide. Peptide mimetics typically arise from the modification of an existing peptide to alter the properties of the molecule. For example, they may arise from modifications to alter the stability or biological activity of the molecule. This may play a role in the development of drug-like compounds from existing peptides. These modifications involve changes to the peptide that will not occur naturally (such as altered core structures and the incorporation of non-natural amino acids).

[00147] The term “target” refers to a molecule or a portion of a molecule capable of being bound by an antigen-binding protein. In certain embodiments, a target may have one or more epitopes. In some embodiments, the target is an antigen. The use of “antigen” in the phrase “antigen-binding protein” simply indicates that the protein sequence comprising the antigen can be bound by an antibody. In this context, it does not require that the protein be foreign or capable of inducing an immune response.

[00148] The term “recombinant” refers to an amino acid sequence or a nucleotide sequence that is intentionally modified by recombinant methods. The term “recombinant nucleic acid,” as used herein, refers to a nucleic acid that is formed in vitro and optionally further manipulated by endonucleases to form a nucleic acid molecule that is not normally found in nature. Examples of recombinant nucleic acids include cDNA, in a linear form, as well as vectors formed in vitro by ligating DNA molecules that are not normally linked. It is understood that once a recombinant nucleic acid is produced and introduced into a host cell, it will be replicated in a non-recombinant manner, i.e., using the cell's in vivo cellular machinery. Petition 870260062263, dated 06 / 25 / 2026, p. 71 / 317 61 / 148 host instead of in vitro manipulations. Consequently, nucleic acids that have been produced recombinantly can be subsequently replicated in a non-recombinant manner. A “recombinant protein” is a protein produced using recombinant techniques, for example, through the expression of a recombinant nucleic acid as described above. The term “recombinant vector” used in the present invention includes any vectors known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenoviral or baculoviral vectors, or artificial chromosomal vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC). These vectors include expression vectors as well as cloning vectors.Expression vectors comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the operationally linked expression of the coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. Cloning vectors are generally used to manipulate and amplify a specific desired DNA fragment and may lack functional sequences necessary for the expression of the desired DNA fragments.

[00149] The term “host cell” refers to a cell that harbors a vector (e.g., a plasmid or virus). Such a host cell can be a prokaryotic cell (e.g., a bacterial cell) or a eukaryotic cell (e.g., a fungal, plant, or animal cell). Host cells include both unicellular prokaryotes and eukaryotic organisms (e.g., bacteria, yeasts, and actinomycetes), as well as isolated cells from higher-order plants or animals when cultured. Petition 870260062263, dated 06 / 25 / 2026, page 72 / 317 62 / 148 in cell culture. The term “recombinant host cell,” as used herein, refers to a host cell comprising a polynucleotide encoding a polypeptide fragment of interest, namely, the viral PA subunit fragment or variants thereof according to the invention. This polynucleotide may be found within the host cell (i) freely dispersed as such, (ii) incorporated into a recombinant vector, or (iii) integrated into the host cell genome or mitochondrial DNA. The recombinant cell may be used for expression of a polynucleotide of interest or for amplification of the recombinant polynucleotide or vector of the invention. The term “recombinant host cell” includes the progeny of the original cell that has been transformed, transfected, or infected with the recombinant polynucleotide or vector of the invention. A recombinant host cell may be a bacterial cell, such as an E. coli cell.E. coli, a yeast cell such as Saccharomyces cerevisiae or Pichia pastoris, a plant cell, an insect cell such as SF9 or High Five cells, or a mammalian cell. Preferred examples of mammalian cells are Chinese hamster ovary cells (CHO), African green monkey kidney cells (COS), human embryonic kidney cells (HEK293), HELA cells, and the like.

[00150] The terms “individual”, “subject” or “patient” are used interchangeably and refer to any mammal, reptile or bird that may benefit from the present invention. In particular, an individual is selected from the group consisting of laboratory animals (e.g., rat, mouse or rabbit), domestic animals (including, for example, guinea pig, rabbit, horse, donkey, cow, sheep, goat, pig, chicken, duck, camel, cat, dog, turtle, tortoise, snake or lizard), or primates, including chimpanzees, bonobos, gorillas and humans. In particular, the “individual” is a human being. Petition 870260062263, dated 06 / 25 / 2026, p. 73 / 317 63 / 148

[00151] The terms “disease” and “disorder” are used interchangeably in the present invention, referring to an abnormal condition, especially an abnormal medical condition, such as a disease or injury, in which a tissue, organ, or individual is no longer able to efficiently perform its function. Typically, but not necessarily, a disease is associated with specific symptoms or signs that indicate the presence of such a disease. The presence of such symptoms or signs may therefore be indicative of a tissue, organ, or individual suffering from a disease. A change in these symptoms or signs may be indicative of the progression of such a disease. The progression of a disease is typically characterized by an increase or decrease in these symptoms or signs, which may indicate an “aggravation” or “improvement” of the disease.The "worsening" of a disease is characterized by a decreasing ability of a tissue, organ, or organism to perform its function efficiently, while the "improvement" of a disease is typically characterized by an increase in the ability of a tissue, organ, or individual to perform its function efficiently. A tissue, organ, or individual at "risk of developing" a disease is in a healthy state but exhibits the potential for an emerging disease. Typically, the risk of developing a disease is associated with early or mild signs or symptoms of that disease. In this case, the onset of the disease can still be prevented by treatment. Examples of diseases include, but are not limited to, infectious diseases, traumatic diseases, inflammatory diseases, skin conditions, endocrine diseases, intestinal diseases, neurological disorders, joint diseases, genetic disorders, autoimmune diseases, and various types of cancer.

[00152] By “tumor” we mean an abnormal group of cells or tissue that grows by rapid and uncontrolled cell proliferation and continues to grow after the stimuli that initiated the new growth have ceased. The Petition 870260062263, dated 06 / 25 / 2026, p. 74 / 317 64 / 148 tumors show a partial or total lack of structural organization and functional coordination with normal tissue and generally form a distinct mass of tissue, which may be benign or malignant.

[00153] By “metastasis” is meant the spread of cancer cells from their original location to other parts of the body. Metastasis formation is a very complex process and depends on the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of endothelial basement membranes to enter the body cavity and vessels, and then, after transport by the blood, infiltration of target organs. Finally, the growth of a new tumor at the target site depends on angiogenesis. Tumor metastasis often occurs even after removal of the primary tumor because tumor cells or components may remain and develop metastatic potential. In one embodiment, the term “metastasis” according to the invention refers to “distant metastasis,” which refers to a metastasis that is distant from the primary tumor and the regional lymph node system.

[00154] The “symptoms” of a disease or disorder are implications of the disease or disorder perceptible to the tissue, organ, or organism that has that disease or disorder and include, but are not limited to, pain, weakness, tenderness, tension, stiffness, and spasm of the tissue, an organ, or an individual, as well as the presence, absence, increase, or decrease of specific indicators, such as biomarkers or molecular markers. The terms “disease” and “disorder,” as used in the present invention, refer to an abnormal condition, especially an abnormal medical condition, such as a disease or injury, in which a tissue, organ, or individual is no longer able to efficiently perform its function. Typically, but not necessarily, a disease or disorder is associated with specific symptoms or signs that indicate the presence of such a disease or disorder. Diseases or disorders include, Petition 870260062263, dated 06 / 25 / 2026, p. 75 / 317 65 / 148 but are not limited to, autoimmune diseases, allergic diseases, cancer-like diseases, skin conditions, endocrine diseases, blood diseases and disorders, eye diseases and disorders, genetic diseases, inflammatory diseases, infectious diseases, bowel diseases, neurological disorders and mental illness. For example, cancer-type diseases include, but are not limited to, basal cell carcinoma, bladder cancer, bone cancer, brain tumor, breast cancer, Burkitt's lymphoma, cervical cancer, colon cancer, cutaneous T-cell lymphoma, esophageal cancer, retinoblastoma, gastric (stomach) cancer, gastrointestinal stromal tumor, glioma, Hodgkin's lymphoma, Kaposi's sarcoma, leukemias, lymphomas, melanoma, oropharyngeal cancer, ovarian cancer, pancreatic cancer, pleuropulmonary blastoma, prostate cancer, throat cancer, thyroid cancer, and urethral cancer.

[00155] As used in this invention, “treating”, “treating”, “treatment” or “therapy” of a disease or disorder means performing one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing the development of symptoms characteristic of the disorder(s) to be treated; (c) inhibiting the worsening of symptoms characteristic of the disorder(s) to be treated; (d) limiting or preventing the recurrence of the disorder(s) in an individual who has previously had the disorder(s); and (e) limiting or preventing the recurrence of symptoms in individuals who were previously symptomatic for the disorder(s). Consequently, a portion with a therapeutic effect treats the symptoms of a disease or disorder by performing one or more of the effects mentioned above (a)-(e).

[00156] As used in the present invention, “preventing”, “preventing”, “prevention” or “prophylaxis” of a disease or disorder means preventing such disease or disorder from occurring in the patient.

[00157] The expression “pharmaceutically acceptable” means approved by a regulatory agency of the Federal Government or a government Petition 870260062263, dated 06 / 25 / 2026, page 76 / 317 66 / 148 state or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, and more specifically for use in humans.

[00158] The term “pharmaceutically active portion,” as used in the present invention, refers to a portion or part of a macromolecule or complex, i.e., a polypeptide, polynucleotide, or complex thereof, that mediates a pharmaceutical effect including, but not limited to, prophylactic, therapeutic, and / or diagnostic effects. Pharmaceutically active portions typically comprise a biological and / or chemical drug, for example, ligands, effector molecules, half-life extension modules, and imaging molecules. The term “ligand” refers to a chemical or biological substance that forms a complex with another molecule to perform a specific biological function. Ligands include, but are not limited to, substrates, inhibitors, and activators, such as antigen-binding molecules, scaffold proteins, natural ligands, ligand-binding receptor fragments, and aptamers.The term "effector molecule" typically refers to small molecules, peptides, or polypeptides that bind to a protein and thereby alter the activity of that protein. These include, but are not limited to, cytokines, chemokines, immunostimulatory molecules, immunosuppressive molecules, death ligands, apoptosis-inducing proteins, kinases, prodrug-converting enzymes, RNases, antibody or antibody fragment agonist, antibody or antibody fragment antagonist, toxins, growth factors, hormones, coagulation factors, fibrinolytic proteins, peptide mimetics and fragments, fusion proteins or their derivatives. "Half-life extension modules" prolong the half-life, for example, the "plasma half-life" or the "serum half-life" of a chemical or biological substance. Imaging molecules are those that bind to specific target molecules, thus allowing visualization of the target. Petition 870260062263, dated 06 / 25 / 2026, page 77 / 317 67 / 148 location of this molecule.

[00159] The terms “pharmaceutical”, “medicine” and “drug” are used interchangeably, referring to a substance and / or a combination of substances that are used for the identification, prevention or treatment of a disease or disorder.

[00160] The terms “preparation” and “composition” are intended to include the formulation of the active compound with encapsulating material as a carrier, providing a capsule in which the active component, with or without other vehicles, is surrounded by a carrier or vehicle, which is thus associated with the active compound.

[00161] “Chemical pharmaceuticals” are typically understood to be artificially synthesized chemical compounds that are effective in the prevention, treatment, or diagnosis of disorders or diseases.

[00162] “Biological products” are typically understood to be medical drugs produced using biotechnological means and are used for prophylactic, therapeutic and / or in vivo diagnostic purposes. Biological products include, but are not limited to, peptides, polypeptides, proteins and nucleic acids (e.g., DNA, RNA, or their hybrids).Approved therapeutic biological products include, among others, hormones (e.g., insulin, hGH, FSH, glucagon-like peptide 1, parathyroid hormone, calcitonin, lutropin, glucagon), growth factors (e.g., erythropoietin, G-CSF / GM-CSF, IGF-1), interferons (e.g., IFN-α, IFN-β, IFN-γ), interleukins (e.g., IL-2, IL-11, IL-1Ra), coagulation factors (e.g., factor VIII, factor IX, factor VIIa, thrombin), thrombolytics and anticoagulants (e.g., t-PA, hirudin, activated protein C), enzymes (e.g., α-glucosidase, glucocerebrosidase, iduronate-2-sulfatase, galactosidase, urate oxidase, DNase), antigen-binding molecules, such as antibodies and antibody fragments (e.g., IgG, Fab) and proteins. Petition 870260062263, dated 06 / 25 / 2026, page 78 / 317 68 / 148 fusion of the same (e.g., TNFR2-Fc, TMP-Fc, CTLA-4-Fc, IL-1R-Fc, LFA-3-Fc, IL-2-DT).

[00163] The term “active ingredient” refers to the substance in a pharmaceutical composition or formulation that is biologically active, that is, that provides pharmaceutical value. A pharmaceutical composition may comprise one or more active ingredients that may act together or independently of each other. The active ingredient may be formulated as neutral or saline forms. Pharmaceutically acceptable salts include those formed with free amino groups, such as, but not limited to, derivatives of hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups, such as, but not limited to, derivatives of sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine and the like.

[00164] The term “vehicle” or “carrier,” as used in the present invention, refers to a pharmacologically inactive substance such as, but not limited to, a diluent, excipient, surfactant, stabilizer, physiological buffer solutions, or vehicles with which the therapeutically active ingredient is administered. Such pharmaceutical vehicles may be liquid or solid. Liquid vehicles include, but are not limited to, sterile liquids such as saline solutions in water and oils, including, but not limited to, those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Saline solutions and aqueous dextrose and glycerol solutions may also be used as liquid vehicles, especially for injectable solutions. A saline solution is a preferred vehicle when the pharmaceutical composition is administered intravenously.Examples of suitable pharmaceutical vehicles are described in “Remington's. Petition 870260062263, dated 06 / 25 / 2026, page 79 / 317 69 / 148 Pharmaceutical Sciences” by EW Martin.

[00165] Suitable pharmaceutical “excipients” include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, calcium carbonate, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene glycol, water, ethanol, etc.

[00166] “Surfactants” include anionic, cationic and non-ionic surfactants such as, but not limited to, sodium deoxycholate, sodium dodecyl sulfate, Triton X-100 and polysorbates such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65 and polysorbate 80.

[00167] “Stabilizers” include, but are not limited to, mannitol, sucrose, trehalose, albumin, as well as protease and / or nuclease antagonists.

[00168] A “physiological buffer solution” includes, but is not limited to, sodium chloride solution, demineralized water, as well as suitable organic or inorganic buffer solutions, such as, but not limited to, phosphate buffer, citrate buffer, tris buffer (tris(hydroxymethyl)aminomethane), HEPES buffer ([4-(2-hydroxyethyl)piperazine]ethanesulfonic acid) or MOPS buffer (3-morpholino-1-propanesulfonic acid). The choice of buffer generally depends on the molarity of the buffer desired. Phosphate buffer is suitable, for example, for injection and infusion solutions.

[00169] The term “adjuvant” refers to agents that enhance, stimulate, activate, potentiate, or modulate the immune response to the active ingredient of the composition at the cellular or humoral level; for example, immunological adjuvants stimulate the immune system's response to the antigen but have no immunological effect. Examples of such adjuvants include, but are not limited to, inorganic adjuvants (e.g., inorganic metal salts such as aluminum phosphate or aluminum hydroxide), organic adjuvants (e.g., saponins or squalene), oil-based adjuvants (e.g., Petition 870260062263, dated 06 / 25 / 2026, p. 80 / 317 70 / 148 example, complete Freund's adjuvant and incomplete Freund's adjuvant) cytokines (e.g., IL-1β, IL-2, IL-7, IL-12, IL-18, GM-CFS and IFN-γ), particulate adjuvants (e.g., immunostimulatory complexes (ISCOMS), liposomes or biodegradable microspheres), virosomes, bacterial adjuvants (e.g., monophosphoryl-lipid A or muramyl peptides), synthetic adjuvants (e.g., non-ionic block copolymers, muramyl peptide analogs or synthetic lipid A) or synthetic polynucleotide adjuvants (e.g., polyarginine or polylysine).

[00170] An “effective amount” or “therapeutically effective amount” is an amount of a therapeutic agent sufficient to achieve the intended purpose. The effective amount of a given therapeutic agent will vary with factors such as the nature of the agent, the route of administration, the size and species of the animal to receive the therapeutic agent, and the purpose of administration. The effective amount in each individual case can be determined empirically by a person skilled in the art, according to methods established in the state of the art. Examples of Achievement

[00171] In a first aspect, the present invention provides an antigen-binding protein that specifically binds to a conformational epitope formed by domains III and IV of the human epidermal growth factor receptor 3 (HER3). The phrase "conformational epitope formed by domains III and IV" means that at least one amino acid from domain III and at least one amino acid from domain IV is linked by the antigen-binding protein. Thus, it does not imply that all amino acids from domains III and IV are part of the conformational epitope, but that amino acid(s) in both domains are linked. Typically, the epitope of an antibody comprises between 12 and 20 amino acids and, therefore, in a specific embodiment, between 1 and 19 amino acids from domain III and Petition 870260062263, dated 06 / 25 / 2026, page 81 / 317 71 / 148 Between 1 and 19 amino acids of domain IV are linked by the antigen-binding protein, preferably between 3 and 17 amino acids of domain III, and between 3 and 17 amino acids of domain IV are linked by the antigen-binding protein. In each case, it is preferred that the linked epitope comprise between 12 and 20 amino acids.

[00172] In realization examples, the conformational epitope is formed by the complete domain III and the complete domain IV of HER3. In alternative realization examples, the conformational epitope is formed by the complete domain III and a fragment of domain IV of HER3. In alternative realization examples, the conformational epitope is formed by a fragment of domain III and the complete domain IV of HER3. In alternative realization examples, the conformational epitope is formed by a fragment of domain III and a fragment of domain IV of HER3.

[00173] In specific embodiments, domain III consists of amino acids 329 to 531 of HER3 according to SEQ ID NO: 1.

[00174] In specific embodiments, the domain IV fragment comprises or consists of amino acids 532-587 of HER3 according to SEQ ID NO: 1.

[00175] In specific embodiments, domain IV consists of amino acids 532-643 of HER3 according to SEQ ID NO: 1.

[00176] Thus, in specific embodiments, the present invention provides an antigen-binding protein that specifically binds to a conformational epitope formed by domains III and IV of HER3, wherein domain III consists of amino acids 329 to 531 of HER3 according to SEQ ID NO: 1, and wherein a fragment of domain IV comprises or consists of amino acids 532-587 of HER3 according to SEQ ID NO: 1.

[00177] In specific embodiment examples, the present Petition 870260062263, dated 06 / 25 / 2026, p. 82 / 317 72 / 148 invention provides an antigen-binding protein that specifically binds to a conformational epitope formed by domains III and IV of HER3, wherein domain III consists of amino acids 329 to 531 of HER3 according to SEQ ID NO: 1, and wherein domain IV consists of amino acids 532 to 643 of HER3 according to SEQ ID NO: 1.

[00178] In a second aspect, the present invention provides an antigen-binding protein that competes with the antigen-binding protein of the first aspect of the present invention for binding to HER3.

[00179] In specific embodiments, the present invention provides an antigen-binding protein that competes with the antigen-binding protein that specifically binds to a conformational epitope formed by domains III and IV of the human epidermal growth factor receptor 3 (HER3).

[00180] In realization examples, the conformational epitope is formed by the complete domain III and the complete domain IV of HER3. In alternative realization examples, the conformational epitope is formed by the complete domain III and a fragment of domain IV of HER3. In alternative realization examples, the conformational epitope is formed by a fragment of domain III and the complete domain IV of HER3. In alternative realization examples, the conformational epitope is formed by a fragment of domain III and a fragment of domain IV of HER3.

[00181] In specific embodiments, domain III consists of amino acids 329 to 531 of HER3 according to SEQ ID NO: 1.

[00182] In specific embodiments, the domain IV fragment comprises or consists of amino acids 532-587 of HER3 according to SEQ ID NO: 1.

[00183] In specific embodiments, domain IV consists of amino acids 532-643 of HER3 according to SEQ ID NO: 1. Petition 870260062263, dated 06 / 25 / 2026, page 83 / 317 73 / 148

[00184] Thus, in specific embodiments, the present invention provides an antigen-binding protein that competes with the antigen-binding protein that specifically binds to a conformational epitope formed by domains III and IV of HER3, wherein domain III consists of amino acids 329 to 531 of HER3 according to SEQ ID NO: 1, and wherein a fragment of domain IV comprises or consists of amino acids 532-587 of HER3 according to SEQ ID NO: 1.

[00185] In specific embodiments, the present invention provides an antigen-binding protein that competes with the antigen-binding protein that specifically binds to a conformational epitope formed by domains III and IV of HER3, wherein domain III consists of amino acids 329 to 531 of HER3 according to SEQ ID NO: 1, and wherein domain IV consists of amino acids 532 to 643 of HER3 according to SEQ ID NO: 1.

[00186] In a specific embodiment, the aforementioned antigen-binding protein of the second aspect competes with the antigen-binding protein of the first aspect for binding to the conformational epitope formed by domains III and IV of HER3.

[00187] In specific embodiments, the antigen-binding protein of the second aspect competes for binding to the conformational epitope formed by domains III and IV of HER3, exhibiting a higher affinity for the epitope than the antigen-binding protein of the first aspect.

[00188] In further embodiments, the antigen-binding protein of the second aspect competes for binding to the conformational epitope formed by domains III and IV of HER3, sterically preventing the binding of the antigen-binding protein of the first aspect. In embodiments, the antigen-binding protein of Petition 870260062263, dated 06 / 25 / 2026, page 84 / 317 74 / 148 The second aspect stereochemically prevents the antigen-binding protein of the first aspect from binding to the identical epitope or prevents binding to an adjacent epitope such that the antigen-binding protein of the first aspect is unable to bind to the conformational epitope formed by domains III and IV of HER3.

[00189] In specific embodiments of the first or second aspect of the present invention, the antigen-binding protein has one or more of the following characteristics: (a) The antigen-binding protein binds to HER3 with an EC50 value below 15 nM (in particular as analyzed by flow cytometry in cells expressing HER3). In particular, the antigen-binding protein binds to HER3 with an EC50 value below 10 nM, below 5 nM, below 1 nM, below 500 pM, below 100 pM, below 50 pM, or below 30 pM; (b) The antigen-binding protein binds to monomeric HER3 with a KD less than 100 nM (in particular as analyzed by quartz crystal microbalance measurements, surface plasmon resonance, optical interferometry (Octet) or competitive ELISA). In particular, the antigen-binding protein binds to monomeric HER3 with a KD value less than 50 nM, less than 30 nM or less than 20 nM; (c) The antigen-binding protein inhibits heregulin-induced HER3 phosphorylation with an IC50 value below 10 nM. In particular, the antigen-binding protein inhibits heregulin-induced HER3 phosphorylation with an IC50 value below 5 nM, below 1 nM, below 500 pM, below 300 pM, below 200 pM, or below 100 pM. In particular, the antigen-binding protein inhibits heregulin-induced HER3 phosphorylation with an IC50 value of 80 pM.

[0190] Consequently, in examples of implementation Petition 870260062263, dated 06 / 25 / 2026, page 85 / 317 75 / 148 specific to the first or second aspect of the present invention, the antigen-binding protein: (a) binds to HER3 with an EC50 value below 15 nM (as analyzed by flow cytometry in HER3-expressing cells), in particular with an EC50 value below 10 nM, below 5 nM, below 1 nM, below 500 pM, below 50 pM, or below 30 pM; and / or (b) binds to monomeric HER3 with a KD value below 100 nM (as analyzed by quartz crystal microbalance measurements), in particular with a KD value below 50 nM, below 30 nM, or below 20 nM; and / or (c) inhibits heregulin-induced HER3 phosphorylation with an IC50 value below 10 nM, in particular with an IC50 value below 5 nM, below 1 nM, below 500 pM, below 300 pM, below 200 pM or below 100 pM, in particular with an IC50 value of 80 pM.

[0191] In specific embodiments of the first or second aspect of the present invention, the antigen-binding protein inhibits or performs one or more of the following functions: (i) HER3 binding to its ligand, (ii) receptor signaling and / or activation, (iii) induces HER3 internalization, (iv) inhibits cell proliferation and / or (v) inhibits tumor growth. [019 2] In particular embodiments of the first or second aspect of the present invention, the antigen-binding protein is selected from the group consisting of; a) an antibody or antigen-binding fragment thereof, b) antibody-like protein, and Petition 870260062263, dated 06 / 25 / 2026, p. 86 / 317 76 / 148 c) a mimetic peptide.

[0193] In specific embodiments of the first or second aspect of the present invention, the antigen-binding protein is an antibody selected from the group consisting of a polyclonal antibody, a monoclonal antibody, monovalent antibodies, a bispecific antibody, heteroconjugated antibodies, multispecific antibodies, deimmunized antibodies, a chimeric antibody, a humanized antibody and a human antibody (in particular a human IgG1 antibody).

[0194] In specific embodiments, the antigen-binding fragment of the antibody is selected from the group consisting of a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fd fragment, an Fv fragment, a disulfide-linked Fv (dsFv), a single-domain antibody, a single-chain Fv antibody (scFv), and a single-domain antibody (VH, VL, VHH, Nanobody, VNAR).

[0195] In specific embodiments, the antibody-like protein is selected from the group consisting of a lipoprotein-associated coagulation inhibitor (LACI-D1); afilins, for example, human crystalline B γ or human ubiquitin; cystatin; Sac7D of Sulfolobus acidocaldarius; lipocalin and lipocalin-derived anticalins; manipulated ankyrin repeat domains (DARPins); Fyn SH3 domain; Kunits domain of protease inhibitors; monobodies, for example, the 10° type III domain of fibronectin; adnectins; cystine knot miniproteins; atrimers; evibodies, for example, CTLA4-based ligands; affibodies, for example, the three-helix bundle of the Z domain of Staphylococcus aureus protein A; Trans-bodies, for example, human transferrin; Tetranectins, for example, monomeric or trimeric human C-type lectin domain; microorganisms, for example, trypsin II inhibitor; aphyllins; armadillo repeat proteins. Petition 870260062263, dated 06 / 25 / 2026, p. 87 / 317 77 / 148

[0196] In particular embodiments of the first or second aspect of the present invention, the antigen-binding protein is monospecific, bispecific, or multispecific. In certain embodiments, the bispecific or multispecific antigen-binding protein binds specifically to a second cellular target.In specific embodiments, the second cellular target is selected from the group consisting of a protein expressed on the surface of an immune cell, preferably CD3, a protein expressed on the surface of tumor cells, in particular the extracellular region of a growth receptor, in specific epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 4 (HER4), insulin-like growth factor receptor 1 (IGF-1R), hepatocyte growth factor receptor (HGFR, c-MET) and their derivatives, in particular EGFR or HER2.

[0197] In particular embodiments of the first or second aspect of the present invention, the antigen-binding protein is trivalent or tetravalent. In specific embodiments, the antigen-binding protein comprises an effector domain that is particularly bound to Fc receptors, the neonatal Fc receptor (FcRn), or the complement system. In specific embodiments, the Fc domain is a domain bound to Fc gamma receptors, in particular CD16, CD32, and / or CD64. In specific embodiments, the Fc domain is a domain that activates the complement system, in particular by binding to C1q of the complement system.

[0198] In a preferred embodiment of the present invention, the antigen-binding protein is bivalent. Unless otherwise indicated, the configurations of the antigen-binding protein embodiments below are written from the N-terminal end to the Petition 870260062263, dated 06 / 25 / 2026, page 88 / 317 78 / 148 left to the C-terminal end of the right. It is further preferred that the antigen-binding protein be bivalent and bispecific. In a further embodiment, the bivalent and bispecific antigen-binding protein is a diabody. The bispecific diabody comprises two chains, each comprising a VH and VL domain of different antibodies. The two variable VH and VL domains are preferably linked by a short linker of 3 to 5 residues.

[0199] The diabody can be a two-chain diabody (Db) or a single-chain diabody (scDb). For the two-chain diabody, the two chains can have the configuration VHA-VLB and VHB-VLA or VLA-VHB and VLB-VHA, where A and B represent the two different specificities. For the single-chain diabody, the first chain, VHA-VLB or VLA-VHB, and the second chain, VHB-VLA or VLB-VHA, are covalently connected. Preferably, the first and second chains are linked by a peptide linker with a length of 10 to 15 amino acids. Preferably, the bispecific diabody is an scDb. Preferably, the antigen-binding protein has the configuration (VHA-VLB-VHB-VLA)scDb. In a particularly preferred embodiment, the antigen-binding protein comprises or consists of the amino acid sequence SEQ ID NO: 12 or SEQ ID NO: 34.

[0200] In another preferred embodiment, the antigen-binding protein is a bispecific Db or bispecific scDb, preferably a bispecific scDb, linked to one or more scFvs, preferably to one or two scFvs. Two or more scFvs may be tandemly connected. An scFv comprises the VH and VL domains of the same antibody, preferably linked to a peptide linker of about 10 to 25 amino acids. An scFv may have the VH-VL or VL-VH configuration. Preferably, one or more scFvs have one or both of the specificities of the bispecific Db or Petition 870260062263, dated 06 / 25 / 2026, p. 89 / 317 79 / 148 bispecific scDb. Thus, the scFvs preferably have the VHAVLA or VLA-VHA configuration, or preferably have the VHB-VLB or VLBVHB configuration. In another preferred embodiment, one or more scFv may have a specificity different from the specificities of the bispecific Db or bispecific scDb. Consequently, one or more scFvs may have the VHC-VLC or VLC-VHC configuration, or VHD-VLD or VLD-VHD, and so on. In a preferred embodiment, the antigen-binding protein is a bispecific trivalent antigen-binding protein. Preferably, the antigen-binding protein has the configuration (VHAVLB-VHB-VLA)scDb-(VHA-VLA)scFv. In a particularly preferred embodiment, the antigen-binding protein comprises or consists of the amino acid sequence SEQ ID NO: 13. In a preferred embodiment, the antigen-binding protein is a tetravalent bispecific antigen-binding protein.In a preferred embodiment, the antigen-binding protein has the configuration (VHAVLA)scFv-(VHA-VLB-VHB-VLA)scDb-(VHA-VLA)scFv. In a particularly preferred embodiment, the antigen-binding protein comprises or consists of the amino acid sequence SEQ ID NO: 35.

[0201] In another preferred embodiment, the antigen-binding protein comprises two bispecific Dbs or bispecific scDbs, preferably bispecific scDbs, each linked to an Fc region, wherein the Fc region serves as a homodimerization domain. In a preferred embodiment, the antigen-binding protein comprises two portions of the (VHA-VLB-VHB-VLA)scDb-Fc configuration. The two portions may be covalently or non-covalently linked. In a particularly preferred embodiment, the antigen-binding protein comprises two portions comprising or consisting of the amino acid sequence SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or Petition 870260062263, dated 06 / 25 / 2026, p. 90 / 317 80 / 148 SEQ ID NO: 33.

[0202] In another example of a preferred embodiment of a bispecific antigen-binding protein, an additional VH domain and VL domain of a second specificity are linked to a light chain and a heavy chain, respectively, wherein the Fc region of the heavy chain serves as a dimerization domain. The two VH domains and two VL domains of different specificities can be connected in various combinations to the light chain and heavy chain, respectively, resulting in different configurations. In one example of a preferred embodiment of the antigen-binding protein, the light chain has the configuration VHA-VHB-CLk and the heavy chain has the configuration VLA-VLB-CH1-CH2-CH3. Some configurations allow for cross-pairing of the VH and VL domains. In one example of a preferred embodiment, the light chain has the configuration VHA-VLB-CLk and the heavy chain has the configuration VHB-VLA-CH1-CH2-CH3.In a preferred embodiment, the light chain has the configuration VLA-VLB-CLk and the heavy chain has the configuration VHB-VHA-CH1-CH2-CH3. In a particularly preferred embodiment, the antigen-binding protein comprises a chain with SEQ ID NO: 31 and a chain with SEQ ID NO: 32.

[0203] In each of the examples above, the letters “A”, “B”, “C”, and “D” symbolize an antigen specificity of the antigen-binding proteins of the present invention. At least one of “A”, “B”, “C”, and “D” within each antigen-binding protein of the invention binds specifically to a conformational epitope formed by domains III and IV of the human epidermal growth factor receptor 3 (HER3). The other specificities may be the same or different. The second specificity and the preferred additional specificities are described below.

[0204] Other examples of bispecific antibodies are described in Brinkmann U. & Kontermann RE, MABS, 2017, 9(2),182-212 and Petition 870260062263, dated 06 / 25 / 2026, page 91 / 317 81 / 148 specifically incorporated herein.

[0205] In a particular embodiment, the antigen-binding proteins of the present invention comprise multimerization domains. Preferred examples are dimerization domains, trimerization domains, or tetramerization domains. If two protein chains are linked, each comprises at least one dimerization domain capable of binding to at least one dimerization domain on the other protein. Consequently, if the antigen-binding protein comprises three protein chains, each comprises at least one trimerization domain capable of interacting with the respective other trimerization domain.In one specific embodiment, dimerization domains are selected from the group consisting of the IgM heavy chain 2 (CH2) domain (MHD2) or IgE heavy chain 3 (CH3) domain, immunoglobulin Fc region, IgG or IgA heavy chain 3 (CH3) domain, IgM or IgE heavy chain 4 (CH4) domain, Fab, Fab2, leucine zipper motifs, barnasebarstar dimers, miniantibodies, and ZIP minibodies; the trimerization domain is selected from the group consisting of tenascin C (TNC), the trimerization region of the non-collagenous C-terminal domain (NC1) of collagen XVIII, Fab3-like molecules, and TriBi minibodies; or tetramerization domains are selected from the group consisting of the p53 tetramerization domain, the tetramerization domain of general control protein 4 (GCN4), the VASP (vasodilator-stimulated phosphoprotein) tetramerization domain, tandem diabodies (diabodies), and di-diabodies.In some preferred embodiments, the use of heterodimerization domains is preferred, particularly if two protein chains with different antigenic specificities are used.

[0206] In specific embodiments of the first or second aspect of the present invention, the antigen-binding protein Petition 870260062263, dated 06 / 25 / 2026, page 92 / 317 82 / 148 comprises a heavy chain sequence with enhanced ADCC.

[0207] In specific embodiments of the first or second aspect of the present invention, the antigen-binding protein comprises: (a) a CDRH1 comprising amino acids 32-37 according to SEQ ID NO: 2 and variants thereof comprising an amino acid exchange, a CDRH2 comprising amino acids 52-69 according to SEQ ID NO: 2 and variants thereof comprising an amino acid exchange and a CDRH3 comprising amino acids 102-112 according to SEQ ID NO: 2 and variants thereof comprising an amino acid exchange, and / or (b) a CDRL1 comprising amino acids 23-33 according to SEQ ID NO: 3 and variants thereof comprising an amino acid exchange, a CDRL2 and variants thereof comprising an amino acid exchange comprising amino acids 49-55 according to SEQ ID NO: 3 and a CDR3L comprising amino acids 88-98 according to SEQ ID NO: 3 and variants thereof comprising an amino acid exchange. [02 08] In specific embodiments of the first or second aspect of the present invention, the antigen-binding protein comprises: (a) an FRH1 comprising amino acids 1-31 according to SEQ ID NO: 2 and its variants comprising at least 80%, preferably 90%, more preferably at least 95% sequence identity, an FRH2 comprising amino acids 38-51 and its variants comprising at least 80%, preferably 90%, more preferably at least 95% sequence identity, an FRH3 comprising amino acids 70-101 and its variants comprising by Petition 870260062263, dated 06 / 25 / 2026, page 93 / 317 83 / 148 less 80%, preferably 90%, more preferably at least 95% sequence identity, and an FRH4 comprising amino acids 113-123 and their variants comprising at least 80%, preferably 90%, more preferably at least 95% sequence identity and / or (b) an FRL1 comprising amino acids 1-22 according to SEQ ID NO: 3 and their variants comprising at least 80%, preferably 90%, more preferably at least 95% sequence identity, an FRL2 comprising amino acids 34-48 according to SEQ ID NO: 3 and their variants comprising at least 80%, preferably 90%, more preferably at least 95% sequence identity, an FRL3 comprising amino acids 56-87 according to SEQ ID NO: 3 and their variants comprising at least 80% preferably 90%, more preferably at least 95% sequence identity,and an FRL4 comprising amino acids 99-109 according to SEQ ID NO: 3 and its variants comprising at least 80%, preferably 90%, more preferably at least 95% sequence identity. [02 09] In specific embodiments of the first or second aspect of the present invention, the antigen-binding protein comprises: (a) a heavy chain comprising CDRH1 consisting of amino acids 32-37 according to SEQ ID NO: 2 and its variants comprising an amino acid exchange, a CDRH2 consisting of amino acids 52-69 according to SEQ ID NO: 2 and its variants comprising an amino acid exchange and a CDRH3 consisting of amino acids 102-112 according to SEQ ID NO: 2 and its variants comprising an amino acid exchange, an FRH1 consisting of amino acids 1-31 according to SEQ ID NO: 2 and its variants Petition 870260062263, dated 06 / 25 / 2026, page 94 / 317 84 / 148 comprising at least 80%, preferably 90%, more preferably at least 95% sequence identity, an FRH2 comprising amino acids 38-51 according to SEQ ID NO: 2 and its variants comprising at least 80%, preferably 90%, more preferably at least 95% sequence identity, an FRH3 comprising amino acids 70-101 according to SEQ ID NO: 2 and its variants with at least 80%, preferably 90%, more preferably at least 95% sequence identity, and an FRH4 comprising amino acids 113-123 according to SEQ ID NO: 2 and its variants comprising at least 80%, preferably 90%, more preferably at least 95% sequence identity; (b) a light chain comprising CDRL1 consisting of amino acids 23-33 according to SEQ ID NO: 3 and variants thereof comprising an amino acid exchange, a CDRL2 comprising amino acids 49-55 according to SEQ ID NO: 3 and variants thereof comprising an amino acid exchange, and a CDR3L comprising amino acids 88-98 according to SEQ ID NO: 3 and variants thereof comprising an amino acid exchange, an FRL1 comprising amino acids 1-22 according to SEQ ID NO: 3 and its variants comprising at least 80%, preferably 90%, more preferably at least 95% sequence identity, an FRL2 comprising amino acids 34-48 according to SEQ ID NO: 3 and its variants comprising at least 80%, preferably 90%, more preferably at least 95% sequence identity, an FR3L comprising amino acids 56-87 according to SEQ ID NO: 3 and its variants comprising at least 80%, preferably 90%,more preferably at least 95% sequence identity, and a FR4L comprising amino acids 99-109 according to SEQ ID NO: 3 and its, Petition 870260062263, dated 06 / 25 / 2026, page 95 / 317 85 / 148 variants comprising at least 80%, preferably 90%, more preferably at least 95% sequence identity.

[0210] In specific embodiments of the first or second aspect of the present invention, the antigen-binding protein comprises a variable domain comprising a heavy chain corresponding to amino acids 1-123 according to SEQ ID NO: 2 or variants having at least 80%, preferably 90%, more preferably at least 95% identity with the amino acid sequence according to SEQ ID NO: 2.

[0211] In specific embodiments of the first or second aspect of the present invention, the antigen-binding protein comprises a variable domain comprising a light chain corresponding to amino acids 1-109 according to SEQ ID NO: 3 or variants having at least 80%, preferably 90%, more preferably at least 95% identity with the amino acid sequence according to SEQ ID NO: 3.

[0212] In specific embodiments of the first or second aspect of the present invention, the antigen-binding protein comprises a variable domain comprising a heavy chain according to amino acids 1-123 according to SEQ ID NO: 2 and a light chain according to amino acids 1-109 according to SEQ ID NO: 3 or variants having at least 80%, preferably 90%, more preferably at least 95% identity with the amino acid sequence according to SEQ ID NO: 3.

[0213] In specific embodiments of the first or second aspect of the present invention, the antigen-binding protein comprises a heavy chain comprising or consisting of amino acids 1-453 according to SEQ ID NO: 4 or variants having Petition 870260062263, dated 06 / 25 / 2026, page 96 / 317 86 / 148 at least 80%, preferably 90%, more preferably at least 95% identity with the amino acid sequence according to SEQ ID NO: 4.

[0214] In specific embodiments of the first or second aspect of the present invention, the antigen-binding protein comprises a light chain comprising or consisting of amino acids 1-215 according to SEQ ID NO: 5 or variants having at least 80%, preferably 90%, more preferably at least 95% identity with the amino acid sequence according to SEQ ID NO: 5.

[0215] In specific embodiments of the first or second aspect of the present invention, the antigen-binding protein comprises a heavy chain comprising or consisting of amino acids 1-453 according to SEQ ID NO: 4 or variants having at least 80%, preferably 90%, more preferably at least 95% identity with the amino acid sequence according to SEQ ID NO: 4, and a light chain comprising or consisting of amino acids 1215 according to SEQ ID NO: 5 or variants having at least 80%, preferably 90%, more preferably at least 95% identity with the amino acid sequence according to SEQ ID NO: 5.

[0216] In specific embodiments, the antigen-binding protein further comprises a ligand, in particular a peptide ligand. In specific embodiments, the peptide ligands have a length between 5 and 40 amino acids (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 32, 33, 34, 35, 36, 37, 38, 39, 40 amino acids), in particular between 5 and 20 amino acids (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids), in particular 8 to 15 amino acids (i.e., 8, 9, 10, 11, 12, 13, 14, 15 amino acids). Petition 870260062263, dated 06 / 25 / 2026, p. 97 / 317 87 / 148

[0217] Flexible peptide linkers are particularly preferred. Flexible linkers are composed of amino acids without bulky side chains that prevent rotation or bending of the amino acid chain. Flexible linkers preferably comprise G, S, T, and A residues. In specific embodiments, at least 50% of the amino acids in the flexible linker peptide consist of amino acids selected from the group consisting of G, S, T, and A. In specific embodiments, at least 60%, 70%, 80%, 90%, 95%, or 100% of the amino acids in the linker consist of amino acids selected from the group consisting of G, S, T, and A. A large number of peptide linkers are described in the state of the art (Robinson & Sauer, 1998; Volkel et al., 2001; Kavoosi et al., 2007; Watanabe et al., 2011).In specific embodiments, peptide ligands include, but are not limited to, peptide ligand 1: GGGGS (SEQ ID NO: 14), peptide ligand 2: GGGGSGGGGS (SEQ ID NO: 15), peptide ligand 3: GGGGSGGGGSGGGGS (SEQ ID NO: 16), peptide ligand 4: GSLGGSGG (SEQ ID NO: 17), peptide ligand 5: GGGSGGGT (SEQ ID NO: 18), peptide ligand 6: GGGSGGGTGS (SEQ ID NO: 19), peptide ligand 7: GGGSGGGTGSGG (SEQ ID NO: 20), peptide ligand 8:. GGGGSGGRASGGGGSGGGGS (SEQ ID NO: 21), peptide linker 9: GGGSGGGS (SEQ ID NO: 22), peptide linker 10: EFTRG (SEQ ID NO: 23), and peptide linker 11: AAA (SEQ ID NO: 24), or multimers, derivatives and fragments thereof.

[0218] In specific embodiments of the first or second aspect of the present invention, the antigen-binding protein comprises a variable domain comprising a heavy chain corresponding to amino acids 1-123 according to SEQ ID NO: 2 and a light chain corresponding to amino acids 1-109 according to SEQ ID NO: 3 or variants having at least 80% identity with the sequence of Petition 870260062263, dated 06 / 25 / 2026, page 98 / 317 88 / 148 amino acids, and a peptide linker, in particular a peptide linker according to SEQ ID NO: 16.

[0219] In specific embodiments, the peptide linker is the position between the heavy chain and the light chain of the variable domain.

[0220] In specific embodiments of the first or second aspect of the present invention, the antigen-binding protein comprises or consists of scFv according to SEQ ID NO: 6.

[0221] In other preferred embodiments, one or more ligands comprise one or more cleavage sites, that is, one or more sequence areas where the ligand sequence can be chemically or enzymatically cleaved by the cleavage of one or more peptide bonds. Enzymatic cleavage can be achieved by proteolytic enzymes including, but not limited to, restriction endonucleases (e.g., type I, type II, type III, type IV, or artificial restriction enzymes) and endo- or exopeptidases or proteinases (e.g., serine proteinases, cysteine ​​proteinases, metalloproteinases, threonine proteinases, aspartate proteinases, glutamic acid proteinases).In particularly preferred embodiments, one or more cleavage sites comprise one or more endopeptidase cleavage sites, i.e., wherein the sequence is cleaved or cleavageable by an endopeptidase such as, but not limited to, trypsin, pepsin, elastase, thrombin, collagenase, furin, thermolysin, endopeptidase V8 and / or cathepsins.

[0222] In specific embodiments of the first or second aspect of the present invention, the antigen-binding protein further comprises one or more markers. In specific embodiments, the one or more markers are selected from the group consisting of affinity marker, solubilization marker, chromatography marker, epitope marker, and fluorescence marker. In specific embodiments, the marker is selected from FLAG-tag (SEQ Petition 870260062263, dated 06 / 25 / 2026, p. 99 / 317 89 / 148 ID NO: 25), His-tag (SEQ ID NO: 26) and Myc-tag (SEQ ID NO: 27).

[0223] In specific embodiments of the first or second aspect of the present invention, the antigen-binding protein further comprises a leader sequence. In specific embodiments, the leader sequence may be a PelB leader sequence (in particular according to SEQ ID NO: 28) for bacterial expression, an IgK leader sequence (in particular according to SEQ ID NO: 29) or an IL-2 leader sequence (SEQ ID NO: 30) for expression in mammalian cells.

[0224] In specific embodiments of the first or second aspect of the present invention, the antigen-binding protein comprises scFv according to SEQ ID NO: 6, a Myc marker (Myctag) and a His marker (His-tag). In specific embodiments of the first or second aspect of the present invention, the antigen-binding protein comprises or consists of the amino acid sequence 23-310 according to SEQ ID NO: 7.

[0225] In specific embodiments of the first or second aspect of the present invention, the antigen-binding protein comprises scFv according to SEQ ID NO: 6, a Myc marker (Myctag) and a His marker (His-tag), and a leader sequence, in particular a PelB, an IgGK, or IL-2 leader sequence. In specific embodiments, the antigen-binding protein comprises or consists of amino acids 1-310 according to SEQ ID NO: 7.

[0226] In examples of embodiments of the first or second aspect of the present invention, the antigen-binding protein is a bispecific antigen-binding protein directed against the conformational epitope formed by domains III and IV of HER3 and EGFR. In specific embodiments, the antigen-binding protein is a single-stranded diabody in which an antigen-binding site is Petition 870260062263, dated 06 / 25 / 2026, page 100 / 317 90 / 148 directed against the conformational epitope formed by domains III and IV of HER3 as described in detail above, and the second antigen-binding site is directed against EGFR. In specific embodiments, the second antigen-binding site is directed against EGFR and is derived from the EGFR-specific humanized antibody, hu225, i.e., the humanized version of C225 (cetuximab, Erbitux). In other embodiments, the antigen-binding protein is trifunctional and additionally comprises an Fc domain, in particular an Fc domain recognized by Fc gamma receptors, in particular CD16, CD32 and / or CD64. In specific embodiments, the antigen-binding protein comprises or consists of an amino acid sequence according to amino acids 23-738 of SEQ ID NO: 8. In other embodiments, the antigen-binding protein further comprises a leader sequence, in particular an IgK leader sequence.In specific embodiments, the antigen-binding protein comprises or consists of an amino acid sequence corresponding to amino acids 1-738 of SEQ ID NO: 8.

[0227] In examples of embodiments of the first or second aspect of the present invention, the antigen-binding protein is a bispecific antigen-binding protein directed against the conformational epitope formed by domains III and IV of HER3 and HER2. In specific embodiments, the antigen-binding protein is a single-chain diabody in which one antigen-binding site is directed against the conformational epitope formed by domains III and IV of HER3 as described in detail above, and the second antigen-binding site is directed against HER2. In specific embodiments, the second antigen-binding site is of the HER2-specific antibody 2-35. In other embodiments, the antigen-binding protein is trifunctional and further comprises an Fc domain, in particular a Petition 870260062263, dated 06 / 25 / 2026, page 101 / 317 91 / 148 Fc domain recognized by Fc gamma receptors, in particular CD16, CD32 and / or CD64. In specific embodiments, the antigen-binding protein comprises or consists of an amino acid sequence corresponding to amino acids 23-744 of SEQ ID NO: 9. In other embodiments, the antigen-binding protein further comprises a leader sequence, in particular an IgK leader sequence. In specific embodiments, the antigen-binding protein comprises or consists of an amino acid sequence corresponding to amino acids 1744 of SEQ ID NO: 9.

[0228] In examples of embodiments of the first or second aspect of the present invention, the antigen-binding protein comprises a bispecific antigen-binding protein directed against the conformational epitope formed by domains III and IV of HER3 and HER2. In specific embodiments, the antigen-binding protein is a single-chain diabody in which one antigen-binding site is directed against the conformational epitope formed by domains III and IV of HER3 as described in detail above, and the second antigen-binding site is directed against HER2. In specific embodiments, the second antigen-binding site is directed against HER2 and is derived from the HER2-specific 4D5 antibody (trastuzumab, Herceptin).In other embodiments, the antigen-binding protein is trifunctional and additionally comprises an Fc domain, in particular an Fc domain recognized by Fc gamma receptors, in particular CD16, CD32 and / or CD64. In specific embodiments, the antigen-binding protein comprises or consists of an amino acid sequence according to amino acids 23-477 of SEQ ID NO: 10. In other embodiments, the antigen-binding protein further comprises a leader sequence, in particular an IgK leader sequence. In specific embodiments, a. Petition 870260062263, dated 06 / 25 / 2026, page 102 / 317 92 / 148 antigen-binding protein comprises or consists of an amino acid sequence according to amino acids 1-477 of SEQ ID NO: 10.

[0229] In examples of embodiments of the first or second aspect of the present invention, the antigen-binding protein comprises an antigen-binding site directed against the conformational epitope formed by domains III and IV of HER3 and further comprises a single-stranded TRAIL domain (scTRAIL). In specific embodiments, the antigen-binding protein comprises an antigen-binding site against the conformational epitope formed by domains III and IV of HER3 as described above in detail. In specific embodiments, the antigen-binding protein further comprises scFv 3-43, in particular according to SEQ ID NO: 6. In specific embodiments, the antigen-binding protein further comprises a Flag-tag marker. In specific embodiments, the antigen-binding protein comprises or consists of an amino acid sequence according to amino acids 23-1020 of SEQ ID NO: 11.In other embodiments, the antigen-binding protein further comprises a leader sequence, in particular an IgK leader sequence. In specific embodiments, the antigen-binding protein comprises or consists of an amino acid sequence in accordance with amino acids 1-1020 of SEQ ID NO: 11.

[0230] In examples of embodiments of the first or second aspect of the present invention, the antigen-binding protein comprises a bispecific antigen-binding site directed against the conformational epitope formed by domains III and IV of HER3 and CD3. In specific embodiments, the antigen-binding protein is a single-stranded diabody in which one antigen-binding site is directed against the conformational epitope formed by domains III and IV of HER3 as described in detail above, and the second antigen-binding site is Petition 870260062263, dated 06 / 25 / 2026, page 103 / 317 93 / 148 directed against CD3. In specific embodiments, the second antigen-binding site is directed against CD3 and is derived from the humanized version of UCHT1 specific for CD3. In specific embodiments, the antigen-binding protein further comprises a His-tag marker. In specific embodiments, the antigen-binding protein comprises or consists of an amino acid sequence corresponding to amino acids 23-515 of SEQ ID NO: 12. In other embodiments, the antigen-binding protein further comprises a leader sequence, in particular an IgK leader sequence. In specific embodiments, the antigen-binding protein comprises or consists of an amino acid sequence corresponding to amino acids 1515 of SEQ ID NO: 12.

[0231] In specific embodiments, the antigen-binding protein further comprises scFv 3-43, in particular according to SEQ ID NO: 6. In specific embodiments, the antigen-binding protein comprises or consists of an amino acid sequence according to amino acids 23-776 of SEQ ID NO: 13. In other embodiments, the antigen-binding protein further comprises a leader sequence, in particular an IgK leader sequence. In specific embodiments, the antigen-binding protein comprises or consists of an amino acid sequence according to amino acids 1776 of SEQ ID NO: 13.

[0232] In specific embodiments, the antigen-binding protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 6, amino acids 23-310 of SEQ ID NO: 7, amino acids 23-738 of SEQ ID NO: 8 amino acids 23-724 of SEQ ID NO: 9, amino acids 23-744 of SEQ ID NO: 10, amino acids 23-1020 of SEQ ID NO: 11, amino acids 23-515 of SEQ ID NO: Petition 870260062263, dated 06 / 25 / 2026, p. 104 / 317 94 / 148 12, and amino acids 23-776 of SEQ ID NO: 13.

[0233] In specific embodiments, the antigen-binding protein comprises or consists of amino acid sequences selected from the group consisting of sequences SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13.

[0234] It will be appreciated by those skilled in the art that, in particular, the CDR sequences, variable and hypervariable regions can be modified without losing the ability to bind to HER3. For example, the CDR regions will be identical or highly homologous to the regions specified in the present invention. The term “highly homologous” includes that 1 to 5, preferably 1 to 4, as well as 1 to 3 or 1 or 2 substitutions, deletions or additions can be made to the CDRs. Additionally, the hypervariable and variable regions can be modified to exhibit substantial homology with the regions specifically disclosed in the present invention.

[0235] Furthermore, it may be desirable, according to the present invention, to modify the amino acid sequences described herein, in particular those of constant regions of the human heavy chain, to adapt the sequence to a desired allotype, for example, an allotype found in the Caucasian population.

[0236] The present invention further comprises antibodies in which alterations have been made to the Fc region to change the functional or pharmacokinetic properties of the antibodies. Such alterations may result in a decrease or increase in the binding of C1q and CDC or the binding of FcyR and ADCC. The substitutions may, for example, be made to one or more amino acid residues of the constant region of the heavy chain, thus causing a change in effector function while retaining the ability to bind to the antigen compared to the modified antibody, cf. US Patent Petition 870260062263, dated 06 / 25 / 2026, p. 105 / 317 95 / 148 5,624,821 and US Patent 5,648,260.

[0237] The in vivo half-life of antibodies can be improved by modifying the epitope of the salvage receptor of the Ig constant domain or of an Ig-like constant domain, so that the molecule does not comprise an intact CH2 domain or intact Ig Fc region, cf. U.S. Patent 6,121,022 and U.S. Patent 6,194,551. In addition, the in vivo half-life can be increased by introducing mutations in the Fc region, for example, by substituting threonine for leucine at position 252, by substituting threonine for serine at position 254, or by substituting threonine for phenylalanine at position 256, cf. U.S. Patent 6,277,375.

[0238] Furthermore, the glycosylation pattern of antibodies can be modified to alter the effector function of antibodies. For example, antibodies can be expressed in a transfectome that does not add the fucose unit normally bound to Asn at position 297 of the Fc region, in order to increase the affinity of the Fc region for Fc receptors, which in turn will result in an increased ADCC of the antibodies in the presence of NK cells, cf. Shield et al. (2002) JBC, 277: 26733. In addition, galactosylation modification can be done to modify CDC.

[0239] Consequently, in specific embodiments of the first or second aspect of the present invention, the variant exhibits at least 85% sequence identity with a given amino acid sequence. In specific embodiments, the variant exhibits at least 90%, 95%, or 98% sequence identity with the given amino acid sequence.

[0240] In a third aspect, the present invention provides a fusion protein comprising the antigen-binding protein according to the first and / or second aspect of the present invention as described in detail above and further comprising at least one portion Petition 870260062263, dated 06 / 25 / 2026, page 106 / 317 96 / 148 pharmaceutically active.

[0241] In specific embodiments, at least one pharmaceutically active portion is a pharmaceutical chemical or a biological substance. In embodiments where at least one pharmaceutically active portion is a biological product, it is preferred that such biological product be a peptide, polypeptide, protein and / or nucleic acid (e.g., DNA, RNA, or hybrids thereof). In specific embodiments, such biological product is selected from the group consisting of hormones (e.g., insulin, hGH, FSH, glucagon-like peptide 1, parathyroid hormone, calcitonin, lutropin, glucagon); growth factors (e.g., erythropoietin, thrombopoietin, G-CSF / GM-CSF, IGF1); cytokines (e.g., TNF, TRAIL, FasL, TGF-β), such as interferons (e.g., IFN-α, IFN-β, IFN-γ) and interleukins (e.g., IL-2, IL-11, IL1Ra); costimulatory and immunostimulatory ligands (e.g., 4-1BBL, CD40L, CD27L, OX40L, GITRL, LIGHT);Coagulation factors (e.g., factor VIII, factor IX, factor VIIa, thrombin); thrombolytics and anticoagulants (e.g., t-PA, hirudin, activated protein C); enzymes (e.g., α-glucosidase, glucocerebrosidase, iduronate-2-sulfatase, galactosidase, urate oxidase, DNase); antigen-binding molecules, such as antibodies and antibody fragments (e.g., IgG, Fab, Fc); and antibody fusion proteins (e.g., TNFR2-Fc, TMP-Fc, CTLA-4-Fc, IL-1R-Fc, LFA-3-Fc, IL-2-DT).

[0242] In specific embodiments, at least one pharmaceutically active moiety is selected from the group consisting of ligands, effector molecules, half-life extension modules and molecules for imaging.

[0243] In specific embodiments, ligands are any chemical or biological substances that form a complex with Petition 870260062263, dated 06 / 25 / 2026, page 107 / 317 97 / 148 another molecule to fulfill a specific biological function, such as substrates, inhibitors, and activators. In particular, ligands include, but are not limited to, antigen-binding molecules, scaffold proteins, natural ligands (e.g., EGF, VEGF, PDGF, FGF, EPO, TPO, TGF-β, TNF, TRAIL), ligand-binding receptor fragments (e.g., TNFR1, TNFR2, VEGFR, CTLA-4, LFA-3, BR3, CD95R, IL-1R, FGFR1), and apatamers (e.g., anti-thrombobin, anti-FIXa, anti-C3b, anti-VEGF, anti-CD40L). Scaffold proteins are regulators of key signaling pathways including, but not limited to, KSR, MEKK1, BCL-10, MAPK, AHNAK-1, HOMER, Pellino, NLRP, DLG1, Spinophilin, and Plant FLU regulatory protein.

[0244] In specific embodiments, the antigen-binding molecule is selected from the group consisting of an antibody fragment, a Fab fragment (excluding those of IgM or IgE), a Fab' fragment (excluding those of IgM or IgE), a heavy chain antibody, a single-domain antibody (sdAb), a variable domain of a heavy chain antibody, VHH, nanobodies, a single-chain variable fragment (scFv), a tandem scFv, a bispecific T cell promoter (BITEs), a diabody, a single diabody, a DART molecule, a triple antibody, a nanoantibody, an alternative scaffold protein (e.g., DARPins, Anticalins, Affibody molecules, Microbodies, Monobodies, Fynomers, Adnetins, Tetranectins, Kunitz domains, Afilins, Avimers), and a fusion protein thereof.It is preferable that the antigen-binding molecule binds to an antigen that is pharmaceutically relevant, that is, suitable for preventing, diagnosing, and / or treating a disease or the symptoms of a disease or disorder. In a preferred embodiment, the disease is a cancer-type disease. Preferably, the antigen-binding molecule recognizes a... Petition 870260062263, dated 06 / 25 / 2026, p. 108 / 317 98 / 148 tumor-associated antigen such as, but not limited to, EGFR, HER2, HER4, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), CA-125, epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE) and abnormal products of ras and p53, estrogen receptors, 5-alpha reductase, prostaglandin endoperoxide synthase 2, VEGFRs, integrin receptor family, fibroblast activating protein, galectin, EpCAM, CEA, CD44, CD44v, CD2, CD5, CD7, CD19, CD20, CD21, CD22, CD24, CD25, CD30, CD33, CD38, CD40, CD52, CD56, CD71, CD72, CD73, CD105, CD117, CD123, claudins, c-Met, PDGFR, IGF1-R, HMW-MAA, TAG72, GD2, GD3, GM2, folate receptor, Ley, MUC-1, MUC-2, PSMA, PSCA, and uPAR. In preferred embodiments, the antigen-binding molecule is considered to be neither a Fab nor an Fc fragment of IgM or IgE.

[0245] In specific embodiments, the antigen-binding molecule is an scFv, preferably an anti-HER2 scFv or an anti-EGFR scFv.

[0246] In specific embodiments, effector molecules, i.e., small molecules, peptides or polypeptides that bind to a protein and thus alter the activity of that protein, include but are not limited to cytokines, chemokines, immunostimulatory molecules, immunosuppressive molecules, death ligands, apoptosis-inducing proteins, enzymes (e.g., kinases), prodrug-converting enzymes, RNases, antibody or antibody fragment agonist, antibody or antibody fragment antagonist, toxins, growth factors, hormones, coagulation factors, fibrinolytic proteins, peptides mimicking these and fragments, fusion proteins or derivatives thereof.

[0247] In specific realization examples, cytokines are interleukins and / or interferons. Interleukins (IL) include, but are not limited to Petition 870260062263, dated 06 / 25 / 2026, page 109 / 317 99 / 148 a, Interleukin-1, Interleukin-2, Interleukin-3, Interleukin-4, Interleukin-5, Interleukin-6, Interleukin-7, Interleukin-8, Interleukin-9, Interleukin-10, Interleukin-11, Interleukin-12, Interleukin-13, Interleukin-14, Interleukin-15, Interleukin-16, Interleukin-17, Interleukin-18, Interleukin-19, Interleukin-20, Interleukin-21, Interleukin-22, Interleukin-23, Interleukin-24, Interleukin-25, Interleukin-26, Interleukin-27, Interleukin-28 Interleukin-29, Interleukin-30, Interleukin-31, Interleukin-32, Interleukin-33, Interleukin-34, and Interleukin-35. Interferons (IFNs) include, but are not limited to, type I interferons (e.g., IFN-α, IFN-β, and IFN-ω), type II interferons (e.g., IFN-γ), and type III interferons. In particular, Interferon A1, Interferon A2, Interferon A4, Interferon A5, Interferon A6, Interferon A7, Interferon A8, Interferon A10, Interferon A13, Interferon A14, Interferon A16, Interferon A17, Interferon A21, Interferon B1, TNF, TRAIL, and FasL are included.

[0248] In specific embodiments, chemokines include, but are not limited to, CC chemokines, CXC chemokines, C chemokines, and CX3C chemokines. In particular, the chemokine includes, but is not limited to, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL9, CCL11, CCL11, CCL12, CCL13, CCL16, CCL16, CCL16, CCL16, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL24, CCL24, CCL24, CCL24, CCL24, CCL27, CCL27, CCL27, CCL27, CXCL3, CXCL3, CXCL3, CXCL5, CXCL5, CXCL5, CXCL7, CXCL9, CXCL16, CXCL16, CXCL16, CXCL16, XCL2 and CX3CL1.

[0249] In specific embodiments, immune-(co)stimulatory proteins include but are not limited to B7.1, B7.2, 4-1BBL, LIGHT, ICOSL, GITRL, CD40L, OX40L and CD70.

[0250] Immunosuppressive proteins can be selected from the group consisting of IL1-Ra, IL-10, CTLA-4, PD-L1, and PD-L2. The Petition 870260062263, dated 06 / 25 / 2026, page 110 / 317 100 / 148 toxins can be selected from the group consisting of Pseudomonas exotoxin A, diphtheria toxin, and ricin.

[0251] In specific embodiments, apoptosis-inducing proteins may be selected from the group consisting of Bid, Bik, Puma, and Bim, and pro-apoptotic cytokines (death ligands) such as, but not limited to, TNF, scTNF, TRAIL, scTRAIL, and FasL. In specific embodiments, the cytokine is TNF. In other embodiments, the cytokine is TRAIL or scTRAIL.

[0252] In specific embodiments, enzymes may be selected from the group consisting of oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases. Kinases include, but are not limited to, AGC kinases such as PKA, PKC, and PKG; CaM kinases such as calcium / calmodulin-dependent protein kinases and serine / threonine protein kinases (e.g., DAPK2); CK1 kinases such as casein group kinase 1; CMGC kinases such as CDK, MAPK, GSK3, and CLK; STE kinases such as yeast Sterile 7, Sterile 11, and Sterile 20; tyrosine kinases (TK); tyrosine kinase-like kinases (TKL); receptor-associated tyrosine kinases; MAP kinases; and histidine kinases.

[0253] Prodrug converting enzymes may be selected from the group consisting of esterases such as, but not limited to, acetylesterase, thiolester hydrolases, monoester phosphoric hydrolases, diester phosphoric hydrolases, monoester triphosphoric hydrolases, sulfuric ester hydrolases (sulfatases), monoester diphosphoric hydrolases and triester phosphoric hydrolases; phosphatases, such as, but not limited to, tyrosine-specific phosphatases, serine / threonine-specific phosphatases, dual-specificity phosphatases, histidine phosphatase and lipid phosphatase; and reductases such as, but not limited to, 5-alpha reductase, dihydrofolate reductase, HMG-CoA reductase, methemoglobin reductase, ribonucleotide reductase, thioredoxin Petition 870260062263, dated 06 / 25 / 2026, page 111 / 317 101 / 148 reductase, E. coli nitroreductase, methylenetetrahydrofolate reductase and carboxypeptidase G2, cytosine deaminase, nitroreductase, thymidine kinase.

[0254] RNases include endoribonucleases, in particular those selected from the group consisting of RNase A, RNase H, RNase I, RNase III, RNase L, RNase P, RNase PhyM, RNase T1, RNase T2, RNase U2, RNase V1 and RNase V, and exoribonucleases, such as, but not limited to, Polynucleotide Phosphorylase (PNPase), RNAase PH, RNAase II, RNAase R, RNAase D, RNAase T, Exceberonuclease Oloribonuclease I and Exoribonuclease II.

[0255] Agonist antibodies or antibody fragments include those that cause an action in a tissue, organ, or individual, such as, but not limited to, receptor signaling, gene expression, protein synthesis, and protein degradation, for example, directed against TRAIL receptors, antiglucocorticoid-induced tumor necrosis factor (GITR) family receptor, and CD40. The agonist antibody or antibody fragment acts by binding to the active site or allosteric sites of a receptor molecule, triggering a specific reaction.

[0256] Antagonist antibodies or antibody fragments include those that block the action of an agonist. Typically, antagonist antibodies or antibody fragments act by binding to the active site or allosteric sites of a receptor molecule, or interact with unique binding sites not normally involved in regulating receptor activity, for example, anti-CTLA-4, anti-TNFR1, anti-VEGFR, anti-PDGFR, anti-EGFR, anti-Her2. Typically, an antagonist antibody or antibody fragment competes with the agonist for structurally defined binding sites or alters the agonist's binding site in a way that the agonist is unable to cause the action it would normally cause due to its binding. Petition 870260062263, dated 06 / 25 / 2026, p. 112 / 317 102 / 148

[0257] In particular, growth factors may be selected from the group consisting of Adrenomedullin (AM), Angiopoietin (Ang), autocrine motility factor, bone morphogenetic proteins (BMPs), brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), Erythropoietin (EPO), fibroblast growth factor (FGF), glial cell lineage-derived neurotrophic factor (GDNF), granulocyte colony-stimulating factor (G-CSF), macrophage-granulocyte colony-stimulating factor (GM-CSF), growth differentiation factor-9 (GDF9), hepatocyte growth factor (HGF), hepatoma-derived growth factor (HDGF), insulin-like growth factor (IGF), myostatin migration-stimulating factor (GDF-8), nerve growth factor (NGF) and other neurotrophins, growth factor derived from platelets (PDGF), thrombopoietin (TPO), transforming growth factor-alpha (TGF-α),Transforming growth factor-beta (TGF-β), vascular endothelial growth factor (VEGF), Wnt signaling pathway, and placental growth factor (PlGF).

[0258] In specific embodiments, coagulation factors may be selected from the group consisting of Thrombin, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII and Factor XIII, and active fragments thereof.

[0259] In specific embodiments, fibrinolytic proteins may be selected from the group consisting of plasmin, urokinase, plasminogen, antiplasmin-2, tissue plasminogen activator (t-PA), and plasminogen activator inhibitor-1 (PAI-1).

[0260] Mimetic peptides and mimetic proteins include peptides and proteins that mimic the activities of other peptides or proteins, in particular peptides or proteins mentioned in the present invention above or below, in particular mimetic peptides of Petition 870260062263, dated 06 / 25 / 2026, page 113 / 317 103 / 148 thrombopoietin, erythropoietin mimetic peptides.

[0261] In other embodiments, half-life extension modules are chemical or biological substances that alter the half-life, for example, the “plasma half-life” or the “serum half-life” of the polypeptide of the present invention. In particular, the half-life extension module is selected from the group consisting of immunoglobulin-binding domains (IgBD), albumin, albumin-binding domains (ABD), peptides, small molecules, fatty acids, antibody fragments, single-domain antibodies, VHH, scaffold proteins and natural ligands exhibiting affinity for a long-circulating plasma protein, any of which are optionally PEGylated, HEsylated, Polysialylated, N-glycosylated, O-glycosylated or PEG-mimicking polypeptides.Preferably, an IgBD can bind to any of the domains of an Ig molecule, namely the variable VH or VL domains and / or the constant CH1, CH2, CH3CH4 and / or CL domains of an Ig molecule. IGBDs include, but are not limited to, domains derived from Staphylococcus aureus protein A (SpA), streptococcal protein G (SpG), Peptococcus magnus protein L (PpL), Escherichia coli protein Eib, Staphylococcus protein Sbi, and streptococcal proteins MAG, MIG, H, M, and ZAG.

[0262] In further embodiments, imaging molecules are those that bind to specific target molecules, thus allowing visualization of the location of that molecule. In particular, the imaging molecule is selected from the group consisting of bioluminescent reagents, chemiluminescent reagents, fluorescent imaging reagents, photosensitizers, chelating reagents, and radioactive moieties.

[0263] The imaging molecule includes bioluminescent, chemiluminescent and fluorescent imaging reagents, such as, but not limited to, luciferase from Renilla reniformis and / or Metridia Longa, peroxalate, Petition 870260062263, dated 06 / 25 / 2026, page 114 / 317 104 / 148 polymethines (e.g., cyanine dyes such as Cy3, Cy5, Cy5.5, Cy7), squarain derivatives, phthalocyanine, porphyrin derivatives, and BODIPY analogs (BODIPY FL, BODIPY R6G, BODIPY TR, BODIPY TMR, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665), as well as fluorescent proteins such as, but not limited to, GFP, EGPF, CFP, BFP, YFP, DsRED (Chudakov et al. (2010) Physiol. Rev. 90: 1103-1163).

[0264] Chelating agents are capable of binding at least one metal ion, such as, but not limited to, calcium, magnesium, iron, aluminum, zinc, copper, arsenic, lead, thallium, and mercury ions, by chelation. These chelating agents may include ethylenediaminetetraacetic acid (EDTA), ethylenediaminetetraacetic acid (disodium calcium versicolor) (CaNa2-EDTA), dimercaprol (BAL), dimercaptosuccinic acid (DMSA), dimercaptopropane sulfonate (DMPS), ferritin, deferoxamine and deferasirox, deferiprone (1,2-dimethyl-3-hydroxyl-4-pyridinone), DOTA, DTPA, DADT, DADS, DO3A, N2S2MAMA, triamidothiol, phosphonates, gadolinium organic complexes, penicillamine, and tetracycline family antibiotic drugs.

[0265] In specific embodiments, the radioactive portion comprises a radionuclide. The radioactive portion may be an isotope of F, Br, Mn, Co, Ga, As, Zr, P, C, S, H, I, In, Lu, Cu, Rh, Bi, At, Y, Re, Ac, Tc, or Hg. The radioactive portion labels the polypeptide of the present invention allowing its detection in a radioactive manner, for example, in the human body, making it not only useful for diagnostic approaches (radioimmunodetection: RAID), but also suitable in therapeutic applications (radioimmunotherapy: RAIT).

[0266] Photosensitizers are chemical compounds capable of emitting light or forming free radicals and singlet oxygen after being excited by light of a specific wavelength. Petition 870260062263, dated 06 / 25 / 2026, pp. 115 / 317 105 / 148 Photosensitizers are used, for example, for photodynamic therapy. In preferred embodiments, photosensitizers include, but are not limited to, compounds from the porphyrin family, texafirin family, chlorine family, and phthalocyanine family, in particular including HpD, ALA, M-ALA, Vertiporphine, Lutexafirin, Temoporphine, Talaporphine, HPPH, Phthalocyanine, and Naphthalocyanine.

[0267] In a fourth aspect, the present invention provides a nucleic acid molecule comprising a sequence encoding the antigen-binding protein of the first or second aspect of the present invention and / or the fusion protein of the third aspect of the present invention. In specific embodiments, this nucleic acid molecule comprises a DNA and / or RNA molecule.

[0268] In a fifth aspect, the present invention provides a vector comprising the nucleic acid molecule of the fourth aspect of the present invention. In specific embodiments, the vector is selected from the group consisting of plasmids, cosmids, phages, viruses and / or artificial chromosomes.

[0269] In a sixth aspect, the present invention provides a recombinant host cell comprising the antigen-binding protein of the first or second aspect of the present invention, the fusion protein of the third aspect of the present invention, the nucleic acid of the fourth aspect of the present invention, and / or a vector of the fifth aspect of the present invention. In specific embodiments, the host cell is a HEK293, CHO, BHK, or PerC6 cell.

[0270] In a seventh aspect, the present invention provides a pharmaceutical composition comprising the antigen-binding protein of the first or second aspect of the present invention, the fusion protein of the third aspect of the present invention, the nucleic acid of the fourth aspect of Petition 870260062263, dated 06 / 25 / 2026, pp. 116 / 317 106 / 148 present invention and / or a vector of the fifth aspect of the present invention, and further comprising one or more vehicles or carriers, diluents, excipients, fillers, binders, lubricants, glidants, disintegrants, adsorbents and / or pharmaceutically acceptable preservatives.

[0271] In specific embodiments, the composition of the seventh aspect contains a therapeutically effective amount of the active ingredient, namely the antigen-binding protein of the first or second aspect of the present invention, the fusion protein of the third aspect of the present invention, the nucleic acid of the fourth aspect of the present invention and / or a vector of the fifth aspect of the present invention, preferably in purified form, together with an adequate amount of vehicle or carrier and / or excipient, so as to provide the appropriate administration form for the patient. The formulation must be suitable for the administration method.

[0272] Pharmaceutical compositions can take the form of solutions, suspensions, emulsions, tablets, coated tablets, capsules, powders, controlled-release formulations, etc. The pharmaceutical composition can be formulated as a suppository, with traditional binders and vehicles, such as triglycerides.

[0273] To prepare pharmaceutical compositions of the present invention, the pharmaceutically acceptable vehicles can be solid or liquid. Compositions in solid form include powder, tablets, pills, capsules, lozenges, pouches, suppositories, and dispersible granules. A solid excipient can be one or more substances, which can also act as a diluent, flavoring agent, binder, preservative, tablet disintegrant, or encapsulation material. In the case of the powder presentation, the excipient is preferably a finely divided solid, which is mixed with the finely divided inhibitor of the present invention. Petition 870260062263, dated 06 / 25 / 2026, pp. 117 / 317 107 / 148 invention. In tablets, the active ingredient is mixed with a vehicle possessing the necessary binding properties in suitable proportions and compacted into the desired shape and size. Suitable excipients are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low melting point wax, cocoa butter and the like. To prepare suppositories, a low melting point wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted and the active component is homogeneously dispersed in it, such as by stirring. The melted homogeneous mixture is then poured into molds of convenient size, allowed to cool and thus solidify. Tablets, powders, capsules, pills, lozenges and pastilles can be used as suitable solid dosage forms for oral administration.

[0274] Liquid compositions include solutions, suspensions and emulsions, for example, water, saline solutions, aqueous dextrose, glycerol solutions or water / propylene glycol solutions. For parenteral injections (e.g., intravenous, intra-arterial, intraosseous, intramuscular, subcutaneous, intraperitoneal, intradermal and intrathecal injections), liquid preparations may be formulated in solution, for example, in aqueous polyethylene glycol solution. A saline solution is a preferred vehicle when the pharmaceutical composition is administered intravenously.

[0275] In specific embodiments, the pharmaceutical composition is in unit dosage form. In such form, the composition can be subdivided into unit doses containing appropriate amounts of the active component. The unit dosage form may be a packaged composition, wherein the packaging contains discrete amounts of the composition, such as packaged tablets, capsules, and powder in vials or ampoules. Also, the unit dosage form may be a capsule, a Petition 870260062263, dated 06 / 25 / 2026, pp. 118 / 317 108 / 148 vial of injection, one tablet, one packet or lozenge, or it may be the appropriate number of any of these in packaged form.

[0276] The composition may also contain smaller amounts of humectants or emulsifying agents, or pH buffering agents, if desired.

[0277] In addition, such a pharmaceutical composition may also comprise another pharmacologically active substance, such as, but not limited to, adjuvants and / or additional active ingredients. Adjuvants in the context of the present invention include, but are not limited to, inorganic adjuvants, organic adjuvants, oil-based adjuvants, cytokines, particulate adjuvants, virosomes, bacterial adjuvants, synthetic adjuvants, or synthetic polynucleotide adjuvants.

[0278] In an eighth aspect, the present invention provides the antigen-binding protein of the first or second aspect of the present invention, the fusion protein of the third aspect of the present invention, the nucleic acid of the fourth aspect of the present invention, the vector of the fifth aspect of the present invention, or the pharmaceutical composition of the seventh aspect of the present invention, for use in medicaments. In specific embodiments, use in medicaments is use in the prophylaxis, treatment, or diagnosis of a disorder or disease, in particular in the prophylaxis, treatment, or diagnosis of proliferative disorders or diseases.

[0279] In specific embodiments, the antigen-binding protein of the first or second aspect of the present invention, the fusion protein of the third aspect of the present invention, the nucleic acid of the fourth aspect of the present invention, the vector of the fifth aspect of the present invention, or the pharmaceutical composition of the seventh aspect of the present invention is for use in inhibiting tumor growth or treating cancer. Petition 870260062263, dated 06 / 25 / 2026, pp. 119 / 317 109 / 148

[0280] Proliferative disorders or disturbances include, but are not limited to, basal cell carcinoma, bladder cancer, bone cancer, brain tumor, breast cancer, Burkitt lymphoma, cervical cancer, colon cancer, cutaneous T-cell lymphoma, esophageal cancer, retinoblastoma, gastric (stomach) cancer, gastrointestinal stromal tumor, glioma, Hodgkin lymphoma, Kaposi's sarcoma, leukemias, lymphomas, melanoma, oropharyngeal cancer, ovarian cancer, pancreatic cancer, pleuropulmonary blastoma, prostate cancer, throat cancer, thyroid cancer, and urethral cancer.

[0281] In a ninth aspect, the present invention provides a method of inhibiting tumor growth or treating cancer, comprising administering a therapeutically effective amount of the antigen-binding protein of the first or second aspect of the present invention, the fusion protein of the third aspect of the present invention, the nucleic acid of the fourth aspect of the present invention, the vector of the fifth aspect of the present invention, or the pharmaceutical composition of the seventh aspect of the present invention to a patient with such a need.

[0282] In the practice of any aspect of the present invention, a pharmaceutical composition as described above or a binding moiety (for example, an antibody or antigen-binding fragment thereof) may be administered to a patient by any route established in the art that provides a sufficient level of the binding moiety to the patient. It may be administered systemically or locally. Such administration may be parenteral, transmucosal, for example, oral, nasal, rectal, intravaginal, sublingual, submucosal, transdermal, or by inhalation. Preferably, administration is parenteral, for example, by intravenous or intraperitoneal injection, and also including, but not limited to, intra-arterial, intramuscular, intradermal, and subcutaneous administration. If the pharmaceutical composition of the present invention is administered locally, it may be Petition 870260062263, dated 06 / 25 / 2026, page 120 / 317 110 / 148 injected directly into the organ or tissue to be treated, for example, the organ affected by a tumor.

[0283] Pharmaceutical compositions adapted for oral administration may be supplied as capsules or tablets; as powder or granules; as solutions, syrups or suspensions (in aqueous or non-aqueous liquids); as edible foams or creams; or as emulsions. Hard gelatin tablets or capsules may comprise lactose, starch or its derivatives, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, stearic acid or its salts. Soft gelatin capsules may comprise vegetable oils, waxes, fats, semi-solid or liquid polyols, etc. Solutions and syrups may comprise water, polyols and sugars.

[0284] An active agent intended for oral administration may be coated or mixed with a material that delays the disintegration and / or absorption of the active agent in the gastrointestinal tract (for example, glyceryl monostearate or glyceryl distearate may be used). Thus, prolonged release of an active agent can be achieved over many hours and, if necessary, the active agent can be protected from being degraded in the stomach. Pharmaceutical compositions for oral administration may be formulated to facilitate the release of an active agent at a particular gastrointestinal location due to specific pH or enzymatic conditions.

[0285] Pharmaceutical compositions adapted for transdermal administration may be supplied as discreet patches intended to remain in intimate contact with the recipient's epidermis for an extended period of time. Pharmaceutical compositions adapted for topical administration may be supplied as ointments; creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, Petition 870260062263, dated 06 / 25 / 2026, pp. 121 / 317 111 / 148 aerosols or oils. For topical administration to the skin, mouth, eyes, or other external tissues, a topical ointment or cream is preferably used. When formulated as an ointment, the active ingredient can be used with any paraffinic base or water-miscible ointment. Alternatively, the active ingredient can be formulated in an oil-in-water or water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops. In these compositions, the active ingredient can be dissolved or suspended in a suitable vehicle, for example, an aqueous solvent. Pharmaceutical compositions adapted for topical administration to the mouth include lozenges and mouthwashes.

[0286] Pharmaceutical compositions adapted for nasal administration may comprise solid vehicles, such as powder (preferably with a particle size in the range of 20 to 500 micrometers). The powder may be administered by inhalation and ingestion, i.e., by rapid inhalation through the nose from a powder container held close to the nose. Alternatively, compositions adapted for nasal administration may comprise liquid vehicles, for example, nasal sprays or nasal drops. These compositions may comprise aqueous or oily solutions of the active ingredient. Compositions for inhalation administration may be supplied in specially adapted devices including, but not limited to, pressurized aerosols, nebulizers or insufflators, which may be constructed to provide predetermined dosages of the active ingredient.In a preferred embodiment, the pharmaceutical compositions of the invention are administered through the nasal cavity to the lungs.

[0287] Pharmaceutical compositions adapted for rectal administration may be supplied as suppositories or enemas. Pharmaceutical compositions adapted for vaginal administration may be Petition 870260062263, dated 06 / 25 / 2026, page 122 / 317 112 / 148 supplied as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

[0288] Pharmaceutical compositions adapted for parenteral administration include sterile aqueous and non-aqueous injectable solutions or suspensions, which may contain antioxidants, buffers, bacteriostatics, and solutes that make the compositions substantially isotonic with the blood of an intended recipient. Other components that may be present in such compositions include water, alcohols, polyols, glycerin, and vegetable oils, for example. Compositions adapted for parenteral administration may be presented in single-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a lyophilized condition requiring only the addition of a sterile liquid vehicle, for example, sterile saline solution for injections, immediately before use. Solutions and suspensions for extemporaneous injection may be prepared from sterile powders, granules, and tablets.

[0289] In a preferred embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile aqueous isotonic buffer. When necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to relieve pain at the injection site. Generally, the ingredients are supplied separately or mixed in unit dosage form, for example, as a lyophilized powder or a waterless concentrate in a hermetically sealed container, such as an ampoule or sachet indicating the amount of active agent. When the composition is to be administered by infusion, it may be dispensed with an infusion bottle containing grade 2 water. Petition 870260062263, dated 06 / 25 / 2026, page 123 / 317 113 / 148 pharmaceutical or sterile saline. When the composition is administered by injection, an ampoule of sterile water for injection or sterile saline solution may be provided so that the ingredients can be mixed before administration.

[0290] In another embodiment, for example, a chemoattractant inhibitor may be administered in a controlled-release system. For example, the inhibitor may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump can be used (see Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14: 201; Buchwald et al. (1980) Surgery 88:507; Saudek et al. (1989) N. Eng. J. Med. 321: 574). In another embodiment, the compound can be administered into a vesicle, in particular a liposome (see Langer (1990) Science 249:1527-1533; Treat et al. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, NY, 353365; WO document 91 / 04014; US Patent 4,704,355).In another example of embodiment, polymeric materials can be used (see Controlled Release Medical Applications (1974) Langer and Wise (eds.), CRC Press: Boca Raton, Fla.; Controlled Drug Bioavailability, Drug Product Design and Performance, (1984) Smolen and Ball (eds.), Wiley: NY; Ranger and Peppas (1953) J. Macromol. Sci. Rev. Macromol. Chem. 23: 61; see also Levy et al.

[0291] In another embodiment, a controlled-release system can be placed in close proximity to the therapeutic target, i.e., target cells, tissue or organ, thus requiring only a fraction of the systemic dose (see, for example, Goodson (1984) 115-138) in Medical Controlled Release Applications, vol. 2). Other release systems Petition 870260062263, dated 06 / 25 / 2026, p. 124 / 317 114 / 148 controlled are discussed in Langer's review (1990, Science 249: 1527-1533).

[0292] In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area requiring treatment; this may be achieved, for example, but not limited to, local infusion during surgery, topical application, for example, in conjunction with a dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous or gelatinous material, including membranes, such as silastic membranes, or fibers.

[0293] The selection of the preferred effective dose will be determined by a person skilled in the art, based on consideration of several factors known to those skilled in the art. Such factors include the particular form of the pharmaceutical composition, for example, polypeptide or vector, and its pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc., which will be established during the usual development procedures typically used in obtaining regulatory approval for a pharmaceutical compound. Other factors in dose consideration include the condition or disease to be prevented and / or treated or the benefit to be achieved in a normal individual, the patient's body mass, the route of administration, whether administration is acute or chronic, concomitant medications, and other factors well known to affect the efficacy of the administered pharmaceutical agents.Therefore, the precise dosage should be decided according to the professional's judgment and the circumstances of each patient, for example, depending on the individual patient's condition and immune status, and in accordance with standard clinical techniques.

[0294] The following examples are merely illustrative of Petition 870260062263, dated 06 / 25 / 2026, page 125 / 317 115 / 148 present invention and should not be interpreted in any way as limiting the scope of the invention as indicated by the appended claims. Examples Example 1 Antibody 3-43 Epitope Binding and Specificity

[0295] A fully human IgG1 molecule (IgG 3-43) comprising the 3-43 variable domain sequence optimized for eukaryotic expression was cloned and expressed in HEK 293-6E cells adapted for suspension culture. The protein was purified from the supernatant of transiently transfected cells by protein A affinity chromatography. SDS-PAGE analysis and size exclusion chromatography confirmed the integrity of the protein. SDS-PAGE analysis of purified IgG 3-43 showed a single band under non-reducing conditions with an intact IgG molecular mass (approximately 150 kDa) and two bands under non-reducing conditions corresponding to the heavy chain (50 kDa) and the light chain (25 kDa) (Fig. 1A). Size exclusion chromatography confirmed the purity of IgG 3-43 (Fig. 1B). Binding to IgG 3-43 antigen was analyzed by ELISA using immobilized HER3-Fc fusion comprising the extracellular domain (aa 27-599) of human HER3.The HER3-Fc fusion protein was coated onto polystyrene microtiter plates at 3 μ / mL in PBS. The remaining binding sites were blocked with PBS, 2% skim milk (MPBS). The plates were then incubated with a serial dilution of IgG 3-43 in MPBS. After washing, the bound antibody was detected with an HRP- and TMB-conjugated anti-human Fc antibody, H2O2 as substrate. IgG 3-43 showed specific, concentration-dependent binding to HER3 with an EC50 value in the subnanomolar range (0.4 ± 0.2 nM) (Fig. 1C). The affinity of IgG 3-43... Petition 870260062263, dated 06 / 25 / 2026, page 126 / 317 The 116 / 148 concentration for the monomeric HER3 receptor was determined by quartz crystal microbalance measurements using an Attana 200 instrument. IgG 343 was immobilized on the surface of a non-specific low-binding carboxyl-linked chip by amine coupling, at a density that resulted in a frequency shift of approximately 90 Hz. The measurement was performed at 25°C with a flow rate of 25 pL / min of PBST (0.1% Tween) pH 7.4. Binding regeneration was performed twice with 3M MgCl2 for 15 sec. After each second measurement, a buffer injection was performed to determine the baseline, subsequently subtracted from neighboring measurements. Soluble His-labeled HER3 was injected in serial two-fold dilutions in PBST in random order, with concentrations between 2.5 and 20 nM (Fig. 1D). A Kd value of 11 nM was determined (Table 2). Example 2 Epitope Mapping and Cross-Reactivity of Antibody 3-43 AntiHER3

[0296] To locate the antibody epitope, full-length (aa 20-643) and truncated forms of the human HER3 extracellular domain (DII-DIV aa 208-643, DIII-DIV aa 329-643, DIV aa 532-643) were cloned and produced as Fc fusion proteins in transfected HEK293 cells. An SDS-PAGE of the fusion proteins purified by protein chromatography A, under non-reducing and reducing conditions, confirmed the correct size and dimeric assembly of the fusion proteins. The binding capacity of IgG 3-43 to different HER3 fusion proteins deleted in the domain was evaluated in ELISA and immunoblotting assays under non-reducing conditions (summarized in Fig. 2A). For ELISA, HER3-Fc fusion proteins were coated onto polystyrene microtiter plates at 10 pg / mL in PBS. The remaining binding sites were blocked with PBS, 2% skim milk (MPBS). The plates were then incubated with a Petition 870260062263, dated 06 / 25 / 2026, page 127 / 317 117 / 148 serial dilution of IgG 3-43 in MPBS. After washing, bound antibody was detected with an HRP- and TMB-conjugated human anti-Fab antibody, H2O2 as substrate. Binding was detected for the full-length HER3Fc fusion protein (aa 20-643) as well as the Fc DII-DIV (aa 208-643) and DIII-DIV (aa 329-643) fusion proteins, but not with DIV-Fc (aa 532-643). This result indicates that the IgG 3-43 epitope resides in domain III of HER3. Fragments comprising part of DIII and the entire DIV (aa 359-643, 395-643, aa 458-643) did not show binding, indicating that the complete DIII domain is required for antibody binding. Surprisingly, no binding was observed when testing an Fc fusion protein containing DI-DIII (aa 20-531; no DIV) or DI-DII and a small portion of DIII (aa 20-358). This finding indicates that DIV is also required for antibody binding.Test fragments comprising DI-III plus parts of DIV (aa 20-587, 20-550) showed binding from 3-43 to aa 20-587, but not to aa 20-550, indicating that the epitope resides in and at least requires aa 328587. This was confirmed using a fragment composed of aa 329-587, which showed binding in ELISA. In contrast, fragments composed of aa 359587 or aa 329-550 did not show binding, thus confirming that the epitope resides in and requires aa 329-587.

[0297] IgG 3-43 was unable to detect denatured and reduced HER3-Fc fusion proteins in immunoblotting experiments, while binding was observed with denatured but not reduced fragments, indicating an epitope for IgG 3-43 sensitive to reduction, i.e., stabilized by disulfide bonds. Furthermore, we analyzed binding to human and mouse HER3-Fc fusion proteins by ELISA (Figure 2B). Binding to both HER3-Fc fusion proteins was detected, demonstrating that IgG 3-43 exhibits cross-reactivity with HER3. Petition 870260062263, dated 06 / 25 / 2026, p. 128 / 317 118 / 148 thus, the IgG 3-43 epitope is conserved in these two species. Example 3 Binding of Anti-HER3 Antibody 3-43 to HER3-Expressing Tumor Cell Lines

[0298] Flow cytometry studies were performed with MCF-7, FaDu, BT474, A431, NCI-N87, and A549 cells expressing HER3 (Fig. 3). Cells were rapidly trypsinized at 37°C. Trypsin was quenched with SBF-containing medium and removed by centrifugation, and cells were seeded at 200,000 cells per probe. Cells were then incubated with varying concentrations of IgG 3-43 for at least one hour at 4°C. Washing was performed twice with PBA (2% (v / v) SBF, 0.02% (w / v) NaN3 in 1x PBS). Mouse PE-labeled anti-Fc human antibody was incubated for a further hour to visualize bound antibody molecules. After two more washing steps, fluorescence was measured using a MACSQuant® Analyzer 10 device, and the median fluorescence intensities relative to unstained cells were calculated using the FlowJo program.These experiments demonstrated the binding of IgG 3-43 to the cellular receptor with surprisingly low EC50 values ​​in a range between 26 and 74 pM. Example 4 IgG 3-43 inhibits the binding of the heregulin ligand.

[0299] Human HRG-βI labeled with recombinant His was incubated with MCF-7 cells and the bound protein was detected via PE-conjugated anti-His antibody. Pre-incubation with excessive amounts of IgG 3-43 (3 μM) strongly reduced the fluorescence intensity of the cells, indicating blockage of HRG binding, while pre-incubation with Cetuximab as a negative control did not have the same effect. Petition 870260062263, dated 06 / 25 / 2026, page 129 / 317 119 / 148 (Figure 4). Example 5 Heregulin-Induced Inhibition of HER3 Phosphorylation and Signal Transduction by Anti-HER3 Antibody 3-43

[0300] IgG 3-43 was further analyzed for its ability to prevent HRG-induced HER3 phosphorylation. Semiconfluent cells were incubated with IgG 3-43 for one hour, followed by 15 minutes of stimulation with HRG (50 ng / ml). Western blot analyses of cell lysates revealed efficient blockade of HER3 phosphorylation, as well as repression of HRG-induced Erk and Akt phosphorylation in different cell lines (MCF-7, BT-474, NCI-N87, A431, A549, FaDu) (Figure 5). Titration of IgG 3-43 also revealed an IC50 value in the lower picomolar range for the blockade of HRG-induced HER3 phosphorylation. Band density was analyzed using the Fusion Solo S program (Vilber), and values ​​related to tubulin load control were used to calculate IC50 values. For MCF-7 cells, an IC50 value of 80 pM was determined.A comparison with IgG 3M6 anti-HER3, which comprises the same variable domain as seribantumab, in MCF-7 cells, demonstrated superior IgG 3-43 activity. Here, 3M6 inhibited HER3 phosphorylation with an IC50 value of 270 pM. Example 6 HER3 Internalization Induced by IgG 3-43 anti-HER3

[0301] Cellular expression levels of HER3 and IgG localization after incubation with IgG 3-43 were analyzed by Western blot and immunofluorescence microscopy, respectively. For Western blot analysis, MCF-7 cells were seeded in 6-well plates two days prior to be semi-confluent on the day of the experiment. Cells were serum-deprived overnight and incubated with 100 nM IgG 3-43. Petition 870260062263, dated 06 / 25 / 2026, page 130 / 317 120 / 148 during the indicated time points. HER3 levels were analyzed by western blot. Band density was analyzed using the Fusion Solo S program (Vilber). Values ​​were corrected for loading differences relative to the tubulin loading control and normalized to the relative values ​​of untreated probes. IgG 3-43 rapidly leads to a reduction in HER3 levels in MCF-7 cells (Figure 6). Furthermore, Cy5-labeled IgG 3-43 was rapidly internalized into MCF-7 cells as confirmed by confocal microscopy (data not shown). After one hour, a strong intracellular accumulation of IgG 3-43 was detected. Example 7 Inhibition of Cellular Proliferation by IgG 3-43 Anti-HER3

[0302] IgG 3-43 was further evaluated for its ability to reduce tumor cell proliferation in vitro. To monitor this effect, several human cancer cell lines (MCF-7, BT-474, NCI-N87, FaDu) were seeded at low density in 96-well plates, allowed to adhere overnight, and then incubated under low serum concentration with IgG 3-43 or other antibodies as a control. Proliferation was determined after 1 week of incubation. For all four cell lines, a reduction in proliferation was observed compared to the control antibody (Figure 7). For FaDu cells, an IC50 value of 273 pM was determined under these conditions. Example 8 IgG 3-43 Effectively Inhibits the Growth of SC Xenografted FaDu Tumors in SCID Mice.

[0303] The antitumor activity of IgG 3-43 was tested in a subcutaneous xenograft model of FaDu cells in SCID mice. 5x106 cells were injected into both flanks of the mice and treatment was initiated when the tumors reached a volume of Petition 870260062263, dated 06 / 25 / 2026, page 131 / 317 121 / 148 approximately 80 mm3 (14 days after tumor cell inoculation). Mice received intravenous injections twice weekly for 3 weeks with doses of 30, 100, and 300 pg, including PBS as a negative control. Antitumor effects were observed for all three IgG 3-43 dosage regimens with increased survival (median survival increased only for the two highest concentrations) and significant inhibition of tumor growth (Figure 8). Example 9 A bispecific Fc-specific single-chain Diabody fusion protein targeting EGFR and HER3 induces potent inhibition of EGFR and HER3 activation.

[0304] We generated a bispecific antibody directed against EGFR and HER3 in single-chain Fc diabody format (Figure 9A, C) comprising the antibody portions of hu225 (humanized version of C225 (cetuximab, Erbitux)) and 3-43. The scDb-Fc fusion protein was produced in HEK293-6E cells in suspension and purified from the cell culture supernatant by protein A affinity purification. SDSPAGE analysis of scDb hu225x3-43-Fc revealed single bands at an apparent molecular mass of approximately 82 kDa under reducing conditions and 200 kDa under non-reducing conditions corresponding to monomeric and dimeric assembly of the construct (Figure 9B). In contrast, cetuximab and IgG 3-43 showed two bands under reducing conditions representing the heavy and light chains. Purity was confirmed by size exclusion chromatography (Figure 9C). The binding activity of scDb-Fc to its antigens and cells expressing ErbB receptors was evaluated by ELISA and flow cytometry, respectively.ELISA analysis revealed that the binding activity of parental antibodies to the extracellular domain (ECD) of EGFR and HER3 is retained in the scDb-Fc format (see Figure 10A). The scDb-Fc molecule and parental antibodies bind at similar EC50 values ​​in the subnanomolar range. Petition 870260062263, dated 06 / 25 / 2026, page 132 / 317 122 / 148 their corresponding antigens (Table 4). Flow cytometry analysis showed that cetuximab and scDb hu225x3-43-Fc bound to FaDu cells with an EC50 value of 0.2 nM, while IgG 3-43 bound with an EC50 value of 0.006 nM (Figure 10B).

[0305] Next, signaling inhibition assays in MCF-7 cells were performed to determine whether receptor activation is inhibited by treatment with scDb hu225x3-43-Fc (Figure 11). Heregulin induced HER3 phosphorylation and activation of downstream effectors Akt and Erk1 / 2. Pretreatment with IgG 3-43 and the combination of IgG 3-43 and cetuximab efficiently blocked HER3 phosphorylation and Akt and Erk1 / 2 activation, and HER3 was degraded. Treatment with scDb hu225x3-43-Fc also showed strong inhibition of HER3 signaling and resulted in HER3 degradation. Signaling inhibition assays were also performed in other ErbB overexpressing cell lines (A-431, A549, FaDu, NCI-N87, SK-BR-3) and EGF-stimulated EGFR inhibition was further evaluated (Figure 12A-F).The bispecific scDb hu225x3-43-Fc antibody, as well as the combination of cetuximab and IgG 3-43, inhibited EGFR phosphorylation in the presence of EGF and HER3 phosphorylation in the presence of heregulin in all cell lines. To further investigate possible differences in HER3 signaling inhibition between scDb-Fc and parental antibodies, signaling inhibition assays were performed with serial dilutions of the antibodies in FaDu cells in the presence of heregulin (see Figure 13). The scDb-Fc antibody inhibited HER3 phosphorylation with an IC50 value of 0.008 nM, while the combination of IgG 3-43 and cetuximab blocked HER3 phosphorylation with an IC50 value of 0.081 nM, demonstrating that the bispecific antibody has superior inhibitory activity for HER3 phosphorylation compared to the combination of parental antibodies. Petition 870260062263, dated 06 / 25 / 2026, page 133 / 317 123 / 148 monospecific. Example 10 A Bispecific Fc-Single Chain Diabody Fusion Protein Targeting HER2 and HER3 Derived from Antibodies 2-35 and 3-43

[0306] We constructed a bispecific antibody targeting HER2 and HER3 in single-chain Fc diabody format containing the antibody portions of antibodies 2-35 and 3-43. The 2-35 portion was also identified by display in phages and is specific for the extracellular domain of HER2. The scDb-Fc fusion protein was produced in HEK293-6E cells in suspension and purified from the cell culture supernatant by protein A affinity purification. SDS-PAGE analysis of scDb 2-35x3-43-Fc revealed a single band at an apparent molecular weight of approximately 82 kDa under reducing conditions and 200 kDa under non-reducing conditions (see Figure 14A). Purity was confirmed by size exclusion chromatography (see Figure 14B). The binding of scDb-Fc to the extracellular domain of HER2 and HER3 compared to parental antibodies IgG 2-35 and IgG 3-43 was determined by ELISA. ELISA analysis revealed that the binding activity of parental antibodies to the ECD of HER2 and HER3 is retained in the scDb-Fc format (see Figure 15).The scDb 235x3-43-Fc fusion protein and IgG 2-35 bind with an EC50 value of approximately 1.5 nM to the HER2 ECD, while the bispecific antibody binds with an EC50 value of 0.24 nM and the IgG 3-43 antibody binds to the HER3 ECD with an EC50 value of 0.33 nM. Signaling inhibition assays in MCF-7 cells showed that IgG 2-35 only slightly decreased HER3 phosphorylation, which is likely due to inhibition of HER3 heterodimerization with HER2 (see Figure 11). The combination of the 2-35 and 3-43 portions in the bispecific antibody format showed potent inhibition of HER3 signaling. These results... Petition 870260062263, dated 06 / 25 / 2026, p. 134 / 317 Figures 124 / 148 demonstrate that scDb 2-35x3-43-Fc may be another candidate for blocking compensatory signaling axes and thus preventing tumor escape. Example 11 Anti-HER3 fusion proteins such as scTRAIL mediate target-dependent cytotoxicity.

[0307] TNF-related apoptosis-inducing ligand (TRAIL) is considered a promising effector molecule due to its selective toxicity in cancer cells. A single-stranded version of TRAIL was fused to the C-terminal of a human IgG1 Fc portion (FcscTRAIL) to induce dimeric assembly, which greatly enhances antitumor effects. To further improve bioactivity, scFv 343 was N-terminally fused to the Fc portion, generating scFv3-43-FcscTRAIL. The fusion protein was produced in stably transfected HEK293 cells and purified from the supernatant by anti-FLAG affinity chromatography. SDS-PAGE analysis and size exclusion chromatography confirmed the purity and integrity of the protein (see Figure 16).

[0308] The novel scFv3-43-Fc-scTRAIL fusion protein was evaluated for its ability to bind to the corresponding target antigen and human TRAIL-R2 by ELISA, as well as by flow cytometry using intact Colo205 and HCT-116 cells. Binding to the antigen and TRAIL receptor was analyzed by ELISA using Fc fusion proteins from the corresponding extracellular domains. ScFv3-43-FcscTRAIL showed concentration-dependent specific binding to HER3 with an EC50 value in the subnanomolar range (see Figure 17A, Table 5). Further studies using ELISA revealed potent binding to human TRAIL-R2 with an EC50 value of 2.84 nM Petition 870260062263, dated 06 / 25 / 2026, page 135 / 317 125 / 148 (see Figure M2B, Table M1). Thus, fusion with scFv3-43 does not prevent binding to TRAIL-R2. The ELISA results were confirmed by flow cytometry studies with Colo205 and HCT-116 cells expressing the TRAIL antigen and receptor (see Figure 17C, D). These data show that scFv3-43-Fc-scTRAIL has full functionality with respect to binding to purified HER3 and TRAIL-R2 expressed on the cell surface.

[0309] The induction of cell death by scFv3-43-Fc-scTRAIL was analyzed using Colo205 cells and compared with untargeted Fc-scTRAIL. One day before treatment, 50,000 Colo205 cells / well were seeded in 96-well plates. After pretreatment of the cells with the sensitizer bortezomib (650 nM) or medium for 30 min., the cells were incubated with serial dilutions of the fusion proteins for 16 h. Cell death was analyzed by crystal violet staining. The fusion protein ScFv3-43-Fc-scTRAIL showed strong induction of cell death in Colo205 cells, which could be further intensified in the presence of bortezomib (see Figure 18). Comparison with untargeted Fc-scTRAIL revealed better effects of scFv3-43-Fc-scTRAIL in the absence and presence of bortezomib (see Figure 18, Table 6).To confirm that this superiority is caused by the scFv3-43 targeting fraction, experiments were repeated by adding the corresponding blocking antibody scFv3-43-Fc (200-fold molar excess) to the pretreatment of cells. In the presence of the blocking antibody, the effects of scFv3-43-Fc-scTRAIL were reduced to the same level as untargeted Fc-scTRAIL (see Figure 18, Table 6). This confirms the suitability of the 3-43 antibody portion for targeting cytotoxic fusion proteins to enhance antitumor effects. Petition 870260062263, dated 06 / 25 / 2026, page 136 / 317 126 / 148 Example 12 Bispecific Anti-HER3 x Anti-CD3 Antibody for T-Cell Redirection

[0310] A bispecific scDb molecule was generated by combining the 3-43 anti-HER3 binding site with a humanized version of the human anti-CD3 antibody UCHT1. Thus, scDb 3-43xCD3 exhibits a binding site for HER3 and a binding site for CD3 (Figure 19A and B). The scDb 3-43xCD3 protein was produced in HEK293 cells and purified by IMAC. SDS-PAGE analysis of scDb 3-43xCD3 revealed a single band at an apparent molecular weight of approximately 55 kDa under reducing conditions and 50 kDa under non-reducing conditions (Figure 19C). Size exclusion chromatography confirmed the purity and integrity of the protein with an apparent molecular mass of approximately 50 kDa (hydrodynamic radius: 2.96 nm) (Figure 19D).

[0311] The binding of the new scDb construct was evaluated by ELISA and flow cytometry.

[0312] The binding to scDb3-43xCD3 antigen was analyzed by ELISA using immobilized HER3-Fc fusion comprising the extracellular domain (aa 27-599) of human HER3. The HER3-Fc fusion protein was coated onto polystyrene microtiter plates at 2 μg / mL in PBS. The remaining binding sites were blocked with PBS, 2% skim milk (MPBS). The plates were then incubated with a serial dilution of scDb3-43xCD3 in MPBS. After washing, the bound antibody was detected with an anti-His antibody conjugated with HRP and TMB, H2O2 as substrate. scDb3-43xCD3 showed specific, concentration-dependent binding to HER3 with an EC50 value in the lower nanomolar range (3.3 nM) (Fig. 19E). Petition 870260062263, dated 06 / 25 / 2026, page 137 / 317 127 / 148

[0313] Flow cytometry studies were performed with MCF-7 expressing HER3 (Figure 19F) and Jurkat expressing CD3 (Figure 19G). For adherent MCF-7, cells were rapidly trypsinized at 37°C and trypsin was blocked with SBF-containing medium and removed by centrifugation. For both MCF-7 and Jurkat cells, 100,000 cells per well were seeded and incubated with a titer of scDb 3-43xCD3 in PBA (2% (v / v) SBF, 0.02% (w / v) NaN3 in 1x PBS) for one hour at 4°C. Washing was performed twice with PBA. Bound protein was detected using PE-conjugated anti-His antibody incubated for another hour at 4°C. After washing, fluorescence was measured using a MACSQuant® Analyzer 10 device. Median relative fluorescence intensities (compared to unstained cells) were calculated using the FlowJo program.Similar binding activities were observed for both antigen-binding sites in the lower nanomolar range, with EC50 values ​​of 1.1 nM for MCF-7 and 3.1 nM for Jurkat (Figure 19 F and G).

[0314] T cell activation was analyzed in an IL-2 release assay using HER3-expressing Colo205 cells and PBMCs. One day before treatment, 20,000 Colo205 cells were seeded per well in a 96-well plate. The medium was removed and replaced with a titration of scDb 3-43xCD3 in fresh medium. After 1 hour of incubation at room temperature, 200,000 PBMCs per well were added and incubated for a further 24 hours at 37°C. The supernatant was collected and the IL-2 concentration was determined by ELISA (human IL-2 kit, R&D) according to the manufacturer's instructions. ScDb 3-43xCD3 showed dose-dependent IL-2 release (T cell activation) in the subnanomolar range with an EC50 value of 0.3 nM (Figure 19H). Petition 870260062263, dated 06 / 25 / 2026, page 138 / 317 128 / 148 Example 13 Bispecific Anti-HER3 Antibody x Trivalent Anti-CD3 Antibody for T-CELL REDIRECTION

[0315] A bispecific trivalent scDb3-43xCD3scFv343 molecule (scDb-scFv) was generated by combining the scDb molecule, specific for HER3 (3-43) and CD3 (humanized version of UCHT1), with an anti-HER3 specific scFv (3-43). Thus, scDb-scFv exhibits two binding sites for HER3 and one binding site for CD3 (Figure 20A). The scDb-scFv protein was produced in HEK293 cells and purified by IMAC. SDS-PAGE analysis of scDb-scFv revealed a single band at an apparent molecular weight of approximately 80 kDa under reducing conditions and 75 kDa under non-reducing conditions (Figure 20B). Size exclusion chromatography confirmed the purity and integrity of the protein with an apparent molecular mass of approximately 62 kDa (hydrodynamic radius: 3.83 nm) (Figure 20C).

[0316] Binding to scDb3-scFv antigen was analyzed by ELISA using immobilized HER3-Fc fusion comprising the extracellular domain (aa 27-599) of human HER3. The HER3-Fc fusion protein was coated onto 2 μg / mL polystyrene microtiter plates in PBS. The remaining binding sites were blocked with PBS, 2% skim milk (MPBS). The plates were then incubated with a serial dilution of scDb-scFv and the bivalent and bispecific scDb3-43xCD3 as a control in MPBS. After washing, bound antibody was detected with an anti-His antibody conjugated with HRP and TMB, H2O2 as substrate. The ScDb-scFv molecule showed specific concentration-dependent binding to HER3 with an EC50 value in the subnanomolar range (0.81 nM), while the scDb3-43xCD3 molecule showed an EC50 value in the lower nanomolar range (4.87 nM) (Figure 20D). Petition 870260062263, dated 06 / 25 / 2026, page 139 / 317 129 / 148

[0317] Flow cytometry studies were performed with MCF-7 expressing HER3 (Figure 20E) and Jurkat expressing CD3 (Figure 20F). For adherent MCF-7, cells were rapidly trypsinized at 37°C and trypsin was blocked with SBF-containing medium and removed by centrifugation. For both MCF-7 and Jurkat cells, 100,000 cells per well were seeded and incubated with a titration of scDb-scFv in PBA (2% (v / v) SBF, 0.02% (w / v) NaN3 in 1x PBS) for one hour at 4°C. Washing was performed twice with PBA. Bound protein was detected using PE-conjugated anti-His antibody incubated for another hour at 4°C. After washing, fluorescence was measured using a MACSQuant® Analyzer 10 device. Median relative fluorescence intensities (compared to unstained cells) were calculated using the FlowJo program.Binding to MCF-7 cells was observed in the picomolar range with an EC50 value of 31.6 pM, comparable to the binding properties of the complete IgG3-43 molecule, and binding to Jurkat cells was in the nanomolar range with an EC50 value of 13.2 nM (Figure 20 E and F). Example 14 A Bispecific Fc-Single Chain Diabody Fusion Protein Targeting HER2 and HER3 Derived from Antibodies 4D5 and 3-43

[0318] We generated a bispecific antibody targeting HER2 and HER3 in single-chain Fc diabody format (Figure 21) comprising the 4D5 (trastuzumab, Herceptin) and 3-43 antibody portions. The scDb-Fc fusion protein was produced in HEK293-6E cells in suspension and purified from the cell culture supernatant by protein A affinity purification followed by FPLC SEC. SDS-PAGE analysis of the scDb 4D5x3-43-Fc revealed single bands at an apparent molecular mass of approximately 85 kDa under reducing conditions and Petition 870260062263, dated 06 / 25 / 2026, p. 140 / 317 130 / 148 200 kDa under non-reducing conditions corresponding to the monomeric and dimeric polypeptides of the construct (Figure 21A). Purity and integrity were confirmed by size exclusion chromatography with an apparent molecular mass of approximately 175 kDa (Figure 21B). Antigen binding of the scDb 4D5x3-43-Fc molecule was analyzed by ELISA using immobilized HER2-His or HER3-His comprising the extracellular domain of human HER2 or HER3. The antigens were coated onto polystyrene microtiter plates at 2 pg / ml in PBS. Remaining binding sites were blocked with PBS, 2% skim milk (MPBS). The plates were then incubated with a serial dilution of the scDb-Fc fusion protein in MPBS. After washing, bound antibody was detected with an anti-human Fc antibody conjugated with HRP and TMB, H2O2 as substrate.ELISA analysis revealed nanomolar-range binding of the scDb 4D5x3-43-Fc fusion protein with EC50 values ​​of 2.5 nM for HER2-His and 1.9 nM for HER3-His (see Figure 21C).

[0319] Flow cytometry studies were performed with FaDu cells expressing HER2 and HER3 (Figure 21D). FaDu cells were rapidly trypsinized at 37°C, trypsin was stopped with SBF-containing medium and removed by centrifugation. 100,000 cells per well were seeded and incubated with a scDb-Fc titration in PBA (2% (v / v) SBF, 0.02% (w / v) NaN3 in 1x PBS) for one hour at 4°C. Washing was performed twice with PBA. Bound protein was detected using PE-conjugated human anti-Fc antibody incubated for another hour at 4°C. After washing, fluorescence was measured using a MACSQuant® Analyzer 10. Median relative fluorescence intensities (relative to unstained cells) were calculated using the FlowJo program. Binding to FaDu cells was observed in the nanomolar range with an EC50 value of 2.9 nM, comparable to Petition 870260062263, dated 06 / 25 / 2026, page 141 / 317 131 / 148 binding properties obtained from ELISA analysis. Example 15 Ligand-Independent Inhibition of Colony Formation in SKBR3 and BT474 Tumor Cells Incubated with IgG 3-43

[0320] SKBR3 and BT474 cells express high levels of HER2 and can thus proliferate in a ligand-independent manner. The potential of IgG 3-43 to inhibit colony formation, as a marker for cell proliferation, was analyzed in these two cell lines (Fig. 22). Cells (1,000 cells per well) were seeded in a 12-well plate in RPMI medium. The following day, cells were incubated with antibody (IgG 3-43 or Trastuzumab) at a concentration of 50 nM in RPMI medium containing 2% SBF. After 7 days, the medium was removed and fresh medium with antibody at the same concentration was added. On day 12, cells were fixed with Histofix for 10 min at room temperature and stained with crystal violet for 10 min (Fig. 22A). Untreated (con) cells were included as a negative control. All incubations were performed in triplicate. Trastuzumab, targeting HER2, was included as a positive control.A potent inhibition of colony formation was observed for IgG 3-43 and trastuzumab in both cell lines (Fig. 22B). These findings indicate that HER3 forms heterodimer-competent signaling with HER2 even in the absence of heregulin, which can be inhibited by IgG 3-43. Example 16 A tetravalent and bispecific Diabody-Ig fusion protein (Db-Ig) targeting EGFR (hu225) and HER3 (3-43)

[0321] A tetravalent and bispecific Db3-43xhu225-Ig molecule was generated by combining a Db molecule, specific for EGFR (hu225; humanized version of C225 (cetuximab, Erbitux)) and HER3 (3-43), with Petition 870260062263, dated 06 / 25 / 2026, page 142 / 317 132 / 148 the constant domains of an IgG antibody. Thus, the Db343xhu225-Ig molecule consists of two different polypeptides, VH3-43xVLhu225-CL (light chain, SEQ ID NO: 31) and VHhu225xVLhu3-43-CH1-CH2-CH3 (heavy chain, SEQ ID NO: 32) (Fig. 23A). The bispecific Db3-43xhu225-Ig exhibits two antigen-binding sites for EGFR and two antigen-binding sites for HER3 (Fig. 23B). The Db3-43xhu225-Ig molecule was transiently expressed in transfected HEK293-6E cells after co-administration of two plasmids encoding the light chain or heavy chain, using polyethyleneimine as a transfection reagent. The protein secreted in the cell culture supernatant was purified using CHI-CaptureSelect affinity chromatography.SDS-PAGE analysis revealed two bands under reducing conditions at approximately 65 kDa and 35 kDa corresponding to the heavy and light chains, and a main band under non-reducing conditions at approximately 220 kDa corresponding to the bispecific Db-Ig molecule (Fig. 23C). The purity, integrity, and homogeneity of the Db3-43xhu225Ig molecule were confirmed by size exclusion chromatography (Fig. 23D). The binding of Db3-43xhu225-Ig and the monospecific parental antibodies (cetuximab (anti-EGFR) and 3-43-IgG (anti-HER3)) to the extracellular domain (ECD) of EGFR (aa 20-643) and HER3 (aa 27-599) was determined by ELISA. The EGFR or HER3 fusion protein labeled with His was coated onto polystyrene microtiter plates at a concentration of 2 μg / ml diluted in PBS. The remaining binding sites were blocked with PBS, 2% skim milk (MPBS).The plates were then incubated with serial dilutions of the bispecific Db3-43xhu225-Ig construct or monospecific parental antibodies. After washing, bound antibodies were detected with an HRP- and TMB-conjugated human anti-Fc antibody, H2O2 as substrate. ELISA analysis revealed that the binding activity of the parental antibodies to the extracellular domain (ECD) of EGFR and HER3 is retained in the format. Petition 870260062263, dated 06 / 25 / 2026, page 143 / 317 133 / 148 Db-Ig. The tetravalent, bispecific Db3-43xhu225-Ig showed concentration-dependent binding to EGFR and HER3 with an EC50 value in the subnanomolar range (0.19 nM for EGFR; 0.26 nM for HER3) (Fig. 23E). The parental antibodies bound with EC50 values ​​similar to their corresponding antigens (Table 7). Simultaneous binding to both antigens, EGFR and HER3, was confirmed by a second ELISA binding analysis. As the first antigen, the EGFR-Fc fusion protein was coated in polystyrene microtiter plates at 2 μg / mL in PBS. The remaining binding sites were blocked with PBS, 2% skim milk (MPBS). The plates were then incubated with a serial dilution of the bispecific Db3-43xhu225-Ig construct diluted in MPBS. After washing, the second antigen, HER3-His (HER3 (aa 27-599 of the extracellular domain fused C-terminally with hexahistidyl-tag; diluted to 300 nM in MPBS), was added to the plates.After washing, the bound HER3-His (second antigen) was detected with an HRP- and TMB-labeled anti-His antibody, H2O2 as substrate. The second antigen was bound to the bispecific Db3-43xhu225-Ig in a concentration-dependent manner with an EC50 value in the subnanomolar range (0.85 nM) (Fig. 23F), similar to the binding of Db3-43xhu225-Ig to the coated HER3-Fc. Thus, this result demonstrates the unrestricted accessibility of both antigen-binding sites within the Db-Ig molecule.

[0322] In addition, binding studies of Db3-43xhu225-Ig and parental monoclonal antibodies (cetuximab and 3-43-IgG) to EGFR and / or HER3-expressing cells (MCF-7, SKBR-3, and FaDu) (Figure 23G) were analyzed by flow cytometry. Adherent cells were washed with PBS and rapidly trypsinized at 37°C. Trypsin was blocked with SBF-containing medium and removed by centrifugation (500xg, 5 minutes). 100,000 cells per well were seeded and incubated with a serial dilution of Db3-43xhu225-Ig or parental monoclonal antibodies diluted in PBA (PBS). Petition 870260062263, dated 06 / 25 / 2026, page 144 / 317 134 / 148 cells containing 2% (v / v) SBF and 0.02% (w / v) NaN3 were incubated for one hour at 4°C. The cells were washed twice using PBA. Bound antibodies were detected using PE-labeled anti-Fc secondary antibody, which was incubated for another hour at 4°C. After washing, the median fluorescence intensity (MFI) was measured using a MACSQuant® Analyzer 10. The relative MFI (compared to unstained cells) was calculated using the MACSQuant® program and Excel. For HER3-positive MCF-7 cell lines, bispecific Db3-43xhu225-Ig binding occurred in the subnanomolar range with an EC50 value of 0.054 nM. Since the parental anti-HER3 343-IgG bound with similar EC50 values ​​(0.021 nM), the binding activity of the parental anti-HER3 antibodies is retained in the Db-Ig format. No binding to MCF-7 cells was observed for the anti-EGFR antibody cetuximab.Regarding the SKBR-3 cell line, which expresses EGFR and HER3 in similar ranges, the bispecific molecule Db3-43xhu225-Ig bound at an EC50 value of 0.047 nM, similar to the binding of both parental antibodies cetuximab (0.031 nM) and 3-43-IgG (0.022 nM). Regarding binding to FaDu cells, Db3-43xhu225-Ig bound with EC50 values ​​of 0.14 nM. As the parental anti-EGFR antibody cetuximab bound to the cells with a similar EC50 value (0.13 nM), the binding activity of the parental antibody cetuximab is also retained in the Db-Ig format. Since FaDu cells express a very high amount of EGFR and a comparatively low amount of HER3, Db3-43xhu225-Ig likely binds more preferentially to the hu225 portions of the cells. However, parental anti-HER3 3-43-IgG also bound with a comparatively low fluorescence signal to the cells with an EC50 value of 0.003 nM. (Figure 23G, Table 2).

[0323] The pharmacokinetic profile of the bispecific and tetravalent molecule Db3-43xhu225-Ig was analyzed in SWISS mice. 25 μg of Petition 870260062263, dated 06 / 25 / 2026, page 145 / 317 135 / 148 proteins were diluted in 100 μL of sterile PBS and injected intravenously into the tail. After different times (3 minutes, 30 minutes, 1 hour, 2 hours, 6 hours, 1 day, 3 days, and 7 days), blood samples were collected from the tail and incubated on ice for 10 minutes. The clotted blood was centrifuged (16,000xg, 20 minutes, 4°C) and the serum samples were stored at -20°C. EGFR-Fc or His-labeled HER3-Fc fusion protein was coated onto polystyrene microtiter plates at a concentration of 2 μg / ml diluted in PBS. The remaining binding sites were blocked with PBS, 2% skim milk (MPBS). The plates were then incubated with serum diluted in MPBS. After washing, bound antibodies were detected with an HRP- and TMB-conjugated anti-human Fab antibody, H2O2 as substrate. The serum concentration of the Db3-43xhu225-Ig molecule was interpolated from a purified fusion protein standard curve (Figure 24).No differences were observed for serum concentrations of Db3-43xhu225-Ig using the EGFR-Fc or HER3-Fc fusion protein as coated antigen. The bispecific molecule Db343xhu225-Ig had an initial half-life of approximately 2.7 hours and a terminal half-life in the range of 87 to 92 hours. Example 17 The scFv-Fc-scTRAIL Anti-HER3 Fusion Proteins Mediate Target-Dependent Cytotoxicity In Vitro Against Various Melanoma Cell Lines

[0324] A panel of melanoma cell lines was screened by flow cytometry for HER3 expression, which was quantified using a QIFIKIT (Dako) (Figure 25A). Most of the melanoma cell lines analyzed exhibited HER3 expression to varying degrees. A375 with moderate HER3 expression was chosen to analyze the binding of an anti-HER3 fusion protein scFv-Fc-scTRAIL (see example 11; Petition 870260062263, dated 06 / 25 / 2026, page 146 / 317 136 / 148 Figure 25B, C). The binding of scFv3-43-Fc-scTRAIL to the HER3-positive A375 cell line was analyzed using flow cytometry. Concentration-dependent binding of scFv3-43-Fc-scTRAIL was observed with an EC50 value of 1.19 ± 0.31 nM (Figure 25D). Competitive inhibition of scFv3-43Fc-scTRAIL with the fusion protein scFv3-43-Fc was performed to verify that enhanced target-format binding resulted from the 3-43 binding domain (Fig. 25E). For this reason, the binding domain of scFv3-43-Fc-scTRAIL was blocked with a 200-fold molar excess of scFv3-43-Fc (inhibitor). The binding of scFv3-43-Fc-scTRAIL was reduced to the same level as the non-targeted protein in the presence of the inhibitor, while the binding of Fc-scTRAIL itself was not affected. These results confirmed that the targeting portion (scFv3-43) of the TRAIL fusion protein increases binding to HER3-positive cells.

[0325] The anti-HER3 fusion protein scFv-Fc-SCTRAIL was then analyzed in vitro for its ability to kill different melanoma cell lines in the presence or absence of bortezomib (Figure 26). One day before treatment, 30,000 to 60,000 cells / well were seeded in 96-well plates. For the combined treatment with bortezomib, cells were pretreated with bortezomib at 250 ng / ml (650 nM), except for A375 which were pretreated with 50 ng / ml bortezomib for 30 min. Cells were then incubated with serial dilutions of the fusion proteins for 16 h. Cell viability was analyzed by crystal violet staining.

[0326] In the absence of bortezomib, scFv3-43-Fc-scTRAIL showed strong induction of cell death in W793 (EC50 value of 4.25 pM), MW1366 (EC50 value of 48.2 pM), and WM35 (EC50 value of 4.96 pM) cells, and partial induction of cell death (not reaching 50% death) in A375, MelJuso, and MeWo cells (see Figure 26). In contrast, the addition of bortezomib sensitized all melanoma cell lines tested, showing Petition 870260062263, dated 06 / 25 / 2026, page 147 / 317 137 / 148 deaths with EC50 values ​​between 0.17 and 4.63 pM (Figure 26). Compared to untargeted Fc-scTRAIL, EC50 values ​​decreased both in the absence and presence of bortezomib (Table 8). Additionally, HER3 expression in melanoma cells, which were used in the cell viability assay, was analyzed in the absence or presence of bortezomib by flow cytometry using a QIFIKIT (Dako). Treatment with bortezomib had no effect (or only a marginal effect) on HER3 expression by melanoma cells (Figure 26). Example 18 The anti-HER3 fusion protein scFv-Fc-scTRAIL exhibits potent antitumor activity and is well tolerated in an in vivo tumor xenograft model with colony-associated cells.205

[0327] The scFv3-43-Fc-scTRAIL fusion protein (see example 11) was evaluated for its antitumor activity, safety, and pharmacokinetic profile in tumor-bearing mice. 3 x 10⁶ Colo205 cells (in 100 μL of DPBS) were subcutaneously injected into the left and right flanks of female nude NMRI mice. Tumor growth was monitored by measuring the length (a) and width (b) of the tumors with a caliper to calculate tumor volume (V = a x b² / 2). Treatment began when the tumors reached a size of approximately 100 mm³. Fusion protein injections (in 150 μL of DPBS) were administered intravenously. Control animals received respective injections of 150 μL of DPBS. Mice (9 or 11 weeks old, 6 mice per group) were treated with 0.2 nmol protein (0.4 nmol scTRAIL units) twice a week for three weeks (days 14, 18, 21, 25, 28, 32).Blood samples were collected 4 hours and 24 hours after the last treatment to analyze protein concentration and ALT levels.

[0328] Complete tumor remission was observed for the Petition 870260062263, dated 06 / 25 / 2026, page 148 / 317 138 / 148 treatment with scFv3-43-Fc-scTRAIL and untargeted Fc-scTRAIL. Tumor remission was stable throughout the nearly 100-day monitoring period for animals treated with scFv3-43-Fc-scTRAIL, and only marginal regrowth was detected for Fc-scTRAIL at the end of the experiment (Figure 27A, B). No toxic effects on the liver were observed for the groups treated with the TRAIL fusion protein, as serum ALT activity at 4 and 24 hours after the last treatment was similar compared to the untreated group or the PBS-treated group (Figure 27C). Serum protein concentrations were determined 4 and 24 hours after the last treatment (Figure 27D). No differences were observed between scFv3-43-Fc-scTRAIL and Fc-scTRAIL. Example 19 A Bispecific Fc-Single Chain Diabody Fusion Protein Containing a G4S Ligand Targeting EGFR and HER3

[0329] The L2 ligand of the bispecific single-chain diabody Fc fusion protein targeting EGFR and HER3 that links VL3-43 to VH3-43 was modified from the sequence GGGGSGGRASGGGGS (SEQ ID NO: 21) to GGGGSGGGGSGGGGS (SEQ ID NO: 16) (Figure 28A, B). The modified scDb-Fc fusion protein was produced in HEK293-6E cells in suspension and purified from the cell culture supernatant by protein A affinity purification and fast protein liquid chromatography. SDS-PAGE analysis of the purified scDbhu225x3-43-Fc revealed single bands at an apparent molecular mass of approximately 82 kDa under reducing conditions and 200 kDa under non-reducing conditions corresponding to monomeric and dimeric assembly of the molecule (Figure 28C). Purity was confirmed by size exclusion chromatography (Figure 28D).The binding of scDb-Fc to the His-tagged extracellular domain of EGFR (aa 20-643) and HER3 (aa 27599) was evaluated by ELISA in comparison with parental antibodies. Petition 870260062263, dated 06 / 25 / 2026, page 149 / 317 139 / 148 (hu225-IgG and 3-43-IgG). The EGFR-His or HER3-His fusion protein was coated onto polystyrene microtiter plates at a concentration of 2 μg / mL diluted in PBS. The remaining binding sites were blocked with PBS, 2% skim milk (MPBS). The plates were then incubated with serial dilutions of the bispecific scDb-Fc molecule or the monospecific parental antibodies. After washing, the bound antibodies were detected with an HRP- and TMB-conjugated human anti-Fc antibody, H2O2 as substrate. ELISA analysis revealed that the binding activity of the parental antibodies to the extracellular domain (ECD) of EGFR and HER3 is retained in the modified scDb-Fc format (see Figure 28E).The scDb-Fc molecule bound to EGFR with EC50 values ​​of 0.16 nM and to HER3 with EC50 values ​​of 0.20 nM, while the parental antibodies bound at similar EC50 values ​​in the subnanomolar range to their corresponding antigens (hu225-IgG: 0.20 nM; 3-43-IgG: 0.54 nM). Example 20 Comparison of the Diabody-Ig Fusion Protein and the Bispecific Fc-Cycle Diabody Targeting EGFR and HER3

[0330] Two different formats of bispecific, tetravalent antibodies targeting EGFR and HER3, single-chain diabody-Fc (scDb-Fc) (see example 19) and diabody-Ig (Db-Ig) (example 16), were analyzed for inhibitory activity against EGFR, HER2, HER3, Akt, and Erk. Signal inhibition assays were performed using FaDu cells. Cells were treated with 50 nM of the parental antibodies (alone or in combination (50 nM of each antibody)), the bispecific antibodies (scDbhu225x3-43-Fc (GGGGS) SEQ ID NO: 33, Db3-43xhu225-Ig) for 1 hour before stimulation with heregulin (50 ng / mL) for 15 min at 37°C. The cells were lysed using RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 10 mM NaF, 20 mM β-glycerophosphate, 1 mM EDTA, 1% NP-40, 1 mM NaaVO4, 0.5% PMSF). Petition 870260062263, dated 06 / 25 / 2026, page 150 / 317 140 / 148 mM, 0.25% DOC, 0.1% SDS) containing a protease inhibitor cocktail, and the lysates were analyzed by immunoblotting. HER2 and HER3 phosphorylation was inhibited by the bispecific antibodies, as well as by 3-43-IgG in the presence of heregulin stimulation, while receptor phosphorylation was inhibited more efficiently in the presence of heregulin by both bispecific antibodies compared to 3-43-IgG. Furthermore, the bispecific antibodies efficiently inhibited Akt and Erk phosphorylation in the absence and presence of heregulin (Figure 29).

[0331] Next, proliferation assays were performed on colon cancer cell lines (SW620, HCT116, and LoVo) using two different bispecific antibody formats or parental antibodies (hu225-IgG and 3-43-IgG), alone or in combination. Cells were cultured in 2D or 3D cultures. 2000 cells / well were seeded in 96-well plates (for 3D culture: 1:2 Matrigel:collagen mixture, RPMI or DMEM + 10% SBF + 2% Matrigel). After 24 hours the medium was discarded and deprivation medium (RPMI or DMEM + 0.2% SBF + 1% P / S) was added. After a further 24 hours of incubation, the cells were treated with / without MEK inhibitor (AZD6244, Selumetinib) and / or antibody (50 nM, combination: 50 nM each). After 1 hour, the cells were stimulated with HRG (6 ng / well) or kept unstimulated. On day 8 after cell seeding, the assay was performed using the CelltiterGlo 3D kit (25 μL of deprivation medium mixed with 25 μL of reagent from the CelltiterGlo 2 kit).0 per well) and luminescence was measured with a plate reader (Tecan Infinite). For SW620 and HCT116 cells, only marginal differences in proliferation were observed for all antibodies. However, when cells were treated in combination with the MEK inhibitor in the presence of heregulin, the bispecific antibodies showed a reduced proliferative effect compared to the other antibodies. For LoVo cells not stimulated by... Petition 870260062263, dated 06 / 25 / 2026, page 151 / 317 141 / 148 In the HRG study, strongly reduced proliferative effects were observed for both bispecific antibodies, as well as for hu225-IgG or the combination of both parental antibodies in 3D culture, whether in the presence or absence of MEK inhibitor. After stimulation with HRG, only the bispecific antibodies were able to efficiently reduce cell proliferation, whether in the presence or absence of MEK inhibitor. Example 21 A Bivalent and Bispecific Single-Chain Diabody (bDb) Targeting HER2 and HER3

[0332] A bispecific scDb molecule was generated by combining the binding site of the 3-43 anti-HER3 antibody with that of the humanized 4D5 anti-HER2 antibody (Trastuzumab). scDb4D5x3-43-LL exhibits a HER3 binding site and a HER2 binding site (Figure 31A and B). The linker (L2) that links VL3-43 with VH3-43 consists of 20 amino acids (GGGGSGGRASGGGGSGGGGGS, SEQ ID NO: 21). scDb4D5x3-43-LL (SEQ ID NO: 34) was produced in HEK293E cells in suspension and purified by IMAC and fast protein liquid chromatography (FPLC). SDSPAGE analysis of the purified scDb4D5x3-43-LL molecule revealed a single band with an apparent molecular mass of approximately 53 kDa under reducing conditions and 50 kDa under non-reducing conditions, respectively (Figure 31C). Size exclusion chromatography confirmed the purity and integrity of the protein (Figure 31D).

[0333] The binding of scDb4D5x3-43-LL compared to parental antibodies (trastuzumab and 3-43 IgG) was evaluated by ELISA using immobilized HER2-Fc or HER3-Fc fusion proteins comprising the extracellular domain (ECD) of human HER2 (aa 23-652) or HER3 (aa 27599). The ECD-Fc fusion proteins were coated onto polystyrene microtiter plates at 2 pg / mL in PBS. The binding sites Petition 870260062263, dated 06 / 25 / 2026, page 152 / 317 142 / 148 remaining plates were blocked with PBS, 2% skim milk (MPBS). The plates were then incubated with a serial dilution of scDb4D5x343-LL or parental antibody in MPBS. After washing, bound antibody was detected with an HRP-conjugated anti-His antibody in the case of scDb4D5x343-LL or with an HRP-conjugated anti-human Fab antibody and TMB, H2O2 as substrate. scDb4D5x343-LL exhibited concentration-dependent binding to HER2 and HER3 with EC50 values ​​in the lower nanomolar range (HER2: 1.54 nM; HER3: 0.93 nM) (Figure 31E). Parental antibodies showed binding to the respective antigen (Trastuzumab for HER2: 0.80 nM; 3-43-IgG for HER3: 0.27 nM), thus, the binding of the scDb4D5x3-43-LL protein is maintained.

[0334] Signal inhibition assays in MCF-7 cells were performed to determine whether HER2, HER3, Akt, and Erk receptor activation is inhibited by treatment with the bispecific molecule scDb4D5x3-43-LL compared to parental antibodies as a single or combined treatment. Additionally, the bispecific and tetravalent fusion protein scDb4D5x3-43-Fc was also used in this experiment to determine receptor phosphorylation. Cells were treated with 50 nM of the parental antibodies (alone or in combination (50 nM of each antibody)), or the bispecific antibody scDb4D5x3-43-LL for 1 hour before stimulation with heregulin (50 ng / mL) for 15 min at 37°C. The cells were lysed using RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 10 mM NaF, 20 mM β-glycerophosphate, 1 mM EDTA, 1% NP-40, 1 mM NaaVO4, 0.5 mM PMSF, 0.25% DOC, 0.1% SDS) containing a protease inhibitor cocktail, and the lysates were analyzed by immunoblotting.For cells not stimulated with HRG, only the bispecific molecule, scDb4D5x3-43-LL bivalent, showed reduced phosphorylation of HER2, Akt, and Erk, while a. Petition 870260062263, dated 06 / 25 / 2026, page 153 / 317 143 / 148 Akt activation was observed for parental antibodies as a single or combined treatment. Furthermore, Erk activation was detected for the bispecific, but tetravalent, fusion protein scDb4D5x3-43-Fc (Figure 32). Similar results were observed in HRG-stimulated cells. Again, only the bispecific and bivalent molecule scDb4D5x3-43 showed efficient reduction of HER2, HER3, Akt, and Erk phosphorylation. Example 22 Bispecific Anti-HER3 x Anti-CD3 Antibodies with Different Valences for T-Cell Redirection

[0335] We generated bispecific antibodies that bind monovalently to human CD3 (humanized version of UCHT1) on one side and to HER3 on the other, in monovalent formats such as scDb3-43xhuU3 (see also Example 12), bivalent formats such as scDb3-43xhuU3-scFv3-43 (see also Example 13), or trivalent formats such as scFv3-43-scDb3-43xhuU3-scFv3-43 (SEQ ID NO: 35, Figure 33A, B). The bispecific and multivalent antibodies were produced in HEK293E cells and purified by IMAC. The trivalent (scDb3-43xhuU3-scFv3-43) and tetravalent (scFv3-43-scDb343xhuU3-scFv3-43) antibodies were further purified by fast protein liquid chromatography, resulting in a homogeneous antibody population.

[0336] The binding of bispecific antibodies was analyzed by flow cytometry using Jurkat cells expressing CD3 (Figure 33C) and MCF-7 cells expressing HER3 (Figure 33D). For adherent MCF-7 cells, they were rapidly trypsinized at 37°C, the trypsin was blocked with SBF-containing medium, and removed by centrifugation. For both Jurkat and MCF-7 cell lines, 100,000 cells per well were seeded and incubated with a titration of the different bispecific and multivalent antibodies in PBA (2% (v / v) SBF, 0.02% (w / v) NaN3 in 1x PBS) during Petition 870260062263, dated 06 / 25 / 2026, page 154 / 317 144 / 148 one hour at 4°C. Washing was performed twice with PBA. The bound protein was detected using anti-His antibody conjugated with PE incubated for another hour at 4°C. After washing, fluorescence was measured using a MACSQuant® Analyzer 10 device. Median relative fluorescence intensities (relative to unstained cells) were calculated using the FlowJo program. Binding to CD3-positive Jurkat cells was observed in a concentration-dependent manner for all three antibodies, resulting in similar EC50 values ​​in the nanomolar range (scDb3-43xhuU3: 2.4 nM; scDb3-43xhuU3-scFv3-43: 4.2 nM; scFv3-43-scDb3-43xhuU3-scFv3-43: 5.2 nM). Binding to MCF-7 cells was also observed in a concentration-dependent manner; however, the binding of bispecific antibodies was dependent on the HER3-binding valence.The EC50 value of the monovalent HER3 ligand (scDb3-43xhuU3) was determined to be 1.1 nM, while the divalent and trivalent HER3 ligands showed binding in the picomolar range with EC50 values ​​of 31.4 pM for scDb3-43xhuU3-scFv3-43 and 17.3 pM for scFv3-43-scDb3-43xhuU3-scFv3-43.

[0337] T cell activation was analyzed in an IL-2 release assay using HER3-expressing MCF-7 cells and PBMCs. One day before treatment, 20,000 MCF-7 cells were seeded per well in a 96-well plate. The medium was removed and replaced with a titration of different bispecific antibodies in fresh medium. After 1 hour of incubation at room temperature, 200,000 PBMCs per well were added and incubated for a further 24 hours at 37°C. The supernatant was collected and the IL-2 concentration was determined by ELISA (human IL-2 kit, R&D) according to the manufacturer's instructions. All three bispecific antibodies showed dose-dependent release of IL-2 (T cell activation) in the subnanomolar range with EC50 values ​​of 0.48 nM (scDb3-43xhuU3), 0.29 nM (scDb3-43xhuU3-scFv3-43) and 0.22 nM (scFv3-43). Petition 870260062263, dated 06 / 25 / 2026, page 155 / 317 145 / 148 scDb3-43xhuU3-scFv3-43) (Figure 33E).

[0338] The killing of target cells by different bispecific antibodies was analyzed using MCF-7 cells expressing HER-3 and human PBMCs. One day before treatment, 20,000 MCF-7 cells were seeded per well in a 96-well plate. The medium was removed and replaced with a titration of different bispecific and multivalent antibodies in fresh medium. After 1 hour of incubation at room temperature, 200,000 PBMCs per well were added and incubated for a further 48 hours at 37°C. Cell viability was measured by MTT assay. All three bispecific antibodies showed dose-dependent ability to kill MCF-7 cells in the picomolar range with EC50 values ​​of 84 pM (scDb3-43xhuU3), 34 pM (scDb3-43xhuU3-scFv3-43), and 32 pM (scFv3-43 scDb3-43xhuU3-scFv3-43) (Figure 33F; black lines). In the presence of PBMCs, no reduced cell viability of MCF-7 cells was observed (Figure 33F; gray lines). Example 23 IgG 3-43 binds to HER3-Fc fusion proteins mutated in domains III and IV.

[0339] Somatic HER3 mutations in domains III and IV were cloned via the Q5® site-directed mutagenesis kit (NEB). In addition to a hot spot mutation (T335A), six other mutations in domains III and IV (T389I, M406K, R453H, Y464C, D492H, K498I) were expressed as HER3-Fc fusion proteins, which were produced in transiently transfected HEK 293-6E cells and purified via protein chromatography A. SDS-PAGE analysis confirmed the purity of the proteins and showed a single band of approximately 140 kDa under reducing conditions (Figure 34A). The binding capacity of 3-43-IgG to different mutated HER3-Fc fusion proteins was analyzed by ELISA and compared with the binding to the protein of Petition 870260062263, dated 06 / 25 / 2026, page 156 / 317 146 / 148 wild-type (non-mutated) HER3-Fc fusion proteins. The different HER3-Fc fusion proteins were coated onto polystyrene microtiter plates at a concentration of 2 μg / mL diluted in PBS. The remaining binding sites were blocked with PBS, 2% skim milk (MPBS). The plates were then incubated with serial dilutions of anti-HER3 antibodies, 3-43-IgG and 3M6-IgG. After washing, bound antibodies were detected with an anti-human Fab antibody conjugated with HRP and TMB, H2O2 as substrate. IgG 3-43 showed binding to all mutations tested, including the T335A hot spot mutation. IgG 3M6, which binds to HER3 domain I, was included as a positive control and showed binding at 100 nM similar to the signals observed for wild-type HER3-Fc.Although IgG 3-43 was able to bind to all mutant forms of HER3, for some of the mutants a reduced saturation binding was observed in ELISA with immobilized HER3-Fc mutants (Figure 34B), although the EC50 values ​​were in a similar range for all HER3 mutants (0.1 to 0.3 nM). Tables Table 2 Monovalent and Bivalent Affinity

[00340] KDs were measured using the Attana system. nalito max (Signal) a (1 / (M*s)) d (1 / s) D (nM) I (Signal) hi2 (SignalA2) ER3-his 3.55 22000 .00696 1.2 .45 ER3-Fc 37.32 68000 .000103 .22 .14 Table 3 Cellular Binding Properties of IgG 3-43.

[00341] The EC50 values ​​for binding to the indicated cells were evaluated by flow cytometry: Petition 870260062263, dated 06 / 25 / 2026, page 157 / 317 147 / 148 Cell Lines EC50 Value (pM) MCF-7 37 FaDu 30 BT474 74 A431 45 NCI-N87 27 A549 53 Table 4 Binding Properties of scDb hu225x3-43-Fc.

[00342] EC50 values ​​[nM dimer] for protein binding EGFR-ECD and HER3-ECD were determined by ELISA. EC50 [nM dimer] values ​​for binding to FaDu cells were evaluated by flow cytometry. ELISA FACS EGFR-ECD HER3-ECD FaDu cetuximab IgG 3-43 0.18 - 0.2 - 0.33 0.30 scDb hu225x3-43-Fc 0.2 0.24 0.23 Table 5 Properties of scFv3-43-Fc-scTRAIL.

[00343] EC50 values ​​[nM monomer] for binding to HER3 and Human TRAIL-R2 antigens were determined by ELISA. Fc fusion proteins of the extracellular domains of HER3 or human TRAIL-R2 were used as antigens. Construction HER3 TRAIL-R2 scFv3-43-Fc-scTRAIL 0.33 2.84 Table 6 Induction of Cell Death by scFv3-43-Fc-scTRAIL.

[00344] The EC50 [nM monomer] values ​​of cell death induction in Colo205 cells were determined in the absence and presence of bortezomib (650 nM) and in the absence and presence of a blocking antibody (200x molar excess). The effects of scFv3-43-Fc-scTRAIL were Petition 870260062263, dated 06 / 25 / 2026, pp. 158 / 317 148 / 148 compared to the non-targeted Fc-scTRAIL fusion protein. Construction EC50 value (pM) without blocking antibody EC50 value (pM) with blocking antibody without BZB 650 nM of BZB without BZB 650 nM of BZB ScFv3-43-Fc-scTRAIL 31.0 6.7 217.5 32.6 Fc-scTRAIL 97.6 21.4 129.5 34.2 Table 7 Binding properties of Db3-43xhu225-Ig.

[00345] ECso [nM] values ​​of binding to the extracellular domain (ECD) of EGFR and HER3 fusion proteins were determined by ELISA. ECso [nM] values ​​of binding to MCF-7, SKBR-3, and FaDu cells were evaluated by flow cytometry. ELISA Flow Cytometry Construction GFR-ECD ER3-ECD CF-7 KBR-3 aDu Db3-43xhu225-lg Table 8 ECso values ​​from scFv3-43-FcscTRAIL and Fc-scTRAIL cell death induction assays in the presence or absence of bortezomib (BZB)

[00346] ECso [pM] values ​​were determined after cells were treated for 16 hours with the protein alone or in combination with bortezomib. less than 50% cell death; mean ± SD. scFv3-43-Fc-scTRAIL Fc-scTRAIL* - BZB + BZB - BZB + BZB WM793 4.25 ± 2.40 0.39 ±0.18 22.0 + 1.75 2.87 ± 1.51 WM1366 48.2 + 7.29 3.58 ± 1.40 20.1 + 4.77 4.84 + 1.00 WM35 4.96 ± 1.81 0.17 + 0.17 691 + 342 0.64 ±0.11 A375 - 0.72 ±0.41 5003 + 4376 1.06 ±0.32 Mel-Juso - 1.00 + 0.04 - 4.10 ± 1.60 MeWo - 4.63 + 2.89 - 4.88 ± 0.87 Petition 870260062263, dated 06 / 25 / 2026, page 159 / 317

Claims

1 / 3 Claims 1. ANTIGEN-BINDING PROTEIN, characterized by specifically binding to a conformational epitope formed by domains III and IV of the human epidermal growth factor receptor 3 (HER3), comprising: (a) a CDRH1 comprising amino acids 32-37 according to SEQ ID NO: 2, a CDRH2 comprising amino acids 52-69 according to SEQ ID NO: 2, and a CDRH3 comprising amino acids 102-112 according to SEQ ID NO: 2, and / or (b) a CDRL1 comprising amino acids 23-33 according to SEQ ID NO: 3, a CDRL2 comprising amino acids 49-55 according to SEQ ID NO: 3, and a CDRL3 comprising amino acids 88-98 according to SEQ ID NO:

3.

2. PROTEIN, according to claim 1, characterized in that the conformational epitope is formed by amino acids 329 to 531 of domain III of HER3, according to SEQ ID NO: 1 and by amino acids 532 to 587 of domain IV of HER3, according to SEQ ID NO:

1.

3. PROTEIN, according to any one of claims 1 to 2, characterized by: (a) binding to cells expressing HER3 with an EC50 value below 15 nM; and / or (b) binding to monomeric HER3 with a KD less than 100 nM; and / or (c) inhibiting heregulin-induced HER3 phosphorylation with an IC50 value below 10 nM.

4. PROTEIN, according to any one of claims 1 to 3, characterized by inhibiting (i) the binding of HER3 to its ligand, (ii) the activation and / or signaling of the receptor, (iii) inducing the internalization of HER3, (iv) inhibiting cell proliferation and / or (v) tumor growth.

5. PROTEIN, according to any one of claims 1 to 4, characterized by being monospecific, bispecific or multispecific.

6. FUSION PROTEIN, characterized by comprising the antigen-binding protein, as defined in any one of claims 1 to 5, further comprising at least one pharmaceutically active portion, wherein the pharmaceutically active portion comprises a single-chain TRAIL domain (scTRAIL), wherein the fusion protein comprises SEQ ID NO:

11.

7. Fusion protein, according to claim 6, characterized by further comprising a dimerization domain.

8. PHARMACEUTICAL COMPOSITION, characterized by comprising the antigen-binding protein, as defined in any one of claims 1 to 5, or the fusion protein, as defined in any one of claims 6 to 7, and further comprising one or more pharmaceutically acceptable carriers, diluents, excipients, fillers, binders, lubricants, glidants, disintegrants, adsorbents and / or preservatives.

9. COMPOSITION, according to claim 8, characterized by being for medical use, in particular for inhibiting tumor growth or treating cancer.

10. ANTIGEN-BINDING PROTEIN, as defined in any one of claims 1 to 5, or fusion protein, as defined in any one of claims 6 to 7, characterized by being for medical use, in particular for inhibiting tumor growth or treating cancer. Petition 870260062263, dated 06 / 25 / 2026, p. 161 / 317 3 / 3 11. USE OF AN ANTIGEN-BINDING PROTEIN, as defined in any one of claims 1 to 5, or of a fusion protein, as defined in any one of claims 6 to 7, characterized in that it is for the manufacture of a medicament to inhibit tumor growth or to treat cancer.

12. USE OF THE PHARMACEUTICAL COMPOSITION, as defined in claim 8, characterized by being for the manufacture of a medicament to inhibit tumor growth or to treat cancer. Petition 870260062263, dated 06 / 25 / 2026, p. 162 / 317

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