Bispecific antibodies that bind to HER-3 and either HER-2 or EGFR

Abstract

The present invention relates to bispecific binding molecules, in particular antibodies, targeting HER3 and another antigen selected from HER-2 and EGFR antigens, methods for the production of such molecules, compositions and their uses.

Description

[Technical Field]

[0001] The present invention relates to bispecific antigen-binding molecules, in particular antibodies, that target HER-3 and another antigen selected from HER-2 and EGFR, methods for the production of such molecules, compositions, and uses thereof. [Background technology]

[0002] The HER family, which includes four tyrosine kinase receptors (EGFR / HER1, HER2, HER3, and HER4), activates multiple, partially redundant, interconnected downstream signaling cascades involved in cell proliferation, such as the MAPK and PI3K / AKT pathways. HER receptors share a common general structure consisting of an extracellular domain, a single transmembrane domain, and an intracellular domain containing a conserved catalytic kinase domain and a carboxy-terminal tail. A fundamental aspect of signaling in this family is the dimerization of two receptors. It is the homodimerization and heterodimerization of two HER family members and transphosphorylation of their intracellular regions that generates the initial signal that results in the activation of multiple downstream signaling pathways. The four members of the HER family can form 28 homo- and heterodimers.

[0003] Deregulation of HER signaling due to gene amplification or mutation is observed in many human tumors, and a wealth of experimental evidence supports the pathogenic role of these events in cancer pathogenesis. Aberrant HER signaling has been observed in numerous solid tumors (e.g., lung, colorectal, pancreatic). For example, there is evidence that the HER family is deregulated in pancreatic cancer. EGFR is expressed in 45–95% of pancreatic cancers, and its expression generally correlates with a worse outcome in resected pancreatic cancers. HER2 overexpression has also been described in 7–58% of pancreatic cancers, and HER2-amplified pancreatic cancers exhibit atypical metastatic patterns, suggesting that HER2 may also be an important driver of tumorigenesis in pancreatic cancer. In addition, HER3 expression correlates with tumor progression and reduced survival in pancreatic cancer patients.

[0004] Pancreatic cancer is the fourth leading cause of cancer deaths in Europe, with the number of cases continuing to increase annually (+2% in men and +10% in women). It has a very poor prognosis, even when diagnosed early. It is one of the few cancers for which survival has not improved significantly over the past 40 years: survival is poorer than 20% and 5% at 1 and 5 years, respectively. Pancreatic cancer remains a rare disease, with as many deaths recorded worldwide in 2020 (466,003) as newly diagnosed cases (495,773), due to the lack of effective treatments. Currently, pancreatic adenocarcinoma (90% of pancreatic cancers) is treated surgically, with chemotherapy, or with a combination of radiation and chemotherapy, but with limited results. The launch of gemcitabine in 1996 as a first-line treatment improved recurrence-free survival by a median of 1.3 months, with overall survival (OS) at 1 year ranging from 2% to 18%. Since 2005, despite various clinical trials mainly involving combinations but little innovation, only two drugs, Tarceva® (erlotinib) and Abraxane® (nab-paclitaxel), have been approved for pancreatic cancer. Erlotinib, the first targeted therapy, improved recurrence-free survival in association with gemcitabine for a median of just one month. Regarding HER3 inhibitors, various molecules are being studied and evaluated in clinical trials (Phase I / II).

[0005] Treatment with therapeutic agents that directly target HER receptors or downstream kinases often faces acquired resistance or is limited by the inherent robustness of the signaling network.

[0006] In such cases, combination therapy has emerged as a natural countermeasure, but its optimal design is not straightforward and may depend on the tumor or its subtype, thus requiring prior patient stratification (Fitzgerald JB, Schoeberl B, Nielsen UB, Sorger PK. Systems biology and combination therapy in the quest for clinical efficacy. Nat Chem Biol. 2006;2:458-66). Currently available options for inhibiting HER signaling include small molecule tyrosine kinase inhibitors (TKIs), such as lapatinib or erlotinib, and therapeutic antibodies, such as cetuximab or trastuzumab. Indeed, in contrast to TKIs, which are limited to signaling modulation, antibodies represent a powerful approach that induces immunological effects in addition to signaling reduction, which contributes to tumor elimination.

[0007] Combined antibody-based therapies have been proposed by many authors. Targeting HER dimers, especially EGFR / HER2 heterodimers, with mAb combinations has been demonstrated to be advantageous for inhibiting pancreatic tumor growth.

[0008] Bispecific antibodies (BsAbs) that combine the targets of two mAbs have also been designed, but they have complex design issues that usually result in poor manufacturability and stability, and in an in vivo pancreatic cancer model, tumors continued to grow even during treatment with the bispecific antibodies, leaving room for improvement in the efficacy of such antibodies.

[0009] For example, Liu and colleagues (Liu et al., A Novel Antibody Engineering Strategy for Making Monovalent Bispecific Heterodimeric IgG Antibodies by Electrostatic Steering Mechanism., J Biol. Chem. 2015;290(12):7535-62) described the construction and characterization of bispecific anti-EGFR and anti-HER2 antibodies utilizing panitumumab and trastuzumab sequences, respectively. The antibodies showed improved activity against EGFR+ / HER2+ cell lines in vitro and in vivo, although the activity of these antibodies still needs to be improved.

[0010] MM-111, an anti-HER2 / anti-HER3 bispecific antibody developed by Merrimack Pharma (McDonagh et al., 2012; Spiess et al., 2015), was studied in patients with HER2-positive cancer of the distal esophagus, gastroesophageal (GE) junction, and stomach. The phase II clinical trial was discontinued due to non-significant results.

[0011] Durigotuzumab, an anti-EGFR / anti-HER3 bispecific antibody (Schaefer et al., 2011b; Spiess et al., 2015), was compared with cetuximab in combination with FOLFIRI as second-line therapy in patients with metastatic colorectal cancer. In this trial, durigotuzumab did not demonstrate benefit over cetuximab in combination with FOLFIRI and was abandoned. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] EP0359282 [Patent Document 2] EP0667165 [Patent Document 3] US6217866 [Patent Document 4] EP0590058 [Patent Document 5] US5821337 [Patent Document 6] US8075890 [Patent Document 7] US6407213 [Patent Document 8] US6054297 [Patent Document 9] US5772997 [Patent Document 10] US6165464 [Patent Document 11] US6399063 [Patent Document 12] US6639055 [Patent Document 13] U.S. Patent No. 5,225,539 [Patent Document 14] WO2012020059 [Patent Document 15] WO2020 / 136566 [Patent Document 16] U.S. Patent Application No. 2014 / 0200331 [Patent Document 17] U.S. Patent Application No. 2014 / 150973 [Patent Document 18] U.S. Patent Application No. 2014 / 0154254 [Patent Document 19] WO2007 / 147901 [Patent Document 20] WO2013 / 005194 [Non-patent literature]

[0013] [Non-Patent Document 1] Fitzgerald JB, Schoeberl B, Nielsen UB, Sorger PK., Systems biology and combination therapy in the quest for clinical efficacy., Nat Chem Biol. 2006; 2:458~66 pages

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[0014] In view of the above, there remains a need for improved bispecific antibodies that target HER receptors for treating tumors. [Means for solving the problem]

[0015] The present inventors have therefore designed novel bispecific antibodies targeting HER3 and another antigen selected from either EGFR or HER2, which are useful in the treatment of various cancers.

[0016] More specifically, the present invention relates to (i) a Fab fragment comprising the VH-CH1 heavy chain associated with the VL-CL light chain of antibody 1 (Ab1), and (ii) a Fab fragment comprising a VH-CH1 heavy chain associated with a VL-CL light chain of antibody 2 (Ab2); a bispecific antigen-binding fragment capable of simultaneously binding to HER-3 and to another antigen selected from either a HER-2 or EGFR antigen, comprising: the N-terminal end of the VH domain of the Fab fragment of Ab1 is linked to the C-terminal end of the CH1 domain of the Fab fragment of Ab2 via a polypeptide linker; one of Ab1 or Ab2 is patritumab or a functional derivative thereof; the other of Ab1 or Ab2 is selected from the group consisting of trastuzumab or a functional derivative thereof, matuzumab or a functional derivative thereof, and cetuximab or a functional derivative thereof; Bispecific antigen-binding fragments are provided.

[0017] In a preferred embodiment, Ab2 is patritumab or a functional derivative thereof and Ab1 is selected from the group consisting of trastuzumab or a functional derivative thereof, matuzumab or a functional derivative thereof, and cetuximab or a functional derivative thereof.

[0018] In certain embodiments, a functional derivative of cetuximab is a humanized form comprising VH and VL chain amino acid sequences that are at least 80% identical to the VH and VL chain amino acid sequences of cetuximab, respectively.

[0019] In certain embodiments, the CH1 and CL domains of Ab1 have sequences that differ from the CH1 and CL domains of Ab2.

[0020] In certain embodiments, the Fab CH1 domain of one of Ab1 or Ab2 is a mutated domain derived from the CH1 domain of an immunoglobulin by substitution of a threonine residue at position 192 of said CH1 domain with glutamic acid, and the cognate CL domain is a mutated domain derived from the CL domain of an immunoglobulin by substitution of an asparagine residue at position 137 of said CL domain with a lysine residue and a serine residue at position 114 of said CL domain with an alanine residue, and / or one or the other of Ab1 or Ab2 is a mutated domain derived from the CL domain of an immunoglobulin by substitution of a lysine residue at position 137 of said CL domain with an alanine residue. The CH1 domain is a mutated domain derived from an immunoglobulin CH1 domain by substitution of a glutamine for the leucine residue at position 143 of the CH1 domain and a valine for the serine residue at position 188 of the CH1 domain, and the cognate CL domain is a mutated domain derived from an immunoglobulin CL domain by substitution of a threonine for the valine residue at position 133 of the CL domain and a valine for the serine residue at position 176 of the CL domain.

[0021] In certain embodiments, the polypeptide linker sequence comprises or consists of the amino acid sequence: EPKX1CDKX2HX3X4PPX5PAPELLGGPX6X7PPX8PX9PX10GG (SEQ ID NO: 33) (in the sequence, X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, which may be identical or different, are any amino acid).

[0022] The present invention also provides a bispecific molecule comprising two identical antigen-binding arms, each consisting of an antigen-binding fragment as defined above.

[0023] In a preferred embodiment, the bispecific molecule of the invention is a full-length antibody comprising two heavy chains and four light chains, Each heavy chain is a. comprises an immunoglobulin Fc region including the hinge-CH2-CH3 domain; b. the Fc region is linked to the Ab1 Fab VH-CH1 heavy chain by the hinge domain, which connects the N-terminal end of the CH2 domain to the C-terminal end of the Ab1 CH1 domain; c. which is then linked to the Fab VH-CH1 heavy chain of Ab2 by a polypeptide linker sequence connecting the N-terminal end of the VH domain of Ab1 to the C-terminal end of the CH1 domain of Ab2; The four light chains include two Fab VL-CL light chains of Ab1 and two Fab VL-CL light chains of Ab2 associated with their cognate heavy chain domains.

[0024] In another embodiment, the bispecific molecule comprises a) two heavy chains each comprising, preferably consisting of, SEQ ID NO: 27, and b) four light chains, two comprising, preferably consisting of, SEQ ID NO: 14 and the other two comprising, preferably consisting of, SEQ ID NO: 18.

[0025] In another embodiment, the bispecific molecule comprises a) two heavy chains each comprising, preferably consisting of, SEQ ID NO: 29, and b) four light chains, two comprising, preferably consisting of, SEQ ID NO: 14 and the other two comprising, preferably consisting of, SEQ ID NO: 16.

[0026] In another embodiment, the bispecific molecule comprises a) two heavy chains each comprising, preferably consisting of, SEQ ID NO: 31, and b) four light chains, two comprising, preferably consisting of, SEQ ID NO: 14 and the other two comprising, preferably consisting of, SEQ ID NO: 20.

[0027] The present invention also provides a method for producing a bispecific molecule of the invention, comprising the steps of: a. culturing in suitable medium and culture conditions a host cell expressing an antibody heavy chain as defined above and an antibody light chain as defined above; b. recovering the produced antibody from the culture medium or the cultured cells; The present invention relates to a method comprising:

[0028] The present invention also relates to a bispecific antigen-binding fragment or a bispecific molecule of the invention for use as a medicament.

[0029] The present invention also relates to a bispecific antigen-binding fragment or a bispecific molecule of the invention for use in the treatment of cancer, preferably a solid tumor, such as pancreatic cancer, head and neck cancer, colorectal cancer, breast cancer or lung cancer, preferably for use in the treatment of pancreatic cancer. [Brief explanation of the drawings]

[0030] [Figure 1] Schematic diagram of a bispecific antibody of the present invention. [Figure 2] Dual engagement of two antigens simultaneously by ELISA. Binding curves showing simultaneous binding of (A) Patri-Trastu-Fc to immobilized HER3-Fc and HER2-His (left panel) and immobilized HER2-Fc and HER3-His (right panel), (B) Patri-Matu-Fc to immobilized HER3-Fc and EGFR-His (left panel) and immobilized EGFR-Fc and HER3-His (right panel), or (C) Patri-Cetu-Fc to immobilized HER3-Fc and EGFR-His (left panel) and immobilized EGFR-Fc and HER3-His (right panel). Half-maximal efficient concentrations (EC50) obtained from ELISA binding curves are shown. [Figure 3]Biological properties of bispecific antibodies inhibiting AKT (pAKT) and ERK (pERK) phosphorylation. Cell lines (A) were pre-stimulated with bispecific antibodies (B) for 20 min, followed by addition of the NRG1 / EGF ligand mix for another 10 min. After cell lysis, pAKT and pERK expression levels were quantified by HTRF. The TR-FRET signal (665 nm / 620 nm emission ratio) was measured in a Pherastar reader relative to the maximal phosphorylation obtained in NRG1 / EGFR-stimulated cells without bispecific antibodies (100%; medium). Phosphorylation levels are presented as percentages ranging from 0% (white) to 100% (black) (corresponding to phosphorylation in the absence of BsAb "culture medium"). [Figure 4] In vivo efficacy of bispecific antibodies in a nude mouse model. Tumor growth (left panel) and survival (center panel) of bispecific antibody-treated mice xenografted with Sw1990 (A) and PDX P2846 (B) PDAC cells. The % tumor growth inhibition (TGI) and median survival benefit (days) at the end of treatment are shown in the right panel. The gray area corresponds to the duration of antibody treatment. [Figure 5] Intratumoral penetration of bispecific antibodies in Sw-1990 xenografts. Immunohistochemical analysis of Sw1990 tumor penetration by bispecific antibodies. Bispecific antibodies are labeled using peroxidase-conjugated anti-human Fc. BsAb IRR is a control bispecific antibody targeting CD19 and CD3. 9‰NaCl solution was used as a negative control. Tumor penetration of Patri-Cetu-Fc was visualized in whole tumor slices grossly (left) and under a ×40 microscope (right). [Figure 6-1]Anti-tumor features of the TME are observed in a Sw1990 xenograft pancreatic mouse model after treatment with bispecific antibodies of the present invention. (A) Immunohistochemical analysis to monitor CD31+ angiogenesis in excised Sw1990 xenografts from treated mice. Microscopic images of Patri-Cetu-Fc vs. BsAb IRR and NaCl-treated xenograft sections are shown as examples (left panel). The average size of CD31+ microvessels in the tumor was quantified using Qupath software in whole tumor sections. (B) NK cell immunophenotyping by flow cytometry of excised Sw1990 xenografts from BiXAb-treated mice. Dissociated cells were labeled with human L / D, CD45, CD3, CD19, CD49, NKp46, IFNg, and CD107a-specific antibodies. After negative selection of CD45+CD3+CD19+ cells, positive gated CD49+NKp46+ NK cells were selected before CD107a and intracellular IFNg labeling. C) Analysis of in vivo ErbB degradation. Immunofluorescence microscopy images of EGFR staining in Patri-Cetu-Fc versus BsAb IRR and NaCl-treated Sw1990 xenograft sections (top panel). Western blot analysis of ErbB receptor expression in extracts of Sw1990 xenografts treated with NaCl solution, Patri-Trastu-Fc, Patri-Matu-Fc, and Patri-Cetu-Fc, respectively. Tubulin was used as a loading control. Western blots were visualized and quantified using a LI-COR Odyssey imaging system (middle panel). Quantification of ErbB expression normalized to tubulin in each group of excised xenografts (bottom panel). Each experiment was performed in three Sw1990 xenograft mice. BsAb IRR is a negative control that simultaneously binds to human CD19 and CD3. [Figure 6-2]Anti-tumor features of the TME are observed in a Sw1990 xenograft pancreatic mouse model after treatment with bispecific antibodies of the present invention. (A) Immunohistochemical analysis to monitor CD31+ angiogenesis in excised Sw1990 xenografts from treated mice. Microscopic images of Patri-Cetu-Fc vs. BsAb IRR and NaCl-treated xenograft sections are shown as examples (left panel). The average size of CD31+ microvessels in the tumor was quantified using Qupath software in whole tumor sections. (B) NK cell immunophenotyping by flow cytometry of excised Sw1990 xenografts from BiXAb-treated mice. Dissociated cells were labeled with human L / D, CD45, CD3, CD19, CD49, NKp46, IFNg, and CD107a-specific antibodies. After negative selection of CD45+CD3+CD19+ cells, positive gated CD49+NKp46+ NK cells were selected before CD107a and intracellular IFNg labeling. C) Analysis of in vivo ErbB degradation. Immunofluorescence microscopy images of EGFR staining in Patri-Cetu-Fc versus BsAb IRR and NaCl-treated Sw1990 xenograft sections (top panel). Western blot analysis of ErbB receptor expression in extracts of Sw1990 xenografts treated with NaCl solution, Patri-Trastu-Fc, Patri-Matu-Fc, and Patri-Cetu-Fc, respectively. Tubulin was used as a loading control. Western blots were visualized and quantified using a LI-COR Odyssey imaging system (middle panel). Quantification of ErbB expression normalized to tubulin in each group of excised xenografts (bottom panel). Each experiment was performed in three Sw1990 xenograft mice. BsAb IRR is a negative control that simultaneously binds to human CD19 and CD3. [Figure 7]In vivo efficacy of bispecific antibody Patri-Cetu-Fc versus monospecific antibodies. Tumor growth (upper panel) and survival (lower panel) of treated mice xenografted with Sw1990 cells. All antibodies in different mouse cohorts contained the same molar antibody concentration based on their Fc content: Patri-Cetu-Fc at 17 mg / kg, monoclonal controls (patritumab or cetuximab) at 10 mg / kg, and combinations (patritumab + cetuximab) at 5 mg / kg each. The gray area corresponds to the duration of antibody treatment. [Figure 8] Binding profiles of BMX003-001 (Patri-Cetu-Fc), BMX003-010 (humanized Patri-Cetu-Fc), and BMX003-011 (humanized Patri-Cetu-Fc modified) in HER3 antigen binding ELISA (n=1). [Figure 9] Binding profiles of BMX003-001 (Patri-Cetu-Fc), BMX003-010 (humanized Patri-Cetu-Fc), and BMX003-011 (humanized Patri-Cetu-Fc modified) in EGFR antigen binding ELISA (n=3). [Figure 10] The ability of BMX003-001 (Patri-Cetu-Fc), BMX003-010 (humanized Patri-Cetu-Fc), and BMX003-011 (humanized Patri-Cetu-Fc modified) to bind to HER3 and EGFR proteins expressed on the cell surface of SW-1990 cells as measured using flow cytometry (n=3). [Figure 11] Biological properties of BMX003-001 (Patri-Cetu-Fc), BMX003-010 (humanized Patri-Cetu-Fc), and BMX003-011 (humanized Patri-Cetu-Fc modified), which inhibit phosphorylation of AKT (A) and ERK (B) in response to growth factors. [Figure 12]The ability of BMX003-001 (Patri-Cetu-Fc), BMX003-010 (humanized Patri-Cetu-Fc), and BMX003-011 (humanized Patri-Cetu-Fc modified) to induce NK cell degranulation (CD107a expression) in the presence of HCT-116 cancer cells (two independent donors A and B). [Figure 13] In vivo efficacy study of BMX003-001 (Patri-Cetu-Fc), BMX003-010 (humanized Patri-Cetu-Fc), and BMX003-011 (humanized modified Patri-Cetu-Fc) in the SW1990 pancreatic cancer xenograft (xenograph) model (BMX003-01, BMX003-010, and BMX003-011 were used at 8.5 mg / kg, and BMX control was used at 17 mg / kg). DETAILED DESCRIPTION OF THE INVENTION

[0031] Definition: The basic structure of a naturally occurring antibody molecule is a Y-shaped tetrameric quaternary structure consisting of two identical heavy chains and two identical light chains held together by non-covalent interactions and interchain disulfide bonds.

[0032] In mammalian species, there are five types of heavy chains: α, δ, ε, γ, and μ, which determine the immunoglobulin classes (isotypes): IgA, IgD, IgE, IgG, and IgM, respectively. The heavy chain N-terminal variable domain (VH) is followed by a constant region containing three domains (numbered CH1, CH2, and CH3 from N- to C-terminus) in heavy chains γ, α, and δ, while the constant region of heavy chains μ and ε is composed of four domains (numbered CH1, CH2, CH3, and CH4 from N- to C-terminus). The CH1 and CH2 domains of IgA, IgG and IgD are separated by a flexible hinge that varies in length between different classes and, in the case of IgA and IgG, different subtypes: IgG1, IgG2, IgG3 and IgG4 have hinges of 15, 12, 62 (or 77) and 12 amino acids, respectively, and IgA1 and IgA2 have hinges of 20 and 7 amino acids, respectively.

[0033] There are two types of light chains: λ and κ, which can associate with any of the heavy chain isotypes, but both will be of the same type in a given antibody molecule. Both light chains appear to be functionally identical. Their N-terminal variable domain (VL) is followed by a constant region consisting of a single domain designated CL.

[0034] Heavy and light chains pair by protein / protein interactions between the CH1 and CL domains and by VH / VL interactions, and the two heavy chains associate by protein / protein interactions between their CH3 domains. The structure of immunoglobulin molecules is generally stabilized by interchain disulfide bonds between the CH1 and CL domains and between the hinges.

[0035] The antigen-binding region or antigen-binding fragment corresponds to the arms of a Y-shaped structure, each consisting of an intact light chain paired with the VH and CH1 domains of a heavy chain, and is referred to as a Fab fragment (for fragment antigen-binding). Fab fragments were initially generated from native immunoglobulin molecules by papain digestion, which cleaves the antibody molecule at the hinge region, amino-terminal to the interchain disulfide bond, thereby releasing two identical antigen-binding arms. Other proteases, such as pepsin, also cleave antibody molecules at the hinge region, but carboxy-terminal to the interchain disulfide bond, releasing a fragment consisting of two identical Fab fragments linked via disulfide bonds and the remainder; reduction of the disulfide bond in the F(ab')2 fragment generates a Fab' fragment.

[0036] The part of the antigen-binding region corresponding to the VH and VL domains is called the Fv fragment (for fragment variable); it contains the CDRs (complementarity-determining regions) that form the antigen-binding site (also named paratope).

[0037] The effector region of an antibody responsible for its binding to effector molecules or cells corresponds to the stem of the Y-shaped structure and contains the paired CH2 and CH3 domains of the heavy chain (or CH2, CH3 and CH4 domains, depending on the class of the antibody) and is called the Fc (fragment crystallizable) region.

[0038] Due to the identity of the two heavy chains and two light chains, a naturally occurring antibody molecule has two identical antigen-binding sites and therefore binds simultaneously to two identical epitopes.

[0039] An antibody "specifically binds" to a target antigen if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances. "Specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding refers to preferential binding.

[0040] The terms "subject," "individual," and "patient" are used interchangeably herein and refer to a mammal being assessed for treatment and / or treated. The subject may be a human, but also includes other mammals, particularly mammals useful as experimental models for human disease, such as mice, rats, rabbits, dogs, etc.

[0041] The term "treatment" or "treating" refers to an act, application, or therapy by which a subject, including a human, is subjected to medical assistance for the purpose of directly or indirectly improving the subject's condition. In particular, the term, in some embodiments, refers to reducing the incidence or alleviating symptoms, eliminating recurrence, preventing recurrence, preventing the incidence, improving symptoms, improving prognosis, or a combination thereof. One of skill in the art will understand that treatment does not necessarily result in the complete absence or elimination of symptoms. For example, with respect to cancer, "treatment" or "treating" can refer to slowing neoplastic or malignant cell growth, proliferation, or metastasis, preventing or delaying the onset of neoplastic or malignant cell growth, proliferation, or metastasis, or some combination thereof.

[0042] The term "tumor" is used interchangeably herein with the term "cancer," e.g., both terms encompass solid and liquid, e.g., diffuse or circulating, tumors. As used herein, the term "cancer" or "tumor" includes pre-malignant as well as malignant cancers and tumors.

[0043] The terms "mutated derivative," "variant," or "functional derivative" designate a sequence that differs from a reference parent sequence by the deletion, substitution, or insertion of one or several amino acids. Preferably, a mutated derivative has an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the native sequence.

[0044] Cetuximab (Erbitux®; ImClone / Lilly, Merck-Serono) is a chimeric mouse-human monoclonal antibody (ATCC HB-9764 & ATCC-97-63) that targets the epidermal growth factor receptor (EGFR). See also EP 0359282, EP 0667165 and US 6217866. Cetuximab is approved for use as a treatment for colorectal cancer and squamous cell carcinoma of the head and neck.

[0045] Trastuzumab (Herceptin®; Genentech / Roche) is a humanized IgG1 that interferes with the HER2 / neu receptor. See also EP0590058, US5821337, US8075890, US6407213, US6054297, US5772997, US6165464, US6399063, and US6639055. Its indications are as an adjuvant and in the treatment of metastatic breast and metastatic gastric cancer.

[0046] Patritumab (U1-59 / U3-1287 / AMG888) is a fully human anti-HER3 monoclonal antibody with an IgG1-like isotype that possesses 1-3 nM affinity for its target and is directed against the juxtamembrane portion of HER3. Patritumab has shown promising antitumor effects in vitro and in vivo in lung, head and neck, and breast cancer models, where it effectively blocks HER3 phosphorylation, degrades the HER3 receptor, and reduces tumor burden.

[0047] Matuzumab (EMD72000) is a humanized IgG1 derived from the murine antibody MAb425 (EMD55900) produced by lymphocyte hybridization from the spleens of BALB / c mice immunized with human A431 squamous cell carcinoma cells. Matuzumab has been tested in Phase I clinical trials against a number of cancers, both alone and in combination with chemotherapy.

[0048] 1. Design of bispecific antigen-binding fragments The present invention provides bispecific, bivalent antigen-binding fragments comprising one binding site for HER-3 and one binding site for another antigen selected from either a HER-2 or EGFR antigen. In certain embodiments, the bispecific antigen-binding fragment is capable of simultaneous binding to HER-3 and HER-2. In another specific embodiment, the bispecific antigen-binding fragment is capable of simultaneous binding to HER-3 and EGFR.

[0049] The antigen-binding fragment of the present invention essentially consists of tandemly arranged Fab fragments. The present invention particularly relates to a bispecific antigen-binding fragment constructed using the amino acid sequences of the heavy chain (VH) and light chain (VL) variable regions of two monoclonal antibodies, "Ab1" and "Ab2," wherein one of Ab1 or Ab2 is patritumab or a functional derivative thereof, and the other of Ab1 or Ab2 is selected from the group consisting of trastuzumab or a functional derivative thereof, matuzumab or a functional derivative thereof, and cetuximab or a functional derivative thereof.

[0050] The bispecific antigen-binding fragment comprises: (i) a Fab fragment comprising the VH-CH1 heavy chain associated with the VL-CL light chain of antibody 1 (Ab1), and (ii) a Fab fragment comprising a VH-CH1 heavy chain associated with a VL-CL light chain of antibody 2 (Ab2); the N-terminal end of the VH domain of the Fab fragment of Ab1 is linked to the C-terminal end of the CH1 domain of the Fab fragment of Ab2 via a polypeptide linker; one of Ab1 or Ab2 is patritumab or a functional derivative thereof; The other of Ab1 or Ab2 is selected from the group consisting of trastuzumab or a functional derivative thereof, matuzumab or a functional derivative thereof, and cetuximab or a functional derivative thereof.

[0051] In certain embodiments, the bispecific antigen-binding fragment comprises two Fab fragments with different CH1 and CL domains. In a preferred embodiment, the bispecific antigen-binding fragment comprises: (i) a Fab fragment having CH1 and CL domains derived from human IgG1 / kappa and VH and VL domains of Ab1; (ii) a Fab fragment having CH1 and CL domains derived from human IgG1 / kappa and VH and VL domains of Ab2; It contains two Fab fragments with different CH1 and CL domains, consisting of:

[0052] In a specific embodiment, Ab1 is patritumab or a functional derivative thereof and Ab2 is trastuzumab or a functional derivative thereof, hi another specific embodiment, Ab1 is trastuzumab or a functional derivative thereof and Ab2 is patritumab or a functional derivative thereof.

[0053] In a specific embodiment, Ab1 is patritumab or a functional derivative thereof and Ab2 is cetuximab or a functional derivative thereof, hi another specific embodiment, Ab1 is cetuximab or a functional derivative thereof and Ab2 is patritumab or a functional derivative thereof.

[0054] In a specific embodiment, Ab1 is patritumab or a functional derivative thereof and Ab2 is matuzumab or a functional derivative thereof, hi another specific embodiment, Ab1 is matuzumab or a functional derivative thereof and Ab2 is patritumab or a functional derivative thereof.

[0055] In a preferred embodiment, Ab2 is patritumab or a functional derivative thereof and Ab1 is selected from the group consisting of trastuzumab or a functional derivative thereof, matuzumab or a functional derivative thereof, and cetuximab or a functional derivative thereof.

[0056] In a particular embodiment, the bispecific antigen-binding fragment comprises a Fab fragment comprising a VH-CH1 heavy chain associated with a VL-CL light chain of patritumab or a functional derivative thereof, - the VH domain comprises, preferably consists of, SEQ ID NO: 5 or a mutated derivative thereof; - the VL domain comprises, preferably consists of, SEQ ID NO: 6 or a mutated derivative thereof; the CH1 domain is preferably the CH1 constant domain of human IgG1 or a mutated derivative thereof; The CL domain is preferably the human kappa constant domain or a mutated derivative thereof.

[0057] In a particular embodiment, the bispecific antigen-binding fragment comprises a Fab fragment comprising a VH-CH1 heavy chain associated with a VL-CL light chain of cetuximab or a functional derivative thereof, - the VH domain comprises, preferably consists of, SEQ ID NO: 7 or a mutated derivative thereof; - the VL domain comprises, preferably consists of, SEQ ID NO: 8 or a mutated derivative thereof; the CH1 domain is preferably the CH1 constant domain of human IgG1 or a mutated derivative thereof; The CL domain is preferably the human kappa constant domain or a mutated derivative thereof.

[0058] In a particular embodiment, the bispecific antigen-binding fragment comprises a Fab fragment comprising a VH-CH1 heavy chain associated with a VL-CL light chain of trastuzumab or a functional derivative thereof, - the VH domain comprises, preferably consists of, SEQ ID NO: 9 or a mutated derivative thereof; - the VL domain comprises, preferably consists of, SEQ ID NO: 10 or a mutated derivative thereof; the CH1 domain is preferably the CH1 constant domain of human IgG1 or a mutated derivative thereof; The CL domain is preferably the human kappa constant domain or a mutated derivative thereof.

[0059] In a particular embodiment, the bispecific antigen-binding fragment comprises a Fab fragment comprising a VH-CH1 heavy chain associated with a VL-CL light chain of matuzumab or a functional derivative thereof, - the VH domain comprises, preferably consists of, SEQ ID NO: 11 or a mutated derivative thereof; - the VL domain comprises, preferably consists of, SEQ ID NO: 12 or a mutated derivative thereof; the CH1 domain is preferably the CH1 constant domain of human IgG1 or a mutated derivative thereof; The CL domain is preferably the human kappa constant domain or a mutated derivative thereof.

[0060] Mutated derivatives The present invention utilizes the wild-type sequences (of patritumab, matuzumab, cetuximab or trastuzumab) or mutated derivatives thereof.

[0061] The terms "mutated derivative," "variant," or "functional derivative" designate a sequence that differs from a reference parent sequence by the deletion, substitution, or insertion of one or several amino acids. Preferably, a mutated derivative or functional variant has an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the native sequence. In certain embodiments, the mutations do not substantially affect the function of the antibody.

[0062] Mutated derivatives or functional variants can include a VH chain comprising an amino acid sequence at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to any of the reference sequences listed herein, a VL chain having an amino acid sequence at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to any of the reference sequences listed herein, or both. Functional variants of patritumab are capable of binding to HER3. Functional variants of cetuximab are capable of binding to EGFR. Functional variants of matuzumab are capable of binding to EGFR. Functional variants of trastuzumab are capable of binding to HER2.

[0063] In some examples, the variants retain a similar antigen binding affinity compared to the reference antibodies described above (e.g., 1×10 -8 Less than 1 x 10, preferably -9 or 1 x 10 -10 In some embodiments, a functional variant of an original antibody has the same binding specificity and an affinity for its target that is at least 50%, e.g., at least 60%, 70%, 80%, or at least 90% of the affinity of the original antibody.

[0064] The affinity of binding is defined by the terms ka (association rate constant), kd (dissociation rate constant) or KD (equilibrium dissociation). Typically, specifically binding, when used in reference to antibodies, is greater than 10 -8 Less than M, e.g., 10 -9 M or 10 -10 refers to an antibody that specifically binds to ("recognizes") its target with an affinity (KD) value of less than 10 M. A lower KD value represents a higher binding affinity (i.e., stronger binding), and therefore, -9 The KD value of -8 The binding affinity is higher than the KD value of

[0065] The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al., J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein molecule of interest. When gaps exist between the two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used.

[0066] In other embodiments, the functional variants described herein may contain one or more mutations (e.g., conservative substitutions) that preferably do not occur in residues predicted to interact with one or more of the CDRs.

[0067] Mutated derivatives or functional variants that are substantially identical to the reference antibodies are described herein.

[0068] The terms "substantially identical" or "instantial" mean that the relevant amino acid sequence of the variant (e.g., in the framework regions (FR), CDRs, VH, or VL domains) differs slightly (e.g., includes conservative amino acid substitutions) compared to the reference antibody, such that the variant has substantially similar binding activity (e.g., affinity, specificity, or both) and biological activity compared to the reference antibody. Such variants can include minor amino acid changes, e.g., one or two substitutions, in the five amino acid sequence of the specified region. Generally, more substitutions can be made in the FR regions as opposed to the CDR regions, as long as they do not adversely affect the binding function of the antibody (e.g., a reduction in binding affinity of more than 50% compared to the original antibody). In some embodiments, the sequence identity can be about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more between the original antibody and the modified antibody. In some embodiments, the modified antibody has the same binding specificity and at least 50% of the affinity of the original antibody. In some embodiments, the mutations do not occur within the CDR regions.

[0069] Conservative substitutions will produce molecules with similar functional and chemical characteristics as the original molecule in which such alteration is made. For example, a "conservative amino acid substitution" can involve replacing a native amino acid residue with another residue that has little or no effect on the polarity or charge of the amino acid residue at that position. Desired amino acid substitutions (whether conservative or non-conservative) can be determined by one of skill in the art. For example, amino acid substitutions can be used to identify critical residues in a molecular sequence or to increase or decrease the affinity of a molecule described herein. Variants containing one or more conservative amino acid substitutions can be prepared according to methods for altering polypeptide sequences known to those of skill in the art, such as those found in references compiling such methods, e.g., Molecular Cloning: A Laboratory Manual, edited by J. Sambrook et al., 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, edited by F.M. Ausubel et al., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0070] The present disclosure also provides antibody variants with improved biological properties of the antibody, for example, having higher or lower binding affinity, or having altered ADCC properties, or having altered efficacy in inhibiting the viability of HER3-, EGFR-, and / or HER2-expressing cells.

[0071] Amino acid sequence variants of antibodies can be prepared by introducing appropriate nucleotide changes into the antibody nucleic acid, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics. Nucleic acid molecules encoding amino acid sequence variants of antibodies can be prepared by a variety of methods known in the art. Such methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of earlier prepared variant or non-variant (natural) versions of the antibody. In one embodiment, the equilibrium dissociation constant (KD) value of an antibody of the invention is 10 -8 Less than M, especially 10 -9 M or 10 -10 The binding affinity is less than M. Binding affinity can be determined using techniques known in the art, such as ELISA or biospecific interaction analysis, or other techniques known in the art.

[0072] Any of the antibodies described herein can be tested according to routine methods to determine its properties, such as antigen-binding activity, antigen-binding specificity, and biological function.

[0073] Any of the antibodies described herein can be modified to contain additional nonproteinaceous moieties known and readily available in the art, for example, by pegylation, hyperglycosylation, conjugation of toxins, radiolabeling, etc. Of interest are modifications that can enhance serum half-life.

[0074] The antibodies of the invention can be glycosylated or non-glycosylated, or can exhibit different glycosylation profiles. In a preferred embodiment, the antibodies are aglycosylated in the variable regions of the heavy and light chains, but glycosylated in the Fc region.

[0075] For example, to remove an N-glycosylation site in the VH domain of cetuximab, Asn at Kabat position H85 is mutated to aspartic acid (D) according to the sequence of SEQ ID NO: 77, or Asn at Kabat position H85 is mutated to glutamic acid (E) according to the sequence of SEQ ID NO: 78.

[0076] Certain mutated derivatives of cetuximab are humanized versions of the reference cetuximab antibody, which in its original form is a chimeric antibody with heavy and light chain variable regions of murine origin. In the humanization approach, the complementarity-determining regions (CDRs) and certain other amino acids from the donor mouse variable region are grafted into a human variable acceptor region and then joined to a human constant region; some CDR amino acids may further be replaced with amino acids found in the human germline sequence of the acceptor region. See, e.g., Riechmann et al., Nature 332:323-327 (1988); U.S. Patent No. 5,225,539.

[0077] The present invention further encompasses bispecific antigen-binding fragments containing humanized versions of the light and / or heavy chains of cetuximab.

[0078] In certain embodiments, Ab1 or Ab2 is cetuximab or a humanized version of cetuximab. In certain embodiments, the humanized form of cetuximab has a VH domain having an amino acid sequence at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or at least 99% identical to SEQ ID NO:7; and a VL domain having an amino acid sequence at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or at least 99% identical to SEQ ID NO:8.

[0079] In certain embodiments, the humanized form of cetuximab is any of the humanized forms described in patent application WO2012020059.

[0080] In certain embodiments, Ab1 or Ab2 is capable of binding to EGFR, - a heavy chain variable region (VH) comprising a CDR1 having the amino acid sequence of SEQ ID NO: 49, a CDR2 having the amino acid sequence of SEQ ID NO: 50, and a CDR3 having the amino acid sequence of SEQ ID NO: 51; and / or - a light chain variable region (VL) comprising a CDR1 having the amino acid sequence of SEQ ID NO: 52, a CDR2 having the amino acid sequence of SEQ ID NO: 53, and a CDR3 having the amino acid sequence of SEQ ID NO: 54; is a humanized form of cetuximab, including

[0081] In one embodiment, at least two, and preferably all, of framework regions 1, 2, and 3 of the heavy chain variable region (VH) of the humanized cetuximab are derived from or correspond to human germline VH gene 4-59*01, which encodes an amino acid sequence comprising SEQ ID NO: 55. Furthermore, framework region 4 of the heavy chain variable region (VH) is preferably derived from or corresponds to human germline gene JH1, which encodes an amino acid sequence comprising SEQ ID NO: 56. In one embodiment, at least one of framework regions 1, 2, and 3 of the light chain variable region (VL) of the humanized cetuximab is derived from or corresponds to human germline VL gene 6-21*01, which encodes an amino acid sequence comprising SEQ ID NO: 57. Preferably, at least two, and more preferably all three, of framework regions 1, 2, and 3 of the light chain variable region are derived from or correspond to human germline VL gene 6-21*01, which encodes an amino acid sequence comprising SEQ ID NO: 57. Furthermore, framework region 4 of the light chain variable region (VL) is preferably derived from or corresponds to the human germline gene JK2, which encodes an amino acid sequence comprising SEQ ID NO:58.

[0082] Preferably, the humanized cetuximab comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 59 and / or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 60. The heavy chain variable region (VH) preferably comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 61 to 68, particularly the amino acid sequence of SEQ ID NO: 63. In a specific embodiment, the heavy chain variable region (VH) of the humanized cetuximab preferably comprises the amino acid sequence of SEQ ID NO: 81. Furthermore, the light chain variable region (VL) preferably comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 69 to 72, particularly the amino acid sequence of SEQ ID NO: 71. Particularly preferred is a humanized antibody comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 63 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 71. Another particularly preferred is a humanized cetuximab comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 81 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 71.

[0083] In certain embodiments, humanized cetuximab is - a VH domain according to SEQ ID NO: 81 and a CH1 domain according to SEQ ID NO: 2 (corresponding to the VH-CH1 sequence according to SEQ ID NO: 83), - a VL domain according to SEQ ID NO: 71 and a CL domain according to SEQ ID NO: 4 (corresponding to the VL-CL sequence according to SEQ ID NO: 84) Includes:

[0084] In certain embodiments, the humanized cetuximab comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 59 or SEQ ID NOs: 61-68, particularly SEQ ID NO: 63. In certain embodiments, the humanized cetuximab comprises a light chain variable region (VL) comprising an amino acid sequence having at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 60 or SEQ ID NOs: 69-72, particularly SEQ ID NO: 71.

[0085] In some examples, Ab1 or Ab2 is humanized cetuximab, including: - a light chain humanized version of cetuximab based on the human immunoglobulin gene kappa variable 6-11 allele 02 (IGKV6-21*02) as defined in the IMGT / gene database; EIVLTQSPDFQSVTPKEKVTITCRASQSIGTNIHWYQQKPDQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQNNNWPTTFGQGTKLEIK (SEQ ID NO: 73) In the sequence, at the following Kabat positions, the amino acid residues are as follows: Kabat position L31 is Thr or Ser Kabat position L32 is Asn or Ser Kabat position L33 is Ile or Leu Kabat position L53 is Glu or Gln Kabat position L89 is Gln or His Optionally, Kabat position L91 can be Asn, Ser, His, Lys or Arg Kabat position L92 is Asn, Ser, His, Lys or Arg Kabat position L93 is Asn, Ser, His, Lys or Arg Kabat position L94 is Trp, Tyr or Phe Kabat position L96 is Thr or Tyr.

[0086] For purposes of clarity, L31 refers to residue 31 in the light chain. - a light chain humanized version of cetuximab based on the human immunoglobulin gene kappa variable 3-11 allele 01 (IGKV3-11*01) as defined in the IMGT / gene database; EIVLTQSPATLSLSPGERATLSCRASQSIGTNIHWYQQKPGQAPRLLIKYASESISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQNNNWPTTFGQGTKLEIK (SEQ ID NO: 74) In the sequence, at the following Kabat positions, the amino acid residues are as follows: Kabat position L29 is Ile or Val Kabat position L30 is Gly or Ser Kabat position L31 is Thr or Ser Kabat position L32 is Asn or Tyr Kabat position L33 is Ile or Leu Kabat position L34 is His or Ala Kabat position L49 is Lys or Tyr Kabat position L50 is Tyr or Asp Kabat position L53 is Glu or Asn Kabat position L54 is Ser or Arg Kabat position L55 is Ile or Ala Kabat position L56 is Ser or Thr Optionally, Kabat position L91 is Asn, Arg, His or Lys Kabat position L92 is Asn, Ser, His, Lys or Arg Kabat position L94 is Trp, Tyr or Phe Kabat position L96 is Thr or Tyr. - a heavy chain humanized version of the cetuximab mAb based on the human immunoglobulin gene heavy variable 4-59 allele 01 (IGHV4-59*01) as defined in the IMGT / gene database; QVQLQESGPGLVKPSETLSLTCTVSGFSLTNYGVHWVRQPPGKGLEWLGVIWSGGNTDYNTPLTSRLTISKDNSKNQVSLKLSSVTAADTAVYYCARALTYYDYEFAYWGQGTLVTVSS (SEQ ID NO: 75) In the sequence, at the following Kabat positions, the amino acid residues are as follows: Kabat position H29 is Leu or Ile Kabat position H30 is Thr or Ser Kabat position H31 is Asn or Ser Kabat position H33 is Gly or Tyr Kabat position H35 is His or Ser Kabat position H37 is Val or Ile Kabat position H48 is Leu or Ile Kabat position H50 is Val or Tyr Kabat position H52 is Trp, Tyr or Phe Kabat position H53 is Ser or Tyr Kabat position H54 is Gly or Ser Kabat position H56 is Asn or Ser Kabat position H58 is Asp or Asn Kabat position H61 is Thr or Pro Kabat position H62 is Pro or Ser Kabat position H64 is Thr or Lys Kabat position H67 is Leu or Val Kabat position H73 is Asn or Thr Kabat position H78 is Val or Phe.

[0087] For purposes of clarity, H29 refers to residue 29 in the heavy chain. - a heavy chain humanized version of the cetuximab mAb based on the human immunoglobulin gene heavy variable 3-33 allele 01 (IGHV3-33*01) as defined in the IMGT / gene database; QVQLVESGGGVVQPGRSLRLSCAVSGFSLTNYGVHWVRQAPGKGLEWLGVIWSGGNTDYNTPVTSRFTISKDNSKNTVYLQMNSLRAEDTAVYYCARALTYYDYEFAYWGQGTLVTVSS (SEQ ID NO: 76) In the sequence, at the following Kabat positions, the amino acid residues are as follows: Kabat position H28 is Ser or Thr Kabat position H30 is Thr or Ser Kabat position H48 is Leu or Val Kabat position H49 is Gly or Ala Kabat position H53 is Ser or Asp Kabat position H55 is Gly or Ser Kabat position H57 is Lys or Thr Kabat position H58 is Asp or Tyr Kabat position H60 is Asn or Ala Kabat position H61 is Thr or Asp Kabat position H62 is Pro or Ser Kabat position H64 is Thr or Lys Kabat position H65 is Ser or Gly Kabat position H78 is Val or Leu.

[0088] In certain embodiments, the Fab fragment of Ab1 and / or the Fab fragment of Ab2 comprise mutations in the VH / VL domains to facilitate pairing of the Fab heavy and light chains, specific mutations being described in International Patent Application WO2020 / 136566, all of which are incorporated herein by reference.

[0089] In certain embodiments, the Fab fragment of Ab1 and / or the Fab fragment of Ab2 comprise a mutated VH domain in which the residue at Kabat position 39 has been mutated from glutamine to lysine; and a VL domain in which the residue at Kabat position 38 has been mutated from glutamine to glutamic acid.

[0090] In certain embodiments, the Fab fragment of Ab1 and / or the Fab fragment of Ab2 comprise a mutated VH domain in which the residue at Kabat position 39 has been mutated from glutamine to lysine; and a VL domain in which the residue at Kabat position 38 has been mutated from glutamine to aspartic acid.

[0091] In certain embodiments, the Fab fragment of Ab1 and / or the Fab fragment of Ab2 comprise a mutated VH domain in which the residue at Kabat position 39 has been mutated from glutamine to glutamic acid; and a VL domain in which the residue at Kabat position 38 has been mutated from glutamine to lysine.

[0092] CH1 / CL mutated domain Fab domain assembly is achieved by natural pairing of the light and heavy chains without the use of a peptide linker. To maximize the likelihood of cognate pairing between the light and heavy chains, one can attempt to introduce mutations at the light and heavy chain interface (CL / CH1 interface) in the Fab fragment.

[0093] In a specific embodiment, the CH1 domain of Ab1 and / or Ab2 is a CH1 constant domain from human IgG1 of any allotype or a mutated derivative thereof. In a specific embodiment, the CH1 domain of Ab1 and / or Ab2 is a CH1 domain of human IgG1 of the G1m(1,17) allotype or a mutated derivative thereof. In another specific embodiment, the CH1 domain of Ab1 and / or Ab2 is a CH1 domain of human IgG1 of the G1m(3) allotype or a mutated derivative thereof.

[0094] In a preferred embodiment, each CH1 domain carries at least one mutation and each CL1 domain also carries at least one mutation, which mutations are selected to improve correct cognate pairing of the CH1 and CL1 domains.

[0095] These mutations can be selected from the following list: - de novo introduced ion pairs or opposite polarity charge mutations of native ion pairs already present at the interface of the heavy and light chains of the Fab fragment; - "knobs-into-holes" mutation; - Mutations that resurface the constant regions opposite the heavy and light chain interface in the Fab fragment to change them from strongly polar to highly hydrophobic (or vice versa).

[0096] Thus, several sets of mutations are preferred, as described in more detail below.

[0097] Of note, throughout this specification, the amino acid sequences and sequence position numbers used herein for the CH1 and CL domains are defined according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0098] Residues in the CL kappa domain that can be mutated can be selected from the group consisting of, for example, S114, F116, F118, E123 (e.g., E123K), Q124, T129, S131, V133, L135, N137, Q160, S162, S174, S176, T178, and T180.

[0099] Residues in the CH1 domain that can be mutated can be selected from the group consisting of, for example, L124, A139, L143, D144, K145, D146, H172, F174, P175, Q179, S183, S188, V190, T192 and K221 (e.g., K221E).

[0100] Specific mutations are described in U.S. Patent Application Nos. 2014 / 0200331, 2014 / 150973, 2014 / 0154254, and International Patent Application No. WO2007 / 147901, all of which are incorporated herein by reference.

[0101] Examples of CH1 / CL mutated derivatives are described below.

[0102] According to certain embodiments, the bispecific antigen-binding fragment comprises: - a mutated CH1 constant domain from human IgG1 corresponding to SEQ ID NO: 1 (the residue at Kabat position 192 was mutated from threonine to glutamic acid (E)); - a mutated CH1 constant domain from human IgG1 corresponding to SEQ ID NO: 45 (the residue at Kabat position 192 was mutated from threonine to aspartic acid (D)); - a mutated CH1 constant domain from human IgG1 corresponding to SEQ ID NO: 2 (the residue at Kabat position 143 was mutated from leucine to glutamine (Q); the residue at Kabat position 188 was mutated from serine to valine (V)); - a mutated CH1 constant domain from human IgG1 corresponding to SEQ ID NO: 46 (the residue at Kabat position 124 was mutated from leucine to glutamine (Q); the residue at Kabat position 188 was mutated from serine to valine (V)); - a mutated human kappa constant domain corresponding to SEQ ID NO: 3 (the residue at Kabat position 114 was mutated from serine to alanine (A) and the residue at Kabat position 137 was mutated from asparagine to lysine (K)); - a mutated human kappa constant domain corresponding to SEQ ID NO: 4 (the residue at Kabat position 133 was mutated from valine to threonine (T) and the residue at Kabat position 176 was mutated from serine to valine (V)); Includes:

[0103] In a preferred embodiment, a pair of interacting polar interface residues is exchanged for a pair of neutral and salt-bridge forming residues: substitution of Thr192 in the CH1 chain with glutamic acid or aspartic acid, and exchange of Asn137 in the CL chain with Lys, optionally together with substitution of the serine residue at position 114 of the CL domain with an alanine residue, can be selected.

[0104] In another set of mutations, Leu143 in the CH1 domain can be replaced by a Gln residue, while the facing residue of the CL chain, Val33, is replaced by a Thr residue. This first double mutation constitutes a switch from hydrophobic to polar interactions. Simultaneously, mutation of two interacting serines (Ser18 in the CH1 chain and Ser176 in the CL chain) to valine residues can achieve a switch from polar to hydrophobic interactions.

[0105] In yet another embodiment, the mutations can include a substitution of a leucine residue at position 124 of the CH1 domain with a glutamine and a substitution of a serine residue at position 188 of the CH1 domain with a valine residue; and a substitution of a valine residue at position 133 of the CL domain with a threonine residue and a substitution of a serine residue at position 176 of the CL domain with a valine residue.

[0106] The "knob-into-hole" mutations include a set of mutations (KH1) in which Leu124 and Leu143 in the CH1 domain were replaced by Ala and Glu residues, respectively, while Val33 in the CL chain was replaced by a Trp residue, while in the set of mutations designated H2, Val90 in the CH1 domain was replaced by an Ala residue, and Leu135 and Asn137 in the CL chain were replaced by Trp and Ala residues, respectively.

[0107] Preferred mutations are disclosed in the table below:

[0108] [Table 1]

[0109] In certain embodiments, the multispecific antibody can carry a double mutation, for example, one Fab carries a CR3 mutation and the other Fab carries a Mut4 mutation.

[0110] Bispecific antigen-binding fragments are more particularly described, according to which Ab1 or Ab2 are - a VH domain consisting of SEQ ID NO: 5 or a mutated derivative thereof, - a CH1 domain selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47, - a VL domain consisting of SEQ ID NO: 6 or a mutated derivative thereof, - a CL domain selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 48 and wherein the CL and CH1 domains are preferably associated as follows: SEQ ID NO: 48 and SEQ ID NO: 47, - SEQ ID NO: 3 and either SEQ ID NO: 1 or SEQ ID NO: 45, - SEQ ID NO: 4 and either SEQ ID NO: 2 or SEQ ID NO: 46.

[0111] Preferably, Ab1 or Ab2 is - a VH domain consisting of SEQ ID NO: 5 or a mutated derivative thereof, - a CH1 domain consisting of SEQ ID NO: 1 or a mutated derivative thereof, - a VL domain consisting of SEQ ID NO: 6 or a mutated derivative thereof, - a CL domain consisting of SEQ ID NO: 3 or a mutated derivative thereof or a mutated derivative thereof.

[0112] Preferably, Ab1 or Ab2 is - a VH-CH1 heavy chain comprising or consisting of SEQ ID NO: 13 or a functional variant having at least 80%, 85%, 90%, 92%, 94%, 96%, 98% or at least 99% sequence identity to SEQ ID NO: 13; and - a VL-CL light chain comprising or consisting of SEQ ID NO: 14 or a functional variant having at least 80%, 85%, 90%, 92%, 94%, 96%, 98% or at least 99% sequence identity to SEQ ID NO: 14 or a mutated derivative thereof.

[0113] Bispecific antigen-binding fragments are more particularly described, according to which Ab1 or Ab2 are - a VH domain consisting of SEQ ID NO: 7, a mutated derivative thereof or a humanized derivative thereof, - a CH1 domain selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47, - a VL domain consisting of SEQ ID NO: 8, a mutated derivative thereof or a humanized derivative thereof, - a CL domain selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 48 wherein the CL and CH1 domains are preferably associated as follows: SEQ ID NO: 48 and SEQ ID NO: 47, - SEQ ID NO: 3 and either SEQ ID NO: 1 or SEQ ID NO: 45, - SEQ ID NO: 4 and either SEQ ID NO: 2 or SEQ ID NO: 46.

[0114] Preferably, Ab1 or Ab2 is - a VH domain consisting of SEQ ID NO: 7, a mutated derivative thereof or a humanized derivative thereof, - a CH1 domain consisting of SEQ ID NO: 2 or a mutated derivative thereof, - a VL domain consisting of SEQ ID NO: 8, a mutated derivative thereof or a humanized derivative thereof, - a CL domain consisting of SEQ ID NO: 4 or a mutated derivative thereof or a mutated derivative thereof.

[0115] Preferably, Ab1 or Ab2 is - a VH-CH1 heavy chain comprising or consisting of SEQ ID NO: 15 or a functional variant having at least 80%, 85%, 90%, 92%, 94%, 96%, 98% or at least 99% sequence identity to SEQ ID NO: 15; and - a VL-CL light chain comprising or consisting of SEQ ID NO: 16 or a functional variant having at least 80%, 85%, 90%, 92%, 94%, 96%, 98% or at least 99% sequence identity to SEQ ID NO: 16 or a mutated derivative thereof.

[0116] Bispecific antigen-binding fragments are more particularly described, according to which Ab1 or Ab2 are - a VH domain consisting of SEQ ID NO: 9 or a mutated derivative thereof, - a CH1 domain selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47, - a VL domain consisting of SEQ ID NO: 10 or a mutated derivative thereof, - a CL domain selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 48 or a mutated derivative thereof comprising: SEQ ID NO: 48 and SEQ ID NO: 47, - SEQ ID NO: 3 and either SEQ ID NO: 1 or SEQ ID NO: 45, - SEQ ID NO: 4 and either SEQ ID NO: 2 or SEQ ID NO: 46.

[0117] Preferably, Ab1 or Ab2 is - a VH domain consisting of SEQ ID NO: 9 or a mutated derivative thereof, - a CH1 domain consisting of SEQ ID NO: 2 or a mutated derivative thereof, - a VL domain consisting of SEQ ID NO: 10 or a mutated derivative thereof, - a CL domain consisting of SEQ ID NO: 4 or a mutated derivative thereof or a mutated derivative thereof.

[0118] Preferably, Ab1 or Ab2 is - a VH-CH1 heavy chain comprising or consisting of SEQ ID NO: 17 or a functional variant having at least 80%, 85%, 90%, 92%, 94%, 96%, 98% or at least 99% sequence identity to SEQ ID NO: 17; and - a VL-CL light chain comprising or consisting of SEQ ID NO: 18 or a functional variant having at least 80%, 85%, 90%, 92%, 94%, 96%, 98% or at least 99% sequence identity to SEQ ID NO: 18 or a mutated derivative thereof.

[0119] Bispecific antigen-binding fragments are more particularly described, according to which Ab1 or Ab2 are - a VH domain consisting of SEQ ID NO: 11 or a mutated derivative thereof, - a CH1 domain selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47, - a VL domain consisting of SEQ ID NO: 12 or a mutated derivative thereof, - a CL domain selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 48 or a mutated derivative thereof comprising: SEQ ID NO: 48 and SEQ ID NO: 47, - SEQ ID NO: 3 and either SEQ ID NO: 1 or SEQ ID NO: 45, - SEQ ID NO: 4 and either SEQ ID NO: 2 or SEQ ID NO: 46.

[0120] Preferably, Ab1 or Ab2 is - a VH domain consisting of SEQ ID NO: 11 or a mutated derivative thereof, - a CH1 domain consisting of SEQ ID NO: 2 or a mutated derivative thereof, - a VL domain consisting of SEQ ID NO: 12 or a mutated derivative thereof, - a CL domain consisting of SEQ ID NO: 4 or a mutated derivative thereof or a mutated derivative thereof.

[0121] Preferably, Ab1 or Ab2 is - a VH-CH1 heavy chain comprising or consisting of SEQ ID NO: 19 or a functional variant having at least 80%, 85%, 90%, 92%, 94%, 96%, 98% or at least 99% sequence identity to SEQ ID NO: 19; and - a VL-CL light chain comprising or consisting of SEQ ID NO: 20 or a functional variant having at least 80%, 85%, 90%, 92%, 94%, 96%, 98% or at least 99% sequence identity to SEQ ID NO: 20 or a mutated derivative thereof.

[0122] In certain embodiments, the bispecific antigen-binding fragment comprises: (i) a Fab fragment comprising a VH-CH1 heavy chain associated with a VL-CL light chain of trastuzumab or a functional derivative thereof, and (ii) a Fab fragment comprising a VH-CH1 heavy chain associated with a VL-CL light chain of patritumab or a functional derivative thereof; wherein the N-terminal end of the VH domain of the Fab fragment of trastuzumab is linked to the C-terminal end of the CH1 domain of the Fab fragment of patritumab via a polypeptide linker; The VH-CH1 heavy chain of trastuzumab preferably consists of SEQ ID NO: 17, the VL-CL light chain of trastuzumab preferably consists of SEQ ID NO: 18, the VH-CH1 heavy chain of patritumab preferably consists of SEQ ID NO: 13, and / or the VL-CL light chain of patritumab preferably consists of SEQ ID NO: 14.

[0123] In a preferred embodiment, the bispecific antigen-binding fragment comprises one heavy chain comprising or consisting of SEQ ID NO: 21 or a functional variant thereof, and two different light chains comprising or consisting of SEQ ID NO: 18 and SEQ ID NO: 14 or functional variants thereof.

[0124] In certain embodiments, the bispecific antigen-binding fragment comprises: (i) a Fab fragment comprising a VH-CH1 heavy chain associated with a VL-CL light chain of patritumab or a functional derivative thereof, and (ii) a Fab fragment comprising a VH-CH1 heavy chain associated with a VL-CL light chain of trastuzumab or a functional derivative thereof; wherein the N-terminal end of the VH domain of the Fab fragment of patritumab is linked to the C-terminal end of the CH1 domain of the Fab fragment of trastuzumab via a polypeptide linker; The VH-CH1 heavy chain of trastuzumab preferably consists of SEQ ID NO: 17, the VL-CL light chain of trastuzumab preferably consists of SEQ ID NO: 18, the VH-CH1 heavy chain of patritumab preferably consists of SEQ ID NO: 13, and / or the VL-CL light chain of patritumab preferably consists of SEQ ID NO: 14.

[0125] In a preferred embodiment, the bispecific antigen-binding fragment comprises one heavy chain comprising or consisting of SEQ ID NO: 22 or a functional variant thereof, and two different light chains comprising or consisting of SEQ ID NO: 18 and SEQ ID NO: 14 or functional variants thereof.

[0126] In certain embodiments, the bispecific antigen-binding fragment comprises: (i) a Fab fragment comprising a VH-CH1 heavy chain associated with a VL-CL light chain of cetuximab or a functional or humanized derivative thereof, and (ii) a Fab fragment comprising a VH-CH1 heavy chain associated with a VL-CL light chain of patritumab or a functional derivative thereof; wherein the N-terminal end of the VH domain of the Fab fragment of cetuximab is linked to the C-terminal end of the CH1 domain of the Fab fragment of patritumab via a polypeptide linker; The VH-CH1 heavy chain of cetuximab preferably consists of SEQ ID NO: 15, the VL-CL light chain of cetuximab preferably consists of SEQ ID NO: 16, the VH-CH1 heavy chain of patritumab preferably consists of SEQ ID NO: 13, and / or the VL-CL light chain of patritumab preferably consists of SEQ ID NO: 14.

[0127] In a preferred embodiment, the bispecific antigen-binding fragment comprises one heavy chain comprising or consisting of SEQ ID NO: 23 or a functional variant thereof, and two different light chains comprising or consisting of SEQ ID NO: 16 and SEQ ID NO: 14 or functional variants thereof.

[0128] In certain embodiments, the bispecific antigen-binding fragment comprises: (i) a Fab fragment comprising a VH-CH1 heavy chain associated with a VL-CL light chain of patritumab or a functional derivative thereof, and (ii) a Fab fragment comprising the VH-CH1 heavy chain associated with the VL-CL light chain of cetuximab or a functional or humanized derivative thereof; wherein the N-terminal end of the VH domain of the Fab fragment of patritumab is linked to the C-terminal end of the CH1 domain of the Fab fragment of cetuximab via a polypeptide linker; The VH-CH1 heavy chain of cetuximab preferably consists of SEQ ID NO: 15, the VL-CL light chain of cetuximab preferably consists of SEQ ID NO: 16, the VH-CH1 heavy chain of patritumab preferably consists of SEQ ID NO: 13, and / or the VL-CL light chain of patritumab preferably consists of SEQ ID NO: 14.

[0129] In a preferred embodiment, the bispecific antigen-binding fragment comprises one heavy chain comprising or consisting of SEQ ID NO: 24 or a functional variant thereof, and two different light chains comprising or consisting of SEQ ID NO: 16 and SEQ ID NO: 14 or functional variants thereof.

[0130] In certain embodiments, the bispecific antigen-binding fragment comprises: (i) a Fab fragment comprising a VH-CH1 heavy chain associated with a VL-CL light chain of matuzumab or a functional derivative thereof, and (ii) a Fab fragment comprising a VH-CH1 heavy chain associated with a VL-CL light chain of patritumab or a functional derivative thereof; wherein the N-terminal end of the VH domain of the Fab fragment of matuzumab is linked to the C-terminal end of the CH1 domain of the Fab fragment of patritumab via a polypeptide linker; The VH-CH1 heavy chain of matuzumab preferably consists of SEQ ID NO: 19, the VL-CL light chain of matuzumab preferably consists of SEQ ID NO: 20, the VH-CH1 heavy chain of patritumab preferably consists of SEQ ID NO: 13, and / or the VL-CL light chain of patritumab preferably consists of SEQ ID NO: 14.

[0131] In a preferred embodiment, the bispecific antigen-binding fragment comprises one heavy chain comprising or consisting of SEQ ID NO: 25 or a functional variant thereof, and two different light chains comprising or consisting of SEQ ID NO: 20 and SEQ ID NO: 14 or functional variants thereof.

[0132] In certain embodiments, the bispecific antigen-binding fragment comprises: (i) a Fab fragment comprising a VH-CH1 heavy chain associated with a VL-CL light chain of patritumab or a functional derivative thereof, and (ii) a Fab fragment comprising a VH-CH1 heavy chain associated with a VL-CL light chain of matuzumab or a functional derivative thereof; wherein the N-terminal end of the VH domain of the Fab fragment of patritumab is linked to the C-terminal end of the CH1 domain of the Fab fragment of matuzumab via a polypeptide linker; The VH-CH1 heavy chain of matuzumab preferably consists of SEQ ID NO: 19, the VL-CL light chain of matuzumab preferably consists of SEQ ID NO: 20, the VH-CH1 heavy chain of patritumab preferably consists of SEQ ID NO: 13, and / or the VL-CL light chain of patritumab preferably consists of SEQ ID NO: 14.

[0133] In a preferred embodiment, the bispecific antigen-binding fragment comprises one heavy chain comprising or consisting of SEQ ID NO: 26 or a functional variant thereof, and two different light chains comprising or consisting of SEQ ID NO: 20 and SEQ ID NO: 14 or functional variants thereof.

[0134] Linker design A polypeptide linker is used to join the N-terminal end of the VH domain of the Fab fragment of Ab1 to the C-terminal end of the CH1 domain of the Fab fragment of Ab2.

[0135] In the context of the present invention, the term "polypeptide linker sequence" refers to a polypeptide of approximately 20 to 80 amino acids, preferably between 30 and 60 amino acids, and more preferably between 30 and 40 amino acids. A polypeptide linker sequence typically consists of fewer than 80 amino acids, preferably fewer than 60 amino acids, and even more preferably fewer than 40 amino acids. Advantageously, the linker sequence is "hinge-derived," meaning that the polypeptide linker comprises all or part of the sequence of the hinge region of one or more immunoglobulins selected from IgA, IgG, and IgD, preferably of human origin. This is also termed a "hinge-derived polypeptide linker sequence" or a "pseudo-hinge linker." The polypeptide linker can comprise all or part of the sequence of the hinge region of only one immunoglobulin. In this case, the immunoglobulin can belong to the same isotype and subclass as the immunoglobulin from which the adjacent CH1 domain is derived, or to a different isotype (isotype 45pe) or subclass. Alternatively, the polypeptide linker may comprise all or part of the sequences of hinge regions of at least two immunoglobulins of different isotypes or subclasses, in which case the N-terminal portion of the polypeptide linker immediately following the CH1 domain preferably consists of all or part of a hinge region of an immunoglobulin belonging to the same isotype and subclass as the immunoglobulin from which the CH1 domain is derived.

[0136] If necessary, the polypeptide linker can further comprise a sequence of 2 to 15, preferably 5 to 10, N-terminal amino acids of the CH2 domain of an immunoglobulin.

[0137] In some cases, sequences from the native hinge region can be used; in other cases, point mutations can be made in these sequences, in particular substitution of one or more cysteine ​​residues in the native IgG1, IgG2 or IgG3 hinge sequence with alanine or serine to avoid unwanted intra- or inter-chain disulfide bonds.

[0138] In certain embodiments, the polypeptide linker sequence comprises or consists of the amino acid sequence EPKX1CDKX2HX3X4PPX5PAPELLGGPX6X7PPX8PX9PX10GG (SEQ ID NO: 33) (in the sequence, X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, which may be identical or different, are any amino acid).

[0139] In certain embodiments, X1, X2, and X3, which are the same or different, are threonine (T) or serine (S).

[0140] In another specific embodiment, the identical or different X1, X2, and X3 are selected from the group consisting of Ala (A), Gly (G), Val (V), Asn (N), Asp (D), and Ile (I), and more preferably, the identical or different X1, X2, and X3 can be Ala (A) or Gly (G).

[0141] Alternatively, X1, X2 and X3, which may be the same or different, may be Leu (L), Glu (E), Gln (Q), Met (M), Lys (K), Arg (R), Phe (F), Tyr (T), His (H), Trp (W), preferably Leu (L), Glu (E) or Gln (Q).

[0142] In certain embodiments, X4 and X5, which may be the same or different, are any amino acid selected from the group consisting of serine (S), cysteine ​​(C), alanine (A), and glycine (G).

[0143] In a preferred embodiment, X4 is serine (S) or cysteine ​​(C).

[0144] In a preferred embodiment, X5 is alanine (A) or cysteine ​​(C).

[0145] In certain embodiments, the identical or different X6, X7, X8, X9, and X10 are any amino acid other than threonine (T) or serine (S). Preferably, the identical or different X6, X7, X8, X9, and X10 are selected from the group consisting of Ala (A), Gly (G), Val (V), Asn (N), Asp (D), and Ile (I).

[0146] Alternatively, the same or different X6, X7, X8, X9, and X10 may be Leu (L), Glu (E), Gln (Q), Met (M), Lys (K), Arg (R), Phe (F), Tyr (T), His (H), or Trp (W), preferably Leu (L), Glu (E), or Gln (Q).

[0147] In a preferred embodiment, the same or different X6, X7, X8, X9, and X10 are selected from the group consisting of Ala (A) and Gly (G).

[0148] In yet a preferred embodiment, X6 and X7 are the same and are preferably selected from the group consisting of Ala (A) and Gly (G).

[0149] In a preferred embodiment, the polypeptide linker sequence comprises or consists of the sequence of SEQ ID NO: 33, wherein: X1, X2 and X3, which may be the same or different, are threonine (T), serine (S); X4 is serine (S) or cysteine ​​(C); X5 is alanine (A) or cysteine ​​(C); X6, X7, X8, X9, and X10, which may be the same or different, are selected from the group consisting of Ala (A) and Gly (G).

[0150] In another preferred embodiment, the polypeptide linker sequence comprises or consists of the sequence of SEQ ID NO: 33, wherein: X1, X2 and X3, which may be the same or different, are Ala (A) or Gly (G); X4 is serine (S) or cysteine ​​(C); X5 is alanine (A) or cysteine ​​(C); X6, X7, X8, X9, and X10, which may be the same or different, are selected from the group consisting of Ala (A) and Gly (G).

[0151] In particular, the polypeptide linker sequence is EPKSCDKTHTSPPAPAPELLGGPAAPPAPAPAGG (SEQ ID NO: 34); EPKSCDKTHTAPPAPAPELLGGPAAPPAPAPAGG (SEQ ID NO: 35); EPKSCDKTHTSPPAPAPELLGGPGGPPGPGPGGG (SEQ ID NO: 36); EPKSCDKTHTSPPAPAPELLGGPAAPPGPAPGGG (SEQ ID NO: 37); EPKSCDKTHTCPPCPAPELLGGPSTPPTPSPSGG (SEQ ID NO: 38); and EPKSCDKTHTSPPSPAPELLGGPSTPPTPSPSGG (SEQ ID NO: 39) It may comprise or consist of a sequence selected from the group consisting of:

[0152] Preferably, the polypeptide linker sequence comprises or consists of the amino acid sequence of SEQ ID NO:34 or SEQ ID NO:35.

[0153] In embodiments in which the antibody comprises different Fab fragments, the polypeptide linkers separating the Fab fragments may be the same or different.

[0154] 2. Design of bispecific antibodies The present invention also relates to a bispecific molecule comprising two identical antigen-binding arms, each consisting of an antigen-binding fragment as defined above. In a preferred embodiment, the bispecific molecule is a full-length antibody.

[0155] If one wishes to obtain an antibody that is devoid of Fc-mediated effects, or that is monovalent for each of the two antigens that it targets, the antibody will not contain an Fc region. In this case, the two antigen-binding arms will be, for example, - by homodimerization of the antigen-binding arms through interchain disulfide bonds provided by the polypeptide linker separating the Fab fragments; and / or - by the addition at the C-terminal end of each antigen-binding arm of a polypeptide extension containing cysteine ​​residues that allow for the formation of interchain disulfide bonds, which may be, by way of non-limiting example, the hinge sequence of IgG1, IgG2 or IgG3, and homodimerization of said polypeptide extensions resulting in a hinge-like structure; - by a linker, preferably a semi-rigid linker, which joins the C-terminal ends of the heavy chains of the two antigen-binding arms to form a single polypeptide chain and maintains the antigen-binding arms at a sufficient distance from each other; They may be connected together.

[0156] Alternatively, if effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and / or antibody-dependent phagocytosis (ADP), or bivalent binding to each of two antigens, are desired, the multispecific antibodies of the invention can further comprise an Fc domain that provides these effector functions. The choice of Fc domain will depend on the type of effector function desired.

[0157] The present invention also provides bispecific tetravalent antibodies comprising two binding sites for HER-3 and two binding sites for another antigen, either HER-2 or EGFR. More specifically, the present invention relates to bispecific antibodies comprising two identical antigen-binding arms (thereby providing a symmetric antibody format), each antigen-binding arm comprising one binding site for HER-3 and one binding site for HER-2. The present invention also relates to bispecific antibodies comprising two identical antigen-binding arms (thereby providing a symmetric antibody format), each antigen-binding arm comprising one binding site for HER-3 and one binding site for EGFR.

[0158] In certain embodiments, each antigen-binding arm consists of a bispecific antigen-binding fragment as defined above.

[0159] In a preferred embodiment, the linker connects two pairs of Fab domains in a tetra-Fab bispecific antibody format, the amino acid sequence of which comprises the heavy chain sequences of at least two Fabs joined by a linker, followed by a hinge sequence, followed by an Fc sequence, co-expressed with the appropriate light chain sequence.

[0160] An example of an antibody of the invention having an IgG-like structure is illustrated in FIG.

[0161] In certain embodiments, the bispecific antibody has an immunoglobulin-like structure: - two identical antigen-binding arms, each consisting of a bispecific antigen-binding fragment as described herein, and - an Fc domain, which is preferably functional, i.e. allows the activation of effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis and complement-dependent cytotoxicity (CDC), Includes:

[0162] In certain embodiments, the bispecific antibody of the invention comprises: - A continuous heavy chain (hinge-CH2-CH3) constructed from Fc followed by a Fab heavy chain (CH1-VH) of antibody 1 (Ab1) and a consecutive Fab heavy chain (CH1-VH) of antibody 2 (Ab2), the latter are connected by a polypeptide linker sequence as described above, - upon protein expression, the resulting heavy chains assemble into homodimers, while the two co-expressed light chains (VL-CL) of Ab1 and the two light chains (VL-CL) of Ab2 associate with their cognate heavy chains to form the final tandem F(ab)'2-Fc molecule; one of Ab1 or Ab2 is patritumab or a functional derivative thereof; The other of Ab1 or Ab2 is selected from the group consisting of trastuzumab or a functional derivative thereof, matuzumab or a functional derivative thereof, and cetuximab or a functional derivative thereof.

[0163] It is understood that the hinge domain connects the N-terminal end of the CH2 domain of Ab1 to the C-terminal end of the CH1 domain of Ab1, and the polypeptide linker sequence connects the N-terminal end of the VH domain of Ab1 to the C-terminal end of the CH1 domain of Ab2.

[0164] In certain embodiments, the bispecific antibody comprises: - two identical antigen-binding arms, each consisting of a bispecific antigen-binding fragment as described herein; - Dimerized CH2 and CH3 domains of immunoglobulins - an immunoglobulin hinge region that connects the C-terminal end of the CH1 domain of the antigen-binding arm to the N-terminal end of the CH2 domain It has an immunoglobulin-like structure containing

[0165] In a specific embodiment, the CH2 and / or CH3 domains are those of an IgG1 or IgG4 isotype, or mutated derivatives thereof. Preferably, the CH2 domain is the CH2 domain of human IgG1 (SEQ ID NO: 41). Preferably, the CH3 domain is the CH3 domain of human IgG1 of the G1m(3) allotype (SEQ ID NO: 42). In another specific embodiment, the CH3 domain is the CH3 domain of human IgG1 of the G1m(1,17) allotype.

[0166] In certain embodiments, the hinge region is that of IgA, IgG1, IgG4, IgD, or a mutated derivative thereof, which connects the C-terminal end of the CH1 domain of the antigen-binding arm to the N-terminal end of the CH2 domain. In certain embodiments, the hinge region is that of IgG4 with an S228P substitution. Preferably, the hinge region is that of IgG1 of SEQ ID NO: 40.

[0167] In certain embodiments, the bispecific antibody comprises an Fc domain that has an immunoglobulin-like structure and can be a wild-type immunoglobulin Fc domain or a mutated derivative thereof. In certain embodiments, the Fc domain is a mutated derivative of an IgG1 Fc domain or a mutated derivative of an IgG4 Fc domain. In certain embodiments, said mutated derivative is an Fc domain that has altered affinity for at least one Fc gamma receptor (FcgR) compared to the parent Fc domain.

[0168] In certain embodiments, the bispecific antibody comprises an Fc domain that has an immunoglobulin-like structure and that has reduced binding to Fc gamma receptors compared to a wild-type Fc domain, e.g., an IgG1 or IgG4 Fc domain, resulting in a bispecific antibody with reduced effector function.

[0169] In certain embodiments, the bispecific antibody comprises an Fc domain, e.g., an IgG1 or IgG4 Fc domain, which has an immunoglobulin-like structure and exhibits no or substantially no binding to Fc gamma receptors compared to a wild-type Fc domain, resulting in a bispecific antibody with muted or reduced effector function.

[0170] The Fc domain can contain one or more mutations that reduce or eliminate its binding to Fc gamma receptors, such as mutations in the CH2 and / or CH3 domains. The mutations include amino acid substitutions, insertions, and / or deletions. Illustrative mutations that reduce or eliminate the binding activity of the Fc domain to Fc gamma receptors include, but are not limited to, L / F234A, L235A / E, G236R / del, G237A, P238S, D265A, H / Q268A, L328R, P329G, S / A330R / S, and / or P331S. All mutated residues in the Fc domain are numbered herein according to the EU nomenclature convention.

[0171] For purposes of clarification, "L / F234A" indicates substitution of leucine or phenylalanine (whichever is present at position 234 of IgG1 or IgG4, respectively) with alanine. For purposes of clarification, "L235A / E" indicates substitution of leucine at position 235 with either alanine or glutamic acid. For purposes of clarification, "G236R / del" indicates either substitution of glycine at position 236 with arginine or deletion of the glycine residue at position 236. For purposes of clarification, "A / S330R / S" indicates substitution of alanine or serine (whichever is present at position 330 of IgG1 or IgG4, respectively) with either arginine or serine.

[0172] Illustrative mutations that reduce or eliminate binding activity of an IgG1 Fc domain to an Fc gamma receptor include, but are not limited to, L234A, L235A / E, G236R / del, G237A, P238S, D265A, H268A, L328R, P329G, A330R / S and / or P331S. Illustrative mutations that reduce or eliminate binding activity of an IgG4 Fc domain to an Fc gamma receptor include, but are not limited to, F234A, L235A / E, G236R / del, G237A, P238S, D265A, Q268A, L328R, P329G and / or S330R.

[0173] In another specific embodiment, the bispecific antibody comprises an Fc domain that has an immunoglobulin-like structure and has increased binding to Fc gamma receptors compared to a wild-type Fc domain, e.g., an IgG1 or IgG4 Fc domain, resulting in a bispecific antibody with improved effector function.

[0174] The Fc domain may comprise one or more mutations that increase its binding to an Fc gamma receptor, such as mutations in the CH2 and / or CH3 domains. Illustrative mutations that increase the binding activity of an Fc domain, e.g., an IgG1 or IgG4 Fc domain, to an Fc gamma receptor include, but are not limited to, S239D, I332E, S298A, E333A, K334A, D280H, K290S, S298D, F243L, R292P, Y300L, V305I, P396L, A330L, G236A, L234Y, G236W, and / or S298A. In a preferred embodiment, the Fc domain, e.g., an IgG1 or IgG4 Fc domain, comprises the mutations S239D and I332E.

[0175] In certain embodiments, the Fc domain, eg, an IgG1 or IgG4 Fc domain, has a reduced fucose content in the Fc glycan at position 297 of the CH2 domain.

[0176] According to certain embodiments, the Fc domain, e.g., an IgG1 or IgG4 Fc domain, has an N-glycan at the glycosylation site (Asn297), said N-glycan having a degree of fucosylation of less than 65%, preferably less than 60%, preferably less than 55%, preferably less than 50%, more preferably less than 45%, preferably less than 40%, preferably less than 35%, preferably less than 30%, preferably less than 25%, preferably less than 20%, preferably less than 10%.

[0177] According to a more particular embodiment, the Fc domain carries an N-glycan at the glycosylation site (Asn297), said N-glycan having a degree of fucosylation equal to 0%.Thus, the present invention provides a bispecific antibody comprising an Fc domain carrying an N-glycan at its glycosylation site Asn297, characterized in that said N-glycan of the Fc domain does not contain fucose.

[0178] Advantageously, Fc domains with altered glycosylation at the glycosylation site at position 297, in particular with reduced fucosylation, show increased binding to Fc-gamma receptors.

[0179] In certain embodiments, the Fc domain comprises: - a hinge domain of SEQ ID NO: 40; and - CH2-CH3 domain of SEQ ID NO: 43 It comprises, preferably consists of,

[0180] In a specific embodiment, the Fc domain comprises, preferably consists of, SEQ ID NO: 44 or a functional variant having at least 80%, 90%, 95%, 96%, 97%, 98% or at least 99% sequence identity to SEQ ID NO: 44. In another specific embodiment, the Fc domain comprises, preferably consists of, SEQ ID NO: 82 or a functional variant having at least 80%, 90%, 95%, 96%, 97%, 98% or at least 99% sequence identity to SEQ ID NO: 82.

[0181] A particular embodiment is a bispecific antibody comprising two heavy chains and four light chains, wherein each heavy chain comprises: a. An immunoglobulin Fc region comprising the hinge-CH2-CH3 domain or a mutated derivative thereof described above; b. the Fc region is linked to the Fab CH1-VH heavy chain of Antibody 1 (Ab1) by the hinge domain; c. which is then linked to the Fab CH1-VH heavy chain of antibody 2 (Ab2) by a polypeptide linker sequence connecting the N-terminus of the VH domain of Ab1 to the C-terminus of the CH1 domain of Ab2; the four light chains include two Fab CL-VL light chains of Ab1 and two Fab CL-VL light chains of Ab2 associated with their cognate heavy chain domains; one of Ab1 or Ab2 is patritumab or a functional derivative thereof as described above, the other of Ab1 or Ab2 is selected from the group consisting of trastuzumab or a functional derivative thereof, matuzumab or a functional derivative thereof, and cetuximab or a functional or humanized derivative thereof, as described above; This relates to bispecific antibodies.

[0182] A particular embodiment is a bispecific antibody comprising two heavy chains and four light chains, wherein each heavy chain comprises: a. An immunoglobulin Fc region comprising the hinge-CH2-CH3 domain or a mutated derivative thereof described above; b. the Fc region is linked to the Fab CH1-VH heavy chain of Antibody 1 (Ab1) by the hinge domain; c. which is then linked to the Fab CH1-VH heavy chain of antibody 2 (Ab2) by a polypeptide linker sequence connecting the N-terminus of the VH domain of Ab1 to the C-terminus of the CH1 domain of Ab2; the four light chains include two Fab CL-VL light chains of Ab1 and two Fab CL-VL light chains of Ab2 associated with their cognate heavy chain domains; Ab2 is patritumab or a functional derivative thereof as described above, Ab1 is selected from the group consisting of trastuzumab or a functional derivative thereof, matuzumab or a functional derivative thereof, and cetuximab or a functional or humanized derivative thereof, as described above; This relates to bispecific antibodies.

[0183] In certain embodiments, the bispecific antibody comprises: a) two heavy chains each comprising, preferably consisting of, SEQ ID NO: 27 or a variant derivative thereof, and b) four light chains, two of which comprise, preferably consist of, SEQ ID NO: 14 or a variant thereof, and the other two of which comprise, preferably consist of, SEQ ID NO: 18 or a variant thereof; It comprises, preferably consists of,

[0184] The heavy chain of SEQ ID NO: 27 is - VH of patritumab (SEQ ID NO: 5) - CH1 domain of patritumab Fab (human IgG1 of G1m(1,17) allotype with mutation T192E) (SEQ ID NO: 1) - AP linker (SEQ ID NO: 34) - VH of trastuzumab (SEQ ID NO: 9) - CH1 domain of trastuzumab Fab (human IgG1 of the G1m(1,17) allotype with the mutations L143Q and S188V) (SEQ ID NO: 2) - human IgG1 hinge (SEQ ID NO: 40) CH2 domain of human IgG1 (SEQ ID NO: 41) - CH3 domain of human IgG1 of the G1m(3) allotype (SEQ ID NO: 42) Includes:

[0185] In certain embodiments, the bispecific antibody comprises: a) two heavy chains each comprising, preferably consisting of, SEQ ID NO: 28 or a variant derivative thereof, and b) four light chains, two of which comprise, preferably consist of, SEQ ID NO: 14 or a variant thereof, and the other two of which comprise, preferably consist of, SEQ ID NO: 18 or a variant thereof; It comprises, preferably consists of,

[0186] The heavy chain of SEQ ID NO: 28 is - VH of trastuzumab (SEQ ID NO: 9) - CH1 domain of trastuzumab Fab (human IgG1 of the G1m(1,17) allotype with the mutations L143Q and S188V) (SEQ ID NO: 2) - AP linker (SEQ ID NO: 34) - VH of patritumab (SEQ ID NO: 5) - CH1 domain of patritumab Fab (human IgG1 of G1m(1,17) allotype with mutation T192E) (SEQ ID NO: 1) - human IgG1 hinge (SEQ ID NO: 40) CH2 domain of human IgG1 (SEQ ID NO: 41) - CH3 domain of human IgG1 of the G1m(3) allotype (SEQ ID NO: 42) Includes:

[0187] In certain embodiments, the bispecific antibody comprises: a) two heavy chains each comprising, preferably consisting of, SEQ ID NO: 29 or a variant derivative thereof, and b) four light chains, two of which comprise, preferably consist of, SEQ ID NO: 14 or a variant thereof, and two of which comprise, preferably consist of, SEQ ID NO: 16 or a variant thereof; It comprises, preferably consists of,

[0188] The heavy chain of SEQ ID NO: 29 is - VH of patritumab (SEQ ID NO: 5) - CH1 domain of patritumab Fab (human IgG1 of G1m(1,17) allotype with mutation T192E) (SEQ ID NO: 1) - AP linker (SEQ ID NO: 34) - VH of cetuximab (SEQ ID NO: 7) - CH1 domain of trastuzumab Fab (human IgG1 of the G1m(1,17) allotype with the mutations L143Q and S188V) (SEQ ID NO: 2) - human IgG1 hinge (SEQ ID NO: 40) CH2 domain of human IgG1 (SEQ ID NO: 41) - CH3 domain of human IgG1 of the G1m(3) allotype (SEQ ID NO: 42) Includes:

[0189] In certain embodiments, the bispecific antibody comprises: a) two heavy chains each comprising, preferably consisting of, SEQ ID NO: 30 or a variant derivative thereof, and b) four light chains, two of which comprise, preferably consist of, SEQ ID NO: 14 or a variant thereof, and two of which comprise, preferably consist of, SEQ ID NO: 16 or a variant thereof; It comprises, preferably consists of,

[0190] The heavy chain of SEQ ID NO: 30 is - VH of cetuximab (SEQ ID NO: 7) - CH1 domain of cetuximab Fab (human IgG1 of the G1m(1,17) allotype with the mutations L143Q and S188V) (SEQ ID NO: 2) - AP linker (SEQ ID NO: 34) - VH of patritumab (SEQ ID NO: 5) - CH1 domain of patritumab Fab (human IgG1 of G1m(1,17) allotype with mutation T192E) (SEQ ID NO: 1) - human IgG1 hinge (SEQ ID NO: 40) CH2 domain of human IgG1 (SEQ ID NO: 41) - CH3 domain of human IgG1 of the G1m(3) allotype (SEQ ID NO: 42) Includes:

[0191] In certain embodiments, the bispecific antibody comprises: a) two heavy chains each comprising, preferably consisting of, SEQ ID NO: 31 or a variant derivative thereof, and b) four light chains, two of which comprise, preferably consist of, SEQ ID NO: 14 or a variant derivative thereof, and the other two of which comprise, preferably consist of, SEQ ID NO: 20 or a variant derivative thereof; It comprises, preferably consists of,

[0192] The heavy chain of SEQ ID NO: 31 is - VH of patritumab (SEQ ID NO: 5) - CH1 domain of patritumab Fab (human IgG1 of G1m(1,17) allotype with mutation T192E) (SEQ ID NO: 1) - AP linker (SEQ ID NO: 34) - VH of matuzumab (SEQ ID NO: 11) - CH1 domain of trastuzumab Fab (human IgG1 of the G1m(1,17) allotype with the mutations L143Q and S188V) (SEQ ID NO: 2) - human IgG1 hinge (SEQ ID NO: 40) CH2 domain of human IgG1 (SEQ ID NO: 41) - CH3 domain of human IgG1 of the G1m(3) allotype (SEQ ID NO: 42) Includes:

[0193] In certain embodiments, the bispecific antibody comprises: a) two heavy chains each comprising, preferably consisting of, SEQ ID NO: 32 or a variant derivative thereof, and b) four light chains, two of which comprise, preferably consist of, SEQ ID NO: 14 or a variant derivative thereof, and the other two of which comprise, preferably consist of, SEQ ID NO: 20 or a variant derivative thereof; It comprises, preferably consists of,

[0194] The heavy chain of SEQ ID NO: 32 is - VH of matuzumab (SEQ ID NO: 11) - CH1 domain of cetuximab Fab (human IgG1 of the G1m(1,17) allotype with the mutations L143Q and S188V) (SEQ ID NO: 2) - AP linker (SEQ ID NO: 34) - VH of patritumab (SEQ ID NO: 5) - CH1 domain of patritumab Fab (human IgG1 of G1m(1,17) allotype with mutation T192E) (SEQ ID NO: 1) - human IgG1 hinge (SEQ ID NO: 40) CH2 domain of human IgG1 (SEQ ID NO: 41) - CH3 domain of human IgG1 of the G1m(3) allotype (SEQ ID NO: 42) Includes:

[0195] The light chain of SEQ ID NO: 14 is - VL of patritumab (SEQ ID NO: 6) - C kappa domain with mutations S114A and N137K (SEQ ID NO: 3) Includes:

[0196] The light chain of SEQ ID NO: 16 is - VL of cetuximab (SEQ ID NO: 8) - C kappa domain with mutations V133T and S176V (SEQ ID NO: 4) Includes:

[0197] The light chain of SEQ ID NO: 18 is - VL of trastuzumab (SEQ ID NO: 10) - C kappa domain with mutations V133T and S176V (SEQ ID NO: 4) Includes:

[0198] The light chain of SEQ ID NO: 20 is - VL of matuzumab (SEQ ID NO: 12) - C kappa domain with mutations V133T and S176V (SEQ ID NO: 4) Includes:

[0199] In certain embodiments, the bispecific antibody comprises: a) two heavy chains each comprising, preferably consisting of, SEQ ID NO: 85 or a variant derivative thereof, and b) four light chains, two of which comprise, preferably consist of, SEQ ID NO: 14 or a variant thereof, and two of which comprise, preferably consist of, SEQ ID NO: 84 or a variant thereof; It comprises, preferably consists of,

[0200] In certain embodiments, the bispecific antibody comprises: a) two heavy chains each comprising, preferably consisting of, SEQ ID NO: 86 or a variant derivative thereof, and b) four light chains, two of which comprise, preferably consist of, SEQ ID NO: 14 or a variant thereof, and two of which comprise, preferably consist of, SEQ ID NO: 84 or a variant thereof; It comprises, preferably consists of,

[0201] 3. Bispecific Antibody Production Those skilled in the art can refer to International Patent Application WO2013 / 005194, which is incorporated herein by reference, for general techniques for expressing multispecific antibodies.

[0202] Also described herein are polynucleotides comprising sequences encoding the protein chains of the molecules or antibodies of the invention. The polynucleotides may also comprise additional sequences; in particular, they may advantageously comprise sequences encoding leader or signal peptides that allow secretion of the protein chains. Host cells transformed with the polynucleotides are also disclosed.

[0203] Typically, the amino acid sequence of the bispecific antibody is used to design the DNA sequence, after codon optimization for mammalian expression if necessary. For the heavy chain, DNA encoding the signal peptide, the variable region and constant CH1 domain of Fab1, followed by a hinge linker and the variable region and constant CH1 domain of Fab2, and flanking sequences for restriction enzyme digestion are synthesized. For the light chain, DNA encoding the signal peptide and the variable and constant kappa regions are synthesized.

[0204] Nucleic acids encoding the heavy and light chains of the bispecific antigen-binding fragments or antibodies of the present invention are inserted into an expression vector. The light and heavy chains can be cloned into the same or different expression vectors. The DNA segments encoding immunoglobulin chains are operably linked to control sequences in the expression vector that ensure the expression of immunoglobulin polypeptides. Such control sequences include signal sequences, promoters, enhancers, and transcription termination sequences. Expression vectors are typically replicable in host organisms either as episomes or as an integral part of the host chromosomal DNA. Generally, expression vectors will contain a selectable marker, such as tetracycline or neomycin, to enable detection of cells transformed with the desired DNA sequences.

[0205] In one example, sequences encoding both the heavy and light chains (e.g., sequences encoding VH and VL, VH-CH1 and VL-CL, or full-length heavy and full-length light chains) are contained in a single expression vector. In another example, each of the heavy and light chains of an antibody is cloned into a separate vector. In the latter case, expression vectors encoding the heavy and light chains can be co-transfected into a single host cell for expression of both chains, and both chains can be assembled either in vivo or in vitro to form an intact antibody. Alternatively, an expression vector encoding the heavy chain and an expression vector encoding the light chain can be introduced into different host cells for expression of each heavy and light chain, which can then be purified and assembled in vitro to form an intact antibody.

[0206] In certain embodiments, host cells are co-transfected with three independent expression vectors, e.g., plasmids, resulting in the simultaneous production of all three chains (i.e., the heavy chain HC and the two light chains LC1 and LC2, respectively) and secretion of the bispecific antibody. More specifically, the three vectors can advantageously be used in a molecular ratio of 2:1:1 (HC:LC1:LC2).

[0207] Recombinant vectors for expressing the antibodies described herein typically contain a nucleic acid encoding the antibody amino acid sequence operably linked to a promoter, either constitutive or inducible. The vector may be suitable for replication and integration in prokaryotes, eukaryotes, or both. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the antibody-encoding nucleic acid. The vectors optionally contain at least one independent terminator sequence, sequences that allow replication of the cassette in both eukaryotes and prokaryotes, i.e., shuttle vectors, and generic expression cassettes containing selectable markers for both prokaryotic and eukaryotic systems.

[0208] The bispecific antibodies described herein can be produced in prokaryotic or eukaryotic expression systems, such as bacteria, yeast, filamentous fungi, plants, insects (e.g., using baculovirus vectors), and mammalian cells. It is not necessary for the recombinant antibodies of the invention to be glycosylated or expressed in eukaryotic cells; however, expression in mammalian cells is generally preferred. Examples of useful mammalian host cell lines are the human embryonic kidney line (293 cells), baby hamster kidney cells (BHK cells), Chinese hamster ovary cells (CHO, CHO-S, CHO-DG44, Flp-in CHO cells), African green monkey kidney cells (VERO cells), and human hepatocytes (Hep G2 cells).

[0209] Mammalian tissue cell culture is preferred for expression and production of polypeptides, as many suitable host cell lines capable of secreting intact immunoglobulins have been developed in the art, including CHO cell lines, various Cos cell lines, HeLa cells, preferably myeloma cell lines (such as NS0) or transformed B cells or hybridomas.

[0210] In a most preferred embodiment, the bispecific antibodies of the present invention are produced using a CHO cell line, most preferably the CHO-S or CHO-DG-44 cell line or a derivative thereof. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter and an enhancer, as well as necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papillomavirus, cytomegalovirus, and others. Vectors containing polynucleotide sequences of interest (e.g., heavy and light chain coding sequences and expression control sequences) can be transfected into host cells by well-known methods that vary depending on the type of cellular host. For example, calcium phosphate treatment or electroporation can be used for other cellular hosts (see generally Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 2nd ed., 1989)). When heavy and light chains are cloned on separate expression vectors, the vectors are co-transfected to obtain expression and assembly of intact immunoglobulins.

[0211] Host cells are transformed or transfected with the vector (e.g., by chemical transfection or electroporation methods) and cultured in conventional nutrient media (or modified as appropriate) to induce promoters, select transformants, or amplify the genes encoding the desired sequences.

[0212] Expression of the antibody can be transient or stable.

[0213] Preferably, bispecific antibodies are produced by a stable expression method, in which cell lines stably transfected with DNA encoding all polypeptide chains of the bispecific antibody are capable of sustained expression, thereby enabling the production of therapeutics. For example, stable expression in CHO cell lines is particularly advantageous.

[0214] Once expressed, whole antibodies of the invention, their dimers, individual light and heavy chains, or other immunoglobulin forms can be further isolated or purified to obtain substantially homogeneous preparations for further assays and applications. Standard protein purification methods known in the art can be used. For example, suitable purification procedures can include fractionation on immunoaffinity or ion exchange columns, ethanol precipitation, high-performance liquid chromatography (HPLC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), ammonium sulfate precipitation, and gel filtration (see generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)). For pharmaceutical uses, substantially pure immunoglobulins of at least about 90-95% homogeneity are preferred, with 98-99% or greater homogeneity being most preferred.

[0215] In vitro production allows for scale-up to obtain large quantities of the desired bispecific antibodies of the invention. Such methods can use, for example, homogenous suspension cultures in airlift reactors or continuous stirrer reactors, or immobilized or entrapped cell cultures, for example, in hollow fibers, in microcapsules, on agarose microbeads or on ceramic cartridges.

[0216] 4.Therapeutic uses The bispecific antibodies of the present invention have been shown to induce tumor growth inhibition.

[0217] The bispecific antigen-binding fragments or antibodies of the present invention are particularly useful as medicaments in the treatment of cancer.

[0218] The term "cancer", as used herein, includes any cancer characterized by HER3, EGFR, or HER2 expression or overexpression, particularly pancreatic cancer, and any other cancer, particularly cancer characterized by co-expression of both HER3 and EGFR, or HER3 and HER2.

[0219] In some embodiments, the cancer comprises cells that have a wild-type KRAS gene.

[0220] Examples of cancers are solid tumors, such as pancreatic cancer, head and neck cancer including squamous cell carcinoma, colorectal cancer, breast cancer, lung cancer, gastric cancer, esophageal cancer, ovarian cancer.

[0221] Preferably, the cancer is pancreatic cancer.

[0222] Thus, a method of treating a patient suffering from cancer by administering an antibody according to the invention to said patient in need of such treatment is described. Thus, another aspect of the invention is the use of a bispecific antibody according to the invention for the manufacture of a medicament for the treatment of cancer.

[0223] One aspect of the invention is a pharmaceutical composition comprising a bispecific molecule according to the invention. Another aspect of the invention is the use of a bispecific molecule according to the invention for the manufacture of a pharmaceutical composition. A further aspect of the invention is a method for the manufacture of a pharmaceutical composition comprising a bispecific molecule according to the invention.

[0224] In another aspect, the present invention provides compositions, eg, pharmaceutical compositions, containing a bispecific molecule as defined herein formulated with a pharmaceutical carrier.

[0225] As used herein, a "pharmaceutical carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).

[0226] The compositions of the present invention can be administered by a variety of methods known in the art. The route and / or mechanism of administration will vary depending on the desired results.

[0227] To administer a bispecific molecule or antibody of the invention by a certain route of administration, it may be necessary to coat the bispecific molecule or antibody of the invention with, or co-administer it with, a material to prevent its inactivation. For example, the bispecific molecule or antibody of the invention can be administered to a subject in an appropriate carrier, for example, liposomes or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art.

[0228] Such compositions can also contain adjuvants, such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured by both sterilization procedures and the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include an isotonic agent, such as sodium chloride, in the composition. In addition, prolonged absorption of injectable pharmaceutical forms can be brought about by the inclusion of agents that delay absorption.

[0229] Actual dosage levels of the active ingredient in the pharmaceutical compositions of the present invention may be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without toxicity to the patient.

[0230] The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition of the invention employed, the route of administration, the time of administration, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, overall health and previous medical history of the patient being treated, and similar factors well known in the medical arts. For example, bispecific molecules or antibodies of the invention can be administered at dosages of 0.2 to 20 mg / kg, from three times per week to once per month.

[0231] The invention thus generally described above will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limitations of the invention. [Example]

[0232] Example 1 Preparation of bispecific antibodies of the present invention The bispecific antibody (BsAb) of the present invention comprises two heavy chains and four light chains. In particular, a BsAb has four antigen-binding sites (Fabs), two Fabs derived from Ab1 and two other Fabs derived from Ab2 (Figure 1). It therefore has dual binding capacity for each of two target antigens (tetravalent). The Fab distal to the Fc domain is designated the "outer Fab," and the Fab proximal to the Fc domain is designated the "inner Fab" (Figure 1).

[0233] Six antibodies were designed based on four parent antibodies: patritumab (anti-HER3), atezolizumab (anti-PDL1), cetuximab (anti-EGFR), and matuzumab (anti-EGFR). The nomenclature and sequences of the heavy and light chains of each construct are detailed in Table 1:

[0234] [Table 2]

[0235] As shown in Figure 1, each bispecific antibody: - two identical heavy chains, each heavy chain comprising (from N-terminus to C-terminus) the VH and CH1 domains of an outer Fab, a linker, the VH and CH1 domains of an inner Fab, a constant Fc domain (including hinge-Ch2-Ch3), - two identical light chains of an outer Fab, each light chain comprising (from N-terminus to C-terminus) the VL and CL domains of an outer Fab, - two identical light chains of an inner Fab, each light chain comprising (from N- to C-terminus) the VL and CL domains of an inner Fab Includes:

[0236] Details regarding the sequences making up the heavy and light chains of each construct are presented in Table 2.

[0237] [Table 3]

[0238] Gene synthesis The amino acid sequences of patritumab (anti-HER3), atezolizumab (anti-PDL1), cetuximab (anti-EGFR), and matuzumab (anti-EGFR) were used to design DNA sequences after codon optimization for mammalian expression. These antibodies are referred to as the "parent" antibodies.

[0239] The heavy chain DNA construct was designed as follows: - signal peptide (SEQ ID NO: 79), followed by a sequence consisting of the variable region of the outer Fab, followed by the constant CH1 domain of the outer Fab. - followed by the linker followed by the variable region of the inner Fab, followed by the constant CH1 domain of the inner Fab.

[0240] Flanking sequences for restriction enzyme digestion were introduced at both ends of the heavy chain DNA construct.

[0241] The DNA construct for the light chain of the outer Fab was designed as follows: - signal peptide (SEQ ID NO: 80), - followed by the variable region of the outer Fab, followed by the constant kappa region of the outer Fab.

[0242] The DNA construct for the light chain of the inner Fab was designed as follows: - signal peptide (SEQ ID NO: 80), - followed by the variable region of the inner Fab, followed by the constant kappa region of the inner Fab.

[0243] All DNA constructs were synthesized by GeneWis or Eurofins. PCR reactions using PfuTurbo Hot Start were performed to amplify the inserts, which were then digested with NotI and ApaI, and NotI and HindIII for the heavy and light chains, respectively. The double-digested heavy chain fragment was ligated into a NotI- and ApaI-digested Icosagen proprietary pQMCF expression vector into which a human IgG1 hinge followed by the CH2-CH3 domains had already been inserted. The double-digested light chain fragment was ligated into a NotI- and HindIII-digested Icosagen proprietary pQMCF expression vector. Plasmid DNA was verified by double-stranded DNA sequencing.

[0244] Expression and purification Transient gene expression was used to produce bispecific antibodies by co-transfecting the three genes encoded on separate vectors at a 2:1:1 = HC:LC1:LC2 molecular ratio (one continuous heavy chain (HC) and two light chains (LC)). For 50 mL-scale expression, a total of 50 μg of plasmid DNA (25 μg heavy chain + 12.5 μg each of light chains, LC1 and LC2) in Icosagen's proprietary pQMCF vector was mixed with 1 mL of CHO TF (Xell AG) growth medium containing Icosagen's proprietary transfection reagent 007 in a 1.5 mL Eppendorf tube and incubated for 20 min at RT. The mixture was cultured at 1-2 × 10 6 cells / ml in CHO TF (Xell AG) growth medium in a 125 mL shake flask. 6 49 mL of CHOEBNALT85 1E cells / mL 9 The cells were shaken at 37°C for 2 days; on day 3, the temperature was shifted to 32°C and the culture was fed; on day 4, the temperature was reduced to 30°C; the duration of full production was 10 days. The cells were centrifuged at 3,000 rpm for 15 minutes to collect the supernatant. The bispecific antibody was purified from the collected supernatant using Protein A resin (MabSelect SuRe 5mL column). The antibody was eluted from Protein A using 0.1M glycine pH 3.5, and the eluate was neutralized with 1M TRIS. The bispecific antibody was further purified using gel filtration chromatography using a Superdex 200 HiLoad 26 / 60pg prep column, while the Fab-Fab antibody was purified using Superdex 200 Increase 10 / 300 GL; all antibodies were buffer exchanged into PBS pH 7.4. All samples were sterile filtered using a 0.2μm ULTRA Capsule GF. Apparent MW was determined using Ladder Precision Plus Protein Unstained Standards (Biorad). Bispecific antibodies typically showed excellent expression titers in transient CHO expression.

[0245] SDS polyacrylamide gel electrophoresis To assess the quality of the purified antibodies, we performed SDS-PAGE. In the presence of sodium dodecyl sulfate (SDS) in the running buffer, the speed at which antibodies migrate through the gel depends primarily on their size, allowing for molecular weight determination. This assay was performed under non-reducing and reducing conditions; the latter allows for the disruption of disulfide bonds and therefore the visualization of individual polypeptide chains (light and heavy chains).

[0246] SDS-PAGE of all bispecific antibodies showed the expected profiles under both non-reducing and reducing conditions, consistent with the calculated theoretical molecular weights.

[0247] Size exclusion chromatography analysis Protein aggregation is frequently observed in engineered protein molecules. Applicants performed analytical size exclusion chromatography (SEC) to assay the high molecular weight species content of the final antibody preparation after gel filtration chromatography.

[0248] SEC chromatograms demonstrated that the percentage content of higher molecular weight species was minor (less than 5%), similar to that of conventional monoclonal antibodies produced in CHO expression systems. The results confirmed the absence or minor content of aggregates. The results also showed that each antibody assembled correctly.

[0249] Example 2 Monospecific binding of an antibody to its cognate antigen The apparent affinities of the BsAbs "Patri-Trastu-Fc," "Patri-Matu-Fc," and "Patri-Cetu-Fc" for EGFR, HER2, and HER3 were assessed by direct ELISA. The monospecific binding of each BsAb, i.e., binding to one antigen at a time, was compared to that of the parent MAb. The EC50 (antibody concentration that induces 50% median binding between baseline and maximum binding) was then defined for each BsAb and the parent antibody.

[0250] Materials and Methods 800 ng / ml of EGFR His-tag, HER2 His-tag, or HER3 His-tag antigen was diluted in 50 μl of 1x PBS in a Nunc Maxisorp 96 plate. The plate was incubated overnight at 4°C, then washed four times with 0.1% PBS-Tween (PBS-T) and saturated with PBS-T containing 1% BSA for 1 hour at 37°C. After four washes with 0.1% PBS-T, antibodies (BsAb, Mab, and control) were diluted in 1x PBS to a final volume of 50 μl and incubated for 1 hour at 37°C. After four washes with 0.1% PBS-T, detection antibody (HRP-coupled anti-human Fc) was added at 1 / 40,000 in a 50 μl volume of 1x PBS and incubated for 1 hour at 37°C. The plate was washed four times with 0.1% PBS-T, and 50 μl of TMB was added to each well for 10–30 minutes at room temperature, protected from light. The reaction was stopped with 50 μl of 1 M hydrochloric acid, after which the optical density was read at a wavelength of 450 nm.

[0251] result The results are shown in Table 3 below.

[0252] [Table 4]

[0253] ELISA analysis for monospecific binding to HER3, HER2 or EGFR. Determination of half-maximal effective concentration (EC50) for each parental antibody (as positive control) or BiXAb against HER3, HER2 or EGFR antigens.

[0254] All bispecific antibodies were able to bind antigen and displayed binding profiles very similar to those of the corresponding parent antibodies: subnanomolar apparent affinities for their cognate antigens were measured for all constructs and were nearly identical to those of their parent antibodies.

[0255] Example 3 Dual binding by bispecific antibodies to their cognate antigens The bispecific binding of the antibody to HER3 / HER2 and HER3 / EGFR was assessed by an ELISA sandwich assay. Briefly, untagged antigen was coated onto a plate and incubated first with a BsAb, then with a second antigen bearing a His tag; finally, the assay was developed using an anti-HIS peroxidase antibody. This design allows for simultaneous binding by the bispecific antibody to both antigens to be assessed.

[0256] Materials and Methods 1000 ng / ml of EGFR, HER2, or HER3 untagged antigen was diluted in 50 μl of 1× PBS in a Nunc Maxisorp 96 plate. The plate was incubated overnight at 4°C, then washed four times with 0.1% PBS-Tween and saturated with PBS-T containing 1% BSA for 1 hour at 37°C. After four washes with 0.1% PBS-T, antibodies (BsAb or IRR) were added at various dilutions in a final volume of 50 μl of 1× PBS and incubated for 1 hour at 37°C. After four washes with 0.1% PBS-T, 1000 ng / ml of EGFR His-tag, HER2 His-tag, or HER3 His-tag antigen in 50 μl of PBS 1× was incubated for 1 hour at 37°C. The plate was washed four times with 0.1% PBS-T, and an HRP-coupled anti-His-tag detection antibody was added at 1 / 2000 in 50 μl of 1× PBS. The plate was incubated for 1 hour at 37°C. The plate was washed four times with 0.1% PBS-T, and 50 μl of TMB color development solution was added for 10-30 minutes at room temperature, protected from light. The reaction was stopped with 50 μl of 1 M hydrochloric acid, and then the optical density was read at a wavelength of 450 nm.

[0257] result The binding curves in Figure 2(A) show the simultaneous binding profiles of Patri-Trastu-Fc to immobilized HER3 generated by soluble HER2-His (left panel) and to immobilized HER2 generated by soluble HER3-His (right panel) in a dose-dependent manner. The binding curves in Figure 2(B) show the simultaneous binding of Patri-Matu-Fc to immobilized HER3-Fc and EGFR-His (left panel) and to immobilized EGFR-Fc and HER3-His (right panel). This also applies to the binding curves shown in Figure 2(C) for Patri-Cetu-Fc.

[0258] The results therefore demonstrate that the BsAb of the present invention is capable of binding to both antigens simultaneously.

[0259] Example 4 Bispecific antibodies inhibit phosphorylation of AKT and ERK The primary antitumor effect of antibodies targeting the HER family of receptors is their ability to inhibit signaling associated with the receptor. Therefore, we investigated two major signaling pathways associated with EGFR, HER2, and HER3 signaling: MAPK / ERK and PI3K / AKT. Using HTRF techniques, we assessed the inhibition of pERK and pAKT phosphorylation induced by the addition of bispecific antibodies after stimulation with EGF plus NRG1 ligand.

[0260] Materials and Methods On day 0, 50,000 cells per well were seeded in 100 μl of complete medium (DMEM or RPMI 10% SVF) in a 96-well flat-bottom plate. On day 1, cells were FCS-depleted by replacing the 10% FCS culture medium with 2% FCS medium. On day 2, the culture medium was removed for all conditions, and cells were treated with 70 nM of antibody (BsAb or MAb) diluted in 100 μl of 2% FCS culture medium for 20 min, followed by the addition of two ligands: 16.6 nM (100 ng / ml) EGF and 3.71 nM (100 ng / ml) NRG1 for an additional 10 min at 37°C. At the end of the 30-min treatment, the medium was then removed, and the cells were rinsed once with cold 1× PBS. Cells were then lysed for 1 hour under agitation, and pERK and pAKT levels were measured using the HTRF kits pERK Thr202 / Tyr204 (CisBio®, #64ERKPEG) and pAKT Ser473 (CisBio®, #64AKSPET) according to the manufacturer's recommendations. Plates were then read at 665 nm and 620 nm on a Pherastar reader (BMG LabTech).

[0261] result In the three PDAC cell lines tested (BxPC-3, AsPC-1, and CFPAC), all BsAbs (Patri-Trastu-Fc, Patri-Cetux-Fc, and Patri-Matu-Fc) showed strong inhibition of pAKT and pERK signaling after ligand induction (Figure 3B). Both EGFR / HER3 bispecific antibodies blocked nearly 100% of basal pAKT signaling in BxPC-3 cells and approximately 85–90% in ASPC1 and CFPAC cells, similar to the HER2 / HER3 antibodies. They also efficiently inhibited pERK signaling in BxPC3 cells (85 and 99% for Patri-Matu-Fc and Patri-Cetux-Fc, respectively, compared to 50% for Patri-Trastu-Fc). All antibodies inhibited the signaling pathway most strongly in the BxPC3 cell line, because this cell line was the only one expressing WT Kras. pERK inhibition was weaker in ASPC1 and CFPAC, which expressed mutated Kras. In all three cell lines, EGFR density was much higher than that of HER2 or HER3 (Figure 3A). The superiority of anti-EGFR-containing BiXAbs over anti-HER2 / HER3 antibodies was also expected.

[0262] Example 5 In vivo effects of Patri-Trastu-Fc, Patri-Matu-Fc and Patri-Cetu-Fc Tumor growth and survival were assessed in bispecific antibody-treated mice xenografted with Sw1990 and PDX P2846 PDAC cells, models that express mutated Kras and similar antigen densities of EGFR, HER2, or HER3 antigens.

[0263] Materials and Methods On day 0, 5-week-old Swiss nude immunodeficient mice were implanted subcutaneously with 3 million Sw-1990 cells (n = 9 mice / group). Treatment began when tumors averaged 90 mm3. Mice were treated with 17 mg / kg of bispecific antibody administered intraperitoneally in a 200 μl injection. The treatment schedule was two injections per week for 4 weeks. Tumor size was measured with a caliper. Mice were sacrificed when tumor size reached 1500-2000 mm3.

[0264] P2846 P7 with a size of 150 mm was implanted intrascapularly under the brown body. The treatment regimen remained the same as in the Sw-1990 mouse model.

[0265] result In mice xenografted with Sw1990 cells, tumor growth was inhibited by 42%, 62%, and 69% by Patri-Trastu-Fc, Patri-Matu-Fc, and Patri-Cetu-Fc, respectively (Fig. 4A).

[0266] Survival of Sw-1990 xenografted mice was improved by 11 days in the Patri-Trastu-Fc treatment group, 15 days in the Patri-Matu-Fc treatment group, and 15 days in the Patri-Cetu-Fc treatment group. EGFR / HER3 BsAb induced the strongest tumor growth inhibition (875 mm³ for 3Patri-1Matu-Fc and 3Patri-1Cetu-Fc on day 46) compared with 3Patri-2Trastu-Fc EGFR / HER2 BsAb (1200 mm³ on day 46). BsAb 3Patri-1Cetu-Fc provided superior overall survival. No signs of toxicity or weight loss were observed in this experiment. Negative control vehicle vs. irrelevant bispecific antibody (BsAb IRR) showed similar tumor growth and survival.

[0267] In mice xenografted with PDX P2846, tumor growth was inhibited by 87% with Patri-Cetu-Fc, 77% with Patri-Trastu-Fc, and 63% with Patri-Matu-Fc (45 days after grafting) (Figure 4B).

[0268] At day 80, survival of mice xenografted with P2846 P7 PDX was improved by 70% in the Patri-Trastu-Fc and Patri-Cetu-Fc treatment groups and by 20% in the Patri-Matu-Fc treatment group compared with the BsAb IRR group. BsAbs Patri-Trastu-Fc and Patri-Cetu-Fc induced the strongest tumor growth inhibition compared with Patri-Matu-Fc. Survival was improved by 34 days in the Patri-Trastu-Fc group and 41 days in the Patri-Cetu-Fc group compared with the control BsAb IRR group. No signs of toxicity or weight loss were observed in this study.

[0269] Example 6 Intratumoral penetration of BsAb in Sw-1990 xenografts A common question regarding the large molecular weight of BsAbs (250 kDa) compared with conventional antibodies (150 kDa) concerns their ability to penetrate tumors, especially pancreatic tumors, which are known to have a rich and dense stroma. Histological sections were prepared from excised tumors; Sw1990 tumor penetration by the bispecific antibody was analyzed by immunohistochemistry. Peroxidase-conjugated anti-human Fc was used to label the bispecific antibody. BsAb IRR was a control bispecific antibody targeting CD19 and CD3. NaCl was used as a negative control (Figure 5).

[0270] Specific labeling of BsAb was observed in tumors treated with Patri-Trastu-Fc, Patri-Matu-Fc, and Patri-Cetu-Fc. 40x images show that labeling was uniform between the center and periphery of the tumor despite the dense stroma, suggesting that BsAb penetrates the tumor. In addition, significant labeling was observed in the membrane of tumor epithelial cells in the tumor center, demonstrating BsAb binding and accumulation in the membrane of tumor cells within the tumor mass. No nonspecific labeling was observed for tumors treated with NaCl. BsAb IRR was weakly detected in the tumor periphery or stroma but not in tumor epithelial cells, indicating that BsAb IRR does not bind to tumor cells.

[0271] Example 7 Patri-Trastu-Fc, Patri-Matu-Fc, and Patri-Cetu-Fc reduce angiogenesis Angiogenesis or neovascularization has been studied using anti-CD31 staining by immunohistochemistry (IHC). CD31 is expressed on endothelial cells, allowing for the labeling of blood vessels. Angiogenesis is a key parameter in tumor transformation, particularly in pancreatic cancer. The proportion of blood vessels was assessed after treatment with three BsAbs compared with controls (NaCl and BsAb IRR). For each treatment, three tumor sections corresponding to three different mice were labeled, and the images were analyzed using ImageJ software. Quantification results are presented in histograms highlighting the variation in vessel size according to treatment (Figure 6A).

[0272] IHC results demonstrate a reduction in vessel size from 75 μm in the control group (NaCl, BsAb IRR) to 55 μm in the group treated with the BsAb of the invention, suggesting inhibition of xenograft neovascularization by the BsAb.

[0273] Example 8 Ex vivo analysis of NK infiltration in tumors treated with Patri-Trastu-Fc, Patri-Matu-Fc, and Patri-Cetu-Fc Materials and Methods NK cell immunophenotyping was performed by flow cytometry in excised Sw1990 xenografts from BsAb-treated mice. Dissociated cells were labeled with live / dead, mouse CD45, CD3, CD19, CD49, NKp46, IFNγ, and CD1070-specific antibodies. NK cells were isolated for immunophenotyping using a panel containing phycoerythrin-conjugated anti-CD3 and APC-Cy7-conjugated anti-CD19 antibodies for negative selection, and AF700-conjugated anti-CD45, APC-conjugated anti-CD49b, and fluorescein-conjugated anti-NKp46 for positive selection. Following BV786-conjugated anti-CD107a and intracellular BV421-conjugated anti-IFNγ labeling, CD45+CD49b+NKp46+ NK cell activation was analyzed by flow cytometry.

[0274] result NK infiltration was increased twofold in tumors treated with the BsAb of the present invention compared with the NaCl and BsAb IRR group, as shown by CD49+NKp46+ analysis (Figure 5B). The increase in IFNγ+ and CD107a+ in NK cells suggested that these intratumoral NK cells were also activated compared with untreated tumors and thus could mediate ADCC.

[0275] Example 9 Ex vivo efficacy of Patri-Trastu-Fc, Patri-Matu-Fc, and Patri-Cetu-Fc in EGFR, HER2, and HER3 receptor degradation Materials and Methods EGFR staining was performed on Patri-Cetu-Fc versus BsAb IRR and NaCl-treated Sw1990 xenograft sections. HER receptor expression was determined by Western blot analysis of each Sw1990 xenograft extract treated with NaCl, Patri-Trastu-Fc, Patri-Matu-Fc, and Patri-Cetu-Fc, respectively. Tubulin was used as a loading control. Western blots were visualized using primary rabbit monoclonal antibodies against EGFR, HER2, and HER3, followed by secondary IRDye800-conjugated goat anti-rabbit IgG (1 / 20,000 dilution). Fluorescence was quantified using a LI-COR Odyssey imaging system. HER receptor expression in each group of excised xenografts was then quantified and normalized to tubulin. Each experiment was performed on three Sw1990 xenografted mice. BsAb IRR is a negative control that simultaneously binds to human CD19 and CD3.

[0276] result Images acquired at ×20 and ×40 magnification (Fig. 5C, top panel) showed a clear reduction in EGFR signal in tumors treated with Patri-Cetu-Fc compared with tumors treated with NaCl or BsAb IRR.

[0277] EGFR, HER2, and HER3 total protein levels were assessed by Western blot in protein extracts from Sw-1990 xenografts and revealed by immunofluorescence. Total protein levels were quantified and normalized to tubulin. The results in Figure 5C (middle and upper panels) show that the three BsAbs of the present invention induce EGFR, HER2, or HER3 degradation compared to the NaCl and BsAb IRR groups.

[0278] Example 10 The bispecific antibody Patri-Cetu-Fc more potently reduced tumor growth compared with monospecific antibodies or their combination in the Sw1990 xenograft model Materials and Methods Mice xenografted with Sw1990 cells were treated (9 mice per group) with (1) BisAb IRR anti-CD19-CD3, (2) bispecific antibody Patri-Cetu-Fc (17 mg / kg), (3) cetuximab parental antibody (10 mg / kg), (4) patritumab parental antibody (10 mg / kg), or the combination (cetuximab + patritumab, 5 + 5 mg / kg). The doses of bispecific and parental antibodies had the same molar concentration based on their Fc content.

[0279] On day 0, 3 million Sw-1990 cells were subcutaneously implanted into Swiss nude immunodeficient mice. Treatment began when tumors reached an average size of 140 mm. The treatment schedule was two injections per week for 4 weeks. Tumor growth and survival of treated mice were assessed (Figure 7).

[0280] result The BsAb Patri-Cetu-Fc induced a strong inhibition of tumor growth in vivo (77% tumor growth inhibition at 38 days post-graft compared to IRR-treated mice). The monospecific antibodies cetuximab and patritumab induced 50% and 43% tumor growth inhibition, respectively, at 38 days post-graft. Finally, the combination of the two monoclonal antibodies showed 65% tumor growth inhibition compared to IRR-treated mice. Thus, the bispecific antibody Patri-Cetu-Fc is much more potent than the parent monospecific antibodies in terms of tumor growth inhibition.

[0281] Patri-Cetu-Fc also induced a strong survival benefit in vivo, demonstrating that the bispecific antibody Patri-Cetu-Fc is more potent than the parent monospecific antibody in terms of survival benefit.

[0282] Example 11 Two additional bispecific antibodies containing humanized cetuximab Fab were designed: - "BMX003-010" antibody: it contains the patritumab Fab domain (as the outer Fab) and a humanized version of the cetuximab Fab domain (as the inner Fab). - The "BMX003-011" antibody, which is identical to BMX003-010 except that it further contains two mutations in the Fc domain (S239D and I332E).

[0283] Both BMX003-010 and BMX003-011 are - a VH domain according to SEQ ID NO: 81 and a CH1 domain according to SEQ ID NO: 2 (corresponding to the VH-CH1 sequence according to SEQ ID NO: 83) - a VL domain according to SEQ ID NO: 71 and a CL domain according to SEQ ID NO: 4 (corresponding to the VL-CL sequence according to SEQ ID NO: 84) The humanized cetuximab Fab comprises

[0284] BMX003-011 differs from BMX003-010 in that it contains two mutations (S239D and I332E) in the Fc domain (mutated Fc domain of SEQ ID NO: 82).

[0285] [Table 5]

[0286] The activity of BMX003-010 and BMX003-011, which contain humanized cetuximab Fab fragments, was compared to the activity of "BMX003-001," which contains the parent cetuximab Fab ("BMX003-001" corresponds to the "Patri-cetu-Fc" construct described above).

[0287] SDS-polyacrylamide gel electrophoresis and size-exclusion chromatography analysis To assess the quality of the purified antibodies, SDS-PAGE and size-exclusion chromatography (SEC) were performed as described above. SDS-PAGE of the BMX003-010 and BMX003-011 bispecific antibodies showed the expected profiles. SEC chromatograms confirmed the absence or minor content of aggregates, indicating that each antibody was correctly assembled.

[0288] Affinity for HER3 and EGFR assessed by ELISA The apparent affinities of the BsAbs BMX003-010, BMX003-011, and BMX003-001 (Patri-Cetu-Fc) antibodies for HER3 and EGFR were assessed by direct ELISA. The monospecific binding of each BsAb, i.e., binding to one antigen at a time, was compared to that of the parent MAb. The EC50 (antibody concentration that induces 50% median binding between baseline and maximum binding) was then defined for each BsAb and parent antibody. ELISA assays were performed as described above.

[0289] Figures 8 and 9 show that all bispecific antibodies were able to bind to their antigens (HER3 and EGFR, respectively) with binding profiles very similar to the corresponding parent antibodies.

[0290] Flow cytometry binding profile The ability of BMX003-010, BMX003-011 and BMX003-001 (Patri-Cetu-Fc) to bind to HER3 and EGFR proteins expressed on the cell surface of SW-1990 cells was measured by flow cytometry (n=3).

[0291] The results in Figure 10 show that BMX-003-001 and humanized BMX-003-010 and BMX-003-011 have very similar EC50 and ECmax.

[0292] Phosphorylation assay Based on Perkin Elmer's Advanced Phospho-ERK and Advanced Phospho-AKT kits. HCT-116 colon cancer cells were plated in cDMEM medium at a density of 50,000 cells / well in 96-well flat-bottom microtiter plates and grown overnight at 37°C (5% CO). Tissue culture medium was removed and replaced with serum-free medium (DMEM + GlutaMAX), and the cells were incubated for an additional 24 hours at 37°C (5% CO). Tissue culture medium was removed and replaced with test article (BiXAb, Mab, or control) resuspended in DMEM and incubated for 20 minutes at 37°C (5% CO). Growth factors (16.6 nM EGF and 3.71 nM NRG1) were added to the wells and incubated for an additional 10 minutes. The medium was removed, and the cells were then washed once with cold PBS. Cells were then lysed by adding lysis buffer (provided in the Perkin Elmer kit). Quantification of the phosphor signal was achieved by following the FRET assay kit instructions provided by Perkin Elmer. Essentially, 4 microliters of premixed europium-labeled antibody was incubated with the cell lysate for 4 hours at room temperature. The supernatant was then transferred to a fluorescence plate reader and HTRF was measured at 665 nm and 620 nm.

[0293] The results show that all bispecific antibodies showed strong inhibition of pAKT (FIG. 11A) and pERK (FIG. 11B) signaling after ligand induction.

[0294] Degranulation assay PBMCs from two independent donors were prepared according to standard procedures and incubated overnight in complete RPMI medium. The HCT116 colon cancer cell line was grown in RPMI tissue culture medium supplemented with 10% FBS. 20,000 HCT116 cells were plated with 100,000 PBMCs in a U-bottom 96-well microtiter plate (resulting in a 1:2 effector-to-target (E:T) ratio based on the NK population being approximately 10% of the PBMCs). BiXAb test articles and controls were then added to the cell mixture along with anti-CD107a-AF488 mAb and incubated for 1 hour at 37°C (5% CO). GolgiStop was then added to the wells, and the plates were incubated for an additional 4 hours. Anti-CD45, anti-CD3, and anti-CD56 mAbs were then added to the wells to allow identification of NK cells by typical FACs staining. NK degranulation was assessed by FACS analysis by gating on CD107a on NK cells.

[0295] The results in Figure 12 show that cetuximab, BMX003-001, and BMX003-010 are comparable. Much greater degranulation was observed in the presence of BMX003-011 (Fc modified), suggesting that the BMX003-011 mutant Fc domain has increased binding to Fc gamma receptors compared to the wild-type Fc domain, resulting in a bispecific antibody with improved effector function.

[0296] In vivo efficacy The in vivo efficacy of BMX003-001 (Patri-Cetu-Fc), BMX003-010 (humanized Patri-Cetu-Fc), and BMX003-011 (humanized Patri-Cetu-Fc modified) was assessed in the SW1990 pancreatic cancer xenograft model. BMX003-01, BMX003-010, and BMX003-011 were used at 8.5 mg / kg, and BMX control was used at 17 mg / kg. Methods were as described above.

[0297] On day 35 (after the last injection on day 32), all groups treated with HER3xEGFR BiXAb exhibited significantly lower tumor volumes than the control group PBS. No significant differences were observed between BMX003-001, BMX-003-010, and BMX-003-011 BiXAbs.

[0298] conclusion The results show that the bispecific antibodies of the invention were efficiently produced and properly assembled. The results also demonstrate the therapeutic properties of the bispecific antibodies: - simultaneously binds to its target with superior affinity compared to the parent antibody; - Inhibits the activation of HER3 / HER2 / EGFR receptors by inhibiting the phosphorylation signaling pathway; - Degrades two targeted receptors simultaneously; - penetrates into the tumor; - Inhibits neovascularization: - Modulating immune cell infiltration in tumors; - Significantly inhibiting tumor growth and improving survival in vivo.

Claims

1. (i) a Fab fragment comprising the VH-CH1 heavy chain associated with the VL-CL light chain of antibody 1 (Ab1), and (ii) a Fab fragment comprising a VH-CH1 heavy chain associated with a VL-CL light chain of antibody 2 (Ab2); a bispecific antigen-binding fragment capable of simultaneously binding to HER-3 and to another antigen selected from HER-2 and EGFR antigens, comprising: the N-terminal end of the VH domain of the Fab fragment of Ab1 is linked to the C-terminal end of the CH1 domain of the Fab fragment of Ab2 via a polypeptide linker; one of Ab1 or Ab2 is patritumab or a functional derivative thereof; the other of Ab1 or Ab2 is selected from the group consisting of trastuzumab or a functional derivative thereof, matuzumab or a functional derivative thereof, and cetuximab or a functional derivative thereof; Bispecific antigen-binding fragment.

2. 2. The bispecific antigen-binding fragment of claim 1, wherein Ab2 is patritumab or a functional derivative thereof, and Ab1 is selected from the group consisting of trastuzumab or a functional derivative thereof, matuzumab or a functional derivative thereof, and cetuximab or a functional derivative thereof.

3. 2. The bispecific antigen-binding fragment of claim 1, wherein the functional derivative of cetuximab is a humanized form comprising VH and VL chain amino acid sequences that are at least 80% identical to the VH and VL chain amino acid sequences of cetuximab, respectively.

4. 2. The bispecific antigen-binding fragment of claim 1, wherein the CH1 and CL domains of Ab1 have sequences that differ from the CH1 and CL domains of Ab2.

5. the Fab CH1 domain of one of Ab1 or Ab2 is a mutated domain derived from the CH1 domain of an immunoglobulin by substitution of a threonine residue at position 192 of said CH1 domain with a glutamic acid residue, and the cognate CL domain is a mutated domain derived from the CL domain of an immunoglobulin by substitution of an asparagine residue at position 137 of said CL domain with a lysine residue and a serine residue at position 114 of said CL domain with an alanine residue; and / or one or the other of Ab1 or Ab2 is a mutated domain derived from the CL domain of an immunoglobulin by substitution of a lysine residue at position 137 of said CL domain with a serine residue at position 114 of said CL domain with an alanine residue.

2. The bispecific antigen-binding fragment of claim 1, wherein the CH1 domain is a mutated domain derived from an immunoglobulin CH1 domain by substitution of a glutamine for the leucine residue at position 143 of the CH1 domain and a valine for the serine residue at position 188 of the CH1 domain, and the cognate CL domain is a mutated domain derived from an immunoglobulin CL domain by substitution of a threonine for the valine residue at position 133 of the CL domain and a valine for the serine residue at position 176 of the CL domain.

6. The bispecific antigen-binding fragment of claim 1, wherein the polypeptide linker sequence comprises or consists of the amino acid sequence: EPKX1CDKX2HX3X4PPX5PAPELLGGPX6X7PPX8PX9PX10GG (SEQ ID NO: 33), in which X1, X2, X3, X4, X5, X6, X7, X8, X9, and X10, which may be identical or different, are any amino acid.

7. A bispecific molecule comprising two identical antigen-binding arms, each consisting of the antigen-binding fragment of claim 1.

8. a full-length antibody comprising two heavy chains and four light chains; Each heavy chain is d. comprising an immunoglobulin Fc region including the hinge-CH2-CH3 domain; e. the Fc region is linked to the Ab1 Fab VH-CH1 heavy chain by the hinge domain, which connects the N-terminal end of the CH2 domain to the C-terminal end of the Ab1 CH1 domain; f. which is then linked to the Fab VH-CH1 heavy chain of Ab2 by a polypeptide linker sequence connecting the N-terminal end of the VH domain of Ab1 to the C-terminal end of the CH1 domain of Ab2; 8. The bispecific molecule of claim 7, wherein the four light chains comprise two Fab VL-CL light chains of Ab1 and two Fab VL-CL light chains of Ab2 associated with their cognate heavy chain domains.

9. 8. The bispecific molecule of claim 7, comprising a) two heavy chains each comprising, preferably consisting of, SEQ ID NO: 27, and b) four light chains, two comprising, preferably consisting of, SEQ ID NO: 14 and two comprising, preferably consisting of, SEQ ID NO:

18.

10. 8. The bispecific molecule of claim 7, comprising a) two heavy chains each comprising, preferably consisting of, SEQ ID NO: 29, and b) four light chains, two comprising, preferably consisting of, SEQ ID NO: 14 and two comprising, preferably consisting of, SEQ ID NO:

16.

11. 8. The bispecific molecule of claim 7, comprising a) two heavy chains each comprising, preferably consisting of, SEQ ID NO: 31, and b) four light chains, two comprising, preferably consisting of, SEQ ID NO: 14 and two comprising, preferably consisting of, SEQ ID NO:

20.

12. 12. A method for producing a bispecific molecule according to any one of claims 7 to 11, comprising: a. culturing a host cell expressing an antibody heavy chain as defined in any one of claims 6 to 10 and an antibody light chain as defined in any one of claims 6 to 10 in a suitable medium and culture conditions; b. recovering the produced antibody from the culture medium or the cultured cells; A method comprising:

13. 12. A bispecific antigen-binding fragment according to any one of claims 1 to 6 or a bispecific molecule according to any one of claims 7 to 11 for use as a medicament.

14. 12. The bispecific antigen-binding fragment of any one of claims 1 to 6 or the bispecific molecule of any one of claims 7 to 11 for use in the treatment of cancer, preferably a solid tumor, such as pancreatic cancer, head and neck cancer, colorectal cancer, breast cancer or lung cancer, preferably for use in the treatment of pancreatic cancer.

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