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

Abstract

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

Description

[0001] The present invention relates to bispecific antigen binding molecules (particularly antibodies) targeting HER-3 and another antigen selected from HER-2 and EGFR, methods for producing these molecules, compositions and uses thereof. Background Art

[0002] The HER family, which includes four tyrosine kinase receptors (EGFR / HER1, HER2, HER3 and HER4), activates multiple partially redundant, interconnected downstream signaling cascades, such as MAPK and PI3K / AKT pathways, which are involved in cell proliferation. The general structure shared by HER receptors consists of an extracellular domain, a single transmembrane domain, and an intracellular domain containing a conserved catalytic kinase domain and a carboxyl-terminal tail. The fundamental aspect of signaling in this family is the dimerization of the two receptors. The homodimerization and heterodimerization of the two HER family members and the transphosphorylation of their intracellular regions generate the initial signals that lead to the activation of many downstream signaling pathways. The four members of the HER family are able to form 28 homodimers and heterodimers.

[0003] Dysregulation of HER signaling through gene amplification or mutation has been observed in many human tumors, and a large body of experimental evidence supports the causative role of these events in the pathogenesis of cancer. HER abnormal signaling has been observed in a large number of solid tumors (lung, colorectal, pancreatic, etc.). For example, there is evidence that the HER family is dysregulated in pancreatic cancer. EGFR is expressed in 45%-95% of pancreatic cancers, and expression is often associated with poor outcomes in resected pancreatic cancers. Overexpression of HER2 has also been described in 7%-58% of pancreatic cancers, and HER2-amplified pancreatic cancers show atypical metastatic patterns, suggesting that HER2 may also be an important driver of tumorigenesis in pancreatic cancer. In addition, HER3 expression is associated with reduced tumor progression and survival in patients with pancreatic cancer.

[0004] Pancreatic cancer is the fourth most common cause of cancer death in Europe, with an increasing number of cases every year. (+2% in men and +10% in women). It has a very poor prognosis, even when diagnosed early. It is one of the only cancers for which survival has barely improved over the past 40 years: less than 20% and 5% after 1 and 5 years, respectively. Pancreatic cancer remains a rare disease, with as many deaths recorded worldwide in 2020 (466,003) as new diagnoses (495,773) due to the lack of effective treatments. Currently, pancreatic adenocarcinoma (90% of pancreatic cancers) is treated either with chemotherapy followed by surgery or with a combination of radiotherapy and chemotherapy, but with limited results. The introduction of gemcitabine as first-line treatment in 1996 increased the median recurrence-free survival by 1.3 months and the one-year overall survival (OS) from 2% to 18%. Since 2005, only (erlotinib) and (nab-paclitaxel) Two drugs are approved for pancreatic cancer, and despite various clinical trials, they mainly involve combinations but little innovation. Erlotinib was the first targeted therapy and, in combination with gemcitabine, improved the median recurrence-free survival by only 1 month. Regarding HER3 inhibitors, multiple 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 intrinsic robustness of the signal transduction network.

[0006] In this context, combination therapies have emerged as a natural countermeasure, but their optimal design is not simple and may depend on the tumor or its subtype, so patients need to be stratified in advance (Fitzgerald JB, SchoeberlB, Nielsen UB, Sorger PK. Systems biology and combination therapy in the questfor clinical efficacy. Nat Chem Biol. 2006; 2: 458–66.). Weapons currently available for inhibiting HER signaling include small molecule tyrosine kinase inhibitors (TKIs), such as lapatinib or erlotinib, and therapeutic antibodies, such as cetuximab or trastuzumab. In fact, compared to TKIs, which are limited to signaling regulation, antibodies represent a powerful approach that, in addition to signaling reduction, can also induce immunological effects to help clear tumors.

[0007] Many authors have proposed antibody-based combination therapies. Targeting HER dimers, especially EGFR / HER2 heterodimers, by mAb combinations has been shown to be beneficial in inhibiting pancreatic tumor growth.

[0008] Bispecific antibodies (BsAbs) have been further designed, which combine the targets of two mAbs. However, they are subject to complex design, which often leads to poor manufacturability and stability, and the efficacy of these antibodies can still be improved, as in vivo pancreatic cancer model tumors continued to grow even when treated with bispecific antibodies.

[0009] For example, 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, which utilized panitumumab and trastuzumab sequences, respectively. The antibody showed improved activity against EGFR+ / HER2+ cell lines in vitro and in vivo, however, the activity of the antibody 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), has been studied in patients with HER2-positive cancers of the distal esophagus, gastroesophageal (GE) junction, and stomach. A phase II clinical trial was stopped due to insignificant results.

[0011] Duligotuzumab, an anti-EGFR / anti-HER3 bispecific antibody (Schaefer et al., 2011b; Spiess et al., 2015), was compared with cetuximab in combination with FOLFIRI as a second-line therapy for patients with metastatic colorectal cancer. In this trial, duligotuzumab did not show benefit over cetuximab in combination with FOLFIRI and was therefore abandoned.

[0012] In view of the above, there remains a need for improved bispecific antibodies targeting HER receptors for the treatment of tumors. SUMMARY OF THE INVENTION

[0014] The inventors have now designed novel bispecific antibodies targeting HER3 and another antigen selected from EGFR or HER2, which can be used to treat a variety of cancers.

[0015] The present invention more specifically provides a bispecific antigen-binding fragment capable of simultaneously binding to HER-3 and another antigen selected from HER-2 and EGFR antigens, the bispecific antigen-binding fragment comprising:

[0016] (i) a Fab fragment comprising the VH-CH1 heavy chain associated with the VL-CL light chain of Antibody 1 (Ab1), and

[0017] (ii) a Fab fragment comprising the VH-CH1 heavy chain associated with the VL-CL light chain of Antibody 2 (Ab2),

[0018] The N-terminal end of the VH domain of the Fab fragment of Ab1 is connected to the C-terminal end of the CH1 domain of the Fab fragment of Ab2 through a polypeptide linker.

[0019] wherein one of Ab1 or Ab2 is pertuzumab or a functional derivative thereof;

[0020] And the other one of Abl 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.

[0021] In a preferred embodiment, Ab2 is Pertratuzumab or a functional derivative thereof, and wherein 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.

[0022] In a specific embodiment, 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.

[0023] In certain embodiments, the CH1 and CL domains of Abl have different sequences than the CH1 and CL domains of Ab2.

[0024] In a specific embodiment, the Fab CH1 domain of one of Ab1 or Ab2 is a mutant domain derived from a CH1 domain of an immunoglobulin by replacing a threonine residue at position 192 of the CH1 domain with a glutamic acid, and the cognate CL domain is a mutant domain derived from a CL domain of an immunoglobulin by replacing an asparagine residue at position 137 of the CL domain with a lysine residue and replacing a serine residue at position 114 of the CL domain with an alanine residue, and / or wherein the Fab of one or the other of Ab1 or Ab2 is The CH1 domain is a mutant domain derived from the CH1 domain of an immunoglobulin, which is achieved by replacing the leucine residue at position 143 of the CH1 domain with glutamine and replacing the serine residue at position 188 of the CH1 domain with a valine residue, and the homologous CL domain is a mutant domain derived from the CL domain of an immunoglobulin, which is achieved by replacing the valine residue at position 133 of the CL domain with a threonine residue and replacing the serine residue at position 176 of the CL domain with a valine residue.

[0025] In a specific embodiment, the polypeptide linker sequence comprises or consists of the amino acid sequence: EPKX1CDKX2HX3X4PPX5PAPELLGGPX6X7PPX8PX9PX10GG (SEQ ID NO: 33), wherein X1, X2, X3, X4, X5, X6, X7, X8, X9, and X10 are the same or different and are any amino acids.

[0026] The invention also provides a bispecific molecule comprising two identical antigen binding arms, each antigen binding arm consisting of an antigen binding fragment as defined above.

[0027] In a preferred embodiment, the bispecific molecules of the invention are full length antibodies comprising two heavy chains and four light chains.

[0028] Each heavy chain contains

[0029] a. The Fc region of an immunoglobulin, which contains the hinge-CH2-CH3 domain

[0030] b. The Fc region is connected to the Fab VH-CH1 heavy chain of Ab1 through the hinge domain, wherein the hinge domain connects the N-terminal end of the CH2 domain of Ab1 and the C-terminal end of the CH1 domain;

[0031] c. which is further connected to the Fab VH-CH1 heavy chain of Ab2 via a polypeptide linker sequence, wherein the polypeptide linker sequence connects the N-terminal end of the VH domain of Ab1 and the C-terminal end of the CH1 domain of Ab2,

[0032] And wherein the four light chains comprise two Fab VL-CL light chains of Abl and two Fab VL-CL light chains of Ab2 associated with their cognate heavy chain domains.

[0033] In another embodiment, the bispecific molecule comprises a) two heavy chains, each heavy chain comprising, preferably consisting of, SEQ ID NO:27, and b) four light chains, two of which comprise, preferably consist of, SEQ ID NO:14 and the other two comprise, preferably consist of, SEQ ID NO:18.

[0034] 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 of which comprise, preferably consist of, SEQ ID NO:14 and the other two comprise, preferably consist of, SEQ ID NO:16.

[0035] In another embodiment, the bispecific molecule comprises a) two heavy chains, each heavy chain comprising, preferably consisting of, SEQ ID NO:31, and b) four light chains, two of which comprise, preferably consist of, SEQ ID NO:14 and the other two comprise, preferably consist of, SEQ ID NO:20.

[0036] The present invention also relates to a method for producing a bispecific molecule of the present invention, said method comprising the steps of:

[0037] a. cultivating a host cell in a suitable medium and culture conditions, the host cell expressing the antibody heavy chain as defined above and the antibody light chain as defined above, and

[0038] b. recovering the produced antibody from the culture medium or from the cultured cells.

[0039] The present invention also relates to a bispecific antigen-binding fragment or a bispecific molecule of the present invention for use as a medicament.

[0040] The present invention also relates to a bispecific antigen-binding fragment or a bispecific molecule of the present invention for use in treating cancer, preferably solid tumors, such as pancreatic cancer, head and neck cancer, colorectal cancer, breast cancer or lung cancer, preferably for use in treating pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 . Schematic diagram of the bispecific antibody of the present invention.

[0042] Figure 2. Dual binding of 2 antigens simultaneously by ELISA. Binding curves show the following simultaneous binding: (A) Patri-Trastu-Fc with fixed HER3-Fc and HER2-His (left figure) and with fixed HER2-Fc and HER3-His (right figure), (B) Patri-Matu-Fc with fixed HER3-Fc and EGFR-His (left figure) and with fixed EGFR-Fc and HER3-His (right figure), or (C) Patri-Cetu-Fc with fixed HER3-Fc and EGFR-His (left figure) and with fixed EGFR-Fc and HER3-His (right figure). The half-maximal effective concentration (EC50) from the ELISA binding curve is indicated.

[0043] Figure 3 .Biological properties of bispecific antibodies that inhibit AKT phosphorylation (pAKT) and ERK phosphorylation (pERK). Cell lines (a) were pre-stimulated with bispecific antibodies (B) for an additional 10 minutes 20 minutes before adding a mixture of NRG1 / EGF ligands. After cell lysis, the expression levels of pAKT and pERK were quantified by HTRF. The TR-FRET signal (665nm / 620nm emission ratio) was measured on a Pherastar reader relative to the maximum phosphorylation obtained in cells stimulated with NRG1 / EGFR without bispecific antibodies (100%; medium). The phosphorylation levels are expressed as percentages, ranging from 0% (white) to 100% (black), corresponding to the phosphorylation in the absence of BsAb "medium".

[0044] Figure 4 . In vivo efficacy of bispecific antibodies in nude mouse models. Tumor growth (left) and survival (middle) of mice treated with bispecific antibodies xenografted with Sw1990 (A) and PDX P2846 (B) PDAC cells. The % of tumor growth inhibition (TGI) at the end of treatment and 50% survival benefit (days) are indicated in the right figure. The gray area corresponds to the duration of antibody treatment.

[0045] Figure 5 .Intratumoral penetration of bispecific antibodies in Sw-1990 xenografts. Immunohistochemical analysis of Sw1990 tumor penetration by bispecific antibodies. The bispecific antibodies were 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 sections (left) and under a x40 microscope (right).

[0046] Figure 6.Anti-tumor characteristics of the TME were observed in a pancreatic mouse model of Sw1990-xenografts after treatment with the bispecific antibodies of the present invention. (A) Immunohistochemical analysis to monitor CD31+ angiogenesis in resected Sw1990 xenografts from treated mice. Microscopic images of xenograft sections treated with Patri-Cetu-Fc- and BsAb IRR and NaCl are shown as examples (left). The average size of CD31+ microvessels in tumors was quantified on whole tumor sections using Qupath software. (B) NK cell immunophenotyping of resected Sw1990 xenografts from mice treated with BiXAbs by flow cytometry. 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 then labeled with CD107a and intracellular IFNg. (C) ErbB degradation analysis in vivo. Immunofluorescence microscopy images of EGFR staining of Sw1990 xenograft sections treated with Patri-Cetu-Fc- and BsAb IRR and NaCl (upper figure). Western blot analysis of ErbB receptor expression in each Sw1990 xenograft extract 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 the LI-COR Odyssey imaging system (middle figure). ErbB expression was quantified in each group of excised xenografts, normalized to tubulin (lower figure). Each experiment was performed on three Sw1990- xenograft mice. BsAb IRR is a negative control that binds to both human CD19 and CD3.

[0047] Figure 7 . In vivo efficacy of the 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 concentration of antibodies based on their Fc content: Patri-Cetu-Fc was used at 17 mg / kg, monoclonal controls (Patrituzumab or Cetuximab) were used at 10 mg / kg, and the combination (Patrituzumab + Cetuximab) was used at 5 mg / kg each. The gray area corresponds to the duration of antibody treatment.

[0048] Figure 8.Binding profiles of BMX003-001 (Patri-Cetu-Fc), BMX003-010 (humanized Patri-Cetu-Fc) and BMX003-011 (modified humanized Patri-Cetu-Fc) in HER3 antigen binding ELISA (n=1).

[0049] Fig. 9 . Binding profiles of BMX003-001 (Patri-Cetu-Fc), BMX003-010 (humanized Patri-Cetu-Fc) and BMX003-011 (modified humanized Patri-Cetu-Fc) in EGFR antigen binding ELISA (n=3).

[0050] Fig.10 The ability of BMX003-001 (Patri-Cetu-Fc), BMX003-010 (humanized Patri-Cetu-Fc), and BMX003-011 (modified humanized Patri-Cetu-Fc) to bind to HER3 and EGFR proteins expressed on the cell surface of SW-1990 cells measured using flow cytometry (n=3).

[0051] Fig.11 . Biological properties of BMX003-001 (Patri-Cetu-Fc), BMX003-010 (humanized Patri-Cetu-Fc) and BMX003-011 (modified humanized Patri-Cetu-Fc) in inhibiting AKT (A) and ERK (B) phosphorylation in response to growth factors.

[0052] Fig.12 .The ability of BMX003-001 (Patri-Cetu-Fc), BMX003-010 (humanized Patri-Cetu-Fc) and BMX003-011 (modified humanized Patri-Cetu-Fc) to induce NK cell degranulation (expression of CD107a) in the presence of HCT-116 cancer cells (2 independent donors, A and B).

[0053] Fig.13 .In vivo efficacy study of BMX003-001 (Patri-Cetu-Fc), BMX003-010 (humanized Patri-Cetu-Fc) and BMX003-011 (modified humanized Patri-Cetu-Fc) with 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). DETAILED DESCRIPTION OF THE INVENTION

[0055] definition:

[0056] The basic structure of a naturally occurring antibody molecule is a Y-shaped tetrameric quaternary structure composed of two identical heavy chains and two identical light chains, held together by non-covalent interactions and by interchain disulfide bonds.

[0057] In mammalian species, there are five types of heavy chains: α, δ, ε, γ and μ, which determine the class (isotype) of immunoglobulins: IgA, IgD, IgE, IgG and IgM, respectively. The heavy chain N-terminal variable domain (VH) is followed by a constant region, which contains three domains (numbered from N to C terminus as CH1, CH2 and CH3) in heavy chains γ, α and δ, while the constant region of heavy chains μ and ε contains four domains (numbered from N to C terminus as CH1, CH2, CH3 and CH4). The CH1 and CH2 domains of IgA, IgG and IgD are separated by a flexible hinge, the length of which varies between the different classes, and in the case of IgA and IgG, have hinges of 15, 12, 62 (or 77) and 12 amino acids between the different subtypes: IgG1, IgG2, IgG3 and IgG4, respectively, and IgA1 and IgA2 have hinges of 20 and 7 amino acids, respectively.

[0058] There are two types of light chains: λ and κ, which can associate with any heavy chain isotype, but both are of the same type in a given antibody molecule. The two light chains appear to be functionally identical. Their N-terminal variable domain (VL) is followed by a constant region consisting of a single domain, called CL.

[0059] The heavy and light chains are paired by protein / protein interactions between the CH1 and CL domains and via VH / VL interactions, and the two heavy chains are associated by protein / protein interactions between their CH3 domains. The structure of the immunoglobulin molecule is usually stabilized by interchain disulfide bonds between the CH1 and CL domains and between the hinges.

[0060] The antigen binding region or antigen binding fragment corresponds to the arm of the Y-shaped structure, which consists of each complete light chain paired with the VH and CH1 domains of the heavy chain, and is called a Fab fragment (for antigen binding fragment). Fab fragments are first generated from natural immunoglobulin molecules by papain digestion, which cuts the antibody molecule in the hinge region on the amino-terminal side of the interchain disulfide bond, thereby releasing two identical antigen binding arms. Other proteases such as pepsin also cut the antibody molecule in the hinge region, but on the carboxyl-terminal side of the interchain disulfide bond, releasing a fragment consisting of two identical Fab fragments and kept connected by disulfide bonds; reduction of disulfide bonds in F(ab')2 fragments generates Fab' fragments.

[0061] The part of the antigen binding region corresponding to the VH and VL domains is called the Fv fragment (for variable fragment); it contains the CDRs (complementarity determining regions) which form the antigen binding site (also called the paratope).

[0062] The effector region of the antibody, which is 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 (or CH2, CH3 and CH4 domains, depending on the class of the antibody) of the heavy chain and is called the Fc (for crystallizable fragment) region.

[0063] Due to the identity of the two heavy chains and the two light chains, a naturally occurring antibody molecule has two identical antigen-binding sites and therefore binds two identical epitopes simultaneously.

[0064] An antibody "specifically binds" a target antigen if it binds with greater affinity, avidity, more readily, and / or for a longer duration than it binds to other substances. "Specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Typically, but not necessarily, reference to binding means preferential binding.

[0065] The terms "subject," "individual," and "patient" are used interchangeably herein and refer to a mammal being evaluated for therapy and / or being treated. The subject can be a human, but also includes other mammals, particularly those that can be used as laboratory models of human disease, such as mice, rats, rabbits, dogs, etc.

[0066] The term "treatment" or "treating" refers to an action, application or therapy in which a subject (including humans) receives medical assistance for the purpose of directly or indirectly improving the subject's condition. In particular, in some embodiments, the term refers to reducing the incidence of disease or alleviating symptoms, eliminating recurrence, preventing recurrence, preventing the incidence of disease, improving symptoms, improving prognosis or a combination thereof. It will be understood by those skilled in the art that treatment does not necessarily result in the complete absence or elimination of symptoms. For example, with respect to cancer, "treatment" or "treating" may refer to slowing down tumor or malignant cell growth, proliferation or metastasis, preventing or delaying the development of tumor or malignant cell growth, proliferation or metastasis, or some combination thereof.

[0067] The term "tumor" is used interchangeably herein with the term "cancer", e.g., both terms encompass solid tumors and liquid tumors, e.g., diffuse or circulating tumors. As used herein, the term "cancer" or "tumor" includes precancerous lesions as well as malignant cancers and tumors.

[0068] The term "mutant derivative", "mutant" or "functional derivative" refers to a sequence that differs from the parent sequence to which it refers by deletion, substitution or insertion of one or several amino acids. Preferably, the mutant 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.

[0069] Cetuximab ( ImClone / Lilly, Merck-Serono) is a chimeric mouse-human monoclonal antibody targeting epidermal growth factor receptor (EGFR) (ATCC HB-9764 & ATCC-97-63). See also EP0359282, EP0667165 and US6217866. Cetuximab is approved for the treatment of colorectal cancer and head and neck squamous cell carcinoma.

[0070] Trastuzumab ( 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 the treatment of adjuvant and metastatic breast cancer and metastatic gastric cancer.

[0071] Pertuzumab (U1-59 / U3-1287 / AMG888) is a fully human anti-HER3 monoclonal antibody with an IgG1-like isotype, with 1-3 nM affinity for its target, and is directed against the juxtamembrane portion of HER3. Pertuzumab has shown promising in vitro and in vivo antitumor effects in lung cancer, head and neck cancer, and breast cancer models, where it effectively blocked HER3 phosphorylation, degraded HER3 receptors, and reduced tumor burden.

[0072] Matuzumab (EMD72000) is a humanized IgG1 derived from the murine antibody MAb 425 (EMD55900), which was generated 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 variety of cancers (alone and in combination with chemotherapy).

[0073] 1. Design of bispecific antigen-binding fragments

[0074] The invention provides bispecific bivalent antigen binding fragments comprising a binding site for HER-3 and a binding site for another antigen selected from HER-2 or EGFR antigens. In a specific embodiment, the bispecific antigen binding fragment can simultaneously bind HER-3 and HER-2. In another specific embodiment, the bispecific antigen binding fragment can simultaneously bind HER-3 and EGFR.

[0075] The antigen-binding fragment of the present invention is essentially composed of Fab fragments arranged in series. The present invention specifically 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 pertuzumab 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.

[0076] The bispecific antigen-binding fragment comprises:

[0077] (i) a Fab fragment comprising the VH-CH1 heavy chain associated with the VL-CL light chain of Antibody 1 (Abl), and

[0078] (ii) a Fab fragment comprising the VH-CH1 heavy chain associated with the VL-CL light chain of Antibody 2 (Ab2),

[0079] The N-terminal end of the VH domain of the Fab fragment of Ab1 is connected to the C-terminal end of the CH1 domain of the Fab fragment of Ab2 through a polypeptide linker.

[0080] wherein one of Ab1 or Ab2 is pertuzumab or a functional derivative thereof;

[0081] And the other one of Abl 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.

[0082] In a specific embodiment, 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 two Fab fragments with different CH1 and CL domains, and the two Fab fragments consist of:

[0083] (i) a Fab fragment having CH1 and CL domains derived from human IgG1 / κ, and VH and VL domains of Ab1,

[0084] (ii) A Fab fragment having CH1 and CL domains derived from human IgG1 / κ and the VH and VL domains of Ab2.

[0085] In a specific embodiment, Abl is Pertrastuzumab or a functional derivative thereof, and Ab2 is Trastuzumab or a functional derivative thereof. In another specific embodiment, Abl is Trastuzumab or a functional derivative thereof, and Ab2 is Pertrastuzumab or a functional derivative thereof.

[0086] In a specific embodiment, Abl is Pertratuzumab or a functional derivative thereof, and Ab2 is Cetuximab or a functional derivative thereof. In another specific embodiment, Abl is Cetuximab or a functional derivative thereof, and Ab2 is Pertratuzumab or a functional derivative thereof.

[0087] In a specific embodiment, Abl is Pertratuzumab or a functional derivative thereof, and Ab2 is Matuzumab or a functional derivative thereof. In another specific embodiment, Abl is Matuzumab or a functional derivative thereof, and Ab2 is Pertratuzumab or a functional derivative thereof.

[0088] In a preferred embodiment, Ab2 is Pertratuzumab or a functional derivative thereof, and Abl 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.

[0089] In a specific embodiment, the bispecific antigen-binding fragment comprises a Fab fragment comprising a VH-CH1 heavy chain associated with a VL-CL light chain of Pertrastuzumab or a functional derivative thereof, wherein:

[0090] - the VH domain comprises, preferably consists of, SEQ ID NO: 5 or a mutant derivative thereof; and

[0091] - the VL domain comprises, preferably consists of, SEQ ID NO: 6 or a mutant derivative thereof;

[0092] Wherein, the CH1 domain is preferably the CH1 constant domain of human IgG1 or a mutant derivative thereof;

[0093] And wherein the CL domain is preferably a human κ constant domain or a mutant derivative thereof.

[0094] In a specific 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, wherein:

[0095] - the VH domain comprises, preferably consists of, SEQ ID NO: 7 or a mutant derivative thereof; and

[0096] - the VL domain comprises, preferably consists of, SEQ ID NO: 8 or a mutant derivative thereof;

[0097] Wherein, the CH1 domain is preferably the CH1 constant domain of human IgG1 or a mutant derivative thereof;

[0098] And wherein the CL domain is preferably a human κ constant domain or a mutant derivative thereof.

[0099] In a specific 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, wherein:

[0100] - the VH domain comprises, preferably consists of, SEQ ID NO: 9 or a mutant derivative thereof; and

[0101] - the VL domain comprises, preferably consists of, SEQ ID NO: 10 or a mutant derivative thereof;

[0102] Wherein, the CH1 domain is preferably the CH1 constant domain of human IgG1 or a mutant derivative thereof;

[0103] And wherein the CL domain is preferably a human κ constant domain or a mutant derivative thereof.

[0104] In a specific 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, wherein:

[0105] - the VH domain comprises, preferably consists of, SEQ ID NO: 11 or a mutant derivative thereof; and

[0106] - the VL domain comprises, preferably consists of, SEQ ID NO: 12 or a mutant derivative thereof;

[0107] Wherein, the CH1 domain is preferably the CH1 constant domain of human IgG1 or a mutant derivative thereof;

[0108] And wherein the CL domain is preferably a human κ constant domain or a mutant derivative thereof.

[0109] Mutation derivatives

[0110] The present invention utilizes the wild-type sequence (the wild-type sequence of pertuzumab, matuzumab, cetuximab or trastuzumab) or a mutant derivative thereof.

[0111] The term "mutant derivative", "mutant" or "functional derivative" refers to a sequence that differs from the parent sequence to which it refers by deletion, substitution or insertion of one or several amino acids. Preferably, the mutant 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 a specific embodiment, the mutation does not significantly affect the function of the antibody.

[0112] The mutant derivative or functional variant may comprise a VH chain, a VL chain, or both, wherein the VH chain comprises 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 reference sequence listed herein, and the VH chain has 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 reference sequence listed herein. The functional variant of Pertuzumab is capable of binding to HER3. The functional variant of Cetuximab is capable of binding to EGFR. The functional variant of Matuzumab is capable of binding to EGFR. The functional variant of Trastuzumab is capable of binding to HER2.

[0113] In some examples, the variant has a similar antigen binding affinity to the reference antibody (e.g., having less than 1 x 10 -8 , preferably less than 1x 10 -9 or 1x 10 -10 In some embodiments, the functional variant of the original antibody has the same binding specificity and has an affinity for its target that is at least 50%, such as at least 60%, 70%, 80%, or at least 90% of the affinity of the original antibody. The affinity of the binding is defined by the terms ka (association rate constant), kd (dissociation rate constant), or KD (equilibrium dissociation). Generally, when used with respect to an antibody, specific binding refers to an antibody that binds to the target with an affinity of less than 10 -8 M, for example, less than 10 -9 M or 10 -10The affinity (KD) value of an M specifically binds ("recognizes") its target. A lower KD value represents a higher binding affinity (i.e., stronger binding), so 10 -9 The KD value indicates a ratio of 10 -8 The higher the KD value, the higher the binding affinity.

[0114] 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, which was modified 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, word length = 3, to obtain amino acid sequences identical to the protein molecule of interest. Where there is a gap between the two sequences, Gapped BLAST can be used 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.

[0115] In other embodiments, the functional variants described herein may contain one or more mutations (eg, conservative substitutions) that preferably do not occur at residues predicted to interact with one or more CDRs.

[0116] Described herein are mutational derivatives or functional variants that are substantially identical to a reference antibody.

[0117] The term "substantially identical" or "insubstantial" means that the relevant amino acid sequence of the variant (e.g., in the framework region (FR), CDR, VH or VL domain) is substantially dissimilar (e.g., including conservative amino acid substitutions) compared to the reference antibody, so that the variant has substantially similar binding activity (e.g., affinity, specificity or both) and biological activity relative to the reference antibody. Such variants may include minor amino acid changes, such as 1 or 2 substitutions in the 5 amino acid sequences of a specified region. Generally, more substitutions may be made in the FR region compared to the CDR region, as long as they do not adversely affect the binding function of the antibody (e.g., reducing the binding affinity by more than 50% compared to the original antibody). In some embodiments, between the original antibody and the modified antibody, the sequence identity may be about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more. In some embodiments, the modified antibody has the same binding specificity as the original antibody and has at least 50% of the affinity of the original antibody. In some embodiments, the mutation does not occur in the CDR region.

[0118] Conservative substitutions will produce molecules with functions and chemical features similar to those of the molecules subjected to such modifications. For example, "conservative amino acid substitutions" may involve replacing a natural amino acid residue with another residue so that the polarity or charge of the amino acid residue at the position is little or not affected. The desired amino acid substitution (whether conservative or non-conservative) may be determined by those skilled in the art. For example, amino acid substitutions may be used to identify important residues of a molecular sequence, or to increase or reduce the affinity of a molecule as described herein. Variants comprising one or more conservative amino acid substitutions may be prepared according to methods known to those of ordinary skill in the art for changing a polypeptide sequence, such as those found in the references for compiling such methods, such as Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, FM Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions 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.

[0119] The present disclosure also provides antibody variants having improved antibody biological properties, such as higher or lower binding affinity, or having altered ADCC properties, or having altered viability inhibitory effects on cells expressing HER3, EGFR and / or HER2.

[0120] The amino acid sequence variants of the antibody can be prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or via peptide synthesis. Such modifications include, for example, deletions, and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions and substitutions is performed to obtain the final construct, provided that the final construct has the desired characteristics. Nucleic acid molecules encoding the amino acid sequence variants of the antibody can be prepared by a variety of methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis and cassette mutagenesis of an earlier prepared variant or non-variant (natural) version of the antibody. In one embodiment, the equilibrium dissociation constant (KD) value of the antibody of the present invention is less than 10 -8 M, especially less than 10 -9 M or 10 -10 M. Binding affinity can be determined using techniques known in the art, such as ELISA or biospecific interaction assays, or other techniques known in the art.

[0121] Any antibody described herein can be tested according to conventional methods to determine its properties, such as antigen binding activity, antigen binding specificity, and biological function.

[0122] Any of the antibodies described herein may be modified to contain additional non-proteinaceous moieties as are known in the art and readily available, for example, by pegylation, hyperglycosylation, conjugation of toxins, radiolabeling, etc. Modifications that can enhance serum half-life are of interest.

[0123] The antibodies of the invention may be glycosylated or not glycosylated, or may display a variety of glycosylation profiles. In a preferred embodiment, the antibody is not glycosylated on the variable regions of the heavy and light chains, but is glycosylated on the Fc region.

[0124] For example, to remove the N-glycosylation site in the cetuximab VH domain, the Asn at Kabat position H85 is mutated to aspartic acid (D) according to the sequence of SEQ ID NO:77, or the Asn at Kabat position H85 is mutated to glutamic acid (E) according to the sequence of SEQ ID NO:78.

[0125] Certain mutant derivatives of cetuximab are humanized forms of the reference cetuximab antibody, the original form of which is a chimeric antibody with murine heavy and light chain variable regions. In the humanization process, the complementary determining regions (CDRs) and certain other amino acids from the donor mouse variable region are transplanted into the human variable acceptor region and then linked to the human constant region; some CDR amino acids can be further replaced by amino acids found in the human germline sequence of the acceptor region. See, for example, Riechmann et al., Nature 332: 323-327 (1988); U.S. Patent No. 5,225,539.

[0126] The invention further encompasses bispecific antigen-binding fragments containing humanized versions of the light and / or heavy chains of cetuximab.

[0127] In a specific embodiment, Ab1 or Ab2 is cetuximab or a humanized version of cetuximab. In a specific embodiment, the humanized form of cetuximab has a VH domain and a VL domain, wherein the VH domain has an amino acid sequence that is at least 80%, 85%, 90%, 92%, 94%, 96%, 98% or at least 99% identical to SEQ ID NO: 7; the VL domain has an amino acid sequence that is at least 80%, 85%, 90%, 92%, 94%, 96%, 98% or at least 99% identical to SEQ ID NO: 8.

[0128] In a specific embodiment, the humanized form of cetuximab is any of the humanized forms described in patent application WO2012020059.

[0129] In a specific embodiment, Abl or Ab2 is a humanized form of cetuximab capable of binding to EGFR and comprises:

[0130] - 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

[0131] - 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.

[0132] In one embodiment, at least two and preferably all of the framework regions 1, 2, and 3 of the heavy chain variable region (VH) of the humanized cetuximab are derived from or correspond to the human germline VH gene 4-59*01 encoding the amino acid sequence comprising SEQ ID NO: 55. In addition, the framework region 4 of the heavy chain variable region (VH) is preferably derived from or corresponds to the human germline gene JH1 encoding the amino acid sequence comprising SEQ ID NO: 56. In one embodiment, at least one of the framework regions 1, 2, and 3 of the light chain variable region (VL) of the humanized cetuximab is derived from or corresponds to the human germline VL gene 6-21*01 encoding the amino acid sequence comprising SEQ ID NO: 57. Preferably, at least two, more preferably all three, of the framework regions 1, 2, and 3 of the light chain variable region are derived from or correspond to the human germline VL gene 6-21*01 encoding the amino acid sequence comprising SEQ ID NO: 57. Furthermore, the framework region 4 of the light chain variable region (VL) is preferably derived from or corresponds to the human germline gene JK2 encoding the amino acid sequence comprising SEQ ID NO:58.

[0133] Preferably, the humanized cetuximab comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 59 and the light chain variable region comprises 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 NO: 61 to 68, in particular an amino acid sequence of SEQ ID NO: 63. In a specific embodiment, the heavy chain variable region (VH) of the humanized cetuximab preferably comprises an amino acid sequence of SEQ ID NO: 81. In addition, the light chain variable region (VL) preferably comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 69 to 72, in particular an amino acid sequence of SEQ ID NO: 71. Particularly preferred is a humanized antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 63 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 71. Another particularly preferred is a humanized cetuximab comprising a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 81 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 71.

[0134] In a specific embodiment, the humanized cetuximab comprises:

[0135] - VH domain of SEQ ID NO: 81 and CH1 domain of SEQ ID NO: 2 (corresponding to the VH-CH1 sequence of SEQ ID NO: 83)

[0136] - the VL domain of SEQ ID NO: 71 and the CL domain of SEQ ID NO: 4 (corresponding to the VL-CL sequence of SEQ ID NO: 84).

[0137] In a specific embodiment, 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 with SEQ ID NO: 59 or with the amino acid sequence of SEQ ID NO: 61 to 68, in particular with SEQ ID NO: 63. In a specific embodiment, 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 with SEQ ID NO: 60 or with the amino acid sequence of SEQ ID NO: 69 to 72, in particular with SEQ ID NO: 71.

[0138] In some examples, Abl or Ab2 is a humanized cetuximab comprising:

[0139] - a humanized version of the light chain of cetuximab based on the human immunoglobulin gene kappa variable 6-11 allele 02 (IGKV6-21*02) as defined in the IMGT / gene database,

[0140] EIVLTQSPDFQSVTPKEKVTITCRASQSIGTNIHWYQQKPDQSPKLLI KYASESISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQNNNWPTTFG QGTKLEIK(SEQ ID NO:73)

[0141] wherein at the following Kabat positions, the amino acid residue is:

[0142] Kabat position L31 Thr or Ser

[0143] Kabat position L32 Asn or Ser

[0144] Kabat position L33 Ile or Leu

[0145] Kabat position L53 Glu or Gln Kabat position L89 Gln or His

[0146] and optionally, Kabat position L91 Asn, Ser, His, Lys or Arg

[0147] Kabat position L92 Asn, Ser, His, Lys or Arg

[0148] Kabat position L93 Asn, Ser, His, Lys or Arg

[0149] Kabat position L94 Trp, Tyr or Phe

[0150] Kabat position L96 Thr or Tyr.

[0151] For clarity, L31 refers to residue 31 in the light chain.

[0152] - a humanized version of the light chain of cetuximab based on the human immunoglobulin gene kappa variable 3-11 allele 01 (IGKV3-11*01) as defined in the IMGT / gene database,

[0153] EIVLTQSPATLSLSPGERATLSCRASQSIGTNIHWYQQKPGQAPRLLIK YASESISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQNNNWPTTFGQG TKLEIK(SEQ ID NO:74)

[0154] wherein at the following Kabat positions, the amino acid residue is:

[0155] Kabat Location L29 Ile or Val

[0156] Kabat position L30 Gly or Ser

[0157] Kabat position L31 Thr or Ser

[0158] Kabat position L32 Asn or Tyr

[0159] Kabat position L33 Ile or Leu

[0160] Kabat position L34 His or Ala

[0161] Kabat position L49 Lys or Tyr

[0162] Kabat position L50 Tyr or Asp

[0163] Kabat position L53 Glu or Asn

[0164] Kabat position L54 Ser or Arg

[0165] Kabat location L55 Ile or Ala

[0166] Kabat position L56 Ser or Thr

[0167] and optionally, Kabat position L91 Asn, Arg, His or Lys

[0168] Kabat position L92 Asn, Ser, His, Lys or Arg

[0169] Kabat position L94 Trp, Tyr or Phe

[0170] Kabat position L96 Thr or Tyr.

[0171] - a humanized version of the heavy chain of cetuximab mAb based on the human immunoglobulin gene heavy variable region 4-59 allele 01 (IGHV4-59*01) as defined in the IMGT / Gene database,

[0172] QVQLQESGPGLVKPSETLSLTCTVSGFSLTNYGVHWVRQPPGKGLE WLGVIWSGGNTDYNTPLTSRLTISKDNSKNQVSLKLSSVTAADTAVYYCA RALTYYDYEFAYWGQGTLVTVSS(SEQ ID NO:75)

[0173] wherein at the following Kabat positions, the amino acid residue is:

[0174] Kabat location H29 Leu or Ile

[0175] Kabat position H30 Thr or Ser

[0176] Kabat position H31 Asn or Ser

[0177] Kabat position H33 Gly or Tyr

[0178] Kabat position H35 His or Ser

[0179] Kabat Location H37 Val or Ile

[0180] Kabat position H48 Leu or Ile

[0181] Kabat position H50 Val or Tyr

[0182] Kabat position H52 Trp, Tyr or Phe

[0183] Kabat position H53 Ser or Tyr

[0184] Kabat position H54 Gly or Ser

[0185] Kabat position H56 Asn or Ser

[0186] Kabat position H58 Asp or Asn

[0187] Kabat Position H61 Thr or Pro

[0188] Kabat position H62 Pro or Ser

[0189] Kabat position H64 Thr or Lys

[0190] Kabat position H67 Leu or Val

[0191] Kabat position H73 Asn or Thr

[0192] Kabat position H78 Val or Phe.

[0193] For clarity, H29 refers to residue 29 in the heavy chain.

[0194] - a humanized version of the heavy chain of cetuximab mAb based on the human immunoglobulin gene heavy variable region 3-33 allele 01 (IGHV3-33*01) as defined in the IMGT / Gene database,

[0195] QVQLVESGGGVVQPGRSLRLSCAVSGFSLTNYGVHWVRQAPGKGL EWLGVIWSGGNTDYNTPVTSRFTISKDNSKNTVYLQMNSLRAEDTAVYY CARALTYYDYEFAYWGQGTLVTVSS(SEQ ID NO:76)

[0196] wherein at the following Kabat positions, the amino acid residue is:

[0197] Kabat position H28 Ser or Thr

[0198] Kabat position H30 Thr or Ser

[0199] Kabat position H48 Leu or Val

[0200] Kabat position H49 Gly or Ala

[0201] Kabat position H53 Ser or Asp

[0202] Kabat position H55 Gly or Ser

[0203] Kabat position H57 Lys or Thr

[0204] Kabat position H58 Asp or Tyr

[0205] Kabat position H60 Asn or Ala

[0206] Kabat position H61 Thr or Asp

[0207] Kabat position H62 Pro or Ser

[0208] Kabat position H64 Thr or Lys

[0209] Kabat position H65 Ser or Gly

[0210] Kabat position H78 Val or Leu.

[0211] In a specific embodiment, the Fab fragment of Ab1 and / or the Fab fragment of Ab2 comprises mutations in the VH / VL domains to promote pairing of the Fab light chain with the Fab heavy chain. Specific mutations are described in international patent application WO2020 / 136566, which is incorporated herein by reference in its entirety.

[0212] In a specific embodiment, the Fab fragment of Abl and / or the Fab fragment of Ab2 comprises a mutated VH domain and a VL domain, wherein the residue at Kabat position 39 of the VH domain has been mutated from glutamine to lysine; and the residue at Kabat position 38 of the VL domain has been mutated from glutamine to glutamic acid.

[0213] In a specific embodiment, the Fab fragment of Abl and / or the Fab fragment of Ab2 comprises a mutated VH domain and a VL domain, wherein the residue at Kabat position 39 of the VH domain has been mutated from glutamine to lysine; and the residue at Kabat position 38 of the VL domain has been mutated from glutamine to aspartic acid.

[0214] In a specific embodiment, the Fab fragment of Abl and / or the Fab fragment of Ab2 comprises a mutated VH domain and a VL domain, wherein the residue at Kabat position 39 of the VH domain has been mutated from glutamine to glutamic acid; and the residue at Kabat position 38 of the VL domain has been mutated from glutamine to lysine.

[0215] CH1 / CL mutation domain

[0216] The assembly of the Fab domain is completed via the natural pairing of the light chain and the heavy chain without the use of a peptide linker. In order to maximize the tendency of homologous pairing between the light chain and the heavy chain, it is contemplated to introduce mutations at the interface (CL / CH1 interface) of the light chain and the heavy chain in the Fab fragment.

[0217] In a specific embodiment, the CH1 domain of Abl and / or Ab2 is a CH1 constant domain of human IgG1 from any allotype or a mutant derivative thereof. In a specific embodiment, the CH1 domain of Abl and / or Ab2 is a CH1 domain of human IgG1 of the Glm(1,17) allotype or a mutant derivative thereof. In another specific embodiment, the CH1 domain of Abl and / or Ab2 is a CH1 domain of human IgG1 of the Glm(3) allotype or a mutant derivative thereof.

[0218] 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 such that correct cognate pairing of the CH1 and CL1 domains is improved.

[0219] These mutations can be selected from the following list:

[0220] - Reintroduced ion pairs or reverse polarity charged mutations of native ion pairs already present at the interface of the heavy and light chains of the Fab fragment;

[0221] -knob-to-mortise" mutation;

[0222] - Mutations that resurface the relative constant regions of the heavy and light chain interface in the Fab fragment to change them from strongly polar to highly hydrophobic or vice versa.

[0223] Therefore, several groups of mutations are suitable, as described in more detail below.

[0224] It is worth noting that 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).

[0225] The residues that may be mutated in the CLκ domain may, for example, be selected from the group consisting of S114, F116, F118, E123 (e.g., E123K), Q124, T129, S131, V133, L135, N137, Q160, S162, S174, S176, T178, and T180.

[0226] The residues that may be mutated in the CH1 domain may, for example, be selected from the group consisting of L124, A139, L143, D144, K145, D146, H172, F174, P175, Q179, S183, S188, V190, T192, and K221 (e.g., K221E).

[0227] Specific mutations are described in US Patent Applications 2014 / 0200331, 2014 / 150973, 2014 / 0154254, and International Patent Application WO 2007 / 147901, all of which are incorporated herein by reference.

[0228] Examples of CH1 / CL mutant derivatives are described below.

[0229] According to certain embodiments, the bispecific antigen-binding fragment comprises:

[0230] - a mutated CH1 constant domain from human IgG1 corresponding to SEQ ID NO: 1 (the residue at Kabat position 192 has been mutated from threonine to glutamic acid (E));

[0231] - a mutated CH1 constant domain from human IgG1 corresponding to SEQ ID NO: 45 (the residue at Kabat position 192 has been mutated from threonine to aspartic acid (D));

[0232] - a mutated CH1 constant domain from human IgG1 corresponding to SEQ ID NO: 2 (the residue at Kabat position 143 has been mutated from leucine to glutamine (Q); and the residue at Kabat position 188 has been mutated from serine to valine (V));

[0233] - a mutated CH1 constant domain from human IgG1 corresponding to SEQ ID NO: 46 (the residue at Kabat position 124 has been mutated from leucine to glutamine (Q); and the residue at Kabat position 188 has been mutated from serine to valine (V));

[0234] - a mutant human kappa constant domain corresponding to SEQ ID NO: 3 (the residue at Kabat position 114 has been mutated from serine to alanine (A) and the residue at Kabat position 137 has been mutated from asparagine to lysine (K));

[0235] - a mutant human kappa constant domain corresponding to SEQ ID NO: 4 (the residue at Kabat position 133 has been mutated from valine to threonine (T) and the residue at Kabat position 176 has been mutated from serine to valine (V));

[0236] In a preferred embodiment, the interacting polar interface residue pairs are exchanged for neutral and salt bridge forming residue pairs. It is possible to choose to replace Thr192 on the CH1 chain by glutamic acid or aspartic acid and replace Asn137 with Lys on the CL chain, optionally replacing the serine residue at position 114 of the CL domain with an alanine residue.

[0237] In another set of mutations, Leu143 of the CH1 domain can be replaced by a Gln residue, whereas the opposite residue of the CL chain, Val33, is replaced by a Thr residue. This first double mutation constitutes a switch from hydrophobic to polar interactions. At the same time, the simultaneous mutation of two interacting serines (Ser188 on the CH1 chain and Ser176 on the CL chain) to valine residues can achieve a switch from polar to hydrophobic interactions.

[0238] In yet another embodiment, the mutation may comprise replacing the leucine residue at position 124 of the CH1 domain with glutamine and replacing the serine residue at position 188 of the CH1 domain with a valine residue; and replacing the valine residue at position 133 of the CL domain with a threonine residue and replacing the serine residue at position 176 of the CL domain with a valine residue.

[0239] The "knob-in-hole" mutations include a mutation group (KH1) in which Leu 124 and Leu 143 of the CH1 domain have been substituted by Ala and Glu residues, respectively, and Val 33 of the CL chain has been substituted by a Trp residue, whereas in a mutation group named H2, Val 90 of the CH1 domain has been substituted by an Ala residue, and Leu 135 and Asn 137 of the CL chain have been substituted by Trp and Ala residues, respectively.

[0240] Preferred mutations are disclosed in the following table:

[0241]

[0242] In certain embodiments, the multispecific antibody may carry double mutations, for example one Fab has a CR3 mutation and another Fab has a Mut4 mutation.

[0243] More specifically described are bispecific antigen-binding fragments, wherein Ab1 or Ab2 is pertrastuzumab or a mutant derivative thereof, comprising:

[0244] - a VH domain consisting of SEQ ID NO: 5 or a mutant derivative thereof,

[0245] - 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 or SEQ ID NO: 47,

[0246] - a VL domain consisting of SEQ ID NO: 6 or a mutant derivative thereof,

[0247] - a CL domain selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 48,

[0248] Wherein the CL and CH1 domains preferably associate as follows:

[0249] - SEQ ID NO:48 and SEQ ID NO:47,

[0250] - SEQ ID NO: 3 and SEQ ID NO: 1 or SEQ ID NO: 45,

[0251] - SEQ ID NO: 4 and SEQ ID NO: 2 or SEQ ID NO: 46.

[0252] Preferably, Ab1 or Ab2 is Pertrastuzumab or a mutant derivative thereof, comprising:

[0253] - a VH domain consisting of SEQ ID NO: 5 or a mutant derivative thereof,

[0254] - a CH1 domain consisting of SEQ ID NO: 1 or a mutant derivative thereof,

[0255] - a VL domain consisting of SEQ ID NO: 6 or a mutant derivative thereof,

[0256] - a CL domain consisting of SEQ ID NO: 3 or a mutant derivative thereof,

[0257] Preferably, Ab1 or Ab2 is Pertrastuzumab or a mutant derivative thereof, comprising:

[0258] - 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 thereto; and

[0259] - 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 thereto.

[0260] More specifically described are bispecific antigen-binding fragments, wherein Ab1 or Ab2 is cetuximab or a mutant derivative thereof, comprising:

[0261] - a VH domain consisting of SEQ ID NO: 7, a mutant derivative thereof or a humanized derivative thereof,

[0262] - 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 or SEQ ID NO: 47,

[0263] - a VL domain consisting of SEQ ID NO: 8, a mutant derivative thereof or a humanized derivative thereof,

[0264] - a CL domain selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 48,

[0265] Wherein the CL and CH1 domains preferably associate as follows:

[0266] - SEQ ID NO:48 and SEQ ID NO:47,

[0267] - SEQ ID NO: 3 and SEQ ID NO: 1 or SEQ ID NO: 45,

[0268] - SEQ ID NO: 4 and SEQ ID NO: 2 or SEQ ID NO: 46.

[0269] Preferably, Ab1 or Ab2 is cetuximab or a mutant derivative thereof, comprising:

[0270] - a VH domain consisting of SEQ ID NO: 7, a mutant derivative thereof or a humanized derivative thereof,

[0271] - a CH1 domain consisting of SEQ ID NO: 2 or a mutant derivative thereof,

[0272] - a VL domain consisting of SEQ ID NO: 8, a mutant derivative thereof or a humanized derivative thereof,

[0273] - a CL domain consisting of SEQ ID NO: 4 or a mutant derivative thereof,

[0274] Preferably, Ab1 or Ab2 is cetuximab or a mutant derivative thereof, comprising:

[0275] - 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 thereto; and

[0276] - 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 thereto.

[0277] More specifically described are bispecific antigen-binding fragments, wherein Ab1 or Ab2 is trastuzumab or a mutant derivative thereof, comprising:

[0278] - a VH domain consisting of SEQ ID NO: 9 or a mutant derivative thereof,

[0279] - 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 or SEQ ID NO: 47,

[0280] - a VL domain consisting of SEQ ID NO: 10 or a mutant derivative thereof,

[0281] - a CL domain selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 48,

[0282] Wherein the CL and CH1 domains preferably associate as follows:

[0283] - SEQ ID NO:48 and SEQ ID NO:47,

[0284] - SEQ ID NO: 3 and SEQ ID NO: 1 or SEQ ID NO: 45,

[0285] - SEQ ID NO: 4 and SEQ ID NO: 2 or SEQ ID NO: 46.

[0286] Preferably, Ab1 or Ab2 is trastuzumab or a mutant derivative thereof, comprising:

[0287] - a VH domain consisting of SEQ ID NO: 9 or a mutant derivative thereof,

[0288] - a CH1 domain consisting of SEQ ID NO: 2 or a mutant derivative thereof,

[0289] - a VL domain consisting of SEQ ID NO: 10 or a mutant derivative thereof,

[0290] - a CL domain consisting of SEQ ID NO: 4 or a mutant derivative thereof,

[0291] Preferably, Ab1 or Ab2 is trastuzumab or a mutant derivative thereof, comprising:

[0292] - 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 thereto; and

[0293] - 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 thereto.

[0294] More specifically described are bispecific antigen-binding fragments, wherein Ab1 or Ab2 is Matuzumab or a mutant derivative thereof, comprising:

[0295] - a VH domain consisting of SEQ ID NO: 11 or a mutant derivative thereof,

[0296] - 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 or SEQ ID NO: 47,

[0297] - a VL domain consisting of SEQ ID NO: 12 or a mutant derivative thereof,

[0298] - a CL domain selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 48,

[0299] Wherein the CL and CH1 domains preferably associate as follows:

[0300] - SEQ ID NO:48 and SEQ ID NO:47,

[0301] - SEQ ID NO: 3 and SEQ ID NO: 1 or SEQ ID NO: 45,

[0302] - SEQ ID NO: 4 and SEQ ID NO: 2 or SEQ ID NO: 46.

[0303] Preferably, Ab1 or Ab2 is Matuzumab or a mutant derivative thereof, comprising:

[0304] - a VH domain consisting of SEQ ID NO: 11 or a mutant derivative thereof,

[0305] - a CH1 domain consisting of SEQ ID NO: 2 or a mutant derivative thereof,

[0306] - a VL domain consisting of SEQ ID NO: 12 or a mutant derivative thereof,

[0307] - a CL domain consisting of SEQ ID NO: 4 or a mutant derivative thereof,

[0308] Preferably, Ab1 or Ab2 is Matuzumab or a mutant derivative thereof, comprising:

[0309] - 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 thereto; and

[0310] - 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 thereto.

[0311] In certain embodiments, the bispecific antigen-binding fragment comprises:

[0312] (i) a Fab fragment comprising the VH-CH1 heavy chain associated with the VL-CL light chain of trastuzumab or a functional derivative thereof, and

[0313] (ii) a Fab fragment comprising the VH-CH1 heavy chain associated with the VL-CL light chain of Pertrastuzumab or a functional derivative thereof,

[0314] The N-terminal end of the VH domain of the Fab fragment of trastuzumab is connected to the C-terminal end of the CH1 domain of the Fab fragment of pertuzumab via a polypeptide linker,

[0315] The VH-CH1 heavy chain of trastuzumab is preferably composed of SEQ ID NO: 17, the VL-CL light chain of trastuzumab is preferably composed of SEQ ID NO: 18, the VH-CH1 heavy chain of pertuzumab is preferably composed of SEQ ID NO: 13, and / or the VL-CL light chain of pertuzumab is preferably composed of SEQ ID NO: 14.

[0316] In a preferred embodiment, the bispecific antigen-binding fragment comprises a heavy chain comprising SEQ ID NO:21 or a functional variant thereof (or consisting of SEQ ID NO:21 or a functional variant thereof), and two different light chains comprising SEQ ID NO:18 and SEQ ID NO:14 or a functional variant thereof (or consisting of SEQ ID NO:18 and SEQ ID NO:14 or a functional variant thereof).

[0317] In certain embodiments, the bispecific antigen-binding fragment comprises:

[0318] (i) a Fab fragment comprising the VH-CH1 heavy chain associated with the VL-CL light chain of Pertrastuzumab or a functional derivative thereof, and

[0319] (ii) a Fab fragment comprising the VH-CH1 heavy chain associated with the VL-CL light chain of trastuzumab or a functional derivative thereof,

[0320] The N-terminal end of the VH domain of the Fab fragment of Pertuzumab is connected to the C-terminal end of the CH1 domain of the Fab fragment of Trastuzumab through a polypeptide linker,

[0321] The VH-CH1 heavy chain of trastuzumab is preferably composed of SEQ ID NO: 17, the VL-CL light chain of trastuzumab is preferably composed of SEQ ID NO: 18, the VH-CH1 heavy chain of pertuzumab is preferably composed of SEQ ID NO: 13, and / or the VL-CL light chain of pertuzumab is preferably composed of SEQ ID NO: 14.

[0322] In a preferred embodiment, the bispecific antigen-binding fragment comprises one heavy chain comprising SEQ ID NO:22 or a functional variant thereof (or consisting of SEQ ID NO:22 or a functional variant thereof), and two different light chains comprising SEQ ID NO:18 and SEQ ID NO:14 or a functional variant thereof (or consisting of SEQ ID NO:18 and SEQ ID NO:14 or a functional variant thereof).

[0323] In certain embodiments, the bispecific antigen-binding fragment comprises:

[0324] (i) 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, and

[0325] (ii) a Fab fragment comprising the VH-CH1 heavy chain associated with the VL-CL light chain of Pertrastuzumab or a functional derivative thereof,

[0326] The N-terminal end of the VH domain of the Fab fragment of cetuximab is connected to the C-terminal end of the CH1 domain of the Fab fragment of pertuzumab through a polypeptide linker,

[0327] The VH-CH1 heavy chain of cetuximab is preferably composed of SEQ ID NO: 15, the VL-CL light chain of cetuximab is preferably composed of SEQ ID NO: 16, the VH-CH1 heavy chain of pertuzumab is preferably composed of SEQ ID NO: 13, and / or the VL-CL light chain of pertuzumab is preferably composed of SEQ ID NO: 14.

[0328] In a preferred embodiment, the bispecific antigen-binding fragment comprises a heavy chain comprising SEQ ID NO:23 or a functional variant thereof (or consisting of SEQ ID NO:23 or a functional variant thereof), and two different light chains comprising SEQ ID NO:16 and SEQ ID NO:14 or a functional variant thereof (or consisting of SEQ ID NO:16 and SEQ ID NO:14 or a functional variant thereof).

[0329] In certain embodiments, the bispecific antigen-binding fragment comprises:

[0330] (i) a Fab fragment comprising the VH-CH1 heavy chain associated with the VL-CL light chain of Pertrastuzumab or a functional derivative thereof, and

[0331] (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,

[0332] The N-terminal end of the VH domain of the Fab fragment of Pertuzumab is connected to the C-terminal end of the CH1 domain of the Fab fragment of Cetuximab via a polypeptide linker.

[0333] The VH-CH1 heavy chain of cetuximab is preferably composed of SEQ ID NO: 15, the VL-CL light chain of cetuximab is preferably composed of SEQ ID NO: 16, the VH-CH1 heavy chain of pertuzumab is preferably composed of SEQ ID NO: 13, and / or the VL-CL light chain of pertuzumab is preferably composed of SEQ ID NO: 14.

[0334] In a preferred embodiment, the bispecific antigen-binding fragment comprises one heavy chain comprising SEQ ID NO:24 or a functional variant thereof (or consisting of SEQ ID NO:24 or a functional variant thereof), and two different light chains comprising SEQ ID NO:16 and SEQ ID NO:14 or a functional variant thereof (or consisting of SEQ ID NO:16 and SEQ ID NO:14 or a functional variant thereof).

[0335] In certain embodiments, the bispecific antigen-binding fragment comprises:

[0336] (i) a Fab fragment comprising the VH-CH1 heavy chain associated with the VL-CL light chain of Matuzumab or a functional derivative thereof, and

[0337] (ii) a Fab fragment comprising the VH-CH1 heavy chain associated with the VL-CL light chain of Pertrastuzumab or a functional derivative thereof,

[0338] The N-terminal end of the VH domain of the Fab fragment of Matuzumab is connected to the C-terminal end of the CH1 domain of the Fab fragment of Pertuzumab through a polypeptide linker,

[0339] The VH-CH1 heavy chain of Matuzumab is preferably composed of SEQ ID NO: 19, the VL-CL light chain of Matuzumab is preferably composed of SEQ ID NO: 20, the VH-CH1 heavy chain of Pertuzumab is preferably composed of SEQ ID NO: 13, and / or the VL-CL light chain of Pertuzumab is preferably composed of SEQ ID NO: 14.

[0340] In a preferred embodiment, the bispecific antigen-binding fragment comprises a heavy chain comprising SEQ ID NO:25 or a functional variant thereof (or consisting of SEQ ID NO:25 or a functional variant thereof), and two different light chains comprising SEQ ID NO:20 and SEQ ID NO:14 or a functional variant thereof (or consisting of SEQ ID NO:20 and SEQ ID NO:14 or a functional variant thereof) or a functional variant thereof.

[0341] In certain embodiments, the bispecific antigen-binding fragment comprises:

[0342] (i) a Fab fragment comprising the VH-CH1 heavy chain associated with the VL-CL light chain of Pertrastuzumab or a functional derivative thereof, and

[0343] (ii) a Fab fragment comprising the VH-CH1 heavy chain associated with the VL-CL light chain of Matuzumab or a functional derivative thereof,

[0344] The N-terminal end of the VH domain of the Fab fragment of Pertuzumab is connected to the C-terminal end of the CH1 domain of the Fab fragment of Matuzumab through a polypeptide linker,

[0345] The VH-CH1 heavy chain of Matuzumab is preferably composed of SEQ ID NO: 19, the VL-CL light chain of Matuzumab is preferably composed of SEQ ID NO: 20, the VH-CH1 heavy chain of Pertuzumab is preferably composed of SEQ ID NO: 13, and / or the VL-CL light chain of Pertuzumab is preferably composed of SEQ ID NO: 14.

[0346] In a preferred embodiment, the bispecific antigen-binding fragment comprises one heavy chain comprising SEQ ID NO:26 or a functional variant thereof (or consisting of SEQ ID NO:26 or a functional variant thereof), and two different light chains comprising SEQ ID NO:20 and SEQ ID NO:14 or a functional variant thereof (or consisting of SEQ ID NO:20 and SEQ ID NO:14 or a functional variant thereof).

[0347] Design of connector

[0348] A polypeptide linker is used to connect 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.

[0349] In the context of the present invention, the term "polypeptide linker sequence" is a polypeptide of about 20 to 80 amino acids, preferably 30 to 60 amino acids, still preferably 30 to 40 amino acids. The polypeptide linker sequence is generally composed of less than 80 amino acids, preferably less than 60 amino acids, and still preferably less than 40 amino acids. Advantageously, the linker sequence is "hinge-derived", which means that the polypeptide linker comprises all or part of the sequence of the hinge region of one or more immunoglobulins, and the one or more immunoglobulins are selected from IgA, IgG and IgD, preferably human. It is also referred to as "hinge-derived polypeptide linker sequence" or "pseudo-hinge linker". The polypeptide linker may comprise all or part of the sequence of the hinge region of only one immunoglobulin. In this case, the immunoglobulin may belong to the same isotype and subclass as the immunoglobulin from which the adjacent CH1 domain is derived, or to different isotypes or subclasses. Alternatively, the polypeptide linker may comprise all or part of the sequence of the hinge region of at least two immunoglobulins of different isotypes or subclasses. In this case, the N-terminal part of the polypeptide linker directly following the CH1 domain preferably consists of all or part of the hinge region of an immunoglobulin belonging to the same isotype and subclass as the immunoglobulin from which said CH1 domain is derived.

[0350] Optionally, the polypeptide linker may further comprise a sequence of 2 to 15, preferably 5 to 10 N-terminal amino acids of the CH2 domain of an immunoglobulin.

[0351] In some cases, sequences from native hinge regions may be used; in other cases, point mutations may be made to these sequences, particularly replacing one or more cysteine ​​residues in the native IgG1, IgG2 or IgG3 hinge sequence with alanine or serine to avoid undesired intra- or inter-chain disulfide bonds.

[0352] In a specific embodiment, the polypeptide linker sequence comprises or consists of the amino acid sequence EPKX1CDKX2HX3X4PPX5PAPELLGGPX6X7PPX8PX9PX10GG (SEQ ID NO: 33), wherein X1, X2, X3, X4, X5, X6, X7, X8, X9, and X10 are the same or different and are any amino acids.

[0353] In certain embodiments, X1, X2 and X3 are the same or different and are threonine (T) or serine (S).

[0354] In another specific embodiment, X1, X2 and X3 are the same or different and are selected from the group consisting of Ala (A), Gly (G), Val (V), Asn (N), Asp (D) and Ile (I). Still preferably, X1, X2 and X3 are the same or different and can be Ala (A) or Gly (G).

[0355] Alternatively, X1, X2 and X3 are the same or different and can 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).

[0356] In a specific embodiment, X4 and X5 are the same or different and are any amino acid selected from the group consisting of serine (S), cysteine ​​(C), alanine (A) and glycine (G).

[0357] In a preferred embodiment, X4 is serine (S) or cysteine ​​(C).

[0358] In a preferred aspect, X5 is alanine (A) or cysteine ​​(C).

[0359] In a specific embodiment, X6, X7, X8, X9, X10 are identical or different and are any amino acid except threonine (T) or serine (S). Preferably, X6, X7, X8, X9, X10 are identical or different and are selected from the group consisting of Ala (A), Gly (G), Val (V), Asn (N), Asp (D) and Ile (I). Alternatively, X6, X7, X8, X9, X10 are identical or different and 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). In a preferred embodiment, X6, X7, X8, X9, X10 are identical or different and are selected from the group consisting of Ala (A) and Gly (G).

[0360] In yet another preferred embodiment, X6 and X7 are identical and are preferably selected from the group consisting of Ala (A) and Gly (G).

[0361] In a preferred embodiment, the polypeptide linker sequence comprises or consists of the sequence SEQ ID NO: 33, wherein

[0362] X1, X2 and X3 are the same or different and are threonine (T) or serine (S);

[0363] X4 is serine (S) or cysteine ​​(C);

[0364] X5 is alanine (A) or cysteine ​​(C);

[0365] X6, X7, X8, X9, and X10 are the same or different and are selected from the group consisting of Ala (A) and Gly (G).

[0366] In another preferred embodiment, the polypeptide linker sequence comprises or consists of the sequence SEQ ID NO: 33, wherein

[0367] X1, X2 and X3 are the same or different and are Ala (A) or Gly (G);

[0368] X4 is serine (S) or cysteine ​​(C);

[0369] X5 is alanine (A) or cysteine ​​(C);

[0370] X6, X7, X8, X9, and X10 are the same or different and are selected from the group consisting of Ala (A) and Gly (G).

[0371] In particular, the polypeptide linker sequence may comprise or consist of a sequence selected from the group consisting of:

[0372] EPKSCDKTHTSPPAPAPELLGGPAAPPAPAPAGG(SEQ ID NO:34);

[0373] EPKSCDKTHTAPPAPAPELLGGPAAPPAPAPAGG(SEQ ID NO:35);

[0374] EPKSCDKTHTSPPAPAPELLGGPGGPPGPGPGGG (SEQ ID NO:36);

[0375] EPKSCDKTHTSPPAPAPELLGGPAAPPGPAPGGG(SEQ ID NO:37);

[0376] EPKSCDKTHTCPPCPAPELLGGPSTPPTPSPSGG(SEQ ID NO:38);

[0377] and EPKSCDKTHTSPPSPAPELLGGPSTPPTPSPSGG (SEQ ID NO: 39).

[0378] Preferably, the polypeptide linker sequence comprises or consists of the amino acid sequence of SEQ ID NO:34 or SEQ ID NO:35.

[0379] In embodiments where the antibody comprises different Fab fragments, the polypeptide linkers separating the Fab fragments may be the same or different.

[0380] 2. Design of bispecific antibodies

[0381] The invention also relates to a bispecific molecule comprising two identical antigen binding arms, each antigen binding arm consisting of an antigen binding fragment as defined above. In a preferred embodiment, the bispecific molecule is a full length antibody.

[0382] If it is desired to obtain an antibody without Fc-mediated effects or an antibody that is monovalent to each of the two antigens it targets, the antibody will not contain an Fc region. In this case, the two antigen-binding arms can be linked together, for example:

[0383] - homodimerization of the antigen binding arms via interchain disulfide bonds provided by the polypeptide linker of the isolated Fab fragment; and / or

[0384] - by adding at the C-terminal end of each antigen binding arm a polypeptide extension containing a cysteine ​​residue allowing the formation of interchain disulfide bonds, and homodimerization of said polypeptide extension, resulting in a hinge-like structure; as a non-limiting example, said polypeptide extension may be, for example, the hinge sequence of IgG1, IgG2 or IgG3;

[0385] - connecting the C-terminal ends of the heavy chains of the two antigen binding arms via a linker, preferably a semi-rigid linker, to form a single polypeptide chain and keeping the antigen binding arms at a sufficient distance from each other.

[0386] 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 the two antigens are desired, the multispecific antibodies of the invention may further comprise an Fc domain that provides these effector functions. The choice of the Fc domain will depend on the type of effector function desired.

[0387] The present invention also provides a bispecific tetravalent antibody comprising two binding sites for HER-3 and two binding sites for another antigen (being HER-2 or EGFR). More particularly, the present invention relates to a bispecific antibody comprising two identical antigen binding arms (thereby providing a symmetrical antibody form), wherein each antigen binding arm comprises one binding site for HER-3 and one binding site for HER-2. The present invention also relates to a bispecific antibody comprising two identical antigen binding arms (thereby providing a symmetrical antibody form), wherein each antigen binding arm comprises one binding site for HER-3 and one binding site for EGFR.

[0388] In a specific embodiment, each antigen binding arm consists of a bispecific antigen binding fragment as defined above.

[0389] In a preferred embodiment, the linker connects two pairs of Fab domains in a four-Fab bispecific antibody format, and its amino acid sequence comprises the heavy chain sequences of at least two Fabs connected by a linker, followed by a hinge sequence, followed by an Fc sequence co-expressed with an appropriate light chain sequence.

[0390] Examples of antibodies of the invention having an IgG-like structure are Figure 1 Shown in.

[0391] In certain embodiments, the bispecific antibody has an immunoglobulin-like structure and comprises:

[0392] - two identical antigen-binding arms, each antigen-binding arm consisting of a bispecific antigen-binding fragment as described herein, and

[0393] - an Fc domain, which is preferably functional, ie it allows the activation of effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis and complement-dependent cytotoxicity (CDC).

[0394] In a specific embodiment, the bispecific antibody of the invention comprises:

[0395] -Continuous heavy chain constructed from Fc (hinge-CH2-CH3)

[0396] - followed by the Fab heavy chains (CH1-VH) of Antibody 1 (Ab1) and the consecutive Fab heavy chains (CH1-VH) of Antibody 2 (Ab2), the latter being connected by a polypeptide linker sequence as described above,

[0397] - and during protein expression, the resulting heavy chains assemble into homodimers, while the co-expressed two light chains (VL-CL) of Ab1 and two light chains (VL-CL) of Ab2 associate with their cognate heavy chains to form the final tandem F(ab)'2-Fc molecule,

[0398] wherein one of Ab1 or Ab2 is pertuzumab or a functional derivative thereof;

[0399] And the other one of Abl 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.

[0400] It is understood that the hinge domain connects the N-terminal end of the CH2 domain to the C-terminal end of the CH1 domain of Abl and the polypeptide linker sequence connects the N-terminal end of the VH domain of Abl to the C-terminal end of the CH1 domain of Ab2.

[0401] In certain embodiments, the bispecific antibody has an immunoglobulin-like structure comprising:

[0402] - two identical antigen-binding arms, each consisting of a bispecific antigen-binding fragment as described herein;

[0403] -Dimerization of CH2 and CH3 domains of immunoglobulins

[0404] -The hinge region of an immunoglobulin, which connects the C-terminal end of the CH1 domain to the N-terminal end of the CH2 domain of the antigen binding arm.

[0405] In a specific embodiment, the CH2 and / or CH3 domains are CH2 and CH3 domains of the IgG1 or IgG4 isotype or mutant 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.

[0406] In a specific embodiment, the hinge region is a hinge region of IgA, IgG1, IgG4, IgD or a mutant 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 a specific embodiment, the hinge region is the hinge region of IgG4 with an S228P substitution. Preferably, the hinge region is the region of IgG1 of SEQ ID NO: 40.

[0407] In a specific embodiment, the bispecific antibody has an immunoglobulin-like structure and comprises an Fc domain, which can be a wild-type immunoglobulin Fc domain or a mutant derivative thereof. In a specific embodiment, the Fc domain is a mutant derivative of an IgG1 Fc domain, or a mutant derivative of an IgG4 Fc domain. In a specific embodiment, the mutant derivative is an Fc domain with a modified affinity for at least one Fc gamma receptor (FcgR) compared to the parent Fc domain.

[0408] In certain embodiments, the bispecific antibody has an immunoglobulin-like structure and comprises an Fc domain with reduced binding to Fcγ receptors compared to a wild-type Fc domain, such as an IgG1 or IgG4 Fc domain, resulting in the bispecific antibody having reduced effector function.

[0409] In certain embodiments, the bispecific antibody has an immunoglobulin-like structure and comprises an Fc domain that exhibits no or substantially no binding to Fcγ receptors compared to a wild-type Fc domain, such as an IgG1 or IgG4 Fc domain, resulting in the bispecific antibody having silent or reduced effector function.

[0410] The Fc domain may include one or more mutations that reduce or eliminate its binding to Fcγ receptors, such as mutations in CH2 and / or CH3 domains. The mutations include amino acid substitutions, insertions and / or deletions. Exemplary mutations that reduce or eliminate the binding activity of the Fc domain to Fcγ 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 mutant residues in the Fc domain are numbered herein according to EU naming conventions.

[0411] For clarity, "L / F234A" indicates that a leucine or phenylalanine (whether present in IgG1 or IgG4, respectively, at position 234) is replaced by an alanine. For clarity, "L235A / E" indicates that a leucine at position 235 is replaced by an alanine or glutamic acid. For clarity, "G236R / del" indicates that a glycine at position 236 is replaced by an arginine, or that a glycine residue at position 236 is deleted. For clarity, "A / S330R / S" indicates that an alanine or serine (whether present in IgG1 or IgG4, respectively, at position 330) is replaced by an arginine or serine.

[0412] Exemplary mutations that reduce or eliminate the binding activity of the IgG1 Fc domain to the Fcγ receptor include, but are not limited to: L234A, L235A / E, G236R / del, G237A, P238S, D265A, H268A, L328R, P329G, A330R / S and / or P331S. Exemplary mutations that reduce or eliminate the binding activity of the IgG4 Fc domain to the Fcγ receptor include, but are not limited to: F234A, L235A / E, G236R / del, G237A, P238S, D265A, Q268A, L328R, P329G and / or S330R.

[0413] In another specific embodiment, the bispecific antibody has an immunoglobulin-like structure and comprises an Fc domain with increased binding to Fcγ receptors compared to a wild-type Fc domain, such as an IgG1 or IgG4 Fc domain, resulting in the bispecific antibody having improved effector function.

[0414] The Fc domain may comprise one or more mutations that increase its binding to Fcγ receptors, such as mutations in the CH2 and / or CH3 domains. Exemplary mutations that increase the binding activity of an Fc domain, such as an IgG1 or IgG4 Fc domain, to an Fcγ 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, an Fc domain, such as an IgG1 or IgG4 Fc domain comprises mutations S239D and I332E.

[0415] In specific embodiments, the Fc domain, such as an IgG1 or IgG4 Fc domain, has reduced fucose content in the Fc glycan at position 297 in the CH2 domain.

[0416] According to a specific embodiment, the Fc domain, e.g., an IgG1 or IgG4 Fc domain, has an N-glycan at the glycosylation site (Asn297) 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%.

[0417] According to a more specific embodiment, the Fc domain has an N-glycan at the glycosylation site (Asn 297), said N-glycan having a degree of fucosylation equal to 0%. Therefore, the present invention provides a bispecific antibody comprising an Fc domain having an N-glycan at its glycosylation site Asn297, characterized in that said N-glycan of the Fc domain does not contain fucose.

[0418] Advantageously, Fc domains with modified glycosylation, particularly hypofucosylation, at the glycosylation site at position 297 show increased binding to the Fc-gamma receptor.

[0419] In a specific embodiment, the Fc domain comprises (preferably consists of):

[0420] - the hinge domain of SEQ ID NO: 40; and

[0421] - the CH2-CH3 domain of SEQ ID NO: 43.

[0422] 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.

[0423] A particular embodiment relates to a bispecific antibody comprising two heavy chains and four light chains, wherein each heavy chain comprises:

[0424] a. an Fc region of an immunoglobulin comprising a hinge-CH2-CH3 domain or a mutant derivative thereof as described above,

[0425] b. the Fc region is connected to the Fab CH1-VH heavy chain of antibody 1 (Ab1) via the hinge domain,

[0426] c. which is further connected to the Fab CH1-VH heavy chain of antibody 2 (Ab2) via a polypeptide linker sequence, wherein the polypeptide linker sequence connects the N-terminus of the VH domain of Ab1 to the C-terminus of the CH1 domain of Ab2,

[0427] 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;

[0428] wherein, as described above, one of Ab1 or Ab2 is pertuzumab or a functional derivative thereof;

[0429] And, as described above, the other of Abl 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.

[0430] A particular embodiment relates to a bispecific antibody comprising two heavy chains and four light chains, wherein each heavy chain comprises:

[0431] a. an Fc region of an immunoglobulin comprising a hinge-CH2-CH3 domain or a mutant derivative thereof as described above,

[0432] b. the Fc region is connected to the Fab CH1-VH heavy chain of antibody 1 (Ab1) via the hinge domain,

[0433] c. which is further connected to the Fab CH1-VH heavy chain of antibody 2 (Ab2) via a polypeptide linker sequence, wherein the polypeptide linker sequence connects the N-terminus of the VH domain of Ab1 to the C-terminus of the CH1 domain of Ab2,

[0434] 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;

[0435] wherein, as described above, Ab2 is pertuzumab or a functional derivative thereof;

[0436] And, as described above, Abl 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.

[0437] In a specific embodiment, the bispecific antibody comprises (preferably consists of):

[0438] a) two heavy chains, each heavy chain comprising (preferably consisting of) SEQ ID NO: 27 or a mutant derivative thereof and

[0439] b) four light chains, two comprising (preferably consisting of) SEQ ID NO: 14 or a mutant derivative thereof, and the other two comprising (preferably consisting of) SEQ ID NO: 18 or a mutant derivative thereof.

[0440] The heavy chain of SEQ ID NO:27 comprises:

[0441] - VH of Pertuzumab (SEQ ID NO: 5)

[0442] - CH1 domain of Pertrastuzumab Fab (human IgG1 of G1m(1,17) allotype with mutation T192E) (SEQ ID NO: 1)

[0443] -AP linker (SEQ ID NO: 34);

[0444] -VH of trastuzumab (SEQ ID NO: 9)

[0445] - CH1 domain of trastuzumab Fab (human IgG1 of G1m(1,17) allotype with mutations L143Q and S188V) (SEQ ID NO: 2)

[0446] -Hinge of human IgG1 (SEQ ID NO: 40)

[0447] -CH2 domain of human IgG1 (SEQ ID NO:41)

[0448] -G1m(3) CH3 domain of human IgG1 of allotype (SEQ ID NO:42)

[0449] In a specific embodiment, the bispecific antibody comprises (preferably consists of):

[0450] a) two heavy chains, each heavy chain comprising (preferably consisting of) SEQ ID NO: 28 or a mutant derivative thereof and

[0451] b) four light chains, two comprising (preferably consisting of) SEQ ID NO: 14 or a mutant derivative thereof, and the other two comprising (preferably consisting of) SEQ ID NO: 18 or a mutant derivative thereof.

[0452] The heavy chain of SEQ ID NO:28 comprises:

[0453] -VH of trastuzumab (SEQ ID NO: 9)

[0454] - CH1 domain of trastuzumab Fab (human IgG1 of G1m(1,17) allotype with mutations L143Q and S188V) (SEQ ID NO: 2)

[0455] -AP linker (SEQ ID NO: 34);

[0456] - VH of Pertuzumab (SEQ ID NO: 5)

[0457] - CH1 domain of Pertrastuzumab Fab (human IgG1 of G1m(1,17) allotype with mutation T192E) (SEQ ID NO: 1)

[0458] -Hinge of human IgG1 (SEQ ID NO: 40)

[0459] -CH2 domain of human IgG1 (SEQ ID NO:41)

[0460] -G1m(3) CH3 domain of human IgG1 of allotype (SEQ ID NO:42)

[0461] In a specific embodiment, the bispecific antibody comprises (preferably consists of):

[0462] a) two heavy chains, each heavy chain comprising (preferably consisting of) SEQ ID NO: 29 or a mutant derivative thereof and

[0463] b) four light chains, two comprising (preferably consisting of) SEQ ID NO: 14 or a mutant derivative thereof, and the other two comprising (preferably consisting of) SEQ ID NO: 16 or a mutant derivative thereof.

[0464] The heavy chain of SEQ ID NO:29 comprises:

[0465] - VH of Pertuzumab (SEQ ID NO: 5)

[0466] - CH1 domain of Pertrastuzumab Fab (human IgG1 of G1m(1,17) allotype with mutation T192E) (SEQ ID NO: 1)

[0467] -AP linker (SEQ ID NO: 34);

[0468] - VH of cetuximab (SEQ ID NO: 7)

[0469] - CH1 domain of trastuzumab Fab (human IgG1 of G1m(1,17) allotype with mutations L143Q and S188V) (SEQ ID NO: 2)

[0470] -Hinge of human IgG1 (SEQ ID NO: 40)

[0471] -CH2 domain of human IgG1 (SEQ ID NO:41)

[0472] -G1m(3) CH3 domain of human IgG1 of allotype (SEQ ID NO:42)

[0473] In a specific embodiment, the bispecific antibody comprises (preferably consists of):

[0474] a) two heavy chains, each heavy chain comprising (preferably consisting of) SEQ ID NO: 30 or a mutant derivative thereof and

[0475] b) four light chains, two comprising (preferably consisting of) SEQ ID NO: 14 or a mutant derivative thereof, and the other two comprising (preferably consisting of) SEQ ID NO: 16 or a mutant derivative thereof.

[0476] The heavy chain of SEQ ID NO:30 comprises:

[0477] - VH of cetuximab (SEQ ID NO: 7)

[0478] - CH1 domain of cetuximab Fab (human IgG1 of G1m(1,17) allotype with mutations L143Q and S188V) (SEQ ID NO: 2)

[0479] -AP linker (SEQ ID NO: 34);

[0480] - VH of Pertuzumab (SEQ ID NO: 5)

[0481] - CH1 domain of Pertrastuzumab Fab (human IgG1 of G1m(1,17) allotype with mutation T192E) (SEQ ID NO: 1)

[0482] -Hinge of human IgG1 (SEQ ID NO: 40)

[0483] -CH2 domain of human IgG1 (SEQ ID NO:41)

[0484] -G1m(3) CH3 domain of human IgG1 of allotype (SEQ ID NO:42)

[0485] In a specific embodiment, the bispecific antibody comprises (preferably consists of):

[0486] a) two heavy chains, each heavy chain comprising (preferably consisting of) SEQ ID NO: 31 or a mutant derivative thereof and

[0487] b) four light chains, two comprising SEQ ID NO: 14 or a mutant derivative thereof (preferably consisting of SEQ ID NO: 14 or a mutant derivative thereof), and the other two comprising SEQ ID NO: 20 or a mutant derivative thereof (preferably consisting of SEQ ID NO: 20 or a mutant derivative thereof).

[0488] The heavy chain of SEQ ID NO:31 comprises:

[0489] - VH of Pertuzumab (SEQ ID NO: 5)

[0490] - CH1 domain of Pertrastuzumab Fab (human IgG1 of G1m(1,17) allotype with mutation T192E) (SEQ ID NO: 1)

[0491] -AP linker (SEQ ID NO: 34);

[0492] - VH of Matuzumab (SEQ ID NO: 11)

[0493] - CH1 domain of trastuzumab Fab (human IgG1 of G1m(1,17) allotype with mutations L143Q and S188V) (SEQ ID NO: 2)

[0494] -Hinge of human IgG1 (SEQ ID NO: 40)

[0495] -CH2 domain of human IgG1 (SEQ ID NO:41)

[0496] -G1m(3) CH3 domain of human IgG1 of allotype (SEQ ID NO:42)

[0497] In a specific embodiment, the bispecific antibody comprises (preferably consists of):

[0498] a) two heavy chains, each heavy chain comprising (preferably consisting of) SEQ ID NO: 32 or a mutant derivative thereof and

[0499] b) four light chains, two comprising SEQ ID NO: 14 or a mutant derivative thereof (preferably consisting of SEQ ID NO: 14 or a mutant derivative thereof), and the other two comprising SEQ ID NO: 20 or a mutant derivative thereof (preferably consisting of SEQ ID NO: 20 or a mutant derivative thereof).

[0500] The heavy chain of SEQ ID NO:32 comprises:

[0501] - VH of Matuzumab (SEQ ID NO: 11)

[0502] - CH1 domain of cetuximab Fab (human IgG1 of G1m(1,17) allotype with mutations L143Q and S188V) (SEQ ID NO: 2)

[0503] -AP linker (SEQ ID NO: 34);

[0504] - VH of Pertuzumab (SEQ ID NO: 5)

[0505] - CH1 domain of Pertrastuzumab Fab (human IgG1 of G1m(1,17) allotype with mutation T192E) (SEQ ID NO: 1)

[0506] -Hinge of human IgG1 (SEQ ID NO: 40)

[0507] -CH2 domain of human IgG1 (SEQ ID NO:41)

[0508] -G1m(3) CH3 domain of human IgG1 of allotype (SEQ ID NO:42)

[0509] The light chain of SEQ ID NO:14 comprises:

[0510] - VL of Pertrastuzumab (SEQ ID NO: 6)

[0511] - CK domain with mutations S114A and N137K (SEQ ID NO: 3)

[0512] The light chain of SEQ ID NO:16 comprises:

[0513] - VL of cetuximab (SEQ ID NO: 8)

[0514] - Cκ domain with mutations V133T and S176V (SEQ ID NO: 4)

[0515] The light chain of SEQ ID NO:18 comprises:

[0516] -VL of trastuzumab (SEQ ID NO: 10)

[0517] - Cκ domain with mutations V133T and S176V (SEQ ID NO: 4)

[0518] The light chain of SEQ ID NO:20 comprises:

[0519] - VL of Matuzumab (SEQ ID NO: 12)

[0520] - Cκ domain with mutations V133T and S176V (SEQ ID NO: 4)

[0521] In a specific embodiment, the bispecific antibody comprises (preferably consists of):

[0522] a) two heavy chains, each heavy chain comprising (preferably consisting of) SEQ ID NO: 85 or a mutant derivative thereof and

[0523] b) four light chains, two comprising (preferably consisting of) SEQ ID NO: 14 or a mutant derivative thereof, and the other two comprising (preferably consisting of) SEQ ID NO: 84 or a mutant derivative thereof.

[0524] In a specific embodiment, the bispecific antibody comprises (preferably consists of):

[0525] a) two heavy chains, each heavy chain comprising (preferably consisting of) SEQ ID NO: 86 or a mutant derivative thereof and

[0526] b) four light chains, two comprising (preferably consisting of) SEQ ID NO: 14 or a mutant derivative thereof, and the other two comprising (preferably consisting of) SEQ ID NO: 84 or a mutant derivative thereof.

[0527] 3. Generation of bispecific antibodies

[0528] For general techniques for expressing multispecific antibodies, the skilled person may refer to international patent application WO2013 / 005194, which is incorporated herein by reference.

[0529] Also described herein are polynucleotides comprising sequences encoding protein chains of molecules or antibodies of the present invention. The polynucleotides may also comprise additional sequences: in particular, it may advantageously comprise sequences encoding leader sequences or signal peptides that allow secretion of the protein chains. Host cells transformed with the polynucleotides are also disclosed.

[0530] Typically, the amino acid sequence of the bispecific antibody is used to design the DNA sequence, optionally after codon optimization for mammalian expression. For the heavy chain, DNA encoding the following is synthesized: signal peptide, DNA encoding the variable region of Fab1 and the constant CH1 domain, followed by hinge linkers and the variable region and constant CH1 domain of Fab2, and flanking sequences for restriction enzyme digestion. For the light chain, DNA encoding the following is synthesized: signal peptide and variable and constant kappa regions.

[0531] The nucleic acid encoding the heavy chain and light chain of the bispecific antigen binding fragment or antibody of the present invention is inserted into an expression vector. The light chain and heavy chain can be cloned in the same or different expression vectors. The DNA fragment encoding the immunoglobulin chain is operably connected to the control sequence that guarantees the expression of the immunoglobulin polypeptides in the expression vector. Such control sequences include signal sequences, promoters, enhancers and transcription termination sequences. The expression vector is usually replicated in a host organism as an episome or as an integral part of the host chromosome DNA. Usually, the expression vector will contain a selection marker, for example, tetracycline or neomycin, to allow detection of those cells transformed with the desired DNA sequence.

[0532] In one example, the heavy chain coding sequence and the light chain coding sequence (e.g., sequences encoding VH and VL, VH-CH1 and VL-CL, or full-length heavy chains and full-length light chains) are all included in one expression vector. In another example, each of the heavy chain and light chain of the antibody is cloned into a separate vector. In the latter case, the expression vector encoding the heavy chain and the light chain can be co-transfected into a host cell for expressing the two chains, which can be assembled in vivo or in vitro to form a complete antibody. Alternatively, the expression vector encoding the heavy chain and the expression vector encoding the light chain can be introduced into different host cells for expressing each of the heavy chain and the light chain, which can then be purified and assembled in vitro to form a complete antibody.

[0533] In a specific embodiment, three independent expression vectors, such as plasmids, are co-transfected into the host cell, resulting in the co-production of all three chains (i.e., the heavy chain HC and the two light chains LC1 and LC2, respectively) and the secretion of the bispecific antibody. More particularly, the three vectors can be advantageously used in a molecular ratio of 2:1:1 (HC:LC1:LC2) as follows.

[0534] Recombinant vectors for expressing antibodies described herein generally contain nucleic acids encoding antibody amino acid sequences, which are operably linked to constitutive or inducible promoters. The carrier may be suitable for replication and integration in prokaryotes, eukaryotes or both. Typical carriers contain transcription and translation terminators, initiation sequences and promoters that can be used to regulate the expression of nucleic acids encoding antibodies. The carrier optionally comprises a universal expression cassette containing at least one independent terminator sequence, a sequence (i.e., shuttle vectors) that allows the box to replicate in eukaryotes and prokaryotes, and a selection marker for prokaryotic and eukaryotic systems.

[0535] Bispecific antibodies as 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. The recombinant antibodies of the present invention need not be glycosylated or expressed in eukaryotic cells; however, it is generally preferred to express in mammalian cells. Examples of useful mammalian host cell lines are human embryonic kidney lines (293 cells), baby hamster kidney cells (BHK cells), Chinese hamster ovary cells / - or + DHFR (CHO, CHO-S, CHO-DG44, Flp-in CHO cells), African green monkey kidney cells (VERO cells) and human hepatocytes (Hep G2 cells).

[0536] Mammalian tissue cell culture is preferred for expressing and producing polypeptides, since many suitable host cell lines capable of secreting intact immunoglobulins have been developed in the art, and include CHO cell lines, various Cos cell lines, HeLa cells, preferably myeloma cell lines (e.g., NS0), or transformed B cells or hybridomas.

[0537] In the most preferred embodiment, the bispecific antibodies of the present invention are produced by using CHO cell lines, most advantageously CHO-S or CHO-DG-44 cell lines or derivatives thereof. The expression vectors of these cells may include expression control sequences, such as replication origins, promoters and enhancers, and necessary processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites and transcription terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, etc. The vector containing the polynucleotide sequence of interest (e.g., heavy chain and light chain coding sequences and expression control sequences) can be transferred to a host cell by a well-known method, which depends on the type of cell host. For example, calcium phosphate treatment or electroporation can be used for other cell hosts. (Generally referring to Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 2nd ed., 1989). When the heavy chain and light chain are cloned on separate expression vectors, the vector is co-transfected to obtain the expression and assembly of the complete immunoglobulin.

[0538] Host cells are transformed or transfected with the vector (eg, by chemical transfection or electroporation) and cultured in a conventional nutrient medium (or modified as appropriate) to induce promoters, select transformants, or amplify the gene encoding the desired sequence.

[0539] Expression of antibodies can be transient or stable.

[0540] Preferably, the bispecific antibody is produced by a stable expression method, wherein a cell line stably transfected with DNA encoding all polypeptide chains of the bispecific antibody is capable of sustained expression, which enables the preparation of therapeutic agents. For example, stable expression in a CHO cell line is particularly advantageous.

[0541] Once expressed, the complete antibodies of the invention, dimers thereof, separate light and heavy chains or other immunoglobulin forms can be further separated or purified to obtain substantially homogeneous preparations for further determination and use. Standard protein purification methods known in the art can be used. For example, suitable purification procedures can include classification on immunoaffinity columns 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). Substantially pure immunoglobulins having at least about 90%-95% homogeneity, and most preferably 98%-99% or higher homogeneity, are preferably used for pharmaceutical purposes.

[0542] In vitro production allows for scale-up to produce large quantities of the desired bispecific antibodies of the invention. Such methods can employ homogenous suspension cultures, for example in airlift reactors or in continuous stirred reactors, or fixed or embedded cell cultures, for example in hollow fibers, microcapsules, agarose microbeads or on ceramic cartridges.

[0543] 4. Therapeutic Uses

[0544] The bispecific antibodies of the invention have been shown to induce tumor growth inhibition.

[0545] The bispecific antigen-binding fragments or antibodies of the invention can be used as medicaments, in particular for the treatment of cancer.

[0546] As used herein, the term "cancer" includes any cancer, particularly pancreatic cancer and any other cancer characterized by expression or overexpression of HER3, EGFR or HER2, and particularly those cancers characterized by co-expression of both HER3 and EGFR or HER3 and HER2.

[0547] In some embodiments, the cancer comprises cells having a wild-type KRAS gene.

[0548] 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.

[0549] Preferably, the cancer is pancreatic cancer.

[0550] Thus described is 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.Thus, another aspect of the invention is the use of a bispecific antibody according to the invention for the preparation of a medicament for the treatment of cancer.

[0551] 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 preparation of a pharmaceutical composition. A further aspect of the invention is a method for preparing a pharmaceutical composition comprising a bispecific molecule according to the invention.

[0552] In another aspect, the invention provides a composition, such as a pharmaceutical composition, comprising a bispecific molecule as defined herein formulated together with a pharmaceutical carrier.

[0553] As used herein, "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).

[0554] The compositions of the present invention can be administered by a variety of methods known in the art. The route and / or mode of administration will vary depending on the desired results.

[0555] In order to administer the bispecific molecules or antibodies of the invention by certain routes of administration, it may be necessary to coat the bispecific molecules or antibodies of the invention with materials that prevent their inactivation, or to co-administer the bispecific molecules or antibodies of the invention with materials that prevent their inactivation. For example, the bispecific molecules or antibodies of the invention can be administered to a subject in an appropriate carrier, e.g., liposomes or diluents. 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 well known in the art.

[0556] These compositions can also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. Can be ensured to prevent the presence of microorganisms by sterilization procedures and by comprising various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.). It may also be desirable to include isotonic agents, such as sodium chloride, in the composition. In addition, the delayed absorption of injectable drug forms can be achieved by comprising agents that delay absorption.

[0557] Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0558] The selected dosage level will depend upon a variety of 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, general health and previous medical history of the patient being treated, and similar factors well known in the medical arts. For example, the bispecific molecules or antibodies of the invention can be administered at a dose of 0.2-20 mg / kg from 3 times per week to once per month.

[0559] Therefore, the present invention as 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 limit the present invention. Example

[0560] Example 1. Preparation of the bispecific antibody of the present invention.

[0561] The bispecific antibody (BsAb) of the present invention comprises 2 heavy chains and 4 light chains. In particular, BsAb has 4 antigen binding sites (Fab), wherein 2 Fabs are from Ab1, and the other 2 Fabs are from Ab2 ( Figure 1 ). Therefore, they have double binding capacity (tetravalent) for each of the two target antigens. The Fab distal to the Fc domain is called the "outer Fab", and the Fab proximal to the Fc domain is called the "inner Fab" ( Figure 1 ).

[0562] Six antibodies were designed based on four parental antibodies: Pertuzumab (anti-HER3), Atezolizumab (anti-PDL1), Cetuximab (anti-EGFR), and Matuzumab (anti-EGFR). The nomenclature and sequences of the heavy and light chains for each construct are detailed in Table 1:

[0563]

[0564] Table 1: Description of bispecific antibody constructs

[0565] like Figure 1 As shown, each bispecific antibody comprises:

[0566] -2 identical heavy chains, wherein each heavy chain comprises (from N-terminus to C-terminus): the VH domain and CH1 domain of the outer Fab, a linker, the VH domain and CH1 domain of the inner Fab, a constant Fc domain (including hinge-Ch2-Ch3).

[0567] - 2 identical light chains of the external Fab, wherein each light chain comprises (from N-terminus to C-terminus): the VL domain and the CL domain of the external Fab.

[0568] - 2 identical light chains of the internal Fab, wherein each light chain comprises (from N-terminus to C-terminus): the VL domain and the CL domain of the internal Fab.

[0569] Details of the sequences making up the heavy and light chains for each construct are provided in Table 2.

[0570]

[0571] Table 2: Details on the sequence of each bispecific construct.

[0572] Gene synthesis

[0573] The amino acid sequences of Pertratuzumab (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.

[0574] The DNA construct of the heavy chain was designed as follows:

[0575] - signal peptide (SEQ ID NO: 79),

[0576] - followed by a sequence consisting of the variable region of the external Fab, followed by the constant CH1 domain of the external Fab

[0577] - Next is the connector

[0578] - followed by the variable region of the inner Fab, followed by the constant CH1 domain of the inner Fab.

[0579] Flanking sequences for restriction enzyme digestion were introduced at both ends of the heavy chain DNA construct.

[0580] The DNA construct of the light chain of the external Fab was designed as follows:

[0581] - signal peptide (SEQ ID NO: 80),

[0582] - followed by the variable region of the outer Fab, followed by the constant kappa region of the outer Fab.

[0583] The DNA construct of the light chain of the internal Fab was designed as follows:

[0584] - signal peptide (SEQ ID NO: 80),

[0585] - followed by the variable region of the inner Fab, followed by the constant kappa region of the inner Fab.

[0586] All DNA constructs were synthesized by GeneWis or Eurofins. PCR reactions were performed using PfuTurbo Hot Start to amplify inserts, followed by digestion of heavy and light chains with NotI and ApaI and NotI and HindIII, respectively. The double-digested heavy chain fragments were ligated to NotI and ApaI digested Icosagen's proprietary pQMCF expression vector, into which the human IgG1 hinge followed by the CH2-CH3 domains had been inserted. The double-digested light chain fragments were ligated to NotI and HindIII digested Icosagen's proprietary pQMCF expression vector. Plasmid DNA was verified by double-stranded DNA sequencing.

[0587] Expression and purification

[0588] Bispecific antibodies were produced using transient gene expression by co-transfecting 3 genes encoded on separate vectors at a molecular ratio of 2:1:1 = HC:LC1:LC2 (1 continuous heavy chain (HC) and 2 light chains (LC)). For 50 mL scale expression, a total of 50 μg of plasmid DNA (25 μg heavy chain + 12.5 μg each light chain LC1 and LC2) in Icosagen's proprietary pQMCF vector was mixed in a 1.5 mL Eppendorf tube, 1 mL of CHO TF (Xell Ag) growth medium containing Icosagen's proprietary transfection reagent 007, and incubated at room temperature for 20 minutes. The mixture was incubated at 1-2x10 6 cells / mL were loaded into 49 mL of CHOEBNaLT85 1E in CHO TF (Xell AG) growth medium in a 125 mL shake flask. 9Cells. The cells were shaken at 37°C for 2 days; on the 3rd day, the temperature was moved to 32°C and the culture was fed; and on the 4th day, the temperature was lowered to 30°C; the duration of full production was 10 days. The supernatant was harvested by centrifuging the cells at 3,000rpm for 15 minutes. The supernatant harvested from the bispecific antibody was purified by protein A resin (MabSelect SuRe5mL column). The antibodies were eluted from protein A using 0.1M glycine pH 3.5, and the eluate was neutralized by 1M TRIS. The bispecific antibodies were further purified by gel filtration chromatography using a Superdex 200HiLoad26 / 60pg preparative column, however, the Fab-Fab antibodies were purified using Superdex 200Increase 10 / 300GL; all antibodies were buffer exchanged into PBS pH 7.4. All samples were sterile filtered using 0.2μm Ultra Capsule GF. The apparent MW was determined using Ladder Precision Plus Protein Unstained Standards (Biorad). Bispecific antibodies generally show good expression titers in transient CHO expression.

[0589] SDS polyacrylamide gel electrophoresis

[0590] 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 rate at which the antibody migrates in the gel depends primarily on its size, allowing the molecular weight to be determined. The assay was performed under non-reducing and reducing conditions; the latter allowed the disruption of disulfide bonds and thus the visualization of individual polypeptide chains (light and heavy chains).

[0591] SDS-PAGE of all bispecific antibodies showed the expected profiles under non-reducing and reducing conditions and were consistent with the calculated theoretical molecular weights.

[0592] Size Exclusion Chromatography Analysis

[0593] Protein aggregation is frequently observed in engineered protein molecules. We performed analytical size exclusion chromatography (SEC) to determine the content of high molecular weight species in the final antibody preparations after gel filtration chromatography.

[0594] The SEC chromatograms demonstrated that the percentage content of higher molecular weight species was very low (less than 5%) and similar to conventional monoclonal antibodies produced in the CHO expression system. The results confirmed that there were no or few aggregates. The results also showed that each antibody was assembled correctly.

[0595] Example 2. Monospecific binding of an antibody to its cognate antigen

[0596] The apparent affinity of BsAb "Patri-Trastu-Fc", "Patri-Matu-Fc" and "Patri-Cetu-Fc" to EGFR, HER2 and HER3 was evaluated by direct ELISA. The monospecific binding (i.e., one antigen at a time) of each BsAb was compared with the parental MAb. The EC50 (antibody concentration that induces 50% median binding between baseline and maximum binding) of each BsAb and the parental antibody was then defined.

[0597] Materials and methods

[0598] In a Nunc Maxisorp 96 plate, 800ng / ml EGFR His tag, HER2 His tag, or HER3 His tag antigen was diluted in 50μl of 1X PBS. The plate was incubated overnight at 4°C, then washed 4 times with 0.1% PBS-Tween (PBS-T) and saturated with PBS-T containing 1% BSA at 37°C for 1h. After washing 4 times in 0.1% PBS-T, antibodies (BsAb, mAb, and control) were diluted in 1X PBS in a final volume of 50μl and incubated at 37°C for 1h. After washing 4 times in 0.1% PBS-T, the detection antibody (HRP-conjugated anti-human Fc) was added at 1 / 40000 in a 50μl volume of 1X PBS and incubated at 37°C for 1h. The plate was washed four times in 0.1% PBS-T, and 50 μl of TMB was added to each well for 10-30 minutes at room temperature and protected from light. The reaction was terminated with 50 μl of 1 M hydrochloric acid, and the optical density was read at a wavelength of 450 nm.

[0599] result

[0600] The results are shown in Table 3 below.

[0601]

[0602] Table 3. ELISA analysis of monospecific binding to HER3, HER2 or EGFR. Measurement of the half maximal effective concentration (EC50) of each parental antibody (as a positive control) or BiXAb against HER3, HER2 or EGFR antigens

[0603] All bispecific antibodies were able to bind antigen and displayed very similar binding profiles to the corresponding parental antibodies. Subnanomolar apparent affinities were measured for all constructs to their cognate antigens, which were almost identical to their parental antibodies.

[0604] Example 3. Dual binding of a bispecific antibody to its cognate antigen

[0605] The bispecific binding of the antibodies to HER3 / HER2 and HER3 / EGFR was evaluated by ELISA sandwich assay. Briefly, the untagged antigen was coated on the plate and incubated with the BsAb first, then the second antigen with the His tag; finally, the anti-HIS peroxidase antibody was used for colorimetric assay. This design allows the evaluation of the simultaneous binding of the bispecific antibody to two antigens.

[0606] Materials and methods

[0607] In a Nunc Maxisorp 96 plate, 1000 ng / ml of antigen without EGFR, HER2 or HER3 tag was diluted in 50 μl of 1X PBS. The plate was incubated overnight at 4°C, then washed 4 times in 0.1% PBS-Tween and saturated with PBS-T containing 1% BSA for 1 h at 37°C. After washing 4 times in 0.1% PBS-T, antibodies (BsAb or IRR) were added at various dilutions in a final volume of 50 μl of 1X PBS and incubated for 1 h at 37°C. After washing 4 times in 0.1% PBS-T, 1000 ng / ml EGFR His tag, HER2 His tag or HER3 His tag antigen in 50 μl PBS1X was incubated for 1 h at 37°C. The plate was washed 4 times with 0.1% PBS-T, and HRP-conjugated anti-His tag detection antibody was added to 50 μl 1X PBS at 1 / 2000. The plate was incubated at 37°C for 1 h. The plate was washed 4 times in 0.1% PBS-T and 50 μl of TMB colorimetric solution was added at room temperature in the dark for 10-30 minutes. The reaction was terminated with 50 μl of 1 M hydrochloric acid and the optical density was read at a wavelength of 450 nm.

[0608] result

[0609] Figure 2 (A) Binding curves showing simultaneous binding profiles of Patri-Trastu-Fc to immobilized HER3 visualized by soluble HER2-His (left panel) and to immobilized HER2 visualized by soluble HER3-His (right panel) in a dose-dependent manner. Figure 2 (B) Binding curves showing 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). Figure 2 This is also the case for the binding curve of Patri-Cetu-Fc shown in (C)

[0610] Therefore, the results show that the BsAb of the present invention is able to bind to two antigens simultaneously.

[0611] Example 4. Bispecific Antibodies Inhibit Phosphorylation of AKT and ERK

[0612] The main anti-tumor effect of antibodies targeting HER family receptors is their ability to inhibit signaling associated with those receptors. Therefore, we investigated two major signaling pathways associated with EGFR, HER2, and HER3 signaling: MAPK / ERK and PI3K / AKT. Using HTRF technology, we evaluated the inhibition of phosphorylation of pERK and pAKT induced by the addition of bispecific antibodies after stimulation with EGF+NRG1 ligand.

[0613] Materials and methods

[0614] On day 0, 50,000 cells per well were seeded in 100 μl complete medium (DMEM or RPMI 10% SVF) in a 96-well flat-bottom plate. On day 1, the cells were depleted of FCS by changing the 10% FCS medium to 2% FCS medium. On day 2, the medium was removed from all conditions and the cells were treated with 70 nM antibody (BsAb or mAb) diluted in 100 μl 2% FCS medium for 20 minutes, to which two ligands, 16.6 nM (100 ng / ml) of EGF and 3.71 nM (100 ng / ml) of NRG1 were added for another 10 minutes at 37°C. Then, at the end of the 30-minute treatment, the medium was removed and the cells were rinsed once with cold 1X PBS. The cells were then lysed for 1 h under agitation and the HTRF kit pERK Thr202 / Tyr204 ( #64ERKPEG) and pAKT Ser473 ( pERK and pAKT levels were measured using a PCR amplification kit (BMG LabTech, #64AKSPET). The plate was then read at 665 nm and 620 nm on a Pherastar reader (BMG LabTech).

[0615] result

[0616] For the three PDAC cell lines tested (BxPC-3, AsPC-1, and CFPAC), all BsAbs (Patri-Trastu-Fc, Patri-Cetu-Fc, and Patri-Matu-Fc) exhibited strong inhibition of pAKT and pERK signaling after ligand induction ( Figure 3B). Similar to the HER2 / HER3 antibodies, both EGFR / HER3 bispecific antibodies blocked almost 100% of basal pAKT signaling in BxPC-3 cells and about 85%-90% in ASPC1 and CFPAC cells. They also effectively 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 strongest signaling pathway in the BxPC3 cell line because this cell line is the only one expressing WT Kras, whereas pERK inhibition was weaker in ASPC1 and CFPAC expressing mutant Kras. In all three cell lines, EGFR density was much higher than that of HER2 or HER3 ( Figure 3 A). BiXAbs containing anti-EGFR are also expected to be superior compared to anti-HER2 / HER3 antibodies.

[0617] Example 5. In vivo efficacy of Patri-Trastu-Fc, Patri-Matu-Fc and Patri-Cetu-Fc.

[0618] Tumor growth and survival were assessed in mice treated with the bispecific antibody xenografted with Sw1990 and PDX P2846 PDAC cells. These models express mutant Kras and similar antigen densities of EGFR, HER2, or HER3 antigens.

[0619] Materials and methods

[0620] On day 0, 3 million Sw-1990 cells were transplanted subcutaneously into 5-week-old Swiss nude immunodeficient mice (n=9 mice / group). 3 Treatment was initiated at 17 mg / kg of the bispecific antibody in 200 μl injections intraperitoneally. The treatment regimen was 2 injections per week for 4 weeks. Tumor size was measured with a caliper. When the tumor size reached 1500 to 2000 mm 3 When , the mice were killed.

[0621] The size is 150mm 3 The P2846 P7 was transplanted subcutaneously through the scapula. The treatment regimen remained the same as the Sw-1990 mouse model.

[0622] result

[0623] In mice xenografted with Sw1990 cells, Patri-Trastu-Fc, Patri-Matu-Fc, and Patri-Cetu-Fc inhibited tumor growth by 42%, 62%, and 69%, respectively ( Figure 4 A).

[0624] The survival of Sw-1990 xenograft mice was improved by 11 days for Patri-Trastu-Fc treatment group, 15 days for Patri-Matu-Fc treatment group, and 15 days for Patri-Cetu-Fc treatment group. 3 ), EGFR / HER3 BsAb induced the strongest tumor growth inhibition (875 mm on day 46 for 3Patri-1Matu-Fc and 3Patri-1Cetu-Fc 3 ). BsAb 3Patri-1Cetu-Fc provided better overall survival. No signs of toxicity or weight loss were observed in this experiment. Negative control vehicle and an irrelevant bispecific antibody (BsAb IRR) showed similar tumor growth and survival.

[0625] 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 transplantation) ( Figure 4 B).

[0626] At day 80, the survival rate of mice xenografted with P2846 P7 PDX was increased by 70% in the Patri-Trastu-Fc and Patri-Cetu-Fc treated groups compared to the BsAb IRR group, and the survival rate of the Patri-Matu-Fc treated group was increased by 20%. The BsAbs Patri-Trastu-Fc and Patri-Cetu-Fc induced the strongest tumor growth inhibition compared to Patri-Matu-Fc. Compared to the control BsAb IRR group, the survival of the Patri-Trastu-Fc group was increased by 34 days, and the survival of the Patri-Cetu-Fc group was increased by 41 days. No signs of toxicity or weight loss were observed in this trial.

[0627] Example 6. Intratumoral penetration of BsAbs in Sw-1990 xenografts.

[0628] A common question regarding the large molecular weight of BsAbs (250 kDa) relative to conventional antibodies (150 kDa) concerns their ability to penetrate into tumors, particularly pancreatic tumors, which are known to have an abundant and dense stroma. Histological sections were prepared from extracted tumors; Sw1990 tumor penetration of the bispecific antibodies was analyzed by immunohistochemistry. The bispecific antibodies were labeled using peroxidase-conjugated anti-human Fc. BsAb IRR is a control bispecific antibody targeting CD19 and CD3. NaCl was used as a negative control ( Figure 5 ).

[0629] Specific labeling of BsAb was observed in tumors treated with Patri-Trastu-Fc, Patri-Matu-Fc, and Patri-Cetu-Fc. The 40X image showed that despite the dense stroma, the labeling between the center and periphery of the tumor was homogeneous, indicating that the BsAb penetrated the tumor. In addition, obvious labeling was observed at the tumor epithelial cell membrane in the center of the tumor, demonstrating the binding and accumulation of BsAb at the tumor cell membrane inside the tumor mass. No nonspecific labeling was observed in tumors treated with NaCl. BsAb IRR was weakly detected at the periphery or stroma of the tumor and was not detected at the tumor epithelial cells, indicating that BsAb IRR did not bind to tumor cells.

[0630] Example 7. Patri-Trastu-Fc, Patri-Matu-Fc and Patri-Cetu-Fc reduce angiogenesis

[0631] Angiogenesis or new blood vessel formation was studied by immunohistochemistry (IHC) using anti-CD31 staining. CD31 is expressed on endothelial cells and therefore allows for the labeling of blood vessels. Angiogenesis is a major parameter for tumor transformation, especially in pancreatic cancer. The proportion of blood vessels was assessed after treatment with 3 BsAbs compared to controls (NaCl and BsAb IRR). For each treatment, 3 tumor sections corresponding to three different mice were marked and the images were analyzed by ImageJ software. The quantitative results are presented in histograms, highlighting the changes in blood vessel size according to treatment ( Figure 6 A).

[0632] IHC results showed that the vessel size decreased from 75 μm in the control group (NaCl, BsAb IRR) to 55 μm in the group treated with the BsAb of the present invention. This indicates that the BsAb inhibits xenograft neovascularization.

[0633] Example 8. Ex vivo analysis of NK infiltration in tumors treated by Patri-Trastu-Fc, Patri-Matu-Fc, and Patri-Cetu-Fc.

[0634] Materials and methods

[0635] NK cell immunophenotyping was performed by flow cytometry on the Sw1990 xenografts from the excision of mice treated with BsAb. The dissociated cells were labeled with live / dead, mouse CD45, CD3, CD19, CD49, NKp46, IFNγ and CD1070 specific antibodies. Anti-CD3 and anti-CD19 antibodies conjugated with APC-Cy7 were used for negative selection, and anti-CD45, anti-CD49b conjugated with APC-, and anti-NKp46 conjugated with fluorescein were used to separate NK cells for immunophenotyping. After anti-CD107a conjugated with BV786 and anti-IFNg conjugated with BV421- in the cell, the activation of CD45+CD49b+NKp46+NK cells was analyzed by flow cytometry.

[0636] result

[0637] As shown in CD49+NKp46+ analysis, NK infiltration in tumors treated with BsAb of the present invention increased 2-fold compared to NaCl and BsAb IRR groups ( Figure 5 B) The increase in IFNγ+ and CD107a+ on NK cells indicates that these intratumoral NK cells are also activated compared to untreated tumors and can therefore mediate ADCC.

[0638] Example 9. In vitro efficacy of Patri-Trastu-Fc, Patri-Matu-Fc and Patri-Cetu-Fc on degradation of EGFR, HER2 and HER3 receptors

[0639] Materials and methods

[0640] Patri-Cetu-Fc- and BsAb IRR- and NaCl-treated Sw1990 xenograft sections were stained for EGFR. 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. Before adding a second IRDye800-conjugated goat anti-rabbit IgG (1 / 20000 dilution), the first rabbit monoclonal antibody for EGFR, HER2 and HER3 was used to visualize the Western blot. Fluorescence was quantified using the LI-COR Odyssey imaging system. HER receptor expression was then quantified and normalized to tubulin in each group of excised xenografts. Each experiment was performed on three Sw1990- xenograft mice. BsAb IRR was a negative control for simultaneous binding of human CD19 and CD3.

[0641] result

[0642] Images acquired at X20 and X40 magnifications ( Figure 5 C, upper panel) shows that EGFR signaling was significantly reduced in tumors treated with Patri-Cetu-Fc compared with tumors treated with NaCl or BsAbIRR.

[0643] EGFR, HER2 and HER3 total protein levels were assessed by Western blotting of protein extracts from Sw-1990 xenografts and revealed by immunofluorescence. Total protein levels were quantified and normalized to tubulin. Figure 5 The results of C (middle and upper panels) show that the three BsAbs of the present invention induced degradation of EGFR, HER2 or HER3 compared with the NaCl and BsAb IRR groups.

[0644] Example 10. The bispecific antibody Patri-Cetu-Fc more effectively reduces tumor growth than monospecific antibodies or combinations thereof in the Sw1990 xenograft model

[0645] Materials and methods

[0646] Mice xenografted with Sw1990 cells were treated with (1) BisAb IRR anti-CD19-CD3 (2) bispecific antibody Patri-Cetu-Fc (17 mg / Kg) (3) Cetuximab parent antibody (10 mg / kg) (4) Patrutuzumab parent antibody (10 mg / kg) or combination (Cetuximab + Patrutuzumab, 5 + 5 mg / kg) (9 mice per group). The doses of bispecific and parent antibodies had the same molar concentration based on their Fc content.

[0647] Swiss nude immunodeficient mice were subcutaneously transplanted with 3 million Sw-1990 cells on day 0. Treatment was initiated when the average tumor size was 140 mm3. The treatment regimen was 2 injections per week for 4 weeks. Tumor growth and survival of treated mice were evaluated ( Figure 7 ).

[0648] result

[0649] The bsAb Patri-Cetu-Fc induced a strong inhibition of tumor growth in vivo (77% tumor growth inhibition at day 38 post-transplantation compared to IRR-treated mice). The monospecific antibodies, cetuximab and patuximab, induced 50% and 43% tumor growth inhibition at day 38 post-transplantation, respectively. Finally, the combination of the two monoclonal antibodies showed 65% tumor growth inhibition when compared to IRR-treated mice. Therefore, the bispecific antibody Patri-Cetu-Fc is more effective than the monospecific parental antibodies in terms of tumor growth inhibition.

[0650] Patri-Cetu-Fc also induced a strong survival benefit in vivo. In terms of survival benefit, the bispecific antibody Patri-Cetu-Fc was shown to be more effective than the monospecific parental antibody.

[0651] Embodiment 11

[0652] Two additional bispecific antibodies comprising humanized cetuximab Fab were designed:

[0653] - "BMX003-010" antibody: it comprises the pertrastuzumab Fab domain (as external Fab) and a humanized version of the cetuximab Fab domain (as internal Fab).

[0654] - The "BMX003-011" antibody, which is identical to BMX003-010, except that it further comprises 2 mutations in the Fc domain (S239D and I332E).

[0655] Both BMX003-010 and BMX003-011 comprise a humanized cetuximab Fab comprising:

[0656] - VH domain of SEQ ID NO: 81 and CH1 domain of SEQ ID NO: 2 (corresponding to the VH-CH1 sequence of SEQ ID NO: 83)

[0657] - VL domain of SEQ ID NO: 71 and CL domain of SEQ ID NO: 4 (corresponding to the VL-CL sequence of SEQ ID NO: 84)

[0658] BMX003-011 differs from BMX003-010 in that it contains 2 mutations (S239D and I332E) in the Fc domain (mutant Fc domain of SEQ ID NO: 82).

[0659] name Heavy chain Light chain (external Fab) Light chain (internal Fab) BMX003-010 SEQ ID NO:85 SEQ ID NO:14 SEQ ID NO:84 BMX003-011 SEQ ID NO:86 SEQ ID NO:14 SEQ ID NO:84

[0660] Table 4: Details of the sequences of the bispecific constructs "BMX003-010" and "BMX003-011".

[0661] The activities of BMX003-010 and BMX003-011 comprising humanized cetuximab Fab fragments were compared to the activity of "BMX003-001" comprising the parental cetuximab Fab ("BMX003-001" corresponds to the "Patri-cetu-Fc" construct described above).

[0662] SDS polyacrylamide gel electrophoresis and size exclusion chromatography analysis

[0663] To assess the quality of the purified antibodies, SDS-PAGE and size exclusion chromatography (SEC) were performed as described above. The SDS-PAGE of the BMX003-010 and BMX003-011 bispecific antibodies showed the expected profiles. The SEC chromatograms confirmed the absence or small amounts of aggregates and indicated that each antibody was assembled correctly.

[0664] Evaluation of affinity to HER3 and EGFR by ELISA

[0665] The apparent affinity of BsAb BMX003-010, BMX003-011 and BMX003-001 (Patri-Cetu-Fc) antibodies to HER3 and EGFR was assessed by direct ELISA. The monospecific binding (i.e., once for an antigen) of each BsAb was compared with the parental MAb. The EC50 (the antibody concentration that induces 50% median binding between baseline and maximum binding) of each BsAb and parental antibody was then defined. ELISA was performed as described above.

[0666] Figure 8 and Fig. 9 It was shown that both bispecific antibodies were able to bind to their antigens (HER3 and EGFR, respectively) with binding profiles very similar to those of the corresponding parental antibodies.

[0667] Flow cytometry binding profile

[0668] 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).

[0669] Fig.10 The results showed that BMX-003-001 and humanized BMX-003-010 and BMX-003-011 had very similar EC50 and ECmax.

[0670] Phosphorylation assay

[0671] Based on Perkin Elmer's Advanced Phospho-ERK and Advanced Phospho-AKT kits. HCT-116 colon cancer cells were plated in cDMEM culture medium at a density of 50,000 cells / well in 96 wells. Flat-bottomed microtiter plates were grown overnight at 37°C (5% CO2). The tissue culture medium was removed and replaced with serum-free culture medium (DMEM+GlutaMAX), and the cells were continued to be incubated for 24hr at 37°C (5% CO2). The tissue culture medium was removed and replaced with the test article (BiXAb, MAb or control) resuspended in DMEM, and incubated for 20 minutes at 37°C (5% CO2). Growth factors (16.6nM EGF and 3.71nM NRG1) were added to the wells and continued to be incubated for 10 minutes. The culture medium was removed, and the cells were then washed once with cold PBS. The cells were then lysed by adding lysis buffer (provided in the Perkin Elmer's kit). By following the FRET assay kit instructions provided by Perkin Elmer, quantification of phosphorescence signals was achieved. In essence, 4 microliters of pre-mixed europium-labeled antibodies were incubated with cell lysates at room temperature for 4 hr. The supernatant was then transferred to a fluorescent plate reader and HTRF was measured at 665nm and 620nm.

[0672] The results showed that after ligand induction, all bispecific antibodies showed inhibition of pAKT ( Fig.11 A) and pERK ( Fig.11 B) Strong inhibition of signaling.

[0673] Degranulation assay

[0674] PBMC from two independent donors were prepared following standard procedures and left to stand overnight in complete RPMI culture medium. HCT116 colon cancer cell line was grown in RPMI tissue culture medium supplemented with 10% FBS. 20,000 HCT116 cells were laid together with 100,000 PBMC in a U-shaped bottom 96-well microtiter plate (based on NK colony accounting for about 10% of PBMC, effector cells and target cells (E: T) ratio of 1: 2 were given). Then BiXAb test article and control were added to the cell mixture together with anti-CD107a-AF488mAb, and incubated for 1hr at 37°C (5% CO2). Then GolgiStop was added to the wells, and the plate was continued to be incubated for 4hr. Then anti-CD45, anti-CD3 and CD56 mAb were added to the wells to allow identification of NK cells by typical FAC staining. NK degranulation was assessed by FACS analysis of CD107a by gating NK cells.

[0675] Fig.12 The results showed that cetuximab, BMX003-001 and BMX003-010 were comparable. Much higher degranulation was observed in the presence of BMX003-011 (Fc modified), indicating that the BMX003-011 mutant Fc domain has increased binding to Fcγ receptors when compared to the wild-type Fc domain, which results in a bispecific antibody with improved effector function.

[0676] In vivo effects

[0677] The in vivo efficacy of BMX003-001 (Patri-Cetu-Fc), BMX003-010 (humanized Patri-Cetu-Fc) and BMX003-011 (modified humanized Patri-Cetu-Fc) was evaluated in the SW1990 pancreatic cancer xenograft model. BMX003-01, BMX003-010 and BMX003-011 were used at 8.5 mg / kg, and the BMX control was used at 17 mg / kg. The method was as described above.

[0678] At day 35 (after the last injection on day 32), all groups treated with HER3x EGFR BiXab presented significantly lower tumor volumes than the control group PBS. No major differences were observed between BMX003-001, BMX-003-010 and BMX-003-011 BiXAb.

[0679] in conclusion

[0680] The results showed that the bispecific antibodies of the present invention were effectively produced and assembled correctly. The results also demonstrated the therapeutic properties of the bispecific antibodies:

[0681] - simultaneously binds its target with good affinity when compared to the parent antibody;

[0682] -Inhibit the activation of HER3 / HER2 / EGFR receptors by inhibiting the phosphorylation signaling pathway;

[0683] -Simultaneous degradation of two targeted receptors;

[0684] - Penetration into the tumor;

[0685] -Inhibits new blood vessel formation;

[0686] - Modulate the infiltration of immune cells in tumors; and

[0687] -Significantly inhibited tumor growth and improved survival in vivo.

Claims

1. A bispecific antigen-binding fragment capable of simultaneously binding to HER-3 and another antigen selected from HER-2 and EGFR antigens, the bispecific antigen-binding fragment comprising: (i) a Fab fragment comprising the VH-CH1 heavy chain associated with the VL-CL light chain of Antibody 1 (Abl), and (ii) a Fab fragment comprising the VH-CH1 heavy chain associated with the VL-CL light chain of Antibody 2 (Ab2), The N-terminal end of the VH domain of the Fab fragment of Ab1 is connected to the C-terminal end of the CH1 domain of the Fab fragment of Ab2 through a polypeptide linker. wherein one of Ab1 or Ab2 is pertuzumab or a functional derivative thereof; And the other of Abl 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.

2. The bispecific antigen-binding fragment of claim 1, wherein Ab2 is pertuzumab or a functional derivative thereof, and wherein 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. The bispecific antigen-binding fragment of any one of claims 1 or 2, 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 . The bispecific antigen-binding fragment of claim 1 , wherein the CH1 and CL domains of Abl have different sequences from the CH1 and CL domains of Ab2.

5. The bispecific antigen-binding fragment of any one of claims 1 to 4, wherein the FabCH1 domain of one of Ab1 or Ab2 is a mutant domain derived from a CH1 domain of an immunoglobulin by replacing a threonine residue at position 192 of the CH1 domain with a glutamic acid, and the cognate CL domain is a mutant domain derived from a CL domain of an immunoglobulin by replacing an asparagine residue at position 137 of the CL domain with a lysine residue and replacing a serine residue at position 114 of the CL domain with an alanine residue, and / or wherein the Fab of one or the other of Ab1 or Ab2 is The CH1 domain is a mutant domain derived from the CH1 domain of an immunoglobulin, which is achieved by replacing the leucine residue at position 143 of the CH1 domain with glutamine and replacing the serine residue at position 188 of the CH1 domain with a valine residue, and the homologous CL domain is a mutant domain derived from the CL domain of an immunoglobulin, which is achieved by replacing the valine residue at position 133 of the CL domain with a threonine residue and replacing the serine residue at position 176 of the CL domain with a valine residue.

6. The bispecific antigen-binding fragment of any one of claims 1 to 5, wherein the polypeptide linker sequence comprises or consists of the following amino acid sequence: EPKX1CDKX2HX3X4PPX5PAPELLGGPX6X7PPX8PX9PX10GG (SEQ ID NO: 33), wherein X1, X2, X3, X4, X5, X6, X7, X8, X9, and X10 are the same or different and are any amino acids.

7. A bispecific molecule comprising two identical antigen binding arms, each antigen binding arm consisting of an antigen binding fragment as defined in any one of claims 1 to 6.

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

9. The bispecific molecule of any one of claims 7 or 8, comprising a) two heavy chains, each heavy chain comprising, preferably consisting of, SEQ ID NO: 27, and b) four light chains, two light chains comprising, preferably consisting of, SEQ ID NO: 14 and the other two light chains comprising, preferably consisting of, SEQ ID NO:

18.

10. The bispecific molecule of any one of claims 7 or 8, comprising a) two heavy chains, each heavy chain comprising, preferably consisting of, SEQ ID NO: 29, and b) four light chains, two of which comprise, preferably consist of, SEQ ID NO: 14 and the other two comprise, preferably consist of, SEQ ID NO:

16.

11. The bispecific molecule of any one of claims 7 or 8, comprising a) two heavy chains, each heavy chain comprising, preferably consisting of, SEQ ID NO: 31, and b) four light chains, two of which comprise, preferably consist of, SEQ ID NO: 14 and the other two comprise, preferably consist of, SEQ ID NO:

20.

12. A method for producing the bispecific molecule of any one of claims 7 to 11, comprising the steps of: a. culturing a host cell in a suitable medium and culture conditions, the 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, and b. recovering the produced antibody from the culture medium or from the cultured cells.

13. 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 as a medicament.

14. 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 treating cancer, preferably solid tumors, such as pancreatic cancer, head and neck cancer, colorectal cancer, breast cancer or lung cancer, preferably for treating pancreatic cancer.

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