Anti-Met Fab-Fc for treatment of tumors and / or metastasis

Abstract

The present disclosure relates to anti-Met Fab-Fc for use in the treatment of tumors and / or metastasis. The present disclosure relates to an anti-Met antibody fragment comprising a single antigen binding arm and an Fc region wherein the antigen binding arm is defined by a variable region having an amino acid sequence as listed in SEQ ID No.: 7, 8, and wherein the anti-Met antibody fragment is useful in the treatment of tumors and / or metastases.

Description

[0001] This application is a divisional application of the patent application "Anti-MetFab-Fc for treating tumors and / or metastases" (application number 2019800806112) filed on October 7, 2019. Technical Field

[0002] The present disclosure relates to novel antibody-derived therapeutics for treating tumors and / or metastases. Background Art

[0003] Cancer is a genetic disease in which endogenous genes in somatic cells mutate. Only a few genes in cancer cells—called oncogenes and tumor suppressor genes—are altered and drive tumorigenesis. Activated oncogenes are promoters, while inactivated tumor suppressor genes are missing brakes on cancer cell growth. After the discovery of oncogenic mutant genes, a new concept about oncogenes emerged: "oncogene addiction," which refers to the fact that cancer cells still rely on a single proto-oncoprotein for their continued proliferation or survival despite the presence of a large number of genetic alterations. Therefore, new therapeutic approaches have been taken to develop "targeted therapies" for the treatment of cancer. In the past 20 years, pharmacological targeting of proteins encoded by oncogenic mutant genes—by chemotherapeutic drugs or antibodies—represents the forefront of weapons to eliminate mutant cells and fight cancer diseases. Targeted therapies are expected to be more effective than conventional cytotoxic chemotherapy and generally have fewer side effects. However, only patients who carry altered specific target genes in their tumors are likely to benefit from targeted therapies. Therefore, personalized medicine is needed in parallel to evaluate druggable genetic lesions through comprehensive genomic profiling of patients' tumors.

[0004] The MET oncogene encodes a unique receptor tyrosine kinase with pleiotropic functions. When genetically altered (through point mutations, gene fusions, translocations, and / or amplification), MET will initiate cellular transformation by virtue of its ability to activate an aggressive growth program. Thus, genetic lesions of MET that result in constitutive overactivation of the Met kinase will initiate and maintain the transformed phenotype (“MET addiction”). 1 .

[0005] MET genetic lesions occur in most solid tumors, with an overall incidence of 1-4%, and are able to upregulate its kinase activity 1 . Point mutations are concentrated in domains that are critical for hepatocyte growth factor (HGF) ligand binding or receptor signaling (SEMA domain, juxtamembrane domain, and catalytic domain). Recently, next-generation sequencing revealed that exon 14 splice site mutations occur in 3% of non-small cell lung cancers. 2 , which results in exon skipping and deletion of the juxtamembrane region of the MET transcript, in which the serine residue (Ser985) negatively regulates Met kinase activity 3And the internalization and degradation of Met requires a tyrosine residue (Tyr1003) 4 .

[0006] In the "invasive growth" program initiated by MET, the proliferative response is linked to migration, survival, extracellular matrix degradation, and cell polarity induction 5 Cells strive to adapt these biological responses to adverse conditions and / or escape to find a more favorable environment. Under hostile conditions, Met is overexpressed by a variety of stimuli such as hypoxia, inflammatory cytokines, proangiogenic factors, mitogens, and even HGF itself - via transcriptional upregulation. Finally, Met is overexpressed in the context of radiation-induced DNA damage and helps resist radiation therapy by promoting the activation of DNA repair and circumvention of programmed cell death in cancer cells.

[0007] Several Met-targeting molecules have been developed to erase hyperactive Met signaling in a selective, robust, and highly effective manner. These drugs include: HGF antagonists (blocking antibodies or decoys), mAbs targeting Met receptors, and chemical tyrosine kinase inhibitors (TKIs). Anti-Met mAbs potentially represent a major step forward in the battle against MET-driven cancers. Today, four anti-Met mAbs have entered early clinical trials: MetMab (onatuzumab, Roche), LY2875358 (imatuzumab, Eli Lilly & Company), ARGX-111 (Argenx), SAIT301 (Samsung), and Sym015 (Symphogen A / S)—a mixture of two antibodies. They work by competitively blocking the binding of HGF to MET (onatuzumab, ARGX-111) and / or downregulating MET (imatuzumab, SAIT301, Sym015).

[0008] Murine DN30 mAb (disclosed in WO 2007 / 090807) is an IgG2A that binds to the extracellular domain of the human Met receptor and induces only some of the biological effects triggered by Met 6 Due to its bivalent nature, which allows simultaneous binding to two different antigen molecules, it will partially activate receptor phosphorylation, leading to stabilization of the receptor complex in a manner similar to that obtained with the natural ligand. This detrimental partial agonist activity towards Met was not observed in the monovalent DN30 Fab fragment (MvDN30). 7. Converting the bivalent DN30 parent antibody into a monovalent Fab fragment would unlock the therapeutic potential of the DN30 anti-Met antibody, thereby generating a full antagonist molecule. However, due to its low molecular weight, the short half-life of Fab is a severe limitation for its deployment in therapy. The inventors have therefore developed new engineered molecules, called DCD (Dual Constant Domain Fab), characterized by the duplication of the constant domains present in the DN30 Fab: DCD-1, in which the duplication is performed in tandem; and DCD-2, in which the constant domains of the light and heavy chains are exchanged with each other (disclosed in WO 2014 / 108829). Both new recombinant molecules showed biochemical properties comparable to those of the original Fab in vitro and could act as full Met antagonists. In vivo, after systemic administration, the new recombinant molecules would reduce the growth of Met-addicted tumors. DCD-1 and DCD-2 showed improved pharmacokinetic characteristics over the original DN30 Fab, but neither achieved a behavior comparable to the original mAb. 8 .

[0009] It has been previously reported that conversion of antibodies from a bivalent form to a monovalent form can also be achieved by deleting - by molecular engineering methods - one of the two antibody arms (disclosed in WO 2005 / 063816). The data reported an improvement in the in vivo stability of the molecule, which was mainly attributed to the activity of the Fc domain binding to Fc receptors expressed in organs. Summary of the invention

[0010] The purpose of the present disclosure is to provide a novel anti-tumor agent derived from a monoclonal antibody, namely, DN30 monoclonal antibody, which can be used for the treatment of tumor patients.

[0011] According to the invention, the above objects are achieved thanks to the subject matter explicitly mentioned in the appended claims, which are to be understood as forming an integral part of the present disclosure.

[0012] The present invention relates to an anti-Met antibody fragment comprising a single antigen binding arm and an Fc region, wherein the Fc region comprises a complex of a first and a second Fc polypeptide. The antibody fragment comprises:

[0013] (i) a first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized light chain variable (VL) domain is fused to the human light chain constant (CL) domain in the N- to C-terminal direction, and wherein the humanized light chain variable (VL) domain contains three complementarity determining regions (CDRs) having the amino acid sequences set forth in SEQ ID No.: 1, 2 and 3, and wherein the humanized VL domain has the amino acid sequence set forth in SEQ ID No.: 7;

[0014] (ii) a second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a human hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein in the N- to C-terminal direction, the humanized heavy chain variable (VH) domain is fused to the human heavy chain constant CH1 domain, the human heavy chain constant CH1 domain is fused to the human hinge region, the human hinge region is fused to the human constant CH2 domain, and the human constant CH2 domain is fused to the human constant CH3 domain, and wherein the humanized heavy chain variable (VH) domain contains three complementarity determining regions (CDRs) having the amino acid sequences set forth in SEQ ID Nos.: 4, 5, and 6, and wherein the humanized VH domain has the amino acid sequence set forth in SEQ ID No.: 8;

[0015] (iii) a third polypeptide comprising a second human Fc polypeptide, wherein the second human Fc polypeptide comprises a hinge region, a human constant CH2 domain and a human constant CH3 domain, wherein in the N- to C-terminal direction, the hinge region is fused to the human CH2 constant domain, and the human CH2 constant domain is fused to the human CH3 constant domain, wherein the hinge region is truncated at the N-terminus.

[0016] The improved therapeutic properties of the above-mentioned antibody fragments depend on the structure of the variable region and are not solely due to the monovalent form of the antibody and / or the presence of the Fc region.

[0017] The present invention also relates to a product comprising, in a single bottle or in two bottles, (a) an anti-Met antibody fragment as defined above and a pharmaceutically acceptable vehicle, and (b) an extracellular portion of human Met and a pharmaceutically acceptable vehicle, wherein the extracellular portion of human Met is capable of binding to hepatocyte growth factor (HGF) in a stable manner and contains at least one amino acid mutation within the epitope recognized by the anti-Met antibody fragment to prevent binding of the anti-Met antibody fragment thereto. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The invention will now be described in detail by way of illustrative and non-limiting examples only and with reference to the accompanying drawings, in which:

[0019] Figure 1 : Schematic diagram of DN30 antibody and its monovalent derivatives. (A) Original murine bivalent mAb; (B) MvDN30, chimeric Fab; (C) chOA-DN30, chimeric monovalent "one-armed" antibody; (D) hOA-DN30, humanized monovalent "one-armed" antibody; (E) OA-DN30 antibody was subjected to SDS-PAGE under reducing and non-reducing conditions. The gel was stained with Gel Code blue.

[0020] Figure 2 : Binding of monovalent DN30-derivative molecules to Met. (A) ELISA binding analysis of MvDN30, chOA-DN30, hOA-DN30 antibodies (liquid phase) and human Met-Fc chimera (solid phase). (B) ELISA binding analysis of hOA-DN30-cl03E08 (liquid phase) and Met-Fc chimera (solid phase); Met from human, mouse, rat and monkey sources were included in the assay. Binding was revealed using anti-human k chain antibodies. OD: optical density; AU: arbitrary units. Each point is the average of three replicates; bars represent standard deviations. The table below the graph reports the Kd values, binding maximum (Bmax) and fit score (R 2 (C) Flow cytometric analysis of hOA-DN30-cl03E08 binding to Met expressed at the surface of a panel of cell lines derived from different species (EBC-1, human lung cancer; C2C12 mouse muscle myoblasts; H9C2 rat cardiac myoblasts; MDCK dog kidney cells; Cos-7 monkey kidney cells). The new molecule binds to human Met-Fc with high affinity; the antibody cross-reacts with human, rat, dog and monkey Met.

[0021] Figure 3 : Agonistic activity of monovalent DN30-derived molecules. Dispersion assay. HPAF-II cells were incubated with indicated concentrations of HGF (Met ligand), DN30 mAb (partial agonist), MvDN30 (full antagonist) and chimeric or humanized one-armed antibodies for 24 hours. All DN30-derived monovalent molecules did not induce cell scattering, a Met-mediated biological response.

[0022] Figure 4 : Shedding and downregulation of Met in cells treated with monovalent DN30-derived molecules. A549 cells were incubated for 48 hours in serum-free medium with the specified increasing concentrations of MvDN30, chOA-DN30 or hOA-DN30 derivatives. Total Met levels were determined by Western blot analysis of cell extracts using anti-Met antibodies. The detected bands correspond to the mature form of the receptor (p145 Met). As a loading control, the filter membrane was probed with an irrelevant protein (vinculin). Shedding of Met was determined by Western blot analysis of conditioned medium using anti-Met antibodies. The detected bands correspond to the shed extracellular domain of the receptor (p80 Met extracellular domain). The table reports the pixel intensity quantification of each western blot band normalized to the corresponding vinculin signal, as measured by densitometry using ImageJ software. All monovalent one-armed antibodies downregulate Met and induce Met shedding.

[0023] Figure 5 : Inhibition of Met activation by monovalent DN30-derived molecules. GTL-16 (MET-addicted cells carrying constitutively active Met due to receptor overexpression maintained by MET gene amplification) were incubated in serum-free medium with hOA-DN30 or chOA-DN30 antibodies (1000 or 250 nM) for 24 h. 1234 / 1235 Met activation in total cell lysates was determined by Western blotting with an anti-Met antibody specific for Met residues (the major phosphorylation site). The same blot was probed again with an anti-Met antibody. To control for protein loading, the filter was also probed with an anti-vinculin antibody. Chimeric and humanized one-armed antibodies inhibited Met phosphorylation.

[0024] Figure 6 : Viability of MET-addicted cells treated with monovalent DN30-derived molecules. GTL-16 human gastric cancer cells were seeded in 10% FCS medium in 96-well plates. After 24 h, cells were treated with increasing concentrations of MvDN30, chOA-DN30 or hOA-DN30 antibodies for another 72 h. Cell number was evaluated by Cell titer-glo. The graph presents the percentage of viable cells relative to untreated controls. Each point is the average of three replicates. The table reports the calculated IC 50 Values ​​and fit scores (R 2 ). The one-armed molecule inhibits cell growth in MET-addicted cells.

[0025] Figure 7: Proliferation and death of MET-addicted cells induced by treatment with monovalent DN30-derived molecules. (A) Proliferation assay: GTL-16 human gastric cancer cells were seeded in 10% FCS medium in 6-well plates. After 24 hours, the cells were treated with a fixed concentration (1 μM) of MvDN30, chOA-DN30 or hOA-DN30-cl03E08 for another 48 hours. EdU (10nμM) was then added to the culture medium for another 2 hours. The percentage of S-phase cells was determined by cytofluorimetry analysis according to the procedure of the ClicK-iT EdU flow cytometry assay. The graph presents cells that are negative and positive for APC staining (EdU+, S-phase cells). The table reports the percentage of S-phase cells. (B) Cytotoxicity assay: GTL-16 human gastric cancer cells were seeded in 10% FCS medium in 96-well plates. After 24 hours, cells were treated with increasing concentrations of MvDN30, chOA-DN30 or hOA-DN30-cl03E08 for an additional 48 hours. Cytotoxicity was evaluated by Cell-Tox Green assay. The graph presents the increase in cytotoxicity relative to untreated controls. Each point is the average of three replicates. The table reports the EC 50 Calculated values ​​and fitting scores (R 2 ). The one-armed molecule effectively blocks proliferation and induces cytotoxicity in MET-addicted cells.

[0026] Figure 8 : HGF-induced cell motility in cells treated with hOA-DN30-cl03E08. HPAF-II cells were treated with the indicated concentrations of hOA-DN30-cl03E08 alone or in combination with DecoyMet K842E In combination (same amount as hOA-DN30-cl03E08) pre-treated for 18 hours, followed by incubation with HGF (6.25 ng / ml) for 24 hours). hOA-DN30-cl03E08 inhibited HGF-induced cell motility; K842E The combination is more effective against cellular responses.

[0027] Fig. 9 : Invasion of cells treated with hOA-DN30-cl03E08. HPAF-II cells were seeded in the upper chamber of a Transwell filter coated with matrigel in serum-free medium containing 0.5 μM hOA-DN30-cl03E08 alone or with 1 μM DecoyMet K842EThe combination of hOA-DN30-cl03E08 and DecoyMet K842E The combination blocks the biological response.

[0028] Fig.10 : Modulation of PD-L1 expression by hOA-DN30-cl03E08. GTL-16 cells were treated with 50 ng / ml IFN-γ alone or in combination with 250 nM hOA-DN30-cl03E08. After 48 hours, cells were lysed; PD-L1 expression and Met activation on total protein extracts were evaluated by Western blotting. As a loading control, filters were probed with anti-GAPDH antibody.

[0029] Fig.11 : In vivo pharmacokinetic profile of MvDN30, DN30 mAb and hOA-DN30-cl03E08. Immunodeficient mice were injected intravenously with a single dose (100 μg) of MvDN30, DN30 mAb or hOA-DN30. Peripheral blood was collected at different time points. Serum concentrations of therapeutic molecules were measured by ELISA. The graph presents the amount of circulating molecules versus time. Samples were performed in triplicate; bars indicate standard deviation.

[0030] Fig.12 : Inhibition of MET-addicted tumor growth in vivo by treatment with monovalent DN30 derivatives. Immunodeficient NOD-SCID mice were inoculated with 1×10 6 GTL-16MET addicted gastric cancer cells. When the average tumor volume reached 68.4±5.4mm 3 Mice were randomly divided into 4 uniform groups and treated with 30 mg / kg of MvDN30, chOA-DN30 or hOA-DN30-cl03E08 by intravenous injection twice a week. As a control, one group was treated with the same volume of PBS (vehicle). Tumor growth was monitored regularly with calipers. After 15 days, mice were sacrificed. (A) Kinetics of tumor growth; (B) Tumor volume reported as a percentage of the untreated group at the end of the experiment. hOA-DN30-cl03E08 inhibited MET-addicted tumor growth more effectively than chOA-DN30.

[0031] Fig.13 : Dose-response inhibition of MET-addicted tumor growth in vivo by hOA-DN30-cl03E08 treatment. Immunodeficient NOD-SCID mice were inoculated with 1×10 6GTL-16MET addicted gastric cancer cells. After 8 days, the average tumor volume reached 80.7±2mm 3 Mice were randomly divided into 5 uniform groups and treated with increasing concentrations of hOA-DN30-cl03E08 by intravenous injection. As a control, one group was treated with the same volume of PBS (vehicle). Tumor growth was monitored regularly with a caliper. After 17 days, mice were sacrificed, and tumors were excised and weighed. (A) Kinetics of tumor growth; (B) Tumor weight. hOA-DN30-cl03E08 inhibited the growth of MET-addicted tumors with a dose-response characteristic.

[0032] Fig.14 : Amino acid sequences of the VL, VH, CL and CH1 domains of chOA-DN30 and hOA-DN30-cl03E08 antibodies. The complementary determining region sequences are highlighted with bold, underlined characters.

[0033] Fig.15 : Amino acid sequences of the first and second Fc polypeptides of OA-DN30. Amino acid mutations within the CH3 domains of the first and second Fc polypeptides - necessary to generate the knob and hole, respectively - are highlighted with bold, underlined characters.

[0034] Fig.16 : Amino acid sequence of a mutant form of DecoyMet. The amino acid mutation K842E is highlighted in bold, underlined characters. DETAILED DESCRIPTION

[0035] In the following description, numerous specific details are given to provide a thorough understanding of the embodiments. The embodiments may be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other cases, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the embodiments.

[0036] Throughout this specification, references to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in conjunction with the embodiment is included in at least one embodiment. Thus, the phrases "in one embodiment" or "in an embodiment" appearing in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0037] The headings provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

[0038] The present disclosure relates to novel therapeutic agents for treating tumors and / or metastases.

[0039] It is estimated that more than 200,000 patients are "MET-addicted" each year and Met inhibition could potentially lead to remission of the disease. Given that mutations accumulate with age and the elderly population will increase in the next 20 years, the burden of cancer is expected to increase and will have a major impact on patient management and healthcare resources worldwide. Genetic alterations leading to "MET addiction" have been found in gastric cancer, oesophageal cancer, colorectal cancer, kidney cancer, lung cancer, melanoma and brain tumors (see COSMIC database: www.sanger.ac.uk). In addition, a number of MET genetic lesions have been found to be an acquired mechanism of resistance to a number of other targeted therapies in colorectal cancer and non-small cell lung cancer (NSCLC). 9 .

[0040] The role of Met in metastasis is also related to its ability to help cells adapt to a hostile environment. Met-driven metastatic capacity depends not only on genetic and epigenetic changes, but also on HGF paracrine secretion by tumor stroma tissue, which is composed of a variety of cell types, including fibroblasts, resident epithelial cells, pericytes, myofibroblasts, vascular and lymphatic endothelial cells, and infiltrating cells of the immune system.

[0041] Met has now been identified as a cancer-specific target to: (i) personalize the treatment of tumors with MET mutation / amplification (“MET addiction”); (ii) prevent / reverse Met-driven primary and secondary resistance to other targeted cancer therapies; (iii) prevent / reverse Met-driven invasive / metastatic phenotypes 1 .

[0042] The inhibitory activity of DN30 depends on its ability to promote the physiological activity of the metalloproteinase ADAM-10 acting at the cell surface 10 , which releases the intact extracellular portion of the receptor from the plasma membrane (Met shedding), followed by rapid degradation of the Met intracellular kinase domain 11 As a result of these processes, Met is physically removed from the cell surface, while the extracellular domain acts as a soluble "decoy" that prevents ligand binding by sequestering HGF. Thus, DN30 has the advantage of inhibiting both HGF-dependent and HGF-independent activation of Met. 12 .

[0043] Compared to other anti-Met antibodies, DN30 blocks receptor function through a unique mechanism that results in distinct and synergistic effects: (i) removal of Met from the cell surface by "shedding" of the ectodomain; (ii) sequestration of HGF ligands; (iii) inhibition of homo- or heterodimerization of Met receptors at the membrane; and (iv) stimulation of receptor degradation. Thus, DN30 offers the opportunity to kill two birds with one stone, allowing for optimal blockade of the Met pathway as it can potentially act not only on cancer cells but also on the tumor microenvironment (HGF-driven biological responses of endothelial cells, fibroblasts, and macrophages).

[0044] The present invention is based on the idea that in order to achieve the highest therapeutic potential against tumors with the help of anti-Met antibodies, it is necessary to use a molecule that: i) does not cause, not even partially cause, the intracellular response induced upon Met activation, i.e. is not an agonist nor a partial agonist; ii) attenuates Met activation by a mechanism of action other than ligand (HGF) displacement; iii) has a favorable pharmacokinetic profile suitable for clinical use; iv) reasonably does not generate an immune response in humans. To meet all the above considerations, the inventors converted the murine DN30 antibody - which blocks Met activation by a shedding mechanism - into a fully humanized monovalent and highly stable molecule, hereinafter named "hOA-DN30", by converting it into a "one-armed" format.

[0045] In one embodiment, the present invention relates to an anti-Met antibody fragment comprising a single antigen binding arm and an Fc region, wherein the Fc region comprises a complex of a first and a second Fc polypeptide. The antibody fragment comprises, preferably consists of:

[0046] (i) a first polypeptide comprising, preferably consisting of: a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized light chain variable (VL) domain is fused to the human light chain constant (CL) domain in the N- to C-terminal direction, wherein the humanized light chain variable (VL) domain contains three complementarity determining regions (CDRs) having the amino acid sequences set forth in SEQ ID Nos.: 1, 2 and 3, and wherein the humanized light chain variable (VL) domain has the amino acid sequence set forth in SEQ ID No.: 7;

[0047] (ii) a second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and a first Fc polypeptide, wherein the first Fc polypeptide comprises a human hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein in the N- to C-terminal direction, the humanized heavy chain variable (VH) domain is fused to the human heavy chain constant CH1 domain, the human heavy chain constant CH1 domain is fused to the human hinge region, the human hinge region is fused to the human constant CH2 domain, and the human constant CH2 domain is fused to the human constant CH3 domain, and wherein the humanized heavy chain variable (VH) domain contains three complementarity determining regions (CDRs) having the amino acid sequences set forth in SEQ ID Nos.: 4, 5, and 6, and wherein the humanized heavy chain variable (VH) domain has the amino acid sequence set forth in SEQ ID No.: 8;

[0048] (iii) a third polypeptide comprising, preferably consisting of: a second human Fc polypeptide, wherein the second human Fc polypeptide comprises, preferably consisting of: a human hinge region, a human constant CH2 domain and a human constant CH3 domain, wherein in the N- to C-terminal direction, the hinge region is fused to the human CH2 constant domain, and the human CH2 constant domain is fused to the human CH3 constant domain, wherein the hinge region is truncated at the N-terminus.

[0049] In one embodiment, the human light chain constant (CL) domain is a human light kappa domain.

[0050] In one embodiment, the human hinge region and human constant domains CH1, CH2 and CH3 are from human IgG1.

[0051] In one embodiment, the two Fc polypeptides are linked by an intermolecular disulfide bond at the hinge region.

[0052] In one embodiment, the first Fc polypeptide and the second Fc polypeptide meet at an interface, and one of the first and second Fc polypeptides comprises a knob at the interface and the other of the first and second Fc polypeptides comprises a hole at the interface, wherein the knob is positionable into the hole.

[0053] In a preferred embodiment, one of the first and second Fc polypeptides comprises a mutated CH3 constant domain, wherein the mutated CH3 constant domain carries an amino acid mutation at position 389, wherein the original amino acid at position 389 has been mutated to introduce an amino acid having a larger side chain volume than the original amino acid, thereby creating a knob at the interface between the Fc polypeptides; and wherein the other of the first and second Fc polypeptides comprises a mutated CH3 constant domain, wherein the mutated CH3 constant domain carries three amino acid mutations at positions 389, 391 and 438, wherein the original amino acids have been mutated to introduce amino acids having a smaller side chain volume than the original amino acids, thereby creating a hole at the interface between the Fc polypeptides, wherein the knob can be positioned in the hole, and wherein the amino acid numbering is according to the EU numbering scheme of Kabat (Sequences of proteins of immunological interest, 5th edition, Vol. 1, 1991 (NIH, Bethesda, Maryland, pages 688-696).

[0054] In a still preferred embodiment, the original amino acids at positions 389, 391 and 438 are threonine, leucine and tyrosine, respectively; and wherein in one of the first and second Fc polypeptides, the threonine at position 389 has been mutated to tryptophan; and wherein in the other of the first and second Fc polypeptides, the threonine at position 389 has been mutated to serine, the leucine at position 391 has been mutated to alanine and the tyrosine at position 438 has been mutated to valine.

[0055] In a preferred embodiment, the human light chain constant (CL) domain has the amino acid sequence set forth in SEQ ID No.:9 and the human heavy chain constant CH1 domain has the amino acid sequence set forth in SEQ ID No.:10.

[0056] In a preferred embodiment, the first human Fc polypeptide has the amino acid sequence set forth in SEQ ID No.:11, and the second human Fc polypeptide has the amino acid sequence set forth in SEQ ID No.:12.

[0057] In one embodiment, the invention relates to an isolated nucleic acid encoding an anti-Met antibody fragment as disclosed above.

[0058] In one embodiment, the invention relates to a composition comprising two or more recombinant nucleic acids which together encode an anti-Met antibody fragment as disclosed above.

[0059] When bound to Met, the anti-Met antibody fragment disclosed herein will induce the shedding of the extracellular domain of Met. When bound to Met, the anti-Met antibody fragment does not exert agonistic activity on Met. When bound to Met, the anti-Met antibody fragment will inhibit Met phosphorylation. When bound to Met, the anti-Met antibody fragment will further block proliferation in MET-addicted cells and induce cytotoxicity. When bound to Met, the anti-Met antibody fragment will further inhibit cell motility and cell invasion induced by hepatocyte growth factor (HGF). The aforementioned properties of the anti-Met antibody fragments for the purpose of this specification provide the antibody fragments with anti-tumor and / or anti-metastatic activity.

[0060] As used herein, the expression "antigen binding arm" refers to the component of the antibody fragment of the present invention that has the ability to specifically bind to the target molecule of interest. The antigen binding arm is a complex of the variable domain sequences (VL and VH) of the immunoglobulin light chain and heavy chain (including CDR and framework regions) and the constant domain sequences (CL and CH) of the immunoglobulin light chain and heavy chain.

[0061] "Humanized" forms of non-human (e.g., mouse) antibodies are non-natural recombinant antibodies that contain very few amino acid residues derived from non-human immunoglobulins. For the most part, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the hypervariable regions (i.e., complementary determining regions—CDRs) of the receptor are replaced with residues from hypervariable regions of non-human species (donor antibodies) having the desired specificity, affinity, and properties. In some cases, framework region (FR) residues of human immunoglobulins may be replaced with corresponding non-human residues.

[0062] In addition, humanized antibodies may contain residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance. Generally, humanized antibodies contain two variable domains in which all or substantially all of the hypervariable loops correspond to those of non-human immunoglobulins, and all or substantially all of the framework residues (FR) are those of human immunoglobulin sequences. Humanized antibodies also contain a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details, see 13-15 . See also the following review articles and references cited therein: 16-18 .

[0063] As used herein, the term "Fc region" generally refers to a dimer complex comprising a C-terminal polypeptide sequence of an immunoglobulin heavy chain, wherein the C-terminal polypeptide sequence is a polypeptide sequence that can be obtained by papain digestion of an intact antibody. The Fc region may comprise a natural or variant Fc sequence. Although the boundaries of the Fc sequence of an immunoglobulin heavy chain may be different, according to the EU numbering scheme of Kabat, the human IgG1 heavy chain Fc sequence is generally defined as extending from the amino acid residues included between Asp 234 and Thr 238 to the carboxyl terminus of the Fc sequence. The Fc sequence of an immunoglobulin generally comprises a hinge region, two constant domains: a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. In this article, "Fc polypeptide" refers to one of the polypeptides that constitute the Fc region. The Fc region increases the stability of the antibody fragment compared to the Fab molecule comprising the antigen binding arm.

[0064] As used herein, "hinge region", "hinge sequence" and variations thereof have the meanings known in the art, which are described in, for example, Janeway et al., Immuno Biology: the immune system in health and disease (Elsevier Science Ltd., NY) (4th edition, 1999) and 19,20 There are instructions in .

[0065] As used herein, the phrase "truncated hinge region" refers to a polypeptide comprising part but not all of the hinge sequence. The truncated hinge region is capable of connecting to the "first" Fc polypeptide. If the wild-type hinge sequence is not present, the remaining sequence in the "second" Fc polypeptide will contain components that are capable of connecting to the "first" Fc polypeptide. For example, the components may be modified residues or additional cysteine ​​residues that are capable of forming disulfide bonds.

[0066] "Knob" refers to at least one amino acid side chain that protrudes from the interface of a first Fc polypeptide and can therefore be positioned in a complementary hole in an adjacent interface (i.e., the interface of a second Fc polypeptide) to stabilize the heteromultimer, thereby favoring the formation of the heteromultimer over the formation of a homomultimer. The knob may be present in the original interface or may be introduced synthetically (e.g., by altering the nucleic acid encoding the interface). Typically, the nucleic acid encoding the interface of the first polypeptide is altered to encode the knob. To do this, the nucleic acid encoding at least one "original" amino acid residue in the interface of the first polypeptide is replaced with a nucleic acid encoding at least one "imported" amino acid residue having a larger side chain volume than the original amino acid residue. It will be understood that there may be more than one original and corresponding imported residue. The number of original residues replaced is capped at the total number of residues in the interface of the first polypeptide.

[0067] "Hole" refers to at least one amino acid side chain that is recessed from the interface of a second Fc polypeptide and thereby accommodates a corresponding knob on the interface of an adjacent first Fc polypeptide. The hole may be present in the original interface or may be introduced synthetically (e.g., by altering the nucleic acid encoding the interface). Typically, the nucleic acid encoding the interface of the second polypeptide is altered to encode the hole. To this end, the nucleic acid encoding at least one "original" amino acid residue in the interface of the second polypeptide is replaced with a nucleic acid encoding at least one "imported" amino acid residue having a smaller side chain volume than the original amino acid residue. It should be understood that there may be more than one original and corresponding imported residue. The number of original residues replaced is upper bounded by the total number of residues in the interface of the second polypeptide.

[0068] The knob is "positionable" into the mortar, meaning that the spatial locations of the knob and mortar, respectively, at the interface of the first Fc polypeptide and the second Fc polypeptide and the dimensions of the knob and mortar are such that the knob can be positioned in the mortar without significantly interfering with the normal association of the first and second polypeptides at the interface. Since the knob generally does not extend perpendicularly from the axis of the interface and has a preferred conformation, the alignment of the knob with the corresponding mortar relies on modeling the knob / mortar pair based on a three-dimensional structure, such as that obtained by X-ray crystallography or nuclear magnetic resonance (NMR). This can be accomplished using techniques generally recognized in the art.

[0069] As used herein, the expression "non-agonist activity" of an anti-Met antibody fragment refers to an anti-Met antibody fragment that does not exert any activity that causes, even partially causes, a cellular response induced upon Met activation. The non-agonist activity of an anti-Met antibody fragment can be measured by conventional techniques such as Western blotting, immunofluorescence, immunohistochemistry, ELISA, cytofluorimetric analysis, or any other method by evaluating the level of phosphorylated Met, including the use of a protein that will specifically recognize Met when cells are incubated with the non-agonist antibody, or will specifically recognize Met residue Tyr if phosphorylated. 1234 -1235 (i.e., the major phosphorylation site of Met 21 ) or if phosphorylated will specifically recognize Met residues Tyr 1349 / 1356 (i.e., the docking site of Met 22 As an alternative, an assessment of the biological response of the cells upon antibody treatment compared to the response induced by HGF treatment can be used.

[0070] The present invention also relates to a product comprising, in a single bottle or in two bottles, (a) an anti-Met antibody fragment as disclosed herein and a pharmaceutically acceptable vehicle, and (b) an extracellular portion of human Met and a pharmaceutically acceptable vehicle, wherein the extracellular portion of human Met is capable of binding to hepatocyte growth factor (HGF) in a stable manner and contains at least one amino acid mutation within the epitope recognized by the anti-Met antibody fragment to prevent binding of the anti-Met antibody fragment thereto.

[0071] In one embodiment, the extracellular portion of human Met contains SEMA, PSI, IPT-1, IPT-2, IPT-3 and IPT-4 domains.

[0072] In a preferred embodiment, the extracellular portion of human Met has an amino acid sequence as set forth in SEQ ID No.: 13, wherein at least one amino acid between position 797 and position 875 of SEQ ID No.: 13 is mutated to prevent binding of anti-Met antibody fragments thereto.

[0073] In a more preferred embodiment, the extracellular portion of human Met has the amino acid sequence set forth in SEQ ID No.:14.

[0074] The present specification also relates to a nucleic acid molecule encoding the extracellular portion of human Met, which extracellular portion can bind to hepatocyte growth factor (HGF) in a stable manner and contains at least one amino acid mutation in the epitope recognized by the anti-Met antibody fragment to prevent the anti-Met antibody fragment from binding thereto, preferably the extracellular portion of human Met has the amino acid sequence set forth in SEQ ID No.: 14.

[0075] As used herein, the expression "the extracellular portion of human Met is capable of binding to human HGF in a stable manner" means that the extracellular portion of human Met binds to HGF with a calculated Kd of no higher than 100 nM.

[0076] As used herein, the expression "the extracellular portion of human Met contains at least one amino acid mutation at the epitope recognized by the anti-Met antibody fragment" means that there are one or more mutations in the extracellular portion of Met that are capable of inducing modifications in the extracellular portion of Met (i.e., amino acid substitutions and / or deletions and / or insertions) that will prevent the above-mentioned region from being occupied by the anti-Met antibody variable domain. Given its common knowledge (represented by the possibility of generating cDNA containing a single nucleotide change in a given DNA sequence using specific primers during DNA replication; see Maniatis T. Molecular cloning: A laboratory manual Cold Spring Harbor Laboratory, 1982), the skilled person does not need further details on achieving a mutant form of the extracellular portion of human Met that retains the ability to bind to human HGF but does not bind to the anti-Met antibody fragment. Therefore, the present invention should by no means be understood as covering only the mutated extracellular portion of human Met as disclosed herein (i.e., SEQ ID No.: 14), because a person skilled in the art can obtain other mutated forms of the extracellular portion of human Met having SEQ ID No.: 13 that will prevent the binding of anti-Met antibodies thereto according to common general knowledge.

[0077] The terms "SEMA", "PSI", "IPT-1", "IPT-2", "IPT-3" and "IPT-4" refer to Met domains that constitute the extracellular region of Met. Such domain names are common knowledge to those skilled in the art, such as those given by 23,24 The SEMA domain is a protein interaction module shared with semaphorins and plexins, covering the region between amino acids 25-516 of Met (SEQ ID No.: 13); PSI is a domain shared with plexins, semaphorins, and integrins, covering the region between amino acids 519-561 of Met (SEQ ID No.: 13); the IPT domain—repeated four times—is an immunoglobulin-like region shared with plexins and transcription factors, covering the region between amino acids 563-934 of MET (SEQ ID No.: 13). In detail, IPT repeat 1 covers amino acid positions 563-656 of SEQ ID No.: 13, IPT repeat 2 covers amino acid positions 657-740 of SEQ ID No.: 13, IPT repeat 3 covers amino acid positions 741-837 of SEQ ID No.: 13, and IPT repeat 4 covers amino acid positions 838-934 of SEQ ID No.: 13.

[0078] The present invention also relates to the therapeutic use of an anti-Met antibody fragment as disclosed herein, optionally in combination with an extracellular portion of human Met as disclosed herein, for treating a patient suffering from a tumor and / or metastasis, wherein said patient carries a genetic alteration of the MET gene.

[0079] The present invention also relates to the therapeutic use of an anti-Met antibody fragment as disclosed herein in combination with an extracellular portion of human Met as disclosed herein for treating a patient suffering from a tumor and / or metastasis, wherein said patient carries a wild-type MET gene.

[0080] In one aspect, the invention provides a method of treating a tumor and / or metastasis in a subject, the method comprising administering to the subject an effective amount of an anti-Met antibody fragment, optionally in combination with an extracellular portion of human Met, thereby treating the condition.

[0081] In one aspect, the invention provides a method of inhibiting the growth of a cell expressing Met, the method comprising contacting the cell with an anti-Met antibody fragment disclosed herein, optionally in combination with an extracellular portion of human Met of the invention, such that the growth of the cell is inhibited.

[0082] In one aspect, the invention provides a method of therapeutically treating a mammal having a cancerous tumor and / or metastasis, the method comprising administering to the mammal an effective amount of an anti-Met antibody fragment disclosed herein, optionally in combination with an extracellular portion of human Met, thereby effectively treating the mammal.

[0083] In one aspect, the present invention provides a method for treating a cell proliferative disorder, the method comprising administering to a subject in need of such treatment an effective amount of an anti-Met antibody fragment of the present disclosure, optionally in combination with an extracellular portion of human Met, thereby effectively treating or preventing the cell proliferative disorder.

[0084] In one aspect, the present invention provides a method for therapeutically treating a tumor and / or metastasis in a mammal, wherein the growth of the tumor is at least partially dependent on the growth enhancing effect of the Met / HGF system, which is the result of increased cell proliferation, protection from apoptosis, or both. The method comprises contacting tumor cells with an effective amount of an anti-Met antibody fragment, optionally in combination with an effective amount of an extracellular portion of human Met of the present invention, thereby effectively treating the tumor and / or metastasis.

[0085] Tumors that can be effectively treated with an anti-Met antibody, optionally in combination with an extracellular portion of human Met, are selected from breast cancer, colorectal cancer, lung cancer, colon cancer, pancreatic cancer, prostate cancer, ovarian cancer, cervical cancer, central nervous system cancer, kidney cancer, hepatocellular carcinoma, bladder cancer, gastric cancer, head and neck tumor cells, papillary carcinoma (e.g., thyroid), melanoma, lymphoma, myeloma, glioma / glioblastoma (e.g., anaplastic astroglioma, glioblastoma multiforme, anaplastic oligodendroglioma, anaplastic oligodendroglial astrocytoma), leukemia cells, sarcoma, rhabdomyosarcoma, or a tumor belonging to carcinoma of unknown primary (CUP).

[0086] In one embodiment, the cells therapeutically targeted in the methods of the invention are hyperproliferative and / or hyperplastic cells. In one embodiment, the cells targeted in the methods of the invention are abnormally proliferative cells. In yet another embodiment, the cells targeted in the methods of the invention are metastatic cells. In yet another embodiment, the cells targeted in the methods of the invention are Met-expressing cells that belong to the microenvironment that sustains tumors and / or metastases.

[0087] The present inventors demonstrated that a monovalent form of the antibody was generated by a one-arm structure obtained by selected point mutations of the CH3 domain and resulting in efficient Fc heterodimerization.

[0088] Monovalency is crucial to avoid the eventual agonistic activity of DN30 attributed to the bivalent native structure of the antibody.

[0089] Unexpectedly and surprisingly, the present inventors discovered that a humanized one-armed form of DN30 (ie, hOA-DN30-cl03E08) exhibited potent inhibitory properties both in vitro and in vivo.

[0090] hOA-DN30-cl03E08 showed superior activity in vitro compared to the corresponding chimeric form (hereinafter referred to as "chOA-DN30") and all other humanized one-armed derivatives disclosed herein (referred to as "hOA-DN30 antibodies") (see the "Materials and Methods" section for a detailed description of all one-armed antibodies derived from DN30). Unexpectedly, hOA-DN30-cl03E08 binds to Met with a higher affinity than other OA-DN30 chimeric or human antibodies tested. Unpredictably, the inventors found that the shedding reaction is enhanced by increasing the affinity of the DN30 antibody for Met. Therefore, the inhibitory effect on Met activation is stronger, as measured by the amount of phosphorylated Met receptors. These activities translate into inhibition of Met-mediated biological responses. Attenuation of the growth of MET-addicted tumor cells occurs at very low doses; hOA-DN30-cl03E08 IC 50All those ICs measured for OA-DN30 chimeric or human antibodies included in the analysis 50 The better inhibition of tumor cell growth may be due to a more extensive blockade of cell proliferation and / or a more pronounced increase in cytotoxicity.

[0091] hOA-DN30-cl03E08 also showed improved therapeutic potential in vivo. This is expected if compared with MvDN30, a molecule with high renal clearance due to its low molecular weight. The poor pharmacokinetic properties of MvDN30 would greatly affect the performance of this molecule in in vivo experimental models.

[0092] One might think that achieving a molecular weight above the renal clearance threshold would in itself be sufficient to elicit an optimal therapeutic response, but this is not the case. In fact, the results obtained with the DCD-1 and DCD-2 derivatives of the DN30 mAb—recombinant molecules with increased size relative to MvDN30—clearly demonstrated that the pharmacokinetic profile was only partially improved and that the therapeutic response was limited to a reduction in tumor growth rate. 8 .

[0093] The superior activity of hOA-DN30-cl03E08 may be due in part to the presence of the Fc region, which produces differences in the tissue / organ distribution of the antibody due to binding to Fc receptors, thereby providing a highly favorable pharmacokinetic profile.

[0094] Positive aspects related to in vivo stability due to the presence of the Fc region have been discussed and published in the application of onatuzumab. This monoclonal antibody is an anti-Met antibody designed in a one-arm format. 25 However, this antibody is produced in bacteria and is therefore not glycosylated. Therefore, it lacks the ability to activate antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). In contrast, hOA-DN30-cl03E08 is produced in mammalian cells and must therefore be considered fully capable of eliciting Fc effector functions.

[0095] hOA-DN30-cl03E08 and onatuzumab also have different mechanisms of action. The first antibody induces Met receptor shedding, wiping Met from the cell surface and absorbing HGF in the extracellular environment like a sponge, while the second antibody competes with HGF for Met binding. Therefore, hOA-DN30 inhibits Met independently of the mechanisms that maintain the abnormal activation of cancer receptors, while onatuzumab is only effective in the context of ligand-dependent activation.

[0096] The presence of the Fc region was not responsible for the significant improvement in the in vivo therapeutic response obtained with hOA-DN30-cl03E08, since this domain was present in all humanized and chimeric one-armed antibodies tested. What the humanized and chimeric one-armed antibodies do not share is the variable region, where the binding site of the antibody is located. In particular, the DN30 CDRs—which are identical in all derivatives—are inserted into the framework regions. These parts of the antibody, even if not directly involved in forming the binding pocket, include specific amino acids known as Vernier residues. 26 , these amino acids can affect the final conformation of the CDR structure and thus determine the final affinity of the antibody for the antigen. The combination of DN30 CDR and framework sequence (i.e., the sequence of the heavy chain and light chain variable domains of cl03E08) will produce an anti-Met antibody with stronger affinity, which is likely because the binding pocket generated by this sequence combination is more compatible with the corresponding epitope on the Met receptor. The more robust interaction between the antibody and Met further achieves receptor shedding and the subsequent weakening of Met-mediated biological responses. This, combined with the improved plasma stability due to the one-armed format, has produced an antibody with unexpectedly superior activity against MET-addicted / driven cancers - namely cl03E08.

[0097] Basilico et al. 27 The data reported in the present study show that simultaneous targeting of HGF and Met results in greater therapeutic robustness in the treatment of tumors and / or metastases. The experimental data presented herein unexpectedly show that, with the aid of hOA-DN30 and DecoyMet K842E The combination of MvDN30 and DecoyMet inhibited the MET / HGF axis better than K842E In fact, the results obtained in the invasion test (here in Fig. 9 reported in ) and Basilico et al. 27 found that if MvDN30 was part of the combination, hOA-DN30 would abrogate HGF-driven cancer cell invasion, while residual activity remained.

[0098] Cancer cells without MET genetic alterations use the "physiological" program triggered by the MET oncogene as an "expedient" to promote the malignant phenotype and release an aggressive metastatic phenotype in response to stress conditions such as hypoxia, ionizing radiation or chemotherapy. The "expedient" requires stimulation of wild-type MET by its ligand HGF. The data provided herein show that in the case of MET "expedient", simultaneous interventions that hit both sides of the MET / HGF axis will lead to improved inhibitory activity. hOA-DN30-cl03E08 induces the physical removal of MET from the cell surface by "shedding" of the extracellular domain. The latter is released in the extracellular environment and acts as a "bait" for HGF. The exogenous supply of recombinant DecoyMet will enhance the HGF chelating activity of endogenous DecoyMet generated by hOA-DN30-cl03E08. hOA-DN30-cl03E08 and DecoyMet K842E The combination of HER2 and HGF acts simultaneously on Met-expressing cancer cells and HGF-secreting tumor stroma. This allows for optimal blockade of HGF-driven Met signaling and is therefore a reliable treatment option for a large number of patients bearing tumors expressing wild-type MET that are dependent on MET to maintain an invasive / metastatic phenotype.

[0099] In view of the common general knowledge in the field related to recombinant DNA technology, as represented by Maniatis T. Molecular cloning: A laboratory manual Cold Spring Harbor Laboratory (1982), the skilled person can easily produce the proteins disclosed herein (i.e., humanized anti-Met antibodies and the extracellular portion of human Met) in the form of proteins or in the form of nucleic acid molecules encoding the proteins. For example, the following standard procedures can be followed: (i) synthesizing the corresponding cDNA sequence, (ii) inserting the cDNA into a plasmid suitable for expression in mammals by conventional recombinant DNA methods, (iii) transiently or stably co-transfecting with the above plasmids of mammalian cell lines, (iv) collecting the culture supernatant, (v) purifying by affinity chromatography of the recombinant protein.

[0100] Therapeutic compositions comprising one or more active ingredients of the invention, i.e., humanized anti-Met antibody fragments alone or in combination with the extracellular portion of human Met, can be prepared in the form of aqueous solutions, lyophilized or other dry preparations with physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A., ed., 1980). Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the doses and concentrations employed and include: buffers; antioxidants; preservatives; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinyl pyrrolidone; amino acids; monosaccharides, disaccharides and other carbohydrates; chelating agents; sugars; salt-forming counterions; metal complexes and / or nonionic surfactants. The formulations may also contain one or more other active compounds necessary for the particular indication being treated, preferably those having complementary activities that do not adversely affect the therapeutic activity of hOA-DN30 alone or in combination with the extracellular portion of human Met. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

[0101] The active ingredient or ingredients may also be entrapped in microcapsules prepared by the techniques disclosed in, for example, Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980).

[0102] Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the active ingredients of the present invention, which matrices are in the form of shaped articles, eg films, or microcapsules.

[0103] One or more active ingredients of the present invention may be used alone or in combination with another antibody, a small molecule tyrosine kinase inhibitor, one or more chemotherapeutic agents (including a mixture of chemotherapeutic agents), one or more other cytotoxic agents, one or more anti-angiogenic agents, cytokines and / or one or more growth inhibitors. Such combination therapies include combined administration (wherein two or more agents are included in the same or separate formulations) and separate administration, in which case the administration of hOA-DN30 may be performed before and / or after one or more adjuvant therapies.

[0104] One or more active components of the present invention (and one or more adjunctive therapeutic agents) are applied by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary and intranasal, and if local treatment is required, intralesional application is possible. One or more active components of the present invention can be applied suitably by pulse infusion, particularly with a decreasing dose of active component. Administration can be carried out by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether application is transient or long-term.

[0105] One or more active ingredients of the present invention will be formulated, dosed and applied in a manner consistent with good medical practice. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the condition, the delivery site of the medicament, the method of application, the time arrangement of application and other factors known to practitioners. One or more active ingredients of the present invention need not but may optionally be formulated with one or more medicaments currently used to prevent or treat the condition in question. The effective amount of such other medicaments depends on the amount of one or more active ingredients of the present invention present in the preparation, the type of condition or treatment and other factors discussed above. These are usually used with the same dosage and route of administration as used above, or with about 1 to 99% of the dosage adopted so far.

[0106] For the treatment of disease, the appropriate dosage of one or more active ingredients of the present invention (when used alone or in combination with one or more other agents, such as one or more chemotherapeutic agents) will depend on the type of disease to be treated, the severity and course of the disease, whether the active ingredient is administered for preventive or therapeutic purposes, the patient's clinical history and response to the active ingredients of the present invention should all be appropriately considered and determined by the attending physician.

[0107] hOA-DN30 is suitable for administration to patients once or through a series of treatments. Depending on the type and severity of the disease, an antibody of about 1 mg / kg to 30 mg / kg is an initial candidate dose for administration to a patient, whether, for example, by one or more separate administrations or by continuous infusion. Depending on the above factors, a typical daily dose may be in the range of about 1 μg / kg to 100 mg / kg or more. For repeated administration for several days or longer, depending on the condition, treatment is continued until the desired disease symptom suppression occurs. An exemplary dose of the antibody fragment is in the range of about 0.05 mg / kg to about 20 mg / kg. Therefore, one or more doses (or any combination thereof) of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg may be administered to the patient. Such a dose may be administered intermittently, for example, weekly or every three weeks (e.g., so that the patient receives about two to about twenty doses of the antibody, such as about six doses of the antibody). An initial higher loading dose may be administered, followed by one or more lower doses. An exemplary dosing regimen includes administering an initial loading dose of about 4 mg / kg, followed by weekly maintenance doses of about 2 mg / kg of the antibody. However, other dosing regimens may be useful. The progress of the therapy is easily monitored by conventional techniques and assays. With regard to combination therapy, the extracellular portion of human Met is preferably delivered at the same time as hOA-DN30 and is administered in proportion to hOA-DN30, preferably but not limited to a molar ratio of 1:1 hOA-DN30:DecoyMet K842E , depending on the type and severity of the disease.

[0108] result

[0109] Generation of chimeric and humanized one-armed forms of the DN30 antibody.

[0110] By classical molecular biology techniques, the present inventors replaced the constant domains of the heavy and light chains of DN30 mAb with constant domains derived from human immunoglobulins. The light chain constant domain was replaced with a human κ-type domain (a more representative domain in natural human antibodies), while the heavy chain constant domain was replaced with a homologous domain derived from human IgG1 (a domain that can induce ADCC / CDC).

[0111] Fair Journey Biologics has achieved humanization of the DN30 mouse antibody using a phage display library approach. They started with the identification of deviating FR (framework) residues and analysis of the nearest human V germline, which was selected from germlines with the same canonical fold combination for CDR1-CDR2, using available tools and databases: abYsistool from Dr. Andrew Martin of UCL plus http: / / www.bioc.uzh.ch / plueckthun / antibody / and http: / / www2.mrc-lmb.cam.ac.uk / vbase / . In generating the phage display library, in which randomization of the VH and VL regions had been performed, four rounds of affinity-driven phage display screening were completed. At the end of this process, the DN30 humanized variants with the highest human identity and homology were selected. The variable regions were connected to constant domain sequences derived from human immunoglobulins (i.e., human kappa light chain and IgG1 heavy chain constant domains) by classical molecular biology techniques.

[0112] To make chimeric and humanized antibodies into one-armed single-chain formats, specific amino acid modifications have been inserted into the CH3 region to create a 'knob-in-hole' structure 28,29 ; One CH3 domain carries a knob mutation: 389 T→W, another CH3 domain carries a hole mutation: 389 T→S; 391 L→A; 438 Y→V. The cDNA encoding the chimeric or humanized light chain, chimeric or humanized heavy chain (mutated in the CH3 domain) and human Fc domain (mutated in the CH3 domain) is cloned into an expression plasmid and then expressed in eukaryotic cells. The assembled one-armed antibody is purified from the cell culture supernatant by affinity chromatography and gel filtration. Figure 1 Schematic diagram showing the SDS-Page separation of DN30 derivatives and purified recombinant OA-DN30 molecules under non-reducing and reducing conditions.

[0113] The DN30 one-armed antibody recognizes Met with high affinity.

[0114] Purified one-armed forms of chimeric or humanized DN30 antibodies were analyzed for binding to the Met receptor by ELISA assay. As a reference, chimeric DN30 Fab (MvDN30) was included in the analysis 12 The assay was assembled to include Met-Fc in the solid phase and the DN30 derivative in the liquid phase. Binding was revealed using an anti-human kappa chain antibody. The data showed that both chimeric and humanized antibodies bound to Met, with hOA-DN30-cl03E08 showing the highest affinity ( Figure 2A).

[0115] Analysis of the cross-reactivity of hOA-DN30-cl03E08 with non-human Met receptors.

[0116] The inventors analyzed the species cross-reactivity of hOA-DN30-cl03E08 by performing the following assays: (i) ELISA assay using purified Met extracellular domains from human, mouse, rat and monkey sources ( Figure 2 B); (ii) flow cytometric analysis of antibody binding to surface Met expressed by cells derived from humans, mice, rats, dogs and monkeys ( Figure 2 C). The results of the experiment showed that hOA-DN30-cl03E08 binds to Met from human, rat, dog and monkey, but has a very weak interaction with mouse Met.

[0117] The DN30 one-armed antibody did not exert agonistic properties.

[0118] The inventors tested whether the new DN30 derivatives would show Met agonist activity in a highly sensitive assay (dispersion assay). HPAF-II human pancreatic cancer cells (which represent a standard system for determining cell motility in response to HGF stimulation) were stimulated for 24 hours with increasing concentrations of the antibody. As positive controls, HGF and a bivalent DN30 mAb were included in the assay. While cells stimulated with the positive control clearly dispersed, the phenotype of cells treated with all monovalent DN30 derivatives (MvDN30, chOA-DN30, and hOA-DN30 antibodies) was indistinguishable from that of untreated cells ( Figure 3 ).

[0119] hOA-DN30 strongly induces Met "shedding".

[0120] The inventors also investigated whether hOA-DN30 retains the ability to promote receptor shedding and downregulation. A549 cells were incubated with increasing concentrations of chOA-DN30 and hOA-DN30 antibodies. As a reference, MvDN30 was included in the assay. After 48 hours, the Met ectodomain released in the conditioned medium was scored by immunoblotting using a monoclonal antibody against the extracellular portion of Met. The total cellular level of Met was also determined on cell lysates using the same antibody. This analysis revealed that all OA-DN30 antibodies induced Met shedding, resulting in the release of soluble Met ectodomain in the extracellular space and physical removal of Met from the cell surface ( Figure 4 ). Unexpectedly, the activity of hOA-DN30-cl03E08 was superior compared to all other DN30 derivatives included in the test.

[0121] hOA-DN30-cl03E08 strongly inhibited Met phosphorylation.

[0122] The present inventors investigated whether chOA-DN30 and hOA-DN30 antibodies inhibit Met phosphorylation in cells derived from human cancer (i.e., GTL-16). These cells have constitutively active Met as a result of overexpression of the receptor due to gene amplification. The activation of Met was determined by immunoblotting with anti-phosphoMet antibodies. Figure 5 As shown in , all molecules attenuated Met phosphorylation / activation in a dose-response pattern. hOA-DN30-cl03E08, which induced the strongest attenuation of Met levels in cells, was the more active DN30 derivative.

[0123] hOA-DN30-cl03E08 strongly inhibits the growth of MET-addicted cells.

[0124] Met inhibitors can only impair cell growth (viability) in MET-addicted cells, which rely on Met signaling for proliferation / survival. To test the activity of DN30 derivatives, exponentially growing GTL-16 cells (MET-addicted human gastric cancer cells) were incubated with increasing concentrations of chOA-DN30 or hOA-DN30 antibodies. MvDN30 was included in the assay as a positive control. After 72 hours, cell viability was determined using a luminescence-based ATP assay. All DN30 derivatives inhibited MET-addicted cell growth in a dose-dependent manner ( Figure 6 ). hOA-DN30-cl03E08 showed the highest inhibition, IC 50 2.5-fold lower than MvDN30, 5.4-fold lower than chOA-DN30, and at least 5.2-fold lower than other hOA-DN30 antibodies (see Figure 6 in the table).

[0125] hOA-DN30-cl03E08 was highly effective in blocking proliferation and inducing cytotoxicity in MET-addicted cells.

[0126] To further analyze the mechanism of the inhibitory effect of DN30 derivatives on the growth of MET-addicted cells, the inventors evaluated cell proliferation and cytotoxicity, comparing MvDN30, chOA-DN30 and hOA-DN30-cl03E08. GTL-16 cells were incubated with a single dose (1 μM) of DN30 derivatives and cell proliferation was assessed by measuring EdU (a thymidine analog) incorporated by cells in the S phase of the cell cycle using the ClickIT Edu flow cytometry assay. MvDN30 was included in the assay as a positive control. The analysis revealed that the DN30 derivatives affected proliferation compared to the control, as the percentage of treated cells in the S phase was drastically reduced. In particular, the most effective molecule was hOA-DN30-cl03E08, of which only 4.9% of the cells were still proliferating (a decrease of 84.2% compared to the control). The proliferating cells in the population treated with chOA-DN30 were 14.97% (a decrease of 52% compared to the control) ( Figure 7 A).

[0127] To test whether the DN30 derivatives only induce a blockade of proliferation or whether they also cause cytotoxicity, exponentially growing MET-addicted GTL-16 cells were incubated with increasing concentrations of chOA-DN30 or hOA-DN30-cl03E08. MvDN30 was included in the assay as a positive control. After 72 h, the cells were incubated with chOA-DN30 or hOA-DN30-cl03E08. TM Cytotoxicity was determined by expressing a green dye and measuring the fluorescence of the cells, which is proportional to the number of dead cells. All DN30 derivatives induced cytotoxicity in GTL-16 cells in a dose-dependent manner ( Figure 7 B). hOA-DN30-cl03E08 showed the highest induction, EC 50 These were 1.3- and 6.1-fold lower than those of MvDN30 and chOA-DN30, respectively.

[0128] hOA-DN30-cl03E08 inhibited HGF-induced cell motility alone and in combination with DecoyMet.

[0129] The inventors tested the ability of hOA-DN30-cl03E08 to inhibit HGF-dependent cell motility of HPAF-II human pancreatic cancer cells. Cells were incubated with or without HGF and treated with increasing doses of hOA-DN30-cl03E08. After 24 hours, cell colonies were stained and analyzed. In this assay, hOA-DN30-cl03E08 strongly reduced HGF-dependent cell scattering ( Figure 8 ).

[0130] In the above cell systems, it has been reported that HGF-induced biological responses can be more effectively inhibited by simultaneously blocking the ligand (HGF) and the receptor (Met) 27 Therefore, the inventors tested hOA-DN30-cl03E08 and DecoyMet K842E Does the combination induce a higher therapeutic response? K842E is a recombinant soluble receptor that encompasses the entire extracellular region of Met, carrying mutations that abolish interaction with DN30; it binds HGF with high affinity and inhibits ligand-driven bioactivity by absorbing and neutralizing HGF like a sponge and by forming heterodimers with intact Met receptors still present on the cell surface, thereby inactivating them. When both molecules were combined in equimolar amounts, the effective dose to completely reverse the scattered phenotype was 4-fold lower than the dose of hOA-DN30-cl03E08 alone that was able to induce a similar cellular phenotype ( Figure 8 ).

[0131] hOA-DN30-cl03E08 inhibited HGF-induced cell invasion alone and in combination with DecoyMet.

[0132] The inventors tested the ability of hOA-DN30-cl03E08 to inhibit HGF-dependent cell invasion of HPAF-II cells. Cells were seeded in serum-free medium containing hOA-DN30-cl03E08 alone (0.5 μM) or in combination with DecoyMetK842E (1 μM) in the upper chamber of a Transwell filter coated with matrigel. The lower chamber was filled with medium containing HGF (25 ng / ml). After 24 hours, cells that migrated to the lower portion of the filter were evaluated by staining and microscopic observation. In this assay, hOA-DN30-cl03E08 strongly reduced HGF-dependent cell invasion; hOA-DN30-cl03E08 and DecoyMetK842E had no significant effect on HGF-dependent cell invasion. K842E The combination almost eliminated the cellular response ( Fig. 9 ).

[0133] hOA-DN30-cl03E08 abolished interferon-γ induction of PD-L1 expression.

[0134] Programmed cell death ligand 1 (PD-L1) and programmed cell death receptor 1 (PD-1) are key regulatory molecules called immune checkpoints that play a central role at the interface between the immune response and the tumor microenvironment. 30 They can significantly impair the ability of the immune system to control tumor progression. Expression of PD-L1 by tumor cells is inducible and interferon gamma (IFNγ) is the most potent inducer 31 .

[0135] The present inventors analyzed whether inhibition of Met by hOA-DN30-cl03E08 could modulate the IFNγ pathway and thus PD-L1 regulation ( Fig.10 ). MET-addicted GTL-16 cells were treated with INFγ for 48 h and analyzed for IFNγ-inducible PD-L1 expression. Under unstimulated conditions, PD-L1 was undetectable, whereas upon exposure to IFNγ, it was consistently upregulated. Treatment with hOA-DN30-Cl03E08 for 48 h significantly attenuated the upregulation of PD-L1 induced by IFNγ.

[0136] hOA-DN30-cl03E08 showed favorable pharmacokinetic characteristics in vivo.

[0137] The present inventors investigated the pharmacokinetic properties of hOA-DN30-cl03E08 compared to MvDN30 and chimeric DN30 mAbs. A single dose of the above molecules was delivered to immunodeficient mice by intravenous injection. Peripheral blood from treated mice was collected at different time points after delivery. Circulating concentrations of the studied molecules were determined by ELISA performed on serum samples. The circulating levels of hOA-DN30-cl03E08 were comparable to those measured for mAbs and were always higher than those for MvDN30 ( Fig.11 ).

[0138] hOA-DN30-cl03E08 attenuates the growth of MET-addicted tumors in vivo

[0139] The ability of DN30 derivatives to inhibit tumor growth in vivo was tested in the MET addiction model. 1 × 10 6 GTL-16 cells. After 1 week, mice bearing palpable tumors were randomized into 4 uniform treatment groups: vehicle (n=6), MvDN30 (n=6), hOA-DN30-cl03E08 (n=5), chOA-DN30 (n=5). The antibody was delivered by intravenous injection at 30 mg / Kg twice a week; to administer the same amount of molecules, MvDN30 was delivered at 15 mg / kg on the same schedule because the molecular weight of the one-armed antibody is twice that of MvDN30 (i.e., the OA-DN30 form is approximately 100 KDa and MvDN30 is approximately 50 KDa). MvDN30 was ineffective—as expected from its short plasma half-life—while the one-armed derivatives all inhibited tumor growth ( Fig.12A) (P = 0.0007 and 0.05 for hOA-DN30-cl03E08 and chOA-DN30 relative to vehicle, respectively). Surprisingly, hOA-DN30-cl03E08 was much more effective than chOA-DN30: in the case of hOA-DN30-cl03E08, the treated mass was 87.8% smaller than the control, whereas in the case of chOA-DN30, the treated mass was 52.9% smaller than the control ( Fig.12 B) (P=0.009 for hOA-DN30-cl03E08 vs. chOA-DN30). The inventors further performed a dose response experiment to analyze the inhibitory activity of hOA-DN30-cl03E08. Maintaining the above experimental conditions, the mice were randomly divided into 5 uniform groups and treated with different doses of hOA-DN30-cl03E08 by intravenous injection three times a week. Doses of 10, 30 and 60 mg / kg were highly effective in blocking tumor growth, while the treatment of 3.3 mg / kg was not statistically different from the control group ( Fig.13 ).

[0140] Materials and methods

[0141] Cell culture

[0142] A549 human lung adenocarcinoma cells, HPAF-II human pancreatic carcinoma cells, C2C12 mouse muscle myoblasts, H9C2(2-1) rat cardiac myoblasts, MDCK dog kidney cells, and Cos-7 monkey kidney cells were obtained from ATCC / LGC Standards Sr1. (Sesto San Giovanni, Italy); the GTL-16 cell line is a clone derived from MKN-45 cells (human gastric carcinoma cells, available from the Japanese Collection of Research Bioresources, Osaka, Japan), and its MET gene copy number differs from that of the parental cell line 32 The GTL-16 cell line can be obtained from the Advanced Biotechnology Center (ABC) of the Interlab Cell Line Collection (ICLC) in Italy, with the accession number of ICLC PD 08003. The human lung cancer EBC-1 is from the Japanese Collection of Research Bioresources). The DN30 hybridoma can be obtained from the Advanced Biotechnology Center (ABC) of the Interlab Cell Line Collection (ICLC) in Italy, with the accession number of ICLC PD 05006.

[0143] All cells were cultured as recommended by the supplier. All cell cultures were tested for mycoplasma contamination.

[0144] Generation of chimeric and human one-armed forms of the DN30 antibody.

[0145] For chimeric DN30 antibodies :Three separate cDNAs encoding light chain (VL-CL), heavy chain (VH-CH1-cH2-CH3) and Fc domain (CH2-CH3) have been generated by gene synthesis. The variable region (mouse sequence) is from DN30 antibody (SEQ ID: 15 and 16); the constant region of human origin is derived from human immunoglobulin, in particular, the light chain includes a human κ type constant domain (GenBank sequence ID#: DI165992), and the heavy chain includes a human IgG1 constant domain (GenBank sequence ID#: DJ392898). At the C-terminal end of the coding sequence, the heavy chain includes His-TAG (SEQ ID: 38, 39) and Fc includes strep-TAG (SEQ ID: 40, 41).

[0146] For humanized DN30 antibody :Fair Journey Biologics has achieved humanization through germline screening of DN30 mouse antibody using phage display library method. They started with the identification of deviated FR (framework) residues and the analysis of the nearest human V germline, which was selected from germlines with the same canonical folding combination for CDR1-CDR2, using available tools and databases: abYsistool from Dr. Andrew Martin of UCL plus http: / / www.bioc.uzh.ch / plueckthun / antibody / and http: / / www2.mrc-lmb.cam.ac.uk / vbase / . In generating phage display libraries, where randomization of VH and VL regions had been performed, four rounds of affinity-driven phage display screening had been completed. At the end of this process, five different DN30 humanized variants with the highest human identity and homology were selected, i.e. clones 03E08, 03G05, 03F04, 03H08 and 04H08. The variable regions were linked to constant domain sequences derived from human immunoglobulins, ie, human kappa light chain and IgG1 heavy chain constant domains, by classical molecular biology techniques.

[0147] To make chimeric and humanized antibodies into one-armed formats (chOA-DN30 and hOA-DN30 antibodies) : Specific amino acid modifications have been inserted in the CH3 region to create a 'knob-in-hole' structure 28,29The “knob-in-hole” design strategy has been shown to be an effective design strategy to enhance heterodimer formation compared to homodimers. The strategy is based on domain interface remodeling and introduces spatially complementary mutations in the CH3 region of the antibody Fc fragment, which is the most extensive site of protein-protein interaction between the H chains of human IgG molecules. One chain, the VH-CH1-CH2-CH3 polypeptide, consists of 389 T→W (knob mutation), in which a small amino acid is replaced by a larger one; conversely, the other chain, the CH2-CH3 polypeptide, involves the replacement of a large residue by a smaller one: 389 T→S; 391 L→A; 438 Y→V (hole mutation). These mutations were generated by phage display technology. 33 The selected one after optimization, in which residues 389, 391 and 438 adjacent to the knob on the partner CH3 domain were randomly replaced. Knob-hole engineering facilitates the assembly of heterodimers, including three different amino acid chains (VL-CL+VH-CH1-CH2-CH3+CH2-CH3) instead of homodimers, i.e., true antibodies (VL-CL+VH-CH1-CH2-CH3) 2 or Fc domain only (CH2-CH3) 2 .

[0148] At the N-terminus, the CH2-CH3 polypeptide includes a sequence corresponding to a truncated human hinge region. At position 233 according to the EU numbering scheme of Kabat, a deletion is necessary to exclude a cysteine ​​involved in forming an interchain disulfide bond with a cysteine ​​at the C-terminal position of the light chain constant domain (CL). In addition, to allow the CH2-CH3 polypeptide to enter the RER / Golgi pathway of protein synthesis—just like the light chain (VL-CL) and heavy chain (VH-CH1-CH2-CH3) of antibodies—at the N-terminus, a sequence corresponding to the signal peptide of the DN30 heavy chain is included before the truncated hinge region.

[0149] The chimeric anti-Met antibody fragment (chOA-DN30) disclosed herein consists of:

[0150] (i) a light chain variable domain having the amino acid sequence set forth in SEQ ID No.: 15;

[0151] (ii) a heavy chain variable domain having the amino acid sequence set forth in SEQ ID No.: 16;

[0152] (iii) a human light chain constant (CL) domain having the amino acid sequence set forth in SEQ ID No.: 9;

[0153] (iv) a human heavy chain constant CH1 domain having the amino acid sequence set forth in SEQ ID No.: 10;

[0154] (v) a first human Fc polypeptide having the amino acid sequence set forth in SEQ ID No.: 11; and

[0155] (vi) a second human Fc polypeptide having the amino acid sequence set forth in SEQ ID No.: 12.

[0156] The humanized anti-Met antibody fragment (hOA-DN30-cl03E08) of the present disclosure consists of:

[0157] (i) a humanized light chain variable domain having the amino acid sequence set forth in SEQ ID No.: 7;

[0158] (ii) a humanized heavy chain variable domain having the amino acid sequence set forth in SEQ ID No.: 8;

[0159] (iii) a human light chain constant (CL) domain having the amino acid sequence set forth in SEQ ID No.: 9;

[0160] (iv) a human heavy chain constant CH1 domain having the amino acid sequence set forth in SEQ ID No.: 10;

[0161] (v) a first human Fc polypeptide having the amino acid sequence set forth in SEQ ID No.: 11; and

[0162] (vi) a second human Fc polypeptide having the amino acid sequence set forth in SEQ ID No.: 12.

[0163] The humanized anti-Met antibody fragment (hOA-DN30-cl03G05) consists of:

[0164] (i) a humanized light chain variable domain having the amino acid sequence set forth in SEQ ID No.: 17;

[0165] (ii) a humanized heavy chain variable domain having the amino acid sequence set forth in SEQ ID No.: 18;

[0166] (iii) a human light chain constant (CL) domain having the amino acid sequence set forth in SEQ ID No.: 9;

[0167] (iv) a human heavy chain constant CH1 domain having the amino acid sequence set forth in SEQ ID No.: 10;

[0168] (v) a first human Fc polypeptide having the amino acid sequence set forth in SEQ ID No.: 11; and

[0169] (vi) a second human Fc polypeptide having the amino acid sequence set forth in SEQ ID No.: 12.

[0170] The humanized anti-Met antibody fragment (hOA-DN30-cl03F04) consists of:

[0171] (i) a humanized light chain variable domain having the amino acid sequence set forth in SEQ ID No.: 19;

[0172] (ii) a humanized heavy chain variable domain having the amino acid sequence set forth in SEQ ID No.: 20;

[0173] (iii) a human light chain constant (CL) domain having the amino acid sequence set forth in SEQ ID No.: 9;

[0174] (iv) a human heavy chain constant CH1 domain having the amino acid sequence set forth in SEQ ID No.: 10;

[0175] (v) a first human Fc polypeptide having the amino acid sequence set forth in SEQ ID No.: 11; and

[0176] (vi) a second human Fc polypeptide having the amino acid sequence set forth in SEQ ID No.: 12.

[0177] The humanized anti-Met antibody fragment (hOA-DN30-cl03E08) of the present disclosure consists of:

[0178] (i) a humanized light chain variable domain having the amino acid sequence set forth in SEQ ID No.: 21;

[0179] (ii) a humanized heavy chain variable domain having the amino acid sequence set forth in SEQ ID No.: 22;

[0180] (iii) a human light chain constant (CL) domain having the amino acid sequence set forth in SEQ ID No.: 9;

[0181] (iv) a human heavy chain constant CH1 domain having the amino acid sequence set forth in SEQ ID No.: 10;

[0182] (v) a first human Fc polypeptide having the amino acid sequence set forth in SEQ ID No.: 11; and

[0183] (vi) a second human Fc polypeptide having the amino acid sequence set forth in SEQ ID No.: 12.

[0184] The objective humanized anti-Met antibody fragment (hOA-DN30-cl04H08) of the present disclosure consists of:

[0185] (i) a humanized light chain variable domain having the amino acid sequence set forth in SEQ ID No.: 23;

[0186] (ii) a humanized heavy chain variable domain having the amino acid sequence set forth in SEQ ID No.: 24;

[0187] (iii) a human light chain constant (CL) domain having the amino acid sequence set forth in SEQ ID No.: 9;

[0188] (iv) a human heavy chain constant CH1 domain having the amino acid sequence set forth in SEQ ID No.: 10;

[0189] (v) a first human Fc polypeptide having the amino acid sequence set forth in SEQ ID No.: 11; and

[0190] (vi) a second human Fc polypeptide having the amino acid sequence set forth in SEQ ID No.: 12.

[0191] Expression and purification of chimeric and human one-armed antibodies

[0192] The cDNAs encoding the above-mentioned light chains (for humanized light chains, the nucleotide sequences set forth in SEQ ID Nos.: 25, 26, 27, 28, 29; for chimeric light chains, the nucleotide sequence set forth in SEQ ID No.: 30), heavy chains (for humanized heavy chains, the nucleotide sequences set forth in SEQ ID Nos.: 31, 32, 33, 34, 35; for chimeric heavy chains, the nucleotide sequence set forth in SEQ ID No.: 36) and Fc (the nucleotide sequence set forth in SEQ ID No.: 37) were cloned into commonly available expression vectors (e.g., pcDNA3.1 plasmid, catalog number V79020, Invitrogen Corporation (Camarillo, California)) and used to transfect ExpiCHO-S cells (catalog number: A29127, ThermoFisherScientific). The carrier ratios were optimized to maximize the output of the desired 97.5 kDa heterodimer (OA-Ab) and minimize the presence of any 150 kDa bivalent homodimer (true Mab). The optimal ratio was 1:1:2 for the light chain, full-length heavy chain ("knob" carrier), and Fc ("hole" carrier), respectively. This resulted in the least amount of homodimer present, with heterodimers accounting for approximately 85% of the total protein. Transient expression was performed by ExpiFectamine TM CHO transfection reagent (Cat. No. A29130, ThermoFisher Scientific) was used to provide power. The supernatant was harvested after 9 days of production. Hitrap MabSelectSure columns (Cat. No. GE29-0491-04, Sigma Aldrich) were used to Purify OA-DN30 antibody on Pure 25 chromatography system. OA-DN30 antibody is eluted with 0.1M citrate buffer of pH 3.0 subsequently. Collect the fraction of elution and neutralize with 1M Tris-HCl of pH 9.0 (ratio of 0.15ml of protein eluted per ml). OA-DN30 antibody is further purified on GE AKTA Prime liquid chromatography system (GE Healthcare Life Sciences) by Superdex 200 26 / 600 size exclusion chromatography column (catalog number GE28-9893-36, Sigma Aldrich) balanced through 1x PBS (pH 7.4), wherein the fraction containing OA-Ab is separated from the homodimer fraction (mAb or Fc). The preparative SEC fraction containing OA-DN30 antibody is merged and analyzed by SDS-PAGE to determine purity. The endotoxin level of the fraction merged is also tested. The final pooled fractions from the preparative SEC purification showed the absence of any divalent homodimer and the presence of a small amount (<5%) of a smaller ≈70 kDa band corresponding to the knob-knob Fc homodimer.

[0193] Generation, expression and purification of mutant Met ectodomain

[0194] Starting from the human Met extracellular domain sequence as disclosed in GenBank with accession number X54559, the cDNA sequence of the human MET extracellular domain (DecoyMet) carrying a single amino acid substitution was synthetically generated using the QuickChange II site-directed mutagenesis kit (Cat. No. 200524, Agilent Technologies (Santa Clara, CA)) according to the manufacturer's instructions. This procedure requires the design of sense and antisense oligonucleotides containing the desired point mutation. The following oligonucleotides were used:

[0195] - Mutation K842E:

[0196] Sense 5′-gtacataatcctgtgtttgagccttttgaaaagccagtg-3′ (SEQ ID No.: 42);

[0197] Antisense 5'-cactggcttttcaaaaggctcaaacacaggattatgtac-3' (SEQ ID No.: 43).

[0198] Engineered soluble receptors were produced by transient transfection of HEK-293T cells with pcDNA3.1 plasmids (Cat. No. V79020, Invitrogen Corporation (California, CA)) expressing cDNA encoding the DecoyMet mutant. The transfected cells were starved for three days and the cell culture supernatant containing the soluble receptor was collected. Recombinant protein was purified by affinity chromatography using a HisTrap HP column (Cat. No. 17524701, GE Healthcare (Freiburg, Germany)) according to the manufacturer's instructions. Large-scale protein production and purification was performed by U-Protein Express BV (Utrecht, The Netherlands).

[0199] Variant of human DecoyMet (DecoyMET K842E ) has the amino acid sequence set forth in SEQ ID No.: 14.

[0200] Analysis of purified proteins by SDS-PAGE + Coomassie Blue

[0201] Purified OA-DN30 antibody (1 μg) was separated into 4-12% acrylamide gradient gels by SDS-PAGE in the presence or absence of β-mercaptoethanol according to standard methods. Molecular weight markers (Cat. No. 1610374, Bio-Rad) were included in the analysis. Gel Code Blue Stain reagent (Cat. No. 24590, Thermo Fisher Scientific) revealed the polypeptides separated into the gel.

[0202] ELISA binding assay

[0203] To analyze the interaction between Met and DN30 derivatives, purified Met-Fc chimeras (Catalog No. 358-MT-100, R&D Systems, 100 ng / well) were immobilized on ELISA plates. After saturation with 0.5% BSA, incubation was performed at 37°C for 1 hour, followed by the addition of increasing concentrations of antibodies in liquid phase (MvDN30, chOA-DN30 or hOA-DN30 antibodies, 0-500 nM, prepared in PBS-BSA 0.5%-Tween 0.1%). Binding was revealed using an anti-human kappa chain antibody conjugated to HRP (Catalog No. A7164, Sigma-Aldrich) followed by incubation with TMB (Catalog No. T8665, Sigma Aldrich). The assay was quantitatively determined by a multi-label microplate reader VICTOR-X4 (Perkin Elmer Instruments INC. (Waterman, MA)). Data were analyzed and fitted using Prism software (GraphPad).

[0204] For ELISA binding analysis of hOA-DN30-c103E08 (liquid phase) with Met-Fc chimeras derived from different species (solid phase), Met from human (Catalog No. 358-MT-100, R&D Systems), murine (Catalog No. 50622-M02H, Sino Biological), rat (Catalog No. 80004-R02H, Sino Biological) and monkey (Catalog No. 90304-C02H, Sino Biological) sources were included in the assay. The procedure was the same as described above.

[0205] Flow cytometric binding analysis

[0206] In order to make hOA-DN30-cl03E08 bind to Met expressed on the cell surface, StemPro TM Accutase TM 2×10 cells were treated with cell dissociation reagent (Catalog No. A1110501, ThermoFisher Scientific) 5EBC-1, C2C12, H9C2, MDCK or Cos-7 cells were collected and incubated in PBS-2% FCS at 4°C for 1 hour. Antibody staining was performed by incubating cells with 10 μg / ml hOA-DN30-cl03E08 in PBS-2% BSA at 4°C for 30 minutes. After washing 6 times with PBS-2% BSA, cells were incubated with 1:100 diluted anti-human IgG-APC (Cat. No. 109-606-088, Jackson Immuno Research) at 4°C for 30 minutes and then washed to remove unbound antibodies. Cells were co-stained with DAPI (3 μl of 1 μg / ml working solution, Sigma-Aldrich, Cat. No. 10236276001) at 4°C for 5 minutes and analyzed for Met expression by Summit 4.3 software (Dako). The fluorescence signal from the isotype control (anti-human APC IgG) was set as the threshold (0 <MFI<10 1 If the mean fluorescence intensity (MFI) is higher than the threshold (MFI>10 1 ), the cells were considered positive for Met expression.

[0207] Analysis of Met shedding

[0208] Nearly confluent A549 monolayer cells were washed twice with PBS and then incubated with increasing concentrations (37, 111, 333, 1000 nM) of DN-30 derivatives in serum-free medium. After 48 hours, the conditioned medium was collected and the cells were lysed with Laemmli buffer. The Met protein level in 15 μg of total cell protein was determined by Western blotting using an anti-Met antibody (3D4, catalog number 08-1366, Invitrogen Corporation); as a loading control, the filter membrane was also probed with an anti-focal adhesion protein antibody (clone hVIN-1, catalog number V9131, Sigma Life Science). The Met extracellular domain level in 15 μl of cell culture supernatant was determined by Western blotting using an anti-human HGFR / Met antibody (catalog number AF276, R&D Systems) directed against the extracellular domain of the receptor. Protein detection was performed using anti-mouse IgG1 HRP-conjugated secondary antibody (Cat. No. JI115035003, Jackson ImmunoResearch) and ECL system (Cat. No. W1015, Promega). Western blot bands were quantified using ImageJ software.

[0209] MET phosphorylation assay

[0210] Serum-starved GTL-16 cells were incubated with DN30 derivatives (1000 or 250 nM) for 24 h. Total cell lysates were analyzed by Western blotting using the following primary antibodies: anti-Met phospho-Tyr 1234 / 1235 (D26, catalog number 3077, Cell Signaling Technology); anti-Met (3D4, catalog number 08-1366, Invitrogen Corporation); and anti-vinculin (clone hVIN-1, catalog number V9131, Sigma Life Science). Protein detection was performed using anti-mouse IgG1 and anti-rabbit IgG (catalog number JI111035003) HRP-conjugated secondary antibodies and ECL system. Western blot bands were quantified using ImageJ software.

[0211] In vitro biological assays

[0212] for Cell scatter assay HPAF-II cells (8000 / well) were seeded in complete medium in 96-well plates. To analyze the agonistic activity of the antibodies, HGF (8 ng / ml, positive control, catalog number 294-HG-025, R&D Systems) or antibodies (DN30 mAb, MvDN30, chOA-DN30, hOA-DN30 antibody, all at a concentration of 200 nM) were added to the culture medium after 24 hours. To analyze the inhibitory activity, the inhibitory activity was analyzed by adding the inhibitory activity alone or with DecoyMet K842E hOA-DN30-cl03E08 of increasing concentration (0-4 μM) was added in 1:1 combination. After another 24 hours, the cells were stimulated for 24 hours with 6.25 ng / ml of HGF. The cells were then fixed with 11% glutaraldehyde (Cat. No. 340855, Sigma-Aldrich), stained with 0.1% crystal violet (Cat. No. C3886, Sigma-Aldrich), and analyzed by microscopic observation (microscope Leica DM2000). Images were captured with a QICAMFast1394 color digital camera (QImaging).

[0213] for Cell invasion assay HPAF-II cells (1.5×10 5 / well) suspended in either alone or with 1 μM DecoyMet K842EThe cells were combined in serum-free medium with 0.5 μM hOA-DN30-cl03E08 and seeded on the upper compartment of a transwell chamber pre-coated with 30 μg / well of Matrigel Matrix (Cat. No. 354234, Corning Inc.). The culture medium supplemented with 2% FBS and 6.25 ng / ml HGF was added to the lower compartment of the chamber. After 24 hours, the cells on the upper side of the transwell filter were mechanically removed, and the cells that migrated through the membrane were fixed with 11% glutaraldehyde and stained with 0.1% crystal violet. Cell invasion was quantified with Image-J software.

[0214] for Viability assay GTL-16 cells were seeded at 2000 cells / well in 10% FBS medium in 96-well plates. After 24 hours, the medium was replaced with fresh medium containing 10% FBS plus the molecule to be tested (increasing concentrations - 0 to 10 μM). Cell viability was assessed after 72 hours using CellTiter-Glo (Catalog No. G7573, Promega Corp) according to the manufacturer's instructions. Chemiluminescence was detected using VICTOR X4.

[0215] for Proliferation assay 350,000 GTL-16 cells were seeded in 10% FCS medium in 6-well plates. After 24 hours, the cells were treated with a fixed concentration (1 μM) of DN30 derivatives for another 48 hours. Then, 10 μM EdU (Catalog No. A10044, Thermo Fisher) was added to the culture medium and treated for another 2 hours. TM EdU AlexaFluor TM The % of cells in S phase was determined by cytofluorimetric analysis using the procedure of the 488 Flow Cytometry Assay (Cat. No. C10425, Thermo Fisher).

[0216] for Cytotoxicity assay 2000 GTL-16 cells were seeded in 10% FCS medium in 96-well plates. After 24 hours, the cells were treated with increasing concentrations of DN30 derivatives for another 48 hours. TM Cytotoxicity was assessed using the ELISA Green cytotoxicity assay (Cat. No. G8741, Promega) according to the manufacturer's instructions.

[0217] PD-L1 expression analysis

[0218] Nearly confluent GTl-16 cells were treated with 50ng / ml IFNγ-1b (Miltenyi Biotec, catalog number 130-096-484) (changed every 24 hours) in combination with 250nM hOA-DN30-cl03E08 for 48 hours. The monolayer cells were then lysed in Laemmli buffer and 45μg of total protein was subjected to 8% SDS-PAGE gel electrophoresis. The proteins were transferred from the gel to iBlot Transfer nitrocellulose membrane (Life Technologies, catalog number IB23001) according to standard methods. PD-L1 expression was detected by anti-PD-L1 antibody (E1L3N, catalog number 13684, Cell Signaling Technology). Anti-MET phospho-Tyr 1234 / 1235 (D26) antibody to check P-Met levels. As a loading control, the filter was probed with anti-GAPDH (D4C6R, catalog number 97166, Cell Signaling Technology). Protein detection was performed using HRP-conjugated goat anti-mouse IgG (catalog number JI115035003) or anti-rabbit IgG (catalog number JI111035144) secondary antibodies (both from Jackson ImmunoResearch) and the ECL system.

[0219] In vivo experiments

[0220] All animal procedures were performed according to protocols approved by the Ethical Committee of Animal Experiments of the Piedmont Cancer Research Foundation and the Italian Ministry of Health. NOD-SCID mice were purchased from Charles River (Calco, Italy).

[0221] for Pharmacokinetic analysis NOD-SCID mice were injected intravenously with a single dose (100 μg) of hOA-DN30-cl03E08. Peripheral blood was collected at different time points: 10 minutes, 30 minutes, 1 hour, 4 hours, 8 hours, 24 hours, 48 ​​hours, 72 hours, 120 hours. Serum concentrations of therapeutic molecules were measured by ELISA as described above in the binding assay section, and the absorbance values ​​of the samples were interpolated on the linear part of the standard curve obtained by serial dilution of different purified molecules. Each time point is the average of 4 mice.

[0222] for Tumor growth analysis 1×10 6GTL-16 cells. Tumor growth was monitored twice a week by caliper measurement. Tumor volume was calculated using the following formula: V = 4 / 3π(x / 2)(y / 2)(z / 2), where x, y, and z are the height, width, and depth of the tumor mass. When the tumor reached 70-100 mm 3 When the volume of 100 mg / kg was reached (approximately one week after cell injection), mice were randomly divided into uniform groups. In one case, 4 groups were generated (PBS-vehicle, MvDN30, chOA-DN30, hOA-DN30-cl03E08; all antibodies 30 mg / kg, 2 times a week); in another case, 5 groups were generated (PBS-vehicle, hOA-DN30-cl03E08 60 mg / kg, 30 mg / kg, 10 mg / kg, 3.3 mg / kg; all doses were 3 times a week). The molecules were administered by intravenous injection. At the end of the experiment (15 or 17 days of treatment), mice were sacrificed, tumors were excised and weighed.

[0223] Statistical analysis

[0224] Microsoft Office Excel 2010 software (Microsoft Corporation) was used to calculate the mean and standard deviation (SD). d Values ​​were calculated using GraphPad Prism software (GraphPad Software) to analyze and fit data from ELISA assays based on nonlinear regression, single-site binding hyperbolic curves. 50 and EC 50 Values ​​were used and GraphPad Prism software was used to analyze and fit the data according to nonlinear regression sigmoidal dose response curves. All experiments were repeated at least twice. The accompanying figure shows a representative experiment.

[0225]

[0226]

[0227] References

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[0229] 2.Kong-Beltran M,Seshagiri S,Zha J,Zhu W,Bhawe K,Mendoza N etal.Somatic mutations lead to an oncogenic deletion of met in lungcancer.Cancer Res 2006;66(1):283-289;doi 10.1158 / 0008-5472.CAN-05-2749.

[0230] 3.Gandino L,Longati P,Medico E,Prat M,Comoglio PM.Phosphorylation ofserine 985negatively regulates the hepatocyte growth factor receptor kinase.JBiol Chem1994;269(3):1815-1820.

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Claims

1. Use of a humanized anti-Met antibody fragment and another antibody, a small molecule tyrosine kinase inhibitor, a chemotherapeutic agent, other cytotoxic agent, an anti-angiogenic agent, a cytokine and / or a growth inhibitor for the preparation of a combination therapy drug, wherein: The humanized anti-Met antibody fragment comprises a single antigen binding arm and an Fc region, wherein the Fc region comprises a complex of a first and a second Fc polypeptide, wherein the antibody fragment comprises: i) a first polypeptide comprising a humanized light chain variable (VL) domain and a human light chain constant (CL) domain, wherein the humanized VL domain is fused to the human CL domain in the N- to C-terminal direction; ii) a second polypeptide comprising a humanized heavy chain variable (VH) domain, a human heavy chain constant CH1 domain, and the first Fc polypeptide, wherein the first Fc polypeptide comprises a human hinge region, a human constant CH2 domain, and a human constant CH3 domain, wherein in the N- to C-terminal direction, the humanized VH domain is fused to the human CH1 domain, the human CH1 domain is fused to the human hinge region, the human hinge region is fused to the human CH2 domain, and the human CH2 domain is fused to the human CH3 domain; and iii) a third polypeptide comprising the second human Fc polypeptide, wherein the second human Fc polypeptide comprises a human hinge region, a human constant CH2 domain and a human constant CH3 domain, wherein in the N- to C-terminal direction, the human hinge region is fused to the CH2 domain, and the CH2 domain is fused to the human CH3 domain, wherein the human hinge region is truncated at the N-terminus.

2. The use according to claim 1, wherein: The humanized VL domain contains three complementarity determining regions (CDRs), the three complementarity determining regions consisting of the amino acid sequences shown in SEQ ID No.: 1, 2 and 3, and wherein the humanized VL domain has the amino acid sequence shown in SEQ ID No.: 7; and The humanized VH domain contains three complementarity determining regions (CDRs), which consist of the amino acid sequences shown in SEQ ID No.: 4, 5 and 6, and wherein the humanized VH domain has the amino acid sequence shown in SEQ ID No.:

8.

3. The use according to claim 1, wherein the humanized anti-Met antibody fragment and the other antibodies, small molecule tyrosine kinase inhibitors, chemotherapeutic agents, other cytotoxic agents, anti-angiogenic agents, cytokines and / or growth inhibitory agents are contained in the same preparation.

4. The use according to claim 1, wherein the humanized anti-Met antibody fragment and the other antibodies, small molecule tyrosine kinase inhibitors, chemotherapeutic agents, other cytotoxic agents, anti-angiogenic agents, cytokines and / or growth inhibitory agents are contained in different preparations.

5. The use according to claim 4, wherein the humanized anti-Met antibody fragment is administered before or after the other antibodies, small molecule tyrosine kinase inhibitors, chemotherapeutic agents, other cytotoxic agents, anti-angiogenic agents, cytokines and / or growth inhibitors. The use according to claim 1 , wherein the human CL domain is a human light κ domain.

7. The use according to claim 1, wherein the human hinge region and the human constant domains CH1, CH2 and CH3 are from human IgG1. The use according to claim 1 , wherein the two Fc polypeptides are linked by an intermolecular disulfide bond in the hinge region.

9. The use of claim 1, wherein the first Fc polypeptide and the second Fc polypeptide meet at an interface, and one of the first and the second Fc polypeptide comprises a knob at the interface, and the other of the first and the second Fc polypeptide comprises a hole at the interface, wherein the knob is positionable into the hole.

10. The use according to claim 9, wherein one of said first and said second Fc polypeptide comprises a mutated CH3 constant domain, wherein said mutated CH3 constant domain carries an amino acid mutation at position 389, wherein the original amino acid at position 389 has been mutated to introduce an amino acid having a larger side chain volume than said original amino acid; and wherein the other of said first and said second Fc polypeptide comprises a mutated CH3 constant domain, wherein said mutated CH3 constant domain carries three amino acid mutations at positions 389, 391 and 438, wherein the original amino acids have been mutated to introduce amino acids having a smaller side chain volume than said original amino acids, wherein said amino acid numbering is according to the EU numbering scheme of Kabat.

11. The use according to claim 10, wherein the original amino acids at positions 389, 391 and 438 are threonine, leucine and tyrosine, respectively; and wherein in one of said first and said second Fc polypeptides, the threonine in position 389 has been mutated to tryptophan; and wherein in the other of said first and said second Fc polypeptides, the threonine at position 389 has been mutated to serine, the leucine at position 391 has been mutated to alanine and the tyrosine at position 438 has been mutated to valine.

12. The use according to claim 1, wherein the human CL domain has the amino acid sequence as set forth in SEQ ID No.: 9 and the human CH1 domain has the amino acid sequence as set forth in SEQ ID No.:

10.

13. The use according to claim 1, wherein the first human Fc polypeptide has an amino acid sequence as set forth in SEQ ID No.: 11, and the second human Fc polypeptide has an amino acid sequence as set forth in SEQ ID No.:

12.

14. The use according to claim 1, wherein when bound to Met, the humanized anti-Met antibody fragment induces shedding of the extracellular domain of Met.

15. The use according to claim 1, wherein the combination therapy drug is used to treat patients with tumors and / or metastases, wherein the patients carry genetic changes in the MET gene.

16. The use according to claim 1, wherein the combination therapy drug is used to treat a patient with a tumor and / or metastasis, wherein the patient carries a wild-type MET gene.

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