TW201716439A - HER3 antibodies - Google Patents

Abstract

The present invention relates to antibodies that bind human HER3, and may be useful for treating cancer alone and in combination with EGFR pathway inhibitors, especially cancer driven by EGFR signaling or that use HER3 as a resistance mechanism, including lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, renal cancer, gastric cancer, breast cancers or hepatocellular carcinoma.

Description

HER3 antibody

The present invention relates to the field of medicine. More specifically, the present invention relates to antibodies that bind to human HER3 and can be applied to solid tumors alone or in combination with other anti-tumor agents, such as for signaling by EGFR and/or using HER3 as a mechanism of resistance. Human epidermal growth factor receptor (EGFR) inhibitors treat cancer (especially tumor metastasis), including hepatocellular carcinoma, gastric cancer, lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer, kidney cancer or breast cancer.

The dysregulation of the epidermal growth factor family of signaling receptors plays a decisive role in the initiation and progression of various human cancers. The EGFR family receptor HER3 and its ligand neuromodulin 1 (NRG1) play a role in a variety of human malignancies and serve as the primary mechanism for EGFR inhibition and other emerging or acquired resistance to targeted therapies. HER3 is the major Akt pathway complement of the EGFR signaling family and is activated by both ligand-dependent (ie, NRG1) and ligand-independent mechanisms.

The generation and functional properties of a different set of ERBB3-specific (ie, HER3-specific) inhibitory monoclonal antibodies with different operating mechanisms and at least 5 independent binding sites have been reported (Vincent, S. et al., US Cancer) American Association for Cancer Res., Bulletin No. 628 (April 2011). Ligands and dimeric inhibitory antibodies and anti-ERBB3 antibodies that potentiate (i.e., potentiate) NRGl binding to ERBB3 but inhibit ERBB3 functional activity are among the different classes of anti-ERBB3 antibodies described by Vincent, S. et al. Furthermore, WO 2015/067986 discloses a mouse anti-human HER3 antibody designated 9F7-F11 which has an affinity for HER3 which is not present when a neuromodulating protein is present Inhibited (ie, the 9F7-F1 1 antibody is non-competitive for neuromodulin), but antibodies to one of the antibody classes reported by Vincent, S. et al., when neuromodulin is present in HER3 positive The affinity of 9F7-F1 1/HER3 is also increased ectopically in the environment of the cells (i.e., 9F7-F1 1 is an atopic anti-HER3 antibody). More specifically, with the incubation with NRG1, the Kd value of 9F7-F11 bound to HER3 was reported to be 0.47 ± 0.07 nM, whereas the Kd was measured to be 2.33 ± 0.30 nM in the absence of NRG1; The NRG1 additive ectopically caused a 6-fold increase in the affinity of 9F7-F1 1 for the HER3 receptor.

Other antibodies against human HER3 have been shown to inhibit tumor growth in a mouse xenograft tumor model, but tumors are usually regenerated shortly afterwards, and are utilized in preclinical models with other EGFR-targeted therapies (such as pertuzumab, Combination therapy with trastuzumab, cetuximab or erlotinib resulted in complete remission (Bossenmaier et al., American Association for Cancer Research, Abstract No. 2668 (April 2014)). Garner, AP et al. also reported that LJM716, a human HER3 IgG antibody, inhibits both ligand-dependent and ligand-independent HER3 activity in vitro, although it does not hinder ligand binding (Garner, AP, Cancer Res 2013; 73 :6024-35).

There is still a need to provide a surrogate antibody that inhibits the HER3/EGFR pathway by binding to high affinity human HER3, which is ectopically elevated in the presence of a neuromodulating protein in the context of HER3-positive cells and can be ligand dependent Both human and ligand-independent models counteract human HER3 activation (ie, block human HER3-mediated signaling). Such anti-human HER3 anti-systems having one or more of the following properties are particularly desirable: i) effective treatment of cancer characterized by one or more KRAS activating mutations; ii) preventing or delaying tumors in patients with HER3 Excellent activity in terms of resistance mechanisms; iii) superior activity in preventing or delaying the development of resistance of a patient's tumor to other therapeutic agents or treatment regimens as compared to the use of such agents or regimens alone in cancer treatment, Such therapeutic agents or treatment regimens such as erlotinib, cisplatin, carboplatin, liposomal doxorubicin, docetaxel, cyclophosphamide and Cyclophosphamide and doxorubicin, velopene, eribulin, paclitaxel-binding particles for injectable suspensions, ixabepilone, capecitabine Capecitabine), ramocirumab, FOLFOX (hyperfluorinated tetrahydrofolate, fluorouracil and oxaliplatin), FOLFIRI (hyperthyroid tetrahydrofolate, Fluorouracil and irinotecan), pertuzumab, trastuzumab, cetuximab, panitumumab, lexizumab (necitumumab), gefitinib, afatinib, neratinib, lapatinib, rociletinib, AZD9291, ASP8273, HM61713 or a pharmaceutically acceptable salt thereof; iv) for improved manufacturability (eg, ease of purification and quality, thermal and chemical stability, solubility, viscosity and/or homogeneity), formulation stability (eg, Alleviating aggregation and self-binding of purified antibodies) and/or pharmacokinetic properties (eg, subtraction) For the purpose of a gap created by non-specific binding in vivo), structurally engineered to reduce overall surface hydrophobicity while maintaining acceptable binding affinity for human HER3-ECD; and/or v) otherwise displaying live In vivo stability, physical and chemical stability, including but not limited to, thermostability, solubility, lower self-binding, and acceptable pharmacokinetic properties for development and/or use in cancer therapy.

Accordingly, embodiments of the present invention provide an antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein HCVR and LCVR together form an antigen binding site that binds to human HER3 Wherein the antigen binding site comprises: a. three complementarity determining regions (CDRs) in HCVR, wherein HCDR1 comprises the amino acid sequence AASGFSLDTYTMI (SEQ ID NO: 8) and HCDR2 comprises the amino acid sequence IIYVTYNTYYANWAKG (SEQ ID NO) :9), and HCDR3 comprises the amino acid sequence TRWGDQNGLDI (SEQ ID NO: 10), and b. three CDRs in LCVR, wherein LCDR1 comprises an amino acid sequence QSSQRVYGNKNLA (SEQ ID NO: 11), LCDR2 comprises the amino acid sequence YFARTLAS (SEQ ID NO: 12), and LCDR3 comprises the amino acid sequence QGTYYGTNWYVG (SEQ ID NO: 13).

Another embodiment of the present invention provides an antibody or antigen-binding fragment thereof comprising a heavy chain (HC) and a light chain (LC), wherein the HC comprises HCVR and the LC comprises LCVR, wherein HCVR and LCVR together form an antigen that binds to human HER3 A binding site, wherein the antigen binding site comprises: a. three CDRs in HCVR, wherein HCDR1 comprises the amino acid sequence AASGFSLDTYTMI (SEQ ID NO: 8) and HCDR2 comprises the amino acid sequence IIYVTYNTYYANWAKG (SEQ ID NO: 9) And HCDR3 comprises the amino acid sequence TRWGDQNGLDI (SEQ ID NO: 10), and b. three CDRs in LCVR, wherein LCDR1 comprises the amino acid sequence QSSQRVYGNKNLA (SEQ ID NO: 11) and LCDR2 comprises the amino acid sequence YFARTLAS (SEQ ID NO: 12) and LCDR3 comprises the amino acid sequence QGTYYGTNWYVG (SEQ ID NO: 13).

In another embodiment, the invention provides an antibody or antigen-binding fragment thereof comprising HC and LC, wherein HC comprises HCVR and LC comprises LCVR, wherein HCVR has the amino acid sequence set forth in SEQ ID NO: LCVR has the amino acid sequence given in SEQ ID NO: 3, and wherein HCVR and LCVR together form an antigen binding site that binds to human HER3.

In another embodiment, the invention provides an antibody or antigen-binding fragment thereof that binds to human HER3, the antibody or antigen-binding fragment comprising HC and LC, wherein HC has the amino acid sequence set forth in SEQ ID NO: LC has the amino acid sequence given in SEQ ID NO:4.

In another embodiment, the invention provides an antibody that binds to human HER3, the antibody comprising two heavy chains and two light chains, wherein each heavy chain has the one set forth in SEQ ID NO: The amino acid sequence and each light chain has the amino acid sequence given in SEQ ID NO:4.

In another embodiment, the invention provides an antibody that binds to human HER3, the antibody comprising two heavy chains and two light chains, wherein each heavy chain has the amino acid sequence set forth in SEQ ID NO: 2 and each A light chain having the amino acid sequence set forth in SEQ ID NO: 4, wherein one of the heavy chains forms an interchain disulfide bond with one of the light chains, and the other heavy chain is A light chain forms an interchain disulfide bond, and one of the heavy chains forms two interchain disulfide bonds with the other heavy chain. In another embodiment of the invention, the antibody that binds to human HER3 is glycosylated.

In another embodiment, the invention provides an antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein HCVR and LCVR together form an antigen binding to human HER3 binding a site, wherein the antigen binding site comprises: a. three complementarity determining regions (CDRs) in HCVR, wherein HCDR1 comprises the amino acid sequence AASGFSLDTYTMI (SEQ ID NO: 8) and HCDR2 comprises the amino acid sequence IIYVTYNTYYANWAKG (SEQ) ID NO: 9), and HCDR3 comprises the amino acid sequence TRWGDQNGLDI (SEQ ID NO: 10), and b. three CDRs in LCVR, wherein LCDR1 comprises the amino acid sequence QSSQRVYGNKNLA (SEQ ID NO: 11), and LCDR2 comprises Amino acid sequence YFARTLAS (SEQ ID NO: 12), and LCDR3 comprises the amino acid sequence QGTYYGTNWYVG (SEQ ID NO: 13), wherein the antibody or antigen-binding fragment thereof is human, macaque and mouse HER3- at 37 °C ECD will have less than about 10nM, 5nM or less than 5nM solution between 1.0nM and dissociation equilibrium constant K _D. In another embodiment, the present invention provides an antibody of the present invention, such as the antibody to SEQ ID NO: human HER3-ECD polypeptide under the display 23 at 37 ℃ solution of about 1.6nM having affinity for the dissociation equilibrium constant K _D . In various embodiments, a further feature of the present invention the antibody is about 2.8 × 10 ⁵ M ^-1 sec ^-1 as the SEQ ID NO: 23 shown in the human HER3-ECD ratio K _on. Further, the antibody of the present invention is characterized by a _Koff ratio of the human HER3-ECD polypeptide as shown in SEQ ID NO: 23 at about 4.4 x 10 ^-4 sec ^-1 at 37 °C. The K _D , K _on and K _off values were established by binding kinetics at 37 ° C as described below in "Binding Kinetics, Affinity and Ligand Binding Enhancement Analysis".

In various embodiments, an antibody of the invention binds to a human HER3-ECD polypeptide as set forth in SEQ ID NO: 23 by high affinity. For purposes of this invention, the term "high affinity" refers to the human HER3-ECD polypeptide (including, but not limited to, such as SEQ ID NO: 23 shown polypeptide of HER3-ECD) of less than about 10nM of K _D.

Certain antibodies of the invention bind to human HER3-ECD by high affinity and do not block the binding of HER3 ligand NRG1 to HER3-ECD after binding to HER3-ECD, but in fact enhance the enhancement of NRG1 and HER3-ECD. Combine. In addition, certain antibodies of the invention show positive responses in tumor xenograft models, such as FaDu and A253 for head and neck cancer, A549 for lung cancer, and BxPC-3 for pancreatic cancer, thereby indicating treatment as a cancer possibility.

In one embodiment, the invention provides a mammalian cell comprising a DNA molecule comprising: a) a polynucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2, and b) encoding having a polynucleotide sequence of a polypeptide of the amino acid sequence of SEQ ID NO: 4, wherein the cell is capable of expressing an antibody comprising i) a heavy chain having the amino acid sequence of SEQ ID NO: 2, and ii) having Light chain of the amino acid sequence of SEQ ID NO: 4.

In one embodiment, the invention provides a method for making an antibody comprising: a) a heavy chain having the amino acid sequence of SEQ ID NO: 2, and b) an amine group having SEQ ID NO: A light chain of an acid sequence, the method comprising culturing a mammalian cell of the invention under conditions such that the antibody behaves and recovering the expressed antibody. In another embodiment, the invention provides an antibody produced by such a method.

In one embodiment, the invention provides a pharmaceutical composition comprising an antibody of the invention and a pharmaceutically acceptable carrier, diluent or excipient.

In a particular embodiment, the invention provides a method of treating cancer, the method comprising A patient in need thereof is administered an effective amount of an antibody of the invention. In other embodiments, the invention provides a method of treating cancer, the method comprising administering to a patient in need thereof an effective amount of an antibody of the invention, wherein the cancer is gastric cancer, head and neck cancer (preferably, head and neck squamous cell carcinoma ( HNSCC)), pancreatic cancer, renal cancer, breast cancer, lung cancer (preferably, NSCLC, and more preferably, sq-NSCLC), colorectal cancer or hepatocellular carcinoma (HCC). In other embodiments, such methods of treating cancer comprise administering an effective amount of an antibody of the invention simultaneously, separately or sequentially in combination with one or more anti-neoplastic agents, the one or more anti-tumor agents being selected from the group consisting of: Groups: cisplatin, carboplatin, lipid doxorubicin, docetaxel, cyclophosphamide and doxorubicin, vonopipine, eribulin, paclitaxel-binding protein particles for injectable suspensions , ixabepilone, capecitabine, remollozumab, vorax (mercaptotetrahydrofolate, fluorouracil, and oxaliplatin), verferoid (hyperthyroid tetrahydrofolate, fluorouracil and Irinotecan), pertuzumab, trastuzumab, cetuximab, panitumumab, lexizumab, gefitinib, afatinib, neratinib, lapata Nisin, roxitinib, AZD9291, ASP8273, HM61713 and erlotinib or a pharmaceutically acceptable salt thereof. In other embodiments, such methods of treating cancer comprise administering an effective amount of a compound of the invention simultaneously, separately or sequentially in combination with one or more tumor immunizing agents, the one or more tumor immunizing agents being selected from the group consisting of: Groups: nivolumab, ipilimumab, pidilizumab, pembrolizumab, and duvalumumab.

In some embodiments, the invention provides a method of treating breast cancer, the method comprising administering to a patient in need thereof an effective amount of an antibody of the invention. In some embodiments, the invention provides a method of treating breast cancer, comprising administering an effective amount of an antibody of the invention simultaneously, separately or sequentially in combination with one or more anti-tumor agents, the one or more anti-tumor agents selected from the group consisting of Groups consisting of: remollozumab, docetaxel, cyclophosphamide and doxorubicin, vinorepine, eribulin, paclitaxel-binding protein particles for injectable suspensions, Shapirone, capecitabine, pertuzumab, trastuzumab, cetuximab, panitux Anti-residumab, gefitinib, afatinib, neratinib, lapatinib, roxitinib, AZD9291, ASP8273, HM61713 and erlotinib or a pharmaceutically acceptable salt thereof .

In some embodiments, the invention provides a method of treating head and neck cancer, preferably HNSCC, comprising administering an effective amount of an antibody of the invention to a patient in need thereof. In various embodiments, the invention provides a method of treating head and neck cancer (preferably, HNSCC), the method comprising administering to the invention simultaneously, separately or sequentially in combination with one or more anti-tumor agents, the one or more anti-tumor agents Choose from a group consisting of: Remoruzumab, Docetaxel, Cyclophosphamide and Doxorubicin, Winopine, Eribulin, Pacific Paclitaxel Binding Protein for Injectable Suspensions Particles, ixabepilone, capecitabine, pertuzumab, trastuzumab, cetuximab, panitumumab, lexizumab, gefitinib, afatinib, come Natinib, lapatinib, roxitinib, AZD9291, ASP8273, HM61713 and erlotinib or a pharmaceutically acceptable salt thereof.

In some embodiments, the invention provides a method of treating ovarian cancer, the method comprising administering to a patient in need thereof an effective amount of an antibody of the invention. In certain embodiments, such methods of treating ovarian cancer comprise administering an effective amount of an antibody of the invention simultaneously, separately or sequentially in combination with one or more anti-neoplastic agents, the one or more anti-tumor agents selected from the group consisting of Group consisting of: remollozumab, cisplatin, carboplatin, lipid doxorubicin, pertuzumab, trastuzumab, cetuximab, panitumumab, lexizumab, Gefitinib, afatinib, neratinib, lapatinib, roxitinib, AZD9291, ASP8273, HM61713 and erlotinib or a pharmaceutically acceptable salt thereof.

In some embodiments, the invention provides a method of treating gastric cancer, the method comprising administering to a patient in need thereof an effective amount of an antibody of the invention. In certain embodiments, the method of treating gastric cancer comprises administering an effective amount of an antibody of the invention simultaneously, separately or sequentially in combination with one or more anti-tumor agents, the one or more anti-tumor agents selected from the group consisting of Group consisting of: Remomitumab, Pertuzumab, Trastuzumab, Cetuximab, Panitumumab, Lai Ciclozum, gefitinib, afatinib, neratinib, lapatinib, roxitinib, AZD9291, ASP8273, HM61713 and erlotinib or a pharmaceutically acceptable salt thereof.

In some embodiments, the invention provides a method of treating HCC comprising administering an effective amount of an antibody of the invention to a patient in need thereof. In certain embodiments, such methods of treating HCC comprise administering an effective amount of an antibody of the invention simultaneously, separately or sequentially in combination with one or more anti-tumor agents, the one or more anti-tumor agents selected from the group consisting of Groups consisting of: remollozumab, pertuzumab, trastuzumab, cetuximab, panitumumab, lexizumab, gefitinib, afatinib, laina Tinidinib, lapatinib, roxitinib, AZD9291, ASP8273, HM61713 and erlotinib or a pharmaceutically acceptable salt thereof.

In some embodiments, the invention provides a method of treating colorectal cancer, the method comprising administering to a patient in need thereof an effective amount of an antibody of the invention. In certain embodiments, such methods of treating colorectal cancer comprise administering an effective amount of an antibody of the invention simultaneously, separately or sequentially in combination with one or more anti-tumor agents, the one or more anti-tumor agents selected from the group consisting of Groups consisting of: Remorifizumab, Forks (Metformin tetrahydrofolate, fluorouracil, and oxaliplatin), Verfury (Methyltetrahydrofolate, Fluorouracil, and Irinotecan) , pertuzumab, trastuzumab, cetuximab, panitumumab, lexizumab, gefitinib, afatinib, neratinib, lapatinib, Rossi Tinidil, AZD9291, ASP8273, HM61713 and erlotinib or a pharmaceutically acceptable salt thereof.

In some embodiments, the invention provides an antibody of the invention for use in therapy. In some embodiments, the invention provides an antibody of the invention for use in the treatment of cancer. In another embodiment, the invention provides an antibody of the invention for use in the treatment of cancer, wherein the cancer is lung cancer (preferably, NSCLC, and more preferably, sq-NSCLC), head and neck cancer (preferably , HNSCC), pancreatic cancer, kidney cancer, stomach cancer, colorectal cancer, breast cancer or HCC. In another embodiment, the invention provides an antibody of the invention for use in KRAS wild type carcinoma Treatment of the disease. In various embodiments, the KRAS wild type cancer is lung cancer (preferably, NSCLC, and more preferably, sq-NSCLC), head and neck cancer (preferably HNSCC), pancreatic cancer, kidney cancer, gastric cancer, colorectal Cancer, breast cancer or HCC. In other embodiments, the invention provides an antibody of the invention for use in the treatment of a cancer comprising a KRAS activating mutation. In various embodiments, the cancer comprising a KRAS activating mutation is lung cancer (preferably, NSCLC, and more preferably, sq-NSCLC), head and neck cancer (preferably HNSCC), pancreatic cancer, renal cancer, gastric cancer, Colorectal cancer, breast cancer or HCC. In other embodiments, the invention provides an antibody of the invention for simultaneous, separate or sequential combination with one or more anti-tumor agents for use in the treatment of cancer, the one or more anti-tumor agents being selected from the group consisting of Groups: cisplatin, carboplatin, lipid doxorubicin, docetaxel, cyclophosphamide and doxorubicin, vinorepine, eribulin, paclitaxel-binding protein particles for injectable suspensions, Ixabepilone, capecitabine, remollozumab, vorax (mercaptotetrahydrofolate, fluorouracil, and oxaliplatin), verapamil (methanoin, tetrahydrofolate, fluorouracil, and y Litcomb), pertuzumab, trastuzumab, cetuximab, panitumumab, lexizumab, gefitinib, afatinib, neratinib, lapatinib , roxitinib, AZD9291, ASP8273, HM61713 and erlotinib or a pharmaceutically acceptable salt thereof.

In other embodiments, the invention provides an antibody of the invention for simultaneous, separate or sequential combination with one or more tumor immunizing agents for use in the treatment of cancer, the one or more tumor immunizing agents being selected from the group consisting of Groups: Navumab, Iplibumab, Pilitizumab, Pacliizumab and Devaluzumab.

In various embodiments, the invention provides antibodies of the invention for use in the treatment of breast cancer. In certain embodiments, the invention provides an antibody of the invention for use in the treatment of KRAS wild-type breast cancer. In other embodiments, the invention provides an antibody of the invention for use in the treatment of breast cancer, wherein the breast cancer contains a KRAS activating mutation. In another embodiment, an antibody of the invention is used in combination with one or more anti-tumor agents for the treatment of breast cancer, the one or more anti-tumor agents selected from the group consisting of: Remo Anti-, docetaxel, cyclophosphamide and doxorubicin, vinorelbine, eribulin, paclitaxel-binding protein particles for injectable suspensions, ixabepilone, capecitabine, patol Chemuzumab, trastuzumab, cetuximab, panitumumab, lexizumab, gefitinib, afatinib, neratinib, lapatinib, roxitinib, AZD9291, ASP8273, HM61713 and erlotinib or a pharmaceutically acceptable salt thereof.

In one embodiment, the invention provides an antibody of the invention for use in the treatment of ovarian cancer. In another embodiment, the invention provides an antibody of the invention for use in the treatment of wild-type ovarian cancer. In another embodiment, the invention provides an antibody of the invention for use in the treatment of ovarian cancer, wherein the ovarian cancer contains a KRAS activating mutation. In another embodiment, an antibody of the invention is used in combination with one or more anti-tumor agents for simultaneous or separate treatment for the treatment of ovarian cancer, the one or more anti-tumor agents being selected from the group consisting of: Remoruba, cisplatin, carboplatin, lipid doxorubicin, pertuzumab, trastuzumab, cetuximab, panitumumab, lexizumab, gefitinib, ar Fatinib, neratinib, lapatinib, roxitinib, AZD9291, ASP8273, HM61713 and erlotinib or a pharmaceutically acceptable salt thereof.

In one embodiment, the invention provides an antibody of the invention for use in the treatment of gastric cancer. In another embodiment, the invention provides an antibody of the invention for use in the treatment of KRAS wild type gastric cancer. In another embodiment, the invention provides an antibody of the invention for use in the treatment of gastric cancer, wherein the gastric cancer contains a KRAS activating mutation. In another embodiment, the antibody of the invention is used in combination with one or more anti-neoplastic agents for simultaneous or separate treatment for the treatment of gastric cancer, the one or more anti-tumor agents being selected from the group consisting of: Molyzumab, pertuzumab, trastuzumab, cetuximab, panitumumab, lexizumab, gefitinib, afatinib, neratinib, lapata Nisin, roxitinib, AZD9291, ASP8273, HM61713 and erlotinib or a pharmaceutically acceptable salt thereof.

In one embodiment, the invention provides an antibody of the invention for use in the treatment of head and neck cancer, preferably HNSCC. In another embodiment, the invention provides an antibody of the invention, For the treatment of KRAS wild type head and neck cancer (preferably, HNSCC). In another embodiment, the invention provides an antibody of the invention for use in the treatment of head and neck cancer, preferably HNSCC, wherein the head and neck cancer (preferably, HNSCC) contains a KRAS activating mutation. In another embodiment, the antibody of the invention is combined with one or more anti-tumor agents simultaneously, separately or sequentially for treatment of head and neck cancer, preferably HNSCC, selected from the group consisting of Groups consisting of: remollozumab, pertuzumab, trastuzumab, cetuximab, panitumumab, lexizumab, gefitinib, afatinib, Linatinib, lapatinib, roxitinib, AZD9291, ASP8273, HM61713 and erlotinib or a pharmaceutically acceptable salt thereof.

In one embodiment, the invention provides an antibody of the invention for use in the treatment of hepatocellular carcinoma, preferably HCC. In another embodiment, the invention provides an antibody of the invention for use in the treatment of KRAS wild-type HCC. In another embodiment, the invention provides an antibody of the invention for use in the treatment of HCC, wherein the HCC comprises a KRAS activating mutation. In another embodiment, the antibody of the invention is combined with one or more anti-neoplastic agents for simultaneous or separate treatment for the treatment of hepatocellular carcinoma, the one or more anti-tumor agents being selected from the group consisting of: : Remoruzumab, Pertuzumab, Trastuzumab, Cetuximab, Panitumumab, Laciciizumab, Gefitinib, Alfatinib, Linatinib, La Patinib, roxitinib, AZD9291, ASP8273, HM61713 and erlotinib or a pharmaceutically acceptable salt thereof.

In one embodiment, the invention provides an antibody of the invention for use in the treatment of colorectal cancer. In another embodiment, the invention provides an antibody of the invention for use in the treatment of KRAS wild-type colorectal cancer. In another embodiment, the invention provides an antibody of the invention for use in the treatment of colorectal cancer, wherein the colorectal cancer contains a KRAS activating mutation. In another embodiment, an antibody of the invention is combined with one or more anti-neoplastic agents for simultaneous or separate or sequential use in the treatment of colorectal cancer, the one or more anti-tumor agents being selected from the group consisting of: : Remoruzumab, Fawkes (Metformin tetrahydrofolate, fluorouracil Pyridine and oxaliplatin), verapamil (hyperilated tetrahydrofolate, fluorouracil and irinotecan), pertuzumab, trastuzumab, cetuximab, panitumumab, Laciciizumab, gefitinib, afatinib, neratinib, lapatinib, roxitinib, AZD9291, ASP8273, HM61713 and erlotinib or a pharmaceutically acceptable salt thereof.

In another embodiment, the invention provides the use of an antibody of the invention for the manufacture of a medicament for the treatment of cancer. In another embodiment, the invention provides the use of an antibody of the invention for the manufacture of a medicament for the treatment of cancer, wherein the cancer is lung cancer (preferably, NSCLC, and more preferably, sq-NSCLC), head and neck cancer (more Good, HNSCC), pancreatic cancer, kidney cancer, stomach cancer, colorectal cancer, breast cancer or HCC. In various embodiments, the invention provides the use of an antibody of the invention for the manufacture of a medicament for the treatment of a wild type cancer of KRAS. In various embodiments, the KRAS wild type cancer is lung cancer (preferably, NSCLC, and more preferably, sq-NSCLC), head and neck cancer (preferably HNSCC), pancreatic cancer, kidney cancer, gastric cancer, colorectal Cancer, breast cancer or HCC. In other embodiments, the invention provides the use of an antibody of the invention for the manufacture of a medicament for the treatment of cancer, wherein the cancer comprises a KRAS activating mutation and the cancer is lung cancer (preferably, NSCLC, and more preferably, sq-NSCLC) Head and neck cancer (preferably HNSCC), pancreatic cancer, kidney cancer, stomach cancer, colorectal cancer, breast cancer or HCC.

In another embodiment, the invention provides the use of an antibody of the invention simultaneously, separately or sequentially in combination with one or more anti-neoplastic agents for the treatment of cancer, the one or more anti-tumor agents selected from the group consisting of Groups of components: cisplatin, carboplatin, lipid doxorubicin, docetaxel, cyclophosphamide and doxorubicin, vonopipine, eribulin, paclitaxel binding protein for injectable suspensions Particles, ixabepilone, capecitabine, remollozumab, vorax (mercaptotetrahydrofolate, fluorouracil, and oxaliplatin), verapamil (methanoin, tetrahydrofolate, fluorouracil) And irinotecan), pertuzumab, trastuzumab, cetuximab, panitumumab, lexizumab, gefitinib, afatinib, natenitny, lapa Tinidil, roxitinib, AZD9291, ASP8273, HM61713 and erlotinib or a pharmaceutically acceptable salt thereof.

In various embodiments, the invention provides an antibody of the invention (preferably, Antibody A), i) for use in therapy, ii) for the treatment of cancer, iii) with an anti-VEGFR2 agent and/or an anti-EGFR agent Simultaneously, separately or sequentially, for the treatment of cancer, iv) combined with anti-VEGFR2 antibodies (including but not limited to) remolizumab and/or anti-EGFR agents simultaneously, separately or sequentially For the treatment of cancer, v) combined with anti-VEGFR2 agent and / or anti-EGFR antibody (including but not limited to, cetuximab, lexizumab, panitumumab) simultaneously, separately or sequentially, For the treatment of cancer, vi) with anti-VEGFR2 antibodies (including but not limited to) remolizumab and/or anti-EGFR antibodies (including but not limited to cetuximab, lexizumab, Pani Monoclonal antibodies) are combined simultaneously, separately or sequentially for the treatment of cancer, vii) simultaneously, separately or sequentially in combination with remollozumab, leprezumab and/or cetuximab for cancer Treatment, viii) simultaneous, separate or sequential combination with remollozumab and cetuximab for the treatment of cancer, ix) simultaneous with remoximab and lexizumab Separately or sequentially for use in the treatment of cancer, x) simultaneous, separate or sequential combination with cetuximab for the treatment of cancer, xi) simultaneous, separate or sequential with remoxeb Combinations for the treatment of cancer, or xii) in combination with levozumab, separately or sequentially, for the treatment of cancer. In various embodiments, the invention provides the above i) to xii), wherein the cancer is KRAS wild type. In certain embodiments, the invention provides the above i) to xii), wherein the KRAS wild type cancer is lung cancer (preferably, NSCLC, and more preferably, sq-NSCLC), head and neck cancer (preferably, HNSCC) , pancreatic cancer, kidney cancer, stomach cancer, colorectal cancer, breast cancer or HCC. In other embodiments, the invention provides the above i) to xii), wherein the cancer contains a KRAS activating mutation. In certain embodiments, the invention provides the above i) to xii), wherein the KRAS mutant cancer is lung cancer (preferably, NSCLC, and more preferably, sq-NSCLC), head and neck cancer (preferably, HNSCC) , pancreatic cancer, kidney cancer, stomach cancer, colorectal cancer, breast cancer or HCC.

The dosage regimen can be adjusted to provide the optimal desired response (eg, therapeutic effect). The therapeutic dose can be titrated using conventional methods known to those skilled in the art to optimize safety and efficacy. Administration schedule for intravenous ( iv ) or non-intravenous administration in topical or systemic or a combination thereof will typically be administered in a single bolus dose or continuous infusion to multiple daily doses (eg, every 4 to 6 hours) Within the scope of, or as directed by the treating physician and the patient. Anti-VEGFR2 Ab (preferably remoleizumab) is generally effective over a wide range of dosages and varies based on the disease condition. An exemplary, non-limiting range of therapeutically effective amounts of ramerizumab is typically in the range of from about 6 mg/kg to 10 mg/kg, preferably from about 8 mg/kg to about 10 mg, for administration every three weeks of circulation. /kg, and optimally about 10 mg/kg. In the combination of the invention, in some cases, a dose lower than the aforementioned lower limit of the anti-VEGFR2 Ab (preferably remolezumab) may be sufficient, and in other cases, it may have acceptable side effects. Smaller or larger doses are used in the case. The amount and frequency of administration can be determined by the physician treating the patient.

In a specific embodiment, the invention provides (a) an antibody of the invention in combination with (b) removumab, simultaneously, separately or sequentially, for use in cancer (preferably, gastric cancer, gastroesophageal junction) Treatment of tumor (GEJ), HCC, lung cancer (preferably, NSCLC), bladder cancer and RCC, and more preferably of its metastatic type, wherein the time period between (a) and (b) administration is For example, less than about 4 hours, less than about 12 hours, about 1 day, about 2 days, about 7 days, about 14 days, about 1 month, or about 2 months. In certain embodiments, prior to the administration of (a) and (b), prior to the administration of (a) or (b), after the administration of (a) or (b), or (a) And after (b), the individual has undergone a surgical resection. In a particular aspect, (a) or (b) or both are preferably administered systemically by means of intravenous infusion. Preferably, a starting dose of (b) between about 200 mg/m ² and about 400 mg/m ² is administered, followed by a weekly infusion between about 150 mg/m ² and 250 mg/m ² . Preferably, a dose ranging from about 6 mg/kg to 10 mg/kg, preferably from about 8 mg/kg to 10 mg/kg, and most preferably about 1 week or biweekly or on the first day of the 21 day cycle, is administered. A dose of 10 mg/kg (b). In other embodiments, the use further comprises another different cancer therapy, such as radiation therapy (preferably, about one week prior to the onset of radiation therapy (b)), another EGFR inhibitor therapy, hormone therapy, or chemotherapy. This includes, but is not limited to, the administration of Verferey, Pacific Paclitaxel or Docetaxel. Any suitable anti-tumor agent can be used, such as a chemotherapeutic agent, radiation, or a combination thereof. The antitumor agents or the evaluated antitumor agents known in the art can be classified into various types including, for example, mitotic inhibitors, alkylating agents, antimetabolites, embedded antibiotics, growth factor inhibitors, cell cycle inhibitors. , aromatase inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormones and anti-angiogenic agents. Examples of alkylating agents include, but are not limited to, cisplatin, cyclophosphamide, melphalan, and dacarbazine. Examples of antimetabolites include, but are not limited to, daunorubicin, gemcitabine, ALIMTA®, and topoisomerase inhibitors including irinotecan (CPT-11), alanine camptothecin (aminocamptothecin), camptothecin, DX-8951f, topotecan (topoisomerase I), etoposide (VP-16) and teniposide (VM-26) (topoisomerase II). Examples of other anti-tumor agents include, but are not limited to, doxorubicin and paclitaxel. When the anti-tumor agent is radiation, the source of the radiation may be external to the patient being treated (external beam radiotherapy - EBRT) or internal (proximity therapy - BT). The dosage of the anti-tumor agent administered will depend, for example, on the type of agent, the type and severity of the tumor being treated, and the factors in which the agent is administered.

In a specific embodiment, the invention provides (a) an antibody of the invention in combination with (b) cetuximab or lesibizumab simultaneously, separately or sequentially for KRAS wild-type colorectal cancer (preferably The treatment of the metastatic type thereof, wherein the period of time between the administration of (a) and (b) is, for example, less than about 4 hours, less than about 12 hours, about 1 day, about 2 days, about 7 Day, about 14 days, about 1 month or about 2 months. In certain embodiments, prior to the administration of (a) and (b), prior to the administration of (a) or (b), after the administration of (a) or (b), or (a) And after (b), the individual has undergone a surgical resection. In a particular aspect, (a) or (b) or both are preferably administered systemically by means of intravenous infusion. Preferably, when (b) is cetuximab, (b) is administered at a starting dose of between about 200 mg/m ² and about 400 mg/m ² , followed by about 150 mg/m ² and about Weekly infusion between 250 mg/m ² . Preferably, when (b) is lacsibizumab, (b) is administered in a dose ranging from 400 mg to 1000 mg on the second or third day of the 21 day cycle, alternatively once a week or Once every two weeks, preferably about 400 mg to 1000 mg is administered on the first, eighth and fifteenth days of the 21 day cycle, more preferably about 600 mg on the first and eighth days of the 21 day cycle. Up to 900 mg, and optimally about 800 mg on the first and eighth days of the 21 day cycle. An additional 21-day cycle can be used to treat patients in need. In other embodiments, the use further comprises another different cancer therapy, such as radiation therapy (preferably, starting about one week prior to initiation of radiation therapy (b)), another EGFR inhibitor therapy, hormone therapy, or chemotherapy ( Includes Frefeld or irinotecan-based chemotherapy. Any suitable anti-tumor agent can be used, such as a chemotherapeutic agent, radiation, or a combination thereof. The antitumor agents or the evaluated antitumor agents known in the art can be classified into various types including, for example, mitotic inhibitors, alkylating agents, antimetabolites, embedded antibiotics, growth factor inhibitors, cell cycle inhibitors. , enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormones and anti-angiogenic agents. Examples of alkylating agents include, but are not limited to, cisplatin, cyclophosphamide, melphalan, and dacarbazine. Examples of antimetabolites include, but are not limited to, daunorubicin, gemcitabine, ALIMTA®, and topoisomerase inhibitors including irinotecan (CPT-11), alanine camptothecin, camptothecin, DX -8951f, topotecan (topoisomerase I), etoposide (VP-16) and teniposide (VM-26) (topoisomerase II). Examples of other anti-tumor agents include, but are not limited to, doxorubicin and paclitaxel. When the anti-tumor agent is radiation, the source of the radiation may be external to the patient being treated (external beam radiotherapy - EBRT) or internal (proximity therapy - BT). The dosage of the anti-tumor agent administered will depend, for example, on the type of agent, the type and severity of the tumor being treated, and the factors in which the agent is administered.

In another specific embodiment, the invention provides (a) an antibody of the invention (preferably, antibody A) in combination with (b) cetuximab or levozumab, simultaneously, separately or sequentially, for use in The treatment of head and neck cancer (preferably, HNSCC), wherein the period of time between (a) and (b) administration is, for example, less than about 4 hours, less than about 12 hours, about 1 day, about 2 days, About 7 days, about 14 days, about 1 month, or about 2 months. In a particular aspect, (a) or (b) or both are preferably administered systemically by means of intravenous infusion. Preferably, when (b) is cetuximab, it is administered at a starting dose of between about 200 mg/m ² and about 400 mg/m ² , followed by about 150 mg/m ² and about 250 mg/m. A weekly infusion between ² . Preferably, when (b) is lacsibizumab, (b) is administered in a dose ranging from 400 mg to 1000 mg on the second or third day of the 21 day cycle, alternatively one dose per week or Once every two weeks, preferably from about 400 mg to 1000 mg on the first, eighth and fifteenth days of the 21 day cycle, more preferably from about 600 mg to 900 mg on the first and eighth days of the 21 day cycle. And preferably about 800 mg on the first and eighth days of the 21 day cycle. An additional 21-day cycle can be used to treat patients in need. In other embodiments, the use further comprises additional different cancer therapies, such as radiation therapy (preferably, starting with (b) about one week prior to initiation of radiation therapy), other EGFR inhibitor therapy, hormone therapy, or chemotherapy ( Includes platinum-based chemotherapy (eg, cisplatin, oxaliplatin or vorax)).

In another specific embodiment, the invention provides (a) an antibody of the invention (preferably, antibody A) in combination with (b) cetuximab or levozumab, simultaneously, separately or sequentially, for use in The treatment of NSCLC (preferably, squamous cell NSCLC (sq-NSCLC)) wherein the period of time between (a) and (b) administration is, for example, less than about 4 hours, less than about 12 hours, about 1 day, about 2 days, about 7 days, about 14 days, about 1 month, or 2 months. In a particular aspect, (a) or (b) or both are preferably administered systemically by means of intravenous infusion. Preferably, when (b) is cetuximab, (b) is administered at a starting dose of between about 200 mg/m ² and about 400 mg/m ² , followed by about 150 mg/m ² and about Weekly infusion between 250 mg/m ² . Preferably, when (b) is lacsibizumab, (b) is administered in a dose ranging from 400 mg to 1000 mg on the second or third day of the 21 day cycle, alternatively one dose per week or Once every two weeks, preferably from about 400 mg to 1000 mg on the first, eighth and fifteenth days of the 21 day cycle, more preferably from about 600 mg to 900 mg on the first and eighth days of the 21 day cycle. And preferably about 800 mg on the first and eighth days of the 21 day cycle. An additional 21-day cycle can be used to treat patients in need.

In other embodiments, the use further comprises additional different cancer therapies, such as radiation therapy (preferably, starting with (b) about one week prior to initiation of radiation therapy), other EGFR inhibitor therapy, hormone therapy, or chemotherapy ( Platinum-based chemotherapy (eg, cisplatin, oxaliplatin or vortex) and/or antimetabolite chemotherapy (eg, gemcitabine) are included. In the above examples, wherein (b) (i.e., cetuximab or leprezumab) is administered at repeated time intervals (e.g., during a standard course of treatment), (a) (i.e., an antibody of the invention, Preferably, antibody A) can be administered prior to each administration of (b). Where (b) is administered at repeated intervals (eg, during a standard course of treatment), (a) may be administered concurrently at each of (b). Where (b) is administered at repeated intervals (eg, during a standard course of treatment), (a) may be administered after each dose of (b). Where (b) is administered at repeated intervals (eg, during a standard course of treatment), (a) may be administered prior to, concurrently with, or after a combination of each of (b). Where (b) is administered at repeated intervals (eg, during a standard course of treatment), (a) may be administered at different time intervals relative to the therapy using (b). Where (b) is administered at repeated intervals (eg, during a standard course of treatment), (a) may be administered in a single or series of doses before, during, or after the treatment with (b). Where (b) is administered at repeated intervals (eg, during a standard course of treatment), (a) may be administered in a single dose before, during, or after the treatment process using (b). Where (b) is administered at repeated intervals (eg, during a standard course of treatment), (a) may be administered in a single dose prior to the course of treatment with (b). Where (b) is administered at repeated intervals (eg, during a standard course of treatment), (a) may be administered in a single dose at any time during the course of treatment with (b). Where (b) is administered at repeated intervals (eg, during a standard course of treatment), (a) may be administered in a single dose following the course of treatment with (b). Where (b) is administered at repeated intervals (eg, during a standard course of treatment), (a) may be administered in a series of doses prior to the course of treatment using (b). Where (b) is repeated at intervals (eg For example, during the standard course of treatment, (a) may be administered in a series of doses following the course of treatment with (b). Where (b) is administered at repeated intervals (eg, during a standard course of treatment), (a) may be administered in a series of doses following the course of treatment with (b).

In another embodiment, the invention provides the use of an antibody of the invention (preferably, antibody A) simultaneously, separately or sequentially in combination with one or more anti-neoplastic agents for the manufacture of a medicament for the treatment of cancer The one or more anti-tumor agents are selected from the group consisting of cisplatin, carboplatin, lipid doxorubicin, docetaxel, cyclophosphamide and doxorubicin, vonopine, and eribulin , paclitaxel protein-binding particles for injectable suspensions, ixabepilone, capecitabine, remollozumab, vorax (mercaptotetrahydrofolate, fluorouracil, and oxaliplatin) Feirui (hyperthyroid tetrahydrofolate, fluorouracil and irinotecan), pertuzumab, trastuzumab, cetuximab, panitumumab, lexizumab, gefitinib, Afatinib, neratinib, lapatinib, roxitinib, AZD9291, ASP8273, HM61713 and erlotinib or a pharmaceutically acceptable salt thereof.

The antibodies of the invention are engineered non-naturally occurring polypeptide complexes. Unless otherwise indicated, the term "antibody" refers to an immunoglobulin molecule comprising two heavy chains and two light chains interconnected by a disulfide bond. The amino terminus portion of each chain includes a variable region of from about 110 to about 120 amino acids which is primarily responsible for recognizing the antigen by virtue of the complementarity determining regions (CDRs) contained therein. The carboxy terminal portion of each chain defines a constant region that is primarily responsible for effector function.

The term "antigen-binding fragment" as used herein refers to any antibody fragment that retains the ability to bind to its antigen. The "antigen-binding fragments" may be selected from the group consisting of Fv, scFv, Fab, F(ab') ₂ , Fab', scFv-Fc fragments and bifunctional antibodies. An antigen-binding fragment of an antibody typically comprises at least one variable region. Preferably, the antigen-binding fragment comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR). More preferably, the antigen-binding fragment as used herein comprises HCVR and LCVR, which confers human HER3 (i.e., "human HER3 binding fragment") or human HER3-ECD (i.e., "human HER3-ECD binding fragment" ) antigen binding specificity.

As used herein, the term "light chain variable region (LCVR)" refers to an antibody molecule comprising an amino acid sequence of a complementarity determining region (CDR; ie, LCDR1, LCDR2, and LCDR3) and a framework region (FR). Part of the LC.

As used herein, the term "heavy chain variable region (HCVR)" refers to an antibody molecule comprising an amino acid sequence of a complementarity determining region (CDR; ie, HCDR1, HCDR2 and HCDR3) and a framework region (FR). Part of HC.

The antibodies of the invention are designed to have engineered CDRs and have a portion of a human original antibody (either all or part of the framework, hinge region and constant region) that is equivalent or substantially identical to the (substantially human) source The framework, hinge region and constant region of the human genome sequence. The whole human framework, hinge region and constant region are sequences of their human germline sequences as well as sequences with naturally occurring somatic mutations and those having engineered mutations. An antibody of the invention may comprise a framework, hinge or constant region derived from a fully human framework, hinge or constant region containing one or more amino acid substitutions, deletions or additions thereto. Furthermore, the antibodies of the invention are substantially preferably non-immunogenic in humans.

The antibodies of the invention are IgG type antibodies and have four amino acid chains (two "heavy" chains and two "light" chains) interconnected by intrachain and interchain disulfide bonds. Each heavy chain consists of an N-terminal HCVR and a heavy chain constant region ("HCCR"). Each light chain consists of an LCVR and light chain constant region ("LCCR"). An antibody having a native human Fc sequence is glycosylated in the Fc region when expressed in certain biological systems. Typically, glycosylation occurs in the highly conserved N-glycosylation site in the Fc region of the antibody. N-glycans are usually linked to asparagine. Antibodies can also be glycosylated at other locations.

In some embodiments, an antibody of the invention comprises an Fc portion derived from a human IgG ₁ Fc region. Furthermore, in some embodiments, the antibodies of the invention comprise an IgG ₁ heavy chain having a C-terminal deaminase.

The HCVR region and the LCVR region can be further subdivided into highly variable regions called complementarity determining regions ("CDRs"). As used herein, the terms "complementarity determining region" and "CDR" refer to a non-contiguous antigen combining site found within the variable regions of the LC and HC polypeptides of the antibody or antigen-binding fragment thereof. Such specific regions have been described by other literature, including: Kabat et al, Ann. NY Acad. Sci. 190:382-93 (1971); Kabat et al, J. Biol. Chem. 252:6609-6616 ( 1977); Kabat et al., Sequences of Proteins of Immunological Interest , Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242 (1991); Chothia et al., J .Mol. Biol. 196:901-917 (1987); MacCallum et al, J. Mol. Biol. 262:732-745 (1996); and North et al, J. Mol. Biol., 406, 228-256 (2011) And wherein the definitions comprise an overlap or subset of amino acid residues when compared to each other.

The CDRs are interspersed with more conserved regions called framework regions ("FR"). Each HCVR and LCVR consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Here, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and HCDR3" and the three CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3." The CDRs contain most of the residues that specifically interact with the antigen. The Kabat CDR definition (Kabat et al., "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991)) is based on antibody sequence variability. Chothia CDR definition (Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins", Journal of Molecular Biology, 196, 901-917 (1987); Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)) is based on the three-dimensional structure of antibodies and the topology of CDR loops. In addition to HCDR1 and HCDR2, the Chothia CDR definition is equivalent to the Kabat CDR definition. North CDR definition (North et al., "A New Clustering of Antibody CDR Loop Conformations, Journal of Molecular Biology, 406, 228-256 (2011)) is based on an affinity-expanded cluster with a large number of crystal structures.

For the purposes of the present invention, the amino acid arrays of the CDR domains within the LCVR and HCVR regions of the antibodies of the invention are based on the recognized Kabat numbering protocol (Kabat et al., Ann. NY Acad. Sci. 190:382-93). (1971); Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242 (1991)) and North Numbering Protocol (North et al., A New Clustering) Of Antibody CDR Loop Conformations, Journal of Molecular Biology, 406:228-256 (2011)). For the light chain CDRs of the antibodies of the invention, the North CDR definition is used. The North definition is also used in both heavy chain HCDR1 and HCDR3. HCDR2 uses a mixture of North and Kabat definitions. The North definition is used to identify the starting N-terminal site, and Kabat is used to define the final position.

The underlined and bolded amino acid shown below was engineered to the CDRs of the antibodies of the invention and produced a hydrophobically modified distribution that exhibited a significant improvement without significant adverse effects on HER3-ECD binding properties (ie, Antibodies that reduce hydrophobicity: HCDR1, AASGFSL D TYTMI (SEQ ID NO: 8); HCDR2, IIYV T YNTYYANWAKG (SEQ ID NO: 9); HCDR3, TRWGD Q NGLDI (SEQ ID NO: 10); LCDR1, QSSQ R VYGNKNLA (SEQ ID NO: 11); and LCDR2, YFA R TLAS (SEQ ID NO: 12).

A DNA molecule of the invention is a non-naturally occurring DNA molecule comprising a polynucleotide sequence encoding a polypeptide having an amino acid sequence of at least one variable region polypeptide of an antibody of the invention. The isolated DNA encoding the HCVR region can be converted to a full-length heavy chain gene by operably linking the DNA encoding HCVR to another DNA molecule encoding the heavy chain constant region. Sequences of human and other mammalian heavy chain constant region genes are known in the art. DNA fragments encompassing such regions can be obtained, for example, by standard PCR amplification.

The isolated DNA encoding the LCVR region can be converted into a full-length light chain gene by operably linked to a DNA encoding a light chain constant region to another DNA molecule by DNA encoding the LCVR. Sequences of human (and other mammalian) light chain constant region genes are known in the art. DNA fragments encompassing such regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

A polynucleotide of the invention can be expressed in a host cell after it has been operably linked to a expression control sequence. The expression vector can typically replicate in the host organism as either an episomal or an integral part of the host chromosomal DNA. Typically, the expression vector will contain a selectable marker (eg, tetracycline, neomycin, and dihydrofolate reductase) to permit detection of the cells of the desired DNA sequence switch.

The antibody of the present invention can be easily produced in mammalian cells such as CHO, NSO, HEK293 or COS cells. Host cells can be cultured using techniques well known in the art.

Vectors containing a polynucleotide sequence of interest (eg, a polypeptide encoding an antibody and a polynucleotide displaying a control sequence) can be transferred to a host cell by well-known methods, depending on the type of cellular host. Variety.

Various methods of protein purification can be employed and such methods are known in the art and are described, for example, in Deutscher, Methods in Enzymology 182:83-89 (1990) and Scopes, Protein Purification: Principles and Practice , 3rd edition, Springer , NY (1994).

In another embodiment of the invention, the antibody or nucleic acid encoding the same is provided in isolated form. As used herein, the term "isolated" refers to a protein, peptide or nucleic acid that is free or substantially free of any other macromolecular species found in the cellular environment. "Substantially free" as used herein means that the protein, peptide or nucleic acid of interest comprises between about 80% and about 99% (in moles) of macromolecular material, preferably about 90. Between about 99% and, more preferably, between about 95% and about 99%.

The antibody of the present invention or a pharmaceutical composition comprising the same can be administered by a parenteral route (for example, subcutaneously and intravenously). The antibodies of the invention may be administered to a patient in single or multiple doses using only pharmaceutically acceptable carriers, diluents or excipients. The pharmaceutical compositions of the present invention can be prepared by methods well known in the art (for example, Remington: The Science and Practice of Pharmacy , 19th Ed. (1995), Gennaro, A. et al., Mack Publishing CO.) and include The antibodies disclosed herein and one or more pharmaceutically acceptable carriers, diluents or excipients.

As used herein, the term "patient" refers to a mammal, preferably a human.

As used herein, the terms "cancer" and "cancerous" refer to or describe a physiological condition in a patient that is typically characterized by unregulated cell proliferation. Benign and malignant cancers are included in this definition. "early cancer" or "early stage tumor" means a cancer that is not advanced or metastatic or classified as stage 0, I, or stage II cancer. Examples of cancer include, but are not limited to, gastric cancer, lung cancer (including NSCLC and sq-NSCLC), head and neck cancer (including HNSCC), pancreatic cancer, kidney cancer, breast cancer, or colorectal cancer or HCC.

The term "treating, treating, treating" refers to inhibiting, delaying, interrupting, arresting, alleviating, terminating, ameliorating or reversing the progression or severity of an existing condition, disorder, condition or disease.

As used herein, the term "K _D" means a particular antibody - antigen interactions equilibrium dissociation constant of - antigen or antibody fragments.

The term "binding" as used herein with reference to the affinity of an antibody to human HER3, unless otherwise indicated, is intended to mean, for example, by conventional methods known in the art (including by substantially as described herein at 25 determined using surface plasmon resonance (SPR) biosensor) or at 37 [℃ ℃ of less than about 1 x 10 ^-8 K _D M of, preferably, less than about 5 x 10 ^-9 M, more Good place, less than about 1 x 10 ^-9 M. The term "selective" or "selectively" used herein with reference to the compounds of the present invention the compound used (such as a human HER3 and / or human HER3-ECD) refers to binding of target, K _D ratio of the compound bound to other proteins ( A compound comprising a member of the HER/EGFR family, such as human HER2), is about 1000-fold, about 500-fold, about 200-fold, about 100-fold, about 50-fold or about 10-fold lower, such as at 25 ° C or 37 ° C by surface The plasmon resonance is measured. Additionally or alternatively, the HER3 selective antibody of the invention binds to human HER3 but does not bind or only minimally binds to human HER2 when analyzed by the methods described in the Examples below.

The terms "HER3", "HER3 polypeptide", "ErbB3" or "ErbB3 polypeptide" are used interchangeably herein and, unless otherwise indicated, refer to human receptor tyrosine protein kinase that binds to human neuromodulin-1. The mature form of erbB-3 (i.e., does not include a signal peptide) as well as its mutant and/or truncated form. Specific examples of HER3 include, for example, a human polypeptide encoded by the nucleotide sequence provided in NCBI GenBank Accession No. M34309, a human HER3 protein encoded by the polypeptide sequence provided in NCBI GenPept Accession No. AAA35979, and/or having, for example, SEQ ID NO: The polypeptide of the amino acid sequence shown in 16. The nucleotide sequences encoding the murine, rhesus and cynomolgus HER3 proteins are correspondingly provided by NCBI Genbank Accession Nos. AY686636.1, AFI35044.1 and EHH66388.1.

The extracellular region of mature human HER3 has an amino acid sequence as shown, for example, in SEQ ID NO: 7. HER3 proteins lacking their signal peptides and/or HER3 proteins fused to one or more other peptides (usually at the C-terminus of the HER3 protein) are frequently produced to aid in purification and/or other laboratory procedures. Thus, unless specifically stated otherwise, the phrase "human HER3-ECD" refers to a HER3 protein with or without a signal peptide, including but not limited to, as shown in SEQ ID NO: 7, 21, 22 or 23. One or more human HER3-ECD proteins.

As used herein, the phrase "effective amount" means the invention that will elicit the biological, medical response or desired therapeutic effect of a tissue, system, animal, mammal or human being sought by a researcher, medical physician or other clinician. The amount of the antibody or pharmaceutical composition comprising the antibody of the invention. The effective amount of the antibody can be based on, for example, the individual's disease condition, age, Gender and body weight and the factors that influence the ability of an antibody to elicit a desired response in an individual. An effective amount is also an amount that is therapeutically beneficial over any toxic or damaging effect of the antibody. The effective amount can be readily determined by the attending medical physician, the diagnostician, or other clinician (e.g., those skilled in the art) by the use of known techniques and by observing results obtained under similar circumstances. In determining the effective amount for the patient, the attending diagnostician considers a number of factors including, but not limited to, the patient's species; its size, age, and general health; the particular disease or condition involved; the degree or disease involved Or the severity of the condition; the response of the individual patient; the particular compound administered; the mode of administration; the bioavailability characteristics of the formulation being administered; the chosen dosing regimen; the use of the concomitant medication; and other relevant circumstances.

As used herein, the phrase "in combination with" refers to the administration of an antibody of the invention, or a pharmaceutically acceptable formulation thereof, concurrently with another therapeutic agent. As used herein, the phrase "in combination with" also means that the antibody of the invention, or a pharmaceutically acceptable formulation thereof, is administered sequentially with another therapeutic agent in any order. As used herein, the phrase "in combination with" also means that the antibody of the invention, or a pharmaceutically acceptable formulation thereof, is administered in combination with another therapeutic agent in any combination thereof. The antibody of the invention or a pharmaceutically acceptable formulation thereof can be administered prior to administration of another therapeutic agent. The antibody of the invention or a pharmaceutically acceptable formulation thereof can be administered concurrently with the administration of another therapeutic agent. The antibody of the invention or a pharmaceutically acceptable formulation thereof can be administered after administration of another therapeutic agent. The antibody of the invention, or a pharmaceutically acceptable formulation thereof, can be administered prior to, concurrently with, or subsequent to the administration of another therapeutic agent, or administered in some combination thereof.

As used herein, the term "effective response" or "response" or "therapeutic effect" of a patient with respect to a therapeutic term using an antibody of the invention means one or more of the patients assigned to the patient after administration of each of the following Clinical or therapeutic benefit: i) an antibody of the invention, preferably antibody A; ii) an antibody of the invention (preferably, antibody A) in combination with an anti-VEGFR2 agent and/or an anti-EGFR agent; iii) the invention Antibody (preferably, antibody A) and anti-VEGFR2 antibody And/or an anti-EGFR agent combination; iv) an antibody of the invention (preferably, antibody A) in combination with an anti-VEGFR2 agent and/or an anti-EGFR antibody; v) an antibody of the invention (preferably, antibody A) and an antibody a VEGFR2 antibody and/or an anti-EGFR antibody combination; vi) an antibody of the invention (preferably, antibody A) in combination with remolizumab, lesibizumab and/or cetuximab; vii) an antibody of the invention (preferably, antibody A) in combination with remolizumab and cetuximab; viii) an antibody of the invention (preferably, antibody A) in combination with remollozumab and lexizumab; ix) The antibody of the present invention (preferably, Antibody A) is combined with cetuximab; x) the antibody of the present invention (preferably, Antibody A) is combined with remollozum; or xi) the antibody of the present invention ( Preferably, antibody A) is combined with lacizumab. In a particular aspect, the present invention is a therapy having a combination of the intrinsic synergies described herein, although in alternative embodiments the combination provides additional benefits for therapy. Such benefits include any one or more of the following: prolonging survival (including overall survival and progression-free survival); producing objective responses (including complete or partial responses); tumor regression, tumor weight or size reduction Increased disease progression time; increased duration of survival; increased progression-free survival; improved overall response rate: increased duration of response and improved quality of life and/or improved signs or symptoms of cancer.

The unexpected therapeutic effect of the combination therapies of the present invention is to produce significant anti-cancer effects in the patient without causing significant toxicity or adverse effects, so that the patient generally benefits from the combined therapeutic approach. Efficacy (i.e., one or more therapeutic effects of the combination therapies of the invention) can be measured by assessing various indicators typically used in cancer treatment, including but not limited to tumor regression, tumor weight, or Size reduction, disease progression time, overall survival, progression free survival, overall response rate, duration of response, and/or quality of life. The antibody of the present invention and the combination therapy of the present invention can cause inhibition of metastatic spread in the case where the primary tumor is not reduced, can cause a reduction in the primary tumor, or can exert a tumor stabilizing effect at all. Because the present invention (in some embodiments) is for the use of a combination of unique anti-tumor agents, novel methods can be used to determine the efficacy (i.e., therapeutic effect) of any particular combination therapy of the present invention, such methods. Including plasma or urinary markers such as angiogenesis The measurement is measured and measured by means of radiographic imaging.

As used herein, the term "complete response" (CR) refers to the disappearance of all target lesions. Any pathological lymph node (whether target or non-target) must reduce the short axis to <10 mm.

As used herein, the term "partial reaction" (PR) refers to a reduction in the sum of the diameters of the target lesions by at least 30%, based on the reference total diameter.

As used herein, the term "progressive disease" (PD) refers to the minimum sum of studies (if the sum of the benchmarks is the smallest sum of the studies, this includes the baseline sum) as a reference, and the sum of the diameters of the target lesions is increased by at least 20 %. In addition to the relative increase of 20%, the sum must also show an absolute increase of at least 5 mm. For the avoidance of doubt, the morphology of one or more new lesions is also considered progressive.

As used herein, the term "stable disease" (SD) refers to the study of the sum of the smallest diameters as a reference, neither sufficiently reduced to qualify for the PR nor adequately increased to qualify the PD.

As used herein, the term "objective response" (OR) refers to the sum of CR plus PR.

Those skilled in the art will appreciate that the terms CR, PR, PD, SD, and OR correspond to the definitions according to RECIST v1.1, Eisenhauer et al, European Journal of Cancer , 2009, 45 , 228-247.

As used herein, the term "time to disease progression" or "TTP" refers to the time from the time of initial treatment until the progression or worsening of the cancer (usually measured in weeks or months). Such progression can be assessed by a skilled clinician.

As used herein, the term "prolonged TTP" refers to increasing the time to disease progression in a treated patient relative to i) an untreated patient or relative to ii) a patient treated with less than all of the anti-tumor agents in a particular combination therapy.

As used herein, the term "survival" refers to a patient remaining alive and includes overall survival as well as progression free survival.

As used herein, the term "overall survival" refers to the time of self-diagnosis or treatment. From time to time, the patient remains alive for a defined period of time, such as 1 year, 5 years, and so on.

As used herein, the term "progression free survival" refers to a patient who remains alive without cancer progressing or becoming worse.

As used herein, the term "prolonged survival" means relative to i) untreated patients, ii) patients treated with less than all antitumor agents in a particular combination therapy, or iii) controlled treatment regimens, increased treatment Overall or progression-free survival of the patient. At least about one month, at least about one month, at least about two months, at least about four months, at least about six months, at least about nine months, or at least about after the initiation of treatment or after the onset of diagnosis of cancer Survival is monitored for 1 year, or at least about 2 years, or at least about 3 years, or at least about 4 years, or at least about 5 years, or at least about 10 years.

As used herein, the term "primary tumor" or "primary cancer" means the original cancer and is not a metastatic disease located in another tissue, organ or location in the patient's body.

As used herein, the term "remolizumab" (also known as Cyramza®, IMC-1121b, and/or CAS Registry Number 947687-13-0) is a human antibody to human VEGFR2, which comprises: Two light chains, each having the amino acid sequence given in SEQ ID NO: 19; and two heavy chains, each having the amino acid sequence given in SEQ ID NO: 20. Remorifuzumab and methods of making and using remollozumab (including treatments for neoplastic diseases such as solid and non-solid tumors) are disclosed in WO 2003/075840. In addition, the clinical activity of remollozumab has also been reported in patients with several cancer types including gastric cancer and GEJ and HCC, NSCLC and RCC. Cyramza® is approved by the US FDA as a single agent or in combination with paclitaxel for the treatment of patients with advanced or metastatic gastric or gastroesophageal (GE)-bound adenocarcinoma, including disease progression At or after fluoropyrimidine or platinum-containing prior chemotherapy; and in combination with docetaxel, indicated for the treatment of patients with metastatic NSCLC, where the disease progresses at or after platinum-based chemotherapy.

As used herein, the term "anti-VEGFR2 Ab" refers to an antibody comprising LCVR and HCVR, wherein anti-VEGFR2 Ab binds to VEGFR2 with sufficient affinity and specificity. In some embodiments, the anti-VEGFR2 Ab is an antibody comprising: a light chain, the amino acid sequence of which is given in SEQ ID NO: 19, and a heavy chain, Its amino acid sequence is given in SEQ ID NO 20. In other embodiments, the anti-VEGFR2 Ab is remollozumab. The selected antibody will have a sufficiently strong binding affinity for VEGFR2. For example, the antibody value of the K _d generally between about 1pM to about 100nM binding VEGFR2. Antibody affinity may be based on the analysis by the surface plasmon resonance (such as those described in PCT Application No. Biacore ^TM Analysis of Publication No. WO2005 / 012359), for example; enzyme-linked immunosorbent assay (ELISA); and competition assays (e.g., Radiolabeled antigen binding assay (RIA) was used to determine. In one embodiment, Kd is measured by RIA performed with anti-VEGFR2 Ab (preferably, remollodomab).

As used herein, the term "DC101" or "LSN3180389" refers to a rat monoclonal antibody against mouse VEGFR2, which can be used as a small anti-VEGFR2 Ab (preferably, remollozumab) in the experiment. An alternative to rats. See, for example, Witte , L. et al., Cancer Metastasis Rev., 17: 155-161 (1998); Prewett , M. et al., Cancer Res., 59: 5209-5218 (1999).

As used herein, the term "lacsibizumab" refers to a recombinant IgG1 human monoclonal antibody that targets the epidermal growth factor receptor (EGFR). Lacepsin monoclonal antibody and methods of making and using the same, including treatments for neoplastic diseases such as solid and non-solid tumors, are disclosed in US 7,598,350. Lacey mAb is also known as IMC-11F8 and/or CAS Registry Number 906805-06-9. In addition, the clinical activity of lexizumab has also been reported in patients with NSCLC (Thatcher, N. et al., J Clin Oncol 32.5 Supplement (2014) and ASCO Demonstration, Abstract 8008, 2014 Discussion "A randomized, multicenter, open -label,phase III study of gemcitabine-cisplatin(GC)chemotherapy plus necitumumab(IMC-11F8/LY3012211)versus GC alone in the first-line treatment of patients(pts)with stage IV squamous non-small cell lung cancer(sq- NSCLC)").

As used herein, the term "cetuximab" (also known as Erbitux®, antibody C225 and/or IMC-C225) refers to a murine anti-EGFR antibody having a human IgG1 heavy chain and a kappa light chain constant region. The Fv region consists of a chimeric mouse/human anti-EGFR monoclonal antibody and has a molecular weight of about 152 kDa. Cetuximab specifically binds to the extracellular domain of human EGFR and is an EGFR inhibitor that prevents ligand binding to EGFR and prevents receptor activation and inhibits the growth of tumor cells that express EGFR. Cetuximab has been approved with or without irinotecan for the treatment of patients with metastatic colorectal cancer with epidermal growth factor receptors, which are refractory or intolerable irinotecan-based chemotherapy .

The invention is further illustrated by the following non-limiting examples.

Example 1: Antibody performance and purification

The complete HC and LC amino acid sequences of the polypeptides of the variable regions of the heavy and light chains, anti-HER3 antibody A (also referred to herein as antibody A), and the nucleotide sequences encoding the above are listed below. In the section "Amino acid and nucleotide sequence". Furthermore, SEQ ID NO of the amino acid sequence corresponding to LC, HC, LCVR and HCVR of Antibody A is shown in Table 1.

Antibodies of the invention, including but not limited to, antibody A, can be prepared and purified essentially as follows. Transiently or stably transfected by a system of expression systems for secreting antibodies using a best predetermined HC:LC vector ratio (such as 1:3 or 1:2) or a single vector system encoding both HC and LC Host cells (such as HEK 293 or CHO). One of a variety of commonly used techniques can be used to purify the clarified medium to which the antibody is secreted. For example, the medium can be suitably applied to a MabSelect column (GE Healthcare) or a KappaSelect column (GE Healthcare) for Fab fragments which have been compatible with, for example, phosphate buffered saline (pH 7.4). The buffer is in equilibrium. The column can be washed to remove non-specific binding components. The cell-binding antibody can be lysed, for example, by a pH gradient such as 20 mM Tris buffer at pH 7 to 10 mM sodium citrate buffer at pH 3.0, or phosphate buffered saline at pH 7.4 to 100 mM glycine buffer at pH 3.0. . Can be (for example) by SDS- Antibody fractions are detected by PAGE and can then be combined. Further purification is optional, depending on the desired use. The antibodies can be concentrated and/or sterile filtered using common techniques. Soluble aggregates and multimers can be efficiently removed by common techniques including size exclusion, hydrophobic interaction, ion exchange, multimodal or hydroxyapatite chromatography. The purity of the antibody after such chromatographic steps is between about 95% and about 99%. The refrigerated product can be kept (freeze immediately at a temperature of -70 ° C) or can be freeze-dried.

Anti-HER3 Antibody Binding Kinetics, Affinity and Ligand Binding Enhancement Analysis A. Determination of binding kinetics by surface plasmon resonance (SPR)

The binding kinetics of the anti-HER3 antibodies of the invention to human, mouse and/or cynomolgus HER3-ECD can be achieved by using surface plasmon resonance biosensors (such as BIAcore® 2000, according to methods known in the art). Determined by BIAcore® 3000 or BIAcore® T100 (GE Health Care, Piscataway, NJ). In addition, as described, all reagents and materials are available from Biacore ^® and may be measured at 25 deg.] C or 37 ℃.

Human and mouse HER3-ECD proteins containing a C-terminal 6x histidine tag (such as those shown in SEQ ID NOs: 22, 23 and 24, respectively) can be expressed in HEK293 cells and purified by charged nickel IMAC . Rhesus mature HER3-ECD, which is 100% identical to rhesus mature HER3-ECD, is commercially available from Sino Biological (Beijing, P.R. China; catalog number; 90043-K08H).

The surface of the protein A used for the extraction of the antibody can be prepared by the following method. Soluble Protein A (Calbiochem, Cat. No. 539202) can be immobilized on CM4 (Biacore #BR-1005-39) using the manufacturer's recommended EDC/NHS amine coupling method (Biacore, catalog number: BR-1000-50). on. For example, the surface of all four flowing cells can be borrowed The cells were cleaned by three consecutive 30 second injections of glycine buffer at pH 1.5. The activated surface can then be injected by a 1:1 mixture of EDC/NHS for seven minutes. Thereafter, soluble protein A (diluted to 50 μg/mL in 10 mM acetate buffer at pH 4.5) was fixed via lateral flow cells (Fc) 1 and 2 injected for three minutes. The unreacted sites remaining on the surface of the sheet can be blocked by a seven minute injection of ethanolamine. The working buffer can be HBS-EP+ (Biacore #BR-1006-69) at 25 °C. Approximately 400 RU to 600 RU of Protein A amine group can be coupled to the CM4 flakes.

Antibody A can be diluted to 1 μg/mL in the operating buffer. Human, mouse, and rhesus monkey (cynomolgus) soluble HER3-ECD ligands at discrete concentrations ranging from 125 nM to 0.24 nM can be prepared using two-fold serial dilutions of the pour into the processing buffer. The meter can be equilibrated to 37 ° C and each power cycle can consist of a series of four separate steps: (1) extracting antibody A only on Fc2; (2) injecting above flow cells 1 and 2 at 100 μL/min (using Kinject) 200 μL (2 min surface contact time) discrete concentrations of HER3-ECD; (3) returned to the operating buffer for 10 minutes to monitor the dissociated phase; and (4) with 10 mM pH 1.5 glycine for 30 sec contact time Regenerate the surface of the wafer. The data obtained were processed using standard double references and adapted to the 1:1 binding model using BiaEvaluation software version 4.1 to determine the association rate (K _on , M ^-1 s ^-1 units), dissociation rate (K _off , s ^{- 1} unit) and the maximum binding level (Rmax). The calculation of the equilibrium dissociation constant (K _D ) can be calculated according to the following relationship: K _D =K _off /K _on and expressed in units of moles.

Antibody A with HER3-ECD as shown in SEQ ID NO: 23, rhesus monkey HER3-ECD (ie, cynomolgus HER3-ECD) and SEQ as SEQ, when substantially determined using the methods described above. ID NO: 24 mice demonstrated the mature HER3-ECD binding, each having three 1.6nM, 2.1nM and 3.0nM of substantially similar K _D (table 2). The binding stoichiometry for all three HER3-ECD ligands was close to 2 (per mole of A2 molar ligand), indicating that Antibody A binds both HER3-ECD ligands simultaneously.

Table 2

B. Anti-HER3 Antibody A Enhances NRG1 Binding

The ability of a HER antibody to inhibit or potentiate the binding of biotinylated NRG1 to a disk-coated HER3-ECD in an ELISA format can be determined substantially as follows.

Human HER3-ECD protein can be conjugated to an ELISA plate at 2 μg/mL overnight at 4 °C. The plates were washed and then blocked with a Blotto (Thermo) blocker for 1 hour at 37 °C. The plate can be washed again and the dose titration of the anti-HER3 antibody can be added at a 2X concentration followed by the addition of biotinylated NRG1 to give a final concentration of 50 ng/mL. The culture plates can be incubated with shaking at room temperature for 2 hours and then washed. The streptavidin-HRP can be added to the culture dish and incubated with shaking for 30 minutes at room temperature. The plates were washed and then developed by the addition of 3,3',5,5'-tetramethylbenzidine (TMB) (SurModics) followed by the addition of a stop solution after 20 minutes. The plate can be read at OD 450 nm.

As depicted in Figure 1, anti-HER3 antibody A does not block the binding of biotinylated NRG1 to culture plate-coated human HER3-ECD (as shown in SEQ ID NO: 23), but the effect is to enhance the organism. The labeled NRG1 binds to the enhanced binding of human HER3-ECD to the culture plate. In contrast, HER3 antibodies 2C2, U1-59 and D11f (see WO 2013/078191, WO 2007/077028 and US 2010/0255010, respectively) inhibit the binding of NRG1 to human HER3-ECD. MOR10703_A50V_N52S (see US 2012/0107306) HER3 antibodies neither inhibit nor enhance the binding of NRG1 to human HER3-ECD.

C. In vitro properties of anti-HER3 antibodies against ligand-dependent and ligand-independent HER3-HER2 heterodimerization

The activity of the anti-HER3 antibody for HER3-HER2 heterodimerization in both ligand-dependent and ligand-independent cases can be substantially measured in the luciferase complementation assay as follows. Briefly, from the HEK293 stable selection (which expresses HER3 and HER2, which uses the N-terminal firefly luciferase-labeled HER3, and the C-terminal firefly luciferase-labeled HER2), 400,000 cells per well can be inoculated in 0.4 mL. 24 well culture plate. After inoculation of the inoculated cells overnight at 37 ° C, 35 μg / mL of test and control antibody can be added and cultured at 37 ° C for 1 hour, followed by 300 ng / ml NRG1 (R & D system; Minneapolis, MN; catalog number 377-HB / CF) was stimulated for 5 minutes, and then luciferase activity for ligand-dependent analysis was measured (Fig. 2A). Alternatively, 35 μg/mL of antibody can be added for incubation for 1 hour at 37 ° C, followed by measurement of ligand-independent analysis of luciferase activity ( FIG. 2B ). Luciferase activity was measured using a Steadylight kit (Perkin Elmer 6016751).

The results show that anti-HER3 antibody A can inhibit the heterodimerization of HER3 and HER2, that is, NRG1 ligand-dependent (Fig. 2A) or ligand-independent (Fig. 2B) and other anti-HER3 antibodies MOR10703_A50V_N52S, U1-59 and Ab#6 (see US 2012/0107306, WO 2007/077028 and WO 2008/100624) is as good or better. When the HER3 intracellular domain does not have potent tyrosine kinase activity, heterodimerization of HER3 has been shown to be essential for its activity. Therefore, heterodimerization inhibition is likely to be a mechanism by which anti-HER3 antibody A can inhibit HER3 activity (and the resulting tumor activity), especially considering that anti-HER3 antibody A does not block NRG1 binding to HER3.

D. Anti-HER3 antibody A causes internalization of HER3 and cell surface loss

In vitro induction of internalization and depletion of cell surface HER3 by anti-HER3 antibodies can be measured in cell-based assays. The foregoing assays can be used to study the ability of the antibodies of the invention to cause clearance of HER3 in cancer cells, potentially eliminating the ability of HER3 to mediate tumor or drug resistance mechanisms in cancer cells.

BT474 cells exhibit relatively high levels of cell surface HER3 and are selected to characterize HER3 internalization and cell surface depletion by anti-HER3 antibody induction. Briefly stated, every 300,000 BT474 cells/well can be seeded in each well of a 6-well culture dish and incubated for 24 hours at 37 ° C, 5% CO ₂ . Each well can be aspirated and 1 mL of fresh growth medium containing 100 nM HER3 test antibody is added. After overnight treatment at 37 ° C, 5% CO ₂ , cells can be removed from the wells using non-enzymatic buffers. The cells were then stained with 1 μg/mL biotinylated 1B4C3 HER3 antibody (BioLegend) for 1 hour at 4 °C. 1B4C3 binds to HER3-ECD but does not compete with any of the test antibodies used in this assay. Cells can be washed and then stained with streptavidin-PE for 30 minutes at 4 °C, followed by characterization by FACS analysis. The percentage of internalization can be determined by comparing the untreated control group. The mean and standard deviation from the three experiments can be calculated and illustrated. Anti-HER3 antibody A caused HER3 internalization and cell surface loss from BT474 in an assay essentially as described above.

Anti-HER3 Antibody A Internalizes HER3 Similar to or preferred to certain other HER3 antibodies known in the art, including MOR10703_A50V_N52S, U1-59, and Ab#6 (see US 2012/0107306, WO 2007/077028, and WO 2008, respectively). /100624) (Figure 3).

E. In vivo efficacy of anti-HER3 antibody A in various cancer xenograft models

The efficacy of antibodies tested alone and in combination with other agents may be subject to approved animal research methods in accordance with international, national and local laws and guidelines (eg, they are approved by the Laboratory Animal Management and Use Committee and are based on current US Department of Agriculture and US National The Institute of Health's regulations and standards are implemented, and are measured in cancer xenograft models such as A549 (lung; KRAS mutation), SCCHN A-253 (head and neck tumor type), FaDu ( Head and neck tumor type), BxPC-3 (pancreatic tumor type).

A control IgG or a combination of test antibodies or test antibodies can be utilized by randomly enrolling a mouse of about 300 mm ³ volumes of xenograft tumors randomly divided into 10 mice per group according to tumor size and body weight according to tumor size and body weight. To treat. The tumor volume was calculated by the equation volume = [(Pi / 6) l x w ² ], where Pi is equal to 3.14, w represents the width and l represents the length. The duration of treatment can be determined by: 1) the achieved research indicators (ie, achieving statistically significant inhibition of tumor growth or ineffective anti-tumor effects), or 2) clinical indicators that have been achieved ( For example, tumor burden affects animal happiness or survival).

Control IgG, anti-HER3 antibody A, cetuximab or lexizumab can be formulated, for example, in phosphate buffered saline. Anti-HER3 antibody A or control isotype IgG formulated with PBS can be administered for 4 weeks at a dose of 20 mg/kg IP twice a week. The EGFR-conjugated anti-EGFR antibody, cetuximab or lexizumab is administered, for example, by intravenous (intravenous (i.v.)) administration of 20 mg/kg once a week. Can span multiple tumor types (eg, squamous NSCLC, squamous bladder, squamous head and neck, squamous thyroid, non-squamous large lung cells, squamous anus, or presence and absence of activating mutations (eg, KRAS mutations) The combination of anti-HER3 antibody A with the human monoclonal anti-EGFR antibody cetuximab or levozumab and its corresponding monotherapy in a xenograft model of non-squamous colorectal cancer. During the course of the study, the antitumor efficacy of the treatment group was assessed twice a week by measuring the tumor volume by means of caliper measurements. For example, body weight can be measured regularly twice a week as a general indicator of tolerance during the course of the study. If p < 0.05, then the difference between treatments can be considered statistically significant. The anti-tumor efficacy of the experimental treatment can be expressed as a T/C ratio (in percent) and can be calculated as outlined below.

%T/C=100 x △T/△C, if △T>0

Where ΔT = average tumor volume of the drug treatment group on the last day of the study minus the average tumor volume of the drug treatment group on the initial administration day; ΔC = control group on the last day of the study (specified in each study) The average tumor volume was subtracted from the mean tumor volume of the control group on the initial dosing day. If ΔT < 0, the regression (% regression) can be calculated instead of %T/C using the equation = 100 × ΔT / T. If the tumor is reached If 50% subsides, it is a partial reaction (PR). If undetectable, the tumor has a complete response (CR).

Tumor volume data were analyzed using the MIXED program of SAS software (version 9.3) using two-way repeated measure difference analysis by time and treatment. The response analyzed was a logarithmic transformation of tumor volume due to the difference between the equilibration time and the treatment group as needed. Space power available Repeat the measurement of the structure associated with the model. Pre-defined pairwise comparisons of one or more treatment groups with one or more control groups were performed for each time point. The mean tumor volume data can be expressed as a geometric mean ± sem.

In experiments essentially as described, when with the xenograft model SCCHN A-253 (part and neck tumor type), A549 (pulmonary tumor with KRAS mutation), FaDu (head and neck tumor type) and BxPC- The anti-HER3 antibody A monotherapy group showed a statistically significant decrease in tumor volume when compared to control IgG in 3 (pancreatic tumor type). For example, anti-HER3 antibody A alone or in combination with cetuximab inhibits tumor growth in SCCHN A-253 xenograft tumors (Figure 4). Anti-HER3 antibody A in combination with cetuximab caused a significant reduction in tumor size, as shown in the waterfall pattern (Figure 5). Treatment with anti-HER3 antibody A and anti-HER3 antibody A in combination with cetuximab had no significant effect on the body weight of xenografted mice (data not shown). Statistical analysis of A-253 compared to control IgG showed a statistically significant reduction in tumor volume in the treatment of tumors in combination with anti-HER3 antibody A and anti-HER3 antibody A in combination with cetuximab. Furthermore, anti-HER3 antibody A alone or in combination with cetuximab inhibited tumor growth in lung A549 xenograft tumors (Figure 6). While cetuximab had no effect on A549 xenograft tumor growth (compared to the control group), anti-HER3 antibody A combined with cetuximab caused a significant reduction in lung A549 xenograft tumor growth and was indeed better than MOR10703_A50V_N52S Combination with cetuximab (Figure 6). Furthermore, the combination of anti-HER3 antibody A and cetuximab produced greater efficacy than either of the individual agents in the xenograft model FaDu (head and neck tumor type) and BxPC-3 (pancreatic tumor type) (data not shown).

Taken together, these results indicate that monotherapy with anti-HER3 antibody A is effective for different tumor types in vivo, including xenograft models of head and neck squamous cell carcinoma, non-small cell lung cancer, and pancreatic cancer. These results also show significant tumor activity in these models after administration of anti-HER3 antibody A in combination with the EGFR inhibitor cetuximab. Sexual regression. Thus, cancer treatments comprising anti-HER3 antibody A in combination with cetuximab show a greater likelihood of efficacy and a more durable response in different tumor types in vivo, including KRAS mutations and KRAS wild-type tumor types.

Amino acid and nucleotide sequence

<SEQ ID NO: 1; PRT1; artificial >

<SEQ ID NO: 2; PRT1; artificial >

<SEQ ID NO: 3; PRT1; artificial >

<SEQ ID NO: 4; PRT1; artificial >

<SEQ ID NO: 5; DNA; artificial>

<SEQ ID NO: 6; DNA; artificial>

<SEQ ID NO: 7; PRT1; Homo sapiens>

<SEQ ID NO: 8; PRT1; artificial >

AASGFSLDTYTMI

<SEQ ID NO: 9; PRT1; artificial >

IIYVTYNTYYANWAKG

<SEQ ID NO: 10; PRT1; artificial >

TRWGDQNGLDI

<SEQ ID NO: 11; PRT1; artificial >

QSSQRVYGNKNLA

<SEQ ID NO: 12; PRT1; artificial >

YFARTLAS

<SEQ ID NO: 13; PRT1; artificial >

QGTYYGTNWYVG

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<SEQ ID NO: 16; PRT1; Homo sapiens>

<SEQ ID NO: 17; PRT1; Homo sapiens>

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CFGPN

<SEQ ID NO: 19; PRT1; Homo sapiens>

<SEQ ID NO: 20; PRT1; Homo sapiens>

<SEQ ID NO: 21; PRT1; Homo sapiens>

<SEQ ID NO: 22; PRT1; artificial >

<SEQ ID NO: 23; PRT1; artificial >

<SEQ ID NO: 24; PRT1; artificial >

Figure 1. Binding of biotinylated NRG1 to culture plate-coated HER3-ECD.

Figure 2 Ligand-dependent HER3-HER2 heterodimerization assay (A) and ligand-independent HER3-HER2 heterodimerization assay (B). The untreated luciferase % represents the mean ± standard deviation from three independent experiments.

Figure 3. Cell surface loss of anti-HER3 antibodies and HER3 induced by internalization.

Figure 4 shows the therapeutic effect of SCCHN A-253 tumor volume (mm ³ ).

Figure 5 Anti-HER3 Antibody A inhibits tumor growth in SCCHN A-253 xenografts in vivo upon binding to cetuximab and causes regression.

Figure 6 shows the therapeutic effect of lung A549 tumor volume (mm ³ ).

<110> American Lilly Pharmaceuticals

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<151> 2015-07-20

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<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 9

<210> 10

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 10

<210> 11

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 11

<210> 12

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 12

<210> 13

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 13

<210> 14

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 14

<210> 15

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 15

<210> 16

<211> 1322

<212> PRT

<213> Homo sapiens

<400> 16

<210> 17

<211> 147

<212> PRT

<213> Homo sapiens

<400> 17

<210> 18

<211> 5

<212> PRT

<213> Homo sapiens

<400> 18

<210> 19

<211> 214

<212> PRT

<213> Homo sapiens

<400> 19

<210> 20

<211> 446

<212> PRT

<213> Homo sapiens

<400> 20

<210> 21

<211> 643

<212> PRT

<213> Homo sapiens

<400> 21

<210> 22

<211> 680

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 22

<210> 23

<211> 661

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 23

<210> 24

<211> 630

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 24

Claims (19)

An antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR together with the LCVR forms an antigen binding site that binds to human HER3, wherein the antigen binding site The locus comprises: a. three complementarity determining regions (CDRs) in the HCVR, wherein HCDR1 comprises the amino acid sequence AASGFSLDTYTMI (SEQ ID NO: 8) and HCDR2 comprises the amino acid sequence IIYVTYNTYYANWAKG (SEQ ID NO: 9), and HCDR3 comprises the amino acid sequence TRWGDQNGLDI (SEQ ID NO: 10), and b. three CDRs in the LCVR, wherein LCDR1 comprises the amino acid sequence QSSQRVYGNKNLA (SEQ ID NO: 11) and LCDR2 comprises the amino acid sequence YFARTLAS ( SEQ ID NO: 12), and LCDR3 comprises the amino acid sequence QGTYYGTNWYVG (SEQ ID NO: 13). An antibody or antigen-binding fragment thereof comprising a heavy chain (HC) and a light chain (LC), wherein the HC comprises a heavy chain variable region (HCVR) and the LC comprises a light chain variable region (LCVR), wherein the HCVR is The LCVR together form an antigen binding site that binds to human HER3, wherein the antigen binding site comprises: a. three complementarity determining regions (CDRs) in the HCVR, wherein HCDR1 comprises the amino acid sequence AASGFSLDTYTMI (SEQ ID NO: 8) HCDR2 comprises the amino acid sequence IIYVTYNTYYANWAKG (SEQ ID NO: 9), and HCDR3 comprises the amino acid sequence TRWGDQNGLDI (SEQ ID NO: 10), and b. three CDRs in the LCVR, wherein the LCDR1 comprises an amine group Acid sequence QSSQRVYGNKNLA (SEQ ID NO: 11), LCDR2 comprises the amino acid sequence YFARTLAS (SEQ ID NO: 12), and LCDR3 comprises the amino acid sequence QGTYYGTNWYVG (SEQ ID NO: 13). An antibody or antigen-binding fragment thereof comprising a heavy chain (HC) and a light chain (LC), wherein The HC comprises a heavy chain variable region (HCVR) and the LC comprises a light chain variable region (LCVR), wherein the HCVR has the amino acid sequence given in SEQ ID NO: 1 and the LCVR has SEQ ID NO: The amino acid sequence given in 3, and wherein the HCVR together with the LCVR forms an antigen binding site that binds to human HER3. The antibody or antigen-binding fragment thereof according to any one of claims 2 to 3, wherein the HC has the amino acid sequence given in SEQ ID NO: 2, and the LC has the SEQ ID NO: 4 Amino acid sequence. The antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof comprises two heavy chains and two light chains, wherein each heavy chain has the SEQ ID NO: 2 The amino acid sequence, and each light chain has the amino acid sequence given in SEQ ID NO:4. The antibody or antigen-binding fragment thereof of claim 5, wherein one of the heavy chains forms an interchain disulfide bond with one of the light chains, and the other heavy chain forms a chain between the other light chain A sulfur bond, and one of the heavy chains forms two interchain disulfide bonds with another heavy chain. The antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the antibody is glycosylated. The antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the antibody binds to human HER3 at the amino acids 198 to 202 (including the terminus) of the polypeptide shown in SEQ ID NO: . The antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the antibody inhibits both neuregulin-1 dependent and non-dependent activation of human HER3. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which is suitable for use in therapy. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which is suitable for the treatment of cancer. The antibody or antigen-binding fragment thereof according to claim 11, wherein the cancer is lung cancer, head and neck cancer, pancreatic cancer, kidney cancer, stomach cancer, colorectal cancer or breast cancer or hepatocellular carcinoma. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier, diluent or excipient. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 for the manufacture of a medicament for the treatment of cancer. The use of claim 14, wherein the cancer is lung cancer, head and neck cancer, pancreatic cancer, kidney cancer, stomach cancer, colorectal cancer or breast cancer or hepatocellular carcinoma. The use of claim 14 or 15, wherein the cancer is head and neck squamous cell carcinoma (HNSCC), NSCLC or squamous non-small cell lung cancer (sq-NSCLC). The use of claim 14 or 15, wherein the cancer is KRAS wild type. The use of claim 14 or 15, wherein the cancer comprises a KRAS activating mutation. The use of claim 14 or 15, wherein the medicament is for use with an effective amount of pertuzumab, trastuzumab, cetuximab, panitumumab ( Panitumumab), necitumumab, gefitinib, afatinib, neratinib, lapatinib, rociletinib, AZD9291, ASP8273, HM61713, erlotinib or a pharmaceutically acceptable salt thereof are administered simultaneously, separately or sequentially.