RU2785866C2 - Methods for treatment of metastatic cancer types, using axl trap receptors - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine and concerns a method for the treatment of human platinum-resistant metastatic ovarian cancer. The method includes administration to a patient of a therapeutically effective dose of a soluble variant of AXL polypeptide, where the soluble variant of AXL polypeptide does not have a transmembrane AXL domain; does not have a fibronectin (FN) domain; has one Ig1 domain and one Ig2 domain; and has a number of amino acid modifications of AXL sequence of a wild type (SEQ ID NO: 1), consisting of: Gly32Ser, Ala72Val, Asp87Gly, Val92Ala, and Gly127Arg; where the specified number of amino acid modifications increases affinity of binding of AXL polypeptide with growth arrest specific protein 6 (GAS6), and where the soluble variant of AXL polypeptide is fused with Fc region with a peptide linker. At the same time, the administration scheme includes 15 mg/kg of the soluble variant of polypeptide AXL, administered every two weeks, and 80 mg/m² of paclitaxel administered once a week.

EFFECT: method provides for effective and safe treatment of platinum-resistant ovarian cancer.

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Description

RELATED PATENT APPLICATIONS

This application claims priority to U.S. Provisional Application No. 62/581,671, filed November 4, 2017, U.S. Provisional Application No. 62/618,916, filed January 18, 2018, and U.S. Provisional Application No. 62/681,944, filed June 7, 2018, each of which is incorporated herein by reference in its entirety.

SEQUENCE LIST

The present application contains a sequence listing in "paper copy" form (PDF file) and a file containing the corresponding sequences (SEQ ID NO: 1) in machine-readable form (ST25 format text file) filed with it. The sequence listing is shown using the standard three letter amino acid code as defined in 37 CFR Part 1.822 (37 C.F.R. 1.822).

STATEMENT REGARDING RESEARCH OR DEVELOPMENT FINANCED FROM THE FEDERAL BUDGET

This work was supported by the Cancer Prevention & Research Institute of Texas, New Company Product Development Award DP150127. The State of Texas, USA, may have rights to any patent issued under this application.

FIELD OF THE INVENTION

Cancer is a group of diseases involving abnormal cell growth with the potential for spread or invasion to other parts of the body. Variants of abnormal growth that form a discrete tumor mass, ie, no cysts or fluid, are defined as solid tumors. Solid tumors can be benign (noncancerous) or malignant (cancerous). Different types of solid tumors are named according to the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas and lymphomas. Cancers derived from either of the two blood cell lines, myeloid and lymphoid, are defined as malignant tumors of the blood. Such malignant neoplasms are also called blood cancers or hemoblastoses. Examples of hemoblastoses include multiple myeloma, acute leukemias (eg, 11q23-positive acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic leukemia, and erythroleukemia), chronic leukemias (eg, chronic myelocytic (granulocytic) ) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma (indolent and highly differentiated forms), Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

The treatment available to patients with cancer is highly dependent on combination therapies, including surgery, cryosurgery, and cytotoxic chemotherapy. Unfortunately, despite the effectiveness in some cases, side effects on normal tissues often manifest themselves as dose-limiting toxicity and do not allow for tumor eradication. And even when the side effects of these various therapies can be controlled, long-term responses are often not achieved, especially in refractory metastatic tumors.

To solve these problems and reduce side effects on normal tissues, the concept of targeted therapy has been developed, that is, a directed effect on a specific molecule or signaling pathway. However, most patients either do not respond to such targeted therapy or relapse rapidly after response. Thus, there is a need for more effective targeted therapies that can be used in conjunction with current standards of care for metastatic and refractory disease.

Therapeutic efforts to prevent and treat cancer have focused on the level of signaling pathways or selective modulator proteins. Protein kinase activities, calcium homeostasis, and oncoprotein activation are stimulatory signals and therefore may be key regulatory points for therapeutic intervention. The AXL receptor and its activating growth arrest-specific 6 (GAS6) ligand are important stimulators of metastasis and cancer resistance to therapy in humans. AXL belongs to the TAM family of receptor tyrosine kinases, including Tyro3 (or SKY), AXL and MER (O'Bryan, JR, Molecular and Cellular Biology, 5016-5031, 1991). GAS6 is a common ligand for all three receptors. Unlike other members of the TAM family that have multiple signal ligands, for AXL GAS6 is the only known ligand (with very high natural binding affinity). Overexpression and activation of the GAS6-AXL signaling transduction pathway have been found to be important in a wide range of human tumors, including cancers of the kidney, pancreas, breast, lung, ovary, and prostate (Rankin, EK, PNAS, 13373-13378, 2014). Overexpression of AXL in highly metastatic cancers is associated with poor prognosis, aggressive tumor behavior, and resistance to therapy. Studies have shown that in solid tumors, activation of the GAS6-AXL signaling pathway promotes tumor invasion and metastasis, as well as the development of resistance to widely used chemotherapeutic agents. Given the critical role that GAS6 and AXL play in advanced and untreatable cancer, this signaling mechanism is an attractive target for therapeutic intervention. Unfortunately, the unusually high binding affinity of GAS6 and AXL of approximately 30 pM has made the development of competitive antagonists very difficult.

The AXL receptor contains two separate epitopes that bind to GAS6: a high affinity site in its N-terminal immunoglobulin-like (Ig) domain and a low affinity site in the second Ig domain. The present inventors engineered the AXL Ig1 core site using rational and combinatorial protein engineering techniques to provide long half-life AXL decoy receptors that bind to GAS6 with higher affinity than endogenous AXL, effectively sequestering GAS6 and preventing AXL signaling . These decoy receptors reduce in vitro invasion/migration of metastatic cells and suppress metastatic disease in aggressive pre-clinical models of human pancreatic, kidney, breast and ovarian cancer, and exhibit a favorable safety profile. When compared directly in preclinical models with the most recent anti-AXL small molecule clinical studies currently under way, decoy receptors have achieved higher antitumor efficacy without demonstrating toxicity in pharmacological studies. It is also important to note that while most agents intended for use in oncology require clinical trials in the cancer patient population due to significant toxicity associated with effective doses, both from direct effects on their targets and off-target effects, the decoy receptors constructed by the present inventors are not a cytotoxic drug.

All ideas of patent documents , US 2013/074809, US 2013/074786, US 2013/074796, US 2015/0315553 are expressly incorporated here by reference.

DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides methods for treating a proliferative disease, such as human metastatic cancer, comprising administering a soluble AXL polypeptide according to a schedule determined to achieve prolonged overall survival compared to controls.

In some embodiments, the proliferative disease is a cancer selected from the group consisting of: B-cell lymphoma; lung cancer (small cell lung cancer and non-small cell lung cancer); bronchial cancer; colorectal cancer; prostate cancer; breast cancer; pancreatic cancer; stomach cancer; ovarian cancer; bladder cancer; cancer of the brain or central nervous system; cancer of the peripheral nervous system; esophageal cancer; cervical cancer; melanomas; uterine or endometrial cancer; cancer of the oral cavity or pharynx; liver cancer; kidney cancer; biliary tract cancer; cancer of the small intestine or appendix; salivary gland cancer; thyroid cancer; adrenal cancer; osteosarcomas; chondrosarcomas; liposarcomas; testicular cancer; malignant fibrous histiocytoma; skin cancer; head and neck cancer; lymphomas; sarcoma; multiple myeloma; and leukemia. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is breast cancer.

In some embodiments, the cancer is a cancer that overexpresses the GAS6 and/or AXL biomarker. In some embodiments, the cancer is recurrent cancer. In some embodiments, the cancer is human metastatic cancer resistant to standard therapies. In some embodiments, the human metastatic cancer is chemoresistant cancer. In some embodiments, the human metastatic cancer is platinum resistant cancer.

In another aspect, the present invention provides methods for treating human metastatic cancer comprising administering a soluble AXL polypeptide lacking an AXL transmembrane domain and having at least one mutation to wild-type AXL that increases the binding affinity of the AXL polypeptide to GAS6 relative to wild-type AXL, in combination with a second therapy selected from the group consisting of targeted therapy with small molecule kinase inhibitors, surgery, cytoreductive therapy, cytotoxic chemotherapy and immunotherapy.

In some embodiments, the second therapy is a cytoreductive therapy and the combination may increase the therapeutic index of the cytoreductive therapy. In some embodiments, cytoreductive therapy may act on the DNA repair pathway. In some embodiments, the cytoreductive therapy is radiation therapy. In some embodiments, the combination may be synergistic.

In some embodiments, the second therapy is a chemotherapeutic agent selected from the group consisting of daunorubicin, adriamycin (doxorubicin), epirubicin, idarubicin, anamycin, MEN 10755, etoposide, teniposide, vinblastine, vincristine, vinorelbine (NAVELBINE), vindesine, vindoline, vincamine , mechlorethamine, cyclophosphamide, melphalan (L-sarcolysine), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozozotocin, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FUdR), thioguanine (β-thioguanine), mercaptopurine (6-MP), pentostatin, fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, cisplatin (cis-DDP), carboplatin, oxaliplatin, hydroxyurea, gemcitabine, and N-methylhydrazine. In some embodiments, the combination may be synergistic.

In some embodiments, the second therapy will include immunotherapy selected from, but not limited to: treatment with depleting antibodies to specific tumor antigens; treatment using antibody-drug conjugates; treatment with agonistic, antagonistic, or blocking antibodies to co-stimulatory or co-inhibitory molecules (immune checkpoints) such as CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), PD-1 (programmed cell death protein 1), OX-40 , CD 137, GITR (glucocorticoid-induced tumor necrosis factor receptor), LAG3 (lymphocyte activation gene 3), TIM-3 (T-cell immunoglobulin and mucin-containing protein 3), and VISTA (V-domain Ig suppressor of T cell activation ); treatments with bispecific antibodies that attract T cells (BiTE®), such as blinatumomab; treatment, including the introduction of biological response modifiers, such as IL-2 (interleukin 2), IL-12, IL-15, IL-21, GM-CSF (granulocyte-macrophage colony stimulating factor), IFN-α (interferon-α), IFN-β and IFN-γ; treatment with therapeutic vaccines such as sipuleucel-T; treatments using dendritic cell vaccines or vaccines based on tumor antigenic peptides; chimeric antigen receptor (CAR) T cell treatments; treatments using CAR-NK cells (natural killer cells); treatment with tumor-infiltrating lymphocytes (TIL); treatments using adoptively transferred antitumor T cells (expanded ex vivo and/or transgenic for TCRs (T cell receptors)); treatment using TALL-104 cells; and treatment with immunostimulatory agents such as CpG Toll-like receptor (TLR) agonists and imiquimod; where the combination therapy provides enhanced destruction of tumor cells by effector cells, that is, when co-administered, there is synergy of the soluble AXL polypeptide and immunotherapy.

In some embodiments, the second therapy will include the administration of a poly(ADP-ribose) polymerase (PARP) inhibitor. In some embodiments, the PARP inhibitor is selected from the group consisting of ABT-767, AZD 2461, BGB-290, BGP 15, CEP 9722, E7016, E7449, flusoparib, INO1001, JPI 289, MP 124, niraparib, olaparib, ONO2231, rucaparib, SC 101914, talazoparib, veliparib, WW 46 or salts or derivatives thereof, olaparib, rucaparib, niraparib, talazoparib and veliparib. In some embodiments, the combination may be synergistic.

In some embodiments, the treatment will include administering a soluble AXL polypeptide variant in combination with pegylated liposomal doxorubicin (PLD). In some embodiments, the method of treatment will include administering a soluble AXL polypeptide variant in combination with paclitaxel. In some embodiments, the combination may be synergistic.

In some embodiments, the soluble AXL polypeptide is a soluble AXL polypeptide variant, wherein said soluble AXL polypeptide variant does not have an AXL transmembrane domain, does not have a functional fibronectin (FN) domain, has one or more Ig1 domains, has one or more Ig2, and wherein said AXL polypeptide variant exhibits increased binding affinity of the AXL polypeptide variant to GAS6 compared to wild-type AXL.

In some embodiments, the soluble AXL polypeptide is a soluble AXL polypeptide variant, wherein said soluble AXL polypeptide variant does not have an AXL transmembrane domain, does not have a functional fibronectin (FN) domain, has a single Ig1 domain, does not have a functional Ig2 domain, and wherein said polypeptide variant AXL shows increased binding affinity of the AXL polypeptide variant to GAS6 compared to wild-type AXL.

In some embodiments, the AXL polypeptide variant is an Fc domain-containing fusion protein. In some embodiments, the polypeptide variant does not have an AXL intracellular domain. In some embodiments, the soluble AXL polypeptide variant also lacks a functional fibronectin (FN) domain, wherein said polypeptide variant exhibits increased polypeptide binding affinity for GAS6. In some embodiments, the soluble AXL polypeptide variant contains at least one amino acid modification relative to the wild-type AXL sequence.

In some embodiments, the soluble AXL polypeptide variant contains at least one amino acid modification in a region selected from the group consisting of (1) 15-50, (2) 60-120, and (3) 125-135 of the wild-type AXL sequence (SEQ ID NO : one).

In some embodiments, the soluble AXL polypeptide variant contains at least one amino acid modification at position 19, 23, 26, 27, 32, 33, 38, 44, 61, 65, 72, 74, 78, 79, 86, 87, 88, 90, 92, 97, 98, 105, 109, 112, 113, 116, 118, or 127 wild-type AXL sequences (SEQ ID NO: 1) or a combination thereof.

In some embodiments, the soluble AXL polypeptide variant contains at least one amino acid modification selected from the group consisting of (1)A19T, (2)T23M, (3)E26G, (4)E27G or E27K 5)G32S, (6)N33S , (7)T38L (8)T44A, (9)H61Y, (10)D65N, (11)A72V, (12) S74N, (13)Q78E, (14)V79M, (15)Q86R, (16)D87G, (17)D88N, (18)I90M or I90V, (19)V92A, V92G or V92D, (20)I97R, (21)T98A or T98P, (22)T105M, (23)Q109R, (24)V112A, (25) )F113L, (26)H116R, (27)T118A, (28) G127R or G127E, (29) G129E and combinations thereof.

In some embodiments, the AXL polypeptide variant contains amino acid changes relative to the wild-type AXL sequence (SEQ ID NO:1) at the following positions: (a) glycine 32; (b) aspartic acid 87; (c) valine 92; and (d) glycine 127.

In some embodiments, the AXL polypeptide variant contains amino acid changes relative to the wild-type AXL sequence (SEQ ID NO: 1) at the following positions: (a) aspartic acid 87 and (b) valine 92.

In some embodiments, the AXL polypeptide variant contains amino acid changes relative to the wild-type AXL sequence (SEQ ID NO:1) at the following positions: (a) glycine 32; (b) aspartic acid 87; (c) valine 92; (d) glycine 127; and (e) alanine 72.

In some embodiments, the AXL polypeptide variant contains amino acid changes relative to the wild-type AXL sequence (SEQ ID NO: 1) at the following position: alanine 72.

In some embodiments, the glycine residue 32 of the AXL polypeptide variant is replaced with a serine residue, the aspartic acid residue 87 is replaced with a glycine residue, the valine residue 92 is replaced with an alanine residue, or the glycine residue 127 is replaced with an arginine residue, or a combination of these substitutions is present.

In some embodiments, the aspartic acid residue 87 of the AXL polypeptide variant is replaced by a glycine residue, or the valine residue 92 is replaced by an alanine residue, or a combination of these substitutions is present.

In some embodiments, the alanine residue of the 72 variant AXL polypeptide is replaced by a valine residue.

In some embodiments, the glycine residue 32 of the AXL polypeptide variant is replaced with a serine residue, the aspartic acid residue 87 is replaced with a glycine residue, the valine residue 92 is replaced with an alanine residue, the glycine residue 127 is replaced with an arginine residue, or the alanine residue 72 is replaced with a valine residue, or a combination of these substitutions is present.

In some embodiments, the AXL variant contains amino acid changes relative to the wild-type AXL sequence (SEQ ID NO: 1) at the following positions: (a) glutamic acid 26; (b) valine 79; (c) valine 92; and (d) glycine 127.

In some embodiments, glutamic acid residue 26 of the AXL polypeptide variant is replaced with a glycine residue, valine 79 residue is replaced with a methionine residue, valine 92 residue is replaced with an alanine residue, or glycine residue 127 is replaced with an arginine residue, or a combination of these substitutions is present.

In some embodiments, the AXL polypeptide variant comprises at least an amino acid region selected from the group consisting of amino acid regions 19-437, 130-437, 19-132, 21-121, 26-132, 26-121, and 1-437 of the AXL polypeptide. wild-type (SEQ ID NO: 1) and wherein one or more amino acid modifications are present in said amino acid region.

In some embodiments, the AXL polypeptide variant contains amino acid changes relative to the wild-type AXL sequence (SEQ ID NO: 1) at the following positions: (a) glycine 32; (b) aspartic acid 87; (c) alanine 72; and valine 92.

In some embodiments, glycine 32 of the AXL polypeptide variant is replaced by a serine residue, aspartic acid 87 is replaced by a glycine residue, alanine 72 is replaced by a valine residue, and valine 92 is replaced by an alanine residue, or a combination of these substitutions is present.

In some embodiments, the soluble AXL polypeptide is an Fc domain-containing fusion protein, and wherein said AXL variant contains amino acid changes relative to the wild-type AXL sequence (SEQ ID NO: 1) at the following positions: (a) glycine 32; (b) aspartic acid 87; (c) alanine 72; and (d) valine 92.

In some embodiments, the soluble AXL polypeptide is a fusion protein containing an Fc domain, and where glycine 32 is replaced by a serine residue, aspartic acid 87 is replaced by a glycine residue, alanine 72 is replaced by a valine residue, and valine 92 is replaced by an alanine residue, or a combination of these substitutions is present.

In some embodiments, the soluble AXL polypeptide is an Fc domain-containing fusion protein, and wherein said AXL variant contains amino acid changes relative to the wild-type AXL sequence (SEQ ID NO: 1) at the following positions: (a) glycine 32; (b) aspartic acid 87; (c) alanine 72; (d) valine 92; and (e) glycine 127.

In some embodiments, the soluble AXL polypeptide is a fusion protein containing an Fc domain, and where glycine 32 is replaced by a serine residue, aspartic acid 87 is replaced by a glycine residue, alanine 72 is replaced by a valine residue, valine 92 is replaced by an alanine residue, and glycine 127 is replaced by an arginine residue, or a combination of these substitutions is present.

In some embodiments, the soluble AXL polypeptide is an Fc domain-containing fusion protein, lacks a functional FN domain, and wherein said AXL variant contains amino acid changes relative to the wild-type AXL sequence (SEQ ID NO: 1) at the following positions: (a) glycine 32; (b) aspartic acid 87; (c) alanine 72; and (d) valine 92.

In some embodiments, the soluble AXL variant is a fusion protein containing an Fc domain, lacks a functional FN domain, and wherein glycine 32 is replaced by a serine residue, aspartic acid 87 is replaced by a glycine residue, alanine 72 is replaced by a valine residue, and valine 92 is replaced by an alanine residue , or a combination of these substitutions is present.

In some embodiments, the soluble AXL polypeptide is an Fc domain-containing fusion protein, lacks a functional FN domain, and wherein said AXL variant contains amino acid changes relative to the wild-type AXL sequence (SEQ ID NO: 1) at the following positions: (a) glycine 32; (b) aspartic acid 87; (c) alanine 72; (d) valine 92; and (e) glycine 127.

In some embodiments, the soluble AXL variant is a fusion protein containing an Fc domain, lacks a functional FN domain, and where glycine 32 is replaced by a serine residue, aspartic acid 87 is replaced by a glycine residue, alanine 72 is replaced by a valine residue, valine 92 is replaced by an alanine residue, and glycine 127 is replaced by an arginine residue, or a combination of these substitutions is present.

In some embodiments, the soluble AXL polypeptide is an Fc domain-containing fusion protein, lacks a functional FN domain, lacks an Ig2 domain, and wherein said AXL variant contains amino acid changes relative to the wild-type AXL sequence (SEQ ID NO: 1) in the following provisions: (a) glycine 32; (b) aspartic acid 87; (c) alanine 72; and (d) valine 92.

In some embodiments, the soluble AXL variant is a fusion protein containing an Fc domain, lacks a functional FN domain, and wherein glycine 32 is replaced by a serine residue, aspartic acid 87 is replaced by a glycine residue, alanine 72 is replaced by a valine residue, and valine 92 is replaced by an alanine residue , or a combination of these substitutions is present.

In some embodiments, the soluble AXL polypeptide is a fusion protein containing an Fc domain, lacking a functional FN domain, lacking an Ig2 domain, and wherein said AXL variant contains amino acid changes relative to the wild-type AXL sequence (SEQ ID NO: 1) in the following provisions: (a) glycine 32; (b) aspartic acid 87; (c) alanine 72; (d) valine 92; and (e) glycine 127.

In some embodiments, the soluble AXL variant is a fusion protein containing an Fc domain, lacks a functional FN domain, lacks an Ig2 domain, and where glycine 32 is replaced by a serine residue, aspartic acid 87 is replaced by a glycine residue, alanine 72 is replaced by a valine residue, valine 92 is replaced by an alanine residue and glycine 127 is replaced by an arginine residue, or a combination of these substitutions is present.

In some embodiments, the soluble AXL polypeptide variant has an affinity of at least about 1 x 10 ^-8 M, 1 x 10 ^-9 M, 1 x 10 ^-10 M, 1 x 10 ^-11 M, or 1 x 10 ^-12 M for GAS6 .

In some embodiments, the soluble AXL polypeptide variant exhibits an affinity for GAS6 that is at least about 5-fold higher than the wild-type AXL polypeptide, at least about 10-fold, or at least about 20-fold.

In some embodiments, the soluble AXL polypeptide variant further comprises a linker. In some embodiments, the linker contains one or more (GLY) ₄ SER units. In some embodiments, the linker contains 1, 2, 3 or 5 units (GLY) ₄ SER.

In some embodiments, the dose of the soluble AXL polypeptide variant administered to the patient is selected from the group consisting of about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3 .5, approximately 4.0, approximately 4.5, approximately 5.0, approximately 5.5, approximately 6.0, approximately 6.5, approximately 7.0, approximately 7.5, approximately 8.0, approximately 8 .5, approximately 9.0, approximately 9.5, approximately 10.0 mg/kg, approximately 10.5, approximately 11.0, approximately 11.5, approximately 12.0, approximately 12.5, approximately 13.0 , approximately 13.5, approximately 14.0, approximately 14.5, approximately 15.0, approximately 15.5, approximately 16.0, approximately 16.5, approximately 17.0, approximately 17.5, approximately 18.0 , about 18.5, about 19.0 mg/kg, about 19.5 and about 20.0 mg/kg. In some embodiments, the soluble AXL polypeptide variant will be administered by intravenous infusion over 30 or 60 minutes at a dose of 10 mg/kg per week. In some embodiments, the soluble AXL polypeptide variant will be administered by intravenous infusion over 30 or 60 minutes at a dose of 5 mg/kg per week. In some embodiments, the soluble AXL polypeptide variant will be administered by intravenous infusion over 30 or 60 minutes at a dose of 2.5 mg/kg per week. In some embodiments, the soluble AXL polypeptide variant will be administered by IV infusion over 30 or 60 minutes at a dose of 1 mg/kg per week. In some embodiments, the soluble AXL polypeptide variant will be administered by intravenous infusion over 30 or 60 minutes at a dose of 20 mg/kg every 14 days. In some embodiments, the soluble AXL polypeptide variant will be administered by intravenous infusion over 30 or 60 minutes at a dose of 10 mg/kg every 14 days. In some embodiments, the soluble AXL polypeptide variant will be administered by IV infusion over 30 or 60 minutes at a dose of 5 mg/kg every 14 days. In some embodiments, the soluble AXL polypeptide variant will be administered by intravenous infusion over 30 or 60 minutes at a dose of 2.5 mg/kg every 14 days. In some embodiments, the soluble AXL polypeptide variant will be administered by intravenous infusion over 30 or 60 minutes at a dose of 1 mg/kg every 14 days.

BRIEF DESCRIPTION OF GRAPHICS

FIG. 1. Inhibition of GAS6-induced invasion/migration using the AXL decoy receptor. (A) Soluble receptor decoy AXL (AVB-S6-500) and MDA-MB-231 Ax1 ⁺ TNBC cells in serum-free medium were placed in the upper compartment of the Boyden chamber coated with matrigel. Serum-containing media as a chemoattractant were introduced into the lower compartment of the chamber. After 24 hours of incubation, the number of cells migrating through Matrigel was determined and expressed as the proportion of invasive cells relative to PBS (phosphate buffered saline) control. (b) AVB-S6-500, OVCAR8 Ax1 ⁺ ovarian cancer cells, type 1 collagen, 50 ng/ml GAS6 and culture media were placed in microwells and incubated. On day 6, the number of cells showing an invasive phenotype was determined and expressed as the proportion of invasive cells relative to PBS control. AVB-S6-500 in the concentration range from 1 to 100 μg/ml significantly inhibited GAS6-induced invasion/migration.

FIG. 2. Representative images obtained from an invasion assay of MDA-MB-231 cells with AVB-S6-500.

FIG. 3. Reducing metastatic tumor burden using the AXL decoy receptor. Mice were intraperitoneally (ip) injected with SKOV3.ip ovarian cancer tumor cells (1×10 ⁶ ), randomized into groups, and treated with AVB-S6-500 at 5, 10, or 20 mg/kg every other day (Q2D). Metastatic tumor burden was assessed after 24 days of dosing by counting all visible metastatic lesions in the peritoneal cavity with excision and weighing of all affected tissue to determine the total mass (A) and number (B) of metastases. AVB-S6-500 significantly reduced the metastatic tumor burden when administered at doses of 10 and 20 mg/kg.

FIG. 4. Improved efficacy with the combination of the AXL decoy receptor and doxorubicin. Mice were intraperitoneally (ip) injected with SKOV3.ip ovarian cancer tumor cells (1×10 ⁶ ), randomized into groups, and treated with AVB-S6-500 at a dose of 20 mg/kg Q2D alone or in combination with doxorubicin (DOX ), 2 mg/kg twice a week. Metastatic tumor burden was assessed after 24 days of dosing. Comparison of the total mass (A) and the number (b) of metastases demonstrated a significant advantage of combination therapy. The combination of AVB-S6-500 and doxorubicin significantly reduced the mean mass of diseased tissue and resulted in a cure in 2 animals.

FIG. 5. Abrupt decrease in serum GAS6 over approximately 1 week in cynomolgus monkeys following a single dose of AXL decoy receptor at 5 mg/kg (human equivalent dose 1.7 mg/kg). In cynomolgus monkeys, AVB-S6-500, 5 mg/kg, resulted in a dramatic decrease in serum GAS6 over at least 168 hours, and in multiple once-weekly dose studies, NOAEL (no adverse effect level) was defined as 150 mg/kg/day or more.

FIG. 6. Abrupt decrease in serum GAS6 over approximately 1 week in human subjects after single administration of the AXL decoy receptor at doses of 1 mg/kg (A) and 2.5 mg/kg (B).

FIG. 7. Abrupt decrease in serum GAS6 over approximately 1 week in humans following single administration of the AXL decoy receptor at doses of 5 mg/kg (A) and 10 mg/kg (B).

FIG. 8. Line graph showing mean (plus/minus) AVB-S6-500 concentrations after a single IV infusion of AVB-S6-500 in healthy subjects (N=6 per dose).

FIG. 9. Line graph showing mean (plus/minus) serum GAS6 concentrations after a single IV infusion of AVB-S6-500 in healthy subjects (N=6 per dose).

OPTION(S) FOR CARRYING OUT THE INVENTION

Definitions

Unless otherwise defined here, the scientific and technical terms used in connection with the present invention have the meaning in which they are usually understood by experts in this field. In addition, unless the context otherwise requires, the singular includes the plural, and the plural includes the singular. In general, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and chemistry, and hybridization of proteins and nucleic acids, and the associated techniques described herein, are widely used and well known in the art. Unless otherwise indicated, the methods and techniques of the present invention are usually carried out in accordance with conventional methods, well known in this field and described in various general and more specialized sources cited and discussed in the present description. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th ^ed ., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012), incorporated herein by reference. Enzymatic reactions and purification procedures are carried out in accordance with the manufacturer's descriptions, as is commonly done in this field, or as described here. The nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry, and the associated laboratory procedures and techniques described herein, are widely used and well known in the art. For chemical synthesis, chemical analysis, manufacture of pharmaceuticals, compositions, their delivery and treatment of subjects, standard techniques are used.

As used herein, the term "tumor" refers to all types of neoplastic cell growth and proliferation, both malignant and benign, and to all precancerous and cancerous cells and tissues.

The terms "cancer", "neoplasm" and "tumor" are used interchangeably herein to refer to cells exhibiting autonomous unregulated growth such that they exhibit an aberrant growth phenotype characterized by a significant decrease in control of cell proliferation. In general, cells of interest herein for detection, analysis, classification, or treatment include precancerous (eg, benign), malignant, pre-metastatic, and non-metastatic cells.

The term "primary tumor" refers to all types of neoplastic cell growth and proliferation, both malignant and benign, and to all precancerous and cancerous cells and tissues located in the anatomical region where autonomous unregulated cell growth began, for example, in the organ of the original cancerous tumor. . Primary tumors do not include metastases.

The "pathology" of cancer includes all phenomena that threaten the well-being of the patient. This includes, but is not limited to, abnormal or uncontrolled cell growth, primary tumor growth and formation, metastasis, malfunction of neighboring cells, abnormal levels of release of cytokines or other secretory products, suppression or exacerbation of an inflammatory or immunological response, neoplasia, precancer, malignancy, invasion of surrounding or distant tissues or organs such as lymph nodes, and so on.

As used herein, the terms "cancer recurrence", "tumor recurrence" and their grammatical variants refer to the continued growth of neoplastic or cancerous cells after a diagnosis of cancer. In particular, recurrence can occur when further growth of cancer cells occurs in the cancerous tissue. Similarly, "tumor spread" occurs when tumor cells disseminate to local or distant tissues and organs; therefore, tumor spread includes tumor metastasis. "Tumor invasion" occurs when a tumor spreads locally and disrupts the function of affected tissues by compressing, destroying, or preventing normal functioning of organs.

As used herein, the term "metastasis/metastasis" refers to the growth of a cancer in an organ or part of the body not directly associated with the organ of the original cancer. Metastasis/metastasis is meant to include micrometastasis/micrometastasis, which is the presence of cancer cells, in an undetectable amount, in an organ or body part not directly associated with the organ of the original cancer (e.g., the organ containing the primary tumor). Metastasis can also be defined as several process steps, such as the release of cancer cells from the area of the original tumor (eg, the area of the primary tumor) and the migration and/or invasion of cancer cells to other parts of the body.

A suitable sample is obtained from the patient, depending on the nature of the cancer. As used herein, the phrase "cancer tissue sample" refers to any cells obtained from a cancerous tumor. In the case of solid tumors that have not metastasized (eg, a primary tumor), a tissue sample will usually be obtained from the surgically removed tumor and prepared for analysis using conventional techniques.

The definition of a suitable patient sample includes blood and other liquid samples of biological origin, solid tissue samples such as a biopsy sample, or tissue cultures, or cells derived from them, and their progeny. The definition includes blood and other liquid samples of biological origin, solid tissue samples such as a biopsy sample, or tissue cultures, or cells derived from them, and their progeny. The definition also includes samples that, after receiving them, have been manipulated in any way, such as treatment with reagents, washing or enrichment with certain cell populations, such as cancer cells. The definition also includes a sample that has been enriched in certain types of molecules, such as nucleic acids, polypeptides, and so on. The term "biological specimen" includes a clinical specimen and also includes tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, and the like. . "Biological sample" includes a sample derived from a patient's cancer cell, such as a sample containing polynucleotides and/or polypeptides derived from a patient's cancer cell (for example, a cell lysate or other cell extract containing polynucleotides and/or polypeptides), and a sample containing patient's cancer cells. A biological sample containing a patient's cancer cell may also contain non-cancerous cells.

Tumors of interest for treatment with the methods of the invention include solid tumors such as carcinomas, gliomas, melanomas, sarcomas, and the like. Of particular interest are ovarian cancer and breast cancer. Carcinomas include many adenocarcinomas such as prostate, lung, etc., adrenal cortex carcinoma, hepatic cell carcinoma, renal cell carcinoma, ovarian carcinoma, carcinoma in situ, ductal carcinoma, breast carcinoma, basal cell carcinoma, squamous cell carcinoma, transitional cell carcinoma, colon carcinoma , nasopharyngeal carcinoma, multilocular cystic renal cell carcinoma, oat cell carcinoma, large cell lung carcinoma, small cell lung carcinoma, and so on. Carcinomas can be found in the prostate, pancreas, colon, brain (eg, glioblastoma), lung, breast, skin, and so on. The definition of soft tissue tumors includes neoplasias derived from fibroblasts, myofibroblasts, histiocytes, vascular/endothelial cells, and perineurium cells. Connective tissue tumors include sarcomas, histiocytomas, fibromas, skeletal chondrosarcomas, extraskeletal myxoid chondrosarcomas, clear cell sarcomas, fibrosarcomas, and so on. Hematologic cancers include leukemias and lymphomas, such as skin T-cell lymphoma, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL) and so on. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is a cancer that overexpresses the GAS6 and/or AXL biomarker. In some embodiments, the patient had previously responded to anti-cancer therapy, but relapsed after therapy was discontinued (hereinafter referred to as "recurrent cancer"). In some embodiments, the cancer is resistant to standard therapies. In some embodiments, the cancer is chemoresistant cancer. In some embodiments, the cancer is a platinum-resistant cancer.

"In combination with", "combination therapy" and "combination products" refer, in certain embodiments, to the simultaneous administration of the first drug and compounds to the patient, as used here. In some embodiments, the combination products are administered non-simultaneously. When administered in combination, the components may be administered at the same time or sequentially in any order at different time points. Thus, the components can be administered separately, but in sufficient temporal proximity to each other to provide the desired therapeutic effect.

As used herein, the phrase "disease-free survival" refers to the absence of such recurrence and/or tumor invasion and death in a patient after diagnosis associated with the impact of cancer on the life expectancy of the patient. The phrase "overall survival" refers to death in a patient after diagnosis, despite the possibility that the cause of the patient's death may not be a direct consequence of cancer. The phrases "likelihood of disease-free survival", "risk of recurrence" and variants thereof refer to the likelihood of a patient having a tumor recurrence or spread after a cancer diagnosis, where the probability is determined in accordance with the method of the invention.

To modulate AXL/GAS6 function, compounds having the desired pharmacological activity may be administered to the recipient in a physiologically acceptable carrier. Therapeutic agents can be administered in a variety of ways, orally, topically, parenterally, for example intravenously, subcutaneously, intraperitoneally, by viral infection, intravascularly, and so on. Of particular interest is intravenous delivery. The compounds may be included in various compositions, depending on the route of administration. The concentration of the therapeutically active compound in the composition may be from about 0.1-100% by weight.

Pharmaceutical compositions may be prepared in various forms such as granules, tablets, pills, suppositories, capsules, suspensions, ointments, lotions, and the like. Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral and topical administration may be used to prepare compositions containing therapeutically active compounds. Diluents known in the art include aqueous media, vegetable and animal oils and fats. As auxiliary agents, stabilizing agents, wetting and emulsifying agents, salts to change the osmotic pressure, or buffers to ensure adequate pH, and transdermal penetration enhancers can be used.

"Inhibitors", "activators", and "modulators" of AXL or its GAS6 ligand are used to refer to inhibitory, activating, or modulating molecules, respectively, identified using in vitro and in vivo receptor or ligand binding or signaling assays, e.g., ligands, receptors, agonists , antagonists and their homologues and mimetics.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of two or more amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. The terms "antibody" and "antibodies" are used interchangeably herein and refer to a polypeptide capable of interacting with and/or binding to another molecule, often referred to as an antigen. Antibodies may include, for example, "antigen-binding polypeptides" or "polypeptides that bind to target molecules." The antigens of the present invention may include, for example, any of the polypeptides described in the present invention.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are amino acids encoded by the genetic code, as well as amino acids modified later, such as hydroxyproline, gamma-carboxyglutamate and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure as naturally occurring amino acids, i.e. α-carbon bonded to hydrogen, carboxyl group, amino group and R group, e.g. homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that differs from the general chemical structure of amino acids, but that function in a manner similar to naturally occurring amino acids. All individual letters used in the present invention to designate amino acids are used in accordance with conventional amino acid symbols widely used in this area, for example, A stands for alanine, C stands for cysteine, and so on. An amino acid represented by a single letter before and after the corresponding position reflects the change from the original amino acid (before the position) to the changed amino acid (after the position). For example, A19T means that the amino acid alanine at position 19 is changed to threonine.

The terms "subject", "individual" and "patient" are used interchangeably herein to refer to a mammal being evaluated for treatment and/or treatment. In one embodiment, the mammal is a human. Thus, the terms "subject", "individual", and "patient" include individuals with cancer, including, but not limited to, ovarian or prostate adenocarcinoma, breast cancer, glioblastoma, and so on, including those who have undergone resection (surgery) to remove cancerous tissue or those who are candidates for resection (surgery) to remove cancerous tissue. Subjects may be humans, but also include other mammals, especially those mammals that can be used as laboratory models of human disease, such as mice, rats, and so on.

The definition of a suitable patient sample includes blood and other liquid samples of biological origin, solid tissue samples such as a biopsy sample, or tissue cultures, or cells derived from them, and their progeny. The definition also includes samples that have been manipulated in any way after they were obtained, such as treatment with reagents, washing, or enrichment with certain cell populations, such as endometrial cells, abnormal kidney cells, abnormal inflammatory cells, and/or transplant rejection cells ( GvHD). The definition also includes a sample that has been enriched in certain types of molecules, such as nucleic acids, polypeptides, and so on. The term "biological specimen" includes a clinical specimen and also includes tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, and the like. . "Biological sample" includes a sample obtained from a cell sample from a patient, such as a sample containing polynucleotides and/or polypeptides obtained from a cell sample from a patient (for example, a cell lysate or other cell extract containing polynucleotides and/or polypeptides), and a sample A that contains sample cells from the patient. A biological sample containing a sample cell from a patient may also contain normal, non-pathological cells.

The term "diagnosis" is used here to refer to the definition of a molecular or pathological condition, disease or situation, such as the definition of a viral infection.

As used herein, the terms "treatment", "treat" and the like refer to administering an agent or performing a procedure to produce an effect. The effect may be prophylactic in the sense of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in the sense of providing a partial or complete cure for the disease and/or symptoms of the disease. As used herein, "treatment" encompasses any treatment of any viral infection or exposure in a mammal, in particular a human, and includes: (a) prevention of infection; (b) suppressing the infection, that is, stopping its development; and (c) attenuating the disease, ie allowing the infection to reverse.

Treatment may refer to any indication of successful treatment, amelioration, or prevention of cancer, including any objective or subjective parameter such as improvement in severity, remission, improvement in symptoms or improvement in patient tolerance, reduction in rate of degradation or worsening, or reduction in severity of outcome of degradation. . Treatment or reduction in the intensity of symptoms may be based on objective or subjective parameters, including the results of an examination by a physician. Accordingly, the term "treatment" includes the administration of the compounds or agents of the present invention to prevent, delay, lessen, or stop or suppress the development of symptoms or conditions. The term "therapeutic effect" refers to the reduction, elimination or prevention of a disease, symptoms of a disease, or side effects of a disease in a subject.

As used herein, the term "correlates" or "correlates with" and similar terms refer to a statistical relationship between the occurrence of two events, where the events include numbers, datasets, and the like. For example, when events involve numbers, positive correlation (also referred to here as "direct correlation") means that as one increases, the other also increases. Negative correlation (also called "inverse correlation" here) means that as one increases, the other decreases.

"Dose unit" refers to physically discrete units suitable for use as unit doses in a particular individual to be treated. Each unit may contain a predetermined amount of the active compound(s) calculated to produce the desired therapeutic effect(s), together with the necessary pharmaceutical carrier. The characteristics of unit dosage forms may be dictated by (a) the unique characteristics of the active compound(s) and the particular therapeutic effect(s) to be achieved, and (b) the limitations inherent in the methods of making such (their) active compound(s).

A "pharmaceutically acceptable excipient" means an excipient useful in formulating a pharmaceutical composition that is generally safe, non-toxic and desirable, and includes excipients acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semi-solid or, in the case of an aerosol composition, gaseous.

The terms "pharmaceutically acceptable", "physiologically tolerable" and their grammatical variants, when referring to compositions, carriers, diluents and reagents, are used interchangeably and reflect that the substances can be administered to a person or used in a person without developing undesirable physiological effects to the extent of , which could make the introduction or use of the composition impossible.

"Therapeutically effective amount" refers to the amount of a compound which, when administered to a subject for the treatment of breast or ovarian cancer, is sufficient to effect such cancer treatment. The "therapeutically effective amount" may vary depending on, for example, the soluble AXL polypeptide variant chosen, the stage of the cancer, the age, body weight and/or health of the patient, and the judgment of the attending physician. In each particular case, the appropriate amount can be readily determined by those skilled in the art or by those skilled in the art through routine experimentation.

The phrase "determining the effectiveness of a treatment" and variations thereof may include any method of determining that a treatment is beneficial to a subject. The term "effectiveness of treatment" and its variants usually indicate a reduction in the severity of one or more of the signs or symptoms associated with the disease, and can be easily determined by a person skilled in the art. "Effectiveness of treatment" may also refer to the prevention or amelioration of the signs or symptoms of toxicity commonly associated with standard or non-standard treatment for a disease. Determining the effectiveness of a treatment will generally depend on the indication for the treatment and the disease being treated, and may include any method known or available in the art for determining that the treatment is beneficial to the patient. For example, indications of the effectiveness of treatment may include, without limitation, remission of the disease or indications. In addition, the efficacy of treatment may also include general improvements in the subject's overall health, such as, but not limited to, improving the patient's quality of life, increasing the subject's estimated survival rate, reducing depression, or reducing the relapse rate of an indication (increased remission time). (See, for example, Physicians' Desk Reference (2010)).

As used herein, the term "progression-free survival" refers to the period of time that a subject having a disease (eg, cancer) survives without a significant worsening of the disease state in question. Progression-free survival can be measured as the period of time during which there is no progression of tumor growth and/or the patient's disease status is not defined as progressive disease. In some embodiments, progression-free survival in a subject with cancer is assessed by tumor size (lesion), number of tumors (lesions), and/or metastasis.

As used herein, "objective response rate (ORR)" is defined as the proportion of patients with a reduction in tumor size by a predetermined amount and over a minimum period of time. Duration of response is usually measured from the time of initial response to documented tumor progression. Typically, ORR can be defined as the sum of partial responses and complete responses.

"Co-administration" of a known cancer drug with a pharmaceutical composition of the present invention means administering the drug and the AXL variant at such a time that both the known drug and the composition of the present invention will have a therapeutic effect. Such concomitant administration may include simultaneous (ie at the same time), prior or subsequent administration of a drug relative to the administration of a compound of the present invention. It will not be difficult for a person skilled in the art to determine the appropriate time, sequence, and doses for the administration of particular drugs and compositions of the present invention.

AXL, MER, Tyro3, and GAS6, and their associated signaling pathways, are described in WO 2011/091305 and United States Patent Applications Nos. 13/554,954 and 13/595,936, all of which are incorporated herein in their entirety by reference for any task.

Typical incarnations

The methods of the present invention include the treatment, reduction or prevention of cancer metastasis by administering a soluble AXL polypeptide variant as described herein. In one aspect, the present invention provides methods for treating human metastatic cancers comprising administering a soluble AXL polypeptide lacking an AXL transmembrane domain and having at least one mutation to wild-type AXL that increases the binding affinity of the AXL polypeptide to GAS6 relative to wild-type AXL. .

In some embodiments, these methods prolong progression-free survival compared to controls. In some embodiments, these methods prolong overall survival compared to controls. In some embodiments, these methods achieve improved progression-free survival compared to controls. In some embodiments, these methods achieve an improved chemotherapy-free interval compared to a control. In some embodiments, these methods achieve improved time to first follow-up therapy compared to controls. In some embodiments, these methods achieve improved time to second follow-up therapy compared to controls. In some embodiments, it is determined that these methods do not have a negative effect on the quality of life, assessed by FOSI and/or EQ-5D-5L.

Cancers of interest include solid tumors and hematologic malignancies. In various embodiments, the cancer is selected from the group consisting of: B-cell lymphoma; lung cancer (small cell lung cancer and non-small cell lung cancer); bronchial cancer; colorectal cancer; prostate cancer; breast cancer; pancreatic cancer; stomach cancer; ovarian cancer; bladder cancer; cancer of the brain or central nervous system; cancer of the peripheral nervous system; esophageal cancer; cervical cancer; melanomas; uterine or endometrial cancer; cancer of the oral cavity or pharynx; liver cancer; kidney cancer; biliary tract cancer; cancer of the small intestine or appendix; salivary gland cancer; thyroid cancer; adrenal cancer; osteosarcomas; chondrosarcomas; liposarcomas; testicular cancer; malignant fibrous histiocytoma; skin cancer; head and neck cancer; lymphomas; sarcoma; multiple myeloma; and leukemia.

Ovarian cancer is the 5th cause of all cancer deaths in women and accounts for 5% of all cancer deaths in women. It is estimated that in 2014 there will be 21980 new cases of ovarian cancer and approximately 14270 women will die from this disease. The estimated incidence of epithelial ovarian cancer in women in the United States in 2012 is approximately 22,280 (15,500 deaths) and 65,538 cases (42,704 deaths) in Europe. High-grade serous ovarian cancer is the most common subtype and exhibits broad genomic instability, indicating a likely defect in homologous recombination (Bowtell D D, Nat Rev Cancer 2010; 10: 803-8). At the time of diagnosis in most women, the disease is widespread, resulting in high mortality. Initial chemotherapy consists of chemotherapy with taxane or platinum or a combination of the two. Despite the fact that approximately 75% of patients respond to first-line therapy, within 1-3 years after it, 70% of these patients relapse. This high recurrence rate, despite an initially high response rate, results in a significant unmet need. Attempts to improve standard dual chemotherapy (carboplatin and paclitaxel) by adding a third cytotoxic drug (topotecan, gemcitabine or doxil) have been unsuccessful (du Bois et al., 2006, and Pfisterer et al., 2006). Maintenance therapy after a response to initial chemotherapy has been achieved may be a way to provide clinical benefit by delaying the side effects of disease progression, delaying the need for toxic chemotherapy, and increasing overall survival. However, there is currently no generally accepted standard for maintenance therapy for ovarian cancer.

In some embodiments, the cancer is ovarian cancer. In some embodiments, the ovarian cancer is resistant to standard therapies. In some embodiments, the recurrent and/or platinum-resistant cancer is ovarian cancer. In some embodiments, at the time of initiation of therapy with a soluble AXL polypeptide variant, the ovarian cancer is platinum-resistant ovarian cancer. In some embodiments, at the time of initiation of therapy with a soluble AXL polypeptide variant, the ovarian cancer is platinum-resistant recurrent ovarian cancer. In some embodiments, the ovarian cancer has responded to the most recent platinum-based chemotherapy regimen prior to initiation of therapy with a soluble AXL polypeptide variant. In some embodiments, the response to the most recent platinum-based chemotherapy regimen is a complete response. In some embodiments, the response to the most recent platinum-based chemotherapy regimen is a partial response. In some embodiments, the ovarian cancer responded to the penultimate platinum-based chemotherapy regimen prior to initiation of therapy with the soluble AXL polypeptide variant.

In another aspect, the present invention provides methods of treating cancer comprising administering a soluble AXL polypeptide lacking an AXL transmembrane domain and having at least one mutation to wild-type AXL that increases the binding affinity of the AXL polypeptide to GAS6 relative to wild-type AXL, in combination with a second therapy selected from the group consisting of surgery, cytoreductive therapy, cytotoxic chemotherapy and immunotherapy. In some embodiments, the combination may be synergistic.

In some embodiments, combination therapy includes antiproliferative or cytoreductive therapy. Antiproliferative or cytoreductive therapy is used therapeutically to eliminate tumor cells and other unwanted cells in a recipient and includes the use of therapies such as delivery of ionizing radiation and administration of chemotherapeutic agents. For example, ionizing radiation (IR) is used to treat approximately 60% of cancer patients by applying energy that damages or destroys cells in the area being treated, and for purposes of the present invention, IR can be delivered at conventional doses and schedules or at reduced doses. Radiation damage to cells is non-specific, with complex effects on DNA. The effectiveness of therapy depends on the fact that cell damage in cancer cells is more pronounced than in normal cells. Radiation therapy can be used to treat any type of cancer. Some types of radiation therapy involve photons, such as x-rays or gamma rays. Another technique for delivering radiation to cancer cells is internal beam radiation therapy, in which radioactive implants are placed directly into a tumor or body cavity, allowing the radiation dose to be concentrated to a small area. A suitable dose of ionizing radiation may be from at least about 2 Gy to no more than about 10 Gy, typically about 5 Gy. A suitable dose of ultraviolet radiation may be from at least about 5 J/m ² to no more than about 50 J/m ² , typically about 10 J/m ² . The sample can be obtained from at least about 4 to no more than about 72 hours after ultraviolet irradiation, usually after about 4 hours.

Chemotherapeutic agents are well known in the art and are used at conventional doses and regimens or at reduced doses or regimens, including, for example, topoisomerase inhibitors such as anthracyclines, including the compounds daunorubicin, adriamycin (doxorubicin), epirubicin, idarubicin, anamycin, MEN 10755 and the like. Other topoisomerase inhibitors include the podophyllotoxin analogs etoposide and teniposide, and the anthracenediones, mitoxantrone and amsacrine. Other antiproliferative agents interfere with microtubule assembly, such as the vinca family of alkaloids. Examples of vinca alkaloids include vinblastine, vincristine, vinorelbine (NAVELBINE), vindesine, vindoline, vincamine, and so on. DNA damaging agents include nucleotide analogs, alkylating agents, and so on. Alkylating agents include: nitrogen mustards such as mechlorethamine, cyclophosphamide, melphalan (L-sarcolysin) and so on; and nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, and so on. Nucleotide analogs include: pyrimidines, eg cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FUdR) and so on; purines, such as thioguanine (6-thioguanine), 6-mercaptopurine (6-MP), pentostatin, fluorouracil (5-FU) and so on; and folic acid analogs, eg methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, and so on. Other chemotherapeutic agents of interest include: metal complexes such as cisplatin (cis-DDP), carboplatin, oxaliplatin, and so on; ureas, for example hydroxyurea; gemcitabine; and hydrazines, for example N-methylhydrazine. In various embodiments, dosages of such chemotherapeutic agents include, without limitation, any of about 10 mg/m ² , 20 mg/m ² , 30 mg/m ² , 40 mg/m ² , 50 mg/m ² , 60 mg/m ² , 75 mg/m ² , 80 mg/m ² , 90 mg/m ² , 100 mg/m ² , 120 mg/m ² , 150 mg/m ² , 175 mg/m ² , 200 mg/m ² , 210 mg/ ^m2 , 220 mg/ ^m2 , 230 mg/ ^m2 , 240 mg/ ^m2 , 250 mg/ ^m2 , 260 mg/ ^m2 and 300 mg/ ^m2 .

In some embodiments, the combination therapy will include immunotherapy. As used herein, the term "immunotherapy" refers to the treatment of cancer, including, without limitation: treatment with depleting antibodies to specific tumor antigens (see, for example, Blattman and Greenberg, Science, 305:200, 2004; Adams and Weiner, Nat Biotech, 23:1147, 2005; Vogal et al. J Clin Oncology, 20:719, 2002; Colombat et al., Blood, 97:101, 2001); treatment using antibody-drug conjugates (see, for example, Ducry, Laurent (Ed.) Antibody Drug Conjugates. In: Methods in Molecular Biology. Book 1045. New York (NY), Humana Press, 2013; Nature Reviews Drug Discovery 12, 259-260, April 2013); treatment with agonist, antagonist, or blocking antibodies to co-stimulatory or co-inhibitory molecules (immune checkpoints) such as CTLA-4 (ipilimumab), PD-1 (nivolumab, pembrolizumab, pidilizumab) and PD-L1 (BMS-936559) , MPLD3280A, MEDI4736, MSB0010718C) (see e.g. Philips and Atkins, International Immunology, 27(1); 39-46, Oct 2014), OX-40, CD137, GITR, LAG3, TIM-3 and VISTA (see ., e.g., Sharon et al., Chin J Cancer., 33(9): 434-444, Sep 2014; Hodi et al., N Engl J Med, 2010; Topalian et al., N Engl J Med, 366: 2443-54, 2012); treatment with bispecific antibodies that attract T cells (BiTE®), such as blinatumomab (see, for example, US patent No. 9260522; application for US patent No. 20140302037); treatment comprising the administration of biological response modifiers such as IL-2, IL-12, IL-15, IL-21, GM-CSF, IFN-α, HN-β and IFN-γ (see e.g. Sutlu T et al., Journ of Internal Medicine, 266(2): 154-181, 2009; Joshi S PNAS USA, 106(29):12097-12102, 2009; Li Y et al., Journal of Translational Medicine, 7:11, 2009); treatment with therapeutic vaccines such as sipuleucel T (see e.g. Kantoff PW New England Journal of Medicine, 363(5):411-422, 2010; Schlom J., Journal of the National Cancer Institutes, 104(8) :599-613, 2012); treatment with dendritic cell vaccines or vaccines based on tumor antigenic peptides; treatment using chimeric antigen receptor (CAR-T) cells (see, e.g., Rosenberg SA Nature Reviews Cancer, 8(4):299-308, 2008; Porter DL et al., New England Journal of Medicine, 365(8):725-733, 2011; Grupp SA et al., New England Journal of Medicine, 368(16): 1509-151, 2013; U.S. Patent No. 9102761; U.S. Patent No. 9101584); treatment using CAR-NK cells (see, for example, Glienke et al., Front Pharmacol, 6(21): 1-7, Feb 2015); treatment using tumor-infiltrating lymphocytes (TIL) (see, for example, Wu et al, Cancer J., 18(2): 160-175, 2012); treatment using adoptively transferred antitumor T cells (cultured ex vivo and/or transgenic for TCR) (see, for example, Wrzesinski et al., J Immunother, 33(1): 1-7, 2010); treatment using TALL-104 cells; and treatment with immunostimulatory agents such as CpG Toll-like receptor (TLR) agonists and imiquimod (see, for example, Krieg, Oncogene, 27:161-167, 2008; Lu, Front Immunol, 5(83): 1- 4, March 2014).

In recent studies, immunotherapy focused on the use of depleting antibodies to specific tumor antigens has been highly successful (see, for example, Blattman and Greenberg, Science, 305:200, 2004; Adams and Weiner, Nat Biotech, 23:1147, 2005) . Some examples of such depleting antibodies specific for tumor antigens are HERCEPTIN® (anti-Her2/neu mAb) (Baselga et al., J Clin Oncology, Vol 14:737, 1996; Baselga et al., Cancer Research, 58: 2825, 1998; Shak, Semin Oncology, 26(Suppl2):71, 1999; Vogal et al J Clin Oncology, 20:719, 2002); and RITUXAN® (anti-CD20 mAb) (Colombat et al., Blood, 97:101, 2001). Unfortunately, despite their undoubted importance in the treatment of cancer, as monotherapy they are usually effective in only about 30% of individuals and with only a partial response. Moreover, after treatment with these antibody regimens, many individuals eventually develop refractoriness or relapses.

Treatment with agonistic, antagonistic, or blocking antibodies to co-stimulatory or co-inhibitory molecules (immune checkpoints) is the subject of active study and clinical research. Under normal physiological conditions, immune checkpoints are key to maintaining self-tolerance (i.e., preventing autoimmunity) and protecting tissues from damage in the immune system's response to pathogenic infection. It is now also clear that tumors recruit certain immune checkpoint pathways as an important mechanism of immune resistance, in particular to T cells specific for tumor antigens (Pardoll DM., Nat Rev Cancer, 12:252-64, 2012). Accordingly, treatment with antibodies to immune checkpoint molecules, including, for example, CTLA-4 (ipilimumab), PD-1 (nivolumab, pembrolizumab, pidilizumab (pidilizumab)) and PD-L1 (BMS-936559; MPLD3280A; MEDI4736; MSB0010718C ) (see, e.g., Philips and Atkins, International Immunology, 27(1); 39-46, Oct 2014), and OX-40, CD137, GITR, LAG3, TIM-3, and VISTA (see, e.g., Sharon et al., Chin J Cancer., 33(9): 434-444, Sep 2014; Hodi et al., N Engl J Med, 2010; Topalian et al., N Engl J Med, 366:2443-54), is being studied as new alternative immunotherapies for the treatment of patients with proliferative diseases such as cancer, and in particular patients with refractory and/or recurrent cancer.

Chimeric antigen receptor (CAR) T cell therapy is an immunotherapy in which a patient's own T cells are isolated in a laboratory, redirected by a synthetic receptor to recognize a specific antigen or protein, and reinfused into the patient. CARs are synthetic molecules containing at least (1) an antigen-binding region, typically derived from an antibody, (2) a transmembrane domain for anchoring the CAR on T cells, and (3) 1 or more intracellular T cell signaling domains. CAR redirects T cell antigen specificity in a manner independent of human leukocyte antigens (HLA) and overcomes problems associated with T cell tolerance (Kalos M and June CH, Immunity, 39(1):49-60, 2013). Over the past 5 years, at least 15 clinical trials of CAR-T cell therapy have been published. A new wave of interest in CAR-T cell therapy emerged in August 2011 when researchers at the University of Pennsylvania (Penn) published a report on 3 patients with refractory chronic lymphocytic leukemia (CLL) who, after a single dose of CAR-T cells, directed to CD 19, a long-term remission was obtained (Porter DL, et al., N Engl J Med., 365(8):725-733, 2011).

Unlike donor T cells, natural killer (NK) cells are known to mediate anti-cancer effects without the risk of inducing graft-versus-host disease (GvHD). Accordingly, alloreactive NK cells are now also in the spotlight as suitable and effective effector cells for cancer cell therapy. Several human NK cell lines have been developed, such as NK-92, HANK-1, KHYG-1, NK-YS, NKG, YT, YTS, NKL, and NK3.3 (Kornbluth, J., et al., J. Immunol 134, 728-735, 1985; Cheng, M. et al., Front.Med. 6:56, 2012), and various CAR-expressing NK cells (CAR-NK cells) have been generated. Immunotherapy with CAR-expressing NK cells (CAR-NK cells) is the subject of active study and clinical research (see, for example, Glienke et al., Front Pharmacol, 6(21):l-7, Feb 2015).

Bispecific T cell recruiting molecules (BiTE®) constitute a class of bispecific single chain antibodies for polyclonal activation of cytotoxic T cells and their redirection against pathogenic target cells. BiTE® are bispecific for surface target antigen on cancer cells and for CD3 on T cells. BiTE® are able to connect any type of cytotoxic T cell to a cancer cell, regardless of T cell receptor specificity, costimulation, or peptide antigen presentation. A unique set of properties, namely very high activity and efficacy against target cells at low numbers of T cells without the need for costimulation of T cells, has not yet been found in any other type of bispecific antibody-based constructs (Baeuerle et al., Cancer Res , 69(12):4941-4, 2009). BiTE antibodies have been constructed to more than 10 different target antigens, including CD19, EpCAM, Her2/neu, EGFR, CD66e (or CEA, CEACAM5), CD33, EphA2, and MCSP (or HMW-MAA) (Id.) . Treatment with BiTE® antibodies such as blinatumomab (Nagorsen, D. et al., Leukemia & Lymphoma 50(6): 886-891, 2009) and solitomab (Amann et al., Journal of Immunotherapy 32(5 ): 452-464, 2009) is undergoing clinical trials.

In some embodiments, the second therapy will include the administration of a PARP inhibitor. Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes involved in various activities in response to DNA damage. PARP-1 is a key DNA repair enzyme mediating single-strand break (SSB) repair by base excision repair (BER). It has been demonstrated that PARP inhibitors selectively kill tumor cells with BRCA1 and BRCA2 mutations. In addition, according to preclinical and preliminary clinical data, PARP inhibitors exhibit selective cytotoxicity against tumors with a deficiency of homologous recombination repair caused by dysfunction of genes other than BRCA1 or BRCA2. In some embodiments, the PARP inhibitor is selected from the group consisting of ABT-767, AZD 2461, BGB-290, BGP 15, CEP 9722, E7016, E7449, flusoparib, INO1001, JPI 289, MP 124, niraparib, olaparib, ONO2231, rucaparib, SC 101914, talazoparib, veliparib, WW 46, or salts or derivatives thereof. In some embodiments, the anti-PARP therapy is administered at a dose equivalent to about 100 mg, about 200 mg, or about 300 mg of niraparib, or a salt or derivative thereof. In some embodiments, the anti-PARP therapy is administered at a dose equivalent to about 100 mg of niraparib, or a salt or derivative thereof. In some embodiments, the anti-PARP therapy is administered at a dose equivalent to about 200 mg of niraparib, or a salt or derivative thereof. In certain embodiments, the anti-PARP therapy is administered at a dose equivalent to about 300 mg of niraparib or a salt or derivative thereof.

The AXL variant may be administered before, at the same time as, or after the second therapy, typically within at least about 1 week, at least about 5 days, at least about 3 days, at least about 1 day. The AXL variant may be delivered via a single dose or may be divided into multiple doses, such as delivery over a period of time, including once a day, once every two days, twice a week, once a week, etc. Further. The effective dose will vary depending on the route of administration, the particular agent, the dose of the cytoreductive agent, and the like, and may be empirically determined by one of ordinary skill in the art. For intravenously administered polypeptides, a useful range can be determined empirically, e.g., at least about 0.1 mg/kg body weight, at least about 0.5 mg/kg body weight, at least about 1 mg/kg body weight , at least about 2.5 mg/kg body weight, at least about 5 mg/kg body weight, at least about 10 mg/kg body weight, at least about 20 mg/kg body weight, or more. In some embodiments, the soluble AXL polypeptide variant will be administered by intravenous infusion over 30 or 60 minutes at a weekly dose of 10 mg/kg. In some embodiments, the soluble AXL polypeptide variant will be administered by IV infusion over 30 or 60 minutes at a weekly dose of 5 mg/kg. In some embodiments, the soluble AXL polypeptide variant will be administered by IV infusion over 30 or 60 minutes at a weekly dose of 2.5 mg/kg. In some embodiments, the soluble AXL polypeptide variant will be administered by intravenous infusion over 30 or 60 minutes at a weekly dose of 1 mg/kg. In some embodiments, the soluble AXL polypeptide variant will be administered by intravenous infusion over 30 or 60 minutes at a dose of 20 mg/kg every 14 days. In some embodiments, the soluble AXL polypeptide variant will be administered by intravenous infusion over 30 or 60 minutes at a dose of 10 mg/kg every 14 days. In some embodiments, the soluble AXL polypeptide variant will be administered by IV infusion over 30 or 60 minutes at a dose of 5 mg/kg every 14 days. In some embodiments, the soluble AXL polypeptide variant will be administered by intravenous infusion over 30 or 60 minutes at a dose of 2.5 mg/kg every 14 days. In some embodiments, the soluble AXL polypeptide variant will be administered by intravenous infusion over 30 or 60 minutes at a dose of 1 mg/kg every 14 days.

In some other embodiments, the therapeutic agents of the present invention are often administered in the form of pharmaceutical compositions, that is, compositions containing the active therapeutic agent and a variety of other pharmaceutically acceptable ingredients (See Remington's Pharmaceutical Science, 15.sup.th ed., Mack Publishing Company, Easton , Pa., 1980). The preferred form depends on the intended route of administration and therapeutic application. The compositions may also contain, depending on the composition desired, pharmaceutically acceptable non-toxic carriers or diluents, which are defined as excipients commonly used in the preparation of pharmaceutical compositions for administration to animals or humans. The diluent is chosen so that it does not affect the biological activity of the combination. Examples of such diluents are distilled water, phosphate buffered saline, Ringer's solutions, dextrose solution and Hank's solution. In addition, the pharmaceutical composition or composition may also contain other carriers, adjuvants or non-toxic non-therapeutic non-immunogenic stabilizers and the like.

In some other embodiments, the pharmaceutical compositions of the present invention may also contain large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids, and copolymers (such as latex-functionalized Sepharose™, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets and liposomes). In addition, these carriers can act as immunostimulatory agents (ie, adjuvants).

In other embodiments, the methods of the present invention comprise administering to a subject in need of treatment a therapeutically effective amount or effective dose of a therapeutic agent (eg, an inhibitory agent) of the present invention. In some embodiments, effective doses of a therapeutic agent of the present invention, for example, for the treatment of primary or metastatic cancer described herein, vary depending on many different factors, including the route of administration, the target area, the physiological state of the patient, whether the patient is human or animal , the administration of other drugs and whether the treatment is prophylactic or therapeutic. Usually the patient is a human, but it is also possible to treat non-human mammals, including transgenic mammals. Doses used in treatment should be titrated to optimize safety and efficacy.

In some embodiments, the dose may be from about 0.0001 to 100 mg/kg, and more commonly from 0.01 to 5 mg/kg, based on the body weight of the recipient. For example, doses may be 1 mg/kg body weight or 10 mg/kg body weight, or in the range of 1-10 mg/kg. In some embodiments, the dose of the soluble AXL polypeptide variant administered to the patient is selected from the group consisting of about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3 .5, approximately 4.0, approximately 4.5, approximately 5.0, approximately 5.5, approximately 6.0, approximately 6.5, approximately 7.0, approximately 7.5, approximately 8.0, approximately 8 .5, approximately 9.0, approximately 9.5, approximately 10.0 mg/kg, approximately 10.5, approximately 11.0, approximately 11.5, approximately 12.0, approximately 12.5, approximately 13.0 , approximately 13.5, approximately 14.0, approximately 14.5, approximately 15.0, approximately 15.5, approximately 16.0, approximately 16.5, approximately 17.0, approximately 17.5, approximately 18.0 , about 18.5, about 19.0 mg/kg, about 19.5 and about 20.0 mg/kg. In some embodiments, the soluble AXL polypeptide variant will be administered by intravenous infusion over 30 or 60 minutes at a weekly dose of 10 mg/kg. In some embodiments, the soluble AXL polypeptide variant will be administered by IV infusion over 30 or 60 minutes at a weekly dose of 5 mg/kg. In some embodiments, the soluble AXL polypeptide variant will be administered by IV infusion over 30 or 60 minutes at a weekly dose of 2.5 mg/kg. In some embodiments, the soluble AXL polypeptide variant will be administered by IV infusion over 30 or 60 minutes at a weekly dose of 1 mg/kg. In some embodiments, the soluble AXL polypeptide variant will be administered by IV infusion over 30 or 60 minutes at a dose of 20 mg /kg every 14 days. In some embodiments, the soluble AXL polypeptide variant will be administered by intravenous infusion over 30 or 60 minutes at a dose of 10 mg/kg every 14 days. In some embodiments, the soluble AXL polypeptide variant will be administered by IV infusion over 30 or 60 minutes at a dose of 5 mg/kg every 14 days. In some embodiments, the soluble AXL polypeptide variant will be administered by intravenous infusion over 30 or 60 minutes at a dose of 2.5 mg/kg every 14 days. In some embodiments, the soluble AXL polypeptide variant will be administered by intravenous infusion over 30 or 60 minutes at a dose of 1 mg/kg every 14 days.

In some embodiments, the treatment regimen provides for the introduction of once every two weeks, or once a month, or once every 3-6 months. Therapeutic agents of the present invention are usually administered multiple times. The intervals between individual injections can be a week, a month or a year. In addition, the intervals may be irregular and depend on the measurement of levels of the therapeutic agent in the blood of the patient. Alternatively, the therapeutic agents of the present invention may be administered in the form of a sustained release formulation, in which case less frequent administration is necessary. The dose and frequency will vary depending on the half-life of the polypeptide in the patient.

In prophylactic use, a relatively low dose is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic use, a relatively high dose is sometimes required at relatively short intervals to reduce or stop the progression of the disease and preferably to partially or completely reduce the intensity of the symptoms of the disease. After this, the introduction to the patient can be carried out according to the prophylactic scheme.

In other embodiments, the methods of the present invention include treating, reducing or preventing primary tumor formation or tumor metastasis or tumor invasion in AML, ovarian cancer, breast cancer, lung cancer, liver cancer, colon cancer, gallbladder cancer, pancreatic cancer. gland, prostate cancer and/or glioblastoma.

In some other embodiments, for prophylactic use, pharmaceutical compositions or medicaments are administered to a patient who is susceptible to or otherwise at risk of developing a disease or condition, in an amount sufficient to eliminate or reduce said risk, reduce the severity or delay the onset of the disease, including biochemical, histological and /or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes that occur during the development of the disease.

In some other embodiments, for therapeutic use, the therapeutic agents of the present invention are administered to a patient suspected of having such a disease or already suffering from such a disease in an amount sufficient to cure or at least partially reduce the symptoms of the disease (biochemical, histological and/or behavioral), including its complications and intermediate pathological phenotypes during the development of the disease. An amount adequate to carry out therapeutic or prophylactic treatment is defined as a therapeutically or prophylactically effective dose. In both prophylactic and therapeutic regimens, agents are typically administered multiple times until a sufficient response is achieved. Response is usually monitored with repeated doses in case of cancer recurrence.

According to the present invention, compositions for the treatment of primary or metastatic cancer can be administered by parenteral, topical, intravenous, intratumoral, oral, subcutaneous, intra-arterial, intracranial, intraperitoneal, intranasal, or intramuscular routes. The most typical route of administration is intravenous or intratumoral, however, other routes may be equally effective.

For parenteral administration, the compositions of the invention may be administered in the form of injectable doses of a solution or suspension of the substance in a physiologically acceptable diluent with a pharmaceutical carrier, which may be a sterile liquid such as water, oils, saline, glycerol or ethanol. In addition, adjuvants such as wetting agents or emulsifiers, surfactants, pH buffering agents, and the like may be present in the compositions. Other components of pharmaceutical compositions are components of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil and mineral oil. Preferred liquid carriers, especially for injectable solutions, are generally glycols such as propylene glycol or polyethylene glycol. Antibodies and/or polypeptides can be administered in the form of a depot injection or implant, which can be designed to provide sustained release of the active ingredient. In some embodiments, the composition contains the polypeptide at a concentration of 1 mg/ml, prepared in an aqueous buffer consisting of 10 mM Tris, 210 mM sucrose, 51 mM L-arginine, 0.01% polysorbate 20 adjusted to pH 7.4 using HCl or NaOH.

Usually the compositions are made in forms for injection in the form of liquid solutions or suspensions; solid forms suitable for dissolution or suspension in liquid vehicles prior to injection may also be made. The drug may also be emulsified or encapsulated in liposomes or microparticles such as polylactide, polyglycolide or copolymer to enhance the adjuvant effect as discussed above. Langer, Science 249: 1527, 1990; and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. The agents of this invention may be administered in the form of a depot injection or implant, which may be designed to provide sustained or pulsatile release of the active ingredient.

Additional formulations suitable for other routes of administration include oral, intranasal, and pulmonary formulations, suppositories, and transdermal formulations.

For suppositories, binding agents and carriers include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in an amount of from 0.5% to 10%, preferably 1-2%. Compositions for oral administration contain excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose and magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10-95% active ingredient, preferably 25-70%.

Topical application may result in transdermal or intradermal delivery.

Local administration may be facilitated by co-administration of the agent with cholera toxin or detoxified derivatives or subunits thereof, or with other similar bacterial toxins. Glenn et al., Nature 391: 851, 1998. Co-administration can be carried out using components in the form of a mixture or in the form of linked molecules obtained by chemical cross-linking or expression as a fusion protein. Alternatively, transdermal delivery can be done using a skin patch or using transferosomes. Paul et al., Eur. J. Immunol. 25:3521-24, 1995; Cevc et al., Biochem. Biophys. Acta 1368: 201-15, 1998.

Pharmaceutical compositions are typically formulated sterile, substantially isotonic, and in full compliance with Good Manufacturing Practice (GMP) requirements of the US Food and Drug Administration. Preferably, a therapeutically effective dose of the polypeptide compositions described herein will provide a beneficial therapeutic effect without causing significant toxicity.

The toxicity of the proteins described herein can be determined by standard pharmaceutical techniques in cell cultures or experimental animals, for example by determining LD50 (50% population lethal dose) or LD ₁₀₀ (100% population lethal dose). The ratio of doses that have a toxic and therapeutic effect is a therapeutic index. Data from such cell culture assays or from animal studies can be used to determine the range of doses that are not toxic when administered to humans. The dose of the proteins described herein preferably falls within a range of circulating blood concentrations including a low toxic or non-toxic effective dose. The dose may vary within this range depending on the dosage form used and the route of administration used. The specific composition, route of administration and dose will be selected by the attending physician based on the condition of the patient (See, for example, Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1).

The scope of the invention also includes kits containing compositions of the invention and instructions for use. The kit may further comprise at least one additional reagent, such as a cytoreductive drug. The compositions may be presented in unit dosage form.

Kits usually contain a label with directions for using the contents of the kit. The term "label" includes any written or recorded material supplied on or with a kit or otherwise accompanying a kit.

All publications and patents cited in this specification are incorporated herein by reference as if it were specifically and individually indicated that each individual publication or patent is incorporated by reference, and is incorporated herein by reference for the disclosure and description of methods and/or substances. for which these publications are cited. The citation of any publication is conditioned on its disclosure prior to the filing date of this application and should not be construed as an admission that the present invention cannot precede such publication by an earlier invention. In addition, publication dates shown may differ from actual publication dates, which may need to be independently verified.

As will be clear to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that can be easily separated from or combined with features of any of a number of other embodiments without departing from the scope or spirit. of the present invention. Any of the above methods can be carried out in the order indicated, or in any other logically possible order. It should also be understood that the terminology used herein is intended to describe specific embodiments.

Although for the sake of clarity of understanding the invention has been described in detail above by means of explanations and examples, it will be clear to a person skilled in the art that, in light of the ideas of this invention, certain changes and modifications of the invention are possible without departing from its essence, and that this is not limits the scope of the present invention, which will be limited only by the appended claims.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, that there are many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the appended claims.

EXPERIMENTAL PART

Example 1

Inhibition of GAS6-induced invasion/migration using the AXL decoy receptor

Inhibition of GAS6-induced invasion/migration was assessed in triple-negative breast cancer (MDA-MB-231) and ovarian cancer (OVCAR8) models using the invasion assay with Corning® Matrigel® or collagen, respectively, and using the AXL decoy receptor , containing a soluble variant of the AXL polypeptide containing amino acid changes relative to the wild-type AXL sequence (SEQ ID NO: 1) at the following positions: (a) glycine 32, (b) aspartic acid 87, (c) alanine 72, (d) valine 92 and (e) glycine 127 lacking an AXL transmembrane domain, lacking a functional FN domain, and containing an Fc domain linked to a soluble AXL polypeptide variant by a peptide linker (hereinafter referred to as AVB-S6-500).

AVB-S6-500 and MDA-MB-231 Ax1 ⁺ TNBC cells in serum-free medium were placed in the upper compartment of the Boyden chamber coated with Matrigel. Medium with serum as a chemoattractant was introduced into the lower compartment of the chamber. After 24 hours of incubation, the number of cells migrating through Matrigel was determined and expressed as the proportion of invasive cells relative to PBS control (FIG. 1A). AVB-S6-500, OVCAR8 Ax1 ⁺ ovarian cancer cells, type 1 collagen, 50 ng/ml GAS6 and culture media were placed in microwells and incubated. On day 6, the number of cells showing an invasive phenotype was determined and expressed as the proportion of invasive cells relative to PBS control (FIG. 1B). AVB-S6-500 in the range of 1 to 100 μg/ml significantly inhibited GAS6-induced invasion/migration.

Example 2

Determination of IC ₅₀ values and comparison of AXL decoy receptor with tyrosine kinase inhibitor in the MDA-MB-231 cell invasion assay IC ₅₀ values of AVB-S6-500 were determined in the MDA-MB-231 cell invasion assay with and without 50 nM GAS6 and cell viability assay and compared them with the IC ₅₀ of bosutinib, an approved tyrosine kinase inhibitor. Representative images of MDA-MB-231 cells treated with AVB-S6-500 are shown in FIG. 2. As shown in Table 1, AVB-S6-500 inhibited cell invasion approximately 100 times more than bosutinib and did not affect cell viability when using a panel of cell lines from 8 different types of cancer (colon cancer, breast cancer, AML, ovarian cancer, pancreatic cancer, and NSCLC (non-small cell lung cancer)) compared to seven standard cytotoxic/chemotherapeutic (SOC) drugs.

Example 3

Reduction of metastatic tumor burden using the AXL decoy receptor

Mice underwent intraperitoneal (ip) administration of SKOVS.ip ovarian cancer tumor cells (1×10 ⁶ ), randomized into groups, and injected with AVB-S6-500 at a dose of 5, 10 or 20 mg/kg every other day (Q2D). Metastatic tumor burden was assessed after 24 days of AVB-S6-500 dosing by counting all visible metastatic lesions in the peritoneal cavity with excision and weighing of all diseased tissue to determine the total mass (FIG. 3A) and number (FIG. 3B) of metastases. AVB-S6-500 significantly reduced the metastatic tumor burden when administered at doses of 10 and 20 mg/kg. In a xenotransplantation model of SKOV3.IP in mice, AVB-S6-500 monotherapy at doses of 10 and 20 mg/kg Q2D (equivalent to 2.5-5 mg/kg per week in humans) significantly reduced the average number and weight of macroscopic metastatic foci and dramatically reduced serum free GAS6 levels.

Example 4

Increased efficacy with combination of AXL decoy receptor and doxorubicin

Mice were intraperitoneally (ip) injected with SKOV3.ip ovarian cancer tumor cells (1×10 ⁶ ), randomized into groups, and injected with AVB-S6-500 at a dose of 20 mg/kg Q2D alone or in combination with doxorubicin ( DOX), 2 mg/kg twice a week. Metastatic tumor burden was assessed after 24 days of dosing. Comparison of total mass (FIG. 4A) and number (FIG. 4B) of metastases demonstrated a significant benefit of combination therapy. The combination of AVB-S6-500 and doxorubicin significantly reduced the mean mass of diseased tissue and resulted in a cure in 2 animals.

Example 5

PK (pharmacokinetic) studies

In studies in mice, a link has been established between a decrease in sGAS6 (a drug target) and an antimetastatic effect. Doses that have been effective in studies in mice have been associated with a dramatic decrease in serum GAS6 levels in mice and correspond to 0.5-1.7 mg/kg in humans. In PK studies in cynomolgus monkeys, AVB-S6-500 demonstrated a dramatic decrease in serum GAS6 in cynomolgus monkeys lasting approximately 1 week after a single dose of 5 mg/kg (human equivalent dose 1.7 mg/kg), which is the desired profile for administration. person by intravenous infusion once a week.

In view of the noted preclinical association between a decrease in serum GAS6 (sGAS6) and antimetastatic activity, sGAS6 has been identified as a useful biomarker in pharmacodynamic (PD) analysis to evaluate sGAS6 levels in all preclinical studies, including GLP toxicology. In the conducted studies, a pronounced constancy of PK / PD was noted.

Example 6

Safety Research

Mice and monkeys well tolerated single and multiple dosing of AVB-S6-500 at doses well in excess of those required for the desired biological effect. After slow bolus intravenous (IV) injections, once or twice a week at doses of 25, 50, and 100 mg/kg (50, 100, and 200 mg/kg per week), in or after CD-1 male and female mice Four 30-minute IV infusions at doses of 30, 100, and 150 mg/kg in monkeys resulted in no deaths or treatment-related adverse effects. All doses resulted in a complete reduction in serum GAS6 levels throughout the study period. No adverse effect levels (NOAEL) were defined as 200 mg/kg per week (highest dose) in mice and 150 mg/kg per week in monkeys, respectively. Based on pharmacokinetic/pharmacodynamic modeling as well as extrapolation of effective doses determined in mice, a dose of 1.5-5 mg/kg of AVB-S6-500 is predicted to be effective in humans. In preclinical studies using sGAS6 as a biomarker, a pronounced persistence of PK/PD has been noted.

AVB-S6-500 was shown to be effective in reducing the metastatic cancer burden in xenotransplant models of human breast and ovarian cancer and safe in cynomolgus monkeys and mice at much higher doses. These results are consistent with previous decoy receptors, demonstrating efficacy and safety in many cancer models and supporting the safe use of AVB-S6-500 in healthy volunteers.

Animal PK/PD modeling has been applied to guide dosing in human studies, taking into account the elevated sGAS6 observed in patients with cancer. Specifically, the toxicological profile allowed dosing to healthy volunteers, and GLP toxicology studies were combined with dose selection based on PD for the first human study. The effect of GAS6 on the clearance of AVB-S6-500 was included in a target-mediated drug distribution (TMDD) model that assumed parallel linear and non-linear clearance of AVB-S6. Simulation of suppression by human GAS6 was performed at dose levels of 1, 2.5, 5 and 10 mg/kg using monkey data. Given the potentially higher levels of sGAS6 in cancer patients and combination chemotherapeutic regimens, various AVB-S6 administration regimens were modeled to predict target effects at doses expected to be used in oncology studies. Using the target-mediated drug distribution (TMDD) model, the human dose calculated to be effective ranged from 1.5 mg/kg (to ensure that GAS6 is maintained at least 50% below baseline) to 5 mg /kg (to provide a 97% reduction in free GAS6 and a possible 3-fold increase in GAS6 levels relative to normal levels).

Example 7

A blinded, randomized, placebo-controlled phase I trial investigating the safety and tolerability of AVB-S6-500 in healthy subjects with a single intravenous dose of increasing dose and multiple intravenous administration

The safety and tolerability of single intravenous administration of AVB-S6-500 at increasing dose (SAD) and multiple intravenous administration of AVB-S6-500 at a single dose (5 mg/kg) (RD) were assessed for a total of four administrations given once a day. week in healthy patients. The pharmacokinetics (PK) and pharmacodynamics (PD) in SAD and RD have been characterized.

Eligible subjects were randomized 3:1 to receive AVB-S6-500 or placebo at the doses shown in Table 2 and Table 3; the subjects did not know which drug they were administered.

For each subject, the study consisted of 3 periods: a pre-treatment period (including a screening visit no more than 28 days prior to day 1), a treatment period, and a follow-up period (end-of-study/early withdrawal visit). At the end of the end-of-study (EOS)/early withdrawal (EW) visit for each cohort, the sponsor, medical monitor (MM), and investigator review the available safety data from all doses to determine whether to continue the AVB- study. S6-500 at the next higher dose.

Subjects included in the escalating single dose groups received a single dose of AVB-S6-500 or placebo according to a randomization scheme; in the multiple dose group, subjects received four injections of AVB-S6-500 or placebo (once a week for 4 weeks) according to the randomization scheme. RD subjects returned to the clinic for each study dose on day 1 of weeks 2 and 3 and continued to attend outpatient visits after each weekly dose. Subjects entered the CRU on day 1 week 4 and remained in the CRU for 24 hours after administration on week 4 to facilitate blood sampling for PK/PD assessment. On day 2 of the week, 4 subjects were discharged from CRU after completion of all examinations scheduled for that day, and they continued to come for outpatient visits until the EOS/EW visit.

All doses of study drug were prepared as infusion solutions in 150 ml diluent (in 250 ml bags) for intravenous infusion over 1 hour. All injections were carried out by medical personnel in a clinic setting. Study drug was supplied in vials containing 10 ml of AVB-S6-500 (concentration 20 mg/ml; total AVB-S6-500 200 mg per vial). AVB-S6-500 was not packaged according to individual subject numbers. Preparation of the investigational medicinal product for intravenous administration was carried out by a pharmacist or a qualified person who performed his duties, based on randomization codes. AVB-S6-500 solution for infusion was packaged and labeled in accordance with current GMP requirements and supplied to the clinic in 20 ml vials (10 ml in each vial).

Blood (serum) samples for analysis of AVB-S6-500 concentration and GAS6 levels (including a pharmacodynamic marker) in subjects included in single dose escalation groups were obtained at the following time points relative to administration: within 45 minutes prior to administration (0 hours ) and approximately 1, 2, 4, 6, 8, 24, 72, 120, 168, and 336 hours after administration. For subjects included in the multiple dose group, serum samples for AVB-S6-500 and GAS6 analysis were obtained at the following time points: study week 1 - within 45 minutes prior to administration (before administration) and approximately 1, 2, 4, 6 , 8, 24, 72 and 120 hours after administration; study week 2 - before administration (within 45 minutes prior to administration; also corresponds to the 168 hour time point for week 1); week 3 of the study - before the introduction (within 45 minutes before the introduction); week 4 of the study - within 45 minutes prior to administration (before administration) and approximately 1, 2, 4, 6, 8, 24, 72, 120, 168, 504, 528 and 696 hours after administration.

For pharmacokinetic and pharmacodynamic analysis, blood samples (4 ml) were placed in serum isolation tubes and processed as described in the PK/PD/ADA assay laboratory manual. Serum samples for pharmacokinetic evaluation of AVB-S6-500 and pharmacodynamic evaluation of GAS6 levels are sent to SNBL. One set of serum samples from each subject is sent on a continuous basis, with multiple shipments per cohort (schedule for shipment is defined in the Study PK/PD Assay Guidelines). The remaining second or third copies of the samples are stored in the CRU as a reserve. Blood samples (4 ml) obtained at a number of time points for PK and PD analysis are analyzed for the presence of anti-drug antibodies (ADA). Pharmacokinetic data will be taken into account when increasing the dose to 10 mg / kg. Specifically, PK data were obtained from the previous administration groups and compared with the current calculated PK values for humans, scaling the data obtained in preclinical studies. Then, as needed, these data were used to reassess the approximate safety margins for subsequent doses.

AVB-S6-500 was well tolerated at all doses. There were no serious adverse events. There were no treatment-related changes on physical examination and assessment of vital signs. None of the laboratory-based AEs (adverse events) were considered clinically significant, none required treatment, and all were asymptomatic. According to the protocol, all laboratory parameters that met the CTCAE v4.03 criteria for subjects receiving the active drug were considered as possibly related. None of these have been found to be likely or definitely related.

Following single IV infusions, AVB-S6-500 PK has shown similar characteristics to other protein-based drugs such as monoclonal antibodies, which typically show small volumes of distribution and biphasic elimination. The maximum serum concentration of AVB-S6-500 (C _max ) and the area under the concentration-time curve (AUC) increased with increasing dose. Over the dose range studied, the increase in C _max was approximately dose-proportional, while the increase in AUC was slightly higher than dose-proportional, indicating a non-linear elimination kinetics, which is characteristic of TMDD. With repeated administration once a week, the last measured concentration, determined immediately before the subsequent infusion (C _trough ), after the 4th administration was approximately 2 times higher than after the 1st administration, indicating a moderate accumulation, which corresponds to the half-life after single injection, amounting to 59 hours. None of the subjects in all of the single and multiple dose groups were positive assays for anti-AVB-S6 antibodies. In addition, reflecting the absence of anti-AVB-S6 antibody positive subjects, there was a prolonged decrease in serum GAS6 with repeated administration of AVB-S6.

As shown in FIG. 6 and 7, serum GAS6 levels were reduced one week after 1 mg/kg (seen in 4 of 6 subjects) (FIG. 6A), one week after 2.5 mg/kg (seen in 6 of 6 subjects) (FIG. 6B), two weeks after administration at a dose of 5 mg/kg (noted in 6 of 6 subjects) (FIG. 7A), two weeks after administration at a dose of 10 mg/kg (noted in 6 out of 6 subjects) and three weeks after administration at a dose of 10 mg/kg (noted in 3 out of 6 subjects).

As shown in FIG. 8 and 9, AVB-S6-500 levels show a dose-dependence and the lowest dose of AVB-S6-500 (1 mg/kg) is pharmacologically active. Mean serum GAS6 levels in subjects were 15.7 plus/minus 3.9 ng/mL. A single infusion of AVB-S6 at doses of 1, 2.5, 5, or 10 mg/kg to healthy subjects resulted in an immediate peak decrease in circulating serum GAS6 concentrations to BLQ levels (below the limit of quantitation) (2 ng/mL). After infusions of AVB-S6-500 at doses of 1 and 2.5 mg/kg, the decrease in GAS6 persisted for 7 days. After infusions at doses of 5 and 10 mg/kg, the decrease in serum GAS6 below detectable levels persisted for 22 and 29 days, respectively. Infusions of AVB-S6-500 to healthy subjects at 5 mg/kg once weekly resulted in an immediate and sustained peak reduction in circulating serum GAS6 concentrations to BLQ levels. The decrease in GAS6 to BLQ levels was maintained in all subjects until 504 hours after the last infusion, when GAS6 was measurable and above the LLOQ (lower limit of quantitation) in 2 of 6 subjects, without however returning to baseline levels. In all other subjects (4 out of 6), GAS6 concentrations remained BLQ. Thus, PK/PD modeling validated the choice of oncology administration regimens that would reduce (greater than 90% reduction) sGAS6 and be comparable to chemotherapeutic regimens.

The PK/PD profile determined in humans was consistent with preclinical and model data.

Example 8

Phase Ib/II study investigating AVB-S6-500 in combination with pegylated liposomal doxorubicin (PLD) or paclitaxel in patients with platinum-resistant recurrent ovarian cancer

Analysis of sGAS6 levels in 48 patients with ovarian cancer suggested that these levels were 2 times higher than those determined in a study in healthy volunteers. Based on data from a PK/PD model in monkeys, a phase I study in healthy volunteers, and a simulated increase in sGAS6 levels, dosing schedules of 5 mg/kg once a week or 10 mg/kg every other week will result in to a dramatic decrease in sGAS6 levels in patients with cancer. PK/PD modeling allowed us to confirm dose selection for a study in patients with ovarian cancer that would result in a reduction in target levels in more than 90% of patients, to identify multiple dosing regimens that would be comparable to chemotherapeutic dosing regimens, and to limit the number of patient visits. The use of proprietary PD analysis, combined with safety profiling and PK/PD in healthy volunteers, allowed rationalization of the clinical program and formed the basis for dose selection for oncological studies.

Phase Ib

In the phase Ib section, an open-label study of the safety and tolerability of AVB-S6-500 in combination with pegylated liposomal doxorubicin (PLD) or paclitaxel in patients with recurrent platinum-resistant ovarian cancer will be conducted.

Patients in the AVB-S6-500 plus PLD group will receive AVB-S6-500 by IV infusion over 30 or 60 minutes at a dose of 10 mg/kg in combination with PLD administered by IV infusion over 60 minutes at a dose of 40 mg /m ² per day 1 of the 1st cycle of therapy. Then AVB-S6-500 will be administered at a dose of 10 mg/kg every 14 days, starting on day 15 of the 1st cycle. In case of poor tolerance of AVB-S6-500 at 10 mg/kg every 2 weeks (q2w) in combination with PLD, the dose of AVB-S6-500 will be reduced to 5 mg/kg once a week. This new cohort will include six new patients.

Patients in the AVB-S6-500 plus paclitaxel group will receive AVB-S6-500 once a week by IV infusion over 30 or 60 minutes at a dose of 10 mg/kg in combination with paclitaxel administered once a week by IV infusion for 60 minutes at a dose of 80 mg / m ² per day 1, 8, 15 and 22 of each cycle of therapy lasting 28 days. In each group, each combination chemotherapy regimen will be first applied to six patients to evaluate the safety of the combination. In case of poor tolerance of AVB-S6-500 at 10 mg/kg in combination with paclitaxel, the dose of AVB-S6-500 will be reduced to 5 mg/kg once a week. This new cohort will include six new patients. With a well tolerated dose of 5 mg/kg in combination with paclitaxel, this regimen will be used in an additional 6 patients.

RP2D will be the dose/schedule of AVB-S6-500 that is found to be safe/tolerated in combination with an appropriate primary chemotherapy regimen and, based on a 1-month PK/PD analysis in a phase lb study, achieves AVB-S6-500 levels in serum above 3720 ng/mL and reduce serum GAS6 in all BLQ patients throughout the administration interval. Study drug administration will continue until residual tumor disappears (in the case of a chemotherapeutic agent; AVB-S6-500 administration should be continued for at least one year after a complete response) or until disease progression, death, withdrawal of informed consent, or unacceptable toxicity .

Phase II

Once the RP2D for AVB-S6-500 has been determined and the tolerable dose of the combination has the desired effect of dramatically lowering serum GAS6, a randomized, double-blind comparison of progression-free survival (PFS) in patients with platinum-resistant recurrent ovarian cancer receiving AVB-S6-500 plus PLD versus placebo plus PLD or AVB-S6-500 plus paclitaxel versus placebo plus paclitaxel. As a 2nd endpoint, objective response rate (ORR) can also be analyzed. AVB-S6-500 will be administered by IV infusion over 60 minutes on an RP2D regimen starting on day 1, with a 28-day therapy cycle. The physician's choice of chemotherapy includes the following options: (1) paclitaxel administered once a week by intravenous infusion over 60 minutes at a dose of 80 mg/m ² with a therapy cycle of 28 days; or (2) PLD administered by IV infusion over 60 minutes at a dose of 40 mg/m ² per day for 1 treatment cycle of 28 days. Study drug administration will continue until residual tumor disappears (in the case of a chemotherapeutic agent; AVB-S6-500 administration should be continued for at least one year after a complete response) or until disease progression, death, withdrawal of informed consent, or unacceptable toxicity .

Patients will be enrolled and randomized 2:1 to one of two large treatment groups: Group A (AVB-S6-500 plus chemotherapy of the physician's choice) and Group B (placebo plus chemotherapy of the physician's choice). Each large arm will have two arms: one combination regimen with PLD and one combination regimen with paclitaxel.

In light of the present disclosure, all of the articles and methods disclosed and claimed herein can be made and practiced without undue experimentation. While the articles and methods of this invention have been described by way of preferred embodiments, those skilled in the art will appreciate that variations may be applied to said articles and methods without departing from the spirit and scope of the invention. It is intended that all such variations and equivalents apparent to those skilled in the art, existing at the time of filing this application or later developed, are included within the spirit and scope of the invention as defined by the appended claims. All patents, patent applications and publications mentioned in this specification are indicative of the level of those skilled in the art to which this invention pertains. All patents, patent applications, and publications are hereby incorporated by reference in their entirety for any purpose, and to the same extent as if it were specifically and individually indicated that each individual publication is incorporated by reference in its entirety for any purpose. The invention pictorially described here can be suitably applied in the absence of any(s) element(s) not disclosed(s) here specifically. Thus, it should be understood that while the present invention has been specifically disclosed by way of preferred embodiments and possible features, modifications and variations on the concepts disclosed herein may be made by those skilled in the art, and that such modifications and variations are contemplated as being included in the scope of this invention as defined by the appended claims.

SEQUENCE LIST

The nucleic acid sequences and amino acid sequences shown in the appended sequence listing are shown using the standard nucleotide base letter abbreviations and the three-letter amino acid code as defined in 37 CFR Part 1.822 (37 C.F.R. 1.822).

SEQ ID NO: 1 is the amino acid sequence of the human AXL polypeptide.

SEQ ID NO: 1 - Amino acid sequence of human AXL polypeptide

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SEQUENCE LIST

SEQUENCE LISTING

<110> Aravive Biologics, Inc.

<120> METHODS OF TREATING METASTATIC CANCERS USING AXL DECOY RECEPTORS

<130> CACAB1.0009WO

<160> 1

<170>PatentIn version 3.5

<210> 1

<211> 887

<212> PRT

<213> Homo sapiens

<400> 1

Met Gly Arg Val Pro Leu Ala Trp Cys Leu Ala Leu Cys Gly Trp Ala

1 5 10 15

Cys Met Ala Pro Arg Gly Thr Gln Ala Glu Glu Ser Pro Phe Val Gly

20 25 30

Asn Pro Gly Asn Ile Thr Gly Ala Arg Gly Leu Thr Gly Thr Leu Arg

35 40 45

Cys Gln Leu Gln Val Gln Gly Glu Pro Pro Glu Val His Trp Leu Arg

50 55 60

Asp Gly Gln Ile Leu Glu Leu Ala Asp Ser Thr Gln Thr Gln Val Pro

65 70 75 80

Leu Gly Glu Asp Glu Gln Asp Asp Trp Ile Val Val Ser Gln Leu Arg

85 90 95

Ile Thr Ser Leu Gln Leu Ser Asp Thr Gly Gln Tyr Gln Cys Leu Val

100 105 110

Phe Leu Gly His Gln Thr Phe Val Ser Gln Pro Gly Tyr Val Gly Leu

115 120 125

Glu Gly Leu Pro Tyr Phe Leu Glu Glu Pro Glu Asp Arg Thr Val Ala

130 135 140

Ala Asn Thr Pro Phe Asn Leu Ser Cys Gln Ala Gln Gly Pro Pro Glu

145 150 155 160

Pro Val Asp Leu Leu Trp Leu Gln Asp Ala Val Pro Leu Ala Thr Ala

165 170 175

Pro Gly His Gly Pro Gln Arg Ser Leu His Val Pro Gly Leu Asn Lys

180 185 190

Thr Ser Ser Phe Ser Cys Glu Ala His Asn Ala Lys Gly Val Thr Thr

195 200 205

Ser Arg Thr Ala Thr Ile Thr Val Leu Pro Gln Gln Pro Arg Asn Leu

210 215 220

His Leu Val Ser Arg Gln Pro Thr Glu Leu Glu Val Ala Trp Thr Pro

225 230 235 240

Gly Leu Ser Gly Ile Tyr Pro Leu Thr His Cys Thr Leu Gln Ala Val

245 250 255

Leu Ser Asn Asp Gly Met Gly Ile Gln Ala Gly Glu Pro Asp Pro Pro

260 265 270

Glu Glu Pro Leu Thr Ser Gln Ala Ser Val Pro Pro His Gln Leu Arg

275 280 285

Leu Gly Ser Leu His Pro His Thr Pro Tyr His Ile Arg Val Ala Cys

290 295 300

Thr Ser Ser Gln Gly Pro Ser Ser Trp Thr His Trp Leu Pro Val Glu

305 310 315 320

Thr Pro Glu Gly Val Pro Leu Gly Pro Pro Glu Asn Ile Ser Ala Thr

325 330 335

Arg Asn Gly Ser Gln Ala Phe Val His Trp Gln Glu Pro Arg Ala Pro

340 345 350

Leu Gln Gly Thr Leu Leu Gly Tyr Arg Leu Ala Tyr Gln Gly Gln Asp

355 360 365

Thr Pro Glu Val Leu Met Asp Ile Gly Leu Arg Gln Glu Val Thr Leu

370 375 380

Glu Leu Gln Gly Asp Gly Ser Val Ser Asn Leu Thr Val Cys Val Ala

385 390 395 400

Ala Tyr Thr Ala Ala Gly Asp Gly Pro Trp Ser Leu Pro Val Pro Leu

405 410 415

Glu Ala Trp Arg Pro Gly Gln Ala Gln Pro Val His Gln Leu Val Lys

420 425 430

Glu Pro Ser Thr Pro Ala Phe Ser Trp Pro Trp Trp Tyr Val Leu Leu

435 440 445

Gly Ala Val Val Ala Ala Ala Cys Val Leu Ile Leu Ala Leu Phe Leu

450 455 460

Val His Arg Arg Lys Lys Glu Thr Arg Tyr Gly Glu Val Phe Glu Pro

465 470 475 480

Thr Val Glu Arg Gly Glu Leu Val Val Arg Tyr Arg Val Arg Lys Ser

485 490 495

Tyr Ser Arg Arg Thr Thr Glu Ala Thr Leu Asn Ser Leu Gly Ile Ser

500 505 510

Glu Glu Leu Lys Glu Lys Leu Arg Asp Val Met Val Asp Arg His Lys

515 520 525

Val Ala Leu Gly Lys Thr Leu Gly Glu Gly Glu Phe Gly Ala Val Met

530 535 540

Glu Gly Gln Leu Asn Gln Asp Asp Ser Ile Leu Lys Val Ala Val Lys

545 550 555 560

Thr Met Lys Ile Ala Ile Cys Thr Arg Ser Glu Leu Glu Asp Phe Leu

565 570 575

Ser Glu Ala Val Cys Met Lys Glu Phe Asp His Pro Asn Val Met Arg

580 585 590

Leu Ile Gly Val Cys Phe Gln Gly Ser Glu Arg Glu Ser Phe Pro Ala

595 600 605

Pro Val Val Ile Leu Pro Phe Met Lys His Gly Asp Leu His Ser Phe

610 615 620

Leu Leu Tyr Ser Arg Leu Gly Asp Gln Pro Val Tyr Leu Pro Thr Gln

625 630 635 640

Met Leu Val Lys Phe Met Ala Asp Ile Ala Ser Gly Met Glu Tyr Leu

645 650 655

Ser Thr Lys Arg Phe Ile His Arg Asp Leu Ala Ala Arg Asn Cys Met

660 665 670

Leu Asn Glu Asn Met Ser Val Cys Val Ala Asp Phe Gly Leu Ser Lys

675 680 685

Lys Ile Tyr Asn Gly Asp Tyr Tyr Arg Gln Gly Arg Ile Ala Lys Met

690 695 700

Pro Val Lys Trp Ile Ala Ile Glu Ser Leu Ala Asp Arg Val Tyr Thr

705 710 715 720

Ser Lys Ser Asp Val Trp Ser Phe Gly Val Thr Met Trp Glu Ile Ala

725 730 735

Thr Arg Gly Gln Thr Pro Tyr Pro Gly Val Glu Asn Ser Glu Ile Tyr

740 745 750

Asp Tyr Leu Arg Gln Gly Asn Arg Leu Lys Gln Pro Ala Asp Cys Leu

755 760 765

Asp Gly Leu Tyr Ala Leu Met Ser Arg Cys Trp Glu Leu Asn Pro Gln

770 775 780

Asp Arg Pro Ser Phe Thr Glu Leu Arg Glu Asp Leu Glu Asn Thr Leu

785 790 795 800

Lys Ala Leu Pro Pro Ala Gln Glu Pro Asp Glu Ile Leu Tyr Val Asn

805 810 815

Met Asp Glu Gly Gly Gly Tyr Pro Glu Pro Pro Gly Ala Ala Gly Gly

820 825 830

Ala Asp Pro Pro Thr Gln Pro Asp Pro Lys Asp Ser Cys Ser Cys Leu

835 840 845

Thr Ala Ala Glu Val His Pro Ala Gly Arg Tyr Val Leu Cys Pro Ser

850 855 860

Thr Thr Pro Ser Pro Ala Gln Pro Ala Asp Arg Gly Ser Pro Ala Ala

865 870 875 880

Pro Gly Gln Glu Asp Gly Ala

885

<---

Claims (4)

1. A method of treating platinum-resistant human metastatic ovarian cancer in a patient, comprising administering to said patient a therapeutically effective dose of a soluble AXL polypeptide variant, wherein the soluble AXL polypeptide variant lacks an AXL transmembrane domain; does not have a fibronectin (FN) domain; has one Ig1 domain and one Ig2 domain; and has a number of amino acid modifications of the wild-type AXL sequence (SEQ ID NO: 1) consisting of: Gly32Ser, Ala72Val, Asp87Gly, Val92Ala and Gly127Arg; wherein said series of amino acid modifications increases the binding affinity of the AXL polypeptide to growth blocking-specific protein 6 (GAS6), and wherein the soluble variant of the AXL polypeptide is fused to the Fc region by a peptide linker; and wherein the administration schedule comprises 15 mg/kg of the soluble AXL polypeptide variant administered once every two weeks and 80 mg/m ^{2 of} paclitaxel administered once a week. 2. The method of claim 1, wherein the platinum-resistant human metastatic ovarian cancer overexpresses the GAS6 or AXL biomarker. 3. The method of claim 1 or 2, wherein the platinum-resistant human metastatic ovarian cancer is a recurrent cancer. 4. The method according to any one of paragraphs. 1-3, where platinum-resistant human metastatic ovarian cancer is resistant to standard therapies.

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