Anti-axl antibodies, antibody fragments and immunoconjugates thereof and uses thereof

Abstract

To provide an antibody specifically binding to Axl protein, an immunoconjugate and a pharmaceutical composition.SOLUTION: Provided are single-chain antigen-binding antibodies that specifically bind to Axl proteins, comprising a heavy chain variable region and a light chain variable region having specific amino acid sequences, wherein all of the amino acid sequence mutations to the specific amino acid sequences occur only in the framework regions, and wherein the antibodies have higher binding affinities to Axl proteins at a pH of a tumor microenvironment ranging from pH7. 8 to 7.0 compared to a non-tumor microenvironment ranging from pH5. 2 to 7.6. Also provided is an immunoconjugate comprising the single-chain antigen-binding antibody, a linker molecule, and one or more cytotoxic agents covalently attached to the single-chain antigen-binding antibody and the linker molecule. Further provided is a pharmaceutical composition comprising said single-chain antigen-binding antibody for use as a medicament for the treatment of carcinoma.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to anti-Axl antibodies, antibody fragments and immunoconjugates of such antibodies and antibody fragments, and the use of the antibodies, antibody fragments and immunoconjugates in diagnostic and therapeutic methods. [Background technology]

[0002] Axl proteins (also known as Ark, UFO, and Tyro-7) are receptor tyrosine kinases in the Tyro-3 family of kinases. Tyro-3 receptor kinases are characterized by two immunoglobin-like domains and double fibronectin type III repeats in the extracellular region combined with a cytoplasmic kinase domain. The ligands for Tyro-3 receptor kinase are Gas6 (growth-arrest-specific 6) and Protein S, two vitamin K-dependent proteins that share 43% amino acid sequence identity and similar domain structures. Each protein has an N-terminal Gla domain containing 11 g-carboxyglutamic acid residues, followed by four epidermal growth factor (EGF)-like modules, and a C-terminal sex hormone-binding globin (SHBG)-like structure consisting of two tandem laminin G domains. The SHBG domain is essential and sufficient for Tyro-3 receptor kinase binding and activation, whereas the GIa domain binds negatively charged membrane phospholipids and plays a key role in Tyro-3 kinase-mediated phagocytosis of apoptotic cells.

[0003] Axl activation leads to signaling through PI-3-kinase / Akt (Franke et al., Oncogene, vol. 22, pp. 8983-8998, 2003) and other major pathways, such as Ras / Erk and β-catenin / TCF (Goruppi et al., Mol. Cell. Biol., vol. 21, pp. 902-915, 2001). Axl is weakly expressed in a range of normal tissues, including the brain, heart, skeletal muscle, organ capsules, and connective tissues of several other organs, and is expressed in monocytes but not lymphocytes. Axl-induced Akt phosphorylation has been reported to be involved in the survival of fibroblasts (Goruppi et al., Mol. Cell. Biol., vol. 17, pp. 4442-4453, 1997), endothelial cells (Hasanbasic et al., Am J Physiol Heart Circ Physiol, vol. 287, H1207-H1213, 2004), vascular smooth muscle cells (Melaragno et al., J. Mol. Cell. Cardiol., vol. 37, pp. 881-887, 2004), and neurons (Allen et al., Mol. Endocrinol., vol. 13, pp. 191-201, 1999). Furthermore, Axl plays a role in cell adhesion and chemotaxis. This is because Axl knockout animals exhibit impaired platelet aggregate stabilization and thrombus formation as a result of reduced activation of platelet integrin IIb3.

[0004] The cause of Axl's insufficiency of Gas6's gas6 and the cause of cancer. Types of cancer, examples, breast cancer (Meric et al., Clin. Cancer Res., vol. 8, pp. 361-367, 2002; Berclaz et al., Ann. Oncol., vol. 12, pp. 819-824, 2001), colon cancer (Chen et al., Int. J. Cancer, vol. 83, pp. 579-584, 1999; Craven et al., Int. J. Cancer, vol. 60, pp. 791-797, 1995), prostate cancer (Jacob et al., Cancer Detect. Prey., vol. 23, pp. 325-332, 1999), lung cancer (Wimmel et al., Eur J. Cancer, vol.37, pp.2264-2274, 2001; gastric cancer (Wu et al., Anticancer Res., vol.22, pp.1071-1078, 2002); ovarian cancer (Sun et al., Oncology, vol.66, pp.450-457, 2004); endometrial cancer (Sun et al., Ann. Oncol., vol.14, pp.898-906, 2003); renal cancer (Chung et al., DNA Cell Biol., vol.22, pp.533-540, 2003); hepatocellular carcinoma (Tsou et al., Genomics, vol.50, pp.331-340, 1998); thyroid cancer (Ito et al., Thyroid, vol.12, pp.971-975, 2002; Ito et al. al.,Thyroid,vol.9,pp.563-567,1999), Chronic osteomyelitis (Janssen et al.,Oncogene,vol.6,pp.2113-2120,1991; Braunger et al.,Oncogene,vol.14,pp.2619-2631 1997; O'Bryan et al.,Mol.Cell.Biol.,vol.11,pp.5016-5031,1991), Acute osteomyelitis (Rochlitz et al.,Leukemia,vol.13,pp.1352-1358,1999), Osteosarcoma (Nakano et al.,J.Biol.Chem.,vol.270,pp.Axl overexpression has been demonstrated in human melanoma (van Ginkel et al., Cancer Res., vol. 64, pp. 128-134, 2004), and head and neck squamous cell carcinoma (Green et al., Br J. Cancer., vol. 94, pp. 1446-1445, 2006).

[0005] Recently, phosphotyrosine signaling profiling has detected activated Axl in approximately 5% of primary tumors of NSCLC (Rikova et al., Cell, vol. 131, pp. 1190-1203, 2007). Axl expression is induced by targeted chemotherapy drugs, and drug-induced Axl expression confers resistance to chemotherapy in acute myeloid leukemia (Hong et al., Cancer Letters, vol. 268, pp. 314-324, 2008), as well as resistance to imatinib and lapatinib / Herceptin in gastrointestinal stromal tumors (Mehadevan et al., Oncogene, vol. 26, pp. 3909-3919, 2007) and breast cancer (Liu et al., Cancer Research, vol. 281, pp. 6871-6878, 2009).

[0006] Furthermore, Axl has been identified as being involved in tumor metastasis, as it is upregulated in invasive breast cancer cell lines compared with non-invasive cells. In vitro, Axl activity was found to be required for migration and invasion, and this activity could be inhibited by antibody treatment (WO 04 / 008147). Similarly, abrogation of Axl activity in vivo via expression of a dominant-negative form of Axl (Vajkoczy, P., et al., Proc. Natl. Acad. Science USA, vol. 103, pp. 5799-5804, 2005) or siRNA-mediated downregulation of Axl (Holland et al., Cancer Res., vol. 65, pp. 9294-9303, 2005) prevented subcutaneous and orthotopic cell growth in murine xenograft experiments.

[0007] Therefore, anti-Axl monoclonal antibodies have been described for use in cancer treatment.For example, publications relating to anti-Axl antibodies include WO2009 / 063965, WO2009 / 062690, WO2011 / 014457, US2014 / 0227283 and US8,853,369.US2014 / 0227283 discloses monoclonal anti-Axl antibodies and their use in diagnostic and therapeutic methods.WO2009 / 062690 discloses antibodies that can bind to the extracellular domain of Axl protein and at least partially inhibit Axl activity.

[0008] These monoclonal anti-Axl antibodies bind to Axl with similar affinity at any location in a patient's body, such as the location of a tumor intended for treatment. It is predicted that binding of such antibodies to Axl in non-tumor environments may have adverse effects on the normal function of Axl in those environments and thus cause significant side effects. The present invention provides conditionally active anti-Axl antibodies and antibody fragments that have higher binding affinity to Axl in tumor microenvironments compared to their binding affinity to Axl in non-tumor environments. The anti-Axl antibodies and antibody fragments of the present invention are predicted to have comparable or greater anti-cancer efficacy with reduced side effects compared to monoclonal anti-Axl antibodies known in the art. This may also allow for the administration of higher doses or more frequent treatments of anti-Axl antibodies and antibody fragments, thus providing a more effective treatment option. Summary of the Invention [Means for solving the problem]

[0009] In one embodiment, the present invention provides an isolated heavy chain variable region polypeptide that specifically binds to an Axl protein. The polypeptide comprises three complementarity determining region H1, H2, and H3 sequences, The H1 sequence is X1GX2X3MX4 (SEQ ID NO: 1); The H2 sequence is LIKX5SNGGTX6YNQKFKG (SEQ ID NO: 2); The H3 sequence is GX7X8X9X 10 X 11 X 12 X 13 X 14 DYX 15 X 16 (SEQ ID NO: 3), X1 is T, A or W; X2 is H or A; X3 is T or I; X4 is N or I; X5 is P or N; X6 is S, I, or T; X7 is H or D or E or P or R or W; X8 is Y or N, X9 is E or A or D or F or G or H or I or L or M or N or R or V or Y; X 10 is S or D or M or N or Q, X 11 is Y or C or E or P, X 12 is F or E or N or S or T or V, X 13 is A or D or G or L or Y, X 14 is M, E, or F, X 15 is W or A or D or H or L or N or P or R or T, X 16 is G or H.

[0010] In another embodiment, the isolated heavy chain variable region polypeptide comprises three complementarity determining region sequences, L1, L2, and L3, L1 sequence is KASQDX 17 X18 SX 19 VX 20 (SEQ ID NO: 4); The L2 sequence is X 21 X 22 X 23 TRX 24 T (SEQ ID NO: 5); L3 array is QEX 25 X 26 SX 27 X 28 X 29 X 30 (SEQ ID NO: 6), X 17 is V or D or G or N or W, X 18 is S or V, X 19 is A, L, or M, X 20 is A or D or N or Q, X 21 is W or F, X 22 is A or I or N or P or Q, X 23 is S or D, X 24 is H or D, X 25 is H or C or F or I or L or Q or S or T or V or Y, X 26 is F or C or D or E or G or N or S, X 27 is T, C, or P, X 28 is P or A or C or D or E or H or K or S or T or V or W, X 29 is L, G, or R, X 30 is T, I or R, Combined with an isolated light chain variable region.

[0011] In yet another aspect, the present invention provides an anti-Axl antibody or antibody fragment comprising an isolated heavy chain variable region polypeptide of the present invention.

[0012] In yet another aspect, the present invention provides an immunoconjugate comprising an antibody or antibody fragment of the present invention, optionally conjugated to an agent selected from a chemotherapeutic agent, a radioactive atom, a cytostatic agent, and a cytotoxic agent.

[0013] In yet another aspect, the present invention provides a pharmaceutical composition comprising a polypeptide, antibody or antibody fragment, or immunoconjugate of the invention together with a pharmaceutically acceptable carrier.

[0014] In yet another aspect, the present invention provides diagnostic or therapeutic kits comprising a polypeptide, antibody or antibody fragment, or immunoconjugate of the invention. [Brief explanation of the drawings]

[0015] [Figure 1-1] 1 shows the sequence alignment of the heavy chain variable region and the light chain variable region of the anti-Axl antibody of the present invention, respectively. [Figure 1-2] 1 shows the sequence alignment of the heavy chain variable region and the light chain variable region of the anti-Axl antibody of the present invention, respectively. [Figure 1-3] 1 shows the sequence alignment of the heavy chain variable region and the light chain variable region of the anti-Axl antibody of the present invention, respectively. [Figure 2] Binding (OD450) of various conditionally active antibodies of the invention to the Axl extracellular domain at pH 6.0 and pH 7.4 is shown. These conditionally active antibodies were more active at pH 6.0 than at pH 7.4. [Figure 3] 1 shows the selectivity of various conditionally active antibodies of the invention for the Axl extracellular domain, measured as the ratio of the binding affinity for a binding partner at pH 6.0 to the binding affinity for the same binding partner at pH 7.4. [Figure 4]1 shows a size exclusion chromatograph demonstrating that the conditionally active antibodies of the invention do not aggregate, as described in Example 1. [Figure 5] 1 shows the thermal stability of conditionally active antibodies of the invention before and after heat shock as measured by ELISA assay as described in Example 1. [Figure 6] 1 shows the selectivity of the conditionally active antibodies of the present invention as measured by SPR assay in Example 1. [Figure 7] 1 shows the pH-dependent binding profile for the binding of anti-Axl antibodies of the present invention to Axl in KREBS buffer. [Figure 8-1] 1 shows the results of another cell killing study using A549 cells and anti-Axl antibodies of the invention at pH 6.0 and pH 7.4 at different antibody concentrations for cell killing. [Figure 8-2] 1 shows the results of another cell killing study using A549 cells and anti-Axl antibodies of the invention at pH 6.0 and pH 7.4 at different antibody concentrations for cell killing. [Figure 8-3] 1 shows the results of another cell killing study using A549 cells and anti-Axl antibodies of the invention at pH 6.0 and pH 7.4 at different antibody concentrations for cell killing. [Figure 9-1] 1 shows the binding affinity to human Axl and cynomolgus monkey Axl for anti-Axl antibodies of the invention in different buffers and at different pH levels. [Figure 9-2] 1 shows the binding affinity to human Axl and cynomolgus monkey Axl for anti-Axl antibodies of the invention in different buffers and at different pH levels. [Figure 10-1] 1 shows cell killing of different cell lines at different pH levels by anti-Axl antibodies of the invention conjugated to duomycin. [Figure 10-2] 1 shows cell killing of different cell lines at different pH levels by anti-Axl antibodies of the invention conjugated to duomycin. [Figure 10-3]1 shows cell killing of different cell lines at different pH levels by anti-Axl antibodies of the invention conjugated to duomycin. [Figure 10-4] 1 shows cell killing of different cell lines at different pH levels by anti-Axl antibodies of the invention conjugated to duomycin. [Figure 11] 1 shows cell killing of A549 cells at different pH levels by anti-Axl antibodies of the invention conjugated to gemcitabine. [Figure 12] 1 shows the effect on tumor volume of treatment of xenograft mice with a duomycin-conjugated anti-Axl antibody of the present invention. [Figure 13] 1 shows the detection of the presence of a duomycin-conjugated anti-Axl antibody of the invention in the blood of cynomolgus monkeys over time following injection of the conjugate. [Figure 14] Figure 14A: shows the detection of the presence of aspartate transaminase (AST) in the blood of cynomolgus monkeys over time, starting just before injection of the conjugate (Before (D-3)) and running through 3 days after injection (After (D-3)). Figure 14B: shows the detection of the presence of alanine aspartate transaminase (ALT) in the blood of cynomolgus monkeys over time, starting just before injection of the conjugate (Before (D-3)) and running through 3 days after injection (After (D-3)). [Figure 15] 1 shows lymphocyte counts over time in the blood of cynomolgus monkeys after injection of the conjugate. [Figure 16] In vivo treatment of mice receiving LCLC103H and DU145, respectively, is shown. DETAILED DESCRIPTION OF THE INVENTION

[0016] definition To facilitate understanding of the examples provided herein, certain frequently used terms are defined herein.

[0017] As used herein, the term "about" in connection with a measured quantity refers to the normal variation in that measured quantity that would be expected by one of ordinary skill in the art making the measurement and exercising a level of care commensurate with the purpose of the measurement and the precision of the measuring device being used. Unless otherwise indicated, "about" refers to a + / - 10% variation of the value provided.

[0018] The term "affinity" as used herein refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (Kd). Affinity can be measured by general methods known in the art, for example, those described herein. Specific descriptions and exemplary embodiments for measuring binding affinity are provided below.

[0019] The term "affinity matured," when used in reference to an antibody, refers to an antibody or antibody fragment with one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody or antibody fragment that does not possess the alterations, which alterations result in an improvement in the affinity of the antibody or antibody fragment for an antigen.

[0020] As used herein, the term "amino acid" refers to any organic compound containing an amino group (--NH2) and a carboxyl group (--COOH), preferably as a free group or, after condensation, as part of a peptide bond. "The 20 naturally encoded polypeptide-forming alpha-amino acids" are understood in the art and refer to alanine (ala or A), arginine (arg or R), asparagine (asn or N), aspartic acid (asp or D), cysteine ​​(cys or C), glutamic acid (glu or E), glutamine (gin or Q), glycine (gly or G), histidine (his or H), isoleucine (ile or I), leucine (leu or L), lysine (lys or K), methionine (met or M), phenylalanine (phe or F), proline (pro or P), serine (ser or S), threonine (thr or T), tryptophan (tip or W), tyrosine (tyr or Y), and valine (val or V).

[0021] As used herein, the term "antiangiogenic agent" refers to a compound that blocks or prevents the development of blood vessels to some extent.Antiangiogenic agents can be, for example, small molecules or antibodies that bind to growth factors or growth factor receptors involved in promoting angiogenesis.In one embodiment, the antiangiogenic agent is an antibody or antibody fragment that binds to vascular endothelial growth factor (VEGF), such as bevacizumab (AVASTIN®).

[0022] As used herein, the term "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; and single-chain antibody molecules (e.g., scFv). These antibody fragments, which retain some ability to selectively bind to the antigen (e.g., polypeptide antigen) of the antibody from which they are derived, can be produced using methods well known in the art (see, e.g., Harlow and Lane, supra).

[0023] As used herein, the term "antibody" refers to an intact immunoglobulin molecule. Antibodies or antibody fragments can be used to isolate preparative amounts of antigens by immunoaffinity chromatography. Various other uses of such antibodies or antibody fragments are for diagnosing and / or staging diseases (e.g., neoplasia), as well as for therapeutic applications to treat diseases such as neoplasia, autoimmune diseases, AIDS, cardiovascular diseases, infectious diseases, etc. Chimeric, human-like, humanized, or fully human antibodies or antibody fragments are particularly useful for administration to human patients.

[0024] A Fab fragment consists of a monovalent antigen-binding fragment of an antibody molecule and can be produced by digestion of whole antibody molecules with the enzyme papain to yield a fragment consisting of an intact light chain and a portion of the heavy chain.

[0025] Fab' fragments of antibody molecules can be obtained by treating whole antibody molecules with pepsin, followed by reduction, to yield molecules consisting of an intact light chain and a portion of the heavy chain. Two Fab' fragments are obtained for each antibody molecule treated in this manner.

[0026] The (Fab')2 fragment of an antibody can be obtained by treating an intact antibody molecule with the enzyme pepsin without subsequent reduction. The (Fab')2 fragment is a dimer of two Fab' fragments held together by two disulfide bonds.

[0027] An Fv fragment is defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains.

[0028] As used herein, the terms "anti-Axl antibody," anti-Axl antibody fragment, and "antibody or antibody fragment that binds to Axl" refer to an antibody or antibody fragment that can bind to Axl with sufficient affinity such that the antibody or antibody fragment is useful as a diagnostic and / or therapeutic agent in targeting Axl. In one embodiment, the extent of binding of an anti-Axl antibody or antibody fragment to an unrelated, non-Axl protein is less than about 10% of the binding of the antibody or antibody fragment to Axl, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody or antibody fragment that binds to Axl has an affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., ≦10 -8 M or less, or 10 -8 M~10 -13 M, or 10 -9 M~10 -13 In certain embodiments, the anti-Axl antibody or antibody fragment binds to an epitope of Axl that is conserved among Axl from different species.

[0029] As used herein, the term "angiogenic disorder" refers to any dysfunction of angiogenesis, including both non-neoplastic and neoplastic conditions, including but not limited to those described below (see, e.g., "cancer"). Non-neoplastic disorders include, but are not limited to, unwanted or abnormal hypertrophy, arthritis, rheumatoid arthritis (RA), psoriasis, psoriatic plaques, sarcoidosis, atherosclerosis, atherosclerotic plaques, diabetic and other proliferative retinopathies, such as retinopathy of prematurity, retrolental fibroplasia, neovascular glaucoma, age-related macular degeneration, diabetic macular edema, corneal neovascularization, corneal graft neovascularization, corneal graft rejection, retinal / choroidal neovascularization, angle neovascularization (rubeosis), ocular neovascular diseases, vascular restenosis, arteriovenous malformations (AVMs), meningiomas, hemangiomas, angiofibromas, thyroid enlargement (e.g., Graves' disease), corneal and other tissue transplants, chronic inflammation, pulmonary inflammation, acute lung injury / ARDS, sepsis, primary pulmonary hypertension, malignant pulmonary effusion , cerebral edema (e.g., associated with acute stroke / closed head injury / trauma), synovitis, pannus formation in RA, myositis ossificans, hypertrophic bone formation, osteoarthritis (OA), refractory ascites, polycystic ovary syndrome, endometriosis, third space fluid disorders (pancreatitis, compartment syndrome, burns, intestinal disease), uterine fibroids, preterm labor, chronic inflammation such as IBD (Crohn's disease and ulcerative colitis), renal allograft rejection, inflammatory bowel disease, nephrotic syndrome, unwanted or abnormal tissue mass growth (non-cancerous), hemophilic joints, hypertrophic scars, hair growth inhibition, Osler-Weber syndrome, pyogenic granuloma, retrolental fibroplasia, scleroderma, trachoma, vascular adhesions, synovitis, dermatitis, preeclampsia, ascites, pericardial effusion (e.g., associated with pericarditis), and pleural effusion.

[0030] As used herein, the term "angiogenesis" refers to all Axl-related processes that contribute to the growth of new blood vessels from existing vessels, particularly, but not limited to, new tumor-supplying vessels. These processes include multiple cellular events, such as proliferation, survival, migration, and sprouting of vascular endothelial cells, attraction and migration of surrounding cells, and basement membrane formation for vascular stabilization, vascular perfusion, or secretion of angiogenic factors by stromal or neoplastic cells, which are stimulated or mediated by the non-catalytic or catalytic activity of Axl, preferably, for example, Axl phosphorylation and / or Axl-mediated signal transduction.

[0031] As used herein, the term "Axl" refers to any native Axl from any vertebrate source, for example, mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. This term encompasses "full-length" unprocessed Axl and any form of Axl resulting from processing in cells. This term also encompasses naturally occurring variants of Axl, such as splice variants or allelic variants. The amino acid sequence of human Axl is well known in the art and is available from public databases, such as GenBank.

[0032] As used herein, the term "Axl activation" refers to the activation or phosphorylation of the Axl receptor. Generally, Axl activation results in signal transduction (e.g., caused by the intracellular kinase domain of Axl or the Axl receptor phosphorylating tyrosine residues in a substrate polypeptide). Axl activation can be mediated by the binding of an Axl ligand (Gas6) to the target Axl receptor. Gas6 binding to Axl can activate the kinase domain of Axl, thereby resulting in the phosphorylation of tyrosine residues in Axl and / or the phosphorylation of tyrosine residues in an additional substrate polypeptide.

[0033] As used herein, the term "Axl-mediated anti-apoptosis" refers to any Axl-involved process that prevents human cells, preferably, but not limited to, human cancer cells, from undergoing programmed cell death (apoptosis). In particular, it refers to any process that prevents human cells, preferably, but not limited to, human cancer cells, from undergoing apoptosis via growth factor withdrawal, hypoxia, exposure to chemotherapeutic agents or radiation, or the initiation of Fas / Apo-1 receptor-mediated signaling, and is stimulated or mediated by the non-catalytic or catalytic activity of Axl, preferably, for example, Axl phosphorylation and / or Axl-mediated signaling.

[0034] As used herein, the term "binding" refers to the interaction of an antibody variable region or Fv with an antigen, where the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the antigen. For example, an antibody variable region or Fv recognizes and binds to a specific protein structure, rather than proteins in general. As used herein, "specifically binding" or "binding specifically" means that an antibody variable region or Fv binds or associates with a specific antigen more frequently, rapidly, for a longer duration, and / or with greater affinity than with other proteins. For example, an antibody variable region or Fv specifically binds to the antigen with greater affinity, avidity, rapidly, and / or for a longer duration than it binds to other antigens. In another example, an antibody variable region or Fv binds to a cell surface protein (antigen) with significantly greater affinity than it binds to related proteins or other cell surface proteins, or to an antigen generally recognized by polyreactive natural antibodies (i.e., naturally occurring antibodies known to bind to various antigens naturally found in humans). However, "specifically binds" does not necessarily require exclusive binding or non-detectable binding of another antigen, which is what the term "selective binding" means. In one example, "specific binding" of an antibody variable region or Fv (or other binding region) that binds to an antigen means that the antibody variable region or Fv binds to the antigen with an equilibrium constant (KD) of 100 nM or less, e.g., 50 nM or less, e.g., 20 nM or less, e.g., 15 nM or less, or 10 nM or less, or 5 nM or less, 2 nM or less, or 1 nM or less.

[0035] As used herein, the term "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation.Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia.More specific examples of such cancer include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

[0036] As used herein, the terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

[0037] As used herein, the term "chemotherapeutic agent" refers to a compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines, such as altretamine, triethylenemelamine, and triethylenephosphoramine. amide, triethylenethiophosphoramide, and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecins (e.g., synthetic analogs topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcannabinol, etc.) camptothecin, scopolectin, and 9-aminocamptothecin; bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; Sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobuenbiquine, fenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;Antibiotics, such as enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin gamma II and calicheamicin omega II (see, e.g., Nicolaou et al., Angew. Chem. Intl. Ed. Engl., 33:183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor; dynemicins, e.g., dynemicin A; esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin (carabicin), caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (e.g., ADRIAMYCIN®), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®), pegylated liposomal doxorubicin (CAELYX®, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozotocin, antimetabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epothilones, and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine;Pyrimidine analogues, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calsterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenal agents, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as florinic acid acid); aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidynin; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidammol; nitraelin; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; schizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinosine docetaxel ("Ara-C"); thiotepa; taxoids such as paclitaxel (TAXOL®), albumin-engineered nanoparticle formulations of paclitaxel (ABRAXANE™), and docetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin;Vincas that interfere with tubulin polymerization to form microtubules, such as vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; the topoisomerase inhibitor RFS2000; difluoromethylornithine (D MF®); retinoids, such as retinoic acid, e.g., bexarotene (TARGRETIN®); bisphosphonates, such as clodronate (e.g., BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines, such as the THERATOPE vaccine and gene therapy vaccines, such as the ALLOVECTIN vaccine, the LEUVECTIN vaccine, and the VAXID vaccine. trademark) vaccines; topoisomerase 1 inhibitors (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT®, Pfizer); perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (R11577); orafenib, ABT510;Bcl-2 inhibitors, such as oblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); serine-threonine kinase inhibitors, such as rapamycin (sirolimus, RAPAMUNE®); farnesyltransferase inhibitors, such as lonafarnib (SCH6636, SARASAR™); and pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above, such as CHOP, which is short for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, which is short for a treatment regimen using oxaliplatin in combination with 5-FU and leucovorin (ELOXATIN™).

[0038] Chemotherapeutic agents, as defined herein, include "antihormonal agents" or "endocrine therapeutic agents" that act to regulate, reduce, block, or inhibit the effects of hormones that may promote cancer growth.These may be hormones themselves, including, but not limited to, antiestrogens with mixed agonist / antagonist profiles, such as tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®), idoxifene, droloxifene, raloxifene (EVISTA®), trioxifene, ketoxifene, and selective estrogen receptor modulators (SERMs), e.g., SERM3; pure antiestrogens that do not block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and / or suppress ER levels, such as fulvestrant (FASLODEX®), and EM800 (such agents may block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and / or suppress ER levels); aromatase inhibitors, including steroidal aromatase inhibitors, such as formestane and exemestane (AROMASIN®), and non-steroidal aromatase inhibitors, such as anastrazole (AR IMIDEX®), letrozole (FEMARA®), and aminoglutethimide, and other aromatase inhibitors, such as vorozole (RIVISOR®), megestrol acetate (MEGASE®), fadrozole, and 4(5)-imidazole; luteinizing hormone-releasing hormone agonists, such as leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin, and tripterelin; sex steroids, such as progestins, such as Examples include megestrol acetate and medroxyprogesterone acetate, estrogens such as diethylstilbestrol and premarin, and androgens / retinoids such as fluoxymesterone, all-trans retinoic acid and fenretinide; onapristone; antiprogesterones; estrogen receptor down-modulators (ERDs); antiestrogens such as flutamide, nilutamide and bicalutamide; and pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above.

[0039] As used herein, the term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0040] As used herein, the term "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five major antibody classes: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively.

[0041] As used herein, the terms "conditionally active antibody" and "conditionally active antibody fragment" refer to an antibody or antibody fragment that is active under conditions in a tumor microenvironment compared to different values ​​of the same conditions in a non-tumor microenvironment. When compared to conditions in a non-tumor microenvironment, conditions in a tumor microenvironment can include lower pH, higher concentrations of lactate and / or pyruvate, low oxygen, lower concentrations of glucose, and slightly higher temperatures. For example, in one embodiment, a conditionally active antibody or antibody fragment may be substantially inactive at normal body temperature but active at higher temperatures than can be found in a tumor microenvironment. In yet another embodiment, a conditionally active antibody or antibody fragment may be less active in normal oxygenated blood than in the hypoxic environment that can exist in a tumor microenvironment. Other conditions in the tumor microenvironment known to those skilled in the art may also be selected for use as conditions in the present invention that can induce an anti-Axl antibody or antibody fragment to have different activities under different values ​​of the conditions.

[0042] As used herein, the term "constitutive," when applied to, for example, Axl activity, refers to the continuous signaling activity of receptor kinases that is not dependent on the presence of ligands or other activating molecules. Depending on the nature of the receptor kinase, all of the activity may be constitutive, or the receptor's activity may be further activated by the binding of other molecules (e.g., ligands). Cellular events that lead to receptor kinase activation are well known to those skilled in the art. For example, activation can include oligomerization into higher-order receptor complexes, such as dimerization and trimerization. A complex may contain a single protein species, i.e., a homocomplex. Alternatively, a complex may contain at least two different protein species, i.e., a heterocomplex. Complex formation can be induced, for example, by overexpression of a normal or mutant form of the receptor on the surface of a cell. Complex formation can also be induced by one or more specific mutations in the receptor.

[0043] The term "cytostatic agent" as used herein refers to a compound or composition that arrests cell growth either in vitro or in vivo. Thus, a cytostatic agent may significantly reduce the proportion of cells in S phase. Further examples of cytostatic agents include agents that block cell cycle progression by inducing G0 / G1 arrest or M-phase arrest. The humanized anti-Her2 antibody trastuzumab (HERCEPTIN®) is an example of a cytostatic agent that induces G0 / G1 arrest. Classical M-phase blockers include vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors, such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Certain agents that arrest G1, such as DNA alkylating agents, e.g., tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C, also cause S-phase arrest. Further information can be found in Mendelsohn and Israel, eds., The Molecular Basis of Cancer, Chapter 1, entitled "Cell cycle regulation, oncogenes, and anticancer drugs," Murakami et al. (WB Saunders, Philadelphia, 1995), e.g., page 13. Taxanes (paclitaxel and docetaxel) are anticancer drugs, both derived from the yew tree. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew tree, is a semisynthetic analog of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, leading to the inhibition of cell mitosis.

[0044] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof, e.g., nucleases; antibiotics; toxins, e.g., small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof; and various anti-tumor or anti-cancer agents disclosed below.

[0045] As used herein, the term "diabody" refers to a diabody in which fragments of a light chain variable domain (V) are combined in the same polypeptide chain. L ) linked to a heavy chain variable domain (V H )(V H -V L (Antibody fragments refer to small antibody fragments having two antigen-binding sites, each containing a nucleotide sequence.) By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain, creating two antigen-binding sites.

[0046] As used herein, the term "detectably labeled" refers to any substance that can be detected or measured directly or indirectly by physical or chemical means to indicate the presence of CTCs in a sample.Representative examples of useful detectable labels include, but are not limited to: molecules or ions that can be directly or indirectly detected based on light absorption, fluorescence, reflection, light scattering, phosphorescence, or luminescence properties; molecules or ions that can be detected by radioactivity; molecules or ions that can be detected by nuclear magnetic resonance or paramagnetics.The group of molecules that can be indirectly detected based on light absorption or fluorescence includes, for example, various enzymes that cause the conversion of suitable substrates from non-light-absorbing to light-absorbing molecules or from non-fluorescent to fluorescent molecules.

[0047] The term "diagnosis" as used herein refers to determining the subject's susceptibility to disease or disorder, determining whether the subject is currently suffering from disease or disorder, prognosticating the subject suffering from disease or disorder (for example, identifying the pre-metastatic or metastatic cancerous state, the stage of cancer, or the response of cancer to treatment), and treatment strategy (for example, monitoring the subject's condition to provide information on the effectiveness or efficacy of treatment).In some embodiments, the diagnostic method of the present invention is particularly useful for detecting early cancer.

[0048] The term "diagnostic agent" as used herein refers to a molecule that can be detected directly or indirectly and used for diagnostic purposes. The diagnostic agent can be administered to a subject or sample. The diagnostic agent can be provided by itself or can be conjugated to a vehicle, such as a conditionally active antibody.

[0049] As used herein, the term "effector function" refers to the biological activity attributable to the Fc region of an antibody and varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0050] As used herein, the term "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0051] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0052] As used herein, the term "framework" or "FR" refers to variable domain residues other than hypervariable region (HVR or H1-3 in the heavy chain and L1-3 in the light chain) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally correspond to the V H (or V L ) appears in the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0053] The terms "full length antibody," "intact antibody," or "complete antibody" refer to an antibody that contains an antigen-binding variable region (V H or V L) and a light chain constant domain (CL) and heavy chain constant domains CH1, CH2, and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. Depending on the amino acid sequence of the constant domain of the heavy chain, full-length antibodies can be assigned to different "classes." There are five major classes of full-length antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to the different classes of antibodies are called alpha, delta, epsilon, gamma, and mu, respectively. The subunits and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0054] As used herein, the terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0055] As used herein, the term "human antibody" refers to an antibody that possesses an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or an antibody derived from a human antibody repertoire or other non-human source that utilizes human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies that comprise non-human antigen-binding residues.

[0056] As used herein, the term "human consensus framework" refers to the human immunoglobulin V L or V HIn selecting framework sequences, the framework represents the most commonly occurring amino acid residues. L or V H The selection of sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda, Md. (1991), vols. 1-3. In one embodiment, V L The subgroup for V is subgroup kappa I as in Kabat et al., supra. H The subgroup for is subgroup III as in Kabat et al., supra.

[0057] As used herein, the term "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, typically two, variable domains in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has been humanized.

[0058] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops ("hypervariable loops"). Generally, naturally occurring four-chain antibodies contain six HVRs; H Three of them (H1, H2, H3), and V LHVRs generally comprise three of these (L1, L2, L3). HVRs generally comprise amino acid residues from the hypervariable loops and / or from the "complementarity-determining regions" (CDRs), the latter of which exhibit the greatest sequence variability and / or are involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol., vol. 196, pp. 901-917 1987). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3 (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. 1991). H Except for CDR1 in the middle, CDRs generally contain amino acid residues that form hypervariable loops. CDRs also contain "specificity-determining residues," or "SDRs," which are residues that contact the antigen. SDRs are contained within regions of CDRs called truncated CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3 (see Almagro and Fransson, Front. Biosci., vol. 13, pp. 1619-1633, 2008). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0059] As used herein, the term "immunoconjugate" refers to an antibody conjugated to one or more heterologous molecules, such as, but not limited to, a cytotoxic agent.

[0060] As used herein, the term "individual" or "subject" refers to a mammal. Mammals include, but are not limited to, livestock animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0061] As used herein, the term "inhibiting cell growth or proliferation" means reducing cell growth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, and includes inducing cell death.

[0062] As used herein, the term "isolated" antibody refers to an antibody that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, as measured, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a general review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B, vol. 848, pp. 79-87, 2007.

[0063] As used herein, the term "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule that is contained in a cell that ordinarily contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0064] As used herein, the term "isolated nucleic acid encoding an anti-Axl antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), and includes such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present in one or more locations in a host cell.

[0065] As used herein, the term "ligand-independent," when applied to, for example, receptor signaling activity, refers to signaling activity that is not dependent on the presence of a ligand. A receptor with ligand-independent kinase activity does not necessarily preclude the binding of a ligand to the receptor to produce additional activation of the kinase activity.

[0066] As used herein, the term "metastasis" refers to all Axl-mediated processes that support cancer cell dispersal from a primary tumor, infiltrate into lymphatics and / or blood vessels, circulate via the bloodstream, and grow in distant foci (metastasis) in normal tissues elsewhere in the body. In particular, it refers to cellular events in tumor cells, such as proliferation, migration, anchorage independence, evasion of apoptosis, or secretion of angiogenic factors, that underlie metastasis and are stimulated or mediated by the noncatalytic or catalytic activity of Axl, preferably, for example, Axl phosphorylation and / or Axl-mediated signaling.

[0067] As used herein, the term "microenvironment" refers to any part or region of a tissue or body that has permanent or temporary physical or chemical differences from other regions of the tissue or body. With respect to tumors, the term "tumor microenvironment" refers to the environment in which the tumor resides, including the acellular areas within the tumor and the area immediately outside the tumor tissue, but not related to the intracellular compartments of the cancer cells themselves. Tumors and the tumor microenvironment are closely related and constantly interact. Tumors can alter their microenvironment, which can affect how tumors grow and spread. Typically, tumor microenvironments have a low pH, ranging from 5.8 to 7.0, more commonly from 6.2 to 6.8, and most commonly from 6.4 to 6.8. On the other hand, normal physiological pH is typically in the range of 7.2 to 7.8. Compared to plasma, tumor microenvironments have low concentrations of glucose and other nutrients, but are also known to have high concentrations of lactate. Furthermore, tumor microenvironments can have temperatures 0.3 to 1°C higher than normal physiological temperatures. The tumor microenvironment is discussed in Gillies et al., "MRI of the Tumor Microenvironment," Journal of Magnetic Resonance Imaging, vol. 16, pp. 430-450, 2002. The term "non-tumor microenvironment" refers to the microenvironment at a site other than the tumor.

[0068] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., each individual antibody in the population is identical and / or binds to the same epitope, except for variant antibodies that contain, for example, naturally occurring mutations or that may arise during production of the monoclonal antibody preparation, with such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies that can be used in accordance with the present invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; these and other exemplary methods for producing monoclonal antibodies are described herein.

[0069] As used herein, the term "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. Naked antibodies can be present in pharmaceutical formulations.

[0070] As used herein, the term "natural antibody" refers to a naturally occurring immunoglobulin molecule with a variable structure. For example, a natural IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain contains a variable region (V), also called a variable heavy domain or heavy chain variable domain. H), followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain contains a variable region (V), also called the variable light domain or light chain variable domain. L ) followed by the constant light chain (C L ) domain. The light chains of antibodies can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domain.

[0071] As used herein, the term "package insert" is used to refer to instructions customarily included in the commercial packaging of a therapeutic product, which contain information about the indications, uses, dosage, administration, concomitant therapy, contraindications and / or precautions regarding the use of such therapeutic product.

[0072] As used herein, the term "percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps if necessary to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity.Alignment for determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine the appropriate parameters for aligning sequences, for example, any algorithm required to achieve maximum alignment across the entire length of the sequences being compared.However, for the purposes herein, the % amino acid sequence identity value is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is authored by Genentech, Inc., and the source code, together with user documentation, has been filed with the U.S. Copyright Office, Washington, DC, 20559, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or can be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, e.g., Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0073] In the context of using ALIGN-2 for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or containing a certain % amino acid sequence identity to, with, or relative to a given amino acid sequence B) is calculated as follows: 100 * (X / Y) (where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and Y is the total number of amino acid residues in B.) It is recognized that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless otherwise specifically stated, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0074] As used herein, the term "pharmaceutical formulation" refers to a preparation that is in a form that allows the effectiveness of the biological activity of the active ingredients contained therein and that does not contain additional components that exhibit unacceptable toxicity to the subject to which the formulation is administered.

[0075] As used herein, the term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation other than the active ingredient that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0076] As used herein, the terms "purified" and "isolated" refer to an antibody or nucleotide sequence according to the present invention in which the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. As used herein, the term "purified" preferably means that at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight of the same type of biological macromolecules are present. An "isolated" nucleic acid molecule encoding a specific polypeptide refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode the polypeptide; however, the molecule may contain some additional bases or moieties that do not adversely affect the basic characteristics of the composition.

[0077] As used herein, the term "recombinant antibody" refers to an antibody (e.g., a chimeric, humanized, or human antibody or antigen-binding fragment thereof) expressed by a recombinant host cell containing nucleic acid encoding the antibody. Examples of "host cells" for producing recombinant antibodies include: (1) mammalian cells, such as Chinese hamster ovary (CHO) cells, COS, myeloma cells (e.g., Y0 and NS0 cells), baby hamster kidney (BHK) cells, Hela and Vero cells; (2) insect cells, such as sf9, sf21 and Tn5; (3) plant cells, such as plants belonging to the genus Nicotiana (e.g., Nicotiana tabacum); (4) yeast cells, such as those belonging to the genus Saccharomyces (e.g., Saccharomyces cerevisiae) or Aspergillus (e.g., Aspergillus niger); and (5) bacterial cells, such as Escherichia coli cells or Bacillus subtilis cells.

[0078] The term "therapeutically effective amount" of an antibody or antibody fragment of the present invention refers to a sufficient amount of the antibody or antibody fragment to treat a disease or disorder at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the antibodies or antibody fragments and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific antibody or antibody fragment used; the specific composition used; the patient's age, weight, general health, sex, and diet; the time of administration, route of administration, and excretion rate of the specific antibody or antibody fragment used; the duration of treatment; drugs used in combination with or concomitantly with the specific antibody used; and similar factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

[0079] As used herein, the term "single-chain Fv" ("scFv") refers to a VFv fragment typically linked by a peptide-encoded linker. H and V L Covalently linked V expressed from a gene fusion containing the encoding gene H ::V L "dsFv" is a V heterodimer stabilized by disulfide bonds. H ::V L Divalent and multivalent antibody fragments can form spontaneously by association of monovalent scFvs or can be generated by coupling monovalent scFvs by peptide linkers (e.g., bivalent sc(Fv)2).

[0080] As used herein, the terms "treatment," "treat," or "treating" refer to clinical interventions that attempt to alter the natural course of the individual being treated and can be performed for prophylaxis or during the course of clinical disease. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, improvement or mitigation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies or antibody fragments of the invention are used to delay the onset of disease or slow the progression of disease.

[0081] As used herein, the term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not intended to be mutually exclusive herein.

[0082] As used herein, the term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (V, V ... H and V L ) generally have a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR) (see, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). H or V L A V domain may be sufficient to confer antigen-binding specificity. Furthermore, an antibody or antibody fragment that binds to a particular antigen may contain a V domain from the antibody that binds to that antigen. H or V L The complementary V domains were isolated using L or V HLibraries of domains can be screened (see, e.g., Portolano et al., J. Immunol., vol. 150, pp. 880-887, 1993; Clarkson et al., Nature, vol. 352, pp. 624-628, 1991).

[0083] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0084] For illustrative purposes, the principles of the present invention will be described with reference to various exemplary embodiments. While certain embodiments of the present invention are specifically described herein, those skilled in the art will readily recognize that the same principles are equally applicable and can be used in other systems and methods. Before describing the disclosed embodiments of the present invention in detail, it should be understood that the present invention is not limited in its application to the details of any particular embodiment shown. Furthermore, the terminology used herein is for purposes of description and not limitation. Furthermore, although certain methods are described herein with reference to steps presented in a certain order, in many cases, as one skilled in the art will recognize, these steps can be performed in any order; thus, the novel methods are not limited to the particular order of steps disclosed herein.

[0085] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Furthermore, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. The terms "comprising," "including," "having," and "consisting of" can also be used interchangeably.

[0086] Unless otherwise indicated, all numbers used in the specification and claims expressing quantities of ingredients, properties, e.g., molecular weights, percentages, ratios, reaction conditions, and the like, should be understood to be modified in all instances by the term "about," whether or not the term "about" is used. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations, which may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0087] It is to be understood that each component, compound, substituent, or parameter disclosed herein should be construed as disclosed for use alone or in combination with each and every other component, compound, substituent, or parameter disclosed herein.

[0088] It should be understood that each amount / value or amount / value range for each component, compound, substituent, or parameter disclosed herein should be construed as also being disclosed in combination with each amount / value or amount / value range disclosed for every other component, compound, substituent, or parameter disclosed herein, and thus any combination of amounts / values ​​or amount / value ranges for two or more components, compounds, substituents, or parameters disclosed herein are also disclosed in combination with each other for purposes of this detailed description.

[0089] It is further understood that each lower limit of each range disclosed herein should be interpreted as being disclosed in combination with each upper limit of each range disclosed herein for the same component, compound, substituent, or parameter. Thus, the disclosure of two ranges should be interpreted as a disclosure of four ranges derived by combining each lower limit of each range with each upper limit of each range. The disclosure of three ranges should be interpreted as a disclosure of nine ranges derived by combining each lower limit of each range with each upper limit of each range, etc. Furthermore, a specific amount / value of a component, compound, substituent, or parameter disclosed in the specification or examples should be interpreted as a disclosure of either the lower or upper limit of a range, and thus may be combined with any other lower or upper limit or specific amount / value of a range for the same component, compound, substituent, or parameter disclosed elsewhere in this application to form a range for that component, compound, substituent, or parameter.

[0090] A. Regions of anti-Axl antibodies or antibody fragments In one aspect, the present invention provides an isolated heavy chain variable region polypeptide that specifically binds to a human Axl protein. The heavy chain variable region polypeptide comprises three complementarity determining region sequences, H1, H2, and H3, The H1 sequence is X1GX2X3MX4 (SEQ ID NO: 1); The H2 sequence is LIKX5SNGGTX6YNQKFKG (SEQ ID NO: 2); The H3 sequence is GX7X8X9X 10 X 11 X 12 X 13 X 14 DYX 15 X 16 (SEQ ID NO: 3), X1 is T, A or W; X2 is H or A; X3 is T or I; X4 is N or I; X5 is P or N; X6 is S, I, or T; X7 is H or D or E or P or R or W; X8 is Y or N, X9 is E or A or D or F or G or H or I or L or M or N or R or V or Y; X 10 is S or D or M or N or Q, X 11 is Y or C or E or P, X 12 is F or E or N or S or T or V, X 13 is A or D or G or L or Y, X 14 is M, E, or F, X 15 is W or A or D or H or L or N or P or R or T, X 16 is G or H.

[0091] An alignment of the heavy chain variable regions is shown in Figure 1A, with the complementarity determining regions H1, H2, and H3 boxed.

[0092] In another aspect, the present invention provides an isolated light chain variable region polypeptide that specifically binds to a human Axl protein. The light chain variable region polypeptide comprises three complementarity determining region sequences, L1, L2, and L3, L1 sequence is KASQDX 17 X 18 SX 19 VX 20 (SEQ ID NO: 4); The L2 sequence is X 21 X 22 X 23 TRX 24 T (SEQ ID NO: 5); L3 array is QEX 25 X 26 SX 27 X 28 X 29 X 30 (SEQ ID NO: 6), X 17 is V or D or G or N or W, X 18 is S or V, X 19 is A, L, or M, X 20 is A or D or N or Q, X 21 is W or F, X 22 is A or I or N or P or Q, X 23 is S or D, X 24 is H or D, X 25 is H or C or F or I or L or Q or S or T or V or Y, X 26 is F or C or D or E or G or N or S, X 27 is T, C, or P, X 28 is P or A or C or D or E or H or K or S or T or V or W, X29 is L, G, or R, X 30 is T, I, or R.

[0093] An alignment of the light chain variable regions is shown in Figure 1B, with the complementarity determining regions L1, L2, and L3 boxed.

[0094] The present invention identifies these isolated heavy and light chain variable region polypeptides from parent antibodies using the methods disclosed in U.S. Patent No. 8,709,755. The heavy and light chain variable regions of the parent antibody (063-hum10F10) are also aligned in Figures 1A-1B to show the mutations in the isolated heavy and light chain variable region polypeptides.

[0095] A mutant antibody library was generated by evolving DNA encoding a wild-type antibody using Comprehensive Positional Evolution (CPE), which randomizes each position in the template antibody one by one. Each mutant antibody in the library has only one single point mutation. The mutant antibodies in the library were generated by simultaneously screening for selective binding affinity to Axl at pH 6.0 compared to pH 7.4 by ELISA. Two dilutions of mutant antibodies were used: 1:3 and 1:9 dilutions. Mutant antibodies with a ratio of binding affinity at pH 6.0 to pH 7.4 of at least 1.5 under either the 1:3 or 1:9 dilution were selected as conditionally active antibodies with the indicated single point mutations in the heavy and light chain variable regions, respectively (Tables 1 and 2).

[0096] [Table 1]

[0097] [Table 2]

[0098] In another aspect, the present invention has identified heavy chain variable regions depicted in Figure 1A and light chain variable regions presented in Figure 1B. Some heavy chain variable regions are encoded by DNA sequences having SEQ ID NOS: 11-13. Some light chain variable regions are encoded by DNA sequences having SEQ ID NOS: 7-10. These heavy and light chain variable regions can specifically bind to Axl. An antibody comprising one of these heavy and light chain variable regions was found to have a higher binding affinity for Axl at a pH found in a tumor microenvironment than at a pH found in a non-tumor microenvironment.

[0099] The present invention also includes variants of the heavy and light chain variable regions encoded by the DNA sequences presented in Figures 1A-1B and having SEQ ID NOS: 9-13, which are capable of specifically binding to Axl. To derive these variants, it was determined that the complementarity-determining regions (CDRs) of the heavy chain variable region (H1-H3) and the CDRs of the light chain variable region (L1-L3) should remain intact.

[0100] The methods described herein are used as a guide for deriving these variants. These variants of heavy and light chain variable regions can be prepared by introducing appropriate modifications into the nucleotide sequences encoding the heavy and light chain variable regions or by peptide synthesis. Such modifications include, for example, deletion from, and / or insertion into, and / or substitution of residues in the amino acid sequence of an antibody or antibody fragment. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct retains at least one desired property, such as antigen binding.

[0101] Substitution, insertion, and deletion variants In some embodiments, antibody or antibody fragment variants are provided that have one or more amino acid substitutions. Target sites for substitutional mutagenesis include CDRs and framework regions (FRs). Conservative substitutions are shown in Table 3 under the heading "Conservative Substitutions." More substantial changes are provided in Table 3 under the heading "Exemplary Substitutions" and are further described below with respect to classes of amino acid side chains. Amino acid substitutions can be introduced into the antibody or antibody fragment of interest, and the products can be screened for desired activity, such as retention / improvement of antigen binding, or reduced immunogenicity.

[0102] [Table 3]

[0103] Amino acids can be grouped according to common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) Residues that influence chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0104] Non-conservative substitutions involve exchanging a member of one of these classes for one from another class.

[0105] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant selected for further testing has an altered (e.g., improved) biological property relative to the parent antibody (e.g., increased affinity, decreased immunogenicity) and / or substantially retains a biological property of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage-display-based affinity maturation techniques, such as those described herein. Briefly, one or more CDR residues are mutated, and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0106] Alterations (e.g., substitutions) can be made in CDRs, for example, to improve antibody affinity. Such alterations can be made in CDR "hot spots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol., vol. 207, pp. 179-196, 2008), and / or in SDRs (a-CDRs), and the resulting variant VH or VL is tested for binding affinity. Affinity maturation by construction of a secondary library and reselection from the secondary library is described, for example, in Hoogenboom et al. in Methods in Molecular Biology, vol. 178, pp. 1-37, 2001). In some affinity maturation embodiments, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves a CDR-directed approach, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. For example, alanine scanning mutagenesis or modeling can be used to specifically identify CDR residues involved in antigen binding. CDR-H3 and CDR-L3 are often targeted in particular.

[0107] In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody or antibody fragment to bind to the antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made in the CDRs. Such changes may be outside the "hot spots" of the CDRs or SDRs. The variant V provided above H and V LIn certain embodiments of the sequences, each CDR is unaltered or contains no more than one, two, or three amino acid substitutions.

[0108] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells, Science, vol. 244, pp. 1081-1085, 1989. In this method, a target residue or group (e.g., charged residues, such as arg, asp, his, lys, and glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody or antibody fragment with the antigen is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of an antigen-antibody complex can be used to identify contact points between the antibody or antibody fragment and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain desired properties.

[0109] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions, ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody of an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0110] Amino acid sequence modifications of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Only the CDRs in the VH and VL of an antibody derived from a non-human animal may be substituted with the VH and VL of a human antibody. H and V LIt is known that when a humanized antibody is produced by simply grafting amino acid residues into the FRs of a non-human antibody, the antibody binding activity is reduced compared to that of the original antibody derived from a non-human animal. It is considered that some amino acid residues in the VH and VL of a non-human antibody, not only in the CDRs but also in the FRs, are directly or indirectly involved in the antibody binding activity. Therefore, replacing these amino acid residues with different amino acid residues from the FRs of the VH and VL of a human antibody reduces the binding activity. To solve this problem, in an antibody into which human CDRs are grafted, it is necessary to attempt to identify amino acid residues in the amino acid sequences of the FRs of the VH and VL of the human antibody that are directly involved in binding to the antibody, interact with amino acid residues in the CDRs, or maintain the three-dimensional structure of the antibody and are directly involved in binding to the antigen. The reduced antigen binding activity can be increased by replacing the identified amino acids with amino acid residues from the original antibody derived from a non-human animal.

[0111] Modifications and changes can be made in the structure of the antibodies of the present invention, and in the DNA sequences that encode them, while still obtaining a functional molecule that encodes an antibody with desired characteristics.

[0112] In making changes in an amino acid sequence, the hydropathic index of amino acids can be taken into consideration. The importance of the hydropathic amino acid index in conferring interactive biological function to a protein is generally understood in the art. It is accepted that the relative hydropathic characteristics of amino acids contribute to the secondary structure of a resulting protein, which in turn determines the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0113] A further object of the present invention also includes function-conservative variants of the antibodies of the invention.

[0114] A "function-conservative variant" is one in which a given amino acid residue in a protein or enzyme is altered without altering the overall conformation and function of the polypeptide, including, but not limited to, the replacement of an amino acid with an amino acid having similar properties (e.g., polarity, hydrogen-bonding potential, acidic, basic, hydrophobic, aromatic, etc.). Amino acids other than those shown to be conserved may differ within a protein, resulting in a variation in the percent protein or amino acid sequence similarity between any two proteins of similar function, e.g., according to an alignment scheme, e.g., similarity may be 70-99% as determined by the Cluster method based on the MEGALIGN algorithm. "Function-conservative variants" also include polypeptides that have at least 60%, preferably at least 75%, more preferably at least 85%, even more preferably at least 90%, and even more preferably at least 95% amino acid identity as determined by the BLAST or FASTA algorithm, and have the same or substantially similar properties or functions as the native or parent protein to which they are compared.

[0115] Two amino acid sequences are "substantially homologous" or "substantially similar" if they are more than 80%, preferably more than 85%, preferably more than 90% amino acid identical, or more than about 90%, preferably more than 95%, similar (functionally identical) over the entire length of the shorter sequence. Preferably, similar or homologous sequences are identified by alignment using, for example, a GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wis.) pileup program, or any sequence comparison algorithm, such as BLAST, FASTA, etc.

[0116] For example, certain amino acids can be substituted for other amino acids in a protein structure without appreciable loss of activity. Because the interaction capacity and properties of a protein determine its biological functional activity, certain amino acid substitutions can be made in a protein sequence, and of course, in its DNA coding sequence, while still obtaining a protein with similar properties. Thus, it is contemplated that various changes can be made in the sequence of an antibody or antibody fragment of the present invention, or the corresponding DNA sequence encoding said antibody or antibody fragment, without appreciable loss of their biological activity.

[0117] It is known in the art that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biologically functional equivalent protein.

[0118] As outlined above, amino acid substitutions are therefore generally based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into account various of the above characteristics are well known to those of skill in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0119] Glycosylation variants In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0120] If the antibody contains an Fc region, the carbohydrate attached thereto can be altered. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, generally attached via an N-linkage to Asn297 in the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH, vol. 15, pp. 26-32, 1997. Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the invention can be made to generate antibody variants with improved properties.

[0121] In one embodiment, antibody variants are provided that have carbohydrate structures lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycans (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at approximately position 297 (EU numbering of Fc region residues) in the Fc region; however, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations within antibodies. Such fucosylation variants may have improved ADCC function. See, for example, US Patent Application Publication No. 2003 / 0157108 (Presta, L.); US Patent Application Publication No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.).Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include U.S. Patent Application Publication No. 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; U.S. Patent Application Publication No. 2003 / 0115614; U.S. Patent Application Publication No. 2002 / 0164328; U.S. Patent Application Publication No. 2004 / 0093621; U.S. Patent Application Publication No. 2004 / 0132140; U.S. Patent Application No. Publication No. 2004 / 0110704; U.S. Patent Application Publication No. 2004 / 0110282; U.S. Patent Application Publication No. 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO 2005 / 053742; WO 2002 / 031140; Okazaki et al. J. Mol. Biol., vol. 336, pp. 1239-1249, 2004; Yamane-Ohnuki et al. Biotech. Bioeng., vol. 87, pp. 614-622, 2004. Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys., vol. 249, pp. 533-545, 1986; U.S. Patent Application Publication No. 2003 / 0157108A; and WO 2004 / 056312A1, particularly Example 11), as well as knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8 knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng., vol. 87, pp. 614-622, 2004; Kanda, Y. et al. al., Biotechnol. Bioeng., vol. 94, pp. 680-688, 2006; and WO 2003 / 085107).

[0122] Further provided are antibody variants having bisected oligosaccharides, for example, biantennary oligosaccharides attached to the Fc region of the antibody, bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878; U.S. Pat. No. 6,602,684; and U.S. Patent Application Publication No. 2005 / 0123546. Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764.

[0123] Fc region variants In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0124] In certain embodiments, the present invention contemplates antibody variants that retain some, but not all, effector functions, making them desirable candidates for applications in which in vivo antibody half-life is important while certain effector functions (e.g., ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 5 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol., vol. 9, pp. 457-492, 1991. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see also, e.g., Hellstrom et al. Proc. Nat'l Acad. Sci. USA, vol. 83, pp. 7059-7063, 1986) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA, vol. 82, pp. 1499-1502, 1985; U.S. Pat. No. 5,821,337 (see also, Bruggemann et al., J. Exp. Med., vol. 166, pp. 1351-1361, 1987). Alternatively, non-radioactive assay methods can be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, Calif.); and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wis.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or in addition, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model, such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA, vol. 95, pp. 652-656, 1998. A C1q binding assay can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To evaluate complement activation, CDC assay can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods, vol. 202, pp. 163-171, 1996; Cragg, MS et al., Blood, vol. 101, pp. 1045-1052, 2003; and Cragg, MS, and MJ Glennie, Blood, vol. 103, pp. 2738-2743, 2004). FcRn binding and in vivo clearance / half-life determination can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol., vol. 18, pp. 1759-1769, 2006).

[0125] Antibodies with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, such as the so-called "DANA" Fc mutant with substitutions of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).

[0126] Certain antibody variants with improved or diminished binding to FcRs have been described (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem., vol. 9, pp. 6591-6604, 2001).

[0127] In one embodiment, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0128] In some embodiments, alterations are made in the Fc region that result in altered (i.e., improved or diminished) C1q binding and / or complement-dependent cytotoxicity (CDC), such as those described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol., vol. 164, pp. 4178-4184, 2000.

[0129] Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol., vol. 117, pp. 587-593, 1976 and Kim et al., J. Immunol., vol. 24, p. 249, 1994), are described in U.S. Patent Application Publication No. 2005 / 0014934. These antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a substitution at Fc region residue 434 (U.S. Pat. No. 7,371,826). For other examples of Fc region variants, see also Duncan & Winter, Nature, vol. 322, pp. 738-740, 1988; U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94 / 29351.

[0130] Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to create cysteine-engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues. In certain embodiments, the substituted residues occur at accessible sites of the antibody. By substituting these residues with cysteine, reactive thiol groups are thereby located at accessible sites of the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates as further described herein. In certain embodiments, any one or more of the following residues can be substituted with cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and 5400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antibodies can be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0131] antibody derivative In certain embodiments, the antibodies or antibody fragments provided herein can be further modified to contain additional non-protein moieties that are readily available and known in the art. Suitable moieties for derivatizing antibodies or antibody fragments include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. Polymers can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody or antibody fragment can vary, and when more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations such as, but not limited to, the particular property or function of the antibody or antibody fragment to be improved, whether the derivative will be used in therapy under defined conditions, etc.

[0132] In another embodiment, a conjugate of an antibody or antibody fragment and a non-protein moiety is provided that can be selectively heated by exposure to radiation. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA, vol. 102, pp. 11600-11605, 2005). The radiation can be of any wavelength, including but not limited to, a wavelength that does not harm normal cells but heats the non-protein moiety to a temperature that kills cells in the vicinity of the antibody-non-protein moiety.

[0133] In another aspect, the present invention provides an anti-Axl antibody or antibody fragment comprising an isolated heavy chain variable region polypeptide or an isolated light chain variable region polypeptide. The isolated heavy chain variable region polypeptide comprises the H1, H2, and H3 regions having SEQ ID NOs: 1-3, respectively. The isolated light chain variable region polypeptide comprises the L1, L2, and L3 regions having SEQ ID NOs: 4-6, respectively.

[0134] The anti-Axl antibodies or antibody fragments of the present invention have a higher binding affinity for Axl under conditions in a tumor microenvironment than under conditions in a non-tumor microenvironment. In one embodiment, the conditions in a tumor microenvironment and the conditions in a non-tumor microenvironment are both pH levels. Thus, the anti-Axl antibodies or antibody fragments of the present invention can selectively bind to Axl at a pH of about 5 to about 6.8, but have a lower binding affinity for Axl at a pH of 7.2 to 7.8, which is found in a normal physiological environment. As shown in Examples 3 and 4, the anti-Axl antibodies or antibody fragments have a higher binding affinity at pH 6.0 than at pH 7.4.

[0135] In certain embodiments, the anti-Axl antibodies or antibody fragments of the invention have a cytotoxicity of about ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, or 10 -8 M~10 -13 M, or 10 -9 M~10-13 The antibody or antibody fragment has a dissociation constant (Kd) for Axl under conditions in a tumor microenvironment of at least about 1.5:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 20:1, at least about 30:1, at least about 50:1, at least about 70:1, or at least about 100:1.

[0136] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab form of the antibody of interest and its antigen using the following assay: The solution binding affinity of the Fab for the antigen is determined by the lowest concentration ( 125 I) Fab is equilibrated with labeled antigen, followed by capturing the bound antigen using an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish assay conditions, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125[I] The antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, incubation can be continued for a longer period (e.g., about 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed, and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μl / well of scintillant (MICROSCINT-20™, Packard) is added, and the plate is counted for 10 minutes on a TOPCOUNT™ gamma counter (Packard). The concentration of each Fab that gives 20% or less of maximum binding is selected for use in the competitive binding assay.

[0137] According to another embodiment, Kd is measured at approximately 10 response units (RU) using a surface plasmon resonance assay with a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C using an immobilized antigen CM5 chip. Briefly, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RU) of bound protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at a flow rate of approximately 25 μl / min at 25° C. The association rate (k on ) and dissociation rate (k off The equilibrium dissociation constant (Kd) is calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® evaluation software version 3.2). off / k on For example, see Chen et al., J. Mol. Biol. 293:865-881 (1999). When the on-rate by the surface plasmon resonance assay is 10 6 M -1 s -1If the on-rate exceeds 100 kJ / s, the on-rate can be measured by using a fluorescence quenching technique to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C in the presence of increasing concentrations of antigen when measured in a spectrometer, for example, a stopped-flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.

[0138] The anti-Axl antibodies of the present invention can be chimeric, humanized, or human. In one embodiment, anti-Axl antibody fragments, such as Fv, Fab, Fab', Fab'-SH, scFv, diabody, triabody, tetrabody, or F(ab')2 fragments and multispecific antibodies formed from antibody fragments, are used. In another embodiment, the antibody is a full-length antibody, as defined herein, such as an intact IgG antibody or other antibody class or isotype. For a review of certain antibody fragments, see Hudson et al. Nat. Med., vol. 9, pp. 129-134, 2003. For a review of scFv fragments, see, e.g., Pluckthuen, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments that contain salvage receptor-binding epitope residues and have increased in vivo half-lives, see U.S. Pat. No. 5,869,046.

[0139] The diabodies of the present invention can be bivalent or bispecific. For example, for examples of diabodies, see EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA, vol. 90, pp. 6444-6448, 1993. Examples of triabodies and tetrabodies are also described in Hudson et al., Nat. Med., vol. 9, pp. 129-134, 2003.

[0140] In some embodiments, the invention includes single-domain antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

[0141] Antibody fragments can be produced by various techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phages) as described herein.

[0142] In some embodiments, the anti-Axl antibody of the present invention can be a chimeric antibody. Some chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, vol. 81, pp. 6851-6855, 1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a "class-switched" antibody in which the class or subclass of the antibody has been changed relative to the class or subclass of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0143] In some embodiments, the chimeric antibody of the present invention is a humanized antibody. Typically, such a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. A humanized antibody may also optionally comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived) to, for example, restore or improve the specificity or affinity of the antibody.

[0144] Humanized antibodies and methods for their production are reviewed, for example, by Almagro and Fransson, Front. Biosci., vol. 13, pp. 1619-1633, 2008, and further described, for example, by Riechmann et al., Nature, vol. 332, pp. 323-329, 1988; Queen et al., Proc. Nat'l Acad. Sci. USA, vol. 86, pp. 10029-10033, 1989; U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al. al., Methods, vol. 36, pp. 25-34, 2005 (describing SDR (a-CDR) grafting); Padlan, Mol. Immunol., vol. 28, pp. 489-498, 1991 (describing "resurfacing"); Dall'Acqua et al., Methods, vol. 36, pp. 43-60, 2005 (describing "FR shuffling"); and Osbourn et al., Methods, vol. 36, pp. 61-68, 2005 and Klimka et al., Br. J. Cancer, vol. 83, pp. 252-260, 2000 (describing a "guided selection" approach to FR shuffling).

[0145] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol., vol. 151, p. 2296, 1993); framework regions derived from consensus sequences of human antibodies in specific subgroups of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, vol. 89, p. 4285, 1992; and Presta et al. J. Immunol., vol. 151, p. 2623, 1993), human mature (somatically mutated) framework regions, or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci., vol. 13, pp. 1619-1633, 2008); and framework regions derived from screening of FR libraries (see, e.g., Baca et al. al., J. Biol. Chem., vol. 272, pp. 10678-10684, 1997 and Rosok et al., J. Biol. Chem., vol. 271, pp. 22611-22618, 1996).

[0146] In some embodiments, the anti-Axl antibody of the present invention is a multispecific antibody, e.g., a bispecific antibody. A multispecific antibody is a monoclonal antibody that has binding specificity for at least two different sites. In some embodiments, one binding specificity is for Axl and the other is for another antigen. In some embodiments, a bispecific antibody can bind to two different epitopes of Axl. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing Axl. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0147] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature, vol. 305, pp. 537-540, 1983; WO 93 / 08829; and Traunecker et al., EMBO J., vol. 10, pp. 3655-3659, 1991), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can be produced by engineering electrostatic steering effects to create antibody Fc heterodimeric molecules (WO 2009 / 089004 A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, vol. 229, pp. 81-83, 1985); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., vol. 148, pp. 1547-1553, 1992); using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, vol. 90, pp. 6444-6448, 1993); and using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., vol. 90, pp. 6444-6448, 1993). al., J. Immunol., vol. 152, pp. 5368-5374, 1994); and, for example, by preparing trispecific antibodies as described in Tutt et al. J. Immunol., vol. 147, pp. 60-69, 1991.

[0148] Engineered antibodies having three or more functional antigen binding sites, such as "octopus antibodies," are also included herein (see, eg, US Patent Application Publication No. 2006 / 0025576A1).

[0149] Antibodies or antibody fragments also include "dual acting Fabs" or "DAFs" that contain antigen binding sites that bind to Axl and another, different antigen (see, e.g., U.S. Patent Application Publication No. 2008 / 0069820).

[0150] The anti-Axl antibodies or antibody fragments of the invention can be produced using the recombinant methods and compositions detailed in U.S. Patent Application Publication No. 2016 / 0017040.

[0151] The physical / chemical properties and / or biological activity of the anti-Axl antibodies or antibody fragments of the present invention can be tested and measured by various assays known in the art, some of which are described in U.S. Patent No. 8,853,369.

[0152] B. Immunoconjugates In another aspect, the present invention also provides an immunoconjugate comprising an anti-Axl antibody herein conjugated to one or more cytotoxic agents, e.g., chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins of bacterial, fungal, plant, or animal origin, enzymatically active toxins, or fragments thereof), or radioactive isotopes.

[0153] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including, but not limited to, maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064, and EP 0425235 B1); auristatins, such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,645, ... 483, 5,780,588, and 7,498,298); dolastatins; calicheamicin or its derivatives (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res., vol. 53, pp. 3336-3342, 1993; and Lode et al., Cancer Res., vol. 58, pp. 2925-2928, 1998); anthracyclines, such as daunomycin or doxorubicin (Kratz et al., Current Med. Chem., vol. 13, pp. 477-523, 2006; Jeffrey et al., Bioorganic & Med. Chem. Letters, vol. 16, pp. 358-362, 2006; Torgov et al., Bioconj. Chem., vol. 16, pp. 717-721, 2005; Nagy et al., Proc. Natl. Acad. Sci. USA, vol. 97, pp. 829-834, 2000; Dubowchik et al. al.,Bioorg.&Med.Chem.Letters,vol.12,vol.1529-1532,2002;King et al.,J.Med.Chem.,vol.45,pp.4336-4343, 2002; and U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes such as decetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

[0154] In another embodiment, the immunoconjugate comprises an antibody described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, or curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and a trichothecene.

[0155] In another embodiment, the immunoconjugate comprises an antibody described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the production of radioconjugates. Examples include At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212and radioactive isotopes of Lu. When a radioactive conjugate is used for detection, it can contain radioactive atoms for scintigraphy tests, such as tc99m or I123, or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123, as well as iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese.

[0156] Conjugates of antibodies and cytotoxic agents can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science, vol. 238, pp. 1098-, 1987. Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See International Publication No. WO 94 / 11026. The linker can be a "cleavable linker" that facilitates the release of cytotoxic drugs in cells. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al., Cancer Res., vol. 52, pp. 127-131, 1992; U.S. Patent No. 5,208,020) can be used.

[0157] The immunoconjugates herein expressly contemplate conjugates prepared using cross-linking reagents such as, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as SVSB (succinimidyl-(4-vinylsulfone)benzoate), which is commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., USA).

[0158] Exemplary embodiments of ADCs include an antibody (Ab) that targets a tumor cell, a drug moiety (D), and a linker moiety (L) that attaches the Ab to D. In some embodiments, the antibody is attached to the linker moiety (L) via one or more amino acid residues, e.g., lysine and / or cysteine.

[0159] An exemplary ADC has the formula I: Ab-(LD) p I, where p is 1 to about 20. In some embodiments, the number of drug moieties that can be conjugated to an antibody is limited by the number of free cysteine ​​residues. In some embodiments, free cysteine ​​residues are introduced into the antibody amino acid sequence by the methods described herein. Exemplary ADCs of Formula I include, but are not limited to, antibodies with one, two, three, or four engineered cysteine ​​amino acids (Lyon et al., Methods in Enzym., vol. 502, pp. 123-138, 2012). In some embodiments, one or more free cysteine ​​residues are already present in the antibody without engineering, in which case the existing free cysteine ​​residues can be used to conjugate the antibody to a drug. In some embodiments, the antibody is exposed to reducing conditions prior to antibody conjugation to generate one or more free cysteine ​​residues.

[0160] a) Exemplary Linkers A "linker" (L) is a bifunctional or polyfunctional moiety that can be used to attach one or more moieties, e.g., a drug moiety (D), to an antibody (Ab) to form an immunoconjugate, e.g., an ADC of Formula I. In some embodiments, an ADC can be prepared using a linker with reactive functional groups for covalently binding to a drug and to an antibody. For example, in some embodiments, a cysteine ​​thiol of an antibody (Ab) can form a bond with a reactive functional group of a linker or a drug-linker intermediate to create an ADC.

[0161] In one embodiment, linker has a functional group that can react with the free cysteine ​​present on antibody to form a covalent bond.Non-limiting examples of such reactive functional groups include maleimide, haloacetamide, α-haloacetyl, activated ester (for example, succinimide ester, 4-nitrophenyl ester, pentafluorophenyl ester, tetrafluorophenyl ester), anhydride, acid chloride, sulfonyl chloride, isocyanate, and isothiocyanate.For example, see the conjugation method in page 766 of Klussman, et al., Bioconjugate Chemistry, vol.15, pp.765-773, 2004.

[0162] In some embodiments, the linker has a functional group that can react with an electrophilic group present on an antibody. Exemplary such electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups. In some embodiments, a heteroatom of the reactive functional group of the linker can react with an electrophilic group on an antibody to form a covalent bond to an antibody unit. Non-limiting exemplary such reactive functional groups include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

[0163] The linker may comprise one or more linker components. Exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), and 4-(N-maleimidomethyl)cyclohexane-1-carboxylate ("MCC"). Various linker components are known in the art, some of which are described below.

[0164] The linker may be a "cleavable linker" that facilitates the release of the drug. Non-limiting examples of cleavable linkers include acid-labile linkers (e.g., containing hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, or disulfide-containing linkers (Chari et al., Cancer Research, vol. 52, pp. 127-131, 1992; U.S. Pat. No. 5,208,020).

[0165] In some embodiments, the linker has the following formula II: -Aa-Ww-Yy-, where A is a "stretcher unit" and a is an integer from 0 to 1; W is an "amino acid unit" and w is an integer from 0 to 12; Y is a "spacer unit" and y is 0, 1, or 2. ADCs containing a linker of formula II have the formula I(A): Ab-(A a -W w -Y y -D) p where Ab, D, and p are as defined above for Formula I. Exemplary embodiments of such linkers are described in U.S. Pat. No. 7,498,298.

[0166] In some embodiments, a linker component comprises a "stretcher unit" (A) that attaches the antibody to another linker component or to a drug moiety. Non-limiting exemplary stretcher units are shown below (where the wavy line indicates the site of covalent attachment to an antibody, drug, or additional linker component): [ka]

[0167] In some embodiments, the linker component comprises an "amino acid unit" (W). In some such embodiments, the amino acid unit allows for cleavage of the linker by a protease, thereby facilitating release of the drug from the immunoconjugate upon exposure to an intracellular protease, e.g., a lysosomal enzyme (Doronina et al., Nat. Biotechnol., vol. 21, pp. 778-784, 2003). Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include, but are not limited to, valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe), phenylalanine-lysine (fk or phe-lys); phenylalanine-homolysine (phe-homolys); and N-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include, but are not limited to, glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). The amino acid unit may contain naturally occurring amino acid residues and / or minor amino acids and / or non-naturally occurring amino acid analogs, such as citrulline. The amino acid unit can be designed and optimized for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C, and D, or plasmin proteases.

[0168] Typically, peptide-type linkers can be prepared by forming a peptide bond between two or more amino acids and / or peptide fragments. Such peptide bonds can be prepared, for example, according to lipid phase synthesis (e.g., E. Schroder and K. Luebke (1965) "The Peptides", volume 1, pp. 76-136, Academic Press).

[0169] In some embodiments, the linker component comprises a "spacer" unit (Y) that links the antibody to the drug moiety directly or via a stretcher unit and / or an amino acid unit. The spacer unit can be "self-immolative" or "non-self-immolative." A "non-self-immolative" spacer unit is one in which some or all of the spacer unit remains attached to the drug moiety upon cleavage of the ADC. Examples of non-self-immolative spacer units include, but are not limited to, a glycine spacer unit and a glycine-glycine spacer unit. In some embodiments, enzymatic cleavage of an ADC containing a glycine-glycine spacer unit by tumor cell-associated proteases results in release of the glycine-glycine-drug moiety from the remainder of the ADC. In some such embodiments, the glycine-glycine-drug moiety is subjected to a hydrolysis step in the tumor cell, thus cleaving the glycine-glycine spacer unit from the drug moiety.

[0170] The "self-immolative" spacer unit allows for the release of the drug moiety. In certain embodiments, the spacer unit of the linker comprises a p-aminobenzyl unit. In some such embodiments, p-aminobenzyl alcohol is attached to the amino acid unit via an amide bond, and a carbamate, methylcarbamate, or carbonate is created between the benzyl alcohol and the drug (Hamann et al. Expert Opin. Ther. Patents, vol. 15, pp. 1087-1103, 2005). In some embodiments, the spacer unit comprises p-aminobenzyloxycarbonyl (PAB). In some embodiments, an ADC comprising a self-immolative linker has the structure: [ka] where Q is -C1-C8 alkyl, -O-(C1-C8 alkyl), -halogen, -nitro, or -cyano; m is an integer ranging from 0 to 4; X can be one or more additional spacer units or can be absent; and p ranges from 1 to about 20. In some embodiments, p ranges from 1 to 10, 1 to 7, 1 to 5, or 1 to 4. Non-limiting exemplary X spacer units include: [ka] wherein R1 and R2 are independently selected from H and C1-C6 alkyl. In some embodiments, R1 and R2 are each -CH3.

[0171] Other examples of self-immolative spacers include, but are not limited to, aromatic compounds that are electronically similar to the PAB group, such as 2-aminoimidazole-5-methanol derivatives (U.S. Pat. No. 7,375,078; Hay et al., Bioorg. Med. Chem. Lett., vol. 9, p. 2237-, 1999) and ortho- or para-aminobenzyl acetals. In some embodiments, spacers that undergo cyclization upon amide bond hydrolysis can be used, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., Chemistry Biology, vol. 2, pp. 223-, 1995), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm et al., J. Amer. Chem. Soc., vol. 94, p. 5815-, 1972), and 2-aminophenylpropionic acid amides (Amsberry et al., J. Org. Chem., vol. 55, p. 5867, 1990). Attachment of a drug to the α-carbon of a glycine residue is another example of a self-immolative spacer that can be useful in ADCs (Kingsbury et al., J. Med. Chem., vol. 27, p. 1447, 1984).

[0172] In some embodiments, the linker L can be a dendritic linker for covalently linking two or more drug moieties to an antibody via a branched, multifunctional linker moiety (Sun et al., Bioorganic & Medicinal Chemistry Letters, vol. 12, pp. 2213-2215, 2002; Sun et al., Bioorganic & Medicinal Chemistry, vol. 11, pp. 1761-1768, 2003). Dendritic linkers can increase the drug-to-antibody molar ratio, i.e., loading, which is related to the efficacy of the ADC. Thus, if an antibody bears only one reactive cysteine ​​thiol group, multiple drug moieties can be attached via a dendritic linker.

[0173] Non-limiting exemplary linkers are shown below in the context of ADCs of Formula I: [ka] wherein R1 and R2 are independently selected from H and C1-C6 alkyl. In some embodiments, R1 and R2 are each -CH3. [ka] (wherein n is 0 to 12). In some embodiments, n is 2 to 10. In some embodiments, n is 4 to 8.

[0174] Further non-limiting exemplary ADCs include those having the structure: [ka] each R is independently H or C1-C6 alkyl; and n is 1 to 12.

[0175] In some embodiments, the linker is substituted with groups that modulate solubility and / or reactivity. Non-limiting examples include charged substituents, such as sulfonates (-SO3 - ) or ammonium may increase the water solubility of the linker reagent and facilitate the coupling reaction of the linker reagent with the antibody and / or drug moiety, or may facilitate the coupling reaction of Ab-L (antibody-linker intermediate) with D, or the coupling reaction of DL (drug-linker intermediate) with Ab, depending on the synthetic route used to prepare the ADC. In some embodiments, a portion of the linker is coupled to the antibody, a portion of the linker is coupled to the drug, and then the Ab-(linker moiety) a Drug-(linker moiety) b to form the ADC of Formula I.

[0176] The compounds of the present invention expressly contemplate ADCs prepared using, but not limited to, the following linker reagents: bis-maleimido-trioxyethylene glycol (BMPEO), N-(β-maleimidopropyloxy)-N-hydroxysuccinimide ester (BMPS), N-(ε-maleimidocaproyloxy)succinimide ester (EMCS), N-[γ-maleimidobutyryloxy]succinimide ester (GMBS), 1,6-hexane-bis-vinylsulfone (HBVS), succinimide Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), succinimidyl 3-(bromoacetamido)propionate (SBAP), succinimidyl iodoacetate (SIA), succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), N-succinimidyl-3-(2- pyridyldithio)propionate (SPDP), N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), succinimidyl 6-[(beta-maleimidopropionamido)hexanoate] (SMPH), iminothiolane (IT), sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, Sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as succinimidyl-(4-vinylsulfone)benzoate (SVSB), and, by way of example, bis-maleimide reagents: dithiobismaleimidoethane (DTME), 1,4-bismaleimidobutane (BMB), 1,4 bismaleimidyl-2,3-dihydroxybutane (BMDB), bismaleimidohexane (BMH), bismaleimidoethane (BMOE), BM(PEG)2 (shown below), and BM(PEG)3 (shown below);Bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), activated esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). In some embodiments, bis-maleimide reagents allow for the attachment of thiol groups of cysteines in antibodies to thiol-containing drug moieties, linkers, or linker-drug intermediates. Other functional groups reactive with thiol groups include, but are not limited to, iodoacetamide, bromoacetamide, vinylpyridine, disulfides, pyridyl disulfides, isocyanates, and isothiocyanates.

[0177] Certain useful linker reagents are available from a variety of commercial sources, e.g., Pierce Biotechnology, Inc. (Rockford, Ill.), Molecular Biosciences Inc. (Boulder, Colo.), or are described in the art, e.g., Toki et al., J. Org. Chem., vol. 67, pp. 1866-1872, 2002; Dubowchik et al., Tetrahedron Letters, vol. 38, pp. 5257-60, 1997; Walker, J. Org. Chem., vol. 60, pp. 5352-5355, 1995; Frisch et al., Bioconjugate Chem., vol. 7, pp. 180-186, 1995; U.S. Pat. No. 6,214,345; WO 02 / 088172; U.S. Pat. App. Pub. No. 2003130189; U.S. Pat. App. Pub. No. 2003096743; WO 03 / 026577; WO 03 / 043583; and WO 04 / 032828.

[0178] Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See, e.g., WO 94 / 11026.

[0179] b) Exemplary Drug Moieties 1) Maytansine and maytansinoids In some embodiments, the immunoconjugate comprises an antibody conjugated to one or more maytansinoid molecules. Maytansinoids are derivatives of maytansine, which are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it was discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are described, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; and 4,309,428. and 4,371,533.

[0180] Maytansinoid drug moieties are attractive drug moieties for antibody-drug conjugates because they are (i) relatively accessible to prepare by fermentation or chemical modification, or derivatization of fermentation products, (ii) amenable to derivatization with functional groups suitable for conjugation to antibodies via non-disulfide linkers, (iii) stable in plasma, and (iv) effective against a variety of tumor cell lines.

[0181] Certain maytansinoids suitable for use as maytansinoid drug moieties are known in the art and can be isolated from natural sources according to known methods or produced using genetic engineering techniques (see, e.g., Yu et al., PNAS, vol. 99, pp. 7968-7973, 2002). Maytansinoids can also be prepared synthetically according to known methods.

[0182] Exemplary maytansinoid drug moieties include, but are not limited to, those with modified aromatic rings, such as C-19-dechloro (U.S. Pat. No. 4,256,746) (e.g., prepared by lithium aluminum hydride reduction of ansamitocin P2); C-20-hydroxy (or C-20-demethyl) + / -C-19-dechloro (U.S. Pat. Nos. 4,361,650 and 4,307,016) (e.g., prepared by demethylation using Streptomyces or Actinomyces or dechlorination using LAH); and C-20-demethoxy, C-20-acyloxy (-OCOR), + / -dechloro (U.S. Pat. No. 4,294,757) (e.g., prepared by acylation using acyl chlorides), as well as those with modifications at other positions on the aromatic ring.

[0183] Exemplary maytansinoid drug moieties include those having modifications such as C-9-SH (U.S. Pat. No. 4,424,219) (e.g., prepared by reaction of maytansinol with H2S or P2S5); C-14-alkoxymethyl (demethoxy / CH2OR) (U.S. Pat. No. 4,331,598); C-14-hydroxymethyl or acyloxymethyl (CH2OH or CHOAc) (U.S. Pat. No. 4,450,254) (e.g., prepared from Nocardia); C-15-hydroxy / acyloxy (U.S. Pat. No. 4,364,866) (e.g., prepared by conversion of maytansinol by Streptomyces); C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (e.g., prepared from Trewia nudolflora nudlflora); C-18-N-demethyl (e.g., U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared by demethylation of maytansinol with Streptomyces); and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared, for example, by titanium trichloride / LAH reduction of maytansinol).

[0184] Many positions on a maytansinoid compound are useful as attachment positions. For example, an ester bond can be formed by reaction with a hydroxyl group using conventional coupling techniques. In some embodiments, the reaction can occur at the C-3 position, which bears a hydroxyl group, the C-14 position, which is modified with hydroxymethyl, the C-15 position, which is modified with a hydroxyl group, and the C-20 position, which bears a hydroxyl group. In some embodiments, the bond is formed at the C-3 position of maytansinol or a maytansinol analog.

[0185] Maytansinoid drug moieties include those having the following structure: [ka] where the wavy line indicates the covalent attachment of the sulfur atom of the maytansinoid drug moiety to the linker of the ADC. Each R can independently be H or C1-C6 alkyl. The alkylene chain attaching the amide group to the sulfur atom can be methanyl, ethanyl, or propyl, i.e., m is 1, 2, or 3 (U.S. Pat. No. 633,410; U.S. Pat. No. 5,208,020; Chari et al., Cancer Res., vol. 52, pp. 127-131, 1992; Liu et al., Proc. Nall. Acad. Sci. USA, vol. 93, pp. 8618-8623, 1996).

[0186] All stereoisomers of the maytansinoid drug moiety, i.e., any combination of R and S configurations at the chiral carbon, are contemplated for the ADCs of the invention (U.S. Pat. No. 7,276,497; U.S. Pat. No. 6,913,748; U.S. Pat. No. 6,441,163; U.S. Pat. No. 633,410 (RE39151); U.S. Pat. No. 5,208,020; Widdison et al. (2006) J. Med. Chem. 49:4392-4408). In some embodiments, the maytansinoid drug moiety has the following stereochemistry: [ka]

[0187] Exemplary embodiments of maytansinoid drug moieties include, but are not limited to, those having the structure: [ka] DM1; DM3 and DM4, each having the formula: (wherein the wavy line indicates the covalent bond of the sulfur atom of the drug to the linker (L) of the antibody-drug conjugate).

[0188] An exemplary antibody-drug conjugate in which DM1 is attached to a thiol group of an antibody via a BMPEO linker has the structure and abbreviation: [ka] where Ab is an antibody; n is 0, 1, or 2; and p is 1 to about 20. In some embodiments, p is 1 to 10, p is 1 to 7, p is 1 to 5, or p is 1 to 4.

[0189] Immunoconjugates containing maytansinoids, methods for their preparation and therapeutic uses, are disclosed, for example, in U.S. Patent Nos. 5,208,020 and 5,416,064; U.S. Patent Application Publication No. 2005 / 0276812A1; and European Patent No. 0425235B1. See also Liu et al., Proc. Natl. Acad. Sci. USA, vol. 93, pp. 8618-8623, 1996; and Chari et al., Cancer Research, vol. 52, pp. 127-131, 1992.

[0190] In some embodiments, antibody-maytansinoid conjugates can be prepared by chemically linking an antibody to a maytansinoid molecule without substantially diminishing the biological activity of either the antibody or the maytansinoid molecule. See, e.g., U.S. Patent No. 5,208,020. In some embodiments, ADCs with an average of 3 to 4 maytansinoid molecules conjugated per antibody molecule have shown efficacy in improving target cell cytotoxicity without adversely affecting antibody function or solubility. In some instances, even a single molecule of toxin / antibody is expected to improve cytotoxicity compared to the use of naked antibodies.

[0191] Exemplary linking groups for producing antibody-maytansinoid conjugates include, for example, those described herein and those disclosed in U.S. Pat. No. 5,208,020; European Patent No. 0425235B1; Chari et al., Cancer Research, vol. 52, pp. 127-131, 1992; U.S. Patent Application Publication No. 2005 / 0276812A1; and U.S. Patent Application Publication No. 2005 / 016993A1.

[0192] (2) Auristatins and dolastatins Drug moieties include dolastatins, auristatins, and their analogs and derivatives (U.S. Pat. Nos. 5,635,483; 5,780,588; 5,767,237; 6,124,431). Auristatins are derivatives of the marine mollusk compound dolastatin-10. Without being bound by any particular theory, dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al., Antimicrob. Agents and Chemother., vol. 45, pp. 3580-3584, 2001), and to have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al., Antimicrob. Agents Chemother., vol. 42, pp. 2961-2965, 1998). Dolastatin / auristatin drug moieties can be attached to antibodies through the N-terminus or C-terminus of the peptidic drug moiety (WO 02 / 088172; Doronina et al., Nature Biotechnology, vol. 21, pp. 778-784, 2003; Francisco et al., Blood, vol. 102, pp. 1458-1465, 2003).

[0193] Exemplary auristatin embodiments include N-terminally linked monomethyl auristatin drug moieties D, as disclosed in U.S. Pat. Nos. 7,498,298 and 7,659,241. E and D F Examples include: [ka] (In the formula, D E and D F The wavy lines indicate the site of covalent attachment to the antibody or antibody-linker component, and independently at each position: R 2 is selected from H and C1-C8 alkyl; R 3 is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle); R 4 is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle); R 5 is selected from H and methyl; or R 4 and R 5 together form a carbocyclic ring of the formula -(CR a R b ) n -(In the formula, R a and R b are independently selected from H, C1-C8 alkyl, and C3-C8 carbocyclic compounds, and n is selected from 2, 3, 4, 5, and 6; R 6 is selected from H and C1-C8 alkyl; R 7is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle); Each R 8 are independently selected from H, OH, C1-C8 alkyl, C3-C8 carbocycle, and O—(C1-C8 alkyl); R 9 is selected from H and C1-C8 alkyl; R 10 is selected from aryl or a C3-C8 heterocycle; Z is O, S, NH, or NR 12 and R 12 is C1-C8 alkyl; R 11 H, C1~C 20 Alkyl, aryl, C3-C8 heterocycle, -(R 13 O) m -R 14 , or (R 13 O) m -CH(R 15 )2 is selected; m is an integer ranging from 1 to 1000; R 13 is a C2-C8 alkyl; R 14 is H or C1-C8 alkyl; Each occurrence of e is independently H, COOH, -(CH2) n -N(R 16 )2, -(CH2) n -SO3H, or (CH2) n -SO3-C1-C8 alkyl; Each occurrence of e is independently H, C1-C8 alkyl, or (CH2) n -COOH; R 18 is -C(R 8 )2-C(R 8 )2-aryl, -C(R 8 )2-C(R 8 )2 (C3-C8 heterocycle), and C(R 8)2-C(R 8 )2 (C3-C8 carbocyclic ring); where n is an integer ranging from 0 to 6.

[0194] In one embodiment, R 3 , R 4 and R 7 are independently isopropyl or sec-butyl, and R 5 is —H or methyl. In an exemplary embodiment, R 3 and R 4 are isopropyl, and R 5 is -H, and R 7 is sec-butyl.

[0195] In yet other embodiments, R 2 and R 6 are methyl, and R 9 is -H.

[0196] In yet other embodiments, R 8 Each occurrence of is -OCH3.

[0197] In an exemplary embodiment, R 3 and R 4 are isopropyl, and R 2 and R 6 are methyl, and R 5 is -H, and R 7 is sec-butyl, and R 8 Each occurrence of is -OCH3 and R 9 is -H.

[0198] In one embodiment, Z is —O— or —NH—.

[0199] In one embodiment, R 10 is aryl.

[0200] In an exemplary embodiment, R 10 is -phenyl.

[0201] In an exemplary embodiment, when Z is —O—, R 11 is -H, methyl or t-butyl.

[0202] In one embodiment, when Z is —NH, R 11 is -CH(R 15 )2 and R 15 is -(CH2) n -N(R 16 )2 and R 16 is -C1-C8 alkyl or (CH2) n -COOH.

[0203] In another embodiment, when Z is -NH, R 11 is -CH(R 15 )2 and R 15 is -(CH2) n -SO3H.

[0204] Formula D E An exemplary auristatin embodiment of is MMAE, where the wavy line indicates the covalent attachment to the linker (L) of the antibody-drug conjugate: [ka]

[0205] Formula D E An exemplary auristatin embodiment of is MMAE, where the wavy line indicates the covalent attachment to the linker (L) of the antibody-drug conjugate: [ka]

[0206] Other exemplary embodiments include monomethylvaline compounds with a phenylalanine carboxy modification at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007 / 008848) and monomethylvaline compounds with a phenylalanine side chain modification at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007 / 008603).

[0207] Non-limiting exemplary embodiments of ADCs of Formula I comprising MMAF and various linker components also include Ab-MC-PAB-MMAF and Ab-PAB-MMAF. Immunoconjugates comprising MMAF attached to an antibody by a non-proteolytically cleavable linker have been shown to possess activity equivalent to that of immunoconjugates comprising MMAF attached to an antibody by a proteolytically cleavable linker (see Doronina et al., Bioconjugate Chem., vol. 17, pp. 114-124, 2006). In some such embodiments, drug release is believed to be influenced by antibody degradation in cells.

[0208] Typically, peptide-based drug moieties can be prepared by forming a peptide bond between two or more amino acids and / or peptide fragments. Such peptide bonds can be prepared, for example, according to solution-phase synthesis methods well known in the field of peptide chemistry (see, for example, E. Schroeder and K. Luebke, "The Peptides," volume 1, pp. 76-136, 1965, Academic Press). Auristatin / dolastatin drug moieties, in some embodiments, can be prepared according to the methods of U.S. Pat. No. 7,498,298; U.S. Pat. No. 5,635,483; U.S. Pat. No. 5,780,588; Pettit et al., J. Am. Chem. Soc., vol. 111, pp. 5463-5465, 1998; Pettit et al., Anti-Cancer Drug Design, vol. 13, pp. 243-277, 1998; Pettit et al., Synthesis, vol. 6, pp. 719-725, 1996; Pettit et al., J. Chem. Soc. Perkin Trans., vol. 15, pp. 859-863, 1996; and Doronina, Nat. Biotechnol., vol. 21, pp. 778-784, 2003.

[0209] In some embodiments, the compound of formula D E auristatin / dolastatin drug moieties, e.g., MMAE, and D E For example, MMAF, and their drug-linker intermediates and derivatives, such as MC-MMAF, MC-MMAE, MC-vc-PAB-MMAF, and MC-vc-PAB-MMAE, can be prepared and then conjugated to an antibody of interest using methods described in U.S. Pat. No. 7,498,298; Doronina et al., Bioconjugate Chem., vol. 17, pp. 114-124, 2006; and Doronina et al., Nat. Biotech., vol. 21, pp. 778-784, 2003.

[0210] (3) Calicheamicin In some embodiments, the immunoconjugate comprises an antibody conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics and their analogs can produce double-stranded DNA breaks at subpicomolar concentrations (Hinman et al., Cancer Research, vol. 53, pp. 3336-3342, 1993; Lode et al., Cancer Research, vol. 58, pp. 2925-2928, 1998). Although calicheamicin has an intracellular site of action, in some instances it does not readily cross the cell membrane. Therefore, cellular uptake of these agents via antibody-mediated internalization can, in some embodiments, greatly enhance their cytotoxic effects. Non-limiting exemplary methods for preparing antibody-drug conjugates having a calicheamicin drug moiety are described, for example, in U.S. Pat. Nos. 5,712,374; 5,714,586; 5,739,116; and 5,767,285.

[0211] (4) Pyrrolobenzodiazepines In some embodiments, the ADC comprises a pyrrolobenzodiazepine (PBD). In some embodiments, the PBD dimer recognizes and binds to a specific DNA sequence. The natural product anthramycin, PBD, was first reported in 1965 (Leimgruber et al., J. Am. Chem. Soc., vol. 87, pp. 5793-5795, 1965; Leimgruber et al., J. Am. Chem. Soc., vol. 87, pp. 5791-5793, 1965). Since then, numerous PBDs, both naturally occurring and analogs, have been reported (Thurston et al., Chem. Rev. vol. 1994, pp. 433-465 1994), including dimers of tricyclic PBD scaffolds (U.S. Pat. Nos. 6,884,799; 7,049,311; 7,067,511; 7,265,105; 7,511,032; 7,528,126; 7,557,099). Without being bound by any particular theory, it is believed that the dimeric structure confers the proper three-dimensional shape for isohelicity with the minor groove of B-form DNA, resulting in a smooth fit at the binding site (Kohn, In Antibiotics III. Springer-Verlag, New York, pp. 3-11 (1975); Hurley and Needham-VanDevanter, Acc. Chem. Res., vol. 19, pp. 230-237, 1986). Dimeric PBD compounds bearing C2 aryl substituents have been shown to be useful as cytotoxic agents (Hartley et al. Cancer Res., vol. 70, pp. 6849-6858, 2010; Antonow, J. Med. Chem. vol. 53, pp. 2927-2941, 2010; Howard et al., Bioorganic and Med. Chem. Letters, vol. 19, pp. 6463-6466, 2009).

[0212] PBD dimers have been conjugated to antibodies, and the resulting ADCs have been shown to have anticancer properties. Non-limiting exemplary binding sites on PBD dimers include the five-membered pyrrolo ring, the tether between PBD units, and the N10-C11 imine group (WO 2009 / 016516; U.S. Patent Application Publication No. 2009 / 304710; U.S. Patent Application Publication No. 2010 / 047257; U.S. Patent Application Publication No. 2009 / 036431; U.S. Patent Application Publication No. 2011 / 0256157; WO 2011 / 130598).

[0213] A non-limiting exemplary PBD dimer component of an ADC has Formula A: [ka] and salts and solvates thereof, wherein The wavy line indicates the site of covalent attachment to the linker; The dotted lines indicate the optional presence of a double bond between C1 and C2 or between C2 and C3; R 2 are H, OH, =O, =CH2, CN, R, OR, =CH-R D , =C(R D )2, O—SO2—R, CO2R and COR, and optionally further selected from halo or dihalo; R D are independently selected from R, COR, COR, CHO, COH, and halo; R 6 and R 9 are independently selected from H, R, OH, OR, SH, SR, NH, NHR, NRR', NO, MeSn, and halo; R 7 are independently selected from H, R, OH, OR, SH, SR, NH, NHR, NRR', NO, MeSn, and halo; Q is independently selected from O, S, and NH; R 11 is either H or R, or when Q is O, SO3M, where M is a metal cation; R and R' are optionally substituted C1-8 alkyl, C1- 12 Alkyl, C3-8 heterocyclyl, C3- 20 Heterocycles, and C5~ 20 aryl groups, optionally in conjunction with the group NRR′, R and R′ together with the nitrogen atom to which they are attached form an optionally substituted 4-, 5-, 6-, or 7-membered heterocyclic ring; R 12 , R 16 , R 19 , and R 17 are R 2 , R 6 , R 9 , and R 7 as defined for R'' is C3~ 12 alkylene groups, the chain of which may be interrupted by one or more heteroatoms, such as O, S, N(H), NMe, and / or aromatic rings, such as benzene or pyridine, which rings are optionally substituted; X and X' are independently selected from O, S, and N(H).

[0214] In some embodiments, R and R′ are optionally substituted C1 12 Alkyl, C3~ 20 Heterocycles, and C5~ 20 aryl groups, and optionally in conjunction with the group NRR′, R and R′ together with the nitrogen atom to which they are attached form an optionally substituted 4-, 5-, 6-, or 7-membered heterocyclic ring. 9 and R 19 is H. In some embodiments, R 6 and R 16 is H.

[0215] In some embodiments, R 7 and R 17 are both OR 7A and R7A is an optionally substituted C1-4 alkyl. In some embodiments, R 7A is Me. In some embodiments, R 7A is ChPh, where Ph is a phenyl group. In some embodiments, X is O. In some embodiments, R 11 is H. In some embodiments, there is a double bond between C2 and C3 in each monomer unit.

[0216] In some embodiments, R 2 and R 12 is independently selected from H and R. In some embodiments, R 2 and R 12 is independently R. In some embodiments, R 2 and R 12 are independently an optionally substituted C 20 Aryl or C5-7 aryl or C8- 10 In some embodiments, R 2 and R 12 is independently an optionally substituted phenyl, thienyl, naphthyl, pyridyl, quinolinyl, or isoquinolinyl. 2 and R 12 are =O, =CH2, =CH-R D , and =C(R D )2. In some embodiments, R 2 and R 12 are each ═CH. In some embodiments, R 2 and R 12 are each H. In some embodiments, R 2 and R 12 are each ═O. In some embodiments, R 2 and R 12 are each ═CF. In some embodiments, R 2 and / or R 12 are independently = C(R D)2. In some embodiments, R 2 and / or R 12 are independently =CH-R D is.

[0217] In some embodiments, R 2 and / or R 12 =CH-R D When , each group can independently have any of the configurations shown below: [ka]

[0218] In some embodiments, =CH-R D is configuration (I). In some embodiments, R'' is a C3 alkylene group or a C5 alkylene group.

[0219] The PBD dimer-val-cit-PAB-Ab and PBD dimer-Phe-Lys-PAB-Ab linkers are protease-cleavable, while the PBD dimer-maleimide-acetal linker is acid-labile.

[0220] PBD dimers and ADCs containing PBD dimers can be prepared according to methods known in the art (see, for example, International Publication No. WO 2009 / 016516; U.S. Patent Application Publication No. 2009 / 304710; U.S. Patent Application Publication No. 2010 / 047257; U.S. Patent Application Publication No. 2009 / 036431; U.S. Patent Application Publication No. 2011 / 0256157; and International Publication No. WO 2011 / 130598).

[0221] (5) Anthracyclines In some embodiments, the ADC may comprise an anthracycline. An anthracycline is an antibiotic compound that exhibits cytotoxic activity. Without being bound by any particular theory, research has shown that anthracyclines can function to kill cells through a number of different mechanisms, for example, 1) intercalation of drug molecules into cellular DNA, thereby inhibiting DNA-dependent nucleic acid synthesis; 2) drug-induced production of free radicals that then react with cellular macromolecules to cause cell damage; and / or 3) interaction of drug molecules with cell membranes (see, for example, C. Peterson et al., "Transport and Storage of Anthracycline in Experimental Systems and Human Leukemia" in Anthracycline Antibiotics in Cancer Therapy; N.R. Bachur, "Free Radical Damage" id. at pp. 97-102). Due to their cytotoxic potential, anthracyclines are used in the treatment of many cancers, including leukemia, breast cancer, lung cancer, ovarian adenocarcinoma, and sarcoma (see, e.g., P.H. Wiernik, in Anthracycline: Current Status And New Developments, p11).

[0222] Non-limiting exemplary anthracyclines include doxorubicin, epirubicin, idarubicin, daunomycin, nemorubicin, and derivatives thereof. Immunoconjugates and prodrugs of daunorubicin and doxorubicin have been prepared and studied (Kratz et al., Current Med. Chem., vol. 13, pp. 477-523, 2006; Jeffrey et al., Bioorganic & Med. Chem. Letters, vol. 16, pp. 358-362, 1996; Torgov et al., Bioconj. Chem., vol. 16, pp. 717-721, 2005; Nagy et al., Proc. Natl. Acad. Sci. USA, vol. 97, pp. 829-834, 2000; Dubowchik et al., Bioorg. & Med. Chem. Letters, vol. 12, pp. 1529-1532, 2002; King et al.). (e.g., J. Med. Chem., vol. 45, pp. 4336-4343, 2002; EP 0328147; U.S. Pat. No. 6,630,579). The antibody-drug conjugate BR96-doxorubicin, which specifically reacts with the tumor-associated antigen Lewis-Y, has been evaluated in phase I and phase II trials (Saleh et al., J. Clin. Oncology, vol. 18, pp. 2282-2292, 2000; Ajani et al., Cancer Jour., vol. 6, pp. 78-81, 2000; Tolcher et al., J. Clin. Oncology, vol. 17, pp. 478-484, 1999).

[0223] PNU-159682 is a potent metabolite (or derivative) of nemorubicin (Quintieri et al., Clinical Cancer Research, vol. 11, pp. 1608-1617, 2005). Nemorubicin is a semisynthetic analog of doxorubicin with a 2-methoxymorpholino group on the glycosidic amino group of doxorubicin, and is currently undergoing clinical evaluation (Grandi et al., Cancer Treat. Rev., vol. 17, pp. 133-138, 1990; Ripamonti et al., Brit. J. Cancer, vol. 65, pp. 703-707, 1992), including Phase II / Phase III clinical trials (Sun et al., Proceedings of the American Society for Clinical Oncology, vol. 22, Abs. 1448, 2003; Quintieri, Proceedings of the American Association of Cancer Research, vol. 44:1st Ed., Abs. 4649, 2003; Pacciarini et al., Jour. Clin. Oncology, vol. 24, p. 14116, 2006).

[0224] Anthracyclines, such as PNU-159682, can be conjugated to antibodies via several binding sites and a variety of linkers (U.S. Patent Application Publication No. 2011 / 0076287; WO 2009 / 099741; U.S. Patent Application Publication No. 2010 / 0034837; WO 2010 / 009124), including those described herein.

[0225] The linker of PNU-159682-maleimide acetal-Ab is acid-labile, while PNU-159682-val-cit-PAB-Ab, PNU-159682-val-cit-PAB-spacer-Ab, and PNU-159682-val-cit-PAB-spacer (R 1 R 2The linker in the )-Ab is protease cleavable.

[0226] (6) Other drug moieties Drug moieties include geldanamycin (Mandler et al., J. Nat. Cancer Inst., vol. 92, pp. 1573-1581, 2000; Mandler et al., Bioorganic & Med. Chem. Letters, vol. 10, pp. 1025-1028, 2000; Mandler et al., Bioconjugate Chem., vol. 13, pp. 786-791, 2002); and enzymatically active toxins and fragments thereof, including, but not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana (Phytolacca Also included are Momordica americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and trichothecenes. See, e.g., WO 93 / 21232.

[0227] Drug moieties also include compounds with nucleolytic activity (eg, ribonucleases or DNA endonucleases).

[0228] In certain embodiments, the immunoconjugate may contain a highly radioactive atom. A variety of radioisotopes are available for the production of radioconjugated antibodies. Examples include At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212, P 32 , Pb 212 In some embodiments, when the immunoconjugate is used for detection, it can be used with a radioactive atom for scintigraphy studies, e.g., Tc 99 Or I 123 or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging (MRI)), such as zirconium-89, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron. For example, zirconium-89 can be complexed to various metal chelators and conjugated to antibodies for PET imaging (WO 2011 / 056983).

[0229] Radiolabels or other labels can be incorporated into the immunoconjugate using known techniques. For example, peptides can be biosynthesized or chemically synthesized using suitable amino acid precursors, e.g., containing one or more fluorine-19 atoms in place of one or more hydrogen atoms. In some embodiments, the label, e.g., Tc 99 , I 123 ,Re 186 ,Re 188 , and In 111 can be attached via a cysteine ​​residue in the antibody. In some embodiments, yttrium-90 can be attached via a lysine residue in the antibody. In some embodiments, iodine-123 can be incorporated using the IODOGEN method (Fraker et al., Biochem. Biophys. Res. Commun., vol. 80, pp. 49-57, 1978). "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes certain other methods.

[0230] In certain embodiments, an immunoconjugate may comprise an antibody conjugated to a prodrug-activating enzyme. In some such embodiments, the prodrug-activating enzyme converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see WO 81 / 01145) into an active drug, e.g., an anticancer drug. Such immunoconjugates are, in some embodiments, useful in antibody-dependent enzyme-mediated prodrug therapy ("ADEPT"). Enzymes that can be conjugated to an antibody include, but are not limited to, alkaline phosphatase, useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase, useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase, useful for converting non-toxic 5-fluorocytosine into the anticancer drug 5-fluorouracil; and proteases, such as Serratia protease, thermolysin, subtilisin, carboxypeptidase, and cathepsins (e.g., cathepsins B and L), useful for converting peptide-containing prodrugs into free drugs. D-alanylcarboxypeptidase, which is useful for converting prodrugs containing D-amino acid substituents; carbohydrate-cleaving enzymes, such as β-galactosidase and neuraminidase, which are useful for converting glycosylated prodrugs into free drugs; β-lactamase, which is useful for converting drugs that are derivatized with β-lactams into free drugs; and penicillin amidases, such as penicillin V amidase and penicillin G amidase, which are useful for converting drugs that are derivatized with phenoxyacetyl or phenylacetyl groups at the amine nitrogen into free drugs. In some embodiments, enzymes can be covalently bound to antibodies by recombinant DNA techniques well known in the art. For example, see Neuberger et al., Nature, vol. 312, pp. 604-608, 1984.

[0231] c) Drug Load Drug loading is represented by the average number of drug moieties per antibody in a molecule of Formula I. Drug loading can range from 1 to 20 drug moieties (D) per antibody. ADCs of Formula I contain a population of antibodies conjugated with drug moieties ranging from 1 to 20. The average number of drug moieties per antibody used in ADC preparations from conjugation reactions can be characterized by conventional means, such as mass spectrometry, ELISA assays, and HPLC. Quantitative distribution of ADCs in units of p can also be measured. In some cases, separation, purification, and characterization of homogeneous ADCs with a certain value of p from ADCs with other drug loads can be achieved by means such as reverse-phase HPLC or electrophoresis.

[0232] For some antibody-drug conjugates, p may be limited by the number of attachment sites on the antibody. For example, if the attachment is a cysteine ​​thiol, as in certain exemplary embodiments described above, the antibody may have only one or a few cysteine ​​thiol groups, or only one or a few sufficiently reactive thiol groups to allow for the attachment of a linker. In certain embodiments, higher drug loading, e.g., p>5, may cause aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates. In certain embodiments, the average drug loading for an ADC ranges from 1 to about 8; from about 2 to about 6; or from about 3 to about 5. Indeed, it has been shown that for certain ADCs, the optimal ratio of drug moieties per antibody may be less than 8, from about 2 to about 5 (U.S. Patent No. 7,498,298).

[0233] In some embodiments, fewer than the theoretical maximum number of drug moieties are conjugated to the antibody during the conjugation reaction.As discussed below, the antibody may contain, for example, lysine residues that do not react with either the drug-linker intermediate or the linker reagent.Generally, antibodies do not contain many free and reactive cysteine ​​thiol groups that can be bound to drug moieties; in fact, most cysteine ​​thiol residues in antibodies exist as disulfide bridges.In some embodiments, the antibody can be reduced under partial or complete reducing conditions with a reducing agent, such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), to generate reactive cysteine ​​thiol groups.In some embodiments, the antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups, such as lysine or cysteine.

[0234] The loading (drug / antibody ratio) of ADCs can be controlled in various ways, for example, by (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limited reduction conditions for cysteine ​​thiol modification.

[0235] When two or more nucleophilic groups react with a drug-linker intermediate or linker reagent, it is understood that the resulting product is a mixture of ADCs with a distribution of one or more drug moieties attached to the antibody. The average number of drugs per antibody can be calculated from the mixture by a double ELISA antibody assay, which is specific for both the antibody and the drug. Individual ADCs can be identified in the mixture by mass spectrometry and separated by HPLC, for example, hydrophobic interaction chromatography (see, for example, McDonagh et al., Prot.Engr.Design & Selection, vol.19, pp.299-307, 2006; Hamblett et al., Clin.Cancer Res., vol.10, pp.7063-7070, 2004). In some embodiments, homogeneous ADCs with a single loading value can be isolated from the conjugation mixture by electrophoresis or chromatography.

[0236] d) Certain methods for preparing immunoconjugates Immunoconjugates that are ADCs of Formula I can be prepared by several routes using organic chemistry reactions, conditions, and reagents known to those skilled in the art, such as, for example, (1) reacting a nucleophilic group on an antibody with a bivalent linker reagent to form Ab-L via a covalent bond, followed by reaction with a drug moiety, D; and (2) reacting a nucleophilic group on a drug moiety with a bivalent linker reagent to form DL via a covalent bond, followed by reaction with a nucleophilic group on an antibody. An exemplary method for preparing ADCs of Formula I via the latter route is described in U.S. Pat. No. 7,498,298.

[0237] Nucleophilic groups on antibodies include, but are not limited to, (i) N-terminal amine groups, (ii) side-chain amine groups, such as lysine, (iii) side-chain thiol groups, such as cysteine, and (iv) sugar hydroxyl or amino groups on glycosylated antibodies. Amine, thiol, and hydroxyl groups are nucleophilic and can react with electrophilic groups on linker moieties and linker reagents to form covalent bonds, such as (i) active esters, such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides, such as haloacetamides; and (iii) aldehyde, ketone, carboxyl, and maleimide groups. Some antibodies have reducible interchain disulfides, i.e., cysteine ​​bridges. Antibodies can be made reactive for conjugation with linker reagents by treatment with a reducing agent, such as DTT (dithiothreitol) or tricarbonylethylphosphine (TCEP), to completely or partially reduce the antibody. Thus, each cysteine ​​bridge theoretically forms two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies through modification of lysine residues, for example, by reacting the lysine residue with 2-iminothiolane (Traut's reagent) to convert the amine to a thiol. Reactive thiol groups can also be introduced into antibodies by introducing one, two, three, four, or more cysteine ​​residues (e.g., by preparing a variant antibody containing one or more non-natural cysteine ​​amino acid residues).

[0238] The antibody-drug conjugates of the present invention can also be produced by the reaction between an electrophilic group on an antibody, such as an aldehyde or ketone carbonyl group, and a nucleophilic group on a linker reagent or drug. Useful nucleophilic groups on a linker reagent include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. In one embodiment, an antibody is modified to introduce an electrophilic moiety that can react with a nucleophilic substituent on a linker reagent or drug. In another embodiment, the sugars of a glycosylated antibody can be oxidized, for example, with a periodate oxidation reagent, to form aldehyde or ketone groups that can react with amine groups on a linker reagent or drug moiety. The resulting imine Schiff base groups can form stable bonds or can be reduced, for example, with a borohydride reagent, to form stable amine bonds. In one embodiment, the reaction of the carbohydrate moiety of a glycosylated antibody with either galactose oxidase or sodium metaperiodate can generate carbonyl (aldehyde and ketone) groups in the antibody that can react with appropriate groups on a drug (Hermanson, Bioconjugate Techniques). In another embodiment, an antibody containing an N-terminal serine or threonine residue can be reacted with sodium metaperiodate to generate an aldehyde in place of the first amino acid (Geoghegan & Stroh, Bioconjugate Chem., vol. 3, pp. 138-146, 1992; U.S. Patent No. 5,362,852). Such aldehydes can react with drug moieties or linker nucleophiles.

[0239] Exemplary nucleophilic groups on drug moieties include, but are not limited to, electrophilic groups on linker moieties and linker reagents, such as (i) active esters, e.g., NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides, e.g., haloacetamides; and (iii) amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups that can react with aldehyde, ketone, carboxyl, and maleimide groups to form covalent bonds.

[0240] Non-limiting exemplary cross-linking reagents that can be used to prepare ADCs are described in the section entitled "Exemplary Linkers" herein. Methods for linking two moieties, such as a proteinaceous moiety and a chemical moiety, using such cross-linking reagents are known in the art. In some embodiments, a fusion protein comprising an antibody and a cytotoxic agent can be produced, for example, by recombinant technology or peptide synthesis. The recombinant DNA molecule can contain regions encoding the antibody and cytotoxic moieties of the conjugate that are adjacent to each other or separated by a region encoding a linker peptide that does not destroy the desired properties of the conjugate.

[0241] In yet another embodiment, the antibody can be conjugated to a "receptor" (e.g., streptavidin) for use in tumor pretargeting, in which the antibody-receptor conjugate is administered to a patient, followed by removal of unbound conjugate from the circulation using a clearing agent, and then administration of a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a drug or radionucleotide).

[0242] C. Methods and Compositions for Diagnostics and Detection In some embodiments, any of the anti-Axl antibodies or antibody fragments provided herein can be used to detect the presence of Axl in a biological sample. As used herein, the term "detecting" encompasses quantitative or qualitative detection. In some embodiments, the biological sample contains cells or tissues, such as breast, pancreatic, esophageal, lung, and / or brain cells or tissues.

[0243] A further aspect of the invention relates to an anti-Axl antibody of the invention for diagnosing and / or monitoring cancer or another disease in which Axl levels are increased or decreased from normal physiological levels in at least one location in the body.

[0244] In a preferred embodiment, the antibody or antibody fragment of the present invention can be labeled with a detectable molecule or substance, such as the above-mentioned fluorescent molecule, radioactive molecule, or any other label known in the art. For example, the antibody of the present invention can be labeled with a radioactive molecule. For example, suitable radioactive molecules include, but are not limited to, radioactive atoms used in scintigraphy tests, such as 123 I, 124 I, 111 In, 186 Re, and 188 The antibody or antibody fragment of the present invention can also be labeled with a spin label for nuclear magnetic resonance (NMR) imaging, such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron. After administration of the antibody, the distribution of the radiolabeled antibody within the patient is detected. Any suitable known method can be used. Some non-limiting examples include computed tomography (CT), positron emission tomography (PET), magnetic resonance imaging (MRI), fluorescence, chemiluminescence, and ultrasound diagnosis.

[0245] The antibodies or antibody fragments of the present invention may be useful for diagnosing and staging cancers and diseases associated with Axl overexpression, including squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer, glial cell tumors such as glioblastoma and neurofibromatosis, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, melanoma, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatoma, sarcoma, blood cancer (leukemia), astrocytoma, and various types of head and neck cancer or other hyperproliferative diseases of Axl expression or overexpression.

[0246] The antibodies or antibody fragments of the present invention may be useful for diagnosing diseases other than cancer in which Axl expression is increased or decreased. (Both soluble and cellular forms of Axl can be used for such diagnosis. Typically, such diagnostic methods involve the use of a biological sample obtained from a patient. As used herein, the term "biological sample" encompasses various sample types obtained from a subject that can be used in diagnostic or monitoring assays. Biological samples include, but are not limited to, blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom, and their progeny. For example, biological samples include cells obtained from tissue samples recovered from individuals suspected of having a cancer associated with Axl overexpression, and in preferred embodiments, glioma, gastric cancer, lung cancer, pancreatic cancer, breast cancer, prostate cancer, kidney cancer, liver cancer, and endometrial cancer. Biological samples include clinical samples, cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples.

[0247] In a particular embodiment, the invention is a method for diagnosing cancer associated with Axl overexpression in a subject by detecting Axl on cells from the subject using an antibody of the invention. In particular, the method comprises: (a) contacting a biological sample of a subject with an antibody or antibody fragment according to the invention under conditions suitable for the antibody or antibody fragment to form a complex with cells of the biological sample that express Axl; and (b) detecting and / or quantifying said complex, wherein detection of said complex is indicative of a cancer associated with Axl overexpression. may include:

[0248] To monitor the progression of cancer, the method can be repeated at different time points to determine whether antibody binding to the sample increases or decreases, and then it can be determined whether the cancer is progressing, regressing, or stable.

[0249] In a specific embodiment, the present invention is a method for diagnosing diseases associated with expression or overexpression of Axl or a decrease or increase in the soluble form of Axl, such as human immune disorders, thrombotic diseases (thrombosis and atherothrombosis), and cardiovascular diseases.

[0250] In one embodiment, an anti-Axl antibody or antibody fragment is provided for use in a diagnostic or detection method. In a further aspect, a method for detecting the presence of Axl in a biological sample is provided. In a further aspect, a method for quantifying the amount of Axl in a biological sample is provided. In certain embodiments, the method comprises contacting a biological sample with an anti-Axl antibody or antibody fragment described herein under conditions that allow binding of the anti-Axl antibody or antibody fragment to Axl, and detecting whether a complex is formed between the anti-Axl antibody or antibody fragment and Axl. Such methods can be performed in vitro or in vivo. In one embodiment, the anti-Axl antibody or antibody fragment is used to select subjects eligible for a therapy. In some embodiments, the therapy comprises administering an anti-Axl antibody or antibody fragment to a subject.

[0251] In one embodiment, a labeled anti-Axl antibody or antibody fragment is provided. Labels include, but are not limited to, labels or moieties that are directly detected (e.g., fluorescent labels, chromophores, electron-dense labels, chemiluminescent labels, and radioactive labels), and moieties such as enzymes or ligands that are indirectly detected, e.g., via enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes. 32 P, 14 C. 125 I, 3 H, and 131 I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, enzymes that oxidize dye precursors using hydrogen peroxide, such as heterocyclic oxidases coupled with HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0252] D. Pharmaceutical Formulations Anti-Axl antibodies or antibody fragments have cell-killing activity. This cell-killing activity extends to multiple different cell lineage types. Furthermore, when conjugated to a cytotoxic agent, these antigens or antibody fragments can reduce tumor size and exhibit reduced toxicity. See Examples 3 and 6-9 of the present application. Thus, anti-Axl antibodies, their fragments, or immunoconjugates can be useful for treating proliferative disorders associated with Axl expression. The antibodies, fragments, or immunoconjugates can be used alone or in combination with any suitable agent or other conventional treatment.

[0253] Anti-Axl antibodies or antibody fragments can be used to treat hyperproliferative diseases associated with Axl and / or Gas6 expression, overexpression, or activation. There are no specific limitations on the type or tissue of cancer that can be treated, other than the requirement for Axl expression. Examples include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer, glial cell tumors (e.g., glioblastoma and neurofibromatosis), cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, melanoma, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatoma, sarcoma, blood cancer (leukemia), astrocytoma, and various types of head and neck cancer. More preferred cancers are glioblastoma, gastric cancer, lung cancer, pancreatic cancer, breast cancer, prostate cancer, kidney cancer, liver cancer, and endometrial cancer.

[0254] Anti-Axl antibodies or antibody fragments are potent activators of the innate immune response and can therefore be used to treat human immune disorders, such as sepsis. The anti-Axl antibodies or antibody fragments of the present invention can also be used as adjuvants for immunization, such as vaccines, and as anti-infective agents against, for example, bacteria, viruses, and parasites.

[0255] Anti-Axl antibodies or antibody fragments can be used to protect against, prevent, or treat thrombotic diseases, such as venous and arterial thrombosis and atherothrombosis. Anti-Axl antibodies or antibody fragments can also be used to protect against, prevent, or treat cardiovascular diseases, as well as to prevent or inhibit the invasion of viruses, such as Lassa and Ebola viruses, and to treat viral infections.

[0256] In each of the embodiments of the therapeutic methods described herein, the anti-Axl monoclonal antibody, antibody fragment, or anti-Axl monoclonal immunoconjugate can be delivered in a manner consistent with conventional methods associated with the management of the disease or disorder for which treatment is sought. In accordance with the disclosure herein, an effective amount of the antibody, antibody fragment, or immunoconjugate is administered to a subject in need of such treatment for a period of time and under conditions sufficient to prevent or treat the disease or disorder. Accordingly, one aspect of the present invention relates to a method of treating a disease associated with Axl expression, comprising administering a therapeutically effective amount of an antibody, antibody fragment, or immunoconjugate of the present invention to a subject in need of treatment for the disease associated with Axl expression.

[0257] For administration, anti-Axl monoclonal antibodies, antibody fragments, or immunoconjugates can be formulated as pharmaceutical compositions. Pharmaceutical compositions containing anti-Axl monoclonal antibodies, antibody fragments, or antibody-drug conjugates can be formulated according to known methods for preparing pharmaceutical compositions. In such methods, therapeutic molecules are typically combined with a mixture, solution, or composition containing a pharmaceutically acceptable carrier.

[0258] A pharmaceutically acceptable carrier is a material that can be tolerated by a recipient patient. Sterile phosphate-buffered saline is an example of a pharmaceutically acceptable carrier. Other suitable pharmaceutically acceptable carriers are well known to those skilled in the art (see, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences (Mack Publishing Company, 19th ed. 1995)). The formulation may further include one or more excipients, preservatives, stabilizers, buffers, albumin to prevent protein loss on the surface of the vial, etc.

[0259] The form, route of administration, dosage, and regimen of a pharmaceutical composition usually depend on the condition to be treated, the severity of the disease, the age, weight, and sex of the patient, etc. These considerations can be taken into account by those skilled in the art to formulate an appropriate pharmaceutical composition. The pharmaceutical composition of the present invention can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration, etc.

[0260] Preferably, the pharmaceutical composition contains a pharmaceutically acceptable vehicle for the formulation to be injected. These may be present in a particular isotonic sterile saline solution (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, etc. or a mixture of such salts), or in a dried, especially lyophilized, composition that allows the constitution of an injectable solution upon addition of, for example, sterile water or saline.

[0261] In some embodiments, tonicity agents, sometimes known as "stabilizers," are present to adjust or maintain the isotonicity of the liquid in the composition. When used with large charged biomolecules, such as proteins and antibodies, they are often referred to as "stabilizers" because they can interact with the charged groups on amino acid side chains, thereby reducing the potential for inter- and intramolecular interactions. Tonicity agents can be present in any amount from 0.1% to 25% by weight of the pharmaceutical composition, preferably 1-5%. Preferred tonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0262] Additional excipients include agents that can function as one or more of the following: (1) bulking agents, (2) solubility enhancers, (3) stabilizers, and (4) agents that prevent denaturation or adhesion to container walls. Such excipients include polyhydric sugar alcohols (listed above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, and the like; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), and the like. ), polyethylene glycol; sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose); disaccharides (e.g., lactose, maltose, sucrose); trisaccharides (e.g., raffinose); and polysaccharides (e.g., dextrin or dextran).

[0263] Non-ionic surfactants or detergents (also known as "wetting agents") can be used to help dissolve the therapeutic agent and protect the therapeutic protein from agitation-induced aggregation, thereby allowing the formulation to be exposed to shear surface stresses without causing denaturation of the active therapeutic protein or antibody. The non-ionic surfactant may be present in a concentration range of about 0.05 mg / ml to about 1.0 mg / ml, preferably about 0.07 mg / ml to about 0.2 mg / ml.

[0264] Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (184, 188, etc.), PLURONIC® polyol, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glycerol monostearate, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0265] The dose used for administration can be adapted depending on various parameters, in particular depending on the mode of administration used, the pathology involved, or alternatively the desired duration of treatment. To prepare a pharmaceutical composition, an effective amount of the antibody or antibody fragment can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0266] Suitable dosage forms for injection use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.In all cases, the dosage form must be sterile and fluid to the extent that easy syringability exists.It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0267] The solution of active compound as free base or pharmacologically acceptable salt can be prepared in the water suitably mixed with surfactant.Dispersion can also be prepared in glycerol, liquid polyethylene glycol and its mixture and in oil.Under normal storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms.

[0268] The antibody or antibody fragment can be formulated into a composition in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein), which are formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases such as potassium, ammonium, calcium, or iron hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0269] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of injectable compositions can be achieved by using agents that delay absorption, for example, aluminum monostearate and gelatin, in the composition.

[0270] Sterile injectable solution is prepared by incorporating the required amount of active compound into a suitable solvent, if necessary, with one or more of the other ingredients listed above, and then sterile filtered.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and the other ingredients required from those listed above.For the preparation of sterile powder for sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying technology, which produces a powder of active ingredient and any additional desired ingredients from its solution that has been previously sterile filtered.

[0271] Preparation of more concentrated or highly concentrated solutions for direct injection is also contemplated, and the use of dimethyl sulfoxide (DMSO) as a solvent is expected to result in extremely rapid penetration, delivering high concentrations of active agent to small tumor areas.

[0272] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be used.

[0273] For parenteral administration in aqueous solution, for example, the solution should be suitably buffered if necessary, and the liquid diluent should first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that can be used will be known to those skilled in the art in light of the present disclosure. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion therapy fluid or injected at the proposed infusion site (see, e.g., "Remington's Pharmaceutical Sciences," 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0274] The antibody or antibody fragment can be formulated to deliver 0.0001 to 10.0 milligrams, or about 0.001 to 5 milligrams, or about 0.001 to 1 milligram, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose in a therapeutic mixture. Multiple doses can also be administered at selected time intervals.

[0275] In addition to compounds formulated for parenteral administration, e.g., intravenous or intramuscular injection, other pharmaceutically acceptable forms include, for example, tablets or other solid forms for oral administration; sustained-release capsules; and any other form currently in use.

[0276] In certain embodiments, the use of liposomes and / or nanoparticles is contemplated for the introduction of antibodies or antibody fragments into host cells. The formation and use of liposomes and / or nanoparticles is known to those of skill in the art.

[0277] Nanocapsules can generally entrap compounds in a stable and reproducible manner.To avoid side effects caused by intracellular polymer overloading, these ultrafine particles (size is about 0.1 μm) are generally designed using polymers that can degrade in vivo.Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are intended for use in the present invention, and these particles can be easily produced.

[0278] Liposomes are formed from phospholipids dispersed in an aqueous medium, spontaneously forming multilamellar concentric bilayer vesicles (also called multilamellar vesicles (MLVs)). MLVs generally have diameters between 25 nm and 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters ranging from 200 to 500 Å, which contain aqueous solution in their cores. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0279] Pharmaceutical formulations containing the anti-Axl antibodies or antibody fragments described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing such antibodies or antibody fragments having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (approximately 10 residues) soluble or unsaturated fatty acids; and the like. (less than 100%) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0280] Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, e.g., rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, e.g., rHuPH20, are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, a sHASEGP is combined with one or more additional glycosaminoglycanases, e.g., chondroitinases.

[0281] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0282] The formulations herein may also contain two or more active ingredients, as needed for the particular indication being treated. Preferably, ingredients with complementary activities that do not adversely affect each other can be combined in a single formulation. For example, it may be desirable to provide an EGFR antagonist (e.g., erlotinib), an anti-androgen (e.g., a VEGF antagonist, which may be an anti-VEGF antibody), or a chemotherapeutic agent (e.g., a taxoid or platinum agent) in addition to the anti-Axl antibody, antibody fragment, or immunoconjugate of the present invention. Such active ingredients are suitably present in a combination in amounts that are effective for the intended purpose.

[0283] Active ingredients can be encapsulated in microcapsules, for example, by coacervation technology or prepared by interfacial polymerization.For example, hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules can be used in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions, respectively.This technology is disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980).

[0284] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody or antibody fragment, which matrices may be in the form of shaped articles, e.g., films, or microcapsules.

[0285] Formulations to be used for in vivo administration are generally sterile. Sterility is readily accomplished, for example, by filtration through sterile filtration membranes.

[0286] E. Therapeutic Methods and Compositions Any of the anti-Axl antibodies or antibody fragments provided herein can be used in therapeutic methods. In one aspect, an anti-Axl antibody or antibody fragment is provided for use as a pharmaceutical. In a further aspect, an anti-Axl antibody or antibody fragment is provided for use in the treatment of cancer (e.g., breast cancer, non-small cell lung cancer, pancreatic cancer, brain tumor, pancreatic, brain, kidney, ovarian, stomach, leukemia, endometrial, colon, prostate, thyroid, liver cancer, osteosarcoma, and / or melanoma). In certain embodiments, an anti-Axl antibody or antibody fragment is provided for use in a therapeutic method. In certain embodiments, the present invention provides an anti-Axl antibody or antibody fragment for use in a method of treating an individual with cancer, comprising administering to the individual an effective amount of an anti-Axl antibody or antibody fragment. In certain embodiments, the present invention provides an anti-Axl antibody or antibody fragment for use in a method for treating an individual having an immune disorder (e.g., an autoimmune disorder), a cardiovascular disorder (e.g., atherosclerosis, hypertension, thrombosis), an infectious disease (e.g., Ebola virus, Marburg virus), or diabetes, comprising administering to the individual an effective amount of an anti-Axl antibody or antibody fragment. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as one described below. In a further embodiment, the present invention provides an anti-Axl antibody or antibody fragment for use in inhibiting angiogenesis, inhibiting cell proliferation, inhibiting immune function, inhibiting inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibiting tumor vasculature (e.g., intratumoral vasculature or tumor-associated vasculature), and / or inhibiting tumor stromal function.

[0287] In certain embodiments, the invention provides an anti-Axl antibody or antibody fragment for use in a method of inhibiting angiogenesis, inhibiting cell proliferation, inhibiting immune function, inhibiting inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibiting tumor vasculature (e.g., intratumor or tumor-associated vasculature), and / or inhibiting tumor stromal function in an individual, comprising administering to the individual an effective anti-Axl antibody or antibody fragment to inhibit angiogenesis, inhibit cell proliferation, inhibit immune function, inhibit inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibit tumor vasculature development (e.g., intratumor or tumor-associated vasculature), and / or inhibit tumor stromal function. An "individual" according to any of the above embodiments is preferably a human.

[0288] In a further aspect, the present invention provides use of an anti-Axl antibody or antibody fragment in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of cancer (in some embodiments, breast cancer, non-small cell lung cancer, pancreatic cancer, brain tumor, pancreatic, brain, kidney, ovarian, stomach, leukemia, endometrial, colon, prostate, thyroid, liver cancer, osteosarcoma, and / or melanoma). In a further embodiment, the medicament is used in a method of treating cancer, comprising administering an effective amount of the medicament to an individual having cancer. In a further embodiment, the medicament is used in a method of treating an immune disorder (e.g., an autoimmune disorder), a cardiovascular disorder (e.g., atherosclerosis, hypertension, thrombosis), an infectious disease (e.g., Ebola virus, Marburg virus), or diabetes, comprising administering an effective amount of an anti-Axl antibody or antibody fragment to the individual. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, for example, In further embodiments, the medicament is for inhibiting angiogenesis, inhibiting cell proliferation, inhibiting immune function, inhibiting inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibiting tumor vasculature (e.g., intratumor vasculature or tumor-associated vasculature), and / or inhibiting tumor stromal function. In further embodiments, the medicament is used in a method for inhibiting angiogenesis, inhibiting cell proliferation, inhibiting immune function, inhibiting inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibiting tumor vasculature (e.g., intratumor vasculature or tumor-associated vasculature), and / or inhibiting tumor stromal function in an individual, comprising administering an effective amount of the medicament to the individual to inhibit angiogenesis, inhibit cell proliferation, inhibit immune function, induce inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibit tumor vasculature development (e.g., intratumor vasculature or tumor-associated vasculature), and / or inhibit tumor stromal function. An "individual" according to any of the above embodiments may be a human.

[0289] In a further aspect, the present invention provides a method of treating cancer. In one embodiment, the method comprises administering to an individual having such cancer an effective amount of an anti-Axl antibody or antibody fragment. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent described below. An "individual" according to any of the above embodiments may be a human.

[0290] In a further aspect, the present invention provides a method of treating an immune disorder (e.g., an autoimmune disorder), a cardiovascular disorder (e.g., atherosclerosis, hypertension, thrombosis), an infectious disease (e.g., Ebola virus, Marburg virus), or diabetes. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent described below. An "individual" according to any of the above embodiments may be a human.

[0291] In a further aspect, the present invention provides methods for inhibiting angiogenesis, inhibiting cell proliferation, inhibiting immune function, inhibiting inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibiting tumor vasculature (e.g., intratumor vasculature or tumor-associated vasculature), and / or inhibiting tumor stromal function in an individual. In one embodiment, the method comprises administering to the individual an effective amount of an anti-Axl antibody or antibody fragment to inhibit angiogenesis, inhibit cell proliferation, promote immune function, induce inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibit tumor vasculature development (e.g., intratumor vasculature or tumor-associated vasculature), and / or inhibit tumor stromal function. In one embodiment, the "individual" is a human.

[0292] In a further aspect, the present invention provides pharmaceutical formulations comprising any of the anti-Axl antibodies or antibody fragments provided herein, e.g., for use in any of the above-described therapeutic methods. In one embodiment, the pharmaceutical formulation comprises any of the anti-Axl antibodies or antibody fragments provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the anti-Axl antibodies or antibody fragments provided herein and at least one additional therapeutic agent, e.g., as described below.

[0293] In each and all of the above treatments, the antibody or antibody fragment of the present invention can be used alone, as an immunoconjugate, or in combination with other agents in the therapy. For example, the antibody of the present invention can be co-administered with at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is an anti-angiogenic agent. In certain embodiments, the additional therapeutic agent is a VEGF antagonist (in some embodiments, an anti-VEGF antibody, e.g., bevacizumab). In certain embodiments, the additional therapeutic agent is an EGFR antagonist (in some embodiments, erlotinib). In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent and / or a cytostatic agent. In certain embodiments, the additional therapeutic agent is a taxoid (e.g., paclitaxel) and / or a platinum agent (e.g., carboplatinum). In certain embodiments, the additional therapeutic agent is an agent that enhances the patient's immunity or immune system.

[0294] Such combination therapy as described above encompasses combined administration (two or more therapeutic agents in the same or separate formulations) and separate administration, in which the antibody or antibody fragment can be administered before, simultaneously with, and / or after the administration of the additional therapeutic agent and / or adjuvant. The antibody or antibody fragment can also be used in combination with radiation therapy.

[0295] The antibody or antibody fragment can be formulated, dosed, and administered in a manner consistent with good medical practice. Relevant factors to consider include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the scheduling of administration, and other factors known to physicians. The antibody or antibody fragment is optionally, but need not be, formulated with one or more agents currently used to prevent or treat the disorder. The effective amount of such other agents depends on the amount of antibody or antibody fragment in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and by the same routes of administration as those described herein, or at about 1-99% of the dosages described herein, or at any dosage and by any route deemed experimentally / clinically appropriate.

[0296] For disease prevention or treatment, the appropriate dosage of an antibody or antibody fragment (when used alone or in combination with one or more other additional therapeutic agents) depends on the type of disease being treated, the type of antibody or antibody fragment, the severity and course of the disease, whether the antibody or antibody fragment is being administered for prophylactic or therapeutic purposes, previous treatments, the patient's medical history and response to the antibody or antibody fragment, and the discretion of the attending physician. The antibody or antibody fragment is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, for example, about 1 μg / kg to 40 mg / kg of the antibody or antibody fragment may be an initial candidate dosage for administration to a patient, whether by one or more separate administrations or by continuous infusion. A typical daily dosage may range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administration over several days or longer, depending on the condition, treatment is generally sustained until a desired suppression of disease symptoms occurs. Such doses can be administered intermittently, for example, weekly or every three weeks (e.g., so that the patient receives from about 2 to about 20, or, for example, about 6, doses of the antibody or antibody fragment). A higher initial loading dose, followed by one or more lower doses, can be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0297] It will be understood that any of the above formulations or methods of treatment can be practiced using antibody fragments or immunoconjugates of the invention in place of, or in addition to, anti-Axl antibodies.

[0298] Improving host immune function to eradicate tumors is a topic of increasing interest. Conventional methods include (i) APC enhancement, such as (a) injecting DNA encoding foreign MHC alloantigens into tumors, or (b) transfecting biopsy-collected tumor cells with genes that increase the probability of tumor immune antigen recognition (e.g., immunostimulatory cytokines, GM-CSF, costimulatory molecules B7.1, B7.2), and (iii) adoptive cellular immunotherapy, or treatment with activated tumor-specific T cells. Adoptive cellular immunotherapy involves isolating tumor-infiltrating host T lymphocytes and expanding their population in vitro, for example, through stimulation with IL-2, tumor, or both. Furthermore, isolated dysfunctional T cells can also be activated by in vitro application of anti-PD-L1 antibodies. The activated T cells can then be readministered to the host. One or more of these methods can be used in combination with the administration of the antibody, antibody fragment, or immunoconjugate of the present invention.

[0299] Conventional cancer treatments include: (i) radiation therapy (e.g., radiotherapy, X-ray therapy, irradiation), or the use of ionizing radiation to kill cancer cells and shrink tumors. Radiation therapy can be administered externally via external beam radiotherapy (EBRT) or internally via brachytherapy; (ii) chemotherapy, or the application of cytotoxic drugs, which generally affect rapidly dividing cells; (iii) targeted therapy, or drugs (e.g., tyrosine kinase inhibitors imatinib, gefitinib; monoclonal antibodies, photodynamic therapy) that specifically affect dysregulated proteins in cancer cells; (iv) immunotherapy, or enhancing the host immune response (e.g., vaccines); (v) hormone therapy, or hormone blockade (e.g., when the tumor is hormone-sensitive); (vi) angiogenesis inhibitors, or blocking blood vessel formation and growth; and (vii) palliative care, or treatments aimed at improving the quality of care to reduce pain, nausea, vomiting, diarrhea, and bleeding. Pain relievers such as morphine and oxycodone, antiemetics such as ondansetron and aprepitant may allow for more aggressive treatment regimens.

[0300] In treating cancer, any of the conventional therapies previously described for the treatment of cancer immunity can be administered before, after, or simultaneously with the administration of the anti-Axl antibody or antibody fragment. Furthermore, the anti-Axl antibody or antibody fragment can be administered before, after, or simultaneously with the administration of conventional cancer therapies, such as tumor-binding antibodies (e.g., monoclonal antibodies, toxin-conjugated monoclonal antibodies) and / or chemotherapeutic agents.

[0301] F. Products and Kits In another aspect of the present invention, an article of manufacture containing materials useful for the treatment, prevention, and / or diagnosis of the above-mentioned disorders is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed from a variety of materials, such as glass or plastic. The container holds the composition by itself or in combination with another composition effective for the treatment, prevention, and / or diagnosis of a condition and can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an antibody or antibody fragment of the present invention. The label or package insert indicates that the composition is used to treat a selected condition. Furthermore, the article of manufacture can include (a) a first container containing a composition comprising the antibody or antibody fragment; and (b) a second container containing a composition comprising an additional cytotoxic or other therapeutic agent. The article of manufacture in this embodiment of the present invention can further include a package insert indicating that the composition can be used to treat a specific condition. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, such as other buffers, diluents, filters, needles, and syringes.

[0302] It is understood that any of the above products may contain an immunoconjugate of the invention in place of, or in addition to, an anti-Axl antibody.

[0303] Finally, the present invention also provides a kit comprising at least one antibody or antibody fragment of the present invention.Kits containing the polypeptides, antibodies or antibody fragments, or antibody-drug conjugates of the present invention are used in detecting Axl expression (increase or decrease) or in therapeutic or diagnostic assays.Kits of the present invention can contain antibodies coupled to solid supports, such as tissue culture plates or beads (e.g., sepharose beads).Kits containing antibodies for in vitro detection and quantification of Axl, for example, in ELISA or Western blot, can be provided.Such antibodies useful for detection can be provided with labels, such as fluorescent or radioactive labels.

[0304] The kit further contains instructions for its use. In some embodiments, the instructions include those required by the US Food and Drug Administration for in vitro diagnostic kits. In some embodiments, the kit further includes instructions for diagnosing the presence or absence of cerebrospinal fluid in a sample based on the presence or absence of Axl in the sample. In some embodiments, the kit includes one or more antibodies or antibody fragments. In other embodiments, the kit further includes one or more enzymes, enzyme inhibitors, or enzyme activators. In still other embodiments, the kit further includes one or more chromatography compounds. In still other embodiments, the kit further includes one or more compounds used to prepare a sample for spectrophotometric assay. In further embodiments, the kit further includes a reference material for interpreting the presence or absence of Axl according to the intensity, color spectrum, or other physical attributes of the indicator.

[0305] The following examples are illustrative of, but not limiting of, the soft gelatin capsules of the present disclosure. Other suitable modifications and adaptations of the variety of conditions and parameters commonly found in the art and obvious to those skilled in the art are within the scope of the present disclosure. [Example]

[0306] Example 1: Conditionally active antibodies against Axl Axl is a transmembrane tyrosine kinase with an extracellular domain that can be accessed by conditionally active antibodies. This cell surface protein is highly expressed in thyroid cancer tissues and is overexpressed in many other cancers, such as myeloproliferative disorders, prostate cancer cells, or breast cancer. A conditionally active antibody against the extracellular domain of Axl protein has been discovered herein.

[0307] A wild-type antibody against Axl (having the heavy chain variable region of 063-hum10F10-HC in Figure 1A and the light chain variable region of 063-hum10F10-HC in Figure 1B) was selected as the template antibody. DNA encoding the wild-type antibody was evolved using comprehensive positional evolution (CPE), a method that randomizes each position in the template antibody one at a time, to generate a mutant antibody library. Each mutant antibody in the library has only one single point mutation. The mutant antibodies in the library were generated by simultaneous screening for preferential binding affinity to Axl at pH 6.0 compared to pH 7.4, as measured by ELISA.

[0308] Simultaneously, the expression level of the mutant antibodies was optimized for higher field applications in the manufacturing process. Screening was performed in serum using the FLAG tag because of the presence of human antibodies in serum, which could cause false-positive results. The screening buffer was carbonate buffer (Krebs buffer with Ringer's—a standard buffer, unlike PBS). The generated conditionally active antibodies were found to have higher affinity for Axl at pH 6.0 but lower affinity for Axl at pH 7.4 compared to the wild-type antibody. Some of the selected mutant antibodies (scFv) are shown in Figure 2. Their activity was higher at pH 6.0 than at pH 7.4, while the activity ratio between pH 6.0 and pH 7.4 was at least 11-fold (Figure 3).

[0309] Furthermore, all of these conditionally active activities have high expression levels as shown in Table 4 below, where the column "Clone" indicates the antibody and the expression level "mg / ml" is shown in the second column.

[0310] These antibody clones were sent to the service provider at the required expression levels ("order amount", expected expression level). However, the actual expression levels of these antibodies ("delivered amount") were significantly higher than the expected expression levels.

[0311] [Table 4]

[0312] The conditionally active antibody showed no aggregation in buffer, as demonstrated in Figure 4 using the BAP063.9-13-1 antibody as a template. The BAP063.9-13-1 antibody was analyzed by size exclusion chromatography. In Figure 4, only one peak was detected, demonstrating that the antibody exhibited little or no aggregation.

[0313] The conditionally active antibodies were also assayed using surface plasmon resonance (SPR) to measure the on- and off-rates for Axl. SPR assays are known to measure the on- and off-rates for conditionally active antibodies. SPR assays were performed in the presence of bicarbonate. The in vivo on- and off-rates (in animals and humans) of conditionally active antibodies are critical characteristics for conditionally active antibodies.

[0314] The conditionally active antibodies were observed to have higher binding affinity at pH 6.0 and lower binding affinity at pH 7.4 compared to the negative control (BAP063 10F10, which has similar binding affinity at both pH 6.0 and pH 7.4) (Figure 5). Furthermore, increasing the temperature from room temperature to 60°C did not significantly alter the ELISA assay results (Figure 5). The ELISA assay also showed that these conditionally active antibodies were highly selective at pH 6.0 compared to pH 7.4 (Figures 6A-6B show one antibody as an example).

[0315] The conditionally active biological antibodies are summarized in Table 5. Two of these antibodies were expressed as scFvs (BAP063.9-13.3 and BAP063.9-48.3). Incubation of the antibodies at 60°C for 1 hour did not change the affinity of most of the antibodies ("thermostability").

[0316] Conditionally active antibodies can be used according to the present invention to detect Axl protein on the surface of CTCs.

[0317] [Table 5]

[0318] Example 2: pH-dependent binding affinity of anti-Axl antibodies Some of the anti-Axl antibodies of the present invention were tested in buffers at different pH levels. One type of buffer was KREBS buffer with 1% bovine serum albumin (BSA). The KREBS buffer was titrated to have a pH ranging from 5 to 7.4. ELISA assays (OD 450 ) was used to measure the binding affinity of the antibodies for Axl, and the results are presented in Figure 7. Two control antibodies (BAP063-3831 and BAP063-3818) were not conditionally active because their binding affinities were not significantly affected by changes in pH. On the other hand, the anti-Axl antibodies of the present invention were conditionally active because their binding affinity for Axl was pH dependent (Figure 7).

[0319] Example 3: Cell killing by anti-Axl antibodies The cell-killing activity of the anti-Axl antibodies of the present invention was tested using A549 cells. The results are shown in Figures 8A-8E. The cell-killing activity was measured at two pH levels: 6.0 and 7.4, representing the pH in the tumor microenvironment and normal physiological pH, respectively. The percentage of cell killing at various antibody concentrations is shown in Figures 8A-8E.

[0320] The two tests yielded consistent cell-killing results. The negative control (anti-Axl humanized WT) showed similar cell-killing activity at pH 6.0 and pH 7.4 for A549 cells (Figure 8A). In contrast, the anti-Axl antibodies of the present invention showed significantly higher cell-killing activity at pH 6.0 compared to pH 7.4, particularly at low antibody concentrations where the antibody did not saturate A549 cells (Figures 8B-8E).

[0321] Example 4: Binding affinity of anti-Axl antibodies to cyno-Axl The binding affinity of the anti-Axl antibodies of the present invention to cyno-Axl was measured and compared with that of human Axl (hAxl) in two different buffers at pH 6.0 and 7.4. The results are shown in Figures 9A-9D. Cyno-Axl is an Axl protein from a non-human primate, i.e., the cynomolgus macaque.

[0322] The control (BA-3831-WT) showed similar binding affinity to both human Axl (hAxl) and cynomolgus monkey Axl (cyno-Axl) at both pH 6.0 and 7.4 in the two buffers (Figure 9A). The anti-Axl antibodies of the present invention showed similar binding affinity profiles for hAxl and cyno-Axl in one of the two buffers, i.e., lower binding affinity to cyno-Axl at pH 7.4 compared to pH 6.0 (Figures 9B-9D). The difference in binding affinity between pH 6.0 and pH 7.0 in the other buffer was not significant.

[0323] Example 5: Cytotoxicity of anti-Axl antibodies conjugated to duomycin Duomycin is cytotoxic because it inhibits cell growth by halting protein synthesis. One of the anti-Axl antibodies of the present invention, BAP063.9 4007, was conjugated to duomycin. Two control antibodies, BAP063humWT and B12 (anti-B12 antibody), were used in this study, both of which were also conjugated to duomycin.

[0324] Several cell lines were treated with three duomycin-conjugated antibodies (BAP063.9 4007, BAP063humWT, and B12) at pH 6.0 and 7.4 (Figures 10A-10H). The duomycin-conjugated antibody BAP063.9 4007 of the present invention exhibited significantly higher cytotoxicity against the cell lines DU145 (prostate cancer cells), MDA-MD-231 (breast cancer cells), PL45 (pancreatic cancer cells), and A549 (adenocarcinoma cells) at pH 6.0 compared to the cytotoxicity against the same cells at pH 7.4.

[0325] Example 6: Anti-Axl antibodies conjugated to model toxins The anti-Axl antibodies of the present invention were conjugated to a model toxin (e.g., gemcitabine) to produce conditionally active antibody-drug conjugates (CAB-Axl-ADCs). The CAB-Axl-ADCs were first tested to confirm that their conditional cell-killing activity was not altered by the drug conjugation process. This test showed that the CAB-Axl-ADCs killed significantly more cells at pH 6.0 than at pH 7.4 (Figure 11).

[0326] The CAB-Axl-ADC was then injected into mice bearing MiaPaCa2 xenograft tumors at a dose of 1 mg / kg twice weekly for 3 weeks. Several controls were used in this study, including naked CAB (anti-Axl antibody without conjugation), vehicle, toxin alone (unconjugated gemcitabine), control ADC, and affinity-matched anti-Axl ADC (AM ADC). The study showed that CAB-Axl-ADC (CAB ADC) and AM ADC provided significantly greater tumor size reduction compared to the control ( FIG. 12 ). Unconjugated anti-Axl antibody did not reduce tumor size. This study demonstrated that anti-Axl antibodies conjugated to toxins were as effective as affinity-matched antibodies in reducing tumor size.

[0327] Example 7: Serum concentrations of anti-Axl antibody drug conjugates in cynomolgus macaques The anti-Axl antibody drug (duomycin) conjugate (CAB-ADC) of the present invention was injected into male and female cynomolgus macaques at three doses: 0.1, 1, and 10 mg / kg. Naked anti-Axl antibody was used as a control. An affinity-matched antibody drug conjugate (AM-ADC) was also used as a control. Serum concentrations of the antibody were measured over a one-week period (168 hours, see Figures 13A-13B). The CAB-ADC persisted in monkey serum longer than the AM-ADC control (Figure 13B). There was no significant difference between male and female monkeys (Figures 13A-13B).

[0328] Example 8: Toxicity of anti-Axl antibody drug conjugates in cynomolgus macaques The toxicity of the CAB-ADC of the present invention was tested in cynomolgus macaques. Aspartate transaminase (AST) and alanine transaminase (ALT) are used by the Food and Drug Administration (FDA) as indicators of drug liver toxicity. Serum AST and ALT levels were measured in both male and female monkeys (Figures 14A-14B). Three days after administration of 10 mg / kg, vehicle (PBS) did not alter serum AST or ALT levels, while the matched antibody-drug conjugate (AM) showed significantly elevated AST and ALT levels. The CAB-ADC showed significantly reduced AST and ALT levels compared with the AM control. This indicated that the anti-Axl antibody-drug conjugate of the present invention has significantly reduced liver toxicity compared with the matched antibody-drug conjugate AM.

[0329] The anti-Axl antibody-drug conjugate of the present invention was also found to cause less inflammation in monkeys (Figure 15). The lymphocyte counts in the blood of monkeys after injection of CAB, AM, and PBS were summarized. Compared to AM, which caused significant inflammation, the anti-Axl antibody-drug conjugate of the present invention (CAB-ADC) caused only mild inflammation in monkeys.

[0330] Example 9: In vivo experiments in mice Mice were implanted with one of two tumor cell lines (LCLC103H or DU145) that developed into tumors. Tumor size was measured after treatment with the antitumor drug monomethyl auristatin E (MMAE). For mice that received LCLC103H, mice were treated with a single dose of vehicle (as a negative control), CAB anti-Axl antibody conjugated MMAE ADC (CAB Axl-MMAE), or non-CAB anti-Axl antibody conjugated MMAE ADCC (non-CAB Axl-MMAE) (Figure 16A). Tumors in mice treated with the ADC shrank, while tumors in mice treated with the vehicle continued to grow.

[0331] Additionally, mice receiving DU145 were treated with vehicle (negative control) or CAB anti-Axl antibody conjugated MMAE ADC (CAB Axl-MMAE) at two different concentrations (6 mg / kg and 10 mg / kg). Tumor volume was measured over time. Tumors continued to grow in the negative control group (vehicle), whereas tumor growth slowed in mice treated with the ADC (Figure 16B).

[0332] However, while numerous features and advantages of the present invention have been set forth in the foregoing detailed description, together with details of the structure and function of the invention, it should be understood that the disclosure is illustrative only, and that changes may be made in the details, particularly in matters of shape, size and arrangement of parts within the principles of the invention, to the fullest extent indicated by the broad general meaning of the terms in which the appended claims are expressed.

[0333] All documents cited herein are incorporated herein by reference in their entirety or for the disclosure on which they are specifically relied upon. Applicants do not intend to publicly offer any disclosed embodiments, and to the extent that any disclosed modifications or variations may not fall literally within the scope of the claims, they are considered part thereof under the doctrine of equivalents.

Claims

1. 1. An isolated polypeptide that specifically binds to an Axl protein, comprising a heavy chain variable region comprising three complementarity determining regions, said regions having H1, H2, and H3 sequences; (a) the H1 sequence is X 1 GX 2 X 3 MX 4 (SEQ ID NO: 1); (b) the H2 sequence is LIKX 5 SNGGTX 6 YNQKFKG (SEQ ID NO: 2); (c) the H3 sequence is GX 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 DYX 15 X 16 (SEQ ID NO: 3), X 1 is T or A or W, X 2 is H or A, X 3 is T or I, X 4 is N or I, X 5 is P or N, X 6 is S or I or T, X 7 is H or D or E or P or R or W, X 8 is Y or N, X 9 is E or A or D or F or G or H or I or L or M or N or R or V or Y, X 10 is S or D or M or N or Q, X 11 is Y or C or E or P, X 12 is F or E or N or S or T or V, X 13 is A or D or G or L or Y, X 14 is M, E, or F, X 15 is W or A or D or H or L or N or P or R or T, X 16 is G or H; Isolated polypeptide.

2. The polypeptide of claim 1, wherein the heavy chain variable region is encoded by a DNA sequence selected from the sequences of SEQ ID NOs: 11 to 13.

3. comprising three complementarity determining regions, L1, L2, and L3 sequences; (a) the L1 sequence is KASQDX 17 X 18 SX 19 VX 20 (SEQ ID NO: 4); (b) the L2 sequence is X 21 X 22 X 23 TRX 24 T (SEQ ID NO: 5); (c) the L3 sequence is QEX 25 X 26 SX 27 X 28 X 29 X 30 (SEQ ID NO: 6), X 17 is V or D or G or N or W, X 18 is S or V, X 19 is A, L or M, X 20 is A or D or N or Q, X 21 is W or F, X 22 is A or I or N or P or Q, X 23 is S or D, X 24 is H or D, X 25 is H or C or F or I or L or Q or S or T or V or Y, X 26 is F or C or D or E or G or N or S, X 27 is T or C or P, X 28 is P or A or C or D or E or H or K or S or T or V or W, X 29 is L or G or R, X 30 is T or I or R; 3. The polypeptide of claim 1 or 2 in combination with an isolated light chain variable region.

4. The polypeptide of claim 3, wherein the light chain variable region is encoded by a DNA sequence selected from SEQ ID NOs: 7-10.

5. 1. An isolated polypeptide that specifically binds to an Axl protein, comprising a light chain variable region comprising three complementarity determining regions having the sequences L1, L2, and L3; (a) the L1 sequence is KASQDX 17 X 18 SX 19 VX 20 (SEQ ID NO: 4); (b) the L2 sequence is X 21 X 22 X 23 TRX 24 T (SEQ ID NO: 5); (c) the L3 sequence is QEX 25 X 26 SX 27 X 28 X 29 X 30 (SEQ ID NO: 6), X 17 is V or D or G or N or W, X 18 is S or V, X 19 is A, L or M, X 20 is A or D or N or Q, X 21 is W or F, X 22 is A or I or N or P or Q, X 23 is S or D, X 24 is H or D, X 25 is H or C or F or I or L or Q or S or T or V or Y, X 26 is F or C or D or E or G or N or S, X 27 is T or C or P, X 28 is P or A or C or D or E or H or K or S or T or V or W, X 29 is L or G or R, X 30 is T or I or R; Isolated polypeptide.

6. The polypeptide of claim 5, wherein the light chain variable region is encoded by a DNA sequence selected from SEQ ID NOs: 7-10.

7. An anti-Axl antibody or antibody fragment comprising the isolated heavy chain variable region polypeptide of claim 1.

8. comprising three complementarity determining regions, L1, L2, and L3 sequences; (a) the L1 sequence is KASQDX 17 X 18 SX 19 VX 20 (SEQ ID NO: 4); (b) the L2 sequence is X 21 X 22 X 23 TRX 24 T (SEQ ID NO: 5); (c) the L3 sequence is QEX 25 X 26 SX 27 X 28 X 29 X 30 (SEQ ID NO: 6), X 17 is V or D or G or N or W, X 18 is S or V, X 19 is A, L or M, X 20 is A or D or N or Q, X 21 is W or F, X 22 is A or I or N or P or Q, X 23 is S or D, X 24 is H or D, X 25 is H or C or F or I or L or Q or S or T or V or Y, X 26 is F or C or D or E or G or N or S, X 27 is T or C or P, X 28 is P or A or C or D or E or H or K or S or T or V or W, X 29 is L or G or R, X 30 is T or I or R; The antibody or antibody fragment of claim 7, further comprising an isolated light chain variable region.

9. 9. The antibody or antibody fragment of claim 7 or 8, which has a higher binding affinity to Axl protein at a value of a condition in a tumor microenvironment compared to a different value of the same condition occurring in a non-tumor microenvironment.

10. 10. The antibody or antibody fragment of claim 9, wherein the condition is pH.

11. 11. The antibody or antibody fragment of claim 10, wherein the pH in the tumor microenvironment is in the range of 5.8 to 7.0 and the pH in the non-tumor microenvironment is in the range of 7.0 to 7.

6.

12. 12. The antibody or antibody fragment of any one of claims 7 to 11, having a ratio of binding affinity for the Axl protein at a value of a condition in a tumor microenvironment to binding affinity for the Axl protein at a different value of the same condition in a non-tumor microenvironment of at least about 1.5:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 20:1, at least about 30:1, at least about 50:1, at least about 70:1, or at least about 100:

1.

13. The antibody or antibody fragment of any one of claims 7 to 12, which is a chimeric antibody, a multispecific antibody, or a humanized antibody.

14. An immunoconjugate comprising the antibody or antibody fragment of any one of claims 7 to 13.

15. 15. The immunoconjugate of claim 14, comprising at least one agent selected from a chemotherapeutic agent, a radioactive atom, a cytostatic agent, and a cytotoxic agent.

16. The immunoconjugate of claim 15 comprising at least two of said agents.

17. The immunoconjugate of claim 15 or 16, wherein the antibody or antibody fragment and the at least one agent are covalently attached to a linker molecule.

18. 18. The immunoconjugate of any one of claims 15 to 17, wherein the at least one agent is selected from maytansinoids, auristatins, dolastatins, calicheamicins, pyrrolobenzodiazepines, and anthracyclines.

19. A polypeptide according to any one of claims 1 to 6, an antibody or antibody fragment according to any one of claims 7 to 13, or an immunoconjugate according to any one of claims 14 to 18; and Pharmaceutically acceptable carrier 10. A pharmaceutical composition comprising:

20. 20. The pharmaceutical composition of claim 19, further comprising a tonicity agent.

21. 21. A method for treating cancer, comprising administering to a patient having cancer the pharmaceutical composition of claim 19 or 20.

22. 21. A diagnostic or therapeutic kit comprising a polypeptide according to any one of claims 1 to 6, an antibody or antibody fragment according to any one of claims 7 to 13, or an immunoconjugate according to any one of claims 14 to 18, or a pharmaceutical composition according to claim 19 or 20, and instructions for using said antibody or antibody fragment, said immunoconjugate and / or said pharmaceutical composition for diagnosis or treatment.

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