Anti-AXL antibodies

Abstract

Antibodies characterized by their variable sequences / CDRs are described, said antibodies specifically binding to the Axl protein. Methods of producing and using said anti-Axl antibodies are also disclosed.

Description

[0001] This application is a divisional application of the Chinese invention patent application (filing date: June 18, 2015; application number: 201580042717.5 (International application number: PCT / EP2015 / 063700); invention name: anti-AXL antibody).

[0002] The present disclosure relates to antibodies that specifically bind to Axl protein. Also disclosed are methods of producing and using anti-Axl antibodies. background

[0003] Axl is a member of the TAM (Tyro3-Axl-Mer) receptor tyrosine kinase (RTK) that shares the vitamin K-dependent ligand Gas6 (growth arrest-specific protein 6). TAM family RTKs regulate a variety of cellular responses, including cell survival, proliferation, autophagy, migration, angiogenesis, platelet aggregation, and natural killer cell differentiation. Axl is expressed in many embryonic tissues and is thought to be involved in mesenchymal and neural development, and its expression in adult tissues is mainly limited to smooth muscle cells (MGI Gene Expression Database; www.informatics.jax.org). Axl activation is associated with several signal transduction pathways, including Akt, MAP kinase, NF-κB, STAT, etc. Originally identified as a transforming gene from patients with chronic myeloid leukemia, Axl has been associated with various high-grade cancers and associated with poor prognosis.

[0004] Axl receptor overexpression has been detected in a variety of solid tumors and myeloid leukemias (Linger et al., Adv Cancer Res. 100:35, 2008; Linger et al., Expert Opin Ther Targets. 14:1073, 2010).

[0005] Axl expression is associated with malignant progression and is an independent predictor of poor patient survival in several malignancies including pancreas (Song et al., Cancer. 117:734, 2011), prostate (Paccez et al., Oncogene. 32:698, 2013), lung (Ishikawa et al., Ann Surg Oncol. 2012; Zhang et al., Nat Genet. 44:852, 2012), breast (Gjerdrum, Proc natl Acad Sci USA 107:1124, 2010), colon cancer (Yuen et al., PLoS One, 8:e54211, 2013), and acute myeloid leukemia (AML) (Ben-Batalla et al., Blood 122:2443, 2013).

[0006] Axl signaling is activated by a protein ligand (Gas6) secreted by tumor-associated macrophages (Loges et al., Blood. 115: 2264, 2010) or an autocrine mechanism (Gjerdrum, Proc natl Acad Sci USA 107: 1124, 2010), which drives receptor dimerization, autophosphorylation and downstream signaling, such as via the PI3 kinase (PI3K)-AKT, particularly AKT and mitogen-activated protein kinase (MAPK) pathways (Korshunov, Clinical Science. 122: 361, 2012). Heterodimerization with other tyrosine kinase receptors, such as epidermal growth factor receptor (EGFR), has also been reported (Linger et al., Expert Opin Ther Targets. 14: 1073, 2010; Meyer et al., Science Signalling 6: ra66, 2013). Abnormal activation of Axl in tumor cells is widely associated with acquired drug resistance to targeted therapeutic agents in vitro and in vivo (Zhang et al., Nat Genet. 44: 852, 2012; Byers et al., Clin Cancer Res. 19: 279, 2013). In several cancer experimental models, including triple-negative breast cancer, hormone-resistant prostate cancer and lung adenocarcinoma, Axl targeting agents blocked tumor formation, metastasis and reversed drug resistance (e.g., to erlotinib) by reversing EMT / CSC characteristics (Holland et al., Cancer Res 70: 1544, 2010; Gjerdrum, Proc natl Acad Sci USA 107: 1124, 2010; Zhang et al., Nat Genet. 44: 852, 2012; Paccez et al., Oncogene. 32: 698, 2013).

[0007] Other applications involving Axl and anti-Axl antibodies include EP2267454A2 [Diagnosis and preventionof cancer cell invasion measuring ... Axl - Max Planck]; WO2009063965 [anti Axl - Chugai Pharmaceutical]; WO2011159980A1 [anti-AxI - Genentech]; WO2011014457A1 [combination treatments Axl and VEGF antagonists - Genentech]; WO2012-175691A1 [Anti Axl20G7-D9-INSERM]; WO2012-175692A1 [Anti Axl 3E3E8-INSERM]; WO2009 / 062690A1 [anti Axl-U3 Pharma] and WO2010 / 130751A1 [humanised anti Axl-U3 Pharma].

[0008] Given the role of Axl in tumorigenesis, it is desirable to identify more antibodies that specifically bind to Axl with favorable properties. The present disclosure relates to such antibodies. Sequence Listing <110> Bergen Biotech AG <120> Anti-Axl antibody <130> RKA / FP7126923 <150> GB 1410826.0 <151> 2014-06-18 <160> twenty two <170> PatentIn Version 3.3 <210> 1 <211> 381 <212> DNA <213> House mouse <400> 1 gaggtgaagc tggtggaatc tgggggagac ttagtgaagc ctggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cactttcagt agctatggca tgtcttgggt tcgccagact 120 ccagacaaga ggctggagtg ggtcgcaacc attagtagtg gtggtagtta cacctactat 180 ccagacagtg tgaaggggcg attcaccatc tccagagaca atgccaagaa caccctgtac 240 ctgcaaatga gcagtctgaa gtctgaggac acagccatgt attactgtgc aagacatccc 300 atctactata cttacgacga tactatggac tactggggtc aaggaacctc agtcaccgtc 360 tcctcagcca aaacgacacc c 381 <210> 2 <211> 351 <212> DNA <213> Mus musculus <400> 2 gacattgtgc tgacccaatc tccagcaatc atggctgcat ctccagggga gaaggtcacc 60 atgacctgca gtgccagctc aagtgtaagt tctggtaact ttcactggta ccagcagaag 120 ccaggcactt ctcccaaact ctggatttat aggacatcca acctggcttc tggagtcccc 180 gctcgcttca gtggcagtgg gtctgggacc tcttactctc ttacaatcag cagcatggag 240 gccgaagatg ctgccactta ttactgccag cagtggagtg gttacccgtg gacgttcggt 300 ggaggcacca agctggaaat caaacgggct gatgctgcac caactgtatc c 351 <210> 3 <211> 122 <212> PRT <213> Mus musculus <400> 3 Glu Val Lys Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg His Pro Ile Tyr Tyr Thr Tyr Asp Asp Thr Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 4 <211> 108 <212> PRT <213> Mus musculus <400> 4 Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ala Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Ser Gly 20 25 30 Asn Phe His Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp 35 40 45 Ile Tyr Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro 85 90 95 Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 5 <211> 10 <212> PRT <213> Mus musculus <400> 5 Gly Phe Thr Phe Ser Ser Tyr Gly Met Ser 1 5 10 <210> 6 <211> 26 <212> PRT <213> Mus musculus <400> 6 Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val Lys 1 5 10 15 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 20 25 <210> 7 <211> 13 <212> PRT <213> House mouse <400> 7 His Pro Ile Tyr Tyr Thr Tyr Asp Asp Thr Met Asp Tyr 1 5 10 <210> 8 <211> 12 <212> PRT <213> House mouse <400> 8 Ser Ala Ser Ser Ser Val Ser Ser Gly Asn Phe His 1 5 10 <210> 9 <211> 7 <212> PRT <213> House mouse <400> 9 Arg Thr Ser Asn Leu Ala Ser 1 5 <210> 10 <211> 9 <212> PRT <213> House mouse <400> 10 Gln Gln Trp Ser Gly Tyr Pro Trp Thr 1 5 <210> 11 <211> 25 <212> PRT <213> House mouse <400> 11 Glu Val Lys Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser 20 25 <210> 12 <211> 14 <212> PRT <213> House mouse <400> 12 Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val Ala 1 5 10 <210> 13 <211> twenty three <212> PRT <213> House mouse <400> 13 Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr 1 5 10 15 Ala Met Tyr Tyr Cys Ala Arg 20 <210> 14 <211> 11 <212> PRT <213> House mouse <400> 14 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 1 5 10 <210> 15 <211> twenty three <212> PRT <213> House mouse <400> 15 Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ala Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys 20 <210> 16 <211> 15 <212> PRT <213> Mus musculus <400> 16 Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr 1 5 10 15 <210> 17 <211> 32 <212> PRT <213> Mus musculus <400> 17 Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser 1 5 10 15 Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys 20 25 30 <210> 18 <211> 10 <212> PRT <213> Mus musculus <400> 18 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 1 5 10 <210> 19 <211> 894 <212> PRT <213> Homo sapiens <400> 19 Met Ala Trp Arg Cys Pro Arg Met Gly Arg Val Pro Leu Ala Trp Cys 1 5 10 15 Leu Ala Leu Cys Gly Trp Ala Cys Met Ala Pro Arg Gly Thr Gln Ala 20 25 30 Glu Glu Ser Pro Phe Val Gly Asn Pro Gly Asn Ile Thr Gly Ala Arg 35 40 45 Gly Leu Thr Gly Thr Leu Arg Cys Gln Leu Gln Val Gln Gly Glu Pro 50 55 60 Pro Glu Val His Trp Leu Arg Asp Gly Gln Ile Leu Glu Leu Ala Asp 65 70 75 80 Ser Thr Gln Thr Gln Val Pro Leu Gly Glu Asp Glu Gln Asp Asp Trp 85 90 95 Ile Val Val Ser Gln Leu Arg Ile Thr Ser Leu Gln Leu Ser Asp Thr 100 105 110 Gly Gln Tyr Gln Cys Leu Val Phe Leu Gly His Gln Thr Phe Val Ser 115 120 125 Gln Pro Gly Tyr Val Gly Leu Glu Gly Leu Pro Tyr Phe Leu Glu Glu 130 135 140 Pro Glu Asp Arg Thr Val Ala Ala Asn Thr Pro Phe Asn Leu Ser Cys 145 150 155 160 Gln Ala Gln Gly Pro Pro Glu Pro Val Asp Leu Leu Trp Leu Gln Asp 165 170 175 Ala Val Pro Leu Ala Thr Ala Pro Gly His Gly Pro Gln Arg Ser Leu 180 185 190 His Val Pro Gly Leu Asn Lys Thr Ser Ser Phe Ser Cys Glu Ala His 195 200 205 Asn Ala Lys Gly Val Thr Thr Ser Arg Thr Ala Thr Ile Thr Val Leu 210 215 220 Pro Gln Gln Pro Arg Asn Leu His Leu Val Ser Arg Gln Pro Thr Glu 225 230 235 240 Leu Glu Val Ala Trp Thr Pro Gly Leu Ser Gly Ile Tyr Pro Leu Thr 245 250 255 His Cys Thr Leu Gln Ala Val Leu Ser Asp Asp Gly Met Gly Ile Gln 260 265 270 Ala Gly Glu Pro Asp Pro Pro Glu Glu Pro Leu Thr Ser Gln Ala Ser 275 280 285 Val Pro Pro His Gln Leu Arg Leu Gly Ser Leu His Pro His Thr Pro 290 295 300 Tyr His Ile Arg Val Ala Cys Thr Ser Ser Gln Gly Pro Ser Ser Trp 305 310 315 320 Thr His Trp Leu Pro Val Glu Thr Pro Glu Gly Val Pro Leu Gly Pro 325 330 335 Pro Glu Asn Ile Ser Ala Thr Arg Asn Gly Ser Gln Ala Phe Val His 340 345 350 Trp Gln Glu Pro Arg Ala Pro Leu Gln Gly Thr Leu Leu Gly Tyr Arg 355 360 365 Leu Ala Tyr Gln Gly Gln Asp Thr Pro Glu Val Leu Met Asp Ile Gly 370 375 380 Leu Arg Gln Glu Val Thr Leu Glu Leu Gln Gly Asp Gly Ser Val Ser 385 390 395 400 Asn Leu Thr Val Cys Val Ala Ala Tyr Thr Ala Ala Gly Asp Gly Pro 405 410 415 Trp Ser Leu Pro Val Pro Leu Glu Ala Trp Arg Pro Gly Gln Ala Gln 420 425 430 Pro Val His Gln Leu Val Lys Glu Pro Ser Thr Pro Ala Phe Ser Trp 435 440 445 Pro Trp Trp Tyr Val Leu Leu Gly Ala Val Val Ala Ala Ala Cys Val 450 455 460 Leu Ile Leu Ala Leu Phe Leu Val His Arg Arg Lys Lys Glu Thr Arg 465 470 475 480 Tyr Gly Glu Val Phe Glu Pro Thr Val Glu Arg Gly Glu Leu Val Val 485 490 495 Arg Tyr Arg Val Arg Lys Ser Tyr Ser Arg Arg Thr Thr Glu Ala Thr 500 505 510 Leu Asn Ser Leu Gly Ile Ser Glu Glu Leu Lys Glu Lys Leu Arg Asp 515 520 525 Val Met Val Asp Arg His Lys Val Ala Leu Gly Lys Thr Leu Gly Glu 530 535 540 Gly Glu Phe Gly Ala Val Met Glu Gly Gln Leu Asn Gln Asp Asp Ser 545 550 555 560 Ile Leu Lys Val Ala Val Lys Thr Met Lys Ile Ala Ile Cys Thr Arg 565 570 575 Ser Glu Leu Glu Asp Phe Leu Ser Glu Ala Val Cys Met Lys Glu Phe 580 585 590 Asp His Pro Asn Val Met Arg Leu Ile Gly Val Cys Phe Gln Gly Ser 595 600 605 Glu Arg Glu Ser Phe Pro Ala Pro Val Val Ile Leu Pro Phe Met Lys 610 615 620 His Gly Asp Leu His Ser Phe Leu Leu Tyr Ser Arg Leu Gly Asp Gln 625 630 635 640 Pro Val Tyr Leu Pro Thr Gln Met Leu Val Lys Phe Met Ala Asp Ile 645 650 655 Ala Ser Gly Met Glu Tyr Leu Ser Thr Lys Arg Phe Ile His Arg Asp 660 665 670 Leu Ala Ala Arg Asn Cys Met Leu Asn Glu Asn Met Ser Val Cys Val 675 680 685 Ala Asp Phe Gly Leu Ser Lys Lys Ile Tyr Asn Gly Asp Tyr Tyr Arg 690 695 700 Gln Gly Arg Ile Ala Lys Met Pro Val Lys Trp Ile Ala Ile Glu Ser 705 710 715 720 Leu Ala Asp Arg Val Tyr Thr Ser Lys Ser Asp Val Trp Ser Phe Gly 725 730 735 Val Thr Met Trp Glu Ile Ala Thr Arg Gly Gln Thr Pro Tyr Pro Gly 740 745 750 Val Glu Asn Ser Glu Ile Tyr Asp Tyr Leu Arg Arg Gly Asn Arg Leu 755 760 765 Lys Gln Pro Ala Asp Cys Leu Asp Gly Leu Tyr Ala Leu Met Ser Arg 770 775 780 Cys Trp Glu Leu Asn Pro Gln Asp Arg Pro Ser Phe Thr Glu Leu Arg 785 790 795 800 Glu Asp Leu Glu Asn Thr Leu Lys Ala Leu Pro Pro Ala Gln Glu Pro 805 810 815 Asp Glu Ile Leu Tyr Val Asn Met Asp Glu Gly Gly Gly Tyr Pro Glu 820 825 830 Pro Pro Gly Ala Ala Gly Gly Ala Asp Pro Pro Thr Gln Pro Asp Pro 835 840 845 Lys Asp Ser Cys Ser Cys Leu Thr Ala Ala Glu Val His Pro Ala Gly 850 855 860 Arg Tyr Val Leu Cys Pro Ser Thr Thr Pro Ser Pro Ala Gln Pro Ala 865 870 875 880 Asp Arg Gly Ser Pro Ala Ala Pro Gly Gln Glu Asp Gly Ala 885 890 <210> 20 <211> 888 <212> PRT <213> Mus musculus <400> 20 Met Gly Arg Val Pro Leu Ala Trp Trp Leu Ala Leu Cys Cys Trp Gly 1 5 10 15 Cys Ala Ala His Lys Asp Thr Gln Thr Glu Ala Gly Ser Pro Phe Val 20 25 30 Gly Asn Pro Gly Asn Ile Thr Gly Ala Arg Gly Leu Thr Gly Thr Leu 35 40 45 Arg Cys Glu Leu Gln Val Gln Gly Glu Pro Pro Glu Val Val Trp Leu 50 55 60 Arg Asp Gly Gln Ile Leu Glu Leu Ala Asp Asn Thr Gln Thr Gln Val 65 70 75 80 Pro Leu Gly Glu Asp Trp Gln Asp Glu Trp Lys Val Val Ser Gln Leu 85 90 95 Arg Ile Ser Ala Leu Gln Leu Ser Asp Ala Gly Glu Tyr Gln Cys Met 100 105 110 Val His Leu Glu Gly Arg Thr Phe Val Ser Gln Pro Gly Phe Val Gly 115 120 125 Leu Glu Gly Leu Pro Tyr Phe Leu Glu Glu Pro Glu Asp Lys Ala Val 130 135 140 Pro Ala Asn Thr Pro Phe Asn Leu Ser Cys Gln Ala Gln Gly Pro Pro 145 150 155 160 Glu Pro Val Thr Leu Leu Trp Leu Gln Asp Ala Val Pro Leu Ala Pro 165 170 175 Val Thr Gly His Ser Ser Gln His Ser Leu Gln Thr Pro Gly Leu Asn 180 185 190 Lys Thr Ser Ser Phe Ser Cys Glu Ala His Asn Ala Lys Gly Val Thr 195 200 205 Thr Ser Arg Thr Ala Thr Ile Thr Val Leu Pro Gln Arg Pro His His 210 215 220 Leu His Val Val Ser Arg Gln Pro Thr Glu Leu Glu Val Ala Trp Thr 225 230 235 240 Pro Gly Leu Ser Gly Ile Tyr Pro Leu Thr His Cys Asn Leu Gln Ala 245 250 255 Val Leu Ser Asp Asp Gly Val Gly Ile Trp Leu Gly Lys Ser Asp Pro 260 265 270 Pro Glu Asp Pro Leu Thr Leu Gln Val Ser Val Pro Pro His Gln Leu 275 280 285 Arg Leu Glu Lys Leu Leu Pro His Thr Pro Tyr His Ile Arg Ile Ser 290 295 300 Cys Ser Ser Ser Gln Gly Pro Ser Pro Trp Thr His Trp Leu Pro Val 305 310 315 320 Glu Thr Thr Glu Gly Val Pro Leu Gly Pro Pro Glu Asn Val Ser Ala 325 330 335 Met Arg Asn Gly Ser Gln Val Leu Val Arg Trp Gln Glu Pro Arg Val 340 345 350 Pro Leu Gln Gly Thr Leu Leu Gly Tyr Arg Leu Ala Tyr Arg Gly Gln 355 360 365 Asp Thr Pro Glu Val Leu Met Asp Ile Gly Leu Thr Arg Glu Val Thr 370 375 380 Leu Glu Leu Arg Gly Asp Arg Pro Val Ala Asn Leu Thr Val Ser Val 385 390 395 400 Thr Ala Tyr Thr Ser Ala Gly Asp Gly Pro Trp Ser Leu Pro Val Pro 405 410 415 Leu Glu Pro Trp Arg Pro Gly Gln Gly Gln Pro Leu His His Leu Val 420 425 430 Ser Glu Pro Pro Pro Arg Ala Phe Ser Trp Pro Trp Trp Tyr Val Leu 435 440 445 Leu Gly Ala Leu Val Ala Ala Ala Cys Val Leu Ile Leu Ala Leu Phe 450 455 460 Leu Val His Arg Arg Lys Lys Glu Thr Arg Tyr Gly Glu Val Phe Glu 465 470 475 480 Pro Thr Val Glu Arg Gly Glu Leu Val Val Arg Tyr Arg Val Arg Lys 485 490 495 Ser Tyr Ser Arg Arg Thr Thr Glu Ala Thr Leu Asn Ser Leu Gly Ile 500 505 510 Ser Glu Glu Leu Lys Glu Lys Leu Arg Asp Val Met Val Asp Arg His 515 520 525 Lys Val Ala Leu Gly Lys Thr Leu Gly Glu Gly Glu Phe Gly Ala Val 530 535 540 Met Glu Gly Gln Leu Asn Gln Asp Asp Ser Ile Leu Lys Val Ala Val 545 550 555 560 Lys Thr Met Lys Ile Ala Ile Cys Thr Arg Ser Glu Leu Glu Asp Phe 565 570 575 Leu Ser Glu Ala Val Cys Met Lys Glu Phe Asp His Pro Asn Val Met 580 585 590 Arg Leu Ile Gly Val Cys Phe Gln Gly Ser Asp Arg Glu Gly Phe Pro 595 600 605 Glu Pro Val Val Ile Leu Pro Phe Met Lys His Gly Asp Leu His Ser 610 615 620 Phe Leu Leu Tyr Ser Arg Leu Gly Asp Gln Pro Val Phe Leu Pro Thr 625 630 635 640 Gln Met Leu Val Lys Phe Met Ala Asp Ile Ala Ser Gly Met Glu Tyr 645 650 655 Leu Ser Thr Lys Arg Phe Ile His Arg Asp Leu Ala Ala Arg Asn Cys 660 665 670 Met Leu Asn Glu Asn Met Ser Val Cys Val Ala Asp Phe Gly Leu Ser 675 680 685 Lys Lys Ile Tyr Asn Gly Asp Tyr Tyr Arg Gln Gly Arg Ile Ala Lys 690 695 700 Met Pro Val Lys Trp Ile Ala Ile Glu Ser Leu Ala Asp Arg Val Tyr 705 710 715 720 Thr Ser Lys Ser Asp Val Trp Ser Phe Gly Val Thr Met Trp Glu Ile 725 730 735 Ala Thr Arg Gly Gln Thr Pro Tyr Pro Gly Val Glu Asn Ser Glu Ile 740 745 750 Tyr Asp Tyr Leu Arg Gln Gly Asn Arg Leu Lys Gln Pro Val Asp Cys 755 760 765 Leu Asp Gly Leu Tyr Ala Leu Met Ser Arg Cys Trp Glu Leu Asn Pro 770 775 780 Arg Asp Arg Pro Ser Phe Ala Glu Leu Arg Glu Asp Leu Glu Asn Thr 785 790 795 800 Leu Lys Ala Leu Pro Pro Ala Gln Glu Pro Asp Glu Ile Leu Tyr Val 805 810 815 Asn Met Asp Glu Gly Gly Ser His Leu Glu Pro Arg Gly Ala Ala Gly 820 825 830 Gly Ala Asp Pro Pro Thr Gln Pro Asp Pro Lys Asp Ser Cys Ser Cys 835 840 845 Leu Thr Ala Ala Asp Val His Ser Ala Gly Arg Tyr Val Leu Cys Pro 850 855 860 Ser Thr Ala Pro Gly Pro Thr Leu Ser Ala Asp Arg Gly Cys Pro Ala 865 870 875 880 Pro Pro Gly Gln Glu Asp Gly Ala 885 <210> 21 <211> 890 <212> PRT <213> Homo sapiens <400> 21 Met Ala Leu Arg Arg Ser Met Gly Arg Pro Gly Leu Pro Pro Leu Pro 1 5 10 15 Leu Pro Pro Pro Pro Arg Leu Gly Leu Leu Leu Ala Ala Leu Ala Ser 20 25 30 Leu Leu Leu Pro Glu Ser Ala Ala Ala Gly Leu Lys Leu Met Gly Ala 35 40 45 Pro Val Lys Leu Thr Val Ser Gln Gly Gln Pro Val Lys Leu Asn Cys 50 55 60 Ser Val Glu Gly Met Glu Glu Pro Asp Ile Gln Trp Val Lys Asp Gly 65 70 75 80 Ala Val Val Gln Asn Leu Asp Gln Leu Tyr Ile Pro Val Ser Glu Gln 85 90 95 His Trp Ile Gly Phe Leu Ser Leu Lys Ser Val Glu Arg Ser Asp Ala 100 105 110 Gly Arg Tyr Trp Cys Gln Val Glu Asp Gly Gly Glu Thr Glu Ile Ser 115 120 125 Gln Pro Val Trp Leu Thr Val Glu Gly Val Pro Phe Phe Thr Val Glu 130 135 140 Pro Lys Asp Leu Ala Val Pro Pro Asn Ala Pro Phe Gln Leu Ser Cys 145 150 155 160 Glu Ala Val Gly Pro Pro Glu Pro Val Thr Ile Val Trp Trp Arg Gly 165 170 175 Thr Thr Lys Ile Gly Gly Pro Ala Pro Ser Pro Ser Val Leu Asn Val 180 185 190 Thr Gly Val Thr Gln Ser Thr Met Phe Ser Cys Glu Ala His Asn Leu 195 200 205 Lys Gly Leu Ala Ser Ser Arg Thr Ala Thr Val His Leu Gln Ala Leu 210 215 220 Pro Ala Ala Pro Phe Asn Ile Thr Val Thr Lys Leu Ser Ser Ser Asn 225 230 235 240 Ala Ser Val Ala Trp Met Pro Gly Ala Asp Gly Arg Ala Leu Leu Gln 245 250 255 Ser Cys Thr Val Gln Val Thr Gln Ala Pro Gly Gly Trp Glu Val Leu 260 265 270 Ala Val Val Val Pro Val Pro Pro Phe Thr Cys Leu Leu Arg Asp Leu 275 280 285 Val Pro Ala Thr Asn Tyr Ser Leu Arg Val Arg Cys Ala Asn Ala Leu 290 295 300 Gly Pro Ser Pro Tyr Ala Asp Trp Val Pro Phe Gln Thr Lys Gly Leu 305 310 315 320 Ala Pro Ala Ser Ala Pro Gln Asn Leu His Ala Ile Arg Thr Asp Ser 325 330 335 Gly Leu Ile Leu Glu Trp Glu Glu Val Ile Pro Glu Ala Pro Leu Glu 340 345 350 Gly Pro Leu Gly Pro Tyr Lys Leu Ser Trp Val Gln Asp Asn Gly Thr 355 360 365 Gln Asp Glu Leu Thr Val Glu Gly Thr Arg Ala Asn Leu Thr Gly Trp 370 375 380 Asp Pro Gln Lys Asp Leu Ile Val Arg Val Cys Val Ser Asn Ala Val 385 390 395 400 Gly Cys Gly Pro Trp Ser Gln Pro Leu Val Val Ser Ser His Asp Arg 405 410 415 Ala Gly Gln Gln Gly Pro Pro His Ser Arg Thr Ser Trp Val Pro Val 420 425 430 Val Leu Gly Val Leu Thr Ala Leu Val Thr Ala Ala Ala Leu Ala Leu 435 440 445 Ile Leu Leu Arg Lys Arg Arg Lys Glu Thr Arg Phe Gly Gln Ala Phe 450 455 460 Asp Ser Val Met Ala Arg Gly Glu Pro Ala Val His Phe Arg Ala Ala 465 470 475 480 Arg Ser Phe Asn Arg Glu Arg Pro Glu Arg Ile Glu Ala Thr Leu Asp 485 490 495 Ser Leu Gly Ile Ser Asp Glu Leu Lys Glu Lys Leu Glu Asp Val Leu 500 505 510 Ile Pro Glu Gln Gln Phe Thr Leu Gly Arg Met Leu Gly Lys Gly Glu 515 520 525 Phe Gly Ser Val Arg Glu Ala Gln Leu Lys Gln Glu Asp Gly Ser Phe 530 535 540 Val Lys Val Ala Val Lys Met Leu Lys Ala Asp Ile Ile Ala Ser Ser 545 550 555 560 Asp Ile Glu Glu Phe Leu Arg Glu Ala Ala Cys Met Lys Glu Phe Asp 565 570 575 His Pro His Val Ala Lys Leu Val Gly Val Ser Leu Arg Ser Arg Ala 580 585 590 Lys Gly Arg Leu Pro Ile Pro Met Val Ile Leu Pro Phe Met Lys His 595 600 605 Gly Asp Leu His Ala Phe Leu Leu Ala Ser Arg Ile Gly Glu Asn Pro 610 615 620 Phe Asn Leu Pro Leu Gln Thr Leu Ile Arg Phe Met Val Asp Ile Ala 625 630 635 640 Cys Gly Met Glu Tyr Leu Ser Ser Arg Asn Phe Ile His Arg Asp Leu 645 650 655 Ala Ala Arg Asn Cys Met Leu Ala Glu Asp Met Thr Val Cys Val Ala 660 665 670 Asp Phe Gly Leu Ser Arg Lys Ile Tyr Ser Gly Asp Tyr Tyr Arg Gln 675 680 685 Gly Cys Ala Ser Lys Leu Pro Val Lys Trp Leu Ala Leu Glu Ser Leu 690 695 700 Ala Asp Asn Leu Tyr Thr Val Gln Ser Asp Val Trp Ala Phe Gly Val 705 710 715 720 Thr Met Trp Glu Ile Met Thr Arg Gly Gln Thr Pro Tyr Ala Gly Ile 725 730 735 Glu Asn Ala Glu Ile Tyr Asn Tyr Leu Ile Gly Gly Asn Arg Leu Lys 740 745 750 Gln Pro Pro Glu Cys Met Glu Asp Val Tyr Asp Leu Met Tyr Gln Cys 755 760 765 Trp Ser Ala Asp Pro Lys Gln Arg Pro Ser Phe Thr Cys Leu Arg Met 770 775 780 Glu Leu Glu Asn Ile Leu Gly Gln Leu Ser Val Leu Ser Ala Ser Gln 785 790 795 800 Asp Pro Leu Tyr Ile Asn Ile Glu Arg Ala Glu Glu Pro Thr Ala Gly 805 810 815 Gly Ser Leu Glu Leu Pro Gly Arg Asp Gln Pro Tyr Ser Gly Ala Gly 820 825 830 Asp Gly Ser Gly Met Gly Ala Val Gly Gly Thr Pro Ser Asp Cys Arg 835 840 845 Tyr Ile Leu Thr Pro Gly Gly Leu Ala Glu Gln Pro Gly Gln Ala Glu 850 855 860 His Gln Pro Glu Ser Pro Leu Asn Glu Thr Gln Arg Leu Leu Leu Leu 865 870 875 880 Gln Gln Gly Leu Leu Pro His Ser Ser Cys 885 890 <210> 22 <211> 823 <212> PRT <213> Homo sapiens <400> 22 Met Lys Ile Asn Asn Glu Glu Ile Val Ser Asp Pro Ile Tyr Ile Glu 1 5 10 15 Val Gln Gly Leu Pro His Phe Thr Lys Gln Pro Glu Ser Met Asn Val 20 25 30 Thr Arg Asn Thr Ala Phe Asn Leu Thr Cys Gln Ala Val Gly Pro Pro 35 40 45 Glu Pro Val Asn Ile Phe Trp Val Gln Asn Ser Ser Arg Val Asn Glu 50 55 60 Gln Pro Glu Lys Ser Pro Ser Val Leu Thr Val Pro Gly Leu Thr Glu 65 70 75 80 Met Ala Val Phe Ser Cys Glu Ala His Asn Asp Lys Gly Leu Thr Val 85 90 95 Ser Lys Gly Val Gln Ile Asn Ile Lys Ala Ile Pro Ser Pro Pro Thr 100 105 110 Glu Val Ser Ile Arg Asn Ser Thr Ala His Ser Ile Leu Ile Ser Trp 115 120 125 Val Pro Gly Phe Asp Gly Tyr Ser Pro Phe Arg Asn Cys Ser Ile Gln 130 135 140 Val Lys Glu Ala Asp Pro Leu Ser Asn Gly Ser Val Met Ile Phe Asn 145 150 155 160 Thr Ser Ala Leu Pro His Leu Tyr Gln Ile Lys Gln Leu Gln Ala Leu 165 170 175 Ala Asn Tyr Ser Ile Gly Val Ser Cys Met Asn Glu Ile Gly Trp Ser 180 185 190 Ala Val Ser Pro Trp Ile Leu Ala Ser Thr Thr Glu Gly Ala Pro Ser 195 200 205 Val Ala Pro Leu Asn Val Thr Val Phe Leu Asn Glu Ser Ser Asp Asn 210 215 220 Val Asp Ile Arg Trp Met Lys Pro Pro Thr Lys Gln Gln Asp Gly Glu 225 230 235 240 Leu Val Gly Tyr Arg Ile Ser His Val Trp Gln Ser Ala Gly Ile Ser 245 250 255 Lys Glu Leu Leu Glu Glu Val Gly Gln Asn Gly Ser Arg Ala Arg Ile 260 265 270 Ser Val Gln Val His Asn Ala Thr Cys Thr Val Arg Ile Ala Ala Val 275 280 285 Thr Lys Gly Gly Val Gly Pro Phe Ser Asp Pro Val Lys Ile Phe Ile 290 295 300 Pro Ala His Gly Trp Val Asp Tyr Ala Pro Ser Ser Thr Pro Ala Pro 305 310 315 320 Gly Asn Ala Asp Pro Val Leu Ile Ile Phe Gly Cys Phe Cys Gly Phe 325 330 335 Ile Leu Ile Gly Leu Val Leu Tyr Ile Ser Leu Ala Ile Arg Lys Arg 340 345 350 Val Gln Glu Thr Lys Phe Gly Asn Ala Phe Thr Glu Glu Asp Ser Glu 355 360 365 Leu Val Val Asn Tyr Ile Ala Lys Lys Ser Phe Cys Arg Arg Ala Ile 370 375 380 Glu Leu Thr Leu His Ser Leu Gly Val Ser Glu Glu Leu Gln Asn Lys 385 390 395 400 Leu Glu Asp Val Val Ile Asp Arg Asn Leu Leu Ile Leu Gly Lys Ile 405 410 415 Leu Gly Glu Gly Glu Phe Gly Ser Val Met Glu Gly Asn Leu Lys Gln 420 425 430 Glu Asp Gly Thr Ser Leu Lys Val Ala Val Lys Thr Met Lys Leu Asp 435 440 445 Asn Ser Ser Gln Arg Glu Ile Glu Glu Phe Leu Ser Glu Ala Ala Cys 450 455 460 Met Lys Asp Phe Ser His Pro Asn Val Ile Arg Leu Leu Gly Val Cys 465 470 475 480 Ile Glu Met Ser Ser Gln Gly Ile Pro Lys Pro Met Val Ile Leu Pro 485 490 495 Phe Met Lys Tyr Gly Asp Leu His Thr Tyr Leu Leu Tyr Ser Arg Leu 500 505 510 Glu Thr Gly Pro Lys His Ile Pro Leu Gln Thr Leu Leu Lys Phe Met 515 520 525 Val Asp Ile Ala Leu Gly Met Glu Tyr Leu Ser Asn Arg Asn Phe Leu 530 535 540 His Arg Asp Leu Ala Ala Arg Asn Cys Met Leu Arg Asp Asp Met Thr 545 550 555 560 Val Cys Val Ala Asp Phe Gly Leu Ser Lys Lys Ile Tyr Ser Gly Asp 565 570 575 Tyr Tyr Arg Gln Gly Arg Ile Ala Lys Met Pro Val Lys Trp Ile Ala 580 585 590 Ile Glu Ser Leu Ala Asp Arg Val Tyr Thr Ser Lys Ser Asp Val Trp 595 600 605 Ala Phe Gly Val Thr Met Trp Glu Ile Ala Thr Arg Gly Met Thr Pro 610 615 620 Tyr Pro Gly Val Gln Asn His Glu Met Tyr Asp Tyr Leu Leu His Gly 625 630 635 640 His Arg Leu Lys Gln Pro Glu Asp Cys Leu Asp Glu Leu Tyr Glu Ile 645 650 655 Met Tyr Ser Cys Trp Arg Thr Asp Pro Leu Asp Arg Pro Thr Phe Ser 660 665 670 Val Leu Arg Leu Gln Leu Glu Lys Leu Leu Glu Ser Leu Pro Asp Val 675 680 685 Arg Asn Gln Ala Asp Val Ile Tyr Val Asn Thr Gln Leu Leu Glu Ser 690 695 700 Ser Glu Gly Leu Ala Gln Gly Ser Thr Leu Ala Pro Leu Asp Leu Asn 705 710 715 720 Ile Asp Pro Asp Ser Ile Ile Ala Ser Cys Thr Pro Arg Ala Ala Ile 725 730 735 Ser Val Val Thr Ala Glu Val His Asp Ser Lys Pro His Glu Gly Arg 740 745 750 Tyr Ile Leu Asn Gly Gly Ser Glu Glu Trp Glu Asp Leu Thr Ser Ala 755 760 765 Pro Ser Ala Ala Val Thr Ala Glu Lys Asn Ser Val Leu Pro Gly Glu 770 775 780 Arg Leu Val Arg Asn Gly Val Ser Trp Ser His Ser Ser Met Leu Pro 785 790 795 800 Leu Gly Ser Ser Leu Pro Asp Glu Leu Leu Phe Ala Asp Asp Ser Ser 805 810 815 Glu Gly Ser Glu Val Leu Met 820 BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1

[0010] Monoclonal antibody (MAb) 1H12 and Axl in flow cytometry + Binding of triple negative breast cancer cell line MDA-MB-231. MAb 1H12 was conjugated with Alexa647 (Invitrogen) and incubated with MDA-MB-231 cells with Axl expression knockdown or with cells transfected with control shRNA. Cell staining was measured using an Accuri C6 flow cytometer (BD Biosciences). Knockdown levels were measured using geometric mean fluorescence intensity values.

[0011] Figure 2

[0012] Overlay of sensorgrams from binding assays showing interaction of MAb 1H12 with recombinant human (rh)Axl, rhMer and rhTyro3. Curves after subtraction of blank surface signal are shown.

[0013] Figure 3

[0014] Biacore analysis of the interaction of ligands (MAb 1H12 and rmGas6) with sensor chip CM5 coated with rhAxl, rmAxl and rhTyro3-Fc. The curves after subtraction of the blank surface signal are shown.

[0015] Figure 4

[0016] Kinetic analysis of the interaction of MAb 1H12 with rhAxl immobilized on the surface of a Biacore sensor chip. Overlays of sensorgrams of MAb 1H12 at different concentrations (1.3-666.7 nM) are shown. Accurate kinetic analysis was performed using BIA evaluation software and curve fitting was performed according to the model '1:1 mass transfer binding'. Affinity constants (dynamic and steady state) and calculated half-lives of antigen binding at 25°C are shown in the insert table.

[0017] Figure 5

[0018] Analysis of competition between MAb 1H12 (1st sample) and anti-Axl MAb 1H12, MAb 1-3, rhGas6 and rmGas6 (2nd samples) using Biacore 3000. Overlay of sensorgrams using different 2nd samples is shown. The starting points of injection of the 1st sample (1H12) and the 2nd sample are indicated by arrows.

[0019] Figure 6

[0020] Western blot analysis of anti-Axl MAb1H12 binding to recombinant human (rh) Mer-Fc and Axl-Fc antigens under reducing and non-reducing conditions. Lanes: M, molecular weight marker (magic marker), molecular weight values ​​in kDa are shown on the left; 1, rhAxl-Fc, non-reducing; 2, rhMer-Fc, non-reducing; 3, rhAxl-Fc, reduced; 4, rhMer-Fc, reduced. The protein band corresponding to rhAxl-Fc is indicated by an arrow.

[0021] Figure 7

[0022] Western blot analysis of the binding of anti-Axl MAb 1H12 to Axl+ and Axl- cell lysates under reducing and non-reducing conditions. Lanes: M, molecular weight marker (magic marker), molecular weight values ​​in kDa are shown on the left; 1, lysate of Axl-LNCaP cells (prostate adenocarcinoma), reduced; 2, lysate of Axl+NCl-H1299 (non-small cell lung cancer), reduced; 3, lysate of Axl+NCl-H1299, non-reducing. The protein band corresponding to the Axl receptor is indicated by an arrow.

[0023] Figure 8

[0024] Amino acid sequences of the VH and VL domains derived from the anti-Axl monoclonal antibody 1H12.

[0025] Fig. 9

[0026] Dose-dependent binding of anti-Axl mouse antibody 1H12 and its chimeric (mouse variable / human constant) counterpart to Axl-positive cells. Different concentrations of mouse (m 1H12) and chimeric (ch 1H12) antibodies were tested in flow cytometry for binding to the triple-negative breast cancer cell line MDA-MB-231. The bound mouse and chimeric antibodies were detected with APC-conjugated donkey F(ab')2 fragments specific for mouse IgG(H+L) (1:500 dilution) or human IgG(H+L) (1:300 dilution) (both from Jackson ImmunoResearch). Cell staining was measured using an Accuri C6 flow cytometer (BD Biosciences). MFI, geometric mean fluorescence intensity.

[0027] Fig.10

[0028] Kinetic analysis of chimeric MAb ch1H12 interacting with rhAxl immobilized on the surface of a Biacore sensor chip. Overlays of sensorgrams of MAb ch1H12 at different concentrations (1.3-666.7 nM) are shown. Accurate kinetic analysis was performed using BIA evaluation software and curve fitting was performed according to a 1:1 Langmuir binding model. Affinity constants (dynamic and steady state) and calculated half-lives of antigen binding at 25°C are shown in the insert table.

[0029] Fig.11

[0030] Biacore analysis of the interaction of murine antibody 1H12 with sensor chips coated with human Axl-Fc, cynomolgus Axl-Fc, and rhesus Axl-Fc.

[0031] Fig.12

[0032] Tumor cell killing using antibody-saporin conjugates. Unconjugated saporin and isotype control antibody (human IgG1) coupled to saporin (control SAP) were used as negative controls. The inset table shows the concentrations that resulted in 50% cell killing (EC 50 , pM) effective concentration.

[0033] Fig.13

[0034] Western blot analysis illustrating the agonistic activity of the 1H12 antibody cross-linked with a secondary anti-mouse antibody. 473 Phosphorylation of Akt was used as an alternative readout for Axl activity. Lanes: 1, molecular weight marker; 2, positive control (lysate from LNCaP cells of prostate adenocarcinoma); 3, lysate of HeLa cells after starvation; 4, HeLa cells after starvation and treated with cross-linked 1H12. 473 ) Immunoblots probed with total cell lysates.

[0035] Fig.14

[0036] Western blot analysis illustrating the dose-dependent agonistic activity of the 1H12 antibody cross-linked with a secondary anti-mouse antibody. 473 Phosphorylation of Akt was used as an alternative readout of Axl activity. Lanes: 1, molecular weight marker; 2, positive control (lysate from LNCaP cells of prostate adenocarcinoma); 3, lysate of HeLa cells after starvation; 4-7, HeLa cells treated with different doses of cross-linked 1H12 (0.2, 0.6, 2.0 and 6.0 μg / ml, respectively) after starvation. 473 ) Immunoblots probed with total cell lysates.

[0037] Fig.15

[0038] Western blot analysis demonstrating the agonistic activity of the 1H12 antibody alone. 473 Phosphorylation of Akt was used as an alternative readout for Axl activity. Lanes: 1, molecular weight marker; 2, positive control (lysate from LNCaP cells of prostate adenocarcinoma); 3, lysate of HeLa cells after starvation; 4, lysate of HeLa cells after starvation and additionally treated with Axl-Fc; 5, HeLa cells after starvation treated with cross-linked 1H12; 6, HeLa cells after starvation treated with cross-linked 1H12 alone. 473 ) Immunoblots probed with total cell lysates.

[0039] Fig.16

[0040] Analysis of competition between MAb 1H12 as the first sample and anti-Axl MAB154 or rmGas6 as the second sample using Biacore 3000. Overlays of sensorgrams using different second samples are shown. The starting points of injection of the first sample (1H12) and the second sample are indicated by arrows.

[0041] Fig.17

[0042] MAb 1H12, commercial antibody polyclonal AF154 and monoclonal MAB154 were used to stain the contrast IHC of Axl+ and Axl-(A) cells. Wild-type (wt) and Axl knock-down type MDA-MB-231 cells were used as Axl+ and Axl- cells, respectively. (B) Axl+ and Axl- cell lysates (wt and Axl knock-down type MDA-MB-231 cells) developed using MAb 1H12 or polyclonal AF154 and monoclonal MAB154 were analyzed by contrast Western blot. As a loading control, GAPDH detection was shown on each blot.

[0043] Disclosure of the Invention

[0044] The following sequences are disclosed herein (for complete sequences, see the 'Sequences' section below):

[0045] SEQ ID NO.1→Nucleotide sequence encoding 1H12 VH

[0046] SEQ ID NO.2→Nucleotide sequence encoding 1H12 VL

[0047] SEQ ID NO.3→Amino acid sequence encoding 1H12 VH

[0048] SEQ ID NO.4→Amino acid sequence encoding 1H12 VL

[0049] SEQ ID NO.5→Amino acid sequence encoding 1H12 VH CDR1

[0050] SEQ ID NO.6→Amino acid sequence encoding 1H12 VH CDR2

[0051] SEQ ID NO.7→Amino acid sequence encoding 1H12 VH CDR3

[0052] SEQ ID NO.8→Amino acid sequence encoding 1H12 VL CDR1

[0053] SEQ ID NO.9→Amino acid sequence encoding 1H12 VL CDR2

[0054] SEQ ID NO.10→Amino acid sequence encoding 1H12 VL CDR3

[0055] SEQ ID NO.11→Amino acid sequence encoding 1H12 VH FR1

[0056] SEQ ID NO.12→Amino acid sequence encoding 1H12 VH FR2

[0057] SEQ ID NO.13→Amino acid sequence encoding 1H12 VH FR3

[0058] SEQ ID NO.14→Amino acid sequence encoding 1H12 VH FR4

[0059] SEQ ID NO.15→Amino acid sequence encoding 1H12 VL FR1

[0060] SEQ ID NO.16→Amino acid sequence encoding 1H12 VL FR2

[0061] SEQ ID NO.17→Amino acid sequence encoding 1H12 VL FR3

[0062] SEQ ID NO.18→Amino acid sequence encoding 1H12 VL FR4

[0063] SEQ ID NO.19→Amino acid sequence encoding human Axl

[0064] SEQ ID NO.20→Amino acid sequence encoding mouse Axl

[0065] SEQ ID NO.21→Amino acid sequence encoding human Tyro3

[0066] SEQ ID NO.22→Amino acid sequence encoding human Mer

[0067] In one aspect, the present invention provides an isolated antibody that binds to Axl and comprises a 1H12 VH domain (SEQ ID NO: 3) and / or a 1H12 VL domain (SEQ ID NO: 4). Preferably, the bound Axl is human Axl.

[0068] Typically, although a single VH domain can be used to bind an antigen as discussed further below, a VH domain is paired with a VL domain to provide an antibody antigen binding site. In a preferred embodiment, a 1H12 VH domain (SEQ ID NO: 3) is paired with a 1H12 VL domain (SEQ ID NO: 4) such that an antibody antigen binding site is formed that includes both the 1H12 VH and VL domains. In other embodiments, a 1H12 VH domain is paired with a VL domain other than the 1H12 VL. Light chain promiscuity is well known in the art.

[0069] One or more CDRs may be taken from the 1H12 VH or VL domain and incorporated into a suitable framework. This is discussed further below. 1H12 VH CDRs 1, 2 and 3 are shown in SEQ ID Nos 5, 6 and 7, respectively. 1H12 VL CDRs 1, 2 and 3 are shown in SEQ ID Nos 8, 9 and 10, respectively.

[0070] In one aspect of the present invention, there is provided an antibody which binds to Axl and which comprises:

[0071] An antibody VH domain selected from the group consisting of a 1H12 VH domain (SEQ ID NO. 3) and a VH domain comprising a VH CDR3 having an amino acid sequence of SEQ ID NO. 7 and optionally one or more VH CDRs having an amino acid sequence selected from the group consisting of SEQ ID NO. 6 and SEQ ID NO. 5; and / or

[0072] An antibody VL domain selected from the group consisting of a 1H12 VL domain (SEQ ID NO.4) and a VL domain comprising one or more VLCDRs having an amino acid sequence selected from the group consisting of SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10.

[0073] For example, the antibody may comprise an antibody VH domain having a VH CDR having an amino acid sequence of SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7. The antibody may further comprise an antibody VL domain comprising a VL CDR having an amino acid sequence of SEQ ID NO. 8, SEQ ID NO. 9, and SEQ ID NO. 10.

[0074] In some embodiments, the antibody comprises: (i) an antibody VH domain comprising a VH CDR having an amino acid sequence of SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, and (ii) an antibody VL domain comprising a VL CDR having an amino acid sequence of SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10.

[0075] The antibody may comprise a 1H12 VH domain (SEQ ID NO. 3) and, optionally, further comprise a 1H12 VL domain (SEQ ID NO. 4).

[0076] Preferably, the antibody competes for binding to human Axl with the Axl binding domain of an antibody comprising the 1H12 VH domain (SEQ ID NO. 3) and the 1H12 VL domain (SEQ ID NO. 4).

[0077] According to another aspect of the present invention, variants of the VH and VL domains are provided, the sequences of which are listed herein and which can be used in antibodies to Axl and can be obtained by means of sequence changes or mutations and screening methods. The present invention also provides such methods.

[0078] As discussed, variable domain amino acid sequence variants of any VH and VL domains whose sequences are specifically disclosed herein may be employed in accordance with the present invention. Specific variants may include one or more amino acid sequence changes (additions, deletions, substitutions and / or insertions of amino acid residues), which may be less than about 20 changes, less than about 15 changes, less than about 10 changes, or less than about 5 changes, 4, 3, 2, or 1. Changes may be made in one or more framework regions and / or one or more CDRs.

[0079] The antibodies according to the present invention may be antibodies that compete with any antibody for binding to an antigen, the antibody binding to an antigen and comprising an antibody VH and / or VL domain disclosed herein or a VH CDR3 disclosed herein or a variant of any of these. That is, in some embodiments, the antibodies according to the present invention are antibodies that bind to the same epitope or an overlapping epitope as an antibody comprising an antibody VH and / or VL domain disclosed herein or a VH CDR3 disclosed herein or a variant of any of these. Competition between antibodies can be easily determined in vitro, for example using ELISA, using binding analysis in a Biacore 3000 machine (see, for example, Examples 15 and Fig.16 ), and / or by labeling an antibody with a specific reporter molecule that can be detected in the presence of an otherwise unlabeled antibody, making it possible to identify antibodies that bind to the same epitope or overlapping epitopes.

[0080] Thus, the present invention includes variants of any of the ones specifically disclosed herein, wherein the variants include one or more amino acid sequence changes in one or more framework regions and / or one or more CDRs. For example, the variant antibody may include no more than 4 sequence changes in any CDR, such as no more than 3, no more than 2, no more than 1 sequence change, or no sequence change in any CDR (such as CDR3 of the VH domain). The variant antibody may compete with the Axl binding domain of an antibody comprising the 1H12 VH domain (SEQ ID NO. 3) and the 1H12 VL domain (SEQ ID NO. 4) for binding to Axl (e.g., human Axl).

[0081] Therefore, another aspect of the present invention provides an antibody comprising a human antibody antigen binding site that competes with 1H12 for binding to human Axl.

[0082] Various methods are available in the art for obtaining antibodies against Axl that can compete with 1H12 for binding to Axl.

[0083] On the other hand, the present invention provides a method for obtaining one or more antibodies capable of binding to an antigen, the method comprising contacting the antibody library according to the present invention with the antigen, and selecting one or more antibody members in the library capable of binding to the antigen.

[0084] The library can be displayed on the surface of phage particles, each particle containing nucleic acid encoding the antibody VH variable domain, and optionally the displayed VL domain (if present), displayed on its surface.

[0085] After selecting an antibody capable of binding to an antigen and displayed on a phage particle, nucleic acid can be obtained from the phage particle displaying the selected antibody. Such nucleic acid can be used for subsequent production of antibodies or antibody VH variable domains (optionally antibody VL variable domains) by expressing a nucleic acid having a sequence taken from a nucleic acid of a phage particle displaying the selected antibody.

[0086] Antibody VH variable domains having the amino acid sequence of an antibody VH variable domain of said selected antibody may be provided in isolated form, as may antibodies comprising such VH domains.

[0087] The ability to bind Axl can be further tested, as well as the ability to compete with 1H12 for binding to Axl.

[0088] The antibodies according to the present invention can bind to Axl with the affinity of 1H12.

[0089] The antibodies of the invention may bind to mouse, rat, monkey, non-human primate and / or human Axl. Preferably, the antibody binds to human and monkey Axl. In some embodiments, the antibody specifically binds to primate Axl. For example, the antibody may specifically bind to human and monkey Axl. In one embodiment, the antibody specifically binds only to human Axl.

[0090] The antibody may be a chimeric, humanized or CDR-grafted anti-Axl antibody. For example, the antibody may be a chimeric human / mouse antibody.

[0091] The binding affinity and neutralization potency of different antibodies can be compared under appropriate conditions.

[0092] In addition to the antibody sequence, the antibodies according to the invention may comprise other amino acids, for example to form peptides or polypeptides, such as folded domains, or to confer other functional characteristics on the molecule in addition to the ability to bind an antigen.

[0093] The antibodies of the invention may carry a detectable label, or may be conjugated (eg, via a peptide bond or a linker) to a toxin (eg, a cytotoxin), an enzyme, or an organic moiety.

[0094] Those skilled in the art will be aware of many methods for chemically conjugating molecules to proteins. In one embodiment of the invention, the antibody may be conjugated to a detectable, fluorescent label, such as fluorescein isothiocyanate (FITC), or to a reporter enzyme such as horseradish peroxidase (HRP).

[0095] In a preferred embodiment, the antibody is conjugated to a cytotoxic drug to form an antibody-drug conjugate (ADC). When the antibody is for pharmaceutical use, the bond connecting the antibody and the drug is preferably stable in the circulation (e.g., blood circulation), but the conjugate is unstable once sequestered in the cell. Therefore, antibodies conjugated to immunoconjugates can be used in methods for treating (e.g.) cancer.

[0096] In a further aspect, the invention provides an isolated nucleic acid comprising a sequence encoding an antibody, VH domain and / or VL domain according to the invention, and provides a method for preparing the antibody, VH domain and / or VL domain of the invention, the method comprising expressing the nucleic acid under conditions that result in the production of the antibody, VH domain and / or VL domain, and recovering it.

[0097] The antibodies according to the invention can be used in methods for treating or diagnosing the human or animal body, such as methods for treating (which may include prophylactic treatment) a disease or condition in a human patient, the method comprising administering to the patient an effective amount of an antibody of the invention or a conjugate or drug conjugate thereof. Disorders that can be treated according to the invention include those discussed elsewhere herein.

[0098] The antibodies according to the invention can be used in imaging methods, for example to determine the presence or location of cells to which the antibody binds.

[0099] In another aspect, the present invention provides a diagnostic kit comprising an antibody according to the present invention and one or more reagents for determining the binding of the antibody to an antigen.

[0100] Another aspect of the invention provides nucleic acids encoding the antibody VH variable domain (SEQ ID NO: 3) and / or VL variable domain (SEQ ID NO: 4) disclosed herein, typically isolated. In some embodiments, the VH encoding nucleic acid has the sequence set forth in SEQ ID NO: 1. In some embodiments, the VL encoding nucleic acid has the sequence set forth in SEQ ID NO: 2.

[0101] Another aspect of the invention provides nucleic acids encoding the VH CDR or VL CDR sequences disclosed herein, in particular VH CDRs selected from SEQ ID NOs 5, 6 and 7 and VL CDRs selected from SEQ ID NOs 8, 9 or 10, most preferably 1H12 CDR3 (SEQ ID NO: 7), typically isolated.

[0102] Another aspect provides a host cell transformed with the nucleic acid of the invention.

[0103] In another aspect, a method of producing an antibody VH variable domain is provided, the method comprising causing expression of an encoding nucleic acid. Such a method may comprise culturing a host cell under conditions for producing the antibody VH variable domain.

[0104] As further aspects of the invention, similar methods of producing VL variable domains and antibodies comprising VH and / or VL domains are provided.

[0105] The production method may include the step of isolating and / or purifying the product.

[0106] The production method may include formulating the product into a composition including at least one additional component, such as a pharmaceutically acceptable excipient.

[0107] These and other aspects of the invention are described in more detail below.

[0108] Antibody properties

[0109] High affinity for Axl

[0110] The 1H12 antibody described herein binds to human Axl with high affinity. As described in Example 4, the 1H12 antibody was determined to have a binding affinity of 4.98×10 -11 M of K DThis is the lowest K value described so far for an anti-Axl antibody. D .

[0111] Unexpectedly, the chimeric MAb ch1H12 (see Examples 11 and Fig.10 ) also has a higher affinity, K D =1.10×10 -11 M; this number is 4.5-fold lower than that of the parental mouse antibody, which may be due to the V H and V L The domains are better oriented.

[0112] Thus, the antibodies described herein bind Axl with high affinity; preferably bind human Axl with high affinity. In some embodiments, the antibodies bind Axl (or human Axl), K D No more than 10 -6 M, if not greater than 5×10 -7 M, not more than 10 -7 M, not more than 5×10 -8 M, not more than 10 -8 M, not more than 5×10 -9 M, not more than 10 -9 M, not more than 5×10 -10 M, not more than 10 -10 M, not more than 5×10 -11 M, not more than 1.5×10 -11 M, not more than 10 -11 M, not more than 5×10 -12 M, not more than 10 -12 M, not more than 5×10 -13 M, not more than 10 -13 M, not more than 5×10 -14 M, not more than 10 -14 M, not more than 5×10 -15 M, not more than 10 -15 M.

[0113] In some embodiments, the antibody binds Axl (or human Axl), K D For 10 -8 M to 10 -10 M, 10 -10 M to 10 -12 , 10 -12 M to 10 -14 , or 10 -14 M to 10 -16 .

[0114] K can be determined and calculated as described in Example 4 D .

[0115] The 1H12 antibody described herein is characterized by having an extremely low off-rate (k 解离 Specifically, in Example 4, it was determined that the 1H12 antibody had an extremely low dissociation rate (k 解离 =1.07×10 -5 s -1 ).

[0116] Unexpectedly, the chimeric MAb ch1H12 (see Examples 11 and Fig.10 ) also has a lower dissociation rate, k 解离 =2.99×10 -6 s -1 ), which results in a half-life of 64.4 hours for the ch1H12 / Axl complex.

[0117] Thus, the antibodies described herein preferably bind to human Axl with a slow off-rate. In some embodiments, the antibodies bind to Axl (or human Axl), k 解离 No more than 10 -3 s -1 , for example, no more than 5×10 -4 s -1 , not more than 10 -4 s -1 , no more than 5×10 -5 s -1 , no more than 2×10 -5 s -1 , not more than 10 -5 s -1 , no more than 3×10 -6 s -1 , no more than 5×10 -6 s -1 , not more than 10 -6 s -1 , no more than 5×10 -7 s -1 , not more than 10 -7 s -1 , no more than 5×10 -8 s -1 , or not more than 10 -8 s -1 .

[0118] Specific binding

[0119] In general, the terms 'specificity' and 'specific binding' may be used to refer to situations where an antibody does not show any significant binding to molecules other than its specific binding partner. For example, an antibody that 'specifically binds' to human Axl will not show any significant binding to mouse Axl.

[0120] The term also applies, for example, when an antibody is specific for a particular epitope carried by a number of antigens, in which case an antibody that 'specifically binds' the epitope will be able to bind all the different antigens carrying the recognised epitope.

[0121] Typically, specificity can be determined by means of a binding assay such as an ELISA using a panel of antigens.

[0122] The 1H12 antibody described herein binds to human Axl with high specificity. This is demonstrated in the Examples, where it is shown that:

[0123] (1) In Example 2, 1H12 did not show significant binding to recombinant antigens derived from hMer and hTyro3 (other members of the human TAM receptor tyrosine kinase family).

[0124] (2) In Example 3, 1H12 binds strongly to human Axl, but shows no binding to mouse Axl (this is in contrast to the mouse Axl ligand, mouse Gas 6, which binds strongly to both mouse and human Axl, and (more weakly) to human Tyro3);

[0125] (3) In Example 9, 1H12 showed no binding or very little binding to the vast majority of the tested tissue samples.

[0126] Thus, the antibodies described herein preferably specifically bind to primate Axl. In some embodiments, the antibodies described herein specifically bind to human and monkey Axl. In one embodiment, the antibodies specifically bind only to human Axl.

[0127] In some embodiments of the invention, the antibodies described herein do not show significant binding to human Tyro3 and / or human Mer. In some embodiments, the antibodies described herein do not show significant binding to mouse Axl. In some embodiments, the antibodies described herein do not show significant binding to any of human Tyro3, ​​human Mer, or mouse Axl.

[0128] A skilled artisan can readily determine whether an antibody does not exhibit "significant binding" to an antigen using, for example, the techniques described in Examples 2 and 3. In some embodiments, if an antibody exhibits "significant binding" to an antigen at a level greater than 10 -3 M, for example, greater than 10 -2 M, greater than 10 -1 M, or K greater than 1M D If the antigen is not bound, it is considered to show no "significant binding" to the specific antigen. K can be determined and calculated as shown in Example 4. D .

[0129] In one aspect, the antibodies of the invention bind to the same epitope as the 1H12 antibody, or to an epitope that overlaps with the epitope bound by the 1H12 antibody. Competition between different antibodies can be easily determined in vitro, for example using ELISA and / or by labeling a specific reporter molecule to a binding member that can be detected in the presence of other unlabeled antibodies, so that it is possible to identify antibodies that bind to the same epitope or overlapping epitopes.

[0130] Antibody internalization

[0131] The 1H12 antibody described herein exhibits good cellular internalization after binding to its target Axl. Internalization was also observed when the antibody was conjugated to a cytotoxin such as saporin (see Examples 13 and Fig.12 ).

[0132] Therefore, the antibody of the present invention or its conjugate is preferably internalized after binding to Axl present on the cell surface.

[0133] Usefulness in Axl detection

[0134] The unexpectedly good binding properties of the antibodies described herein make them particularly useful in applications involving Axl detection. For example, comparative testing has demonstrated that the 1H12 antibody produces significantly stronger signals than commercial anti-Axl antibodies in both immunohistochemistry and Western blot applications for both AF154 and MAB154 (see Examples 16 and Fig.17 ); Competition analysis showed that 1H12 and MAB154 bind to the same or overlapping epitopes (see Fig.16 ). This stronger signal and enhanced Axl detection sensitivity provides significant advantages in detection and analytical assays.

[0135] Agonism of Axl signaling

[0136] The 1H12 antibody described herein can induce Axl signaling when bound to Axl. This is shown in Examples 14 and Figure 13-15 This is demonstrated in the case of 473 As measured by phosphorylation of the Axl effector Akt, 1H12 binding was seen to induce strong Axl signaling in a dose-dependent manner.

[0137] Thus, in one aspect the antibodies described herein agonize Axl signaling; that is, the antibodies described herein are preferably Axl agonists.

[0138] In another aspect, the invention provides antibodies that bind to the same epitope or an overlapping epitope as an antibody comprising an antibody VH and / or VL domain disclosed herein or a VH CDR3 disclosed herein or a variant of any of these, wherein the antibody is an Axl agonist. As described herein, binding to the same or overlapping epitope can be readily determined in vitro by competition studies.

[0139] In some embodiments, Axl signaling is at least 10% stronger in the presence of an antibody of the invention than in the presence of a non-Axl binding control antibody, for example, at least 10% stronger, at least 20% stronger, at least 30% stronger, at least 40% stronger, at least 50% stronger, at least 60% stronger, at least 70% stronger, at least 80% stronger, at least 90% stronger, at least 100% stronger, at least 200% stronger, at least 500% stronger, or at least 1000% stronger in the presence of an antibody of the invention than in the presence of a non-Axl binding control antibody. As described in Example 14 herein, the level of Axl signaling can be measured by measuring Ser 473 The phosphorylation of Akt on Axl was measured.

[0140] Downregulation of Axl expression and / or activity

[0141] In some embodiments, the anti-Axl antibody induces downregulation of Axl receptor expression on the surface of a cell (eg, the surface of a tumor cell).

[0142] In some embodiments, cell surface Axl expression is reduced to less than 80% of Axl cell surface expression without Axl antibody treatment. In some embodiments, cell surface Axl expression is reduced to less than 70%, less than 60%, less than 50% or less than 40% of Axl cell surface expression without Axl antibody treatment.

[0143] In some embodiments, total Axl expression in a cell (e.g., a tumor cell) is reduced to less than 80% of total Axl expression without Axl antibody treatment. In some embodiments, total Axl expression is reduced to less than 70%, less than 60%, less than 50%, or less than 40% of total Axl expression without Axl antibody treatment. In some embodiments, downregulation of Axl expression occurs rapidly and persists for at least 24 hours.

[0144] In some embodiments, the anti-Axl antibody inhibits constitutive Axl activity.

[0145] In some embodiments, the anti-Axl antibody inhibits Axl activity.

[0146] In some embodiments, an anti-Axl antibody promotes cell death, e.g., by apoptosis, of tumor cells, such as A549 tumor cells; this can be measured, e.g., by BrdU incorporation assays, MTT, [3 H]-thymidine incorporation (e.g., TopCount assay (PerkinElmer)), cell viability assays (e.g., CellTiter-Glo (Promega)), DNA fragmentation assays, caspase activation assays, trypan blue exclusion, chromatin morphology assays, and the like.

[0147] In some embodiments, the anti-Axl antibody inhibits Axl downstream signaling. In some embodiments, the anti-Axl antibody inhibits Gas6-dependent cell proliferation.

[0148] In some embodiments, the anti-Axl antibody inhibits inflammatory cytokine expression by tumor-associated macrophages.

[0149] In some embodiments, anti-Axl antibodies inhibit tumor growth and / or metastasis by modulating tumor stromal function.

[0150] definition

[0151] Antibody

[0152] This term describes natural or partially or completely synthesized immunoglobulins. The term also includes any polypeptide or protein comprising an antibody antigen-binding domain. Antibody fragments comprising antibody antigen-binding domains include full antibodies (e.g., IgG antibodies comprising VH, CH1, CH2, CH3, VL and CL domains arranged in a standard manner) or full antibodies that retain their binding activity to target antigens. Such fragments include Fv (variable fragment), Fab (antibody binding fragment) and F (ab') 2 fragments, as well as single-chain Fv antibodies (scFv), dsFv, mini antibodies, double antibodies (diabodies), single-chain double antibodies, tandem scFv, TandAb, double-chain antibodies, three-chain antibodies, κ (λ) antibodies, BiTE, DVD-1g, SIP, SMIP or DART. In addition, antibodies and fragments thereof can be humanized antibodies, such as described in EP239400A. For example: monoclonal and polyclonal antibodies, recombinant antibodies, proteolytic and recombinant fragments of antibodies (Fab, Fv, scFv, diabodies), single domain antibodies (VHH, sdAb, nanobodies, IgNAR, VNAR) and proteins unrelated to antibodies that have been engineered to have antibody-like specific binding (antibody mimetics), such as the following, but not limited to:

[0153]

[0154] The antibody may comprise all or a portion of an antibody heavy chain constant region and / or an antibody light chain constant region.

[0155] Monoclonal and other antibodies may be obtained and engineered antibodies or chimeric molecules that retain the specificity of the original antibody may be produced using recombinant DNA technology. Such technology may involve connecting DNA fragments of immunoglobulin variable regions or complementary determining regions (CDRs) encoding antibodies and genes encoding immunoglobulin constant regions or constant regions plus framework regions of different immunoglobulins. See, for example, EP-A-184187, GB 2188638A or EP-A-239400. Hybridomas or other cells that produce antibodies may undergo genetic mutations or other changes that may or may not change the binding specificity of the produced antibodies.

[0156] Because antibodies can be modified in many ways, the term "antibody molecule" should be interpreted as including any polypeptide or other molecule having an antibody-derived antigen binding domain with the desired specificity. Thus, this term includes antibody fragments and derivatives, including any polypeptide containing an immunoglobulin binding domain, either natural or fully or partially synthesized. Thus, chimeric molecules containing an immunoglobulin binding domain fused to another polypeptide or an equivalent are included. The cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023.

[0157] It has been demonstrated that fragments of whole antibodies can perform the function of binding to antigens. Examples of binding fragments are (i) Fab fragments consisting of VL, VH, CL and CH1 domains; (ii) Fd fragments consisting of VH and CH1 domains; (iii) Fv fragments consisting of VL and VH domains of a single antibody; (iv) dAb fragments consisting of VH domains (Ward, ES et al., Nature 341, 544-546 (1989)); (v) isolated CDR regions; (vi) F(ab')2 fragments, i.e., bivalent fragments comprising two linked Fab fragments; (vii) single-chain Fv molecules (scFv), in which the VH domain and the VL domain are connected by a peptide linker that allows the two domains to associate to form an antigen binding site (Bird et al., Science, 242, 423-426, 1988; Huston et al., PNAS USA, 85, 5879-5883, 1988); (viii) bispecific single chain Fv dimers (PCT / US92 / 09965) and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO94 / 13804; P. Holliger et al., Proc. Natl. Acad. Sci. USA 90, 6444-6448, 1993). Fv, scFv or diabody molecules can be stabilized by incorporating disulfide bridges connecting the VH and VL domains (Y. Reiter et al., Nature Biotech, 14, 1239-1245, 1996). Minibodies comprising scFvs linked to a CH3 domain can also be produced (S. Hu et al., Cancer Res., 56, 3055-3061, 1996).

[0158] Antibodies may be bispecific or multispecific. When bispecific antibodies are to be used, these may be conventional bispecific antibodies, which may be manufactured in various ways (Holliger, P. and Winter G. Current Opinion Biotechnol. 4, 446-449 (1993)), such as chemical preparation or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above. Diabodies and scFvs may be constructed without the use of Fc regions, using only variable domains, which may reduce side effects, such as side effects caused by antibody effector functions, or reduce human anti-mouse antibody (HAMA) responses in the case of using antibodies of murine origin.

[0159] Completely different from bispecific full antibodies, bispecific diabodies can also be particularly useful because they can be easily constructed and expressed in bacteria (e.g., Escherichia coli). Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificity can be easily selected from antibody libraries using phage display (WO94 / 13804). If one arm of the diabody is to be kept constant, for example, with specificity for Axl, a library in which the other arm is changed can be generated and antibodies of appropriate specificity can be selected. Bispecific full antibodies can be prepared by "knobs into holes" engineering (JBBRidgeway et al., Protein Eng., 9, 616-621, 1996).

[0160] Antigen binding domain

[0161] This describes the part of the antibody molecule that contains a part or all of the antigen that recognizes and specifically binds to and is complementary to it. When the antigen is large, the antibody may only bind to a specific part of the antigen, which is called an epitope. The antigen binding domain can be provided by one or more antibody variable domains (e.g., a so-called Fd antibody fragment consisting of a VH domain). Preferably, the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0162] Specific protein

[0163] Axl

[0164] As used herein, 'human Axl' refers to the Axl member of the human TAM receptor tyrosine kinase family. In some embodiments, the human Axl polypeptide corresponds to Genbank accession number AAH32229, version number AAH32229.1 GI: 21619004, record update date: March 6, 2012 01:18 pm (SEQ ID NO.19). In one embodiment, the nucleic acid encoding the human Axl polypeptide corresponds to Genbank accession number M76125, version number M76125.1 GI: 292869, record update date: June 23, 2010 08:53 am.

[0165] Rat Axl

[0166] As used herein, 'murine Axl' refers to the Axl member of the murine TAM receptor tyrosine kinase family. In some embodiments, the murine Axl polypeptide corresponds to Genbank Accession No. AAH46618, Version No. AAH46618.1 GI: 55777082, Record Update Date: March 6, 2012 01:36 PM (SEQ ID NO. 20). In one embodiment, the nucleic acid encoding the murine Axl polypeptide corresponds to Genbank Accession No. NM_009465, Version No. NM_009465.4 GI: 300794836, Record Update Date: March 12, 2014 03:52 PM.

[0167] Tyro3

[0168] As used herein, 'human Tyro3' refers to the Tyro3 member of the human TAM receptor tyrosine kinase family. In some embodiments, the human Tyro3 polypeptide corresponds to Genbank Accession No. Q06418, Version No. Q06418.1 GI: 1717829, Record Update Date: April 22, 2014 12:07 PM (SEQ ID NO.21). In one embodiment, the nucleic acid encoding the human Tyro3 polypeptide corresponds to Genbank Accession No. BC051756, Version No. BC051756.1 GI: 30704372, Record Update Date: March 6, 2010 01:43 PM.

[0169] Mer

[0170] As used herein, 'human Mer' refers to the Mer member of the human TAM receptor tyrosine kinase family (official name = MERTK, Uniprot ID = Q12866). In some embodiments, the human Mer polypeptide corresponds to Genbank accession number AAI14918, version number AAI14918.1 GI: 109732052, record update date: March 6, 2012 04:21 pm (SEQ ID NO. 22). In one embodiment, the nucleic acid encoding the human Mer polypeptide corresponds to Genbank accession number NM_006343, version number NM_006343.2 GI: 66932917, record update date: March 16, 2014 08:52 pm.

[0171] BSA

[0172] As used herein, 'BSA' refers to bovine serum albumin. In some embodiments, BSA corresponds to Genbank Accession No. CAA76847, Version No. CAA76847.1 GI:3336842, Record Update Date: January 7, 2011 02:30 PM.

[0173] Include

[0174] This is usually used in the sense of "comprising", ie allowing for the presence of one or more features or components.

[0175] Separate

[0176] This refers to the state that the antibody of the present invention or the nucleic acid encoding such antibody usually presents according to the present invention. Antibodies and nucleic acids will not contain or substantially contain substances naturally associated therewith, such as other polypeptides or nucleic acids found together therewith in their natural environment or their preparation environment (e.g., cell culture) (when such preparation is by recombinant DNA technology practiced in vitro or in vivo). Antibodies and nucleic acids can be formulated with diluents or adjuvants and can still be separated in fact-for example, antibodies will usually be mixed with gelatin or other carriers when used for coating microtiter plates for immunoassays, or will be mixed with pharmaceutically acceptable carriers or diluents when used for diagnosis or treatment. Antibodies can be naturally or glycosylated by heterologous eukaryotic cells (e.g., CHO or NS0 (ECACC 85110503) cells) systems, or they can be non-glycosylated (e.g., if produced in prokaryotic cells).

[0177] Basically as List

[0178] By 'substantially as listed' it is meant that the relevant CDR or VH or VL domain of the invention will be identical or highly similar to the specified region whose sequence is listed herein. By "highly similar", it is expected that 1 to 5, preferably 1 to 4, such as 1 to 3 or 1 or 2 or 3 or 4 amino acid substitutions may occur in the CDR and / or VH or VL domain.

[0179] The backbone of the CDR

[0180] The structure for carrying the CDR of the present invention will generally have an antibody heavy chain or light chain sequence or a substantial portion thereof, wherein the CDR is located at the position of the CDR of the naturally occurring VH and VL antibody variable domains encoded by the corresponding rearranged immunoglobulin gene. The structure and position of the immunoglobulin variable domain can be determined by reference to (Kabat, EA et al., Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987 and updates thereof, now available on the Internet (http: / / immuno.bme.nwu.edu or use any search engine to find "Kabat").

[0181] The variable domains used in the present invention can be obtained from any germline or rearrangement mouse or people's variable domains, or can be synthetic variable domains based on the consensus sequence of known mouse or people's variable domains. Recombinant DNA technology can be used to introduce CDR sequences of the present invention (e.g., CDR3) into the variable domain library lacking CDR (e.g., CDR3).

[0182] For example, Marks et al. (Bio / Technology, 1992, 10:779-783) described a method for generating an antibody variable domain library, wherein a common primer directed to or adjacent to the 5'-end of the variable domain is used together with a common primer for the third framework region of the human VH gene to provide a VH variable domain library lacking CDR3. Marks et al. also described how to combine the library with the CDR3 of a specific antibody. Using similar techniques, the sequences derived from the CDR3 of the present invention can be shuffled through a VH or VL domain library lacking CDR3, and the shuffled complete VH or VL domain is combined with a cognate VL or VH domain to provide the antibodies of the present invention. The library can then be displayed in a suitable host system, such as the phage display system of WO92 / 01047, so that suitable antibodies can be selected. The library can be composed of 10 4 Individual antibodies, for example, 10 6 Up to 10 8 or 10 10 any part of an antibody.

[0183] Similar shuffling or combinatorial techniques were also disclosed by Stemmer (Nature, 1994, 370:389-391). Stemmer described DNA shuffling techniques for the beta-lactamase gene, but observed that the method could be used to generate antibodies.

[0184] Another alternative is to use random mutagenesis of one or more selected VH and / or VL genes to generate mutations throughout the variable domain, generating new VH or VL regions carrying sequences derived from the CDRs of the invention. Such methods are described by Gram et al. (1992, Proc. Natl. Acad. Sci., USA, 89: 3576-3580), who used error-prone PCR.

[0185] Another method that can be used is to direct mutagenesis to the CDR regions of the VH or VL genes. Such techniques are disclosed by Barbas et al. (1994, Proc. Natl. Acad. Sci., USA, 91: 3809-3813) and Schier et al. (1996, J. Mol. Biol. 263: 551-567).

[0186] The above techniques are all known as in the art and do not themselves form part of the present invention. A skilled person will be able to use such techniques to provide the antibodies of the present invention using routine methods in the art.

[0187] Epitope-specific antibodies

[0188] Another aspect of the present invention provides a method for obtaining an antibody specific for an Axl epitope, the method comprising providing a VH domain that is an amino acid sequence variant of the VH domain by addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of the VH domain listed herein, optionally combining the VH domain thus provided with one or more VL domains, and testing the VH domain or VH / VL combination to identify an antibody or antibody antigen-binding domain that is specific for Axl. The VL domain may have an amino acid sequence substantially as listed herein.

[0189] A similar approach may be employed, wherein one or more sequence variants of the VL domains disclosed herein are combined with one or more VH domains.

[0190] Another aspect of the present invention provides a method for preparing an antibody specific to Axl, the method comprising:

[0191] (a) supply A starting nucleic acid library, wherein the starting nucleic acid library coding A VH domain comprising the CDR3 to be replaced or lacking the CDR3 coding region;

[0192] (b) combining the library with a donor nucleic acid encoding an amino acid sequence substantially as set forth herein for VH CDR3, such that the donor nucleic acid inserts into the CDR3 region of the library, so as to provide a library of product nucleic acids encoding VH domains;

[0193] (c) expressing nucleic acids of the library;

[0194] (d) selecting an antibody specific for Axl; and

[0195] (e) recovering the antibody and / or nucleic acid encoding the antibody.

[0196] Likewise, a similar approach can be employed wherein a VL CDR3 of the invention is combined with a starting nucleic acid library encoding a VL domain that includes the CDR3 to be replaced or lacks the CDR3 encoding region.

[0197] Similarly, one or more or all three CDRs can be grafted into a VH or VL domain library, and the library screened for one or more antibodies specific for Axl.

[0198] The variable domain of immunoglobulin is comprised of at least three CDR districts and its insertion framework district.Preferably, this part also will comprise at least about 50% of any one or two in the first and fourth framework districts, and this 50% is the C-terminal 50% of the first framework district and the N-terminal 50% of the fourth framework district.Other residues at the N-terminal and C-terminal places of the variable domain of a large part can be those residues that are usually unrelated to the naturally occurring variable domain district.For example, the construction of the antibody of the present invention made by recombinant DNA technology can result in the introduction of the N or C-terminal residues encoded by the introduced joint to facilitate cloning or other manipulation steps.Other manipulation steps include introducing joints to connect the variable domain of the present invention to other protein sequences including immunoglobulin heavy chain, other variable domains (for example, in double antibody production) or as discussed in more detail below protein markers.

[0199] Although in preferred aspects of the invention antibodies comprising a pair of VH and VL domains are preferred, single binding domains based on VH or VL domain sequences form other aspects of the invention.It is known that single immunoglobulin domains, particularly VH domains, are capable of binding to a target antigen in a specific manner.

[0200] In the case of either single binding domain, these domains can be used to screen for complementary domains capable of forming two-domain antibodies capable of binding Axl.

[0201] This can be achieved by phage display screening using a so-called hierarchical double combinatorial approach as disclosed in WO92 / 01047, wherein a single colony containing either the H or L chain clone is used to infect the entire library of clones encoding the other chain (L or H) and the resulting double-chain antibodies are selected according to phage display techniques such as those described in that reference. This technique is also disclosed in Marks et al., supra.

[0202] The antibodies of the present invention may also include an antibody constant region or a portion thereof. For example, the antibodies of the present invention may include CL, CH1, CH2 and / or CH3 domains (or any combination thereof). The VL domain may be attached at its C-terminus to an antibody light chain constant domain comprising a human Cκ or Cλ chain, preferably a Cκ chain. Similarly, antibodies based on the VH structure may be attached at its C-terminus to an entire immunoglobulin heavy chain or a portion thereof derived from any antibody isotype, such as IgG, IgA, IgE and IgM and any isotype subclass. Fc regions such as Δnab and Δnac disclosed in WO99 / 58572 may be used.

[0203] Chimeric, humanized and CDR-grafted antibodies

[0204] As used herein, a "chimeric" antibody or a "humanized" antibody or a "CDR-grafted" antibody includes any combination of an anti-Axl antibody described herein or any CDR derived therefrom with one or more proteins or peptides derived from a non-murine, preferably human, antibody.

[0205] Chimeric or humanized antibodies include those in which the CDRs are derived from one or more anti-Axl antibodies described herein and at least a portion or the remainder of the antibody is derived from one or more human antibodies. Thus, the human portion of the antibody may include a framework, CL (e.g., Cκ or Cλ), CH domains (e.g., CH1, CH2, CH3), hinge regions (which are substantially non-immunogenic in humans).

[0206] The antibody region derived from a human antibody does not need to have 100% identity with a human antibody. In a preferred embodiment, as few mouse amino acid residues as possible are retained in order to neglect immunogenicity, but mouse residues may be retained if necessary to support the antigen binding site formed by the CDR, while increasing the humanization of the antibody to a maximum. Relative to non-modified antibodies, such changes or modifications are optional and preferably maintain or reduce immunogenicity in humans or other species.

[0207] It should be noted that humanized antibodies can be produced by non-human animals or prokaryotic or eukaryotic cells that can express functionally rearranged human immunoglobulin (e.g., heavy chain and / or light chain) genes. Further, when the antibody is a single-chain antibody, it may be included in a linker peptide not found in natural human antibodies. For example, scFv may include a linker peptide, such as two to about twenty glycine or other amino acid residues (preferably glycine and serine residues (e.g., Gly4Ser or Gly2Ser repeats)), which connects the variable region of the heavy chain and the variable region of the light chain. Such linker peptides are considered to be non-immunogenic in people. In some embodiments, the linker length is at least 12 amino acids.

[0208] Antibody humanization can be performed by, for example, synthesizing a combinatorial library of all six CDRs of a non-human target monoclonal antibody fused in frame with many individual human frameworks. A human framework library containing genes representing all known heavy and light chain human germline sequences can be used. The resulting combinatorial library can then be screened to bind to the target antigen. This method allows selection of the most favorable full human framework combination in terms of maintaining binding activity with the parent antibody. The humanized antibody can then be further optimized by various techniques.

[0209] For full-length antibody molecules, immunoglobulin genes can be obtained from genomic DNA or mRNA of hybridoma cell lines. Antibody heavy and light chains are cloned in mammalian vector systems. Assembly is confirmed by sequencing using methods known in the art. Antibody constructs can be expressed in other human or mammalian host cell lines. The construct can then be verified by transient transfection assays and Western blot analysis of target expressed antibodies. Rapid assays can be used to separate and screen stable cell lines with the highest productivity.

[0210] The human genes encoding the constant region (C) of humanized antibodies, fragments and regions can be derived from human fetal liver libraries by known methods. Human C region genes can be derived from any human cell, including cells that express and produce human immunoglobulins. Human CH regions can be derived from human heavy chains of any known class or isotype, including γ, μ, α, δ, ε and subclasses thereof, such as G1, G2, G3 and G4. Because the heavy chain isotype is responsible for the various effector functions of antibodies, the selection of CH domains will be guided by the desired effector functions, such as complement fixation or activity in antibody-dependent cellular toxicity (ADCC). Preferably, the CH domain is derived from γ1 (IgG1).

[0211] The human CL region may be derived from any human L chain isotype, kappa or lambda, with kappa being preferred.

[0212] The gene encoding the human immunoglobulin C region is obtained from human cells by standard cloning techniques (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) and Ausubel et al., eds., Current Protocols in Molecular Biology (1987-1993)). The human C region gene can be easily obtained from a known clone containing genes representing two classes of light chains, five classes of heavy chains and their subclasses.

[0213] Chimeric antibody fragments, such as Fab and F(ab')2, can be prepared by designing chimeric heavy chain genes that are appropriately truncated. For example, a chimeric gene encoding the heavy chain portion of a F(ab')2 fragment would include DNA sequences encoding the CH1 domain and hinge region of the heavy chain, followed by a translation stop codon to obtain a truncated molecule.

[0214] Methods for engineering or humanizing non-human or human antibodies can be used and are well known in the art. Typically, humanized or engineered antibodies have one or more amino acid residues from a non-human source, such as, but not limited to, mouse, rat, rabbit, non-human primate or other mammal. These human amino acid residues are often referred to as "import" residues, which are typically taken from "import" variable, constant domains or other domains of known human sequences. Known human Ig sequences are publicly available, for example at www.ncbi.nlm.nih.gov / entrez / query.fcgi; www.atcc.org / phage / hdb.html; www.sciquest.com / ; www.abcam.com / ; www.antibodyresource.com / onlinecomp.html; www.public.iastate.edu / .about.pedro / research_tools.html; www.mgen.uni-heidelberg.de / SD / IT / IT.html; www.whfreeman.com / immunology / CH05 / kuby05.htm; www.library.thinkquest.org / 12429 / lmmune / Antibody.html; www.hhmi.org / grants / lectures / 1996 / vlab / ; www.path.cam.ac.uk / .about.mrc7 / mike images.html; www.antibodyresource.com / ; mcb.harvard.edu / BioLinks / lmmunology.html.www.immunologylink.com / ; pathbox.wustl.edu / .about.hcenter / index.html; www.biotech.ufl.edu / .about.hcl / ; www.pebio.com / pa / 340913 / 340913.htm l; www.nal.usda.gov / awic / pubs / antibody / ; www.m.ehime-u.ac.jp / .about.yasuhito / Elisa.html; www.biodesign.com / table.asp; www.icnet.uk / axp / facs / davies / links.html; www.biotech.ufi.edu / .about.fccl / protocol.html; www.isac-net.org / sites_geo.html;aximt1.imt.uni-marburg.de / .about.rek / AEPStart.htmlbaserv.uci.only.nI / .about.jraats / links1.html;www.recab.uni-hd.de / immuno.bme.nwvu.edu / www.mrc-cpe.cam.ac.uk / imt-doc / public / INTRO.html;ww w.ibt.unam.mx / vir / V_mice.html:imgt.cnusc.fr:8104 / www.biochem.ucl.ac.uk / .about.martin / abs / index.html:a ntibody.bath.ac.uk / abgen.cvm.tamu.edu / lab / wwwabgen.html / www.unizh.ch / .about.honegger / AHOseminar / Slide. 0 1.html / www.cryst.bbk.ac.uk / .about.ubcg07s / www.nimr.mrc.ac.uk / CC / ccaewg / ccaewg.htm / www.path. cam.ac.uk / .about.mrc7 / humanization / TAHHP.html;www.ibt.unam.mx / vir / structure / stat__aim.html;ww w.biosci.missouri.edu / smithgp / index.html;www.cryst.bioc.cam.ac.uk / .about.fmolina / Web-pages / Pe pt / spottech.htmlwww.jerini.de / fr__products.htmwww.patents.ibm.con / ibm.html.KabatPeopleSequences of Proteins of Immunological Interest,USDept.Health(1983).

[0215] As known in the art, such imported sequences can be used to reduce immunogenicity or reduce, enhance or change binding, affinity, association rate, dissociation rate, avidity, specificity, half-life or any other suitable characteristics. Typically, when the non-human sequences of the variable and constant regions are replaced by people or other amino acids, some or all of the non-human or human CDR sequences are maintained.

[0216] Antibodies can also be optionally humanized, maintaining high affinity to antigen and other favorable biological properties. In order to achieve this goal, optionally humanized antibodies can be prepared by using the three-dimensional model analysis of parental sequences and the process of various conceptual humanized products of parental and humanized sequences. The three-dimensional immunoglobulin model is usually available and is familiar to those skilled in the art. The computer program that illustrates and displays the possible three-dimensional conformational structure of selected candidate immunoglobulin sequences is available. Inspection of these displays allows analysis of the possible effects of residues on the function of candidate immunoglobulin sequences, i.e., analyzing the residues that affect the ability of candidate immunoglobulin to bind its antigen. Like this, FR residues can be selected and by a total and imported sequence combination, so as to realize desired antibody characteristics, such as the affinity increased to the target antigen.

[0217] In general, the CDR residues are directly and most significantly involved in influencing antigen binding. Humanized or engineered antibodies can be performed using any known method, such as, but not limited to, those described in Winter et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 333:334 (1989); 239:1534 (1988)), Sims et al., J. Immunol. 151:2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), U.S. Pat. Nos. 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192 , 5,723,323, 5,766,886, 5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539; 4,816,567, PCT / :US98 / 162 80. US96 / 18978, US91 / 09630, US91 / 05939, US94 / 01234, GB89 / 01334, GB91 / 01134, GB92 / 01755; WO90 / 14443, WO90 / 14424, WO90 / 14430, EP 229246.

[0218] The human constant region of a humanized antibody can be of any class or isotype (IgG, IgA, IgM, IgE, IgD, etc.) and can include a kappa or lambda light chain. In one embodiment, the human constant region includes an IgG heavy chain or a defined fragment, such as at least one of the IgG subclasses, IgG1, IgG2, IgG3, or IgG4.

[0219] Labeled Antibodies

[0220] The antibodies of the invention may be labeled with a detectable or functional marker. Detectable markers include radioactive markers such as [ 131 I] or [ 99Tc], conventional chemical methods known in the art of radioimmunoconjugates can be used to attach labels to the antibodies of the invention. Labels also include enzyme labels such as horseradish peroxidase. Labels also include chemical moieties, such as biotin, which can be detected via binding to specific homologous detectable moieties, such as labeled avidin or streptavidin. Preferably, labels also include fluorescent labels such as FITC.

[0221] Organic part

[0222] The modified antibodies and antigen-binding fragments may comprise one or more organic moieties that are covalently bonded directly or indirectly to the antibody. Each organic moiety bonded to the antibodies or antigen-binding fragments described herein may independently be a hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. As used herein, the term "fatty acid" encompasses monocarboxylic acids and dicarboxylic acids. "Hydrophilic polymer group," as the term is used herein, refers to an organic polymer that is more soluble in water than in octane. For example, polylysine is more soluble in water than in octane. Therefore, antibodies modified by covalent attachment of polylysine are encompassed by the present disclosure. Hydrophilic polymers suitable for modifying antibodies described herein may be linear or branched and include, for example, polyalkane diols (e.g., polyethylene glycol (PEG), monomethoxypolyethylene glycol (mPEG), PPG, etc.), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides, etc.), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartic acid, etc.), polyoxyalkylenes (e.g., polyethylene oxide, polypropylene oxide, etc.) and polyvinylpyrrolidone. Preferably, the hydrophilic polymers for modifying antibodies described herein have a molecular weight of about 800 to about 150,000 daltons as a single molecular entity. For example, PEG5000 and PEG20,000 can be used, where the subscript is the average molecular weight of the polymer in daltons. The hydrophilic polymer group may be substituted with one to about six alkyl, fatty acid, or fatty acid ester groups. The hydrophilic polymer substituted with a fatty acid or fatty acid ester group may be prepared using a suitable method. For example, a polymer containing amine groups can be coupled to a carbohydrate of a fatty acid or fatty acid ester, and an activated carbohydrate (eg, activated with N,N-carbonyldiimidazole) on the fatty acid or fatty acid ester can be coupled to a hydroxyl group on the polymer.

[0223] Fatty acids and fatty acid esters suitable for modifying antibodies described herein may be saturated or may contain one or more unsaturated units. Fatty acids suitable for modifying antibodies described herein include, for example, dodecanoic acid (C12, lauric acid), tetradecanoic acid (C14, myristic acid), octadecanoic acid (C18, stearic acid), eicosanoic acid (C20, arachidic acid), docosanoic acid (C22, behenic acid), triacontanic acid (C30), tetracontanic acid (C40), cis-δ9-octadecanoic acid (C18, oleic acid), all cis-δ5,8,11,14-eicosanoic acid (C20, arachidonic acid), octanedioic acid, tetradecanedioic acid, octadecane dioic acid, docosane dioic acid, etc. Suitable fatty acid esters include dicarboxylic acid monoesters containing straight or branched lower alkyl groups. The lower alkyl group may contain one to about twelve, preferably one to about six carbon atoms.

[0224] Modified human antibodies and Fabs can be prepared using a suitable method, for example, by reacting with one or more modifiers. "Modifier", as the term is used in this article, refers to a suitable organic group (for example, hydrophilic polymers, fatty acids, fatty esters) comprising an activated group. "Activated group" is a chemical part or functional group that can react with a second chemical group under appropriate conditions, so as to form a covalent bond between the modifier and the second chemical group. For example, amine reactive activated groups include electrophilic groups such as toluenesulfonate, mesylate, halogen (chlorine, bromine, fluorine, iodine), N-hydroxysuccinimide ester (NHS) etc. The activated group that can react with thiol includes, for example, maleimide, iodoacetyl, acryloyl, pyridyl disulfide, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol) etc. The aldehyde functional group can be coupled to a molecule containing amine or hydrazide, and an azido group can react with a trivalent phosphorus group to form a phosphoramidate or phosphoramidite bond. Suitable methods for introducing activated groups into molecules are known in the art (see, for example, Hernanson, GT, Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996)). The activated group can be directly bonded to an organic group (e.g., a hydrophilic polymer, a fatty acid, a fatty acid ester), or bonded through a linker moiety, such as a divalent C1-C12 group, in which one or more carbon atoms can be replaced by heteroatoms such as oxygen, nitrogen or sulfur. Suitable linker moieties include, for example, tetraethylene glycol, -(CH2)3-, -NH-(CH2)6-NH-, -(CH2)2-NH- and -CH2-O-CH2-CH2-O-CH2-CH2-O-CH-NH-. Modifiers comprising linker moieties can be generated, for example, by reacting mono-Boc-alkyl diamine (e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with fatty acids in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to form amide bonds between free amine and fatty acid carboxylates. The Boc protecting group can be removed from the product by treatment with trifluoroacetic acid (TFA) to expose the primary amine, which can be coupled to the other carboxylate, or can be reacted with maleic anhydride and the resulting product cyclized to generate an activated maleic acid derivative of fatty acid. (See, for example, Thompson et al., WO 92 / 16221).

[0225] Modified antibodies can be produced by reacting a human antibody or antigen-binding fragment with a modifying agent. For example, an amine-reactive modifying agent, such as NHS ester of PEG, can be used to bond an organic moiety to the antibody in a non-site-specific manner. Modified human antibodies or antigen-binding fragments can also be prepared by reducing the disulfide bonds (e.g., intrachain disulfide bonds) of the antibody or antigen-binding fragment. The reduced antibody or antigen-binding fragment can then be reacted with a thiol-reactive modifying agent to produce a modified antibody as described herein. Modified human antibodies and antigen-binding fragments containing organic moieties bonded to specific sites of the antibodies described herein can be prepared using appropriate methods, such as reverse proteolysis (Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al., Bioconjugate Chem., 5:411-417 (1994); Kumaran et al., Protein Sci. 6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1):59-68 (1996); Capellas et al., Biotechnol. Bioeng., 56(4):456-463 (1997)), and the methods described in Hermanson, GT, Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996).

[0226] Immunoconjugates

[0227] The present invention also provides immunoconjugates comprising an anti-Axl antibody conjugated herein to one or more cytotoxic agents (such as 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.

[0228] 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 European Patent EP 0 425 235B1); auristatins, such as the monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin; 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. 53:3336-3342 (1993); and Lode et al., Cancer Res. 53:3336-3342 (1993); Res. 58: 2925-2928 (1998)); anthracyclines such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chern. 13: 477-523 (2006); Jeffrey et al., Bioorganic & Med. Chern. Letters 16: 358-362 (2006); Torgov et al., Bioconj. Chern. 16: 717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97: 829-834 (2000); Dubowchik et al., Bioorg. & Med. Chern. Letters 12:1529-1532 (2002); King et al., J. Med. Chern. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

[0229] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria toxin A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins (P API, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecenes.

[0230] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioimmunoconjugate. Various radioisotopes can be used to produce radioimmunoconjugates. Examples include [ 211 At], [ 131 I]、[ 125 I]、[ 90 Y]、[ 186 Re]、[ 188 Re]、[ 153 Sm]、[ 212 Bi]、[ 32 P]、[ 212 Pb] and radioactive isotopes of Lu. When the radioimmunoconjugate is used for detection, it may contain a radioactive atom for scintillation studies, such as [ 99 Tc] or [ 123 I], or spin labels for nuclear magnetic resonance (NMR) imaging (also called magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

[0231] 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 (such as dimethyl adipimidate HCl), activated esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 3-methyldiethylenetriaminepentanoic acid 1-benzyl isothiocyanate (MXDTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94 / 11026. The linker can be a "cleavable linker" that facilitates the release of the cytotoxic drug in the cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Res. 52: 127-131 (1992); U.S. Pat. No. 5,208,020).

[0232] The immunoconjugates or ADCs herein expressly include, but are not limited to, such conjugates prepared with cross-linking reagents, including, 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-S1AB, sulfo-SMCC and sulfo-SMPB and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA).

[0233] Glycosylation variants

[0234] In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree of antibody glycosylation. The addition or deletion of antibody glycosylation sites can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0235] When the antibody comprises an Fc region, the carbohydrate attached thereto can be changed. The natural antibodies produced by mammalian cells generally comprise branched, biantennary oligosaccharides, which are generally attached to the Asn297 of the CH2 domain of the Fc region by N-linkage. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modification of oligosaccharides in the antibodies of the present invention may be performed to produce antibody variants with certain improved properties.

[0236] In one embodiment, antibody variants having carbohydrate structures lacking fucose attached (directly or indirectly) to the Fc region are provided. For example, the amount of fucose in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. For example, as described in WO 2008 / 077546, the amount of fucose is determined by calculating the average amount of fucose at Asn297 within the sugar link relative to the sum of all glycosyl structures (e.g., complex, hybrid, and high mannose structures) attached to Asn297. Asn297 refers to an asparagine residue located at approximately position 297 in the Fc region (Eu numbering of Fc region residues); however, due to minor sequence variations in antibodies, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, e.g., U.S. Patent Publication Nos. US 2003 / 0157108 (Presta, L.), US 2004 / 0093621 (Kyowa Haklw Kogyo Co., Ltd.). Examples of publications on "defucosylated" or "fucosyl deficient" antibody variants include: US2003 / 01571; WO2000 / 61739; WO2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 010 9865; WO2003 / 085119; WO2003 / 084570; WO2005 / 035586; WO2005 / 035778; WO2005 / 053742; WO2002 / 0311 40; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004).

[0237] Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986); U.S. Patent Application Publication No. US2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al. Biotechnol. Bioeng., 94 (4): 680-688 (2006); and WO2003 / 085107).

[0238] Also provided are antibody variants with bisected oligosaccharides, for example, wherein the biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. For example, in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US2005 / 0123546 (Umana et al.) Examples of such antibody variants are described. Also provided are antibody variants having at least one galactose residue in the oligosaccharides attached to the Fc region. Such antibody variants may have improved CDC function. For example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.) Such antibody variants are described.

[0239] Fc region variants

[0240] In certain embodiments, one or more amino acid modifications may be introduced into the antibody Fc region provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0241] In certain embodiments, the present invention contemplates antibody variants having some but not all effector functions, making them desirable candidates for applications where the half-life of the antibody in vivo is important and certain effector functions (such as complement fixation and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays may be performed to confirm reduction / loss of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays may be performed to ensure that the antibody lacks Fcγ binding (and therefore may lack ADCC activity), but retains FcRn binding ability. Primary cells that mediate ADCC, i.e., NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991) Table 3 on page 464 summarizes FcR expression on hematopoietic cells. In U.S. Pat. Nos. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'I Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al. Proc. Nat'I Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al. J. Exp. Med. 166:1351-1361 (1987)), non-limiting examples of in vitro assays for assessing ADCC activity of target molecules are described. Alternatively, non-radioactive assays (see, e.g., ACTI TM non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA; and CytoTox Non-radioactive cytotoxicity assay (Promega, Madison, Wl). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or in addition, ADCC activity of the target molecule 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 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks complement dependent cytotoxicity (CDC) activity. See, for example, C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, MS et al., Blood 101: 1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance / half-life Fc assays can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18 (12): 1759-1769 (2006)).

[0242] Antibodies with reduced effector function include those in which one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 are substituted (U.S. Pat. No. 6,737,056). Such Fc mutants include those with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine (U.S. Pat. No. 7,332,581).

[0243] 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. 9(2):6591-6604 (2001)).

[0244] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC activity, eg, substitutions at positions 298, 333, and / or at (EU numbering of residues) of the Fc region.

[0245] In some embodiments, alterations are made in the Fc region that result in altered (ie, increased or decreased) CIq binding and / or CDC activity, e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0246] 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. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005 / 0014934A1 (Hinton et al.). Those antibodies comprise an Fc region having 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 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, such as substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826). See also Duncan and Winter, Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94 / 29351 for other examples of Fc region variants.

[0247] Cysteine ​​engineered antibody variants

[0248] In certain embodiments, it may be desirable to generate cysteine ​​engineered antibodies, eg, "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues.

[0249] In certain embodiments, there are substituted residues at accessible sites of the antibody. By replacing those residues with cysteine, reactive thiol groups are located at accessible sites of the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to produce immunoconjugates as further described herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. For example, as described in U.S. Pat. No. 7,521,541, cysteine ​​engineered antibodies may be produced.

[0250] Diagnosis and treatment methods

[0251] The antibodies of the invention are designed for use in diagnostic or therapeutic methods in human or animal subjects, preferably humans.

[0252] Therefore, other aspects of the present invention provide diagnostic methods, which include administration, provided together with one or more reagents, such as antibodies conjugated to detectable labels such as FITC. The antibodies provided can be used to develop rapid and reliable tests for cancer cells derived from biopsy tissue. For example, the antibodies can be used as tests for metastatic cancer cells, such as circulating tumor cells, which can be found to circulate in body fluids such as blood or lymph. Other target cancers include breast cancer, lung cancer, gastric cancer, head and neck cancer, colorectal cancer, kidney cancer, pancreatic cancer, uterine cancer, liver cancer, bladder cancer, endometrial cancer and prostate cancer as well as lymphoma (e.g., non-Hodgkin's lymphoma, NHL) and leukemia (particularly acute myeloid leukemia, AML).

[0253] Other aspects of the invention provide methods of treatment comprising administering provided antibodies, pharmaceutical compositions comprising such antibodies, antibodies for use in methods of treatment as described herein, antibodies for use in methods of treating specific clinical indications as described herein, and use of such antibodies for the manufacture of a medicament for administration, e.g., for use in a method of preparing a medicament or pharmaceutical composition comprising formulating the antibody with a pharmaceutically acceptable excipient.

[0254] Clinical Indications

[0255] Clinical indications for which antibodies with high specificity for human Axl may be used to provide therapeutic benefit include any condition in which Axl is overexpressed, or in which Axl antagonism would provide clinical benefit. These include immune disorders, cardiovascular disorders, thrombosis, diabetes, immune checkpoint disorders, or proliferative diseases such as cancer, particularly metastatic cancer. In addition, Axl is known to play a role in many cancers of epithelial origin.

[0256] Target immune checkpoint conditions include: chronic viral infections, melanoma, colorectal cancer, breast cancer, ovarian cancer, non-small cell lung cancer (NSCLC), prostate cancer, renal cell carcinoma, pancreatic cancer, esophageal cancer, bladder cancer, myeloma, kidney cancer, bladder cancer, brain tumors, and lymphoma.

[0257] Target cancers include: leukemias, such as but not limited to acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia (such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythrocytic leukemia and myelodysplastic syndrome), chronic leukemias, such as but not limited to chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas, such as but not limited to Hodgkin's disease, non-Hodgkin's disease; multiple myeloma, such as but not limited to smoldering multiple myeloma, non-secretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia (Waldenstrom's macroglobulinemia; unidentified monoclonal gammopathy; benign monoclonal gammopathy; heavy chain disease; bone and connective tissue sarcomas such as, but not limited to, skeletal sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft tissue sarcoma, angiosarcoma, (angioendothelioma), fibrosarcoma, Kaposi's sarcoma, sarcoma), leiomyosarcoma, liposarcoma, lymphangiosarcoma, metastatic cancer, neurilemmoma, rhabdomyosarcoma, synovial sarcoma; brain tumors, such as but not limited to glioma, glioblastoma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, non-glioma, acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, pineal cell tumor, pineal cell tumor, primary brain lymphoma; breast cancer, including but not limited to adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary carcinoma of the breast, mucinous breast cancer, tubular carcinoma of the breast, papillary breast cancer, primary carcinoma, Paget's disease (Paget's disease) and inflammatory breast cancer; adrenal cancer, such as, but not limited to, pheochromocytoma and adrenocortical carcinoma; thyroid cancer, such as, but not limited to, papillary or follicular thyroid cancer, medullary thyroid cancer, and anaplastic thyroid cancer; pancreatic cancer, such as, but not limited to, insulinoma, gastrinoma, glucagonoma, vasodilator peptide tumor, somatostatin-secreting tumor, and carcinoid tumor or islet cell tumor; pituitary cancer, such as, but not limited to, Cushing's disease, prolactin-secreting tumor, acrolein-secreting tumor, ulcerative colitis ... Hypertrophy, diabetes insipidus; eye cancer, such as but not limited to eye melanoma such as iris melanoma, choroidal melanoma and ciliary body melanoma, and retinoblastoma; vaginal cancer such as squamous cell carcinoma, adenocarcinoma and melanoma; vulvar cancer such as squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma and Paget's disease; cervical cancer, such as but not limited to squamous cell carcinoma and adenocarcinoma; uterine cancer, such as but not limited to endometrial carcinoma and uterine sarcoma; ovarian cancer, such as but not limited to ovarian epithelial carcinoma, borderline tumors, germ cell tumors and stromal tumors;Esophageal cancer, such as but not limited to squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; gastric cancer, such as but not limited to adenocarcinoma, mycosis fungoides (polypoid), ulcerative, superficial spreading type, extensive spreading type, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancer; rectal cancer; liver cancer, such as but not limited to hepatocellular carcinoma and hepatoblastoma; gallbladder cancer, such as but not limited to adenocarcinoma; bile duct cancer, such as but not limited to papillary, nodular, and diffuse bile duct cancer; lung cancer, such as non-small cell lung cancer (NSCLC), squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma, and small cell lung cancer (SCLC); testicular cancer, such as but not limited to germ cell tumor, seminoma, anaplastic, classic (typical), spermatocytic cell tumor, non-seminomatous ovarian cancer, such as, but not limited to, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, ovarian cancer, Additionally, cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioma endotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial cancer, cystadenocarcinoma, bronchial carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and papillary adenocarcinoma. Preferably, the cancer is selected from breast cancer, melanoma, prostate cancer, ovarian cancer, colorectal cancer, lung cancer, or glioma cancer. More preferably, the cancer is metastatic breast cancer or lung cancer. Targeting and treatment of circulating tumor cells is contemplated. ;

[0258] The treatment of metastatic cancer depends on the location of the primary tumor. When breast cancer spreads to the lungs, for example, it is still breast cancer and is determined by the source of metastatic cancer in the breast, rather than by the fact that it is now in the lungs. About 5% of the time, metastatic cancer is found, but the primary tumor cannot be identified. The treatment of these metastatic cancers is indicated by their location rather than their source. Metastatic cancer is named by the tissue of the original tumor (if known). For example, breast cancer that has spread to the brain is called metastatic breast cancer to the brain.

[0259] Anti-Axl treatments according to the invention can be used to provide significant benefits to patients with conditions in which Axl is overexpressed, or in which Axl antagonism would provide clinical benefit. Treatment can be given by injection (e.g., intravenously) or by local delivery methods. The provided antibodies can be used to direct the delivery of pharmaceutical compositions to target tissues or systemically, so as to target, for example, circulating tumor cells (CTCs) or other metastatic cells.

[0260] In another aspect of the invention, a method of inhibiting cancer stem cells in a subject is provided, the method comprising contacting the subject with an antibody (or a conjugate thereof) as described herein. Antibodies and conjugates for use in such methods are also contemplated.

[0261] EGFR antagonism

[0262] The present invention also provides a method of inhibiting constitutive Axl activation, comprising administering to a subject an effective amount of any one of the anti-Axl antibodies disclosed herein for inhibiting constitutive Axl.

[0263] In one aspect, the invention provides a method for treating a subject having a cancer associated with an EGFR activating mutation or EGFR gene amplification, wherein the subject has developed resistance to treatment with an EGFR antagonist, the method comprising determining whether the subject has Axl expression, Axl activating mutation, or Axl gene amplification, and administering an EGFR antagonist and any anti-Axl antibody described herein to those subjects having Axl activating mutation or Axl gene amplification.

[0264] In one aspect, the invention provides a method for treating a subject having a cancer associated with an EGFR activating mutation or EGFR gene amplification, comprising: (i) monitoring a subject being treated with an EGFR antagonist to determine whether the subject develops Axl expression, an Axl activating mutation, or Axl gene amplification, and (ii) if the subject has developed an Axl activating mutation or Axl gene amplification, modifying the subject's treatment regimen to include any anti-Axl antibody described herein in addition to the EGFR antagonist.

[0265] In one aspect, the invention provides a method for treating a subject having a cancer associated with an EGFR activating mutation or EGFR gene amplification, comprising: (i) monitoring a subject being treated with an EGFR antagonist to determine whether the subject develops resistance to the inhibitor, (ii) testing the subject to determine whether the subject has Axl expression, an Axl activating mutation, or Axl gene amplification, and (iii) if the subject has an Axl activating mutation or Axl gene amplification, modifying the subject's treatment regimen to include any anti-Axl antibody described herein in addition to the EGFR antagonist.

[0266] In one aspect, the invention provides a method for evaluating an EGFR antagonist, comprising: (i) monitoring a population of subjects being treated with an EGFR antagonist to identify those subjects who have developed resistance to the therapeutic agent, (ii) testing resistant subjects to determine whether the subject has Axl expression, an Axl activating mutation, or Axl gene amplification, and (iii) when the subject has Axl expression, an Axl activating mutation, or Axl gene amplification, modifying the subject's treatment regimen to include any anti-Axl antibody described herein in addition to the EGFR antagonist.

[0267] In one aspect, the present invention provides a method for reducing EGFR phosphorylation in cancer cells, wherein the cancer cells have acquired resistance to an EGFR antagonist, and wherein the cells contain an Axl activating mutation or Axl gene amplification, the method comprising the step of contacting the cells with any anti-Axl antibody described herein and an EGFR antagonist.

[0268] In one aspect, the present invention provides a method for reducing PBK-mediated signaling in cancer cells, wherein the cancer cells have acquired resistance to an EGFR antagonist, and wherein the cells comprise Axl expression, an Axl activating mutation, or Axl gene amplification, the method comprising the step of contacting the cells with any anti-Axl antibody described herein and an EGFR antagonist.

[0269] In one aspect, the invention provides a method for reducing EGFR-mediated signaling in cancer cells, wherein the cancer cells have acquired resistance to an EGFR antagonist, and wherein the cells comprise Axl expression, an Axl activating mutation, or Axl gene amplification, the method comprising contacting the cells with any anti-Axl antibody described herein and an EGFR antagonist.

[0270] In one aspect, the present invention provides a method for restoring sensitivity of cancer cells to an EGFR antagonist, wherein the cancer cells have acquired resistance to the EGFR antagonist, and wherein the cells contain Axl expression, Axl activating mutations, or Axl gene amplification, the method comprising contacting the cells with any anti-Axl antibody described herein and an EGFR antagonist.

[0271] In one aspect, the present invention provides a method for reducing the growth or proliferation of cancer cells, wherein the cancer cells have acquired resistance to an EGFR antagonist, and wherein the cells comprise Axl expression, an Axl activating mutation, or Axl gene amplification, the method comprising the step of contacting the cells with any anti-Axl antibody described herein and an EGFR antagonist.

[0272] In one aspect, the present invention provides a method for increasing apoptosis of cancer cells, wherein the cancer cells have acquired resistance to an EGFR antagonist, and wherein the cells contain Axl expression, an Axl activating mutation, or Axl gene amplification, the method comprising the step of contacting the cells with any anti-Axl antibody described herein and an EGFR antagonist.

[0273] In one aspect, the present invention provides a method for reducing resistance of cancer cells to an EGFR antagonist, wherein the cancer cells have acquired resistance to the EGFR antagonist, and wherein the cells contain an Axl activating mutation or Axl gene amplification, the method comprising the step of contacting the cells with any anti-Axl antibody described herein and an EGFR antagonist.

[0274] In one aspect, the invention provides a method for treating acquired EGFR antagonist resistance in cancer cells, wherein the cells comprise an Axl activating mutation or Axl gene amplification, the method comprising contacting the cells with any anti-Axl antibody described herein and an EGFR antagonist.

[0275] In some embodiments, the cancer cell is any EGFR driven cancer. In some embodiments, the cancer cell comprises an EGFR activating mutation. In some embodiments, the cancer cell comprises an EGFR gene amplification. In some embodiments, the EGFR gene amplification is at least 2 times. In some embodiments, the Axl amplification is at least 2 times. In some embodiments, the cancer cell comprises an EGFR gene mutation associated with increased resistance to EGFR antagonists. In some embodiments, the EGFR gene mutation associated with increased resistance to EGFR antagonists is a T790M mutation of EGFR.

[0276] In some embodiments, the EGFR antagonist is a small molecule therapeutic agent, a nucleic acid therapeutic agent or a protein therapeutic agent. In some embodiments, the EGFR antagonist is an antibody, an antisense molecule or a small molecule kinase inhibitor. In some embodiments, the EGFR antagonist is an EGFR kinase inhibitor selected from the following: gefitinib, erlotinib, cetuximab, pantinumumab. In some embodiments, the EGFR antagonist is an anti-EGFR antibody selected from the following: cetuximab, panitumumab. In some embodiments, the nucleic acid therapeutic agent is a siRNA molecule.

[0277] In one aspect, the invention provides a method for identifying a subject who is a candidate for treatment with an EGFR antagonist and any of the anti-Axl antibodies described herein, wherein the subject has been treated with an EGFR antagonist and has a cancer with acquired resistance to the EGFR antagonist, the method comprising detecting Axl expression, Axl activating mutations, or Axl gene amplification in cancer cells from the subject.

[0278] In one aspect, the present invention provides a method for identifying a subject who is being treated with an EGFR antagonist and is at risk of acquiring resistance to the EGFR antagonist, comprising detecting the presence of Axl expression, Axl activating mutation, or Axl gene amplification in cancer cells from the subject, wherein the presence of Axl expression, Axl activating mutation, or Axl gene amplification indicates the risk of acquiring the resistance.

[0279] In one aspect, the invention provides a method of treating a subject having a cancer that is resistant to treatment with an EGFR antagonist, comprising administering to the subject an EGFR antagonist and any of the anti-Axl antibodies described herein.

[0280] In one aspect, the invention provides a method for treating a subject having a cancer associated with an EGFR activating mutation or EGFR gene amplification, wherein the subject has developed resistance to treatment with an EGFR antagonist, the method comprising determining whether the subject has Axl expression, such as elevated Axl levels and / or activity, and administering an EGFR antagonist and any anti-Axl antibody described herein to those subjects having Axl expression, such as elevated Axl activity.

[0281] In one aspect, the invention provides a method for treating a subject having a cancer associated with an EGFR activating mutation or EGFR gene amplification, comprising: (i) monitoring a subject being treated with an EGFR antagonist to determine whether the subject develops Axl expression, such as elevated levels and / or Axl activity, and (ii) if the subject has developed Axl expression, such as elevated Axl levels and / or activity, modifying the subject's treatment regimen to include any anti-Axl antibody described herein in addition to the EGFR antagonist.

[0282] In one aspect, the invention provides a method for treating a subject having a cancer associated with an EGFR activating mutation or EGFR gene amplification, comprising: (i) monitoring a subject being treated with an EGFR antagonist to determine whether the subject develops resistance to the inhibitor, (ii) testing the subject to determine whether the subject has Axl expression, such as elevated Axl levels and / or activity, and (iii) if the subject has elevated Axl levels and / or activity, modifying the subject's treatment regimen to include any anti-Axl antibody described herein in addition to the EGFR antagonist.

[0283] In another aspect, the present invention provides a method for (i) restoring sensitivity of cancer cells to EGFR antagonists, (ii) reducing resistance of cancer cells to EGFR antagonists, and / or (iii) treating acquired EGFR antagonist resistance in cancer cells by contacting the cells with an EGFR antagonist and any anti-Axl antibody described herein.

[0284] In an exemplary embodiment, the cancer cell has acquired resistance to EGFR antagonists and comprises elevated levels of Axl activity and / or expression, for example, associated with activating mutations in the Axl gene, Axl gene amplification, or Gas6-mediated Axl activation. The methods disclosed herein can be used to restore sensitivity of cancer cells, reduce resistance of cancer cells, and / or treat acquired resistance of cancer cells.

[0285] In another aspect, the invention provides a method of reducing the growth and / or proliferation of cancer cells, or increasing apoptosis of cancer cells, by contacting the cells with an EGFR antagonist and any anti-Axl antibody described herein. In an exemplary embodiment, the cancer cells have acquired resistance to EGFR antagonists and comprise elevated Axl activity and / or expression, such as associated with activating mutations in the Axl gene, Axl gene amplification, or Gas6-mediated Axl activation.

[0286] Pharmaceutical composition

[0287] The antibodies of the invention will generally be administered in the form of a pharmaceutical composition comprising at least one component in addition to the antibody.

[0288] Therefore, the pharmaceutical composition described in the present invention and used in accordance with the present invention may contain, in addition to the active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other material known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, such as intravenous injection. The pharmaceutical composition can be used in humans and veterinary medicine for use by humans or animals.

[0289] Examples of such suitable excipients for the various forms of pharmaceutical compositions described herein may be found in "Handbook of Pharmaceutical Excipients", 2nd edition, edited by A Wade and PJ Weller (1994).

[0290] Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro ed. 1985). Examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, etc. Examples of suitable diluents include ethanol, glycerol, water, and buffered saline.

[0291] The choice of pharmaceutical carrier, excipient or diluent may be made for the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may contain any suitable binder, lubricant, suspending agent, coating agent, cosolvent, buffer, flavoring agent, surfactant, thickener, preservative (including antioxidant), etc. as or in addition to the carrier, excipient or diluent, as well as substances included to render the formulation isotonic with the blood of the intended recipient.

[0292] Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flowing lactose, beta-lactose, corn sweeteners, natural and synthetic rubbers such as acacia, tragacanth or sodium alginate, carboxymethylcellulose and polyethylene glycol.

[0293] Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc. Preservatives, stabilizers, dyes, and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid, and parabens. Antioxidants and suspending agents may also be used.

[0294] Pharmaceutical formulations include those suitable for oral, topical (including skin, buccal and sublingual), rectal or parenteral (including subcutaneous, intradermal, intramuscular and intravenous), nasal and pulmonary administration (e.g., by inhalation). The formulation can be conveniently presented in discrete dosage units when appropriate and can be prepared by any method known in the pharmaceutical field. All methods include the step of associating the active compound with a liquid carrier or a subdivided solid carrier or both, and then, if necessary, the product is molded into the desired formulation. Pharmaceutical formulations suitable for oral administration in which the carrier is solid are most preferably presented as unit dose formulations such as pills, capsules or tablets, each of which contains a predetermined amount of active agent. Tablets can be made by compression or molding, optionally with one or more auxiliary ingredients. Compressed tablets can be prepared by compressing an active agent in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricant, surfactant or dispersant in a suitable machine. Molded tablets can be made by molding an active agent with an inert liquid diluent. Tablets may optionally be coated and, if uncoated, may optionally be scored. Capsules may be prepared by filling capsule shells with the active agent, either alone or in admixture with one or more auxiliary ingredients, and then sealing them in the usual manner. Caches are similar to capsules in that the active agent, along with any auxiliary ingredients, is sealed in a rice paper bag. The active agent may also be formulated as dispersible granules, which may, for example, be suspended in water or sprinkled on food prior to administration. Granules may be packaged, for example, in sachets. Formulations suitable for oral administration wherein the carrier is a liquid may be presented as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water liquid emulsion.

[0295] Formulations for oral administration include controlled release dosage forms, such as tablets, in which the active agent is formulated in an appropriate controlled release matrix, or coated with a suitable controlled release film. Such formulations may be particularly convenient for prophylactic use.

[0296] Pharmaceutical formulations suitable for rectal administration in which the carrier is a solid are most preferably presented as unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. Suppositories can be conveniently formed by mixing the active agent with a softened or melted carrier, followed by cooling and molding in a mold.

[0297] Pharmaceutical formulations suitable for parenteral administration include sterile solutions or suspensions of the active agent in aqueous or oily vehicles.

[0298] Injectable formulations may be suitable for rapid bolus injection or continuous infusion. Such formulations are conveniently presented in unit dose or multi-dose containers, which are sealed after the formulation is introduced until required for use. Alternatively, the active agent may be in powder form, which may be reconstituted with a suitable vehicle, such as sterile, pyrogen-free water, prior to use.

[0299] The active compound may also be formulated as a long-acting depot formulation, which may be administered by intramuscular injection or by implantation (e.g., subcutaneously or intramuscularly). The depot formulation may include, for example, a suitable polymeric or hydrophobic material, or an ion exchange resin. Such long-acting formulations are particularly convenient for prophylactic use.

[0300] Formulations suitable for pulmonary administration via the buccal cavity are presented so that particles containing the active compound and ideally having a diameter in the range of 0.5 to 7 microns are delivered in the recipient's bronchial tree. As one possibility, such formulations are presented in the form of a finely pulverized powder that can be conveniently presented in a pierceable capsule, such as a suitable gelatin, for use in an inhalation device, or alternatively as a self-propelling formulation comprising an active agent, a suitable liquid or gas propellant, and optionally other ingredients (such as a surfactant and / or a solid diluent). Suitable liquid propellants include propane and chlorofluorocarbons, and suitable gaseous propellants include carbon dioxide. Self-propelling formulations may also be employed in which the active agent is dispersed in the form of droplets of a solution or suspension.

[0301] Such self-propelling formulations are similar to those known in the art and can be prepared by prescribed procedures. Suitably they are presented in a container provided with a manually operable or automatically operated valve having the desired spray characteristics; advantageously, the valve is of the metered type, which delivers a fixed volume, e.g. 25 to 100 microliters, after each operation.

[0302] As another possibility, the active agent may be in the form of a solution or suspension for use in a nebulizer or atomizer, which uses accelerated airflow or ultrasonic agitation to produce a fine mist of droplets for inhalation.

[0303] Formulations suitable for nasal administration include those generally similar to those described above for pulmonary administration. When dispersed, such formulations should ideally have a particle size in the range of 10 to 200 microns to ensure retention in the nasal cavity; this can be achieved by using a powder of appropriate particle size or selecting an appropriate valve, as appropriate. Other suitable formulations include coarse powders having a particle size in the range of 20 to 500 microns for administration by rapid inhalation through the nasal passage from a container close to the nose, and nasal drops containing 0.2 to 5% w / v of the active agent in an aqueous or oily solution or suspension.

[0304] Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.1M and preferably 0.05M phosphate buffer or 0.8% saline. In addition, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Aqueous carriers include water, alcohol / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, glucose and sodium chloride, lactated Ringer's or fixed oils. Preservatives and other additives may also be present, for example, antibacterial agents, antioxidants, chelating agents, inert gases, etc.

[0305] Formulations suitable for topical preparation may be provided, for example, as a gel, cream or ointment. Such preparations may be applied, for example, to a wound or ulcer, either directly on the surface of the wound or ulcer or on a suitable support such as a bandage, gauze, mesh fabric, etc., which may be applied to or over the area to be treated.

[0306] Liquid or powder formulations can also be provided, which can be sprayed or sprinkled directly on the part to be treated, such as a wound or ulcer. Alternatively, the formulation can be sprayed or sprinkled on a carrier such as a bandage, gauze, mesh fabric, etc., and then applied to the part to be treated.

[0307] According to another aspect of the invention, there is provided a method for preparing a pharmaceutical or veterinary composition as described above, the method comprising associating the active compound with the carrier, for example by mixing. Generally, by uniformly and intimately associating the active agent with a liquid carrier or a finely divided solid carrier or both, and then shaping the product if necessary. The invention extends to a method for preparing a pharmaceutical composition, which comprises associating the agent with a pharmaceutically or veterinarily acceptable carrier or vehicle.

[0308] Application

[0309] The pharmaceutical composition of the present invention can be suitable for oral, rectal, nasal, intrabronchial, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, arterial and intradermal), intraperitoneal and intrathecal administration. Preferably, the formulation is a formulation for intravenous or subcutaneous administration.

[0310] The formulation can be conveniently presented in unit dosage form, i.e., in the form of discrete portions containing one unit dose, multiple unit doses, or subunits of a unit dose. For example, the formulation is in the form of tablets and sustained-release capsules, and can be prepared by any method known in the pharmaceutical field.

[0311] The formulations for oral administration in the present invention may be presented as discrete units each containing a predetermined amount of active agent, such as capsules, gellules, drops, cachets, pills or tablets; as powders or granules; as solutions, emulsions or suspensions of the active agent in aqueous or non-aqueous liquids; or as oil-in-water liquid emulsions or water-in-oil liquid emulsions; or as pills, etc. Preferably, these compositions contain 1 to 250 mg and more preferably 10-100 mg of active ingredient per dose.

[0312] For compositions (e.g., tablets and capsules) for oral administration, the term "acceptable carrier" includes vehicles such as common excipients, e.g., binders, such as syrup, gum arabic, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropyl-methylcellulose, sucrose and starch; fillers and carriers, such as corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid; and lubricants such as magnesium stearate, sodium stearate and other metal stearates, stearyl stearate, silicone oil, talcum wax, oil and colloidal silicon oxide. Flavoring agents such as mint, wintergreen oil, cherry flavor, etc. may also be used. It may be desirable to add coloring agents to prepare dosage forms that are easy to identify. Tablets may also be coated by methods well known in the art.

[0313] Tablets may be made by compression or molding, optionally with one or more auxiliary ingredients. Compressed tablets may be prepared by compressing the active agent in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricant, surfactant or dispersant in a suitable machine. Molded tablets may be made by molding a mixture of powdered compounds moistened with an inert liquid diluent in a suitable machine. Tablets may optionally be coated or scored and may be formulated to provide sustained or controlled release of the active agent.

[0314] Other formulations suitable for oral administration include lozenges comprising the active agent in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active agent in an inert basis such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active agent in a suitable liquid carrier.

[0315] Other administration forms include solutions or emulsions, which can be injected intravenously, intraarterially, intrathecally, subcutaneously, intradermally, intraperitoneally or intramuscularly, and which can be prepared from sterile or sterilizable solutions. Injectable forms usually contain 10-1000 mg, preferably 10-250 mg of active ingredient per dose.

[0316] The pharmaceutical composition of this invention may also be in the form of a suppository, pessary, suspension, emulsion, lotion, ointment, cream, gelatin, spray, solution or dusting powder.

[0317] An alternative to transdermal administration is through the use of a skin patch. For example, the active ingredient may be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycol or liquid paraffin. The active ingredient may also be incorporated into an ointment consisting of a white wax or white soft paraffin base together with such stabilizers and preservatives as may be required at a concentration of 1 to 10% by weight.

[0318] Alternative formulation strategies may provide formulations suitable for oral or suppository routes. The route of administration may be determined by the physicochemical properties of the therapeutic, by particular considerations for the disease, optimizing efficacy or minimizing side effects.

[0319] Another mode of administration employs pre-coating or otherwise incorporation into an internal device, for which the optimal amount of antibody will be determined by appropriate experimentation.

[0320] In some preferred embodiments of the present invention, the antibody molecule is a monomeric fragment, such as Fab or scFv. Such antibody fragments may be characterized by a relatively short half-life.

[0321] dose

[0322] One of ordinary skill in the art can readily determine the appropriate dosage of the present composition to be administered to a subject without undue experimentation. Typically, a physician will determine the actual dosage that will be most suitable for an individual patient and the actual dosage will depend on a variety of factors, including the activity of the specific agent employed, the metabolic stability and duration of action of the agent, age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, severity of the particular condition, and ongoing therapy for the individual.

[0323] According to the present invention, the provided compositions can be administered to individual patients. Administration is preferably in a "therapeutically effective amount," which is sufficient to show benefit to the patient. Such benefit may be at least an improvement in at least one symptom. The actual amount administered, as well as the rate and duration of administration, will depend on the nature and severity of the disease being treated. Treatment prescriptions, such as dosage determinations, are within the responsibility of general physicians and other medical doctors. Appropriate doses of antibodies are well known in the art; see Ledermann J.A. et al. (1991) Int. J. Cancer 47:659-664; Bagshawe, KD et al. (1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4:915-922.

[0324] The exact dosage will depend on many factors, including whether the antibody is used for diagnosis or treatment, the size and location of the area to be treated, the exact characteristics of the antibody (e.g., whole antibody, antibody fragment or diabody) and the characteristics of any detectable label or other molecule attached to the antibody. Typical antibody dosages can be administered intravenously as a pill. Other modes of administration include intravenous infusion over several hours to achieve a similar total cumulative dose. This is a dose for a single treatment of adult patients, which can be adjusted proportionally for children and infants and can also be adjusted proportionally to the molecular weight for other antibody forms. Repeat the treatment at intervals of once a day, twice a day, once a day, or once a month at the discretion of the physician.

[0325] The dosages disclosed herein are examples of the average case. There can, of course, be individual instances where higher or lower dosage ranges are beneficial, and such are within the scope of this invention.

[0326] According to the present invention, an effective amount of the agent may be administered to inhibit Axl. Of course, this dosage will be further modified according to the type of administration of the agent. For example, to achieve an "effective amount" for acute treatment, parenteral administration is preferred. Intravenous infusion of the compound in 5% dextrose in water or normal saline, or a similar formulation with a suitable excipient is most effective, although intramuscular bolus injection is also useful. Typically, the parenteral dose will be about 0.01 to about 100 mg / kg; preferably 0.1 to 20 mg / kg, to maintain the drug concentration in the plasma at a concentration that effectively inhibits the kinase or saturates the target receptor. The agent may be administered one to four times a day at a level that achieves a total daily dose of about 0.4 to about 400 mg / kg / day. The exact amount of therapeutically effective active agent, and the best route of administration of such an agent, is easily determined by a person of ordinary skill in the art by comparing the blood level of the agent with the concentration required for efficacy.

[0327] The agents of the present invention may also be orally administered by a patient in a manner such that the drug concentration is sufficient to achieve one or more therapeutic indications disclosed herein. Typically, a pharmaceutical composition containing the agent is administered in an oral dose of about 0.1 to about 50 mg / kg in a manner consistent with the patient's condition. Preferably, the oral dose will be about 0.5 to about 20 mg / kg.

[0328] The agents of the invention can be tested in any of several biological assays to determine the concentration of the agent required to have a given pharmacological effect.

[0329] Combination therapy

[0330] Compositions can be used alone or in combination with other treatments, simultaneously or sequentially according to the disease to be treated. For example, antibodies of the present invention or their conjugates can be used as anticancer monotherapy or in combination with other cancer treatments as mentioned below. Other treatments may include administration of an appropriate amount of pain relief drugs such as nonsteroidal anti-inflammatory drugs (e.g., aspirin, ibuprofen or ketoprofen) or opiates (e.g., morphine) or antiemetics.

[0331] In a preferred aspect, the antibody of the present invention (or its conjugate) is administered in combination with an immune checkpoint modulator (ICM), such as an immune checkpoint inhibitor (ICI). Typically, an ICM is an agent that binds to a target receptor, such as an aptamer or an antibody.

[0332] The ICM used in combination with the antibody (or its conjugate) of the present invention may be any suitable ICM known in the art. Specifically, suitable immune checkpoint regulators include:

[0333] CTLA-4 targeted agents, including ipilimumab and tremelimumab.

[0334] PD-1 targeted agents, including pembrolizumab, mivolumab, and AMP-514 / MEDI0680.

[0335] BD-L1 targeting agents, including MPDL3280A, MEDI4736, MSB0010718C, and BMS-936559.

[0336] 4-1BB-targeting agents, including Urelumab and PF-05082566.

[0337] OX-40 targeted agents, including MEDI6469, MEDI6383 (rOX40L), and MOXR0916.

[0338] GITR-targeted agents, including TRX518.

[0339] CD27-targeting agents, including CDX-1127.

[0340] CD40 targeting agents, including CP-870,893.

[0341] LAG3-targeted agents, including BMS-986016.

[0342] Immune checkpoints are inhibitory pathways in the immune system that can be occupied by tumors to induce immune resistance. The use of agents to block or modulate immune checkpoints, including T cell stimulatory and inhibitory receptors and dendritic cell stimulatory receptors, and thereby reduce or reverse cancer immune resistance, is an important approach in cancer research.

[0343] T cell stimulatory receptors that can be regulated by using ICM include CD28, ICOS, 4-1BB, OX40, GITR, CD27, TWEAKR, HVEM and TIM-1. T cell inhibitory receptors that can be regulated by using ICM include PD-L1, CTLA-4, PD-1, BTLA, TIM-3, VISTA, LAG-3 and TIGIT. Dendritic cell stimulatory receptors that can be regulated by using ICM include CD40 and 4-1BB.

[0344] Combinations of ICMs When used with an antibody (or conjugate thereof) of the invention, all ICMs may target inhibitory receptors, all ICMs used may target stimulatory receptors, or a combination of ICMs targeting both inhibitory and stimulatory receptors may be used.

[0345] Thus, an antibody (or its conjugate) of the present invention for use in a method of treating cancer is provided, wherein the treatment further comprises administering one or more ICMs. Similarly, there is provided the use of an antibody (or its conjugate) of the present invention in the manufacture of a medicament for treating cancer, wherein the treatment further comprises administering one or more immune checkpoint regulators.

[0346] Also provided are antibodies of the invention (or their conjugates) for use in methods of treating cancer, or use of such antibodies (or their conjugates) in the manufacture of a medicament for treating cancer, wherein the treatment further comprises administering one or more immune checkpoint regulators selected from the group consisting of ipilimumab, tremelimumab, pembrolizumab, nivolumab, AMP-514 / MEDI0680, MPDL3280A, MEDI4736, MSB0010718C, BMS-936559, usrulumab, PF-05082566, MEDI6469, MEDI6383 (rOX40L), MOXR0916, TRX518, CDX-1127, CP-870,893, and BMS-986016.

[0347] The antibodies of the invention (or their conjugates) may be administered prior to one or more ICMs, simultaneously with the one or more ICMs, or after the one or more ICMs.

[0348] Also provided are antibodies of the invention (or their conjugates) for use in cancer treatment, or use of such antibodies (or their conjugates) in the manufacture of a medicament for cancer treatment, wherein the treatment further comprises one or more ICMs, and wherein the cancer is selected from lung cancer, melanoma, breast cancer, ovarian cancer or carcinoma.

[0349] -------------------------------------------------

[0350] Suitable agents for combination therapy

[0351] These include alkylating agents, for example, alkyl sulfonates such as busulfan;

[0352] Nitrogen mustards such as chlorambucil, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, melphalan and uramustine, ethyleneimine derivatives (such as thiotepa);

[0353] Nitrosoureas such as carmustine, lomustine, and streptozocin, triazenes such as dacarbazine, procarbazine, and temozolamide;

[0354] Platinum compounds such as cisplatin, carboplatin, oxaliplatin, satraplatin and picoplatin, onnaplatin, tetraplatin, sprioplatin, iproplatin, chloro(diethylenediamine)platinum(II), dichloro(ethylenediamine)platinum(II), (2-ethylmalonate)diammineplatinum(II), (1,2-diaminocyclohexane)malonateplatinum(II), (4-carboxyphthalate)-(1,2-diaminocyclohexane)platinum(II), (1,2-diaminocyclohexane)-(isocitrate)platinum(II) and (1,2-diaminocyclohexane)-cis-(pyruvate)platinum(II);

[0355] Antimetabolites, including antifolates such as methotrexate, permetrexed, raltitrexed, and trimetrexate;

[0356] Pyrimidine analogs such as azacitidine, capecitabine, cytarabine, edatrexate, floxuridine, fluorouracil, gemcitabine, and troxacitabine;

[0357] Purine analogs such as cladribine, chlorodeoxyadenosine, clofarabine, fludarabine, mercaptopurine, pentostatin, and thioguanine;

[0358] Natural products, including antitumor antibiotics such as bleomycin, dactinomycin, mithramycin, mitomycin, mitoxantrone, porfiromycin, and anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, and valrubicin);

[0359] Mitotic inhibitors such as the vinca alkaloids vincristine, vindesine, vinorelbine, vinblastine, vinvesir, and vincristine;

[0360] Enzymes such as L-asparaginase and PEG-L-asparaginase;

[0361] Microtubule polymer stabilizers such as the taxanes paclitaxel and docetaxel;

[0362] Topoisomerase I inhibitors such as the camptothecins irinotecan and topotecan; topoisomerase II inhibitors such as podophyllotoxin, amsacrine, etoposide, teniposide, losoxantrone, and actinomycin;

[0363] Hormones and hormone antagonists, including androgens such as fluoxymesterone and testolactone,

[0364] Antiandrogens such as bicalutamide, cyproterone, flutamide, and nilutamide;

[0365] Corticosteroids such as dexamethasone and prednisone;

[0366] aromatase inhibitors such as aminoglutethimide, anastrozole, exemestane, formestane, and letrozole;

[0367] Estrogens such as diethylstilbestrol;

[0368] Antiestrogens such as fulvestrant, raloxifene, tamoxifen, and toremifene;

[0369] Luteinizing hormone-releasing hormone (LHRH) agonists and antagonists such as abarelix, buserelin, goserelin, leuprolide, histrelin, desorelin, nafarelin acetate, and triptorelin;

[0370] Progestins such as medroxyprogesterone acetate and megestrol acetate, and thyroid hormones such as levothyroxine and liothyronine;

[0371] PKB pathway inhibitors, including perifosine, enzastaurin hydrochloride, and triciribine;

[0372] PI3K inhibitors such as semaphore and SF1126;

[0373] mTOR inhibitors such as rapamycin and analogs;

[0374] CDK inhibitors, including seliciclib, avocidib, and 7-hydroxystaurosporine;

[0375] COX-2 inhibitors, including celecoxib;

[0376] HDAC inhibitors, including trichostatin A, suberoylanilide hydroxamic acid, and clindamycin;

[0377] DNA methylase inhibitors, including temozolomide; and

[0378] Other agents include altretamine, arsenic trioxide, thalidomide, lenalidomide, gallium nitrate, levamisole, mitotane, hydroxyurea, octreotide, procarbazine, suramin, photodynamic compounds (such as methoxsalen and sodium porfimer), and proteasome inhibitors (such as bortezomib).

[0379] Molecularly targeted therapeutic agents include:

[0380] functional therapeutic agents, such as gene therapy agents;

[0381] antisense therapeutics;

[0382] Tyrosine kinase inhibitors such as erlotinib hydrochloride, gefitinib, imatinib mesylate, and semaxanib;

[0383] RAF inhibitors such as sorafenib;

[0384] Gene expression modulators such as retinoids and retinoids, for example adapalene, bexarotene, trans-retinoic acid, 9-cis-retinoic acid, and N-(4-hydroxyphenyl) retinamide;

[0385] Phenotype-directed therapeutics, including monoclonal antibodies such as alemtuzumab, bevacizumab, cetuximab, ibritumomab tiuxetan, rituximab, and trastuzumab;

[0386] immunotoxins (such as emtansine), radioimmunoconjugates (such as l-tositumobab), binders (such as aptamers, which target any of the molecular targets described herein),

[0387] and

[0388] Cancer vaccines.

[0389] Biological therapeutics include:

[0390] Interferons such as interferon-[alpha]2a and interferon-[alpha]2b, and interleukins such as aldesleukin, denileukin diftitox and oprelvekin. Axl inhibitors include 1-(6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazin-3-yl)-N3-((7-(S)-pyrrolidin-1-yl)-6,7,8,9-tetrahydro-5H-benzo[7]cycloannulene-2-yl)-1H-1,2,4-triazole-3,5-diamine (BGB324 / R428), CH5451098 (Roche), and Axl inhibitors described in PCT / US07 / 089177, PCT / US2010 / 021275, and PCT / EP2011 / 004451, which are incorporated herein by reference.

[0391] In addition to these agents intended to act on cancer cells, anticancer therapy also includes the use of protective or adjuvant agents, including:

[0392] Cytoprotectants such as amifostine and dexrazoxane;

[0393] Phosphonates such as pamidronate and zoledronic acid; and

[0394] Stimulants such as epoetin, darbeopetin, filgrastim, PEG-filgrastim and sargramostim.

[0395] Many combination chemotherapy regimens are known in the art, such as carboplatin / paclitaxel, capecitabine / docetaxel, fluorouracil / levamisole, fluorouracil / leucovorin, methotrexate / leucovorin, and trastuzumab / paclitaxel, alone or in further combination with carboplatin and the like.

[0396] ----------------------------------------------

[0397] Throughout the specification, the methods described herein are preferably performed in vitro or ex vivo.

[0398] ----------------------------------------------

[0399] The invention provides methods of causing or allowing the antibodies as provided herein to bind to Axl. As noted, such binding can occur in vivo, e.g., after administration of the antibody or nucleic acid encoding the antibody, or it can occur in vitro, e.g., in ELISA, Western blot analysis, immunocytochemistry, immunohistochemistry, immunoprecipitation, or affinity chromatography.

[0400] The amount of antibody bound to the Axl receptor can be determined. The quantification can be related to the amount of antigen in the sample, which may be of diagnostic interest.

[0401] The reactivity of the antibody in the sample can be determined by any suitable means. Radioimmunoassay (RIA) is one possibility. Radiolabeled antigen is mixed with unlabeled antigen (sample) and allowed to bind to the antibody. The bound antigen is separated from the unbound antigen by physical means and the amount of radioactive antigen bound to the antibody is determined. The more antigen there is in the sample, the less radioactive antigen will bind to the antibody. Competitive binding assays can also be used, using non-radioactive antigen, using antigen or the like linked to a receptor molecule. The receptor molecule can be a fluorochrome, phosphor or laser dye with spectrally separated absorption or emission characteristics. Suitable fluorochromes include fluorescein, rhodamine, phycoerythrin and Texas Red. Suitable chromogenic dyes include diaminobenzidine.

[0402] Other reporter factors include macromolecular colloidal particles or particulate materials such as colored, magnetic or paramagnetic latex beads, and biological or chemical agents that can directly or indirectly cause a detectable signal to be visually observed, electronically detected or otherwise recorded. These molecules can be enzymes that catalyze reactions that develop or change color or cause changes in electrical properties (for example). They can be molecularly excitable, so that electronic transitions between energy states result in characteristic spectral absorption or emission. They can include chemical entities used in conjunction with biosensors. Biotin / avidin or biotin / streptavidin and alkaline phosphatase detection systems can be used.

[0403] Other reporter factors include DNA tags. These tags can be easily quantified by, for example, qPCR.

[0404] The signal generated by the individual antibody-reporter conjugates can be used to derive quantifiable absolute or relative data on the binding of the relevant antibody in samples (normal and test).

[0405] The present invention also provides the use of antibodies as described above for measuring antigen levels in competitive assays, i.e., methods for measuring antigen levels in samples by using antibodies as provided by the present invention in competitive assays. This may be without physically separating bound and unbound antigens. It is possible to connect a reporter molecule to the antibody so that physical or optical changes occur upon binding. The reporter molecule may directly or indirectly produce a detectable and preferably measurable signal. The connection of the reporter molecule may be direct or indirect, covalent (e.g., via a peptide bond) or non-covalent. Connection via a peptide bond may be due to recombinant expression of a gene fusion encoding the antibody and the reporter molecule.

[0406] The invention also provides for the direct measurement of antigen levels, for example in a biosensor system, by employing the antibodies according to the invention.

[0407] The mode of determining binding is not a feature of the present invention and the skilled person will be able to select a suitable mode according to his or her preference and general knowledge.

[0408] The present invention also extends to antibodies that compete with antigen-binding and include antibody variable domains (VH or VL or both) including CDRs with amino acids substantially as listed herein or any antibody variable domains with amino acid sequences substantially as listed herein for binding Axl. Competition between antibodies can be easily determined in vitro, for example, by labeling specific reporter molecules to a binding member that can be detected in the presence of other unlabeled binding members, so that identification of antibodies in conjunction with the same epitope or overlapping epitopes becomes possible. For example, ELISA or flow cytometry can be used to measure competition. Alternatively, as described in Example 5, Biacore instruments can be used to identify competitive antibodies via surface plasma resonance (SPR) technology.

[0409] In tests for competition, peptide fragments of the antigen may be used, particularly peptides that include the target epitope. Peptides having the epitope sequence plus one or more amino acids at either end may be used. Such peptides may be said to "consist essentially of" the specified sequence. Antibodies according to the invention may be such that their binding to the antigen is inhibited by a peptide having or including a given sequence. In tests for this, peptides having either sequence plus one or more amino acids may be used.

[0410] Antibodies that bind a particular peptide can be isolated from a phage display library, for example, by panning with the peptide.

[0411] The present invention also provides isolated nucleic acids encoding antibodies of the present invention. Nucleic acids include DNA and RNA. In a preferred aspect, the present invention provides nucleic acids encoding CDR, VH or VL domains of the present invention as defined above.

[0412] The present invention also provides a construct in the form of a plasmid, vector, transcription or expression cassette comprising at least one polynucleotide as described above.

[0413] The present invention also provides recombinant host cells comprising one or more constructs as described above. The nucleic acids encoding any of the CDRs, VH or VL domains or antibodies provided themselves form an aspect of the present invention, as do methods for producing encoded products, which methods include expression from their encoding nucleic acids. Expression can be conveniently achieved by culturing a recombinant host cell containing the nucleic acid under appropriate conditions. Following production by expression, the VH or VL domains or antibodies can be isolated and / or purified using any suitable technique known in the art.

[0414] The antibodies, VH and / or VL domains and encoding nucleic acid molecules and vectors according to the invention may be provided, for example, isolated and / or purified from their natural environment, in a substantially pure or homogeneous form, or in the case of nucleic acids, free of or substantially free of nucleic acids or genes from sources other than sequences encoding polypeptides having the desired function. The nucleic acids according to the invention may comprise DNA or RNA and may be fully or partially synthetic. Unless the context requires otherwise, reference to a nucleotide sequence as set forth herein encompasses a DNA molecule having the specified sequence, and encompasses an RNA molecule having the specified sequence in which U is substituted with T.

[0415] Systems for cloning and expressing polypeptides in various host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast, baculovirus and insect cell systems. Mammalian cell lines that can be used to express heterologous polypeptides in the art include Chinese hamster ovary cells (CHO), HeLa cells, baby hamster kidney (BHK) cells, NS0 and SP2 / 0 mouse myeloma cells, YB2 / 0 rat myeloma cells, human cell lines HEK-293 and PER.C6 and many other cells. Common, preferred bacterial hosts are Escherichia coli (E. coli).

[0416] The expression of antibodies and antibody fragments in prokaryotic cells such as E. coli is established in the art. For a review, see, e.g., Plückthun, A. Bio / Technology 9:545-551 (1991). Expression in eukaryotic cells in culture can also be used by those skilled in the art as an option for producing antibodies, see, e.g., Ref, ME (1993) Curr. Opinion Biotech. 4:573-576; Trill JJ et al. (1995) Curr. Opinion Biotech 6:553-560.

[0417] Suitable vectors can be selected or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other appropriate sequences. Depending on the circumstances, the vector can be a plasmid, a virus such as a phage or a phagemid (Sambrook and Russell, 2001, Molecular Cloning: a Laboratory Manual: 3rd edition, Cold Spring Harbor Laboratory Press). Many known techniques and protocols for manipulating nucleic acids, such as the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression and protein analysis are described in detail in Current Protocols in Molecular Biology, 2nd edition, edited by Ausubel et al., John Wiley & Sons, 1992.

[0418] Therefore, another aspect of the invention provides a host cell containing a nucleic acid as disclosed herein. A further aspect provides a method comprising introducing such a nucleic acid into a host cell. The introduction may be carried out using any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retrovirus or other viruses (e.g., vaccinia) (or for insect cells, baculovirus). For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation, and transfection using bacteriophage.

[0419] Following introduction, expression from the nucleic acid may be caused or permitted, for example, by culturing the host cell under conditions for expression of the gene.

[0420] In one embodiment, the nucleic acid of the invention is integrated into the genome (eg, chromosome) of the host cell. Integration may be facilitated by including sequences that promote recombination with the genome, according to standard techniques.

[0421] The present invention also provides a method comprising using a construct as described above in an expression system to express an antibody or polypeptide as described above.

[0422] Aspects and embodiments of the present invention will now be described, by way of example, with reference to the following experiments.

[0423] All documents cited anywhere in this specification are incorporated by reference.

[0424] Claim of Invention

[0425] The following paragraphs describe a number of specifically contemplated embodiments and combinations of the invention.

[0426] 1. An antibody that binds to Axl, comprising:

[0427] An antibody VH domain selected from the group consisting of a 1H12 VH domain (SEQ ID NO. 3) and a VH domain comprising a VH CDR3 having an amino acid sequence of SEQ ID NO. 7 and optionally one or more VH CDRs having an amino acid sequence selected from the group consisting of SEQ ID NO. 6 and SEQ ID NO. 5; and / or

[0428] An antibody VL domain selected from the group consisting of a 1H12 VL domain (SEQ ID NO.4) and a VL domain comprising one or more VLCDRs having an amino acid sequence selected from the group consisting of SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10.

[0429] 2. The antibody according to paragraph 1, comprising an antibody VH domain, wherein the antibody VH domain comprises an antibody VH domain having a VH CDR having an amino acid sequence of SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, and the antibody competes for binding to Axl with the Axl binding domain of an antibody comprising the 1H12 VH domain (SEQ ID NO.3) and the 1H12 VL domain (SEQ ID NO.4).

[0430] 3. The antibody according to paragraph 1 or paragraph 2, comprising the 1H12 VH domain (SEQ ID NO. 3).

[0431] 4. The antibody according to paragraph 3, comprising the 1H12 VL domain (SEQ ID NO. 4).

[0432] 5. A variant of the antibody according to any one of paragraphs 1-4, wherein the variant comprises one or more amino acid sequence changes in one or more framework regions and / or one or more CDRs.

[0433] 6. An antibody according to any one of paragraphs 1-5, which binds Axl with an affinity equal to or better than the Axl antigen binding site formed by the 1H12 VH domain (SEQ ID NO. 3) and the 1H12 VL domain (SEQ ID NO. 4), the affinity of the antibody and the affinity of the antigen binding site being determined under the same conditions.

[0434] 7. The antibody of any one of paragraphs 1-6, comprising a scFv antibody molecule.

[0435] 8. The antibody of any one of paragraphs 1-6, comprising an antibody constant region.

[0436] 9. The antibody according to paragraph 8, which comprises a whole antibody.

[0437] 10. The antibody according to any one of paragraphs 1-6, wherein the antibody is an antibody fragment that binds to an antigen, such as a single domain antibody, Fv, scFv, dsFv, Fd, Fab, F(ab')2, miniantibody, diabody, single-chain diabody, tandem scFv, TandAb, double-chain antibody, three-chain antibody, kappa (lambda) antibody, BiTE, DVD-lg, SIP, SMIP or DART.

[0438] 11. The antibody of any of paragraphs 1-10, comprising additional amino acids that provide other functional characteristics besides the ability to bind antigen.

[0439] 12. The antibody according to any one of paragraphs 1-11, which has a concentration of no more than 5×10 -11 M of K D Combined with Axl.

[0440] 13. The antibody according to any one of paragraphs 1-11, which has a concentration of no more than 1.5×10 -11 M of K D Combined with Axl.

[0441] 14. The antibody according to any one of paragraphs 1-13, which has a concentration of no more than 2×10 -5 s -1 K 解离 Combined with Axl.

[0442] 15. The antibody according to any one of paragraphs 1-14, which has a concentration of no more than 3×10 -6 s -1 K 解离 Combined with Axl.

[0443] 16. The antibody according to any of paragraphs 1-15, wherein the Axl is human Axl.

[0444] 17. The antibody of any of paragraphs 1-16, which specifically binds to primate Axl.

[0445] 18. The antibody according to any one of paragraphs 1-17, which:

[0446] (i) More than 10 -3 M of K D Binds to rat Axl;

[0447] (ii) more than 10 -3 M of K D Combined with Mer; and / or

[0448] (iii) more than 10 -3 M of K D Binds human Tyro3.

[0449] 19. The antibody of any of paragraphs 1-18, wherein the antibody is an Axl agonist.

[0450] 20. The antibody of paragraph 19, wherein Axl signaling is at least 10% stronger.

[0451] 21. The antibody of any of paragraphs 1-20, wherein the antibody is a chimeric antibody.

[0452] 22. The antibody of any of paragraphs 1-20, wherein the antibody is a humanized antibody.

[0453] 23. The antibody of any one of paragraphs 1-22, wherein the antibody binds:

[0454] (i) the same epitope as the 1H12 antibody, or

[0455] (ii) an epitope that overlaps with the epitope bound by the 1H12 antibody.

[0456] 24. The antibody according to any one of paragraphs 1-23, wherein the antibody is internalized after binding to Axl present on the cell surface.

[0457] 25. The antibody according to any of paragraphs 1-24, optionally conjugated to a detectable label, enzyme or toxin via a peptide bond or a linker.

[0458] 26. The antibody according to paragraph 25, wherein the toxin is selected from the group consisting of MMAE and MMAF.

[0459] 27. The antibody of paragraph 25, wherein the detectable label is FITC.

[0460] 28. An isolated nucleic acid comprising a nucleotide sequence encoding the antibody or antibody VH or VL domain of an antibody according to any one of paragraphs 1-24.

[0461] 29. A host cell transformed with the nucleic acid according to paragraph 28.

[0462] 30. A method of producing an antibody or an antibody VH or VL domain, the method comprising culturing the host cell according to paragraph 29 under conditions for producing the antibody or antibody VH or VL domain.

[0463] 31. The method of paragraph 30, further comprising isolating and / or purifying the antibody or antibody VH or VL variable domain.

[0464] 32. The method of paragraph 30 or paragraph 31, further comprising formulating the antibody or antibody VH or VL variable domain into a composition comprising at least one additional component.

[0465] 33. A method for obtaining an antibody that binds to Axl, the method comprising

[0466] Providing one or more VH domains each being an amino acid sequence variant of the 1H12 VH domain by adding, deleting, substituting or inserting one or more amino acids in the amino acid sequence of the 1H12 VH domain (SEQ ID NO. 3), and optionally combining the one or more VH domain amino acid sequence variants thus provided with one or more VL domains to provide one or more VH / VL combinations; and / or

[0467] Providing a VL domain that is an amino acid sequence variant of the 1H12 VL domain by adding, deleting, substituting or inserting one or more amino acids in the amino acid sequence of the 1H12 VL domain (SEQ ID NO. 4), and combining the one or more VL domain amino acid sequence variants provided in this way with one or more VH domains to provide one or more VH / VL domain combinations;

[0468] and

[0469] The VH domain amino acid sequence variants or VH / VL combination or combinations are tested to identify antibodies that bind Axl.

[0470] 34. A method for obtaining an antibody that binds to Axl, the method comprising:

[0471] Providing a starting nucleic acid encoding one or more VH domains comprising the CDR3 to be replaced or lacking a CDR3 coding region, and combining the starting nucleic acid with a donor nucleic acid encoding the VH CDR3 amino acid sequence of SEQ ID NO.7 such that the donor nucleic acid is inserted into the CDR3 region of the starting nucleic acid to provide a product nucleic acid encoding a VH domain; or

[0472] Providing a starting nucleic acid encoding one or more VL domains comprising a CDR3 to be replaced or lacking a CDR3 coding region, and combining the starting nucleic acid with a donor nucleic acid encoding the VL CDR3 amino acid sequence of SEQ ID NO. 10, such that the donor nucleic acid is inserted into the CDR3 region of the starting nucleic acid, so as to provide a product nucleic acid encoding a VL domain;

[0473] expressing nucleic acids encoding said product nucleic acids of VH domains and optionally combining said VH domains so produced with one or more VL domains to provide a VH / VL combination, and / or expressing nucleic acids encoding said product nucleic acids of VL domains and combining said VL domains so produced with one or more VH domains to provide a VH / VL combination;

[0474] selecting an antibody comprising a VH domain or a VH / VL combination, said antibody binding to Axl; and

[0475] The antibody that binds Axl and / or the nucleic acid encoding the antibody that binds Axl are recovered.

[0476] 35. The method of paragraph 33 or paragraph 34, wherein the antibody that binds Axl is an antibody fragment comprising a VH domain and a VL domain.

[0477] 36. The method of paragraph 35, wherein the antibody fragment is a scFv antibody molecule.

[0478] 37. The method of paragraph 35, wherein the antibody fragment is a Fab antibody molecule.

[0479] 38. The method of paragraph 36 or paragraph 37, further comprising providing the VH domain and / or the VL domain of the antibody fragment in a whole antibody.

[0480] 39. The method of any of paragraphs 33-38, further comprising formulating the antibody that binds Axl or the antibody VH or VL variable domain of the antibody that binds Axl into a composition comprising at least one additional component.

[0481] 40. The method of any of paragraphs 30-39, further comprising binding an antibody that binds Axl to Axl or a fragment of Axl.

[0482] 41. A method comprising binding the Axl-binding antibody of any of paragraphs 1-27 to Axl or a fragment of Axl.

[0483] 42. The method of paragraph 40 or paragraph 41, wherein the binding occurs in vitro.

[0484] 43. The method of any of paragraphs 40-42, comprising determining the amount of antibody binding to Axl or a fragment of Axl.

[0485] 44. The method of any of paragraphs 30-39, further comprising using the antibody in the manufacture of a medicament for treating a disease or disorder characterized by overexpression of Axl.

[0486] 45. The antibody of any of paragraphs 1-27, or an immunoconjugate thereof in combination with another therapeutic agent.

[0487] 46. ​​A composition comprising the antibody of any of paragraphs 1-27, or an immunoconjugate thereof, together with a pharmaceutically acceptable excipient.

[0488] 47. The composition of paragraph 46, further comprising another therapeutic agent.

[0489] 48. The antibody of paragraph 45 or the composition of paragraph 47, wherein the additional therapeutic agent is an immune checkpoint modulator (ICM), such as an immune checkpoint inhibitor (ICI).

[0490] 49. An antibody according to any of paragraphs 1-27, 45 or 48, or a composition according to any of paragraphs 46-48, for use in a method of treatment.

[0491] 50. The antibody or composition of paragraph 49 for use in a method of treating a proliferative disease.

[0492] 51. The antibody or composition of paragraph 40, wherein the proliferative disease is cancer, such as AML.

[0493] 52. The antibody or composition of paragraph 51, wherein the cancer is a metastatic cancer.

[0494] 53. Use of an antibody according to any of paragraphs 1-27, 45 or 48, or a composition according to any of paragraphs 46-48, in the manufacture of a medicament for treating a disease or disorder characterized by overexpression of Axl.

[0495] 54. A method of treating a disease or condition characterized by overexpression of Axl, the method comprising administering to a patient having or at risk of developing the disease or condition an antibody according to any one of paragraphs 1-27, 45 or 48, or a composition according to any one of paragraphs 46-48.

[0496] 55. The method of paragraph 50, wherein the antibody directs delivery of the pharmaceutical composition to target metastatic cancer cells.

[0497] 56. Use of an antibody according to any one of paragraphs 1-27, 45 or 48 and one or more reagents allowing the determination of binding of the antibody to metastatic cancer cells in the manufacture of a diagnostic agent for detecting a disease or disorder characterized by overexpression of Axl.

[0498] 57. A method for diagnosing a disease or condition characterized by overexpression of Axl, the method comprising administering to a patient having or at risk of developing the disease or condition an antibody according to any one of paragraphs 1-27, 45 or 48, or a composition according to any one of paragraphs 46-48, and one or more reagents that allow for determination of binding of the antibody to metastatic cancer cells.

[0499] 58. A diagnostic kit comprising the antibody according to any of paragraphs 1-27, 45 or 48 and one or more reagents that allow the determination of binding of the moiety to metastatic cancer cells.

[0500] 59. A kit comprising an antibody according to any of paragraphs 1-27, 45 or 48, or a composition according to any of paragraphs 46-48.

[0501] 60. A pharmaceutical composition comprising an effective amount of the antibody according to any one of paragraphs 1-27 as an active ingredient, together with a pharmaceutically acceptable excipient. Example

[0502] Example 1: Generation of mouse anti-AXL monoclonal antibodies

[0503] Monoclonal antibodies (MAbs) directed against human Axl receptor were generated by immunizing immune competent OF1 mice (Charles River) with a recombinant antigen containing the extracellular domain of human Axl fused to the human IgGl Fc domain (rhAxl-Fc; R&D Systems).

[0504] Splenocytes from mice showing the presence of rhAxl-specific antibodies in the blood were used to fuse with mouse myeloma cells according to standard protocols. The cells were plated (10 5 The hybridoma selection was carried out by culturing the hybridoma with hypoxanthine-aminopterin-thymidine (HAT) medium (cells / well). After 12 days of selection, the supernatant was harvested and tested for Axl binding in enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Five positive clones that showed the highest antigen binding activity after the second round of subcloning by limiting dilution were expanded to carry out large-scale antibody production in vitro. MAbs were purified from the cell culture supernatant by protein G affinity chromatography.

[0505] Select flow cytometry ( Figure 1 ) is shown with Axl + The cell-specific binding antibody clone 1H12 was further characterized.

[0506] For flow cytometry, adherent cells in culture were washed with PBS, detached by trypsin (0.25%) for 1 min and tapping the culture dish to completely detach. Trypsin was quenched by adding complete medium to the tissue culture flask, followed by washing the cells with PBS. During the wash step, cells were collected by centrifugation at 200 g for 5 min. Antibodies were diluted to a total concentration in PBS containing 0.02% bovine serum albumin (BSA). 200 μL of 10 5Cell staining was performed with a cell suspension of 100 cells for 20 min. After two washing steps with PBS 0.02% BSA, cells were resuspended in 200 μL and kept on ice before analysis on an Accuri C6 flow cytometer (BD Biosciences).

[0507] Example 2: Mouse monoclonal antibody 1H12 does not cross-react with other members of the human TAM receptor family

[0508] All binding experiments were performed at 25°C using a Biacore 3000 instrument (GE Healthcare). Soluble recombinant antigens corresponding to human TAM receptor family members, Axl (rhAxl-Fc chimera; R&D Systems, catalog number 154-AL), Mer (rhMer-Fc chimera; R&D Systems, catalog number 891-MR) and Tyro3 (rhTyro3 / Dtk-Fc chimera; R&D Systems, catalog number 859-DK) were immobilized on the surface of a CM5 sensor chip using an amine coupling method at a surface density of 393.0, 303.6 and 364.0 resonance units, respectively. Biacore runs were performed in automatic mode using the Binding Analysis Wizard. Samples containing MAb 1H12 at a concentration of 10 μg / mL in HBS-EP buffer (GE Healthcare) were injected over the surface with immobilized antigen at a flow rate of 30 μL / min for 3 minutes (association) followed by 5 minutes of dissociation.

[0509] Figure 2 The results shown demonstrate specific interaction with human Axl but no binding to recombinant human Mer and Tyro3 antigens.

[0510] Example 3: Mouse monoclonal antibody 1H12 does not cross-react with mouse AXL

[0511] Binding experiments were performed at 25°C using a Biacore 3000 instrument (GE Healthcare). Soluble recombinant antigens corresponding to human Axl (rhAxl-Fc chimera; R&D Systems, catalog number 154-AL), mouse Axl (rmAxl-Fc chimera; R&D Systems, R&D Systems; catalog number 854-AX) and human Tyro3 (rhTyro3 / Dtk-Fc chimera; R&D Systems, catalog number 859-DK) were immobilized on the surface of a CM5 sensor chip using an amine coupling method at surface densities of 1,308.0, 2,115.9 and 1,429.0 RU, respectively. Biacore runs were performed in automatic mode using the Binding Analysis Wizard.

[0512] Samples containing MAb 1H12 or recombinant mouse (rm) Axl-ligand Gas6 (R&D Systems, catalog number 986-GS / CF) at a concentration of 10 μg / mL in HBS-EP buffer (GE Healthcare) were injected over the surface with immobilized antigen at a flow rate of 30 μL / min for 3 minutes (association) followed by 5 minutes of dissociation.

[0513] Figure 3 The results shown demonstrate that MAb 1H12 specifically interacts with human Axl but does not bind to recombinant mouse Axl and human Mer antigen ( Figure 3 , upper panel). In contrast, mouse Gas6, used as a control, demonstrated binding to both human and mouse Axl and slightly weaker binding to human Tyro3 ( Figure 3 , figure below).

[0514] Example 4: Affinity determination of mouse monoclonal antibody 1H12

[0515] Affinity determination of anti-Axl antibody 1H12 was performed by surface plasmon resonance measurement using a Biacore 3000 instrument (GE Healthcare) at 25° C. Sensor chip CM5 with immobilized rhAxl-Fc chimera (R&D Systems, catalog number 154-AL) at a density of 190 RU was used as the solid antibody coating surface.

[0516] For kinetic measurements, different concentrations of anti-Axl MAb 1H12 (1.3 to 666.7 nM) in HBS-EP buffer (Biacore, catalog number BR-1001-88) were injected at 30 μL / min for 3 min, followed by 5 min of dissociation (buffer alone). After each cycle, the surface was regenerated by injecting regeneration solution (10 mM HCl, 1 M NaCl) at 50 μL / min for 30 sec.

[0517] Mass transfer control experiments demonstrated that there was no significant mass transfer limitation for MAb 1H12. Additional conjugation control experiments did not reveal conjugation for MAb 1H12, as the dissociation phases were almost identical or had only one analyte concentration (1.8 μM or 270 μg / mL) after 1, 3, or 20 minutes of injection. Kinetic association (binding rate, k) was calculated using BIA evaluation software and a 1:1 Langmuir binding model. 结合 ) and dissociation (dissociation rate, k 解离 ) rate. Press k 解离 / k 结合 The equilibrium dissociation constant (K D ). Press In2 / k解离 The half-life of the antibody-antigen complex formed (t 1 / 2 ).

[0518] like Figure 4 As shown, mouse MAb 1H12 exhibited extremely high affinity (K D =4.98x10 -11 M), mainly due to the extremely slow dissociation rate (k 解离 =1.07x10 -5 1 / s), which results in a half-life of 1H12 / Axl complex of 18 hours.

[0519] Example 5: Mouse monoclonal antibody 1H12 does not block the binding of GAS6 to AXL

[0520] Competition binding studies were performed using a Biacore 3000 instrument (GE Healthcare) and a binding analysis wizard, with two samples injected in several cycles. As a first sample, a saturating concentration of MAb 1H12 (1.8 μM or 270 μg / mL) was injected at a flow rate of 30 μL / min on the surface of a CM5 sensor chip coated with rhAxl-Fc (using amine coupling method) for 3 minutes, followed by stabilization for 2.5 minutes (HBS-EP buffer alone) before injecting the second sample. The following second samples were used: recombinant human (rh) Gas6 (R&D Systems, catalog number 885-GS), recombinant mouse (rm) Gas6 (R&D Systems, catalog number 986-GS / CF) and a panel of anti-Axl antibodies (MAb1, 2, 3); all at a concentration of 25 μg / mL. As a control, MAb 1H12 was used as a second sample under the same conditions (25 μg / mL). The second sample was injected for 3 minutes, followed by 2.5 minutes of stabilization (buffer alone) and the surface was regenerated by injecting regeneration solution (10 mM HCl, 1 M NaCl) at a flow rate of 50 μL / min for 30 seconds.

[0521] Figure 5 The results shown demonstrate that MAb 1H12 does not compete for Axl binding with Gas6 (both human and mouse) and any other anti-Axl antibodies used in the experiments.

[0522] Example 6: Mouse monoclonal antibody 1H12 binds to denatured reduced and non-reduced AXL in Western blot analysis

[0523] For Western blot analysis, recombinant human (rh) Axl-Fc chimera (R&D Systems, catalog number 154-AL) with a predicted molecular mass of 71.7 kDa (corresponding to 100-110 kDa in SDS-PAGE under reducing conditions) and rhMer-Fc (R&D Systems, catalog number 891-MR) with a predicted molecular mass of 78.9 kDa (corresponding to 100-110 kDa in SDS-PAGE under reducing conditions) were used as antigens. Samples containing the antigens were denatured in the presence or absence of a reducing agent (Life Technologies) and loaded into the wells of a NuPAGE 3-8% Tris-acetate polyacrylamide (PAA) gel, 1.0 mm x 12 well (Invitrogen). As molecular weight markers, SeeBlue Plus2 prestained molecular weight markers (Novex LC5925) were used.

[0524] Electrophoresis was performed using Tris-acetate SDS running buffer under recommended conditions (Life Technologies) and, as described for the XCell II TM Proteins were transferred to nitrocellulose membranes using a transfer buffer with 20% methanol as described in the Blot Module (Invitrogen) manual for both gels. The membranes were incubated in 10 mL of blocking buffer, TBS / 0.1% Tween 20 (TBST) with 5% skim milk for 1 hour at room temperature, followed by overnight incubation at 4°C in 5 mL of incubation buffer (TBST with 3% skim milk) containing 1 μg / mL MAb1H12. The membranes were washed three times with 10 mL of TBST for 5 minutes each, followed by incubation with goat anti-mouse IgG (H+L) HRP-conjugated secondary antibody (1:3000) in 5 mL of incubation buffer for 1 hour at room temperature with gentle shaking. Afterwards, the membranes were washed three times in 10 mL of TBST for 5 minutes each and twice with 10 mL of TBS buffer. The membranes were incubated with 1 mL of ECL substrate for 1 minute at room temperature. Excess substrate solution was aspirated and the membranes were analyzed using ChemiDoc. TM Blots were visualized using an XRS+ imager (Bio Rad) and Image lab software.

[0525] Figure 6 The results shown demonstrate that antibody 1H12 specifically interacts with both reduced and non-reduced denatured Axl antigen. No binding to rhMer-Fc was detected. The results indicate that MAb 1H12 recognizes a linear epitope on the extracellular portion of the Axl receptor.

[0526] Example 7: Mouse monoclonal antibody 1H12 binds to denatured leukocytes expressed on the cell surface in its natural environment Protogenic and non-reduced AXL receptors

[0527] From Axl + and Axl - Cell lysates of cell lines, NCl-H1299 (non-small cell lung cancer, NSCLC) and LNCaP (prostate adenocarcinoma) were prepared according to standard protocols. Aliquots of cell lysates were denatured in the presence or absence of a reducing agent (Life Technologies) and loaded onto the wells of NuPAGE 3-8% Tris-acetate polyacrylamide (PAA) gels, 1.0 mm x 12 wells (Invitrogen). SDS-PAGE and Western blot analysis were performed essentially as described in Example 6.

[0528] Figure 7 The results shown demonstrate that MAb 1H12 binds to Axl + Specific interaction with Axl receptors (reduced and non-reduced) present in NCl-H1299 cells. No interaction with Axl was observed. + or Axl - Interaction with other cellular proteins present in the cell.

[0529] Example 8: Sequencing of mouse monoclonal antibody 1H12

[0530] Hybridoma 1H12 cells were propagated under standard conditions. 5x10 6 The cells were subjected to mRNA separation and cDNA synthesis. For the PCR amplification of the genes encoding the heavy chain and light chain variable regions (VH and VL, respectively), a mouse IgG library primer set (Progen, Heidelberg, Germany, catalog number F2010) was used. PCR amplification using different primer combinations resulted in 14 sequences being generated by PCR using 7 different primer combinations for the VH gene and 7 sequences being generated by PCR using 4 different primer combinations for the VL gene. The sequences of clones VH5 C9-3 and Vκ4G4-1 were selected, as determined by the nucleotide comparison with the IMGT database, and further work was done based on the highest homology with the corresponding germline sequences.

[0531] The amino acid sequences of the 1H12 VH and VL domains are shown in Figure 8 middle.

[0532] Example 9: Anti-AXL mouse monoclonal antibody 1H12 showed similarity to normal human tissues in immunohistochemical analysis Weak or no reaction

[0533] In a validation experiment, the optimal regimen and concentration for antibody 1H12 was determined. For this work, Axl + and Axl -The antibodies were tested at concentrations ranging from 0.05 μg / mL to 16.0 μg / mL (16, 8, 4, 2, 1, 0.5, 0.1, and 0.05 μg / mL). + MAb 1H12 showed mild to strong responses in cells; the response was mild at 0.5 μg / mL. Therefore, an optimal concentration of 1 μg / mL was set for tissue cross-reactivity (TCR) studies. At this concentration, no response was seen in Axl-negative cells.

[0534] TCR studies were performed using commercial frozen tissue microarrays purchased from BioChain (Production No. T6234701-2). All tissues were delivered from BioChain as frozen sections, acetone-fixed frozen TMAs. The experiments were performed as follows: frozen sections (8 μm) were air-dried overnight at room temperature and fixed in acetone for 10 minutes, after which they were blocked in 5% goat normal serum (Jackson ImmunoResearch, 005-000-121) for 30 minutes. The sections were then stained with primary antibodies (1H12) in PBS with 5% goat normal serum for 1 hour, after which they were washed three times in PBS. Subsequently, the sections were stained with EnVision mice (Dako, K4001) for 30 minutes. Finally, the sections were washed three times in Tris-HCl and then stained with 3,3'-diaminobenzidine (DAB) dye for 5 minutes. Images were captured using HTX imaging. Staining intensity was judged as: negative (0), weak reaction (1+), mild reaction (2+), or strong reaction (3+). The results of normal human TCR are shown in Table 2.

[0535] Antibody 1H12 exhibited weak to mild or mild reactions in lymph nodes and spleen cells (membrane staining). In the liver - local mild reactions may be seen in Kuppfer cells (membrane staining). Some local mild or strong intracellular reactions were seen in pancreatic epithelial cells. The following tissues did not show specific positive staining: adrenal glands, bone marrow, various brain tissues and spinal cord, colon, endothelium / aorta, esophagus, fallopian tubes, heart, kidneys, lungs, ovaries, placenta, prostate, skin, spinal cord, striated muscle, stomach, testes, thymus, thyroid, ureters and uterus.

[0536] Table 2.

[0537]

[0538]

[0539]

[0540] Example 10: Generation and testing of chimeric monoclonal antibody 1H12

[0541] The VH and VL sequences retrieved from mouse hybridoma 1H12 are used to produce synthetic genes, which are expressed in mammalian cells (GeneArt) through codon optimization. In an expression vector suitable for antibody production in mammalian cells, these mouse VH and VL genes are connected in frame with genetic elements encoding the constant domains of human IgG1 heavy chain and light chain (C-κ) respectively. The production of chimeric (mouse variable / human constant) IgG1 antibodies is achieved by transient expression in Chinese hamster ovary (CHO) cells, followed by purification using protein A affinity chromatography. The combination of purified chimeric antibodies (>95% purity) and Axl positive breast cancer cell line MDA-MB-231 was analyzed in flow cytometry. For comparison, parental mouse MAb 1H12 was used. For flow cytometry, adherent cells in culture were washed with PBS, separated by trypsin (0.25%) for 1 minute and hit the culture dish to separate completely. Trypsin was quenched by adding complete medium to tissue culture bottles, followed by washing cells with PBS. During the washing step, cells were collected by centrifugation at 200 g for 5 minutes. Antibodies were diluted to a total concentration in PBS containing 0.02% bovine serum albumin (BSA). 200 μL of 10 5 Cell suspensions of 10 cells were subjected to cell staining for 20 minutes. Cell-bound antibodies were detected with APC-conjugated donkey anti-human or anti-mouse IgG (H+L) F(ab')2 fragments (Jackson ImmunoResearch), respectively. After two washing steps with PBS / 0.02% BSA, cells were resuspended in 200 μL and kept on ice before analysis on an Accuri C6 flow cytometer (BD Biosciences). Fig. 9 The results shown demonstrate that the chimeric antibody binds strongly to MDA-MB-231 cells.

[0542] Example 11: Affinity determination of chimeric monoclonal antibody ch1H12

[0543] Affinity determination of anti-Axl chimeric (mouse variable / human constant IgG1) antibody ch1H12 was performed by surface plasmon resonance measurement using a Biacore 3000 instrument (GE Healthcare) at 25° C. A sensor chip CM5 with immobilized rhAxl-Fc chimera (R&D Systems, catalog number 154-AL) at a density of 190 RU was used as a solid antigen-coated surface.

[0544] For kinetic measurements, different concentrations of anti-Axl MAb ch1H12 (1.3 to 666.7 nM) in HBS-EP buffer (Biacore, catalog number BR-1001-88) were injected at 30 μL / min for 3 min, followed by 5 min of dissociation (buffer alone). After each cycle, the surface was regenerated by injecting regeneration solution (10 mM HCl, 1 M NaCl) at 50 μL / min for 30 sec.

[0545] Mass transfer control experiments demonstrated that there was no significant mass transfer limitation for MAb 1H12. Kinetic association (binding rate, k) was calculated using BIA evaluation software and a 1:1 Langmuir binding model. 结合 ) and dissociation (dissociation rate, k 解离 ) rate. Press k 解离 / k 结合 The equilibrium dissociation constant (K D ). Press In2 / k 解离 The half-life of the antibody-antigen complex formed (t 1 / 2 ).

[0546] like Fig.10 As shown, chimeric MAb ch1H12 exhibited extremely high affinity (K D =1.10x10 -11 M), mainly due to the extremely slow dissociation rate (k 解离 =2.99x10 -6 1 / s), which resulted in a half-life of 64.4 hours for the ch1H12 / Axl complex. The affinity values ​​found were comparable to those of the parental murine antibody 1H12 (K D This may indicate that V H and V L The domains are better oriented when anchored on a human constant domain scaffold.

[0547] Example 12: Mouse monoclonal antibody 1H12 cross-reacts with AXL from non-human primates

[0548] Recombinant Axl-Fc chimeric proteins containing the extracellular portion of the Axl receptor from cynomolgus and rhesus monkeys (cyno-Axl and rhe-Axl, respectively) were produced by transient expression in CHO cells. Recombinant cyno-Axl and rhe-Axl antigens were immobilized on the surface of a CM5 sensor chip using an amine coupling method at a surface density of 1,345.0 and 1,515.9 RU, respectively. As a positive control, hu-Axl-Fc chimera produced under the same conditions was immobilized on the same chip at a density of 1,234.9 RU.

[0549] Binding experiments were performed using a Biacore 3000 instrument (GE Healthcare) at 25° C. Biacore runs were performed in automatic mode using the Binding Analysis Wizard.

[0550] Samples containing MAb 1H12 at a concentration of 10 μg / mL in HBS-EP buffer (GE Healthcare) were injected over the surface with immobilized antigen at a flow rate of 30 μL / min for 3 minutes (association) followed by 5 minutes of dissociation.

[0551] Fig.11 The results shown demonstrate that MAb 1H12 binds strongly and specifically to all Axl variants of human origin and from cynomolgus and rhesus monkeys.

[0552] Example 13: Killing tumor cells using chimeric monoclonal antibody ch1H12 conjugated to saporin

[0553] To produce the immunotoxin, the chimeric MAb ch1H12 was non-covalently coupled to the plant toxin saporin using a FabFc-ZAP human conjugate (final concentration 4.5 nM) (AdvancedTargeting Systems, catalog number IT-65). The effect of ch1H12-saporin internalization on tumor cell viability was tested using the Axl-positive tumor cell line MDA-MB-231 (human triple-negative breast cancer). 800 cells were seeded per well in a 96-well plate supplemented with DMEM / F-12 medium with 10% FBS, L-glutamine (4 mM), streptomycin (5 μg / ml) and penicillin (5 U / ml) and allowed to attach for 16 hours. The cells were incubated for 72 hours with different dilutions of the immunotoxin ch1H12-saporin. By using The cell viability was determined by XTT / PMS assay using a microplate reader (BMGLABTECH).

[0554] Fig.12 The results shown demonstrate good internalization and strong cell killing potency of the ch1H12-based immunotoxin, EC 50 The values ​​(effective concentration causing 50% cell killing) were in the picomolar range.

[0555] Example 14: Antibody 1H12 induces AXL downstream signaling

[0556] Human cervical cancer cell line HeLa ( CCL-2 TM) were used for the experiments. Cells were grown to 80% confluence in T175 flasks in MEM medium (Sigma) supplemented with 10% FBS, penicillin-streptomycin, and L-glutamine. Cells were washed with PBS and detached by treatment with 0.25% trypsin / EDTA (Sigma), followed by centrifugation and resuspension in fresh medium (MEM / 0.5% FBS). Cells were plated in Petri dishes (3×10 6 Cells) were added to MEM medium supplemented with 10% FBS. After incubation at 37°C for 7 hours, the cells were washed with PBS and kept in starvation medium (MEM / 0.5% FBS) supplemented with 500ng / ml of Axl-Fc (R&D Systems) to deplete endogenous Gas6. After incubation for 24 hours, the medium was aspirated and fresh MEM / 0.5% FBS medium containing anti-Axl antibody 1H12 at a concentration of 7.5μg / mL alone or 1H12 (antibody cross-linking mixture) pre-mixed with AffiniPure goat anti-mouse IgG (H+L) #2.22 conjugated to biotin-SP was added. After incubation at 37°C for 10-30 minutes, the cells were collected by centrifugation and resuspended in NP40-lysis buffer, followed by incubation on ice for 30 minutes. Cell lysates were cleared by centrifugation (12,000rpm, 4°C, 5min) and protein concentration was determined using the BCA protein assay. Cell lysate samples containing 35 μg of total protein were denatured in the presence of a reducing agent (Life Technologies) and loaded into the wells of a NuPAGE 10% Bis-Tris polyacrylamide (PAA) gel, 1.0 mm x 12 well (Invitrogen). Electrophoresis was performed using Bis-Tris SDS running buffer under the recommended conditions (Life Technologies) and, as described in the XCell II TM Proteins were transferred to PVDF membranes using transfer buffer with 20% methanol as described in the Blot Module (Invitrogen) manual for both gels. The membranes were incubated in 10 mL of blocking buffer, TBS / 0.1% Tween 20 (TBST) with 5% skim milk for 1 hour at room temperature, followed by incubation at 4°C in 5 mL of anti-phospho-Akt (Ser 473) antibody (Cell Signaling) incubation buffer (TBST with 3% skim milk) overnight. The membrane was washed three times with 10 mL of TBST for 5 minutes each time, followed by incubation with goat anti-rabbit HRP-conjugated secondary antibody for 1 hour at room temperature with gentle shaking. Afterwards, the membrane was washed three times in 10 mL of TBST for 5 minutes each time and twice with 10 mL of TBS buffer. The membrane was incubated with 1 mL of ECL substrate for 1 minute at room temperature. Excess substrate solution was aspirated and the membrane was analyzed using ChemiDoc TM The blots were visualized using an XRS+ imager (Bio Rad) and Image lab software. As a loading control, detection was performed using an anti-mouse actin antibody (1:10,000; Sigma) under the same conditions. Anti-phospho-Akt does not distinguish between AKT1, AKT2, and AKT3, so the total level of 'phospho-Akt' is shown in the blot. Detection with an anti-GAPDH antibody (Millipore) was used as a loading control.

[0557] Fig.13 The results shown demonstrate that anti-Axl antibody 1H12 cross-linked with a secondary anti-mouse antibody induces strong Axl signaling in HeLa cells using Ser 473 Downstream phosphorylation of Akt was used as a readout. This effect was shown to be dose-dependent, with higher amounts of cross-linked 1H12 resulting in stronger receptor signaling ( Fig.14 ).also, Fig.15 The data presented demonstrate that the 1H12 antibody can cause Axl receptor activation and downstream signaling alone, without the need for cross-linking with a secondary antibody. The results indicate that the 1H12 antibody has strong agonistic activity.

[0558] Example 15: Mouse monoclonal antibody 1H12 and commercial antibody MAB 154 competes for binding to AXL

[0559] Competition binding studies were performed using a Biacore 3000 instrument (GE Healthcare) and the Binding Analysis Wizard, injecting two samples in several cycles. As the first sample, a saturating concentration of MAb 1H12 (1.8 μM or 270 μg / mL) was injected at a flow rate of 30 μL / min on the surface of a CM5 sensor chip coated with rhAxl-Fc (using amine coupling method) for 3 minutes, followed by stabilization for 2.5 minutes (HBS-EP buffer alone) before injecting the second sample. The following second samples were used: recombinant mouse (rm) Gas6 (R&D Systems, catalog number 986-GS / CF) and commercial anti-Axl monoclonal antibody MAB154 (mouse IgG1, clone #108724; R&D Systems, catalog number MAB154); both at a concentration of 25 μg / mL. As a control, MAb 1H12 was used as the second sample under the same conditions (25 μg / mL). The second sample was injected for 3 minutes, followed by 2.5 minutes of stabilization (buffer alone) and the surface was regenerated by injecting regeneration solution (10 mM HCl, 1 M NaCl) at a flow rate of 50 μL / min for 30 seconds.

[0560] Fig.16 The results shown demonstrate that MAb 1H12 inhibits the binding of MAB154 to Axl and does not compete with mouse Gas6.

[0561] Example 16: Comparison of AXL detection using 1H12 and commercial antibodies

[0562] Immunohistochemistry and Western blot analysis were performed using MAb 1H12 to detect Axl.

[0563] FFPE sections from cell pellets prepared using Axl+ parental MDA-MB-231 cells or Axl-MDA-MB-231shAxl2 cells were stained with MAb1H12, polyclonal AF154, and monoclonal MAb 154 ( Fig.17 A). The MDA-MB-231 shAxl2 cell line has Axl expression knocked down using a retroviral construct expressing a shRNA targeting Axl; this results in a mixed population in which -10% of cells remain Axl+.

[0564] Optimal staining of Axl+ cells with MAb 1H12 was achieved at a concentration of 1 μg / ml (highly positive staining of the cell membrane and weak or no staining of the cytoplasm), while Axl- cells showed mainly negative staining with some expression in dispersed individual cells. The presence of isolated Axl-expressing cells is consistent with the observation of multiple MDA-MB-231shAxl2, as -flow cytometry showed that approximately 90% of the cells carried the retroviral shRNA construct. Counterstaining was performed using AF154 at a dilution of 1:6400 (0.03125 μg / ml03125 μg / mL) and demonstrated slightly stronger membrane staining. However, strong staining was also observed in Axl- cells in the dispersed population, with some weaker expression in most cells.

[0565] Staining with MAB154 demonstrated weaker performance on Axl+ cells than MAb 1H12 at similar concentrations. However, fewer cells in the Axl- population were weakly stained compared to both MAb 1H12 and AF154. This suggests that 1H12 produces improved results with respect to IHC compared to AF154 and MAB154.

[0566] Similar comparisons were performed by Western blot analysis of Axl+ and Axl- cell lysates using MAbs 1H12, AF154, and MAB154 ( Fig.17 B). For recommendation and visualization purposes, all three antibodies were used at a concentration of 1 μg / ml. A 140 kDa Axl protein band was displayed in all Axl+ cell lysates. However, this band was barely detectable in Axl- lysates with AF154, although some weak background staining was detected, which suggests a response to other low-level proteins as previously noted in Ahmed et al., 2015. Blots with MAb 1H12 were consistently stronger than those seen with MAB154. We infer from these results that MAb 1H12 performs significantly better than AF154 and MAB 154 in western blots.

[0567] Specifically, this application involves the following technical solutions:

[0568] 1. An antibody that binds to Axl, comprising:

[0569] An antibody VH domain selected from the group consisting of a 1H12 VH domain (SEQ ID NO. 3) and a VH domain comprising a VH CDR3 having an amino acid sequence of SEQ ID NO. 7 and optionally one or more VH CDRs having an amino acid sequence selected from the group consisting of SEQ ID NO. 6 and SEQ ID NO. 5; and / or

[0570] An antibody VL domain selected from the group consisting of a 1H12 VL domain (SEQ ID NO.4) and a VL domain comprising one or more VLCDRs having an amino acid sequence selected from the group consisting of SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10.

[0571] 2. The antibody according to 1, comprising an antibody VH domain, wherein the antibody VH domain comprises a VH CDR having an amino acid sequence of SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, and the antibody competes for binding to Axl with the Axl binding domain of an antibody comprising the 1H12 VH domain (SEQ ID NO.3) and the 1H12 VL domain (SEQ ID NO.4).

[0572] 3. The antibody according to 1 or 2, comprising the 1H12 VH domain (SEQ ID NO. 3).

[0573] 4. The antibody according to 3, comprising the 1H12 VL domain (SEQ ID NO. 4).

[0574] 5. A variant of the antibody according to any one of 1-4, wherein the variant comprises one or more amino acid sequence changes in one or more framework regions and / or one or more CDRs.

[0575] 6. The antibody according to any one of 1-5, which binds to Axl with an affinity equal to or better than the Axl antigen binding site formed by the 1H12 VH domain (SEQ ID NO. 3) and the 1H12 VL domain (SEQ ID NO. 4), the affinity of the antibody and the affinity of the antigen binding site being determined under the same conditions.

[0576] 7. The antibody according to any one of 1-6, which comprises a scFv antibody molecule.

[0577] 8. The antibody according to any one of 1-6, comprising an antibody constant region.

[0578] 9. The antibody according to 8, comprising a whole antibody.

[0579] 10. The antibody according to any one of 1-6, wherein the antibody is an antibody fragment that binds to an antigen, such as a single domain antibody, Fv, scFv, dsFv, Fd, Fab, F(ab')2, a mini antibody, a diabody, a single-chain diabody, a tandem scFv, TandAb, a double-chain antibody, a three-chain antibody, a κ(λ) antibody, BiTE, DVD-lg, SIP, SMIP or DART.

[0580] 11. The antibody according to any one of 1-10, comprising additional amino acids that provide other functional characteristics besides the ability to bind antigen.

[0581] 12. The antibody according to any one of 1-11, which has a concentration of no more than 5×10 -11 M of K D Combined with Axl.

[0582] 13. The antibody according to any one of 1-11, which has a concentration of no more than 1.5×10 -11 M of K D Combined with Axl.

[0583] 14. The antibody according to any one of 1-13, which has a concentration of no more than 2×10 -5 s -1 K 解离 Combined with Axl.

[0584] 15. The antibody according to any one of 1-14, which has a concentration of no more than 3×10 -6 s -1 K 解离 Combined with Axl.

[0585] 16. The antibody according to any one of 1-15, wherein the Axl is human Axl.

[0586] 17. The antibody according to any one of 1-16, which specifically binds to primate Axl.

[0587] 18. The antibody according to any one of 1-17, which:

[0588] (i) More than 10 -3 M of K D Binds to rat Axl;

[0589] (ii) more than 10 -3 M of K D Combined with Mer; and / or

[0590] (iii) more than 10 -3 M of K D Binds human Tyro3.

[0591] 19. The antibody according to any one of 1-18, wherein the antibody is an Axl agonist.

[0592] 20. The antibody according to 19, wherein Axl signaling is at least 10% stronger.

[0593] 21. The antibody according to any one of 1-20, wherein the antibody is a chimeric antibody.

[0594] 22. The antibody according to any one of 1-20, wherein the antibody is a humanized antibody.

[0595] 23. The antibody according to any one of 1-22, wherein the antibody binds to:

[0596] (i) the same epitope as the 1H12 antibody, or

[0597] (ii) an epitope that overlaps with the epitope bound by the 1H12 antibody.

[0598] 24. The antibody according to any one of 1-23, wherein the antibody is internalized after binding to Axl present on the cell surface.

[0599] 25. The antibody according to any one of 1-24, optionally conjugated to a detectable label, enzyme or toxin via a peptide bond or a linker.

[0600] 26. The antibody according to 25, wherein the toxin is selected from the group consisting of MMAE and MMAF.

[0601] 27. The antibody according to 25, wherein the detectable label is FITC.

[0602] 28. An isolated nucleic acid comprising a nucleotide sequence encoding the antibody or antibody VH or VL domain of an antibody according to any one of 1-24.

[0603] 29. A host cell transformed with the nucleic acid according to 28.

[0604] 30. A method for producing an antibody or an antibody VH or VL domain, the method comprising culturing the host cell according to 29 under conditions for producing the antibody or the antibody VH or VL domain.

[0605] 31. The method of 30, further comprising isolating and / or purifying the antibody or antibody VH or VL variable domain.

[0606] 32. The method of claim 30 or 31, further comprising formulating the antibody or antibody VH or VL variable domain into a composition comprising at least one additional component.

[0607] 33. A method for obtaining an antibody that binds to Axl, the method comprising

[0608] Providing one or more VH domains each being an amino acid sequence variant of the 1H12 VH domain by adding, deleting, substituting or inserting one or more amino acids in the amino acid sequence of the 1H12 VH domain (SEQ ID NO. 3), and optionally combining the one or more VH domain amino acid sequence variants thus provided with one or more VL domains to provide one or more VH / VL combinations; and / or

[0609] Providing a VL domain that is an amino acid sequence variant of the 1H12 VL domain by adding, deleting, substituting or inserting one or more amino acids in the amino acid sequence of the 1H12 VL domain (SEQ ID NO. 4), and combining the one or more VL domain amino acid sequence variants provided in this way with one or more VH domains to provide one or more VH / VL domain combinations;

[0610] and

[0611] The VH domain amino acid sequence variants or VH / VL combination or combinations are tested to identify antibodies that bind Axl.

[0612] 34. A method for obtaining an antibody that binds to Axl, the method comprising:

[0613] Providing a starting nucleic acid encoding one or more VH domains comprising the CDR3 to be replaced or lacking a CDR3 coding region, and combining the starting nucleic acid with a donor nucleic acid encoding the VH CDR3 amino acid sequence of SEQ ID NO.7 such that the donor nucleic acid is inserted into the CDR3 region of the starting nucleic acid to provide a product nucleic acid encoding a VH domain; or

[0614] Providing a starting nucleic acid encoding one or more VL domains comprising a CDR3 to be replaced or lacking a CDR3 coding region, and combining the starting nucleic acid with a donor nucleic acid encoding the VL CDR3 amino acid sequence of SEQ ID NO. 10, such that the donor nucleic acid is inserted into the CDR3 region of the starting nucleic acid, so as to provide a product nucleic acid encoding a VL domain;

[0615] expressing nucleic acids encoding said product nucleic acids of VH domains and optionally combining said VH domains so produced with one or more VL domains to provide a VH / VL combination, and / or expressing nucleic acids encoding said product nucleic acids of VL domains and combining said VL domains so produced with one or more VH domains to provide a VH / VL combination;

[0616] selecting an antibody comprising a VH domain or a VH / VL combination, said antibody binding to Axl; and

[0617] The antibody that binds Axl and / or the nucleic acid encoding the antibody that binds Axl are recovered.

[0618] 35. The method according to 33 or 34, wherein the antibody that binds to Axl is an antibody fragment comprising a VH domain and a VL domain.

[0619] 36. The method of 35, wherein the antibody fragment is a scFv antibody molecule.

[0620] 37. The method according to 35, wherein the antibody fragment is a Fab antibody molecule.

[0621] 38. The method according to 36 or 37 further comprises providing the VH domain and / or the VL domain of the antibody fragment in the whole antibody.

[0622] 39. The method according to any one of 33-38, further comprising formulating the antibody that binds to Axl or the antibody VH or VL variable domain of the antibody that binds to Axl into a composition comprising at least one additional component.

[0623] 40. The method according to any one of 30-39, further comprising binding an antibody that binds Axl to Axl or a fragment of Axl.

[0624] 41. A method comprising binding the Axl-binding antibody according to any one of 1-27 to Axl or a fragment of Axl.

[0625] 42. The method of claim 40 or 41, wherein the binding occurs in vitro.

[0626] 43. The method according to any one of 40-42, comprising determining the amount of antibody binding to Axl or a fragment of Axl.

[0627] 44. The method according to any one of 30-39, further comprising using the antibody for the manufacture of a medicament for treating a disease or disorder characterized by overexpression of Axl.

[0628] 45. An antibody according to any one of 1-27, or an immunoconjugate thereof in combination with another therapeutic agent.

[0629] 46. ​​A composition comprising the antibody according to any one of 1-27, or an immunoconjugate thereof, together with a pharmaceutically acceptable excipient.

[0630] 47. The composition of 46, further comprising another therapeutic agent.

[0631] 48. The antibody of 45 or the composition of 47, wherein the another therapeutic agent is an immune checkpoint modulator (ICM), such as an immune checkpoint inhibitor (ICI).

[0632] 49. An antibody according to any one of 1-27, 45 or 48, or a composition according to any one of 46-48, for use in a method of treatment.

[0633] 50. The antibody or composition of 49, for use in a method for treating a proliferative disease.

[0634] 51. The antibody or composition of 40, wherein the proliferative disease is cancer, such as AML.

[0635] 52. The antibody or composition of 51, wherein the cancer is a metastatic cancer.

[0636] 53. Use of an antibody according to any one of 1-27, 45 or 48, or a composition according to any one of 46-48, in the manufacture of a medicament for treating a disease or disorder characterized by overexpression of Axl.

[0637] 54. A method of treating a disease or condition characterized by overexpression of Axl, the method comprising administering to a patient having the disease or condition or at risk of developing the disease or condition, an antibody according to any one of 1-27, 45 or 48, or a composition according to any one of 46-48.

[0638] 55. The method of 50, wherein the antibody directs delivery of the pharmaceutical composition to target metastatic cancer cells.

[0639] 56. Use of an antibody according to any one of 1-27, 45 or 48 and one or more reagents allowing the determination of binding of the antibody to metastatic cancer cells in the manufacture of a diagnostic agent for detecting a disease or disorder characterized by overexpression of Axl.

[0640] 57. A method for diagnosing a disease or condition characterized by overexpression of Axl, the method comprising administering to a patient having the disease or condition or at risk of developing the disease or condition, an antibody according to any one of 1-27, 45 or 48, or a composition according to any one of 46-48, and one or more reagents that allow for determination of binding of the antibody to metastatic cancer cells.

[0641] 58. A diagnostic kit comprising an antibody according to any one of 1-27, 45 or 48 and one or more reagents allowing the determination of the binding of the moiety to metastatic cancer cells.

[0642] 59. A kit comprising an antibody according to any one of 1-27, 45 or 48, or a composition according to any one of 46-48.

[0643] 60. A pharmaceutical composition comprising an effective amount of the antibody according to any one of 1-27 as an active ingredient, together with a pharmaceutically acceptable excipient.

[0644] sequence

[0645] SEQ ID NO.1 [VH domain (nt)]

[0646]

[0647] SEQ ID NO.2 [VL domain (nt)]

[0648]

[0649] SEQ ID NO.3 [VH domain ( aa )]

[0650]

[0651] SEQ ID NO.4 [VL domain ( aa )]

[0652]

[0653] SEQ ID NO.5 [Heavy chain CDR1]

[0654]

[0655] SEQ ID NO.6 [Heavy chain CDR2]

[0656]

[0657] SEQ ID NO.7 [Heavy chain CDR3]

[0658]

[0659] SEQ ID NO.8 [Light chain CDR1]

[0660]

[0661] SEQ ID NO.9 [Light chain CDR2]

[0662]

[0663] SEQ ID NO.10 [Light chain CDR3]

[0664]

[0665] SEQ ID NO.11 [Heavy chain FR1]

[0666]

[0667] SEQ ID NO.12 [Heavy chain FR2]

[0668]

[0669] SEQ ID NO.13 [Heavy chain FR3]

[0670]

[0671] SEQ ID NO.14 [Heavy chain FR4]

[0672]

[0673] SEQ ID NO.15 [Light chain FR1]

[0674]

[0675] SEQ ID NO.16 [Light chain FR2]

[0676]

[0677] SEQ ID NO.17 [Light chain FR3]

[0678]

[0679] SEQ ID NO.18 [Light chain FR4]

[0680]

[0681] SEQ ID NO.19 [Axl]

[0682]

[0683] SEQ ID NO.20 [Rat Axl]

[0684]

[0685] SEQ ID NO.21 [People Tyro3]

[0686]

[0687] SEQ ID NO.22 [People]

[0688]

Claims

1. A composition comprising an antibody that binds to Axl or an immunoconjugate of said antibody, together with a carrier, a buffer or a stabilizer, wherein said antibody comprises: VH CDR1, VH CDR2 and VH CDR3 of an antibody VH domain, wherein the VH domain consists of the amino acid sequence of SEQ ID NO: 3; and VL CDR1, VL CDR2 and VL CDR3 of the antibody VL domain, wherein the VL domain consists of the amino acid sequence of SEQ ID NO:

4.

2. The composition according to claim 1, wherein the antibody comprises: an antibody VH domain comprising a VH CDR1 of the amino acid sequence of SEQ ID NO.5, a VH CDR2 of the amino acid sequence of SEQ ID NO.6, and a VH CDR3 of the amino acid sequence of SEQ ID NO.7; and An antibody VL domain comprising a VL CDR1 of the amino acid sequence of SEQ ID NO.8, a VL CDR2 of the amino acid sequence of SEQ ID NO.9, and a VL CDR3 of the amino acid sequence of SEQ ID NO.

10.

3. The composition of claim 1, wherein the antibody comprises: The VH domain of the amino acid sequence of SEQ ID NO.3; and / or The VL domain of the amino acid sequence of SEQ ID NO.

4.

4. The composition of claim 2, wherein the antibody comprises: The VH domain of the amino acid sequence of SEQ ID NO.3; and / or The VL domain of the amino acid sequence of SEQ ID NO.

4.

5. The composition of any one of claims 1-4, wherein the antibody is a scFv antibody molecule.

6. The composition of any one of claims 1-4, wherein the antibody comprises an antibody constant region.

7. The composition of claim 6, wherein the antibody is a whole antibody.

8. The composition of any one of claims 1-4, wherein the antibody is a chimeric antibody.

9. The composition of any one of claims 1-4, wherein the antibody is a humanized antibody.

10. The composition of any one of claims 1-4, wherein the antibody is conjugated to a detectable label, an enzyme, or a toxin.

11. The composition according to claim 10, wherein the toxin is selected from the group consisting of MMAE and MMAF.

12. The composition of claim 10, wherein the detectable label is FITC.

13. Use of an antibody that binds Axl in the manufacture of a medicament for treating breast cancer or cervical cancer in a human patient, wherein the antibody comprises: VH CDR1, VH CDR2 and VH CDR3 of an antibody VH domain, wherein the VH domain consists of the amino acid sequence of SEQ ID NO: 3; and VL CDR1, VL CDR2 and VL CDR3 of the antibody VL domain, wherein the VL domain consists of the amino acid sequence of SEQ ID NO:

4.

14. Use of the composition according to claim 1 in the manufacture of a medicament for treating breast cancer or cervical cancer.

15. The use according to claim 13 or 14, wherein the antibody or the composition is formulated for administration in combination with another therapeutic agent, wherein the other therapeutic agent is: an analgesic, an antiemetic or an anticancer agent.

16. The use according to claim 15, wherein the other therapeutic agent is: an EGFR antagonist or an immune checkpoint modulator (ICM).

Images