ANTI-FLT3 ANTIBODY, ITS USE, AS WELL AS COMPOSITION UNDERSTANDING THE REFERRED ANTIBODY AND NUCLEIC

专利摘要:
anti-flt3 antibodies and methods of using them. the present invention relates to anti-flt3 antibodies with a modified fc region comprising amino acid substitutions 239d and 332e to increase antibody-dependent cell cytotoxicity (adcc) of these antibodies. the invention later relates to pharmaceutical compositions containing such antibodies, nucleic acids encoding such antibodies as well as methods of using such antibodies.
公开号:BR112012015740B1
申请号:R112012015740-0
申请日:2010-12-23
公开日:2020-09-29
发明作者:Ludger Grosse-Hovest；Hans-Jörg Bühring；Martin Hofmann；Steffen Aulwurm；Grundram Jung
申请人:Synimmune Gmbh；
IPC主号:

专利说明:

Field of the Invention
[0001] The present invention is in the field of antibodies and refers to FLT3-specific antibodies with a modified Fc region to generate or increase antibody dependent cell cytotoxicity (ADCC) as well as methods of using such antibodies. Background of the Invention
[0002] The tyrosine kinase receptor FLT3 expressed on the cell surface of hematopoietic progenitor cells plays an important role in early hematopoiesis. Due to its essential role in the regulation of survival, proliferation, and differentiation of hematopoietic cells (B and T cells), anomalous FLT3 activity is involved in the development and progression of cancers of the hematopoietic system. For example, internal tandem duplications of FLT3 are the most common mutations associated with acute myelogenous leukemia (AML). There is thus a need in the art for antibodies that can specifically target and destroy FLT3 expression cells.
[0003] Thus, an objective of the inventors of the present invention was to provide anti-FLT3 antibodies that can bind to and kill FLT3 expression cells in vivo. Summary of the Invention
[0004] The present invention relates to antibodies directed against human FLT3 receptor tyrosine kinase and methods of using them. In certain respects, antibodies include a variant Fc region. In other embodiments, the antibodies are chimeric or humanized antibodies. The present invention later relates to pharmaceutical compositions comprising such antibodies and methods of using the antibodies in various indications of disease.
[0005] In a first aspect, the present invention relates to an antibody that binds to human FLT3 receptor tyrosine kinase, wherein said antibody comprises a heavy chain and / or a light chain and has at least one amino acid substitution in the constant region in relation to an anti-FLT3 antibody of origin, in which said at least one amino acid substitution includes amino acid substitutions 239D and 332E, in which the positional numbering is in accordance with the EU index (Kabat et al. , 1983). In a specific aspect, the substitutions are S239D and I332E.
[0006] In one embodiment of the invention, the anti-FLT3 antibody has cell killing activity, such as, for example, antibody dependent cell-mediated cytotoxicity (ADCC) function. That means that in contact with FLT3 expression cells, the antibody is able to facilitate cell death, for example, by triggering the activation of the complement system, phagocytosis or apoptosis.
[0007] In one embodiment, the antibody comprises a heavy chain and a light chain. The heavy chain may comprise a VH CDR1, VH CDR2, and VH CDR3 region and / or the light chain may comprise a VL CDR1, VL CDR2 region, and / or VL CDR3 region.
[0008] In a specific embodiment, a VL CDR1 comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of the amino acid sequences SEQ ID NO: I and SEQ ID NO: 7; a VL CDR2 comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 8; a VL CDR3 comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 3 and SEQ ED NO: 9; a VH CDR1 comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 4 and SEQ ID NO: 10; the VH CDR2 comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 5 and SEQ ID NO: 11; and VH CDR3 comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 6 and SEQ ID NO: 12.
[0009] In another specific embodiment, VL CDR1 comprises, consists essentially of, or consists of the amino acid sequence indicated in SEQ ID NO: I; the VL CDR2 comprises, consists essentially of, or consists of the amino acid sequence indicated in SEQ ID NO: 2; the VL CDR3 comprises, consists essentially of, or consists of the amino acid sequence indicated in SEQ ID NO: 3; the VH CDR1 comprises, consists essentially of, or consists of the amino acid sequence indicated in SEQ ID NO: 4; the VH CDR2 comprises, consists essentially of or consists of the amino acid sequence indicated in SEQ ID NO: 5; and VH CD3 comprises, consists essentially of, or consists of the amino acid sequence indicated in SEQ ID NO: 6.
[00010] In yet another specific embodiment, VL CD1 comprises, consists essentially of, or consists of the amino acid sequence indicated in SEQ ID NO: 7; the VL CDR2 comprises, consists essentially of or consists of the amino acid sequence indicated in SEQ ID NO: 8; the VL CDR3 comprises, consists essentially of, or consists of the amino acid sequence indicated in SEQ ID NO: 9; the VH CDR1 comprises, consists essentially of, or consists of the amino acid sequence indicated in SEQ ID NO: 10; VH CDR2 comprises, consists essentially of, or consists of the amino acid sequence indicated in SEQ ID NO: 11; and VH CDR3 comprises, consists essentially of, or consists of the amino acid sequence indicated in SEQ ID NO: 12.
[00011] In one embodiment of the invention, the inventive antibody heavy chain comprises a VH domain comprising, which essentially consists of or consisting of the amino acid sequence indicated in SEQ ID NO: 14 and / or the light chain of the invented antibody comprises a VL domain comprising, consisting essentially of or consisting of the amino acid sequence indicated in SEQ ID NO: 13.
[00012] In another embodiment of the invention, the heavy chain of the invented antibody comprises a VH domain comprising, consisting essentially of or consisting of the amino acid sequence indicated in SEQ ID NO: 30 and / or the light chain of the invented antibody comprises a VL domain comprising, consisting essentially of or consisting of the amino acid sequence indicated in SEQ ID NO: 29.
[00013] In another embodiment of the invention, the claimed antibody is a chimeric antibody and comprises a heavy chain having the amino acid sequence indicated in SEQ ID NO: 27 and / or a light chain having the amino acid sequence indicated in SEQ ID NO : 23.
[00014] In another embodiment of the invention, the claimed antibody is a chimeric antibody and comprises a heavy chain having the amino acid sequence indicated in SEQ ID NO: 43 and / or a light chain having the amino acid sequence indicated in SEQ ID NO : 39.
[00015] In certain embodiments of the invention, the antibody of the invention comprising amino acid substitutions S239D / I332E binds with increased affinity for the FcRIlIa receptor or has increased ADCC effector function when compared to the parent antibody without said substitution. In this context, the term "augmented" includes arguments where the source antibody does not show any experimentally verifiable ADCC effector function so that the early generated Fc-optimized antibody exhibits, for the first time and in contrast to the source antibody from the which it can be derived, effective function of ADCC.
[00016] In other embodiments, the antibody comprises one or more other amino acid modifications to a position selected from the group consisting of 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 240, 241, 243, 244, 245, 246, 247, 249, 255, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 280, 281, 282, 283, 284, 285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 313, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 333, 334, 335, 336, and 337, in that positional numbering is in accordance with the EU index. These one or more other amino acid modifications can be selected from the group of amino acid substitutions consisting of 221K, 221Y, 222E, 222Y, 223E, 223K, 224E, 224Y, 225E, 225K, 225W, 227E, 227G, 227K, 227Y, 228E, 228G, 228K, 228Y, 230A, 230E, 230G, 230Y, 231E, 231G, 231K, 231P, 231Y, 232E, 232G, 232K, 232Y, 233A, 233D, 233F, 233G, 233H, 233I, 233K, 233L, 233M, 233N, 233Q, 233R, 233S, 233T, 233V, 233W, 233 Y, 234A, 234D, 234E, 234F, 234G, 234H, 234I, 234K, 234M, 234N, 234P, 234Q, 234R, 234S, 234T, 234S, 234 , 234W, 234Y, 235A, 235D, 235E, 235F, 235G, 235H, 235I, 235K, 235M, 235N, 235P, 235Q, 235R, 235S, 235T, 235V, 235W, 235Y, 236A, 236D, 236E, 236F, 236H , 2361, 236K, 236L, 236M, 236N, 236P, 236Q, 236R, 236S, 236T, 236V, 236W, 236Y, 237D, 237E, 237F, 237H, 2371, 237K, 237L, 237M, 237N, 237P, 237Q, 237P, 237Q , 237S, 237T, 237V, 237W, 237Y, 238D, 238E, 238F, 238G, 238H, 2381, 238K, 238L, 238M, 238N, 238Q, 238R, 238S, 238T, 238V, 238W, 238Y, 240A, 240I, 240M, 240M, 240M , 240T, 241D, 241E, 241L, 241 R, 241S, 241W, 241Y, 243E, 243H, 243L, 243Q, 243R, 243W, 243Y, 244H, 245A, 246D, 246E, 246H, 246Y, 247G, 247V, 249H, 249Q, 249Y, 255E, 255Y, 258H, 258S, 258Y, 260D, 260E, 260H, 260Y, 262A, 262E, 262F, 2621, 262T, 263A, 263I, 263 M, 263T, 264A, 264D, 264E, 264F, 264G, 264H, 264I, 264K, 264L, 264M , 264N, 264P, 264Q, 264R, 264S, 264T, 264W, 264Y, 265F, 265G, 265H, 2651, 265K, 265L, 265M, 265N, 265P, 265Q, 265R, 265S, 265T, 265V, 265W, 265, 265, 265 , 2661, 266M, 266T, 267D, 267E, 267F, 267H, 2671, 267K, 267L, 267M, 267N, 267P, 267Q, 267R, 267T, 267V, 267W, 267Y, 268D, 268E, 268F, 268G, 2681, 268G, 2681 , 268L, 268M, 268P, 268Q, 268R, 268T, 268V, 268W, 269F, 269G, 269H, 2691, 269K, 269L, 269M, 269N, 269P, 269R, 269S, 269T, 269V, 269W, 269Y, 270F, 270 , 270H, 2701, 270L, 270M, 270P, 270Q, 270R, 270S, 270T, 270 W, 270 Y, 271 A, 27 ID, 271 E, 271F, 271G, 271H, 2711, 271K, 271L, 271 M, 271N , 271Q, 271R, 271S, 271T, 271V, 271W, 271Y, 272D, 272F, 272G, 272H, 2721, 272K, 272L, 272M, 272P, 272R, 272S, 272T, 272V, 272W, 272Y, 2731, 274D, 274E, 274F, 274G, 274H, 2741, 274L, 274M, 274N, 274P, 274R, 274T, 274V, 274W, 274Y, 275L, 275W, 276D, 276E, 276F, 276G, 276H, 2761, 276L, 276M, 276P, 276R, 276S, 276T, 276V, 276W, 276Y, 278D, 278E, 278G, 278H, 2781, 278K, 278L, 278M, 278N, 278P, 278Q, 278R, 278S, 278T, 278V, 278W, 280G, 280K, 280L, 280P, 280W, 281D, 281E, 281K, 281N, 281P, 281Q, 281 Y, 282E, 282G, 282K, 282P, 282Y, 283G, 283H, 283K, 283L, 283P, 283R , 283Y, 284D, 284E, 284L, 284N, 284Q, 284T, 284Y, 285D, 285E, 285K, 285Q, 285W, 285Y, 286E, 286G, 286P, 286Y, 288D, 288E, 288 Y, 290D, 290H, 290L, 290N, 290W, 29 ID, 291E, 291 G, 291H, 2911, 291Q, 291T, 292D, 292E, 292T, 292Y, 293F, 293G, 293H, 2931, 293L, 293M, 293N / 293P, 293 R, 293S, 293T , 293V, 293 W, 293 Y, 294F, 294G, 294H, 2941, 294K, 294L, 294M, 294P, 294R, 294S, 294T, 294V, 294W, 294Y, 295D, 295E, 295F, 295G, 295H, 2951, 295M , 295N, 295P, 295R, 295S, 295T, 295V, 295W, 295 Y, 296A, 296D, 296E, 296G, 296H, 2961, 296K, 296L, 296M, 296N, 296 Q, 296R, 296S, 296T, 296V, 297D, 297E, 297F, 297G, 297H, 2971, 297K, 297L, 297M, 297P, 297Q, 297R, 297S, 297T, 297V, 297W, 297Y, 298A, 298D, 298E, 298 298F, 298H, 2981, 298K, 298M, 298N, 298Q, 298R, 298T, 298W, 298Y, 299A, 299D, 299E, 299F, 299G, 299H, 2991, 299K, 299L, 299M, 299N, 299P, 299Q, 299R, 299S, 299V, 299W, 299Y, 300A, 300D, 300E, 300G, 300H, 300K, 300M, 300N, 300P, 300Q, 300R, 300S, 300T, 300V, 300W, 301D, 301E, 301H, 301Y, 3021, 303D, 303E, 303Y, 304D, 304H, 304L, 304N, 304T, 305E, 305T, 305Y, 313F, 317E, 317Q, 318H, 318L, 318Q, 318R, 318Y, 320D, 320F, 320G, 320H, 3201,320L, 320N, 320P, 320S, 320T, 320V, 320W, 320Y, 322D, 322F, 322G, 322H, 322 [auction], 322P, 322S, 322T, 322V, 322W, 322Y, 3231, 324D, 324F, 324G, 324H, 3241, 324L , 324M, 324P, 324R, 324T, 324V, 324W, 324Y, 325A, 325D, 325E, 325F, 325G, 325H, 3251, 325K, 325L, 325M, 325P, 325Q, 325R, 325S, 325T, 325V, 325W, 325 Y, 326E, 3261, 326L, 326P, 326T, 327D, 327E, 327F, 327H, 3271, 327K, 327L, 327M, 327, 327P, 327R, 327S, 327 T, 327V, 327W, 327Y, 328A, 328D, 328E, 328F, 328G, 328H, 3281, 328K, 328M, 328N, 328P, 328Q, 328R, 328S, 328T, 328V, 328W, 328Y, 329D, 329E, 329F, 329G, 329H, 329I, 329K, 329L, 329M, 329N, 329Q, 329R, 329S, 329T, 329V, 329, 329Y, 330E, 330F, 330G, 330H, 330I, 330L, 330M, 330N, 330P, 330R, 330S, 330T, 330V, 330W, 330Y, 331D, 331F, 331H, 3311, 331L, 331M, 331Q, 331R, 331T, 331V, 331W, 331Y, 333A, 333F, 333H, 333I, 333L, 333M, 333P, 333T, 333Y, 334A, 334F, 3341, 334L, 334P, 334T, 335D, 335F, 335G, 335H, 335I, 335L, 335M, 335N, 335P, 335R, 335S, 335V, 335W, 335Y, 336E, 336K, 336Y, 337E, 337H, and 337N, where the positional numbering is in accordance with the EU index.
[00017] In another embodiment, one or more other amino acid modifications are at a position selected from the group consisting of 221, 222, 223, 224, 225, 228, 230, 231, 232, 240, 244, 245, 247, 262, 263, 266, 271, 273, 275, 281, 284, 291, 299, 302, 304, 313, 323, 325, 328, and 336, where the positional numbering is in accordance with the EU index . In such an embodiment, the one or more other amino acid modifications can be selected from the group of amino acid substitutions consisting of 221K, 221Y, 222E, 222Y, 223E, 223K, 224E, 224Y, 225E, 225K, 225W, 228E , 228G, 228K, 228Y, 230A, 230E, 230G, 230Y, 231 E, 231 G, 231 K, 231 P, 231 Y, 232E, 232G, 232K, 232 Y, 240A, 2401, 240M, 240T, 244H, 245A , 247G, 247V, 262 A, 262E, 262F, 2621, 262T, 263A, 2631, 263M, 263T, 266A, 2661, 266M, 266T, 271 A, 271 D, 271 E, 271 F, 271G, 271 H, 2711 , 271K, 271 L, 271M, 271N, 271Q, 271R, 271S, 271T, 271V, 271W, 271Y, 2731, 275L, 275W, 281 D, 281 E, 281 K, 281 N, 281P, 281Q, 281 Y, 284D , 284E, 284L, 284N, 284Q, 284T, 284Y, 291 D, 291E, 291G, 291H, 2911, 291Q, 291T, 299A, 299D, 299E, 299F, 299G, 299H, 299I, 299K, 299L, 299M, 299N, 299P, 299Q, 299R, 299S, 299V, 299W, 299Y, 304D, 304H, 304L, 304N, 304T, 313F, 323I, 325A, 325D, 325E, 325F, 325G, 325H, 325I, 325K, 325L, 325M, 325P, 325Q, 325R, 325S, 325T, 325V, 325 W, 325 Y, 328A, 328D, 328E, 328 F, 328G, 328H, 3281, 328K, 328M, 328N, 328P, 328Q, 328R, 328S, 328T, 328V, 328W, 328Y, 336E, 336K, and 336Y.
[00018] In a specific embodiment, the antibody comprises one or more other amino acid modifications selected from the group consisting of: 236 A, 268D, 268E, 330Y, and 330L.
[00019] In another aspect, the present invention depicts nucleic acid molecules encoding the heavy chain and / or the light chain of an antibody of the invention. Such nucleic acid molecules can comprise a nucleotide sequence that encodes the variable domain of the light chain, such as that indicated in SEQ ID NO: 17 or SEQ ID NO: 33, or a nucleotide sequence that encodes the variable domain of the heavy chain , such as that indicated in SEQ ID NO: 18 or SEQ ID NO: 34.
[00020] In a specific aspect, the nucleic acid encoding the antibody light chain of the invention has a nucleotide sequence selected from the group consisting of SEQ ID Nos. 24 and 40.
[00021] In another specific embodiment, the nucleic acid encoding the antibody heavy chain of the invention has a nucleotide sequence selected from the group consisting of SEQ ID Nos. 28 and 44.
[00022] In another aspect, the present invention relates to a method of treating a human receptor tyrosine kinase FLT3 related disorder or disease, wherein said method includes administering the antibody of the invention to an individual who you need it. The individual may, for example, be an animal or a human being, preferably a mammal, such as a human being.
[00023] In one embodiment, said disorder or disease is a cell proliferative disorder or disease.
[00024] In another embodiment, the disease or disorder is a tumor of hematopoietic origin, such as lymphoma or leukemia. The lymphoma or leukemia can be selected from the group consisting of: non-Hodgkin's lymphomas (NHL), chronic lymphocytic leukemia (CLL), acute B-cell lymphoblastic leukemia / lymphoma (B-ALL), mantle cell lymphoma (MCL) , hair cell leukemia (HCL), chronic myeloid leukemia (CML), acute myeloid leukemia, and multiple myeloma (MM). In a preferred embodiment, the lymphoma is acute myeloid leukemia (AML).
[00025] In another embodiment, the disease or disorder is a myelodysplasia syndrome (MDS).
[00026] In several embodiments, lymphoma or leukemia is in the stage of minimal residual disease (MRD), for example, achieved after conventional chemotherapy with or without stem cell transplantation.
[00027] In certain embodiments of the invented methods, the antibody can be administered in combination with at least one agent selected from the group consisting of a cytotoxic agent, a chemotherapeutic agent, a cytokine, an inhibitory growth agent, an anti-hormonal agent , a kinase inhibitor, an anti-angiogenic agent, a cardioprotective agent, an immunostimulatory agent, an immunosuppressive agent, an angiogenesis inhibitor, a protein tyrosine kinase inhibitor, and secondary antibody.
[00028] In yet another aspect, the present invention also includes a pharmaceutical composition comprising an antibody according to the invention and a pharmaceutically acceptable carrier.
[00029] In another aspect, the present invention relates to a method of inhibiting the proliferation of a cell that expresses FLT3, wherein said method comprises contacting said cell with an antibody according to the invention. The method can be an in vitro method.
[00030] In another aspect, the present invention relates to a method of increasing antibody-dependent cell-mediated cytotoxicity relative to a cell expressing FLT3, wherein said method comprises contacting said cell with an antibody according to the invention.
[00031] Yet another aspect of the invention is a method of detecting a mammal from at least one cell that expresses FLT3, wherein said method comprises administering to the mammal an antibody according to the invention.
[00032] The present invention also relates to using an antibody according to the present invention for the treatment of an FLT3-related disorder or disease. The FLT3-related disorder or disease may be a cell proliferative disease or disorder, such as a tumor of hematopoietic origin, for example a lymphoma or leukemia, or myelodysplasia syndrome (MDS). Can lymphoma or leukemia be selected from the group consisting of: non-Hodgkin's lymphomas (NHL), chronic lymphocytic leukemia (CLL), lymphoblastic leukemia lymphoma acute B cell (B-ALL), mantle cell lymphoma (MCL), hair cell leukemia (HCL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), and multiple myeloma (MM) and preferably it is acute myeloid leukemia.
[00033] In another embodiment, the invention relates to using an antibody according to the invention for targeting a cell that expresses FLT3. The targeting may include the use of the antibody to deliver a drug or toxin to the FLT3 expression cell.
[00034] In yet another aspect, the invention includes using an antibody according to the invention for detecting a cell that expresses FLT3 in a biological sample. For such use, the antibody can be labeled with a detectable portion, such as fluorophor, chromophor, immunogenic label and the like.
[00035] The present invention also relates to a monoclonal antibody against FLT3, wherein the antibody is produced by a transfected producer cell line, such as CHO or Sp2 / 0.
[00036] In yet another aspect, the invention depicts a transfected cell line that produces an antibody according to the invention. Brief Description of Drawings
[00037] The invention will be better understood with reference to the detailed description when considered in combination with the non-limiting examples and the accompanying drawings.
[00038] Figure 1 shows a schematic representation of the cloning procedure for chimerization of monoclonal antibodies. Boxes represent exons, circle indicates augmenting elements and UT regions of fine lines and intron sequences. P, promoter; Li and L2, leader sequences encoded by two different exons; And, enhancer; V, variable region; D, region of diversity; J, union region; C (1-3) exons of the constant region; H, main region.
[00039] Figure 2 shows the parental vector containing the VJ region of the mouse light chain and the C region of the human kappa gene. The region relevant for fragment exchange is shown enlarged in Figure 2A. The sequence context generated in the insertion of the VJ region of monoclonal antibodies BV10 or 4G8 into the expression vector chimFLT3 -light is shown in Figure 2B. The cleavage site for secretory signal peptides is indicated by |; and exon-intron boundaries by [,].
[00040] Figure 3 shows the original vector containing the human y 1 isotype 1g heavy chain. The region relevant to the cloning of the VDJ fragment is shown enlarged (a). The Mlul-Spel fragment to be exchanged (shown enlarged as b) contains the entire human y 1 heavy chain constant region and two amino acid modifications in the CH2 domain as indicated (Ser239-Asp; Iso332-Glu). Figure 3B shows the sequence context generated in the insertion of the VDJ region of the BV10 or 4G8 monoclonal antibody heavy chain into the chimFLT3-heavy chain expression vector. The dividing site for secretory signal peptides is indicated by |; and exon-intron boundaries by [,].
[00041] Figure 4 shows the cell killing effects of chimeric antibodies optimized by Fc chim4G8-SDIE (A) and chimBVIO-SDIE (B) respectively and unstimulated human PBMCs against human expression NALM16 leukemia cells of FLT3 cultured compared to chim4G8 and chimBVIO unmodified chimeric antibodies. Fig. 4 C shows the cell killing effects of chimeric antibodies directed to NG2 which was optimized by Fc in the same positions as the above antibodies chim4G8-SDIE and chimBVIO-SDIE on human SKMel63-melanoma cells. Cytotoxicity was determined using a chromium release assay, duration of the assay and target effector ratios are indicated.
[00042] Figure 5 shows the cell killing effect by 4G8-SDIE and human anti-FLT3-optimized anti-FLT3 antibody PBMCs in immature AML precursor cells compared to the unmodified parental mouse antibody.
[00043] Figure 6 shows an amino acid sequence alignment of the variable chain regions of the light (A) and heavy (B) antibody of anti-FLT3 4G8 and BV10 clones.
[00044] Figure 7 shows the connection of 4G8 and BV10 optimized and chimeric mouse to FLT3. NALM16 cells (B, C) or Sp2 / 0 cells transfected with mock and FLT3 (A) were incubated with the indicated antibodies and were analyzed by indirect immunofluorescence and flow cytometry. Open and shaded histograms in (A) represent staining with isotype control and the indicated FLT3 antibodies (10 pg / ml), respectively. MFI = mean fluoro-rescence intensity.
[00045] Figure 8 shows the effect of 4G8SDIEM on proliferation and FLT3 ligand binding (FLT3L) of leukemic cells. (A) NALM16 cells were incubated with 4G8SDIEM or BV10SDIEM at 1 g / ml in the presence of the indicated concentrations of the recombinant FLT3 ligand and the amount of antibody bound was determined by flow cytometry and indirect immunofluorescence. (B) Immature AML precursor cells isolated from the peripheral blood of the three different patients by density, gradient centrifugation were incubated with the indicated concentrations of 4G8SDIEM for 24 hours and proliferation was assessed using a 3 [H] - uptake assay thymidine. Right-hand bars represent proliferation in the absence of the antibody.
[00046] Figure 9 shows ADCC activity of SDIEM-modified and unmodified versions of the FLT3 4G8 and BV10 antibodies. NALM 16 cells marked with 5I [Cr] were incubated for 4 hours with PBMCs from a healthy donor (# 4) in the presence of the indicated concentrations of the SDIEM modified or chimeric (X) unmodified versions of 4G8 and BV10 at a ratio of PBMC: 50: 1 target cell. Target cell death was determined using a standard 51 [Cr] release assay. A representative result results from 6 independent experiences with PBMCs from healthy donors is shown.
[00047] Figure 10 shows the ADCC activity of 4G8SDIEM against leukemic cells. Cytolytic activity of PBMCs from three different healthy donors (PBMC # 1, # 2, # 3) against NALM 16 (A) cells and donor PBMCs # 2 against immature leukemic precursor cells from the three different patients (AML # 1, # 2, # 7) (B) was determined in a 51 [Cr] release assay for 4 hours and 8 hours, respectively. In (C) cytolytic activity after 8 hours against AML immature precursor cells # 1 and # 15 is shown using autologous PBMCs from the respective patients as effector cells. Filled or open symbols indicate 4G8SDIEM-mediated ADCC and non-binding control antibody 9.2.27SDIE, respectively. Bars filled to the right (NK) indicate NK activity in the absence of antibody. Note that PBMC # 1-3 refer to PBMCs from healthy donors and are not related to immature AML # 1-3 precursor cells.
[00048] Figure 11 shows antigen change and FLT3 expression in leukemic cells from NALM16 cells of different origin (A) and immature precursor cells from two different AML patients were incubated with the indicated concentrations of 4G8SDIEM. After 48 hours, cells were incubated, re-incubated with 2 pig / ml of 4G8SDIEM and analyzed by flow cytometry and indirect immunofluorescence. Expression of FLT3 shown in the pre-incubated cells without antibodies was defined as 100%. (B) Immature AML precursor cells from 15 patients were incubated with 4G8 of mouse (10 g / ml), washed and analyzed by flow cytometry and indirect immunofluorescence. The amount of antibody molecules bound was determined by comparison with calibrated beads (QIFIKIT). (C) The immature AML precursor cell used in (B) was incubated with non-binding PE conjugated 9.2.27SDIE antibody or PE conjugated 4G8SDIEM (10 pg / ml) was analyzed by direct immunofluorescence flow cytometry. SFI = specific fluorescence index. The SFI of four samples has not been determined (n.d.) because of the high binding of the control antibody of 9.2.27SDIE.
[00049] Figure 12 shows the expression of FLT3 in DCs and normal bone marrow cells. (A) DCs isolated from peripheral blood from healthy donors by magnetic cell separation were incubated with 4G8 of mice, washed, stained with a secondary labeled antibody, washed again and incubated with a mixture of CD1lc- and CD303 antibodies differently marked. Cells were then analyzed by flow cytometry. Connection of 4G8 to CD303 + pDC and the CDI lc + niDC subpopulation is shown in (B) and (C), respectively. (D, E) Similar to normal bone marrow cells (A-C) isolated by gradient centrifugation density were incubated with mouse 4G8, washed, stained with secondary labeled antibody and a differently labeled mixture of CD34 and CD45 antibodies. Linking 4G8 to the low CD34 + subpopulation is shown in (E). Shaded histograms represent primary staining with isotope control, open histograms with mouse 4G8. Representative results from one of the three experiments with DCs and bone marrow cells from different healthy donors are shown.
[00050] Figure 13 shows the cytotoxic activity of 4G8SDIEM against normal cells. (A) Human bone marrow cells from two different healthy donors (black and shaded bars) were incubated with 5 pg / ml of 4G8 SDIEM and colony-forming units determined after 12 days of incubation of semi-solid medium. Numbers of CFUs were related to untreated controls. (B) DCs isolated from PBMCs from healthy donors by magnetic cell separation and NALM16 cells were used as targets for 4G8 SDIEM in a 51 [Cr] release assay for four hours (PBMC ratio: 100: 1 target). A representative experience of the three with autologous DCs and PBMCs from different donors is shown.
[00051] Figure 14 shows the in vitro effects of 4G8 antibody on a target patient and effector cells. (A) Patient PBMC were analyzed by FACS for FLT3 expression using parental mouse 4G8 isotope and antibody control followed by anti-mouse PE conjugate and double staining for CD34. (B, C) Patient PBMC were incubated with FLT3 positive NALM16 cells labeled with chromium (B) or immature patient precursor cells isolated by CD34 + selection (C). Target cells were pretreated with the indicated concentrations of 4G8-SDIEM or the chimeric, unmodified 4G8 antibody (4G8-ch). ADCC induction was determined by chromium release assays at a 50: 1 PBM: target ratio. Note that PBMC and unpurified NK cells were used.
[00052] Figure 15 shows the half life and binding characteristics of 4G8-SDIEM in vivo. (A) 4G8-SDIEM serum half-life was determined by incubating FLT3 expression NALM16 cells with serum samples at different time points for clinical application. The amount of specifically bound antibody was determined by FACS and compared to binding activity of serum samples containing defined levels of 4G8-SDIEM. ND, not determined. (B) To detect binding of 4G8-SDIEM in vivo, BM immature precursor cells obtained before therapy (dO) and 1 h after application of the 10 mg dose (d5) were incubated with the parental 4G8 mouse antibody, a second anti-FLT3 antibody of non-cross-reactive (BV10) as indicated, or isotype control (open peaks) at 10 pig / ml, followed by a conjugate by an anti-mouse PE conjugate absorbed from human. Complete inhibition of mouse 4G8, but no BV10 binding as determined by FACS indicates binding saturation of 4G8-SDIEM.
[00053] Figure 16 shows the clinical effects of 4G8-SDIEM. (A, B) The percentages of CD34 + immature precursor cells (open circles) and activated (CD69 +) CD56 + CD3-NK cells (diamonds) among mononuclear cells in peripheral blood (PB) (A) or bone marrow (BM) ( B) were determined by FACS at the indicated time during the treatment of evident leukemia (C) TNF serum levels at the times indicated during the treatment of evident leukemia were determined by measuring IMMULITE (R). (D) The percentage of activated NK cells among mononuclear cells in PB (diamonds) and TNF serum levels (circles) were determined as described above at the times indicated during the application of 4G8-SDIEM in complete remission (CR). Detailed Description of the Invention
[00054] The terms used here have the following meanings, unless otherwise explicitly stated.
[00055] By "ADCC" or "antibody dependent cell-mediated cytotoxicity" as used herein is meant that the cell-mediated reaction in which cytotoxic cells expressing FcRs recognize antibody bound in a target cell and subsequently cause lysis of the target cell .
[00056] By "ADCP" or "antibody-dependent cell-mediated phagocytosis" as used herein is meant that the cell-mediated reaction in which non-specific cytotoxic cells expressing FcRs recognize antibody bound in a target cell and subsequently cause phagocytosis of target cell.
[00057] By "amino acid" and "amino acid identity" as used here is meant that one of the 20 naturally occurring amino acids or any unnatural analogues that may be present in a defined, specific position. So "amino acid" as used here is either naturally occurring or synthetic amino acids. For example, homophenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention. "Amino acid" also includes imino acid residues such as proline and hydroxyproline. The side chain can be either in the (R) or (S) configuration. In one embodiment, the amino acids are in the (S) or L configuration. If non-naturally occurring side chains are loaded, non-amino acid substituents can be used, for example, to prevent or delay degradation in vivo.
[00058] By "antibody" here is meant a protein that consists of one or more polypeptides substantially encoded by all or part of the recognized immunoglobulin genes. The immunoglobulin genes recognized, for example, in humans, include the kappa (K), lambda ([À]) genetic sites, and heavy chain genetic sites, which together comprise the myriad variable region genes, and the genes constant region mu (p), delta (δ), gamma (y), epsilon (ε), and alpha (a) that encode the isotypes of lg, IgD, IgG (IgGI, lgG2, lgG3, and lgG4), IgE , and IgA (IgAI and IgA2) respectively. Antibody here is intended to include full-length antibodies and antibody fragments, and may refer to a natural antibody from any organism, an engineered antibody, or an antibody recombinantly generated for experimental, therapeutic or other purposes.
[00059] By "B cell" or "B lymphocyte" as used here it is meant that a type of lymphocyte developed in the bone marrow that circulates in the blood and lymph, and provides humoral immunity. B cells recognize free antigen molecules and differentiate or mature in plasma cells that secrete immunoglobulin (antibodies) that inactivate the antigens. Memory cells are also generated to produce the specific immunoglobulin (antibody) in subsequent encounters with that antigen. B cells are also known as "beta cells" on the islet of Langerhans.
[00060] By "T cell" or "T lymphocyte" as used here is meant that a type of lymphocyte developed in the bone marrow that circulates in the blood and lymph, and provides cellular immunity. T cells comprise a T cell receptor that recognizes antigen molecules bound to the cell. T cells can mature into helper T cells that secrete cytokines and activate other types of cells or cytotoxic T cells that bind to and destroy other cells.
[00061] By "FLT3" (fms-type tyrosine kinase receptor-3), "FLK2" (fetal liver kinase-2), and "CD 135" are used interchangeably here to mean that a cytokine receptor expressed on the surface of hematopoietic progenitor cells. FLT3 is a cell surface marker used to identify certain types of hematopoietic (blood) pro-parents in the bone marrow. Specifically, multipotent parents (MPP) and common lymphoid parents (CLP) express high levels of FLT3 surface. The FLT3 receptor is linked by the cytokine Flt3 ligand (Flt3L). FLT3 is a type III receptor tyrosine kinase. When this receptor is connected by Flt3L it forms a dimer (homodimer) that activates second messenger signaling. FLT3 signaling plays an important role in the survival, proliferation, and differentiation of developing cell lymphocytes (B cell and T cell). As deregulation of FLT3 signaling can cause proliferative diseases, such as cancer, and in particular leukemia, FLT3 is classified as a proto-oncogene. In fact, internal tandem duplications of FLT3 are the most common mutations associated with acute myelogenous leukemia (AML). The use of FLT3 here is intended to include all known or undisclosed alleles and polymorphic forms of FLT3. The human FLT3 antigen sequence is provided in SEQ ID NO: 65.
[00062] By "CDC" or "complement dependent cytotoxicity" as used herein is meant that the reaction in which one or more complementary protein components recognize antibody bound in a target cell and subsequently cause lysis of the target cell.
[00063] By "constant region" of an antibody as defined herein, it is meant that the region of the antibody that is encoded by one of the light or heavy chain immunoglobulin region constant genes.
[00064] By "constant light chain" or "light chain constant region" as used here is meant that the region of an antibody encoded by the cap (CK) OR lambda (CÀ) light chains. The constant light chain typically comprises a single domain, and as defined here refers to positions 108-214 of CK OR lambda CÀ, where the numbering is in accordance with the EU index.
[00065] By "constant heavy chain" or "heavy chain constant region" as used here is meant that the region of an antibody encoded by the mu, delta, gamma, alpha, or epsilon genes to define the antibody's isotype as IgM , IgD, IgG, IgA, or IgE, respectively. For full-length IgG antibodies, the constant heavy chain, as defined here, refers to the N-terminal of the CH1 domain to the C-terminal of the CH3 domain, thus comprising positions 118-447, where the numbering is according to the EU index.
[00066] By "effector function" as used here is meant that a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include FcR-mediated effector functions such as ADCC and ADCP, and complement-mediated effector functions such as CDC.
[00067] By "effector cell" as used here is meant that a cell in the immune system that expresses one or more Fc receptors and mediates one or more effector functions. Effector cells include but are not limited to monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer cells (NK), and T cells and can be of any organism including but not limited to humans, mice, rabbit rats, and monkeys.
[00068] By "Fab" or "Fab region" as used herein is meant that polypeptides comprising the immunoglobulin domains of VH, CHI, VH, and CL. Fabs can refer to that region in isolation, or that region in the context of a full-length antibody or antibody fragment.
[00069] By "Fc" or "Fc region", as used herein, it is meant that the polypeptide comprising the constant region of an antibody excluding the first immunoglobulin domain of constant region. Thus Fc refers to the last two immunoglobulin domains of IgA constant region, IgD, and IgG, and the last three domains of immunoglobulin constant region of IgE and IgM, and N-terminal flexible articulation to these domains. For IgA and IgM, Fc may include the J chain. For IgG, Fc comprises immunoglobulin domains Cy2 and Cy3 and the joint between Cy1 and Cy2. Although the limits of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to contain C226 or P230 residues at its carboxyl terminus, where the numbering is in accordance with the EU index as in Kabat. Fc can refer to that region in isolation, or that region in the context of an Fc polypeptide, for example an antibody.
[00070] By "Fc polypeptide" as used herein is meant that a polypeptide comprising all or part of an Fc polypeptide from the Fc region includes Fc fragments, isolated Fcs and antibody Fc fusions.
[00071] By "gamma Fc receptor" or "FcR" as used herein is meant that any member of the family of proteins that bind to the IgG antibody Fc region and are substantially encoded by the FcR genes. In humans this family includes but is not limited to FcRI (CD64), including FcRIa, FcRIb, and FcRIc isoforms; FcRIl (CD32), including FcyRlla isoforms (including H131 and R131 allotypes), FcRIlb (including FcRIlb-1 and FcRllb-2), and FcRIIc; and FcRIII (CD 16), including FcRIIIa isoforms (including V158 and F158 allotypes) and FcRIIIb (including FcRIIIb-NAI and FcRlllb-NA2) (Jefferis et al., 2002, Immunol Lett 82: 57-65), as well as any isoforms or allotypes of human FcRs or FcRs not disclosed. Mouse FcyRs include but are not limited to FcRI (CD64), FcyRII (CD32), FRUI (CD16), and FcRIII-2 (CD16-2), as well as any non-mouse FcR or FcR isotypes or allotypes revealed. An FcR can be of any organism, including but not limited to humans, mice, rats, rabbits, and monkeys.
[00072] By "Fc ligand" or "Fc receptor" as used here is meant that a molecule, for example, a polypeptide, originates from any organism that binds to the Fc region of an antibody to form a ligand complex of Fc. Fc ligands include, but are not limited to, FcRs, FcRn, Clq, C3, raanan-binding lecithin, mannose receptor, staphylococcal protein A, streptogenic G protein, and viral FcR. Fc ligands also include Fc receptor homologues (FcRH), which are a family of Fc receptors that are homologous to FcRs (Davis et al., 2002, Immunological Reviews 190: 123-136). Fc ligands can include undisclosed molecules that bind Fc.
[00073] By "IgG" as used herein is meant that a polypeptide that belongs to the class of antibodies that is substantially encoded by a recognized gamma immunoglobulin gene. In humans this class comprises IgGI, IgG2, IgG3, and IgG4. In mice, this class comprises IgGI, lgG2a, lgG2b, lgG3.
[00074] By "immunoglobulin (lg)" here is meant a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Immunoglobulins include, but are not limited to, antibodies. Immunoglobulins can have numerous structural forms, including, but not limited to, full-length antibodies, antibody fragments, and individual immunoglobulin domains.
[00075] By "immunoglobulin domain (lg)" here is meant an immunoglobulin that exists as a distinct structural entity as determined by one skilled in the protein structure technique. Lg domains typically have a characteristic beta sandwich folding topology. The Ig domains known in the IgG class of antibodies are VH, Cl, C2, C3, VL, and CL.
[00076] By "amino acid modification" here is meant an amino acid substitution, insertion and / or deletion in a polypeptide sequence.
[00077] By "amino acid substitution" or "substitution" here is meant that the replacement of an amino acid at a particular position in a sequence of polypeptides of origin with another amino acid. For example, substitution I332E refers to a variant polypeptide, in this case the heavy chain constant variant, where isoleucine at position 332 is replaced with glutamic acid. The wild type residue may or may not be designated. For the preceding example, 332E indicates the replacement of position 332 with a glutamic acid. For the purposes here, multiple substitutions are typically separated by a cut. For example, 239D / 332E refers to a double variant comprising substitutions 239D and 332E.
[00078] By "amino acid insertion" or "insertion" as used here is meant that the addition of an amino acid to a particular position in a sequence of origin polypeptides. For example, insert-236G designates a glycine insert at position 236.
[00079] By "amino acid deletion" or "deletion" as used here it is meant that the removal of an amino acid to a particular position in a sequence of polypeptides of origin For example, G236 designates the deletion of glycine at position 236 .
[00080] By "origin polypeptide", "origin protein", "precursor polypeptide", or "precursor protein" as used interchangeably herein means that a polypeptide that is subsequently modified to generate a variant, for example example, any polypeptide that serves as a template and / or base for at least one amino acid modification described here. The originating polypeptide can be a naturally occurring polypeptide, or a variant or engineered version of a naturally occurring polypeptide. Polypeptide of origin can refer to the polypeptide itself, compositions that comprise the polypeptide of origin, or the sequence of amino acids that encode it. Correspondingly, by "source antibody" or "source immunoglobulin" as used herein is meant that an antibody or immunoglobulin that is modified to generate a variant (for example, a source antibody may include, but is not limited to, a protein comprising a naturally occurring 1g constant region).
[00081] By "protein" or "polypeptide" as used here is meant that at least two covalently linked amino acids, which include proteins, polypeptides, oligopeptides and peptides The protein can be formed from naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures, that is, "analogues", such as pepttoids.
[00082] By "position" as used here is meant a location following a protein. Positions can be numbered sequentially, or according to an established format, for example, the EU index as in Kabat (Kabat et al., 1983). If not stated otherwise, all positions mentioned are numbered according to the EU index. Corresponding positions are determined as outlined here, usually through alignment with other source sequences.
[00083] By "residue" as used here is meant that a position on a protein in its associated amino acid identity. For example, Serina 239 (also called Ser239 and S239) is a residue at position 239 in the human antibody IgGI.
[00084] By "target antigen" or "target" or "antigen" as used here is meant that the molecule that is specifically bound by the variable region of a given antibody. A target antigen can be a protein, carbohydrate, lipid, or other chemical compound.
[00085] By "target cell" as used here is meant a cell that expresses a target antigen.
[00086] By "variable region" as used herein is meant that the region of an immunoglobulin comprising one or more lg domains substantially encoded by any of the VK, VÀ, and / or VH genes that form the immunoglobulin genetic sites hood, lambda, and heavy chain respectively.
[00087] By "variant protein", "protein variant", "variant polypeptide", or "polypeptide variant" as used herein it is meant that a sequence of polypeptides that differs from that of a polypeptide sequence of origin by virtue of at least one amino acid modification. Variant polypeptide can refer to the polypeptide itself, a composition comprising the polypeptide, or the amino sequence encoding it. In one embodiment, the variant polypeptide has at least one amino acid modification compared to the parent polypeptide, for example, from about one to about ten amino acid modifications, for example, from about one to about five modifications of amino acid compared to the origin. The variant polypeptide sequence here can have at least about 80% homology with the original polypeptide sequence, for example, at least about 90% homology, at least about 95% homology, etc. Correspondingly, by "variant antibody" or "antibody variant" as used herein is meant that a sequence of antibodies that differs from that of a sequence of antibodies of origin by virtue of at least one amino acid modification. Antibody variant or variant antibody may refer to the polypeptide antibody itself, compositions comprising the antibody variant polypeptide, or the amino acid sequence encoding it. Correspondingly, by "heavy chain constant variant" or "light chain constant variant" or "Fc variant" as used herein is meant that the constant heavy chain, constant light chain, or polypeptide or Fc region sequence, respectively , which differs in sequence from that of a source sequence in that it has at least one amino acid modification.
[00088] By "wild type" or "WT" here is meant an amino acid sequence or a nucleotide sequence that is found in nature, including allelic variations. A wild-type protein, polypeptide, antibody, immunoglobulin, IgG, etc., has an amino acid sequence or a nucleotide sequence that has not been intentionally modified.
[00089] For all immunoglobulin heavy chain constant region positions discussed in the present invention, numbering is in accordance with the EU index as in Kabat (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed, United States Public Health Service, National Institutes of Health, Bethesda). The "EU index as in Kabat" refers to the numbering of the human IgGI EU antibody residue, as described in Edelman et al., 1969, Biochemistry 63 78-85).
[00090] "Antigens" are macromolecules capable of generating an antibody response in an animal and being recognized by the resulting antibody. Both antigens and haptens comprise at least one antigenic determinant or "epitope", which is the region of the antigen or hapten that binds to the antibody. Typically, the epitope in a hapten is the entire molecule.
[00091] The term "sample", as used here, refers to an aliquot of material, often biological matrices, an aqueous solution or an aqueous suspension derived from biological material. Materials that can be evaluated for the presence of analyte by the methods of the present invention include, for example, cells, tissues, homogenates, lysates, extracts, and purified or partially purified proteins and other biological molecules and mixtures thereof.
[00092] Non-limiting examples of samples typically used in the methods of the invention include animal and human body fluids such as whole blood, serum, plasma, cerebrospinal fluid, sputum, bronchial lavage, bronchial aspirations, urine, semen, fluids lymph and various other external secretions from the respiratory, intestinal and genitourinary tracts, tears, saliva, milk, leukocytes, myelomas and the like; biological fluids such as cell culture supernatants; kind of fabric that may or may not be fixed; and cell species that may or may not be fixed. The samples used in the methods of the present invention will vary in the test format and in the nature of the tissues, cells, extracts or other materials, especially biological materials, to be analyzed. Methods for the preparation of protein extracts from cells or samples are well known in the art and can be readily adapted to obtain a sample that is compatible with the methods of the invention.
[00093] "Specifically binding" and "specific binding", as used here, means that an antibody binds to its target (analyte) based on the recognition of an epitope on the target molecule. The antibody preferably recognizes and binds to the target molecule with a higher binding affinity than it binds to other compounds that may be present. In various embodiments of the invention, "specifically binding" may mean that an antibody binds to the target molecule with at least about 106 times greater affinity, preferably at least about 107 times greater affinity, more preferably at least about 108 times greater affinity, and more preferably at least about 109 times greater affinity than it binds molecules unrelated to the target molecule. Typically, specific binding refers to affinities in the range of about 106 times to about 109 times greater than non-specific binding. In some embodiments, specific binding may be characterized by affinities greater than 109 times over non-specific binding. The binding affinity can be determined by any suitable method. Such methods are known in the art and include, without limitation, surface plasmon resonance and isothermal titration colorimetry. In a specific embodiment, the antibody uniquely recognizes and binds to the target analyte.
[00094] The term "monoclonal antibody", as used here, refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical except for possible naturally occurring mutations which may be present in smaller quantities. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant in the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized by hybridoma culture, not contaminated by other immunoglobulins. The "monoclonal" modifier indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and should not be interpreted as producing antibody requirement by any particular method. Monoclonal antibodies can include "chimeric" antibodies (US Patent No. 4,816,567; and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA, 81: 6851-6855) and humanized antibodies (Jones et al. ( 1986) Nature, 321: 522-525; eichmann et al. (1988) Nature, 332: 323- 329; Presta (1992) Curr. Op. Struct. Biol. 2: 593-596).
[00095] Monoclonal antibodies can be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in the culture. These include, but are not limited to, the Koehler and Milstein (1975) hybridoma technique, Nature, 256: 495-7; and U.S. patent no. 4,376,110), the human B-cell hybridoma technique (osbor, et al. (1983), Immunology Today, 4: 72; Cote, et al. (1983), Proc. Natl. Acad. Sci. USA, 80 : 2026-30), and the EBV hybridoma technique (Cole, et al. (1985), in Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., New York, pp. 77-96). The preparation of specific monoclonal antibodies to a target compound is also described in Harlow and Lane, eds. (1988) Antibodies - A Laboratory Manual. Cold Spring Harbor Laboratory, Chapter 6. Such antibodies can be any class of immunoglobulin including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The production of inAb hybridoma can be cultured in vitro or in vivo. Production of high titers of mAbs in vivo produces this a very effective method of production.
[00096] "Polyclonal antibodies" are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, or an antigenic functional derivative thereof. For the production of polyclonal antibodies, host animals such as rabbits, mice and goats can be immunized by injection with an antigen or hapten vehicle conjugate optionally supplemented with auxiliaries.
[00097] Techniques described for the production of single chain antibodies (US patent No. 4,946,778; Bird (1988), Science 242: 423-26; Huston, et al. (1988), Proc. Natl. Acad. USA, 85: 5879-83, and Ward, et al. (1989), Nature, 334: 544-46) can be adapted to produce single gene chain antibodies. Single chain antibodies are typically formed by linking the light and heavy chain fragments of the Fv region via an amino acid dot, resulting in a single chain polypeptide.
[00098] Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, such fragments include but are not limited to: the F (ab ') 2 fragments that can be produced by pepsin digestion of the antibody molecule and the Fab fragments that can be generated by reducing the disulfide bridges of the fragments of F (ab ') 2. Alternatively, Fab expression bi-libraries can be constructed (Huse, et al. (1989), Science, 246: 1275-1281) to allow quick and easy identification of monoclonal Fab fragments with the desired specificity.
[00099] The terms "polynucleotide" and "(nucleic acid molecule)" are used interchangeably here to refer to polymeric forms of nucleotides of any length, including naturally occurring and non-naturally occurring nucleic acids. Polynucleotides may contain deoxyribonucleotides, ribonucleotides and / or their analogs. Methods for the selection and preparation of nucleic acids are diverse and well described in standard biomolecular protocols. A typical way would be preparative PCR PCR and chromatographic purification that starts from existing model DNAs or synthesis by artificial nucleic acid steps. Typically, the nucleic acid molecules mentioned here are DNA molecules.
[000100] The term "at least one" as used herein with respect to amino acid substitutions refers to at least 1, but preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50 or a plurality of amino acid substitutions.
[000101] The terms "contact" or "incubation", as used interchangeably here, generally refer to the provision of access from one component, reagent, analyte or sample to another.
[000102] The term "detection" as used here refers to any method of checking for the presence of a given molecule. Techniques used to accomplish this may include, but are not limited to, immunoassays, such as ELISA and Immuno PCR (IPCR).
[000103] Hematological malignancies are cancers that affect blood, bone marrow, and lymph nodes. Hematological malignancies can be derived from one or two of the main blood cell lines: myeloid and lymphoid cell lines. The myeloid cell line normally produces granulocytes, erythrocytes, thrombocytes, macrophages and mast cells; the lymphoid cell line produces B, T, NK and plasma cells. Lymphomas, lymphocytic leukemia, and myeloma come from the lymphoid line, while chronic and acute myelogenous leukemia, myelodysplastic syndromes and myeloproliferative disease are of myeloid origin.
[000104] Leukemia is a cancer of the blood or bone marrow and is characterized by an abnormal proliferation of blood cells, usually white blood cells (leukocytes). Leukemia is clinically and pathologically subdivided into a variety of large groups. Acute leukemia is characterized by the rapid increase in immature blood cells. This clumping forms a bone marrow that is unable to produce healthy blood cells. Immediate treatment is required in acute leukemia due to the rapid progression and accumulation of malignant cells, which then spill into the bloodstream and spread to other organs in the body. Acute forms of leukemia are the most common forms of leukemia in children. Chronic leukemia is distinguished by the excessive construction of relatively mature but still abnormal white blood cells. Typically taking months or years to progress, cells are produced at a much higher rate than normal cells, resulting in many abnormal white blood cells in the blood. While acute leukemia must be treated immediately, chronic forms are sometimes monitored for some time before treatment to ensure maximum therapy effectiveness. Chronic leukemia mainly occurs in older people, but it can theoretically occur in any age group. In addition, diseases are subdivided according to what kind of blood cell is affected. This division divides leukemias into lymphoblastic or lymphocytic leukemia and myeloid or myelogenous leukemia: In lymphoblastic or lymphocytic leukemia, the cancerous change occurs in a type of marrow cell that normally goes on to form lymphocytes, which are cells of the immune fighting system infection. Most lymphocytic leukemia involves a specific subtype of lymphocyte, the B cell. In myeloid or myelogenous leukemia, the cancerous change occurs in a type of marrow cell that normally follows to form red blood cells, some other types of cells white and platelets.
[000105] Acute myeloid leukemia (AML), also known as acute myelogenous leukemia, is a cancer of the myeloid blood cell line, characterized by the rapid growth of abnormal white blood cells that accumulate in the bone marrow and interfere with the production of normal blood cells. AML is the most common acute leukemia that affects adults, and its incidence increases with age. Although AML is a relatively rare disease, accounting for about 1.2% of cancer deaths in the United States, its incidence is expected to increase as the population ages.
[000106] The symptoms of AML are caused by replacing the normal bone marrow with leukemic cells, which causes a drop in red blood cells, platelets, and normal white blood cells. These symptoms include fatigue, shortness of breath, easy bruising and bleeding, and increased risk of infection. Although several risk factors for AML have been identified, the specific cause of the disease remains unclear. Since acute leukemia, AML progresses rapidly and is fatal within weeks or months if left untreated.
[000107] AML has several subtypes; Treatment and prognosis vary between subtypes. Five-year survival ranges from 15-70%, and relapse rate ranges from 78-33%, depending on the subtype.
[000108] Monoclonal antibodies are a class of therapeutic proteins that can be used to treat diseases and proliferative disorders of cells, in particular those that affect the hematopoietic system. Numerous favorable antibody properties, including but not limited to target specificity, ability to mediate immune effector mechanisms, and long serum half-life, make powerful antibody therapies. The present invention describes antibodies against the FLT3 proto-oncogene.
[000109] FLT3 has been shown to play a significant role in the onset and progression of leukemias, in particular AML, and early experiments with FLT3 inhibitors in patients with AML show promising results. However, there is still a need for anti-FLT3 antibodies that are useful in the treatment of leukemias, such as AML.
[000110] The clinical success of antibodies directed against FLT3 depends on its potential mechanism (s) of action. There are numerous possible mechanisms by which antibodies measure cellular effects, including antiproliferation via blocking of necessary growth trajectories, intracellular signaling that leads to apoptosis, down regulation and / or increased receptor change, complement-dependent cytotoxicity (CDC), cytotoxicity antibody-dependent cell-mediated (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) and promotion of an adaptive immune response (Cragg et al., 1999, Curr Opin Immunol 11 541- 547, Glennie et al., 2000, Immunol Today 21 403- 410). Antibody effectiveness may be due to a combination of these mechanisms, and their relative importance in clinical therapy for oncology appears to be dependent on cancer.
[000111] The importance of FcR-mediated effector functions for the activity of some antibodies has been demonstrated in mice (Clynes et al., 1998, Proc Natl Acad Sei USA 95 652-656, Clynes et al., 2000, Nat Med 6 443- 446,), and correlations observed between clinical efficacy in humans and their allotype of alpha (VI 58) or low (F158) polymorphic FcRIlIa affinity (Cartron et al., 2002, Blood 99 754- 758, Weng & Levy, 2003, Journal of Clinical Oncology, 21 3940-3947). Together, these data suggest that an antibody that is optimized for binding to certain FcRs may better mediate effector functions, thereby destroying target cells more effectively in patients. Thus a promising means for increasing the anti-tumor potency of antibodies is via increased ability to mediate cytotoxic effector functions such as ADCC, ADCP, and CDC In addition, antibodies can mediate anti-tumor mechanisms via inhibitory growth or apoptotic signaling that can occur when an antibody binds to its target in tumor cells. Such signaling can be potentiated when antibodies are presented to tumor cells linked to immune cells via FcR. Therefore, increased antibody affinity for FcRs can result in increased antiproliferative effects.
[000112] Some success has been achieved in modifying antibodies with selectively increased binding to FcRs to provide increased effector function. Antibody engineering for optimized effector function was achieved using amino acid modifications (see, for example, US patent application 2004-0132101 or US patent application 2006- 0024298.
[000113] Unfortunately, it is not known a priori that mechanisms of action can be optimal for a given target antigen. Furthermore, it is not known that antibodies may be able to mediate a given mechanism of action against the target cell. In some cases, a lack of antibody activity, either mediated by Fv or mediated by Fc, may be due to the targeting of an epitope on the target antigen that is poor for mediating such activity. In other cases, the targeted epitope may be receptive to a desired Fc-mediated or Fv-mediated activity, yet the affinity (affinity of the Fv region for the antigen or affinity of the Fc region for Fc receptors) may be insufficient in relation to in addressing this problem, the present invention describes modifications in anti-FLT3 antibodies that provide Fc-mediated activities, for example new or enhanced Fc-mediated activity.
[000114] Antibodies are immune proteins that bind a specific antigen. In most mammals, including humans and mice, antibodies are constructed from paired light and heavy polypeptide chains. The light and heavy variable chain regions show significant sequence diversity between antibodies, and are responsible for binding the target antigen. Each chain is formed and the individual immunoglobulin domain (lg), and thus the generic term immunoglobulin is used for such proteins.
[000115] Natural antibody structural units typically comprise the tetramer. Each tetramer is typically made up of identical pairs of polypeptide chains, each pair having a "light" chain (typically having a molecular weight of about 25 kDa) and a "heavy" chain (typically having a molecular weight of about 50 -70 kDa). Each of the light and heavy chains are formed from two distinct regions, called the variable and constant regions. For the IgG class of immunoglobulins, the heavy chain consists of four immunoglobulin domains linked from N- to C-terminal in the order VH-CH1-CH2-CH3, with reference to a variable domain of heavy chain, domain 1 heavy chain constant, domain 2 heavy chain constant, and domain 3 heavy chain constant respectively (also called VH-Cyl-CY2-CY3, referring to a heavy chain variable domain, domain 1 constant range, domain 2 constant range, and domain 3 constant range respectively). The IgG light chain consists of two immunoglobulin domains linked from N- to C-terminal in the VL-CL order, making reference to the light chain variable domain and the light chain constant domain. The constant regions show less sequence diversity, and are responsible for binding numerous natural proteins to elicit important biochemical events.
[000116] The variable region of an antibody contains the antigen binding determinants of the molecule, and thus determines the specificity of an antibody to its target antigen. The variable region is so named because it is the most distinct in the sequence of other antibodies within the same class. In the variable region, three loops are brought together for each of the heavy and light chain V domains to form an antigen binding site. Each of the loops is called a complementarity determining region (hereinafter referred to as a "CDR"), where the variation in the amino acid sequence is most significant. There are 6 total CDRs, three each per light and heavy chain, designated VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3. The variable region on the outside of the CDRs is called the frame region (FR). Although not as diverse as CDRs, sequence variability does not occur in the FR region between different antibodies, after all, this antibody characteristic architecture provides a stable framework (the FR region) in which substantial antigen diversity ( CDRs) can be exploited by the immune system to obtain specificity for a wide range of antigens. numerous high-resolution structures are available from fragments of variable region of different organisms, some unbound and some in complex with antigen. Sequence and structural characteristics of the variable antibody regions are disclosed, for example, in Morea et al., 1997, Biophys Chem 68: 9-16; Morea et al, 2000, Methods 20: 267-279, and the conserved characteristics of antibodies are disclosed, for example, in Maynard et al., 2000, Annu Rev Biomed Eng 2: 339-376.
[000117] Antibodies are grouped into classes, also called isotypes, as genetically determined by the constant region. Constant human light chains are classified as cape (CK) and lambda (CÀ) light chains. Human heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. The IgG class is the most commonly used for therapeutic purposes.
[000118] By "IgG" as used herein is meant that a polypeptide that belongs to the class of antibodies that is substantially encoded by a recognized gamma immunoglobulin gene. In humans, this class comprises subclasses IgG I, lgG2, lgG3, and lgG4. In mice, this class comprises subclasses IgGI, lgG2a, lgG2b and lgG3. IgM has subclasses, including, but not limited to, IgMI and lgM2. IgA has several subclasses, including but not limited to IgAI and IgA2. Thus, "isotype" as used here means that any of the classes or subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. The known human immunoglobulin isotypes are IgGI, IgG2, IgG3, IgG4, IgAI, IgA2, IgMI, IgM2, IgD, and IgE.
[000119] Also useful for the invention may be IgGs which are hybrid compositions of the natural human IgG isotypes. Effector functions such as ADCC, ADCP, CDC, and serum half-life differ significantly between different classes of antibodies, including, for example, IgGI, lgG2, lgG3, lgG4s IgAI, lgA2, IgD, IgE, IgG, and IgM ser human (Michaelsen et al., 1992, Molecular Immuno-logy, 29 (3): 319-326). Numerous studies have explored variants of IgGI, lgG2, lgG3, and lgG4 in order to investigate the determinants of differences in effector function between them. See for example Canfield & Morrison, 1991, J. Exp. Med. 173: 1483-1491; Chappel et al., 1991, Proc. Natl. Acad. Sci. USA 88 (20): 9036-9040; Chappel et al., 1993, Journal of Biological Chemistry 268: 25124-25131; Tao et al., 1991, J. Exp. Med. 173: 1025-1028; Tao et al., 1993, J. Exp. Med. 178: 661-667; Redpath et al., 1998, Human Immunology, 59, 720-727.
[000120] As described in patent application 2006-0134105 entitled "IgG Immunoglobulin Variants with Optimized Effector Function", it is possible to engineer amino acid modifications in an antibody that comprise constant regions of other classes of immunoglobulin. Such engineered hybrid IgG compositions can provide improved effector function properties, including ADCC, phagocytosis, CDC, and improved serum half-life.
[000121] As is well known in the art, immunoglobulin polymorphisms exist in the human population. Gm polymorphism is determined by the IGHG1, IGHG2 and IGHG3 genes that have alleles that encode allotypic antigenic determinants called Glm, G2m, and G3m allotypes for markers of human IgGI, lgG2 and lgG3 molecules (no Gm allotypes were found in chain 4 gamma). Markers can be classified into "allotypes" and "isoalotypes". These are distinguished on different serological bases depending on the strong sequence homologies between isotopes. Allotypes are specific antigenic determinants for allelic forms of the lg genes. Allotypes represent slight differences in the amino acid sequences of heavy or light chains of different individuals. Even a single amino acid difference can give rise to an allotypic determinant, although in many cases there have been several amino acid substitutions that have occurred. Allotypes are differences in sequence between alleles of a subclass so the antiserum recognizes only allelic differences. An isoalotype is an allele in an isotype that produces an epitope that is shared with a non-polymorphic homologous region of one or more other isotypes and because of that the antiserum will react with both the relevant allotypes and relevant homologous isotypes (Clark, 1997, IgG effector mechanisms, Chem. Immunol. 65-88-110, Gorman & Clark, 1990, Semin. Immunol. 2 (6): 457-66).
[000122] Allelic forms of human immunoglobulins have been well characterized. In addition, other polymorphisms have been characterized (Kim, et al., 2001, J. Mol. Evol. 54 1-9, incorporated here in their entirety by reference) At the moment, 18 Gm allotypes are known: Glm (1, 2, 3 , 17) or Glm (a, x, f, z), G2m (23) or G2m (n), G3m (5, 6, 10, 1 1, 13, 14, 15, 16, 21, 24, 26, 27, 28) or G3m (bl, c3, b5, bO, b3, b4, s, t, gl, c5, u, v, g5) (Lefranc, et al., The human IgG subclasses: molecular analysis of structure, function and regulation Pergamon, Oxford, pp 43-78 (1990), Lefranc, G et al., 1979, Hum. Genet .: 50, 199-21 1). Allotypes that are inherited in fixed combinations are called Gm haplotypes. The antibodies of the present invention can be substantially encoded by any allotype, isoalotype, or haplotype of any immunoglobulin gene antibodies of the present invention can be substantially encoded by genes from any organism, for example, mammals, including, but not limited to humans , rodents including but not limited to mice and rats, lagomorpha including but not limited to rabbits and hares, camelidae including but not limited to camels, llamas, and non-human dromedaries and primates, including but not limited to Prosimians, Platyrrhini (New Monkeys World), Cercopithecoidea (Old World monkeys), and Hominoidea including Gibbons and Lesser and Great Apes.
[000123] In one embodiment, the antibodies of the present invention are substantially human. The antibodies of the present invention can be substantially encoded by immunoglobulin genes that belong to any of the antibody classes. In one embodiment, the antibodies of the present invention comprise sequences that belong to the IgG class of antibodies, including subclasses of human IgGI, IgG2, IgG3, and IgG4. In a substitute embodiment, the antibodies of the present invention comprise sequences that belong to IgA (including human IgAI and IgA2 subclasses), IgD, IgE, IgG, or IgM antibody classes. The antibodies of the present invention can comprise more than one protein chain. That is, the present invention can find use in an antibody that is a monomer or an oligomer, including a homo- or hetero-oligomer.
[000124] In one embodiment, the antibodies of the invention are based on the human IgG sequences, and thus human IgG sequences are used as the "base" sequences against which other sequences are compared, including but not limited to. limited to sequences from other organisms, for example, rodent and primate sequences, as well as sequences from other immunoglobulin classes such as IgA, IgE, IgD, IgM, and the like. It is contemplated that, although the antibodies of the present invention are engineered in the context of an antibody of origin, the variants can be engineered into "transferred to the context of another, according to the antibody of origin. This is done by determining the" equivalent "residues or "matching" and substitutions between the first and second antibodies, typically based on the sequence or structural homology between the sequences of the two antibodies. In order to establish homology, an amino acid sequence of a first antibody outlined here is directly compared to sequence of a secondary antibody.After alignment of the sequences, using one or more of the homology alignment programs known in the art (for example using conserved between species), allowing insertions and deletions necessary to maintain alignment (that is, avoiding the elimination of conserved waste through arbitrary insertion and deletion), equivalent residues for particular amino acids in the primary sequence of the first antibody are defined. Alignment of conserved waste can conserve 100% of such waste. However, alignment of more than 75% or as little as 50% of conserved waste is also appropriate to define equivalent waste. Equivalent residues can also be defined by determining structural homology between a first and second antibody that is at the tertiary structure level for antibodies whose structures have been determined. In this case, equivalent residues are defined as those for which the atomic coordinates of two or more of the main chain atoms of a particular amino acid residue of a origin or precursor (in N, CA in CA, C in C and O in O ) are within 0.13 nm, for example, 0.1 nm, after alignment. Alignment is achieved after the best model has been oriented and has been positioned to give the maximum overlapping of atomic coordinates of non-hydrogen protein atoms of proteins. Regardless of how equivalent or corresponding residues are determined, and regardless of the identity of the source antibody in which the antibodies were made, what is intended to be transported is that the antibodies disclosed by the present invention can be engineered into any second antibody of origin that has significant sequence or structural homology to the antibody. So for example, if a variant antibody is generated in which the source antibody is human IgGI, using the methods described and other methods for determining equivalent residues, the variant antibody can be engineered into a lgG2 antibody of origin of human lgG2, an IgA antibody of human IgA origin, an antibody of mouse lgG2 or lgG2b, and the like Again, as described above, the context of the source antibody does not affect the ability to transfer antibodies of the present invention to other antibodies of origin. For example, variant antibodies that are engineered into a human IgGI antibody that targets an antigen epitope can be transferred into a human IgG2 antibody that targets a different antigen epitope, and so on.
[000125] In the IgG class of immunoglobulins, there are several immunoglobulin domains in the heavy chain. By "immunoglobulin (Ig) domain" here is meant that an region of an immunoglobulin having a distinct tertiary structure. Of interest in the present invention are the constant heavy chain domains, including, the constant heavy domain (CH) and a joint. In the context of IgG antibodies, the IgG isotypes each have three CH regions: "CHI" refers to positions 118-220, "CH2" refers to positions 237-340, and "CH3" refers to to positions 341 -447 according to the EU index as in Kabat. By "hinge" or "major region" or "major antibody region" or "major immunoglobulin region" here is meant that the flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody. Structurally, the IgG domain of CH1 ends at EU position 220, and the IgG domain of CH2 starts at residue EU position 237. Thus for IgG a joint is defined here to include positions from 221 (D221 in IgGI) to 236 (G236 in IgGI ), where the numbering is in accordance with the EU index as in Kabat. In some embodiments, for example, in the context of an Fc region, the lower joint is included, with the "lower joint" generally referring to positions 226 or 230. The constant heavy chain, as defined here, referred to it refers to the N-terminal of the CH1 domain to the C-terminal of the CH3 domain, thus comprising positions 1 18-447, where the numbering is in accordance with the EU index. The constant light chain comprises a single domain, and as defined here refers to positions of 108-214 of C or CK, where the numbering is in accordance with the EU index.
[000126] Specifically included within the definition of "antibody" are full length antibodies. By "full length antibody" here is meant the structure that constitutes the natural biological form of an antibody, including variable and constant regions. For example, in most mammals, including humans and mice, the full-length IgG class antibody is a tetramer and consists of identical pairs of two immunoglobulin chains, each pair having a light and heavy chain, each chain light comprising domains of immunoglobulin VL and CL, and each heavy chain comprising domains of immunoglobulin VH, CHI (Cyl), CH2 (Cy2), and CH3 (Cy3). In some mammals, for example, in camels and llamas, IgG antibodies can consist of just two heavy chains, each heavy chain comprising a variable domain linked to the Fc region.
[000127] Alternatively, antibodies can have a variety of structures, including, but not limited to antibody fragments. Antibody fragments include, but are not limited to, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies, antibody mimetics, chimeric antibodies, antibody fusions (sometimes called "antibody conjugates"), and fragments of each, respectively. Specific antibody fragments include, but are not limited to, (i) the Fab fragment consisting of VL, VH, CL and CH1 domains, (ii) the Fd fragment consisting of VH and CH1 domains, (iii ) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment, which consists of a single variable region, (v) isolated CDR regions, (vi) F (ab ') 2 fragments, a divalent fragment comprising two Fab fragments linked (vii) molecules of Single chain Fv (scFv), in which a VH domain and a VL domain are linked by a peptide linker that allows the two domains to join to form an antigen binding (viii) Fv chain dimers simple bispecific and (ix) "diabody" or "tri-body", multivalent or multispecific fragments constructed by gene fusion. The antibody fragments can be modified. For example, molecules can be stabilized by incorporating disulfide bridges that link the VH and VL domains. Examples of antibody architectures and formats are described in Holliger & Hudson, 2006, Nature Biotechnology 23 (9): 1126-1136, and Carter 2006, Nature Reviews Immunology 6: 343-357 and references cited here.
[000128] Antibodies of the invention can include multispecific antibodies, notably bispecific antibodies, also sometimes called "diabody". There are antibodies that bind to two (or more) different antigens. Diabodies can be manufactured in a variety of media known in the art, for example, prepared chemically or from hybrid hybridomas. In one embodiment, the antibody is a minibody. Minibodies are minimized antibody-like proteins comprising i, scFv joined to a CFI3 domain. In some cases, scFv may be joined to the Fc region, and may include some or all of the main region. For a description of multispecific antibodies see Holliger & Hudson, 2006, Nature Biotechnology 23 (9): 1126-1136 and reference cited here.
[000129] In one embodiment, the antibody of the invention is an antibody fragment. Of particular interest are antibodies that comprise Fc regions, Fc fusions, and the heavy chain constant region (CHI-joint-CH2-CH3). Antibodies of the present invention can comprise Fc fragments. An Fc fragment of the present invention can comprise 1 - 90% of the Fc region, for example, 10 - 90%, 30 - 90%, etc. Thus, for example, an Fc fragment of the present invention can comprise an IgGI domain Cy2, an IgGI Cy2 domain and main region, an IgGI Cy3 domain, and so on. In one embodiment, an Fc fragment of the present invention further comprises a fusion pair, effectively making it an Fc fragment fusion. Fc fragments may or may not contain extra polypeptide sequences.
[000130] Immunogenicity is the result of a series of complex responses to a substance that is perceived as foreign, and may include production of neutralizing and non-neutralizing antibodies, formation of immune complexes, complement activation, mast cell activation, inflammation , hypersensitivity and anaphylaxis responses. Several factors can contribute to protein immunogenicity, including, but not limited to, a protein sequence, route and frequency of administration, and patient population. Immunogenicity can limit the efficacy and safety of a protein therapy in multiple ways. Effectiveness can be reduced directly by the formation of neutralizing antibodies. Efficacy can also be reduced indirectly, as binding to either non-neutralizing antibodies or neutralizing antibodies typically leads to rapid clearance of severe side effects in the serum and even death can occur when an immune reaction is increased. Thus in one embodiment, protein engineering is used to reduce the immunogenicity of the antibodies of the present invention.
[000131] In some embodiments, the frame components can be mixed from different species. Such an antibody can be a chimeric antibody and / or a humanized antibody. In general, both "chimeric antibodies" and "humanized antibodies" refer to antibodies that combine regions of more than one species. "Chimeric antibodies" traditionally comprise variable region (s) of a mouse (or rat) , in some cases) and the constant region (s) of a human being (Morrison et al., 1984, Proc Natl Acad Sei USA 81 6851-6855).
[000132] By "humanized" antibody as used herein is meant that an antibody comprising a human framework (FR) region and one or more complementarity determining regions (CDRs) of a non-human antibody (usually ca - mundongo or rat). The non-human antibody providing the CDRs is called the "donor" and the human immunoglobulin providing the structure is called the "acceptor". Humanization is mainly in the graft of donor CDRs onto acceptor (human) VL and VH structures (Winter US 5,225,539). This strategy is called "CDR grafting". "Retrofitting" of selected acceptor structure residues to the corresponding donor residues is often required to recover affinity that is lost in the initial grafted construct (US 5,693,762). The humanized antibody will also comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin, and thus will typically comprise a human Fc region. A variety of techniques and methods for humanizing and remodeling non-human antibodies are well known in the art (See Tsurushita & Vasquez, 2004, humanization of monoclonal antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA), and references cited here). Humanization or other methods of reducing the immunogenicity of non-human antibody variable regions can include resurgence methods, as described, for example, in Roguska et al., 1994, Proc Natl Acad Sei USA 91 969-973). In one embodiment, selection-based methods can be employed to humanize and / or variable regions of affinity mature antibody, that is, to increase the affinity of the variable region for its target antigen. Other humanization methods may involve grafting only parts of the CDRs, including but not limited to methods described in, Tan et al., 2002, J Immunol 169 1119 -1125, De Pascalis et al., 2002, J Immunol 169 3076-3084. Structure-based methods can be employed for affinity maturation and humanization, for example, as described in U.S. patent no. 7,117,096 and related applications.
[000133] In certain variations, the immunogenicity of the antibody is reduced using a method described in US patent application 2006- 0008883, entitled "Methods of Generating Variant Protein with Increased Host String Content and Thereof Compositions, filed on December 3 of 2004.
[000134] Modifications to reduce immunogenicity may include modifications that reduce binding of processed peptides derived from a source sequence to MHC proteins. For example, amino acid modifications would be engineered such that there are no or a minimal number of immune epitopes that are predicted to bind, with high affinity, to any prevalent MHC alleles. Various methods of identifying MHC-binding epitopes in protein sequences are known in the art and can be used to classify epitopes in an antibody of the present invention. See, for example, US patent applications 2002-0119492, 2004-0230380 or 2006-0148009 and references cited here.
[000135] In an alternative embodiment, the antibodies of the present invention can be fully human, that is the sequences of the antibodies are fully or substantially human. "Fully human antibody" or "complete human antibody" refers to a human antibody having the gene sequence of an antibody derived from a human chromosome with the modifications outlined herein. Numerous methods are known in the art for generating fully human antibodies, including using transgenic mice (Bruggemann et al., 1997, Curr Opin Biotechnol 8: 455-458,) or human antibody libraries coupled with selection methods ( Griffiths et al., 1998, Curr Opin Biotechnol 9: 102-108).
[000136] The antibodies of the present invention target FLT3 and can comprise variable regions (e.g., CDRs) of any known and undisclosed anti-FLT3 antibody. Antibodies of the invention may exhibit selectivity for FLT3. Examples include total length versus splice variants, cell surface vs. Soluble forms, selectivity for various polymorphic variants, or selectivity for specific conformational forms of a target. An antibody of the present invention can bind any epitope or region in FLT3 and can be specific for fragments, mutant forms, joining forms, or anomalous forms of the antigens. Numerous useful antibodies have been disclosed that target FLT3 that can find use in the present invention.
Suitable FLT3 antibodies include anti-FLT3 antibodies 4G8 and BVIO, as disclosed in U.S. patent no. 5,777,084 and U.S. patent no. 6,156,882.
[000138] The antibodies of the present invention can find use in a wide range of products. In one embodiment the antibody of the invention is a therapy, a diagnosis, or a research reagent. In one embodiment, an antibody of the invention is therapeutic. An antibody of the present invention can find use in an antibody composition that is either monoclonal or polyclonal. In one embodiment, the antibodies of the present invention are used to kill target cells that carry the target antigen, for example, cancer cells. In a substitute embodiment, the antibodies of the present invention are used to block, antagonize or agonize the target antigen. In a substitute embodiment, the antibodies of the present invention are used to block, antagonize or agonize the target antigen and kill the target cells that carry the target antigen.
[000139] It will be recognized that the sequences of the variable domains including the CDRs identified here can be combined into any combination in an antibody. In addition, these sequences can be independently modified by adding all or part of an Fc region or Fc variant as disclosed herein. The modified sequences can also be combined into any combination in an antibody.
[000140] The present invention relates to antibodies comprising modifications, where the modifications alter affinity for one or more Fc receptors, and / or alter the ability of the antibody to mediate one or more effector functions. Modifications of the invention include amino acid modifications.
[000141] The inventors of the present invention surprisingly found that by introducing amino acid substitutions 239D and 332E into the CH2 domain of the Fc part of known anti-FLT3 antibodies, such as 4G8 and BV10 (supra), cell killing activity of these antibodies can be significantly increased or even detected and generated for the first time. In one embodiment, the amino acid substitutions are S239D and I332E. This is surprising, as it has been experimentally shown that the same modifications generally do not increase cell killing activity. In other words, on different antibodies directed at a different target antigen, the introduction of these substitutions had no measurable effect on cell death.
[000142] Furthermore, such modified antibodies may further comprise amino acid modifications in the heavy chain constant region at positions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 240, 241, 243, 244, 245, 246, 247, 249, 255, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 280, 281, 282, 283, 284, 285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 313, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 333, 334, 335, 336, and 337, which have been shown to allow modification of FcR binding properties, effector function, and potentially clinical properties of antibodies (See USSN 11 / 124,620, deposited on May 5, 2005, entitled "Optimized Fc Variants").
[000143] In particular, additional variants that alter binding to one or more human Fc receptors may comprise an amino acid modification in the heavy chain constant region, as described here, selected from the group consisting of 221K, 221 Y, 222E , 222Y, 223E, 223K, 224E, 224Y, 225E, 225K, 225W, 227E, 227G, 227K, 227Y, 228E, 228G, 228K, 228Y, 230A, 230E, 230G, 230Y, 231 E, 231 G, 231 K, 231 P, 231 Y, 232E, 232G, 232K, 232Y, 233A, 233D, 233F, 233G, 233H, 233I, 233K, 233L, 233M, 233N, 233Q, 233R, 233S, 233T, 233V, 233W, 233Y, 234A, 233 234D, 234E, 234F, 234G, 234H, 2341, 234K, 234M, 234N, 234P, 234Q, 234R, 234S, 234T, 234V, 234W, 234Y, 235A, 235D, 235E, 235F, 235G, 235H, 2351, 235K, 235M, 235N, 235P, 235Q, 235R, 235S, 235T, 235V, 235W, 235Y, 236A, 236D, 236E, 236F, 236H, 2361, 236K, 236L, 236M, 236N, 236P, 236Q, 236R, 236S, 236T, 236V, 236W, 236Y, 237D, 237E, 237F, 237H, 2371, 237K, 237L, 237M, 237, 237P, 237Q, 237R, 237S, 237T, 237V, 237W, 237Y, 238D, 238E, 238F, 238G, 238H, 238I, 238K, 238L, 238M, 238N, 238Q, 238R, 238S, 238T, 238V, 238W, 238Y, 240A, 240I, 240M, 240T, 241 D, 241E, 241L, 241R, 241S, 241W, 241Y, 243E , 243H, 243L, 243Q, 243R, 243 W, 243Y, 244H, 245A, 246D, 246E, 246H, 246Y, 247G, 247V, 249H, 249Q, 249Y, 255E, 255Y, 258H1258S, 258Y, 260D, 260E, 260H, 260Y, 262A, 262E, 262F, 2621, 262T, 263 A, 2631, 263M, 263T, 264A, 264D, 264E, 264F, 264G, 264H, 2641, 264K, 264L, 264M, 264N, 264P, 264Q, 264R, 264S , 264T, 264 W, 264 Y, 265F, 265G, 265H, 2651, 265, 265L, 265M, 265N, 265P, 265Q, 265R, 265S, 265T, 265V, 265W, 265Y, 266A, 2661, 266M, 266T, 267D , 267E, 267F, 267H, 2671, 267K, 267L, 267M, 267N, 267P, 267Q, 267R, 267T, 267V, 267W, 267Y, 268D, 268E, 268F, 268G, 2681, 268K, 268L, 268M, 268P, 268M, 268P, 268M , 268R, 268T, 268V, 268W, 269F, 269G, 269H, 2691, 269K, 269L, 269M, 269N, 269P, 269R, 269S, 269T, 269V, 269 W, 269 Y, 270F, 270G, 270H, 2701, 270L , 270M, 270P, 270Q, 270R, 270S, 270T, 270W, 270Y, 271 A, 271D, 271E, 271F, 271G, 271H, 2711, 271, 271L, 271M , 271N, 271Q, 271R, 271 S, 271T, 271V, 271 W, 271 Y, 272D, 272F, 272G, 272H, 2721, 272, 272L, 272M, 272P, 272R, 272S, 272T, 272V, 272W, 272Y, 2731, 274D, 274E, 274F, 274G, 274H, 2741, 274L, 274M, 274N, 274P, 274R, 274T, 274V, 274W, 274Y, 275L, 275W, 276D, 276E, 276F, 276G, 276H, 2761, 276L, 2761, 276L, 276M, 276P, 276R, 276S, 276T, 276V, 276W, 276Y, 278D, 278E, 278G, 278H, 2781, 278K, 278L, 278M, 278N, 278P, 278Q, 278R, 278S, 278T, 278V, 278W, 280G, 280K, 280L, 280P, 280W, 281D, 281E, 281 K, 281N, 281P, 281Q, 281Y, 282E, 282G, 282K, 282P, 282Y, 283G, 283H, 283K, 283L, 283P, 283R, 283Y, 284D, 284E , 284L, 284N, 284Q, 284T, 284Y, 285D, 285E, 285K, 285Q, 285W, 285Y, 286E, 286G, 286P, 286Y, 288D, 288E, 288Y, 290D, 290H, 290L, 290N, 290W, 29 ID, 29 IE, 291G, 291 H, 2911, 291Q, 291 T, 292D, 292E, 292T, 292Y, 293F, 293G, 293H, 2931, 293L, 293M, 293N, 293P, 293R, 293S, 293T, 293V, 293 W, 293 Y, 294F, 294G, 294H, 2941, 294K, 294L1294M1294P, 294R, 294S, 294T, 294V, 294W, 294 Y, 295D, 295E, 295F, 295G, 295H, 2951, 295M1295N, 295P1295R1295S, 295T1295V1295W1295Y, 296A, 296D, 296E, 296g, 296H12961, 296K1296L1296M, 296N1296Q1296R1296S1296T1296V, 297D, 297E1297F1297G, 297H, 2971, 297K, 297L, 297M1297P1297Q1297R, 297S1297T, 297V, 297W, 297Y, 298A, 298D, 298E, 298F, 298H, 2981, 298, 298M, 298N, 298Q, 298R, 298T, 298 W, 298Y, 299A, 299D, 299E, 299F, 299G, 299H, 2991,299K, 299L, 299M, 299N, 299P, 299Q, 299R , 299S 1299V, 299W, 299Y, 300A, 300D1300E, 300G, 300H, 300K, 300M, 300N, 300P, 300Q, 300R, 300S, 300T, 300V, 300W, 301D, 301E, 301H, 301Y, 3021,303D, 303E, 303Y, 304D, 304H, 304L, 304N, 304T, 305E, 305T, 305Y, 313F, 317E, 317Q, 318H, 318L, 318Q, 318R, 318Y, 320D, 320F, 320G, 320H, 3201,320L, 320N, 320P, 320S, 320T, 320V, 320W, 320Y, 322D, 322F1322G, 322H13221, 322P1322S1322T, 322V, 322W, 322Y, 3231, 324D, 324F, 324G, 324H, 3241, 324L, 324M, 324P, 324R, 324T, 324V, 324 W, 324 W, 324 W, 324 W , 324 Y, 32 A, 325D, 325E, 325F, 325G, 325H, 3251, 325K, 325L, 325M, 325P, 325Q, 325R, 325S, 325T, 325V, 325W1325Y, 326E, 3261, 3 26L, 326P, 326T, 327D, 327E, 327F, 327H, 3271, 327, 327L, 327M, 327N, 327P, 327R, 327S, 327T, 327V, 327W, 327Y, 328A, 328D, 328E, 328F, 328G, 328H, 3281, 328K, 328M, 328N, 328P, 328Q, 328R, 328S, 328T, 328V, 328W, 328Y, 329D, 329E, 329F, 329G, 329H, 3291, 329, 329L, 329M, 329N, 329Q, 329R, 329S, 329T, 329V, 329W, 329Y, 330E, 330F, 330G, 330H, 3301, 330L, 330M, 330N, 330P, 330R, 330S, 330T, 330V, 330W, 330Y, 331 D, 331 F, 331 H, 3311, 331 L, 331 M, 331Q, 331 R, 331T, 331V, 331W, 331Y, 333A, 333F, 333H, 3331, 333L, 333M, 333P, 333T, 333Y, 334A, 334F, 3341, 334L, 334P, 334T, 335D, 335F, 335G, 335H, 3351, 335L, 335M, 335N, 335P, 335R, 335S, 335V, 335W, 335Y, 336E, 336K, 336Y, 337E, 337H, and 337N, where the numbering is in accordance with the index of I.
[000144] Furthermore, the invented antibodies may further comprise amino acid modifications outside the Fc region, such as those described in U.S. patent no. 7,276,585, filed on March 24, 2005, entitled "Immunoglobulin variants outside the Fc region", including amino acid modifications in the heavy chain constant region positions 118, 119, 120, 121, 122, 124, 126, 129, 131, 132, 133, 135, 136, 137, 138, 139, 147, 148, 150, 151, 152, 153, 155, 157, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 183, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 201,203, 205, 206, 207, 208, 209, 210, 21 1, 212, 213, 214, 216, 217, 218, 219, 221, 222, 223, 224, 225, 226, 227, 228, 229 , 230, 231, 232, 233, 234, 235, and 236 and / or including amino acid modifications in the light chain constant region positions 108, 109, 110, 111, 112, 114, 1 16, 121, 122, 123, 124, 125, 126, 127, 128, 129, 131, 137, 138, 140, 141, 142, 143, 145, 147, 149, 150, 151, 152, 153, 154, 155, 156, 157, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 176, 180, 181, 182, 183, 184, 185, 187, 188, 189, 190, 191, 193, 195, 197, 199, 200, 202, 203, 204, 205, 206, 207, 208, 210, 211,212, e213.
[000145] These modifications may allow for further modification of FcR binding properties, effector function, and potentially clinical properties of antibodies. In particular, variants that alter binding to one or more human Fc receptors may comprise an amino acid modification in the heavy chain constant region, as described here, selected from the group consisting of 118K, 118E, 118Y, 119R, 119E, 119Y, 120R, 120E, 1201, 121 E, 121Y, 121 H, 122E, 122R, 124K, 124E, 124Y, 126K, 126D, 129L, 129D, 131G, 131T, 132D, 132R, 132L, 133R, 133E, 133L, 1351, 135E, 135K, 136E, 136K, 1361, 137E, 138S, 138R, 138D, 1391, 139E, 139K, 147A, 147E, 148Y, 148K, 150L, 150K, 150E, 151 A, 151 D, 152L, 152K, 153L, 153D, 155E, 155K, 1551, 157E, 157, 157Y, 159K, 159D, 159L, 160K, 160E, 160Y, 161 D, 162D, 162K, 162Y, 163R, 164R, 164E, 164Y, 165D, 165R, 165Y , 166D, 167A, 168L, 169E, 171G, 171 H, 172K, 172L, 172E, 173T, 173D, 174E, 174K, 174Y, 175D, 175L, 176D, 176R, 176L, 177R, 177E, 177Y, 178D, 179K, 179Y, 179E, 180, 180L, 180E, 183T, 1871, 187, 187E, 1881, 189D, 189G, 1901, 190K, 190E, 191 D, 191 R, 191Y, 192N, 192R, 192L, 193F, 193E, 1 4R , 194D, 1 95R, 15D, 195Y, 196K, 196D, 196L, 197R, 197E, 197Y, 198L, 199T, 199D, 199K, 201 E, 201 K, 201 L, 203D, 203L, 203K, 205D, 205L, 206A, 206E, 207K, 207D, 208R, 208E, 208 Y, 209E, 209K, 209Y, 210L, 210E, 210Y, 211 R, 211 E, 211Y, 212Q, 212K, 212H, 212L, 212Y, 213N, 213E, 213H, 213L, 213Y , 214N, 214E, 214H, 214L, 214Y, 216N, 216K, 216H, 216L, 216Y, 217D, 217H, 217A, 217V, 217G, 218D, 218E, 218Q, 218T, 218H, 218L, 218Y, 219D, 219 , 219K, 219T, 219H, 219L, 2191, 219Y, 205A, 210A, 213 A, 214A, 218A, 221 K, 221Y, 221 E, 221 N, 221Q, 221 R, 221 S, 22 IT, 221 H, 221 A, 221V, 221 LI 2211, 221 F, 221 M, 221 W, 221 PI 221G, 222E, 222Y1 222D1 222N, 222Q, 222R, 222S, 222T, 222H, 222V, 222L, 2221, 222F, 222M1 222W, 222P, 222G, 222A, 223D, 223N, 223Q, 223R, 223S, 223H, 223A, 223V, 223L, 2231, 223F, 223M, 223Y, 223 Wl 223P, 223G, 223E1 223K, 224D, 224N, 224Q, 224K, 224R, 224K, 224 , 224T, 224V1 224L1 2241, 224F1 224M1 224W, 224P, 224G, 224E, 224Y, 224A, 225D, 225N1 225Q, 225R, 225S, 225H, 225A1 225V, 22 5L, 2251, 225F1 225M, 225Y1 225P1 225G1 225E, 225, 225W, 226S, 227E, 227, 227Y, 227G, 227D, 227N, 227Q, 227R, 227S, 227T, 227H, 227A, 227V, 227L, 2271, 227F, 2271 227M, 227W, 228K, 228Y1 228G, 228D1 228N1 228Q1 228R, 228T, 228H1 228A, 228V, 228L, 2281, 228F, 228M, 228W, 229S, 230A, 230E, 230Y, 230G, 230D, 230N, 230Q, 230K , 230S, 230T, 230H, 230V, 230L, 2301, 230F1 230M1 230W, 231 K, 231 P, 231 D, 231 N, 231Q, 231 R, 231S, 231T, 231 HI 231V1 231 L, 2311, 231 F} 231 M, 231W, 232E, 232K, 232Y, 232G, 232D, 232N, 232Q, 232R, 232S, 232T, 232H, 232A, 232V, 232L, 2321, 232F, 232M1 232W, 233D, 233N1 233Q, 233R, 233, 233R, 233 233H, 233A, 233V, 233L, 2331,233F, 233M, 233Y, 233 W, 233G, 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 2341, 234V, 234F1 234K, 234R, 234S, 234A, 234M, 234A 234G, 235D, 235S, 235N, 235Q, 235T, 235H, 235Y, 2351, 235V, 235F, 235E, 235K, 235R1 235A1 235M, 235W, 235P, 235G, 236D, 236E, 236N, 236Q1 236K, 236R, 236S, 236T , 236H, 236A, 236V, 236L, 2361, 236F1 236M, 236Y, 236W, and 236P, in that the numbering is in accordance with the EU index.
[000146] In particular, variants that alter binding to one or more human Fc receptors may comprise an amino acid modification in the light chain constant region, as described here, selected from the group consisting of 108D, 1081, 108Q, 109D, 109P, 109R, H OE, 1101, 110K, 111 E, 11 1 K, 1 11 LI 112E, 112R, 1 12Y1 114D, 1141, 114K, 116T, 121 Dl 122R, 122S, 122Y, 123L, 123R, 124E, 125E , 125K, 126D, 126L, 126Q, 127A, 127D, 127K, 128N, 129E, 1291, 129K, 131T, 137K, 137S, 138D, 138K, 138L, 140E1 140H, 140K, 141 El 141 K, 142D, 142G, 142L , 143A, 143L, 143R, 145D, 145T, 145Y, 147A, 147E, 147K, 149D, 149Y1 150A, 1511, 151 Kl 152L, 152R, 152S, 153D, 153H, 153S, 154E, 154R, 154V, 155E, 1551, 155K, 156A, 156D, 156R, 157N, 158D, 158L, 158R, 159E1 159K1 159L, 160, 160V, 161 Kl 161 LI 162T, 163E, 163K, 163T, 164Q, 165K, 165P, 165Y, 166E, 166M, 166S1 167K1 167L, 168K, 168Q, 168Y, 169D, 169H, 169S, 1701, 170N, 170R, 171A1 171 N, 171V, 172E1 17211 172K, 173K, 173L, 173Q, 174A, 176T, 18 0E, 180K, 180S, 181 Kl 182E, 182R, 182T, 183D, 183L, 183P, 184E, 184K, 184Y, 1851, 185Q, 185R, 187K, 187Y1 188E, 188S, 188Y, 189D, 189K, 189Y, 190E, 190L , 190R, 191 E, 191 RI 191 S, 193E, 193K, 193S, 1951, 195K, 195Q, 197E, 197K, 197L, 199E, 199K, 199Y, 200S, 202D, 202R, 202Y, 203D, 203L, 203R, 204T , 205E, 205K, 206E, 2061, 206K, 207A, 207E, 207L, 208E, 208K, 208T, 210A, 210E, 210K, 211A, 211 E, 211 P, 212E, 212K, 212T, 213L, 213R, where the numbering is in accordance with the EU index.
[000147] Additional substitutions that can also be used in the present invention include other substitutions that modulate Fc receptor affinity, FcR-mediated effector function and / or complement-mediated effector function include but are not limited to 298A, 298T, 326A, 326D , 326E, 326W, 326Y, 333A, 333S, 334L, and 334A (US 6,737,056; Shields et al. 5 Journal of Biological Chemistry, 2001, 276 (9): 6591-6604; US 6,528,624; Idusogie et al., 2001, J. Immunology 166: 2571-2572), 247L, 255L, 270E, 392T, 396L, and 421 K (USSN 10 / 754,922; USSN 10 / 902,588), and 280H, 280Q, and 280Y (USSN 10 / 370,749).
[000148] In other embodiments, antibodies of the present invention can be combined with heavy chain constant variant that alter FcRn binding. These include modifications that modify the affinity of FcRn in a specific pH manner. In particular, variants that enhance Fc binding to FcRn include but are not limited to: 250E, 250Q, 428L, 428F, 250Q / 428L (Hinton et al., 2004, J. Biol. Chem. 279 (8): 6213-6216 , Hinton et al. 2006 Journal of Immunology 176: 346- 356, USSN 11/102621, PCT US2003 / 033037, PCT / US2004 / 01 1213, USSN 10/822300, USSN 10/687118, PCT / US2004 / 034440, USSN 10 / 966673), 256A, 272A, 286A, 305A, 307A, 31 1 A, 312A, 376A, 378Q, 380A, 382A, 434A (Shields et al., Journal of Biological Chemistry, 2001, 276 (9): 6591-6604, USSN 10/982470, US6737056, USSN 11/429793, USSN 11/429786, PCT / US2005 / 02951 1, USSN 11/208422), 252F, 252T, 252Y, 252W, 254T, 256S, 256R, 256Q, 256E, 256D, 256T , 309P, 311 S, 433R, 433S, 4331, 433P, 433Q, 434H, 434F, 434Y, 252Y / 254T / 256E, 433K7434F / 436H, 308T / 309P / 311S (Dali Acqua and others Journal of Immunology, 2002, 169: 5171-5180, US7083784, PCT US97 / 03321, US6821505, PCT US01 / 48432, USSN 11/397328), 257C, 257M, 257L, 257N, 257Y, 279E, 279Q, 279Y, insertion of Ser after 281, 2 83F, 284E, 306Y, 307V, 308F, 308Y 311V, 385H, 385N, (PCT US2005 / 041220, USSN 1 1/274065, USSN 11 / 436,266) 204D, 284E, 285E, 286D, and 290E (PCT7US2004 / 037929 incorporated here in its entirety by reference).
[000149] In some embodiments of the invention, antibodies may comprise isotypic modifications, that is, modifications in an IgG of origin in the type of amino acid in a substitute IgG.
[000150] The present invention provides variant antibodies that are optimized for numerous therapeutically relevant properties. A variant antibody comprises one or more amino acid modifications in relation to a parent antibody, wherein the amino acid modification (s) provide one or more optimized properties. Thus, the antibodies of the present invention are variant antibodies. An antibody of the present invention differs in the amino acid sequence from its parent antibody by virtue of at least the two amino acid modifications 239D and 332E. In addition, the variant antibodies of the present invention may comprise more than the two amino acid modifications mentioned above when compared to the origin, for example from about three to fifty amino acid modifications, for example, from about three to ten modifications of amino acid, from about three to about five amino acid modifications, etc., compared to the origin. Thus the sequences of the variant antibodies and those of the original antibodies are substantially homologous. For example, the antibody of variant sequences here will have about 80% homology with the original antibody sequence, for example, at least about 90% homology, and at least about 95% homology, etc. .
[000151] The antibodies of the present invention may comprise amino acid modifications, but provide effector function properties optimized in relation to the origin. Substitutions and optimized effector function properties are described in US patent application 2004-0132101, PCT application US03 / 30249, and in US patent application 7,317,091 10 / 822,231, (Properties that can be optimized include but are not limited to increased and reduced affinity for an FcR In one embodiment, the antibodies of the present invention are optimized to have increased affinity for human activation FcR, for example, FcRI, FcRIla, FcRIlc, FcRIlIa, and FcRJIlb. an antibody of the invention is optimized to have increased affinity for a human FcRIlIa In a substitute embodiment, antibodies are optimized to have reduced affinity for the human inhibitory receptor FcRIlb. These embodiments are anticipated to provide antibodies with increased therapeutic properties in human beings, for example, increased effector function and greater anticancer potency.
[000152] In other embodiments, antibodies of the present invention provide increased affinity for one or more FcRs, yet reduced affinity for one or more other FcRs. For example, an antibody of the present invention can have increased binding to FcRIlIa, still reduced binding to FcRIlb. Alternatively, an antibody of the present invention can have increased binding to FcRIla and FcRI, yet reduced binding to FcRIlb.
[000153] Modifications of the invention can increase binding affinity for one or more FcRs. By "higher affinity" or "enhanced affinity" or "increased affinity" or "better affinity" than an immunoglobulin of origin, as used here, it is meant that an Fc variant binds to an Fc receptor with an equilibrium constant significantly higher association (Ka) or a lower association equilibrium constant (Kd) than the parent polypeptide when the amounts of the parent variant and polypeptide in the binding assay are essentially the same. For example, the Fc variant as an enhanced FcR binding affinity may exhibit about 5 times to about 1000 times, for example, about 10 times to about 500 times improvement in the Fc receptor binding affinity in comparison with the parent polypeptide, where Fc receptor binding affinity is determined by methods known in the art. Correspondingly, by "reduced affinity" when compared to a polypeptide of Fc origin as used here is meant that a variant of Fc binds an Fc receptor with significantly low Ka or higher Kd than the original polypeptide.
[000154] Embodiments comprise optimization of Fc that binds to a human FcR, however in embodiments the substitute antibodies of the present invention have reduced or increased affinity for FcRs of non-human organisms, including but not limited to rodents and primates not human. Antibodies that are optimized to bind to a non-human FcR may find use in the experiment. For example, mouse models are available for a variety of diseases that allow testing of properties such as efficacy, toxicity, and pharmacokinetics for a given candidate drug. As is known in the art, cancer cells can be grafted or injected into mice to mimic human cancer, a process called xenograft. Antibody testing, which comprises antibodies that are optimized for one or more mouse FcRs, can provide valuable information regarding the protein's effectiveness, its mechanism of action, and the like. The antibodies of the present invention can also be optimized for increased functionality and / or solution properties in the aglycosylated form. In one embodiment, the aglycosylated antibodies of the present invention bind an Fc linker with greater affinity than the aglycosylated form of the parent antibody. Fc ligands include, but are not limited to, FcRs, Clq, FcRn, and proteins A and G, and can be from any source including but not limited to human, mouse, rat, rabbit, or monkey. In a substitute embodiment, the antibodies are optimized to be more stable and / or more soluble than the aglycosylated form of the parent antibody.
[000155] Antibodies of the invention may comprise modifications that modulate the interaction with Fc ligands other than FcRs, including but not limited to complementary proteins, FcRn, and Fc receptor homologues (FcRHs). FcRHs include but are not limited to FcRHI, FcRH2, FcRH3, FcRH4, FcRH5, and FcRH6 (Davis et al., 2002, Immunol. Reviews 190: 123-136).
[000156] Antibodies of the present invention can comprise one or more modifications that provide optimized properties that are not specifically related to effector function per se. The modifications can be amino acid modifications, or they can be modifications that are made enzymatically or chemically. Such modification (s) are likely to provide some improvement in the antibody, for example an increase in its stability, solubility, function or clinical use. The present invention contemplates a variety of improvements that can be made by coupling the antibodies of the present invention with further modifications.
[000157] In one embodiment, the variable region of an antibody of the present invention can be affinity matured, that is, amino acid modifications have been made in the VH and / or VL domains of the antibody to increase binding of the antibody to its target antigen . Such types of modifications can improve the association and / or dissociation kinetics for binding to the target antigen. Other modifications include those that improve selectivity for target vs. antigen. alternative targets. These include modifications that enhance selectivity for antigen expressed in target cells vs. non-target cells. Further improvements to the target recognition properties can be provided by additional modifications. Such properties can include, but are not limited to, specific kinetic properties (i.e. association and dissociation kinetics), selectivity for the particular target versus alternative targets, and selectivity for a specific target form versus alternative forms. Examples include full length forms versus bonding variants, cell surface vs. soluble forms, selectivity for various polymorphic variants, or selectivity for specific conformational forms of the target antigen.
[000158] Antibodies of the invention can comprise one or more modifications that provide reduced or increased internalization of an antibody. In one embodiment, antibodies of the present invention can be used or combined with additional modifications in order to reduce the cellular internalization of an antibody that occurs via interaction with one or more Fc ligands. This property can be expected to increase effector function, and potentially reduce the immunogenicity of the antibodies of the invention. Alternatively, antibodies of the present invention can be used directly or combined with additional modifications in order to increase the cellular internalization of an antibody that occurs via interaction with one or more Fc ligands.
[000159] In one embodiment, modifications are made to improve the biophysical properties of the antibodies of the present invention, including, but not limited to stability, solubility, and oligomeric status. Modifications may include, for example, substitutions that provide more favorable intramolecular interactions in the antibody as to provide greater stability, or substitutions of exposed non-polar amino acids with polar amino acids for higher solubility. Numerous optimization objectives and methods are described in US patent application 2004-0110226, which can find use for engineering additional modifications to further optimize the antibodies of the present invention The antibodies of the present invention can also be combined with additional modifications that reduce size or oligomeric state, such that tumor preparation is increased, or in vivo clearance rates are increased when desired.
[000160] Other modifications in the antibodies of the present invention include those that allow specific formation or homomimeric or homomultimeric molecules. Such modifications include but are not limited to engineered disulfides, as well as chemical modifications or aggregation methods that can provide a mechanism for the generation of homodimerics or covalent homomultimers. For example, methods of engineering compositions of such molecules are described in Kan et al., 2001, J. Immunol., 2001, 166: 1320-1326; Stevenson et al., 2002, Recent Results Cancer Res. 159 104-12; US 5,681,566; Caron et al., 1992, J. Exp. Med. 176: 1191-1195, and Shopes, 1992, J. Immunol. 148 (9): 2918-22. Additional modifications to the variants of the present invention include those that allow specific formation or heterodimeric, bifunctional, and / or multifunctional molecules. Such modifications include, but are not limited to, one or more amino acid substitutions in the CH3 domain, in which the substitutions reduce the formation of homodimer and increase the formation of heterodimer. For example, engineering methods and compositions of such molecules are described in Atwell et al., 1997, J. Mol. Biol. 270 (l): 26-35, and Carter et al., 2001, J. Immunol. Methods 248: 7-15, each incorporated herein in its entirety by reference. Additional modifications include changes in the joint and CH3 domains, where the modifications reduce the tendency to form dimers.
[000161] In other embodiments, the antibodies of the present invention comprise modifications that remove proteolytic degradation sites. These can include, for example, protease sites that reduce yields, as well as protease sites that degrade the protein administered in vivo. In one embodiment, further modifications are made to remove covalent degradation sites such as deamidation sites (i.e. deamidation of glutaminyl and asparaginyl residues to the corresponding glutamine and aspartate residues), oxidation, and proteolytic degradation. Deamidation sites that are particularly useful for removal are those that increase the tendency for deamidation, including, but not limited to, asparaginyl and glutamyl residues followed by glycines (pieces of NG and HQ, respectively). In such cases, replacing any residue can significantly reduce the tendency to deamidate. Common oxidation sites include cysteine and methionine residues. Other covalent modifications, which can be either introduced or removed, include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the "amine groups of side chains of lysine, arginine and (TE Creighton, Proteins : Structure and Molecular Properties, WH Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group. Additional modifications may also include but are not limited to post-translational modifications such as glycolysis and N or O-linked phosphorylation.
[000162] Modifications may include those that improve yields of express and / or purification of hosts or host cells commonly used for the production of biologies. These include, but are not limited to, various mammalian cell lines (e.g., CHO), yeast cell lines, bacterial cell lines, and plants. Additional modifications include modifications that remove or reduce heavy chain capacity to form interchain disulfide bonds. Additional modifications include modifications that remove or reduce the ability of heavy chains to form intrachain disulfide bonds.
[000163] The antibodies of the present invention may comprise modifications that include using unnatural amino acids incorporated using, for example, technologies developed by Schultz and colleagues, including but not limited to methods described by Cropp & Shultz, 2004, Trends Genet. 20 (12): 625-30, Anderson et al., 2004, Proc. Natl. Acad. Sci. U.S.A. 101 (2): 7566-71, Zhang et al., 2003, 303 (5656): 371-3, and Chin et al., 2003, Science 301 (5635): 964-7. In some embodiments, these modifications allow manipulation of various functional, biophysical, immunological or manufacturing properties discussed above. In additional embodiments, these modifications allow for additional chemical modification for other purposes. Other changes are contemplated here. For example, the antibody can be linked to one of a variety of non-proteinaceous polymers, for example, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. Additional amino acid modifications can be made to allow specific or non-specific chemical or post-translational modification of the antibodies. Such modifications include, but are not limited to, PEGylation and glycosylation. Specific substitutions that can be used to allow PEGylation include, but are not limited to, introduction of new cysteine residues or unnatural amino acids such that effective and specific coupling chemicals can be used to bind a PEG or otherwise polymeric moiety. Introduction of specific glycosylation sites can be achieved by introducing new N-X-T / S sequences into the antibodies of the present invention.
[000164] Covalent modifications of antibodies are included within the scope of this invention, and are in general, but not always, made post-translationally. For example, several types of covalent modifications of the antibody are introduced into the molecule by reacting specific amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or at the N- or C-terminal residues.
[000165] In some embodiments, the covalent modification of the antibodies of the invention comprises the addition of one or more labels. The term "tagging group" is any detectable label. In some embodiments, the labeling group is coupled to the antibody via spacer arms of various lengths to reduce potential spherical obstruction. Various methods for labeling proteins are known in the art and can be used in carrying out the present invention. In general, labels fall within a variety of classes, depending on the assay in which they are to be detected: a) isotopic labels, which can be heavy or radioactive isotopes; b) magnetic labels (for example, magnetic particles); c) redox reactive portions; d) optical dyes; enzyme groups (for example, horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase); e) biotinylated groups; and f) predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.). In some embodiments, the marking group is coupled to the anti-body via spacer arms of various lengths to reduce potential steric obstruction. Various methods for labeling proteins are known in the art and can be used in carrying out the present invention. Specific labels include optical dyes, including, but not limited to, chromophores, matches and fluorophores, with the latter being specific in many cases. Fluorophores can be either "small molecule" fluoro, or proteinaceous fluoro. By "fluorescent label" is meant any molecule that can be detected via its inherent fluorescent properties.
[000166] In one embodiment, the antibodies of the invention are antibody "fusion proteins", sometimes referred to herein as "antibody conjugates". The fusion pair or conjugate pair can be proteinaceous or non-proteinaceous; the latter generally being generated using functional groups in the antibody and the conjugate pair. Fusion and conjugate patterns can be any molecule, including polypeptides and small molecule chemical compounds. For example, a variety of antibody conjugates and methods described in Trail et al., 1999, Curr. Opin. Immunol. 11: 584-588. Possible conjugate pairs include, but are not limited to, cytokines, cytotoxic agents, toxins, radioisotopes, chemotherapeutic agent, antiangiogenesis agents, tyrosine kinase inhibitors, and other therapeutically active agents. In some embodiments, conjugate pairs may be considered too much as payloads, this means that the purpose of a conjugate is the targeted delivery of the conjugate pair to a targeted cell, for example a cancer cell or immune cell, by the antibody . Thus, for example, the conjugation of a toxin to antibody targets of supplying the toxin to cells that express the target antigen. As it will be appreciated by one skilled in the art, in reality the concepts and definitions of fusion and conjugate are overlapping. The designation of an antibody as a fusion or conjugate is not intended to oblige it for any particular embodiment of the present invention. Certainly, these terms are used loosely to convey the broad concept that any antibody of the present invention can be linked genetically, chemically, or otherwise, to one or more polypeptides or molecules to provide some desired property.
[000167] Suitable conjugates include, but are not limited to, labels as described below, cytotoxic drugs and agents including, but not limited to, cytotoxic drugs (e.g., chemotherapeutic agents) or toxins or active fragments of such toxins. Toxins and their corresponding suitable fragments include diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcine, crotin, phenomycin, enomycin and the like. Cytotoxic agents also include radiochemicals made by conjugating radioisotopes to antibodies, or binding a radionuclide to a chelating agent that has been covalently bound to the antibody. Additional embodiments use calicheamicin, aunstatins, geldanamycin, maytansine, and duocarmicins and the like; for the latter, see U.S. patent application 2003/0050331
[000168] In one embodiment, the antibodies of the present invention are fused or conjugated to a cytokine. By "cytokine" as used here it is meant that a generic term for proteins released by a population of cells that act on another cell as intercellular mediators. For example, as described in Penichet et al., 2001, J Immunol Methods 248- 91-101, cytokines can be fused to antibody to provide a set of desirable properties. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormone. Included among cytokines are growth hormone such as ser growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone, thyroxine; insulin; proinsulin; relaxin, prelaxin; glycoprotein hormone such as antigen-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and lutenizing hormone (LH); liver growth factor; fibroblast growth factor, prolactin, placental lactogen; tumor necrosis alpha and beta factor; mulle [i] an inhibiting substance, mouse gonadotropin-associated peptide; inhibin; activin, vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; platelet growth factor, transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor I and II; eitropoietin (EPO); osteoinductive factors, interferons such as interferon-alpha, beta, and -gamma; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF), granulocyte macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF), interleukins (ILs) such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL -8, IL-9, IL-10, IL-11, IL-12, IL-15, a tumor necrosis factor such as TNF-alpha or TNF-beta; C5a; and other polypeptide factors including LIF and kit ligand (KL). As used here, the term cytokine includes proteins from natural sources or from recombinant cell culture, and biologically active equivalent of native sequence cytokines.
[000169] In an alternative embodiment, the antibodies of the present invention are fused, conjugated or operably linked to a toxin, including but not limited to small molecule toxins and enzymatically active toxins from bacteria, fungi, of animal or plant origin , including fragments and / or their variants. For example, a variety of immunotoxins and immunotoxin methods are described in Thrush et al., 1996, Ann. Rev. Immunol. 14: 49-71. Small molecule toxins include, but are not limited to, calicheamicin, maytansine (U.S. Patent No. 5,208,020), trichotene, and CC1065. In one embodiment of the invention, the antibody is conjugated to one or more maytansine molecules (for example, about 1 to about 10 maytansine molecules per antibody molecule). Maytansine can, for example, be converted to May-SS-Me which can be reduced to ay-SH3 and reacted with modified antibody (Chan et al., 1992, Cancer Research 52 127-131) to generate a maytansinoid antibody conjugate. Another conjugate of interest comprises an antibody conjugated to one or more calicheamycin molecules. The calicheamicin family of antibiotics is capable of producing double-stranded DNA that breaks down at sub-picomolar concentrations. Structural analogs of calicheamicin that can be used include, for example, disclosed in Hinman et al., 1 93, Cancer Research 53 3336-3342, Lode et al., 1998, Cancer Research 58 2925-2928, US 5,714,586; US 5,712,374, US 5,264,586; and US 5,773,001. 10 Dolastatin analogs such as auristatin E (AE) and monomethyluristatin E (MMAE) can find use as conjugates for the antibodies of the present invention (Doronina et al., 2003, Nat Biotechnol 21 (7): 778-84; Francisco e others, 2003 Blood 102 (4): 1458-65). Useful enzymatically active toxins include but are not limited to diphtheria A chain, diphtheria toxin non-binding fragments, exotoxin A chain (from Pseudomonas aeruginosa), A ricin chain, abrin A chain, modecin A chain, alpha -sarcina, Aleurites fordii proteins, diantin proteins, American Phytolaca proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcine, crotine, saponaria officinalis inhibitor, gelonin, mitogelin, restrictocine, phenomycin, enomycin and trichothecenes. See, for example, PCT WO 93/21232. The present invention subsequently contemplates a conjugate between an antibody of the present invention and a compound with nucleolytic activity, for example, a DNA ribonuclease or endonuclease such as deoxyribonuclease (DNase).
[000170] In an alternative embodiment, an antibody of the present invention can be fused, conjugated, or operably linked to a radioisotope to form a radioconjugate. A variety of radioactive isotopes are available for the production of antibodies to radioconjugates. Examples include, but are not limited to, At211, 1131, 1125, Y90, Rel86, Relδδ, Sml53, BÍ212, P32, and radioactive isotopes of Lu.
[000171] In yet another embodiment, an antibody of the present invention can be conjugated to a "receptor" (such as strepavidin) for use in tumor targeting in which the antibody receptor conjugate is administered to the patient, followed by removing unbound conjugate from the circulation using a scrubbing agent and then administering a "binder" (e.g. avidin) that is conjugated to a cytotoxic agent (e.g. radionucleotide). In an alternative embodiment, the antibody is conjugated to operably linked to an enzyme in order to employ antibody dependent enzyme prodrug therapy (ADEPT). ADEPT can be used by conjugating or operably binding the antibody to a prodrug activating enzyme that converts a prodrug (for example, a peptide chemotherapeutic agent, see PCT application WO 81/01 145, incorporated here in its entirety by reference) to an anticancer drug. See, for example, PCT application WO 88/07378 or U.S. patent no. 4,975,278, each incorporated herein in its entirety by reference. The enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such a way as to contain it in its most active cytotoxic form. Enzymes that are useful in the method of this invention include but are not limited to alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic 5-fluorocytosine into the anticancer drug, 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), which are useful for converting peptide-containing prodrugs into free drugs; D- alanylcarboxypeptidases, useful for the conversion of prodrugs containing D-amino acid substituents, carbohydrate dividing enzymes such as beta-galactosidase and neuraminidase useful for the conversion of glycosylated prodrugs into free drugs, beta-lactamase useful for the conversion of drugs derivatized with alpha-lactams in free drugs, and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting derivatized drugs into their amine nitrogen with phenoxyacetyl or phenylacetyl groups, respectively, in free drugs. Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes", can be used to convert the prodrugs of the invention into free active drugs (see, for example, Massey, 1987, Nature 328. 457-458, incorporated here in his totality by reference). Antibody abzyme conjugates can be prepared to deliver abzyme to a population of tumor cells. A variety of additional conjugates are contemplated for the antibodies of the present invention. A variety of chemotherapeutic agents, antiangiogenic agents, tyrosine kinase inhibitors, and other therapeutic agents are described below, which can find use as antibody conjugates.
[000172] Also contemplated as conjugate and fusion pairs are Fc polypeptides. Thus, an antibody can be a multimeric Fc polypeptide, comprising two or more Fc regions. The advantage of such a molecule is that it provides multiple binding sites for Fc receptors with a single protein molecule. In one embodiment, Fc regions can be linked using a chemical engineering approach. For example, Fab's and Fc's can be linked by thioether bonds creating cysteine residues in the joints, generating molecules such as FabFc2. Fc regions can be linked using disulfide engineering and / or chemical crosslinking. In one embodiment, Fc regions can be linked genetically. In one embodiment, Fc regions in an antibody are genetically linked to generate tandem Fc regions linked as described in US patent application 2005-0249723, entitled "Fc polypeptides as new Fc ligand binding sites". Linked tandem Fc polypeptides can comprise two or more Fc regions, for example, one to three, two, etc., Fc regions. Can it be advantageous to explore numerous engineering constructs in order to obtain homo- or hetero-antibodies in tandem linked with the most favorable functional and structural properties. Linked tandem antibodies can be linked to linked tandem homo-antibodies, which is an antibody of one isotype is fused genetically to another antibody of the same isotype. It is anticipated that since there are multiple FcR, Clq, and / or FcRn binding sites on linked tandem Fc polypeptides, effector and / or pharmacokinetic functions can be increased. In an alternative embodiment, antibodies of different isotypes can be linked tandem, called linked tandem hetero antibodies. For example, because of the ability to target FcR and FcRI receptors, an antibody that binds both FcRs and FcRI can provide an enhancement. significant clinical.
[000173] Conjugate and fusion pairs can be linked to any region of an antibody of the present invention, including at the N- or C-terminals, or at some residue in between the terminals. In one embodiment, a conjugate or fusion pair is attached at the N- or C-terminal of the antibody, for example, the N-terminal. A variety of linkers can find use in the present invention to covalently attach antibodies to a fusion or conjugate pair. By "ligand", "ligand sequence", "spacer", "stringed sequence" or its grammatical equivalents, here is to say that a molecule or group of molecules (such as monomer or polymer) that connects two molecules and often serves to put the two molecules in a desirable configuration. Ligands are known in the art, for example, homo- or hetero-bifunctional ligands as they are well known (see, 1994 Pierce Chemical Company catalog, technical section on crosslinker, pages 155-200). Numerous strategies can be used to covalently link molecules together. These include, but are not limited to, polypeptide bonds between N- and C-terminal proteins or protein domains, binding via disulfide bonds, and binding via chemical cross-linking reagents. In one aspect of this embodiment, the linker is a peptide bond, generated by matching techniques or peptide synthesis. The linker may contain amino acid residues that provide flexibility. Thus, the ligand peptide can pre-comminently include the following amino acid residues Gly, Ser, Ala, or Thr. The bound peptide will have a length that is suitable for binding two molecules in such a way that they assume the correct conformation with respect to each other so that they retain the desired activity. Suitable lengths for this purpose include at least one and no more than 50 amino acid residues. In one embodiment, the linker is about 1 to 30 amino acids in length, with linkers from 1 to 20 amino acids in length being desirable. Useful linkers include glycine-serine polymers (including, for example, (GS) n, (GSGGS) n (GGGGS) n (GGGS) n, where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible binders, as will be appreciated by those in the art. Alternatively, a variety of non-proteinaceous polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or polyethylene glycol and polypropylene glycol copolymers, can find use as binders, which may find use to bind antibodies of the present invention in addition to conjugate or fusion, or to bind the antibodies of the present invention to a conjugate.
[000174] The present invention provides methods for producing and testing experimentally antibodies. The methods described are intended to restrict the present invention to any application or theory of operation. Certainly, the methods provided are intended to illustrate in general that one or more antibodies can be experimentally tested to obtain variant antibodies. General methods for antibody molecular biology, expression, purification and screening are described in antibody engineering, edited by Duebel & Kontermann, Springer-Verlag, Heidelberg, 2001, and Hayhurst & Georgi- or, 2001, Curr Opin Chem Biol 5 683-689; Maynard & Georgiou, 2000, Annu Rev Biomed Eng 2 339-76, antibodies. A Laboratory Manual by Harlow & Lane, New York: Cold Spring Harbor Laboratory Press, 1988.
[000175] In one embodiment of the present invention, nucleic acids are created that encode the antibodies, and which can then be cloned into host cells, expressed and analyzed, if desired. Thus, nucleic acids, and particularly DNA, can be made by encoding each protein sequence. These doctors are performed using well-known procedures. For example, a variety of methods that can find use in the present invention are described in Molecular Cloning - A Laboratory Manual, 3rd Ed. (Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001), and Current Protocols in Molecular Biology (John Wiley & Sons). As will be appreciated by those skilled in the art, the generation of exact sequences for a library comprising a large number of sequences is potentially expensive and time consuming. By "library" here is meant a set of variants in any form, including but not limited to a list of nucleic acid or amino acid sequences, a list of nucleic acid or amino acid substitutions in variable positions, a physical library comprising nucleic acids encoding library sequences, or a physical library comprising variant proteins, either in purified or unpurified form. Correspondingly, there are a variety of techniques that can be used to effectively generate libraries of the present invention. Such methods that can find use in the present invention are described or referenced in U.S. patent no. 6,403,312; US patent application 2002-0048772, U.S. patent no. 7,315,786; US patent application 2003-030827, PCT application WO 01/40091 or PCT application WO 02/25588. Such methods include but are not limited to gene pool methods, PCR-based methods and methods using PCR variations, ligase chain reaction-based methods, combined oligo methods such as those used in synthetic shuffling, methods error-prone amplification methods and methods using random mutations, classic site-directed mutagenesis methods, packet muatagenesis and other methods of gene synthesis and magnification. As is known in the art, a variety of commercially available kits and methods are available for gene pool, mutagenesis, vector subcloning, and the like, and such commercial products find use in the present invention for the generation of nucleic acids that encode antibodies.
[000176] The antibodies of the present invention can be produced by culturing a host cell transformed with nucleic acid, for example, an expression vector, containing nucleic acid encoding the antibodies, under the appropriate conditions to induce or cause expression of the protein. The appropriate conditions for expression will vary with the choice of the expression vector and the host cell, and will vary easily determined by one skilled in the art through routine experimentation. A wide variety of suitable host cells can be used, including but not limited to mammalian cells, bacteria, insect cells, and yeast. For example, a variety of cell lines that can find use in the present invention are described in the ATCC cell line catalog, available from the American Type Culture Collection.
[000177] In one embodiment, antibodies are expressed in mammalian expression systems, including systems in which expression constructs are introduced into mammalian cells using viruses such as retrovirus or adenovirus. Any mammalian cells can be used, for example, human, mouse, rat, hamster, primate cells, etc. Suitable cells also include known cell screening, including but not limited to Jurkat, NIH3T3, CHO, BHK cells , COS, HEK293, PER C.6, HeLa, Sp2 / 0, NSO cells and their variants. In an alternative embodiment, library proteins are expressed in bacterial cells. Bacterial expression systems are well known in the art, and include Escherichia coli (E. coli). Bacillus subtisels, Streptococcus cremoris, and Streptococcus lividans. In alternative embodiments, antibodies are produced in insect cells (for example Sf21 / Sf) or yeast cells (for example S. cerevisiae, Pichia, etc.). In an alternative embodiment, antibodies are expressed in vitro using cell-free translation systems. In vitro translation systems derived from both prokaryotic (eg E. coli) and eukaryotic cells (eg, wheat germ, rabbit reticulocytes) are available and can be chosen based on the expression levels and functional properties of the protein of interest. For example, as appreciated by those skilled in the art, in vitro translation is required by some display technologies, for example, ribosome display. In addition, antibodies can be produced by chemical synthesis methods. Also transgenic expression systems, both animal (for example cow, sheep or goat milk, embryonated chicken eggs, whole insect larvae, etc.) and plant (for example, corn, tobacco, duckweed, etc.). The nucleic acids encoding the antibodies of the present invention can be incorporated into an expression vector in order to express protein. A variety of expression vectors can be used for protein expression. Expression vectors can comprise self-replicating extrachromosomal vectors or vectors that integrate into a host genome. Expression vectors are constructed to be compatible with the type of host cell. Thus expression vectors that find use in the present invention include but are not limited to those that allow expression of protein in mammalian cells, bacteria, insect cells, yeast, and in in vitro systems. As is known in the art, a variety of vectors of expression are available, commercially or otherwise, that can find use in the present invention for the expression of antibodies.
[000178] Expression vectors typically comprise an operably linked protein with regulatory or control sequences, selectable markers, any fusion pairs, and / or additional elements. By "operably linked" here is meant that the nucleic acid is placed in a functional relationship with another sequence of nucleic acids. In general, these expression vectors include translational and transcriptional regulatory nucleic acid operably linked to the nucleic acid encoding the antibody, and are typically appropriate to the host cell used to express the protein. In general, transcriptive or translational regulatory sequences can include promoter sequences, ribosomal binding sites, translational stop and start sequences, and enhancer or activator sequences. As is also known in the art, expression vectors typically contain a marker or selection gene to allow selection of transformed host cells containing the expression vector. Selection genes are well known in the art and will vary with the host cell used.
[000179] Antibodies can be operably linked to a fusion pair to allow targeting of the expressed protein, purification, screening, display, and the like. Fusion pairs can be linked to the antibody sequence via a ligand sequence. The linker sequence will generally comprise a small number of amino acids, typically less than ten, although longer linkers may also be used. Typically, ligand sequences are selected to be flexible and resistant to degradation. As will be appreciated by those skilled in the art, any of a wide variety of sequences can be used as linkers. For example, a linker sequence common to the GGGGS amino acid sequence. A fusion pair can be a signal or targeting sequence that directs the antibody and any fusion pairs associated with a desired cell location or extracellular medium. As is known in the art, certain signal sequences can direct a protein to be either secreted into the growth medium or into the periplasmic space, located between the cell's outer and inner membrane. A fusion pair can also be a sequence that encodes a peptide or protein that allows purification and / or screening. Such fusion pairs include but are not limited to polyhistidine tags (His tags) (for example H6 and H10 or other tags for use with immobilized metal affinity chromatography (IMAC) systems (for example, Ni2 + affinity columns)), GST fusions, MBP fusions, Strep tag, BSP biotinylation target sequence of BirA bacterial enzymes, and epitope tags that are targeted by antibodies (e.g., c-myc tags, flag labels and the like). As will be appreciated by those skilled in the art, such labels can be useful for purification, for sorting, or both. For example, an antibody can be purified using a His tag by immobilizing it to a Ni + 2 affinity column, and then after purification of the same His tag it can be used to immobilize the antibody to a Ni coated plate. +2 to perform an ELISA or other binding assay (as described below). A fusion pair may allow the use of a selection method to screen antibodies (see below). Fusion pairs that allow for a variety of selection methods are well known in the art, and all of these find use in the present invention. For example, by fusing members of an antibody library for gene III protein, phage display can be employed (Kay et al., Peptide and protein phage display: a laboratory manual, Academic Press, San Diego, CA, 1996; Lowman et al., 1991, Biochemistry 30: 10832-10838; Smith, 1985, Science 228: 1315-1317). Fusion pairs can allow antibodies to be labeled. Alternatively, a fusion pair can be linked to a specific sequence in the expression vector, allowing the associated fusion pair and antibody to be covalently or non-covalently linked to the nucleic acid encoding them.
[000180] Methods of introducing exogenous nucleic acid into host cells are well known in the art, and will vary with the host cell used. Techniques include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, calcium chloride treatment, polybrene mediated transfection, protoplast fusion, electroporation, phage or viral infection, encapsulation of the polynucleotide (s) ( s) in liposomes, and direct microinjection of DNA into nuclei. In the case of mammalian cells, transfection can be either transient or stable.
[000181] In one embodiment, antibodies are purified or isolated after expression. Proteins can be isolated and purified in a variety of ways known to those skilled in the art. Standard purification methods include chromatographic techniques, including ion exchange, hydrophobic interaction, affinity, size classification and gel filtration, and reverse phase, performed at atmospheric pressure or at high pressure using systems such as FPLC and HPLC . Purification methods also include electrophoretic, precipitation, immunological, dialysis and chromatofocal techniques. Ultrafiltration and diafiltration techniques, in combination with protein concentration, are also useful. As is well known in the art, a variety of natural proteins bind Fc and antibodies, and these proteins can find use in the present invention for the purification of antibodies. For example, bacterial proteins A and G bind to the Fc region. Likewise, bacterial L protein binds to the Fab region of some antibodies, with naturally making the target antigen of the antibody. Purification can often be permitted by a particular fusion pair. For example, antibodies can be purified using glutathione resin if a GST fusion is employed, Ni + affinity chromatography if a Hi tag is employed, or immobilized by anti-flag antibody if a flag tag is used. For general guidance on appropriate purification techniques, see, for example, Protein Purification: Principles and Practice, 3rd Ed., Scopes, Springer-Verlag, NY, 1994. The degree of purification required will vary depending on the screening or use of antibodies. In some cases purification is necessary. For example, in one embodiment, if antibodies are secreted, screening can occur directly from the medium. As is well known in the art, some selection methods do not involve protein purification. Thus, for example, if an antibody library is made in the phage display library, protein purification cannot be performed.
[000182] Antibodies can be screened using a variety of methods, including, but not limited to those using in vitro assays, cell-based and in vivo assays, and selection technologies. High-capacity screening and automation technologies can be used in screening procedures. Screening may employ the use of a fusion or label pair. The use of fusion pairs was discussed above. By "marked" here is meant that the antibodies of the invention have one or more elements, isotopes, or chemical compounds linked to allow detection in screening. In general, labels fall into three classes: a) immune labels, which can be an epitope incorporated as a fusion pair that is recognized by an antibody, b) isotopic labels, which can be heavy or radioactive isotopes, and c) small molecule labels, which can include fluorescent and colorimetric dyes, or molecules such as biotin that allow other labeling methods. Labels can be incorporated into the compound at any position and can be incorporated in vitro or in vivo during protein expression.
[000183] In one embodiment, the functional and / or biophysical properties of antibodies are screened in an in vitro assay. In vitro assays can allow a wide dynamic range for the screening properties of interest. Antibody properties that can be screened include but are not limited to stability, solubility, and affinity for Fc ligands, for example FcRs. Multiple properties can be screened simultaneously or individually. Proteins can be purified and not purified, depending on the requirements of the assay. In one embodiment, screening is a qualitative or quantitative binding assay for binding antibodies to a protein or non-protein molecule that is known or thought to bind to the antibody. In one embodiment, screening is a binding assay for measurement that binds to the target antigen. In an alternative embodiment, the screening is an assay for the binding of antibodies to an Fc ligand, including, but not limited to the FcRs family, the neonatal FcRn receptor, the complement protein Clq, and the bacterial proteins A and G. Fc ligands can be from any organism, for example, humans, mice, rats, rabbits, monkeys, etc. Binding assays can be performed using a variety of methods known in the art, including, but not limited to FRET (fluorescence resonance energy transfer) and BRET-based tests (bioluminescence resonance energy transfer, AlfaScreen (R) (extended luminescent homogeneous proximity tests), flicker proximity test, ELISA (linked immunosorbent tests the enzyme), SPR (Surface plasmon resonance, also known as Biacore (R)), isothermal titration calorimetry, differential scanning calorimetry, gel electrophoresis and chromatography including gel filtration. These and other methods may take advantage of some fusion pair or antibody label Assays can employ a variety of detection methods including but not limited to chromogenic labels, fluorescence loved, luminescent or isotopic.
[000184] The biophysical properties of antibodies, for example stability and solubility, can be screened using a variety of methods known in the art. Protein stability can be determined by measuring the thermodynamic balance between folded and unfolded states. For example, antibodies of the present invention can be folded using chemical denaturation, heat, or pH, and this transition can be monitored using methods including, but not limited to, circular dichroism spectroscopy, fluorescence spectroscopy, fluorescence spectroscopy absorbance, NMR spectroscopy, calorimetry, and proteolysis. As will be appreciated by those skilled in the art, the kinetic parameters of folded and unfolded transitions can also be monitored using these and other techniques. The total solubility and integrity of an antibody can be quantitatively or qualitatively determined using a wide range of methods that are known in the art. Methods that may find use in the present invention for the characterization of the biophysical properties of antibodies include gel electrophoresis, isoelectric focusing, capillary electrophoresis, chromatography such as size exclusion chromatography, ion exchange chromatography, and liquid chromatography. high reverse phase performance, peptide mapping, oligosaccharide mapping, mass spectrometry, ultraviolet absorbance spectroscopy, fluorescence spectroscopy, circular dichroism spectroscopy, isothermal titration calorimetry, differential scanning calorimetry, analytical dispersion ultracentrifugation, dynamic light, proteolysis and crosslinking, turbidity measurement, filtration delay assay, immunological assays, fluorescent dye binding assays, protein staining assays, microscopy, and aggregation deletion via ELISA or other binding assay. Structural analysis using X-ray crystallographic techniques and NMR spectroscopy may also find use. In one embodiment, stability and / or solubility can be measured by determining the amount of protein solution after a period of time. In that assay, the protein may or may not be exposed to some extreme condition, for example, high temperature, low pH, or the presence of denaturant. Since function typically requires a stable, soluble and / or well-folded / structured protein, the binding and functional assays as mentioned above also provide a means of making such a measurement. For example, a solution comprising an antibody could be analyzed for its ability to bind target antigen, then exposed to elevated temperature for one or more defined periods of time, then analyzed for antigen binding again. Since unfolded or aggregated proteins are not expected to be able to bind antigen, the amount of activity remaining provides a measure of antibody stability and solubility.
[000185] The biological properties of the antibodies of the present invention can be characterized in cell, tissue and whole organism experiments. As is known in the art, drugs are hotly tested on animals, including but not limited to mice, rats, rabbits, dogs, cats, pigs, and monkeys, to measure drug efficacy for the treatment against a disease or model of disease, or to measure pharmacokinetics of the drug, toxicity, and other properties. Animals can be called a disease model. With respect to the antibodies of the present invention, a particular challenge arises when using animal models to assess the potential for human efficacy of candidate polypeptides - this is due, at least in part, to the fact that antibodies that have a specific effect on the affinity for a human Fc receptor cannot have a similar affinity effect with the orthologous animal receptor. These problems can be further exacerbated by the inevitable ambiguities associated with the correct designation of true orthologists (Mechetina et al., Immunogenetics, 2002 54: 463-468), and the fact that some orthologists simply do not exhibit in animals (for example, human beings have an FcRIla whereas mice do not). Therapeutics are often tested in mice, including but not limited to nude mice, SCID mice, xenograft mice, and transgenic mice (including knockins and inactivations). For example, an antibody of the present invention that is intended as an anticancer therapy can be tested in a mouse cancer model. In this method, a tumor or tumor cell line is grafted onto or injected into a mouse, and subsequently the mouse is treated with therapy to determine the antibody's ability to reduce or inhibit cancer metastasis and growth. An alternative approach is the use of a SCID mouse model in which immune deficient mice are injected with human peripheral blood lymphocytes (PBLs), giving a semi-functional system and a human immune system - with an appropriate assay of human FcRs - to mice that were subsequently injected with antibodies or Fc polypeptides that target injected human tumor cells. In such a model, the Fc polypeptides that target the desired antigen interact with human PBLs within the mice to bind tumoricidal effector functions. Such experimentation can provide significant data for determining the potential of the antibody to be used as a therapy. Any organism, for example, mammals, can be used for testing. For example, because of their genetic similarity to humans, monkeys may be suitable for therapeutic models, and thus can be used to test the effectiveness, toxicity, pharmacokinetics, or other properties of the antibodies of the present invention. Tests of the antibodies of the present invention in humans are routinely required for approval as drugs, and so naturally these experiments are contemplated. Thus, the antibodies of the present invention can be tested in humans to determine their therapeutic efficacy, toxicity, pharmacokinetics, and / or other clinical properties.
[000186] Toxicity studies are performed to determine the antibody or Fc-related effects that cannot be evaluated in the standard pharmacology profile or occur only after repeated administration of the agent. More toxicity tests are performed on two species - a rodent and a non-rodent - to ensure that any unexpected adverse effects are not overlooked before new therapeutic entities are introduced into man. In general, those models can measure a variety of toxicities including genotoxicity, chronic toxicity, immunogenicity, reproductive / developmental toxicity, and carcinogenicity. Included within the parameters mentioned above are standard measurement of food consumption, body weight, antibody formation, clinical chemistry, and macro- and microscopic examination of standard organs / tissues (eg, cardiotoxicity). Additional measurement parameters are injection site trauma and the measurement of neutralizing antibodies, if applicable. Traditionally, monoclonal antibody therapies, naked or conjugated, are evaluated for cross-reactivity with normal tissues, immunogenicity / antibody production, ligand or conjugate ligand toxicity and toxicity of the radiolabeled "spectator" species. However, such studies may need to be individualized to take into account specific concerns and the following set of guidance by ICH S6 (safety studies for biotechnological products also noted above). As such, the general principles are that the products are sufficiently well characterized and for which impurities / contaminants have been removed, that the test material is comparable through all development, and LPG tendency.
[000187] The pharmacokinetics (PK) of the antibodies of the invention can be studied in a variety of animal systems, with the most relevant being non-human primates such as cynomolgous and rhesus monkeys. Single or repeated administrations i.v./s.c. over a 6000-fold dose range (0.05-300 mg / kg) can be assessed for half-life (days to weeks) using plasma concentration and clearance as well as volume of distribution at a constant state and level of systemic absorbance can be measured. Examples of such measurement parameters in general include maximum observed plasma concentration (Cmax), time to reach Cmax (Tmax), area under plasma concentration-time curve 0 to infinity [AUCo-inf] and half-life of evident elimination (Tin). Added measured parameters could include compartmental analysis of concentration-time data obtained following i.v. administration and bioavailability. Examples of pharmacological / toxicological studies using cynomolgus have been established for Rituxan (R) and Zevalm (R) in which monoclonal antibodies to CD20 are cross-reactive. Biodistribution, dosimetry (for radiolabeled antibodies), and PK studies can also be done on rodent models. Such studies would assess tolerance in all doses administered, toxicity to local tissues, localization to animal models of rodent xenograft, depletion of target cells. The antibodies of the present invention can provide superior pharmacokinetics in animal systems or in humans. For example, increased binding to FcRn can increase the half-life and exposure of the therapeutic antibody. Alternatively, decreased binding to FcRn can decrease half-life and exposure to Fc-containing drug in cases where reduced exposure is favorable such as when such drug has side effects. It is known in the art that the set of Fc receptors is differentially expressed in various types of immune cells, as well as in different tissues. Differential tissue distribution of Fc receptors may ultimately have an impact on the pharmacokinetic (PD) and pharmacokinetic (PK) properties of antibodies of the present invention. Since presentation antibodies have varying affinities for the Fc receptor set, further screening of the polypeptides for PD and / or PK properties can be extremely useful in defining the optimal balance of PD, PK, and therapeutic efficacy conferred by each candidate polypeptide.
[000188] Pharmacodynamic studies may include, but are not limited to, targeting specific tumor cells or blocking signaling mechanisms, measuring target antigen depletion that expresses cells or signals, etc. Such pharmacodynamic effects can be demonstrated in animal or human models.
[000189] The antibodies of the present invention can be used for therapeutic purposes. As will be appreciated by those in the art, the antibodies of the present invention can be used for any therapeutic purpose using antibodies and the like. In one embodiment, antibodies are administered to a patient to treat disorders including, but not limited to, cancer.
[000190] A "patient" for the purposes of the present invention includes both humans and other animals, for example, mammals, for example, humans. Thus the antibodies of the present invention have both human therapy and veterinary applications. The term "treatment" or "treating" in the present invention is intended to include therapeutic treatment as well as suppressive or prophylactic measurements for a disease or disorder, so, for example, successful administration of an antibody before the onset of disease results in treatment of the disease.As another example, successful administration of an optimized antibody after the clinical manifestation of the disease to combat the symptoms of the disease comprises treatment of the disease. "Treatment" and "treating" also includes administration of an optimized antibody after appearance of the disease in order to eradicate the disease. Successful administration of an agent after the onset and after the clinical symptoms develop, c Possible abatement of clinical symptoms and perhaps improvement of the disease, comprises treatment of the disease. Those "in need of treatment" include mammals already having the disease or disorder, as well as those prone to disease or disorder, including those in which the disease or disorder must be avoided.
[000191] In one embodiment, an antibody of the present invention is administered to a patient having a disease that involves inappropriate expression of a protein or other molecule, such as FLT3. Within the scope of the present invention this is intended to include diseases and disorders characterized by anomalous proteins, due, for example, to changes in the amount of a protein present, protein location, post-translational modification, conformational state, the presence of a mutant protein, etc. An ultra-abundance may be due to any cause, including, but not limited to, ultra-expression at the molecular level, prolonged or accumulated appearance at the site of action, or increased activity of a protein in relation to normal. Included within this definition are diseases and disorders characterized by a reduction in a protein. This reduction may be due to any cause, including, but not limited to reduced expression at the molecular level, reduced or decreased appearance at the site of action, mutant forms of a protein, or decreased activity of a protein compared to normal. Such ultra-abundance or reduction of a protein can be measured in relation to the normal expression, appearance or activity of a protein, and measurement can play an important role in testing the development and / or clinic of the antibodies of the present invention.
[000192] By "cancer" and "cancerous" here refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to carcinoma, lymphoma, blastoma, sarcoma (including liposarcoma), neuroendocrine tumors, mesothelioma, schwannoma, meningioma, adenocarcinoma, melanoma, and lymphoid leukemia or malignancies.
[000193] More particular examples of such cancers include hematological malignancies, such as non-Hodgkin's lymphomas (NHL), B-cell acute lymphoblastic leukemia / lymphoma (B-ALL), and acute T-cell lymphoblastic leukemia / lymphoma ( T-ALL), thymoma, Langerhans cell histocytosis, multiple myeloma (MM), myeloid neoplasms such as acute myelogenous leukemias (AML), including matured AML, undifferentiated AML, acute promyelocytic leukemia, acute myelomonocytic leukemia, and acute monocytic leukemia, myelodysplasia syndromes, and chronic myeloproliferative disorders (MDS), including chronic myelogenous leukemia (CML).
[000194] If the cancer or tumor is a lymphoma or leukemia, the disease may be in the minimal residual disease (MRD) stage. This stage can, for example, be read after conventional chemotherapy with or without stem cell transplantation. In this context, "MRD" refers to a disease state where small numbers of lymphoma / leukemic cells remain in the patient during treatment or after treatment when the patient is in remission, including complete remission (no symptoms or signs disease). This is the main cause of relapse in cancer and leukemia. At this stage, although the patient may be in complete remission, the disease is still detectable by the state of the art, such as polymerase chain reaction (PCR) and flow cytometry (FACS).
[000195] The target of the antibodies of the present invention can be polymorphic in the human population. For a given patient or patient population, the effectiveness of the antibodies of the present invention can thus be affected by the presence or absence of specific polymorphisms in the proteins. For example, FcRIlIA is polymorphic at position 158, which is commonly either V (high affinity) or F (low affinity). Patients with the homozygous V / V genotype are seen to have a better clinical response to treatment with the anti-CD20 antibody Rituxan (R) (rituximab), probably because these patients mount a stronger N response (Dall'Ozzo and others ( 2004) Cancer Res. 64-4664-9). Additional polymorphisms include but are not limited to FcRIlA R131 or H131, and such polymorphisms are known to either increase or decrease Fc binding and subsequent biological activity, depending on the polymorphism. Antibodies of the present invention can bind to a particular polymorphic form of a receptor, for example, FcyRIIIA 158 V, or to bind with equivalent affinity for all polymorphisms to a particular position on the receptor, for example both 158V and 158F polymorphisms of FcRIlIA. In one embodiment, antibodies of the present invention can have equivalents that bind to polymorphisms that can be used in an antibody to eliminate the differential efficacy seen in patients as different polymorphisms. Such a property can provide greater consistency in the therapeutic response and reduce populations of patients who do not respond. Such an Fc variant with identical binding to receptor polymorphisms may have increased biological activity, such as ADCC, CDC or circulating half-life, or alternatively decreased activity, via modulation of the binding of the relevant Fc receptors. In one embodiment, antibodies of the present invention can bind with a higher or lower affinity for one of the polymorphisms of a receptor, or accentuate the difference in binding or reverse the difference. Such a property can allow the creation of therapies particularly tailored for effectiveness with a patient population having such a polymorphism. For example, a patient population having a polymorphism with a higher affinity for an inhibitory receptor such as FcRIlb could receive a drug containing an antibody with such reduced polymorphic binding to the receptor, creating a more effective drug.
[000196] In one embodiment, patients are screened for one or more polymorphisms in order to predict the effectiveness of the antibodies of the present invention. This information can be used, for example, to select patients to include or exclude from clinical experiences or, post-approval, to provide guidance to doctors and patients regarding appropriate dosages and treatment options. In one embodiment, patients are selected for inclusion in clinical experiments for an antibody of the present invention if their genotype indicates that they are likely to respond significantly better to an antibody of the present invention when compared to one or more antibody therapies currently used. In another embodiment, appropriate dosages and treatment regimens are determined using such genotype information. In another embodiment, patients are selected for inclusion in a clinical experiment or for a post-approval prescription for therapy based on their polymorphism genotype, where such therapy contains a engineered antibody to be specifically effective for such a population, or alternatively where such therapy contains an antibody that does not show differential activity to the different forms of polymorphism.
[000197] Included in the present invention are diagnostic tests to identify patients who are likely to show a favorable clinical response to an antibody of the present invention, or who are likely to show a significantly better response when treated with an antibody of the present invention versus an or more currently used antibody therapies. Any of numerous methods for the determination of FcR polymorphisms in humans known in the art can be used.
[000198] Furthermore, the present invention comprises prognostication tests performed on clinical samples such as tissue and blood samples. Such tests can assess for effector function activity, including, but not limited to ADCC, CDC, phagocytosis, and opsonization, or to kill, regardless of mechanism, cancer cells or otherwise pathogenic. In one embodiment, ADCC assays, such as those described above, are used to predict, for a specific patient, the effectiveness of a given antibody of the present invention. Such information can be used to identify patients for inclusion or exclusion in clinical experiences, or to inform decisions regarding appropriate dosages and treatment regimens. Such information can also be used to select a drug that contains a particular antibody that shows superior activity in such an assay.
[000199] Pharmaceutical compositions are contemplated in which an antibody of the present invention and one or more therapeutically active agents are formulated. Antibody formulations of the present invention are prepared for storage by mixing the antibody having the desired degree of purity with optional pharmaceutically acceptable vehicles, excipients or statilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980), in the form lyophilized formulations or aqueous solutions. Acceptable vehicles, excipients or statilizers are non-toxic to receptors at the dosages and concentrations employed and include buffers such as phosphate, citrate, acetate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyl-dimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl orbenzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m -cresol); low molecular weight polypeptides (less than about 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinyl-pyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; sweeteners and other flavoring agents; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; additions; coloring agents; salt-forming counterions such as sodium; metal complexes (for example, Zn protein complexes); and / or non-ionic surfactants such as TWEEN (R), PLURONICS (R) or polyethylene glycol (PEG). In one embodiment, the pharmaceutical composition comprising the antibody of the present invention can be in a water-soluble form, such as being present as pharmaceutically acceptable salts, which are intended to include both acid and base addition salts. "Pharmaceutically acceptable acid addition salt" refers to those salts that retain the biological effectiveness of free bases and are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, acid nitric, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. "Pharmaceutically acceptable base addition salts" include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly useful are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include primary, secondary and tertiary amine salts, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. The formulations to be used for in vivo administration would be sterile. This is readily accomplished by filtration through sterile filtration membranes or other methods.
[000200] The antibodies disclosed here can also be formulated as immunoliposomes. A liposome is a small vesicle comprising several types of lipids, phospholipids and / or surfactant that is useful for providing a therapeutic agent to a mammal. Liposomes containing the antibody are prepared by methods known in the art, as described in Epstein et al., 1985, Proc Natl Acad Sei USA, 82: 3688; Hwang et al., 1980, Proc Natl Acad Sei USA, 77: 4030; U.S. patent no. 4,485,045; U.S. patent no. 4,544,545; and PCT application WO 97/38731. Liposomes with increased circulation time are disclosed in U.S. patent no. 5,013,556. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidyl tanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to provide liposomes with the desired diameter. A chemotherapeutic agent or other therapeutically active agent is optionally within the liposome (Gabizon et al., 1989, J National Cancer Inst 81: 1484).
[000201] The antibody and other therapeutically active agents can also be trapped in microcapsules prepared by methods including, but not limited to, coacervation techniques, interfacial polymerization (for example using hydroxymethylcellulose or gelatin microcapsules, or microcapsules of poly- (methylmethacrylate), colloidal drug delivery systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), and macroemulsions These techniques are disclosed in Re-mington's Pharmaceutical Sciences 16th edition, Osol , A. Ed., 1980. Sustained-release preparations can be prepared Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymer, matrices that are in the form of shaped articles, for example, films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (eg, poly (2-hydroxyethyl methacrylate), or poly (vinyl alcohol)), polylactides (U.S. patent no. 3,773,919), copolymers of L-glutamic acid and gamma ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic-glycolic acid copolymers such as Lupron Depot (R) (which are injectable microspheres made up of copolymer lactic acid-glycolic acid and leuprolide acetate), poly-D - (-) - 3-hydroxybutyric acid, and ProLease (R) (commercially available from Alkermes), which is a microsphere-based delivery system made up of the molecule desired bioactive substance incorporated in a poly-DL-lactide-coglycolide (PLG) matrix.
[000202] Administration of the pharmaceutical composition comprising an antibody of the present invention, for example, in the form of a sterile aqueous solution, can be done in a variety of ways, including, but not limited to orally, subcutaneously, intravenously, intranasally, intraotically , transdermally, topically (for example, gels, ointments, lotions, creams, etc.), intraperitoneally, intramuscularly, intrapulmonary, vaginally, parenterally, rectally, or intraocularly. As is known in the art, the pharmaceutical composition can be formulated correspondingly depending on the mode of introduction.
[000203] As is known in the art, protein therapies are often provided by infusion or IV bolus. The antibodies of the present invention can also be supplied using such methods. For example, administration can be by intravenous infusion with 0.9% sodium chloride as an infusion vehicle.
[000204] In addition, any of numerous delivery systems are known in the art and can be used to administer the antibodies of the present invention. Examples include, but are not limited to, encapsulation in liposomes, microparticles, microspheres (e.g., PLA / PGA microspheres), and the like. Alternatively, an implant of a gelatinous material, porous or non-porous, including membranes or fibers, can be used. Sustained-release systems may comprise a polymeric matrix or material such as polyesters, hydrogels, poly (vinyl alcohol), polylactides, copolymers of L-glutamic acid and ethyl-L-glutamate, ethylene-vinyl acetate, lactic acid copolymers - glycolic acid such as Lupron Depot (R), and poly-D - (-) - 3-hydroxybutyric acid. It is also possible to administer a nucleic acid encoding the antibody of the present invention, for example, by retroviral infection, direct injection or coating with lipids, cell surface receptors or other transfection agents. In all cases, controlled release systems can be used to release the antibody at or near the desired location of action.
[000205] The quantities and frequencies of dosed administration are, in one embodiment, selected to be therapeutically or prophylactically effective. As is known in the art, protein degradation adjustments, systemic versus localized supply, and rate of new protease synthesis, as well as age, body weight, general health, sex, diet, time of administration, drug interaction and severity of the condition may be necessary, and will be determinable with routine experimentation by those skilled in the art.
[000206] The concentration of the therapeutically active antibody in the formulation can vary from about 0.1 to 100% by weight. In one embodiment, the concentration of the antibody is in the range of 0.003 pM 1.0 molar. In order to treat a patient, a therapeutically effective dose of the antibody of the present invention can be administered. By "therapeutically effective dose" here is meant the dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be determinable by the person skilled in the art using known techniques. Dosages can vary from 0.0001 to 100 mg / kg of body weight or more, for example from 0.1, 1, 10, or 50 mg / kg of body weight, for example, from 1 to 10 mg / kg body weight.
[000207] In some embodiments, only a single dose of the antibody is used. In other embodiments, multiple doses of the antibody are administered. The time between administrations can be less than 1 hour, about 1 hour, about 1-2 hours, about 2-3 hours, about 3-4 hours, about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 2-4 days, about 4-6 days, about 1 week, about 2 weeks, or more than 2 weeks.
[000208] In other embodiments, the antibodies of the present invention are administered in metronome dosing regimens, or by continuous infusion or frequent administration without prolonged rest periods. Such metronomic administration may involve administration dosed at constant intervals without rest periods. Typically such regimens include continuous infusion or chronic low dose over an extended period of time, for example, 1-2 days, 1-2 weeks, 1-2 months, or up to 6 months or more. The use of lower doses can minimize side effects and the need for rest periods.
[000209] In certain embodiments, the antibody of the present invention and one or more other prophylactic or therapeutic agents are cyclically administered to the patient. Cyclization therapy involves administering a first agent at once, a second agent at a second time, optionally additional agents at additional times, optionally a rest period, and then repeating that sequence of administration one or more times. The number of cycles is typically 2 - 10. Cycling therapy can reduce the development of resistance to one or more agents, can minimize side effects, or can improve treatment effectiveness.
[000210] The antibodies of the present invention can be administered concomitantly with one or more other regimens or therapeutic agents. Additional therapeutic agents or regimens can be used to improve the effectiveness or safety of the antibody. Also, additional therapeutic agents or regimens can be used to treat the same disease or a comorbidity for sure than to alter antibody activation. For example, an antibody of the present invention can be administered to the patient in conjunction with chemotherapy, radiation therapy or both chemotherapy and radiation. The antibody of the present invention can be administered in combination with one or more other prophylactic or therapeutic agents, including, but not limited to, cytotoxic agents, chemotherapeutic agents, cytokines, growth inhibitory agents, anti-hormonal agents, kinase inhibitors, anti-angiogenic agents, cardioprotectors, immunostimulating agents, immunosuppressive agents, agents that promote hematological cell proliferation, angiogenesis inhibitors, protein tyrosine kinase (PTK) inhibitors, additional antibodies, FcRIlb or other Fc receptor inhibitors, or other therapeutic agents.
[000211] The terms "in combination with" and "co-administration" are not limited to the administration of prophylactic or therapeutic agents at exactly the same time. Instead, it is meant that the antibody of the present invention and the other agent or agents are administered in a sequence and within such a time interval that they can act together to provide a benefit that is increased versus treatment with only either the antibody of the present invention or the other agent or agents. In one embodiment, that the antibody and the other agent or agents act additively, for example, they act synergistically. Such molecules are suitably present in combination in amounts that are effective for the intended purpose. The skilled clinician can determine empirically, or by consideration of the pharmacokinetics and modes of action of the agents, the appropriate dose or doses of each therapeutic agent, as well as the appropriate timings and methods of administration.
[000212] In one embodiment, the antibodies of the present invention are administered with one or more additional molecules comprising antibodies or Fc. The antibodies of the present invention can be co-administered with one or more other antibodies that are effective in treating the same disease or an additional comorbidity, for example, two antibodies can be administered that recognize two antigens that overlap in a given type of cancer.
[000213] In one embodiment, the antibodies of the present invention are administered with a chemotherapeutic agent. By "chemotherapeutic agent" as used here is meant a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan, androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone, anti-adrenals such as aminoglutetimide, mitotane, trilostane, anti-androgens such as flutamide, nutyl, nutamide, nutamide, ; antibiotics such as aclacinomycins, actinomycin, au- tramycin, azasene, bleomycins, cactinomycin, kallikeamicin, carbabicin, Caminomycin, carzinofilm, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-oxo-lorine-lorine-lorine-norxine-lorine , epirabraicin, esorubicin, idarubicin, marcelomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, chelamycin, rhodububicin, streptonigrin, etreptozocin, tubercidin, ubenimex, zinostatin, zinostatin, zinostatin, zinostatin, zinostatin, zinostatin, zinostatin, zinostatin, zinostatin, zinostatin, zinostatin, zinostatin, zinostatin, zinostatin, zinostatin, zinostatin, zinostatin, zinostatin, zinostatin, zinostatin, zinostatin. anti-estrogens including, for example, tamoxifen, raloxifene, aromatase inhibition of 4 (5) - imidazoles, 4-hydroxy tamoxifen, trioxifene, ceoxifene, LY 117018, onapristone, and toremifene (Fareston); anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as de-dopterin, methotrexate, pteropterin, trimetrexate; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; emylenimines and methylamylamines including altretamine, triethylenomelamine, triethylene phosphoramide, thethylenethiophosphoramide and trimethylolomelamine; replenishing folic acid such as frolinic acid; nitrogen mustards such as chlorambucil, chlornaphazine, colophosphamide, estramustine, ifosfamide, meclorethamine, mecloreamine oxide hydrochloride, melphalan, novembicin, phenesterine, prednimustine, trophosphamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, photemustine, lomustine, nimustine, ranimustine; platinum analogues such as cis-platinum and carboplatin; immature precursor cell; platinum; proteins such as arginine deiminase and asparaginase; purine analogs such as fludarabine, 6-mercaptopurine, tiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, didesoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; taxanes, for example paclitaxel (TAXOL (R), Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel (TAXOTERE (R), Rhne-Poulenc Rorer, Antony, France); topoisomerase inhibitor RFS 2000; thymidylate synthase inhibitor (such as Tomudex); additional chemotherapeutics including aceglatone; glycoside aldophosphamide; aminolevulinic acid; amsacrine; bestrabucila; bisanthrene; edatraxate; defo- famine; demecolcine; diaziquone; difluoromethylornithine (DMFO); elformitin; ellipinium acetate; etoglucid; gallium nitrate; hydroxyurea; slow; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; fenamet; pirarubicin; podophinylic acid; 2-ethylhydrazide; procarbazine; PSK (R); razoxane; sizofuran; spirogermanium; nuazonic acid; triaziquone; 2,2'2 "-trichlorotriethylamine; urethane; vindesine; dacarbazine; manomustine; mitobronitol; mitolactol; pipobroman; gacittosine; arabinoside (" Ara-C "); cyclophosphamide; thiotepa; chlorambucil; gencitabine; 6-thioguanine; 6-thioguanine; methotrexate; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1; retinoic acid; sperminins; capecite; , pharmaceutically acceptable acids or derivatives of any of the above can also be used.
[000214] A chemotherapeutic agent or other cytotoxic agent can be administered as a prodrug. By "prodrug" as used herein is meant that a precursor form or a derivative of a pharmaceutically active substance that is less cytotoxic to tumor cells in comparison to a parent drug is capable of being enzymatically activated or converted to the more original form. active. See, for example, Wilman, 1986, Biochemical Society Transactions, 615th Meeting Belfast, 14: 375-382; Stella et al., "Profármacos: A Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery; and Borchardt et al., (ed.): 247-267, Human Being Press, 1985. Prodrugs that may find use with the present invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulphate-containing prodrugs containing peptide, D-amino acid modified drugs, glycosylated drugs, beta-lactam-containing drugs, optionally substituted phenylacetamide drugs or drugs containing optionally substituted phenylacetamide, 5-fluorocytosine drugs and other 5-fluorotoxic drugs that can be converted into a free fluoridine. more active. Examples of cytotoxic drugs that can be derived in a prodrug form for use with the antibodies of the present invention include, but are not limited to, any of the aforementioned chemotherapeutic agents.
[000215] In another embodiment, the antibody is administered with one or more immunomodulatory agents. Such agents can increase or decrease the production of one or more cytokines, show up or down regulated autoantigen, mask MHC antigens, or promote the state of proliferation, differentiation, migration or activation of one or more types of immune cells. . Immunomodulatory agents include, but are not limited to, non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen, celecoxib, diclofenac, etodolac, fenprofen, indomethacin, ketoralac, oxaprozine, nabumentone, sulindac, tolmentine, rofecox, tolofoxin, rofec, tolofoxin, rofec, tolofoxin, rofec, tolofoxin, and nabumetone, steroids (for example glucocorticoids, dexamethasone, cortisone, hydroxycortisone, methylprednisolone, prednisone, prednisolone, trimcinolone, azulfidinaicosanoids such as progestins, thromboxanes, and leukotrienes, as well as topical steroids, such as anthol, and anthol, such as tazarotene), cytokines such as TGFb, IFNalpha, IFNbeta, IFNgama, IL-2, IL-4, IL-10, cytokine, chemokine, or receptor receptor antagonists including antibodies, soluble receptors, and receptor Fc fusions against BAFF, B7, CCR2, CCR5, CD2, CD3, CD4, CD6, CD7, CD8, CD11, CD14, CD15, CD17, CD18, CD20, CD23, CD28, CD40, CD40L, CD44, CD45, CD402, CD64, CD80 , CD86, CD147, CD152 , complement factors (C5, D) CTLA4, eotaxin, Fas, ICAM, ICOS, IFNa, IFNp.TFNy, IFNAR, IgE, IL-1, IL-2, IL-2R, IL-4, IL-5R, IL -6, IL-8, IL-9 IL-12, IL-13, IL-13R1, IL-15, IL-18R, IL-23, integrins, LFA-1, LFA-3, MHC, selectins, TGFp, TNF, TNFp, TNF-R1, T cell receptor, including Enbrel (R) (etanercept), Humira (R) (adalimumab). and Remicade (R) (infliximab), heterologous anti-lymphocyte globulin; other immunomodulatory molecules such as 2-amino-6-aryl-5-substituted pyrimidines, anti-idiotypic antibodies to MHC-binding peptides and MHC fragments, azathioprine, brequinar, bromocriptine, cyclophosphamide, cyclosporin A, D-penicillamine, deoxispergualm, FK506, glutaraldehyde, gold, hydroxychloroquine, leflunomide, malononitrileamides (e.g. leflunomide), methotrexate, minocycline, mizoribine, mycophenolate mofetil, rapamycin, and sulphasasine.
[000216] In an alternative embodiment, antibodies of the present invention are administered with a cytokine. By "cytokine" as used here is meant a generic term for proteins released by a cell population that act in another cell as intercellular mediators. Examples of such cytokines are lymphokines, monocines, and traditional polypeptide hormone. Included among cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone, thyroxine; insulin; proinsulin, relaxin, prorelaxin, glycoprotein hormone such as antigen-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and lutenization hormone (LH), liver growth factor, fibroblast growth factor; prolactin, placental lactogen, tumor necrosis factor - alpha and - beta; inhibiting substance of mulerian, peptide associated with mouse gonadotropin, inhibin; activin, vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; platelet growth factor, transforming growth factors (TGFs) such as TGF-alpha and TGF-beta, insulin-like growth factor I and II; eitropoietin (EPO), osteoinductive factors, interferons such as interferon-alpha, - beta, -gama, colony-stimulating factors (CSFs) such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM- CSF), and granulocyte-CSF (G-CSF), interleukins (ILs) such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, to tumor necrosis factor such as TNF-alpha or TNF-beta, and other polypeptide factors including LIF and kit binder (KL). As used here, the term cytokine includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents in native sequence cytokines.
[000217] In one embodiment, cytokines or other agents that stimulate cells of the immune system are co-administered with the antibody of the present invention. Such a treatment mode can increase the desired effector function. For example, agents that stimulate N cells, including but not limited to IL-2, can be co-administered. In another embodiment, agents that stimulate macrophages, including but not limited to C5a, formyl peptides such as N-formyl-methionyl-leucyl-phenylalanine (Beigier-Bompadre et al. (2003) Scand J. Immunol. 57. 221-8 ), can be co-administered. Also, agents that stimulate neutrophils, including but not limited to G-CSF, GM-CSF, and the like can be administered. In addition, agents that promote migration of immunostimulatory cytokines can be used. Also additional agents including but not limited to gamma interferon, IL-3 and IL-7 can promote one or more effector functions.
[000218] In an alternative embodiment, cytokines or other agents that inhibit effector cell function are co-administered with the antibody of the present invention. Such treatment can limit unwanted effector function.
[000219] The antibodies of the present invention can be combined with other therapeutic regimens. For example, in one embodiment, the patient to be treated with an antibody of the present invention can also receive radiation therapy. Radiation therapy can be administered according to protocols commonly used in the art and known to the skilled technician. Such therapy includes, but is not limited to, cesium, iridium, iodine or cobalt radiation. Radiation therapy can be irradiation of the whole body, or it can be directed locally to a specific tissue or site in the body, such as the lung, bladder or prostate. Typically, radiation therapy is administered in pulses over a period of about 1 to 2 weeks. Radiation therapy can, however, be administered over longer periods of time. For example, radiation therapy can be administered to patients having head and neck cancer for about 6 to about 7 weeks. Optionally, radiation therapy can be administered as a single dose or as multiple doses, sequential doses. The skilled clinician can empirically determine the appropriate dose (s) of radiation therapy useful here. According to another embodiment of the invention, the antibody of the present invention and one or more other anticancer therapies are employed to treat cancer cells ex vivo. It is contemplated that such ex vivo treatment may be useful in bone marrow transplantation and particularly, autologous bone marrow transplantation. For example, treatment of cells or tissue (s) containing cancer cells with antibody and one or more other anticancer therapies, as described above, can be employed to deplete or substantially deplete cancer cells prior to transplantation in a recipient patient. .
[000220] It is naturally contemplated that the antibodies of the invention can be used in combination with yet other therapeutic techniques such as surgery or phototherapy.
[000221] The present invention is further illustrated by the following examples. However, it would be understood, that the invention is not limited to the exemplified embodiments. EXAMPLES Materials and methods A. Bacterial Strains and Plasmids
[000222] DH5a of Escherichia coli (Invitrogen, Karlsruhe, Germany) was used for the amplification of plasmids and cloning. B. Cell lines
[000223] Sp2 / 0-Agl4 mouse myeloma cell line (ATCC, American Type Culture Collection, Manassas, VA, USA) used for the production of recombinant hum-FLT3 specific antibody derivatives was grown on IMDM (PAN- Biotech, Aidenbach, Germany) was supplemented with 10% fetal calf serum (PAN-Biotech, Aidenbach, Germany), 1% penicillin and streptomycin (Lonza, Basel, Switzerland). Stable transfectants were selected with 1 mg / ml of G418 (Invitrogen, Karlsruhe, Germany).
[000224] BV10 and 4G8 hybridoma cell lines, which secrete anti-human FLT3-specific mouse IgGI / K antibodies (obtained from Dr. HJ. Biihring, UKT Tubingeno, Germany), were grown in RPMI 1640 (PAN-Biotech, Aidenbach, Germany) were supplemented with 10% fetal calf serum (PAN-Biotech, Aidenbach, Germany), 1% penicillin and streptomycin (Lonza, Basel, Switzerland).
[000225] Peripheral blood mononuclear cells (PBMCs), isolated by gradient centrifugation density (LSM 1077, Lonza, Basel, Switzerland), hybridoma cells and NALM16 cells (present species from R. Handgretinger, Department of Pediatrics, University of Tubingen) were maintained in RPMI 1640, mouse Sp2 / 0-Agl4 cells (ATCC, Manassas, USA) in IMDM medium (Lonza). All media were supplemented with 10% heat-inactivated fetal calf serum, 100 U / ml penicillin, 100 pg / ml streptomycin, 1 mM sodium pyruvate, non-essential amino acids, 2 mM L-glutamine and 57 nM beta-mercaptoethanol. C. Transfectant with FLT3
[000226] FLT3 human full length cDNA (GenBank #BC 126350) was obtained from ImaGenes, Saloon, Germany. The cDNA was cloned into the pcDNA3 vector using B HI and Xbal restriction sites added to the sites and were transfected into Sp2 / 0-Agl4 cells by electroporation. D. Antibodies and Flow Cytometry
[000227] Isotype control antibodies, CD33-PE-Cy5 (clone WM53), CD34-APC (clone 581), CD-45-FITC (clone HI30), CD123-PE-Cy5 (clone 9F5), CDI Ic- PE (clone B-ly6) and were acquired from BD Biosciences (Heidelberg, Germany), the CD303-FITC antibody from Miltenyi Biotech (Bergisch-Gladbach, Germany). All antibodies were incubated with cells for 30 minutes at 4 ° C. For indirect immunofluorescence, fragments of F (ab) 2 of goat anti-mouse conjugated with PE or APC, fragments of F (ab) 2 of goat anti-human, were used (Jackson ImmunoResearch, West Grove, USA). In several experiments, we combined indirect or direct immunofluorescence for multidimensional analysis that adds labeled antibodies in a final step. Cells were analyzed on a FACSCanto II or FACSCalibur (Becton Dickinson). Cons for the analysis of quantitative indirect immunofluorescence (QIFI-KIT (R)) were acquired from Dako (Hamburg, Germany) and were used according to the manufacturer's protocol. For quantification of suitable humanized antibodies, beads were not available. Thus, a specific fluorescence index (SFI) was calculated by dividing the average fluorescence intensity obtained with 4G8SDIEM by the fact that it detected with the non-binding, control antibody modified by SDIE 9.2.27. For these experiments of PE-conjugated antibodies generated with the Lynx Rapid PE antibody conjugation kit (AbD Serotec, Dusseldorf, Germany) were used. Recombinant FLT3 ligand (rFLT3L) was purchased from Peprotech EC (London, Great Britain). For competition experiments, various concentrations of rFLT3L were incubated with NALM16 and BV10SDIEM or 4G8SDIEM cells (1 µg / ml) for 30 minutes at 4 ° C and were analyzed by flow cytometry and indirect immunofluorescence. E. 3 [H] -methyl thymidine uptake assays
[000228] 2x105 AML immature precursor cells were seeded in triplicates in 96-well plates and were incubated with various concentrations of optimized antibodies. After 24 hours, cells were pulsed for another 20 hours with 3 [H] -methyl-thymidine (0.5 pCi / well) and were collected in the filtermats. Built-in radioactivity was determined by liquid scintillation counting on a 2450 microplate counter (Perkin Elmer, Waltham, USA). F. 51 [Cr] Release Tests
[000229] NALM16 cells and immature primary AML precursor cells were used as targets. To separate immature precursor cells and effector cells from PBMC preparations of patients with leukemia, cells were labeled with CD34 and CD33 microcounts and separated in LD columns after the manufacturers' protocol (Miltenyi Biotec). The number of cells The number of immature precursor cell contaminants in the negatively selected effector cell population was determined by FACS analysis and varied between 1% and 10% depending on the initial immature precursor cell contamination. In some experiments, labeled DCs were used as target cells. Chromium release assays were performed as previously described (Otz T, Grosse-Hovest L, Hofmann M, Rammensee HG, Jung G. A bispecific singlechain antibody that mediates target-restricted cell, supra-agonistic CD28 stimulation and assassing of lymphoma cells. Leukemia. 2009; 23 (l): 71-77). Briefly, labeled target cells and PBMCs were incubated at 37 ° C for 4 or 8 hours in 96-well flat-bottom plates in the presence of various concentrations of antibodies in a 50: 1 PBM: target ratio. 51 [Cr] specific release percentage was calculated according to the formula [cpm (test) -cpm (spontaneous) / [cpm (triton lysis)] - cpm (spontaneous)]. If immature leukemic precursor cells are used as targets, the addition of effector cells without spontaneous 51 [Cr] release reduced by antibody in some experiments resulting in negative values for the specific release. G. Antigen displacement
[000230] NALM16 cells or immature AML precursor cells were incubated with various concentrations of 4G8SDIEM or BV10SDIEM in the medium of RPMI 1640. After 24 or 48 hours the cells were incubated with ice cold FACS buffer, incubated with ice a saturation concentration of 4G8SDIEM (2 pg / ml) for 30 minutes at 4 ° C, were stained with F-ab fragments of goat anti-human conjugated with PE and analyzed by FACS. Relative surface expression of FLT3 was calculated which defines the average fluorescence intensity of cells preincubated in antibody as 100%. H. Isolation and Maturation of Dendritic Cell (PC)
[000231] DCs were isolated from buffy coat preparations of healthy individuals using the DC blood isolation kit II according to the manufacturer's protocol (Miltenyi Biotec). Myeloid (mDC) and plasmacytoid (pDC) subsets were stained with a mixture of antibodies from CDIIc-PE, CD303-FITC and CD123-PE-Cy5. For in vitro generation of mDC, 1x108 PBMCs from healthy individuals were seeded in 10 ml X-Vivolõ medium (Gibco, Darmstadt, Germany). After 2 hours at 37 ° C, adherent cells were cultured in RPMI 1640 medium supplemented with 50 ng / ml GM-CSF and IL-4 (20 ng / ml) for 5 days. On day 6 LPS (100 ng / ml) was added. Cells were cultured on day 7 and were analyzed by flow cytometry. I. Cologne Training Unit Tests
[000232] Bone marrow cells were obtained by washing the femoral head of patients undergoing hip surgery. The cells were purified by gradient centrifugation density and were seeded at 107 µg / ml in RPMI 1640 medium containing 5 pig / ml 4G8SDIEM or 9.2.27SDIE. After 24 hours of incubation, cells were transfected into antibody containing (5 g / ml) methylcellulose medium (10,000 cells / ml, classic MethoCult H4434, Stemcell Technologies, Grenoble, France). The test was carried out in triplicates. After 12 days colonies were counted and classified. Example 1: Identification of Unknown FLT3-specific Antibody Sequences A. Cloning of DNA encoding V regions
[000233] V regions were cloned by PCR. Most techniques start from mRNA and make use of the antibody similarity of V regions (Kabat, EA, Wu, TT, Reid-MOIIer, M., Perry, HM, Gottsman, KS Sequences of Proteins of immunological interest, 4a ed. US Department of Health and Human Services, Public Health Service, National Institute of Health, Bethesda, MD. 1987) which makes the design of degenerate primers for PCR amplification possible (Larrick, JW, Daniellson, L., Brenner, CA, Wallace, EF, Abrahamson, M., Fry, KE, Borrebaeck, CAK Polymer chain reaction used mixed primers: cloning monoclonal antibody genes variable region of humans from simple hybridoma cells. / Technology 7: 934-938, 1989; Orlandi, R., Giissow, DH, Jones, PT, Winter, G. Cloning of immunoglobulin variable domains for expression by the polymerase chain reaction. Proc. Natl. Acad. USA 86: 3833-3837, 1989). However, the disoriented amplification of complete V repertoires requires many complex sets of degenerate primers (Marks JD, Hoogenboom HR, Bonnert TP, McCafferty J., Griffiths AD, Winter G. By-passing immunization. Human antibodies from V-gene libraries exhibited on phage. J. Mol. Biol. 222: 581-597, 1991). The cloning of V regions with very atypical sequences could still not be possible with this approach. In addition, the original sequence will be ordered in those parts that are covered by the initiators. The amino acids in these regions seem to contribute to the current timing of the CDR regions (Chothia, C, Lesk, AM, Tramontane, A., Levitt, M., Smith-Gill, S J., and other Conformations of immunoglobulin hyperegion variables. Nature , 342: 877-883, 1989). For this reason, cloning of region V by using degenerate primers could lead to reduced antibody affinity. One method to take advantage of these potential problems is to clone both chains of the antibody by reverse polymerase chain reaction (iPCR) with primers that match the antibody's constant region sequences. The cloning procedure is schematically illustrated in figure 1.
[000234] Cytoplasmic RNAs were prepared from the hybridoma cell lines BV10 and 4G8 (Rappold I., Ziegler BL, Kdhler I., Marchetto S., Rosnet 0-, Birnbaum D., Simmons PJ, Zannettino AC, Hill B ., Neu S., Knapp W „Alitalo R., Alitalo K., Ullrich A., Kanz L, Bilhring HJ Functional and phenotypic characterization of bone marrow and cord blood subsets expressing FLT3 receptor tyrosine kinase (CD 135 Blood, 90: 111-125, 1997) using the RNeasy kit (Qiageno, Hildeno, Germany) applying a modified protocol for the isolation of cytoplasmic RNA only
[000235] Using oligo (dT) primer 5, double stranded cDNA (ds-cDNA) was prepared from 0.3-2 g mRNA using the cDNA synthesis system (Roche, Mannheim , Germany). More specifically, to allow abrupt end formation on the DNA strands, the ds-cDNA was incubated with T4-DNA polymerase. The reaction mixture was extracted once with an equal volume of phenol-chloroform-isoamyl alcohol (25: 24: 1) and was precipitated with ethanol. The dissolved ds-cDNA pellet was incubated with T4 DNA ligase (Roche, Mannheim, Germany) to circulate the cDNA (Uematsu Y. A novel and rapid cloning method for the T-cell receptor region variable sequences. Immunogenetics, 34: 174 -178, 1991). The 3 'polyl (A) end is connected to the unknown 5' end of the cDNA by circulation. B. Magnification of PC of variable regions of immunoglobulin cDNA
[000236] The use of two specific external directed constant primers (summarized in table 1) in an iPCR reaction allowed the amplification of the entire cDNA of redisposed heavy and light chain gene segments. 1-5 pl of circulated ds- cDNA were included in a standard 50 µJ PCR reaction (HotStar Taq DNA Polymerase, Qiageno, Hildeno, Germany) with Ck-for and Ck-back primer pair for the light chain and pair of gamma-for and gammal-back initiators for heavy chain amplification. The primers are designed to annex to the constant regions of the cDNAs. Forty cycles of magnification were performed under the following conditions: 30 sec 94 ° C, 1 min 56 ° C, 2 min 30 sec 72 ° C. The resulting amplification product contains the complete V region, 5 'UT region, pA end, 3' UT region and is flanked by the constant region sequences. The DNA obtained from the inverse PCR was separated into 1% agarose gels. DNA strips of corresponding size (light chain approx. 1000 bp; heavy chain about 1600 bp) were cut, isolated by standard techniques (Maniatis and others 1982) and were cloned into the pGEM-T Easy vector (Promega, Madison , WI, USA). For standard sequence determination primers for the vector system and standard sequence determination primers and specific immunoglobulin region constant primers (light chain: k-forl and k-for2; heavy chain: CGI-forl, CGI-for2, CGI-revI, CGI-rev2) were used (see Table 1). Table 1: Primers used for the amplification and sequencing of VJ and VDJ regions of specific FLT3 antibodies


[000237] Thus, the light chains and full heavy chains of mouse 4G8 antibodies (light chain amino acid sequence indicated in SEQ ID NO: 15 including the variable domain (SEQ ID NO: 13), the variable domain including CDR1 (SEQ ID NO: I), CDR2 (SEQ ID NO: 2) and CDR3 (SEQ ID NO: 3); heavy chain amino acid sequence indicated in SEQ ID NO: 16, including the variable domain (SEQ ID NO: 14), the variable domain including CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5) and CDR3 (SEQ ID NO: 6)) and BV10 (light chain amino acid sequence indicated in SEQ ID NO: 31 including the domain variable (SEQ ID NO: 29), the variable domain including CDR1 (SEQ ID NO: 7), CDR2 (SEQ ID NO: 8) and CDR3 (SEQ ID NO: 9); heavy chain amino acid sequence indicated in SEQ ID NO: 32 including the variable domain (SEQ ID NO: 30), the variable domain including CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: II) and CDR3 (SEQ ID NO: 12)) were identified.
[000238] The 4G8 murine antibody light chain is encoded by the nucleotide sequence indicated in SEQ ID NO: 19 (complete cDNA sequence indicated in SEQ ID NO: 20), where the variable domain is encoded by the indicated nucleotide sequence in SED ID NO: 17. The 4G8 antibody heavy chain is encoded by the nucleotide sequence indicated in SEQ ID NO: 21 (full length cDNA sequence indicated in SEQ ID NO: 22), where the variable domain is encoded by the sequence of nucleotides indicated in SEQ ID NO: 18.
[000239] The BV10 murine antibody light chain is encoded by the nucleotide sequence indicated in SEQ ID NO: 35 (complete cDNA sequence indicated in SEQ ID NO: 36), where the variable domain is encoded by the indicated nucleotide sequence in SED ID NO: 33. The BV10 antibody heavy chain is encoded by the nucleotide sequence indicated in SEQ ID NO: 37 (complete cDNA sequence indicated in SEQ ID NO: 38), where the variable domain is encoded by the nucleotide sequence indicated in SEQ ID NO: 34. Example 2: Construction and expression of FLT3-specific chimeric antibodies and their derivatives
[000240] In the second stage of construction of recombinant antibodies, the cloned V regions were combined with the desired C regions in an expression vector. The cloning procedure performed here allows the introduction of complete lg V regions and their expression in lymphoid cells without any changes in their amino acid sequence. Therefore, the nucleotide sequence of the ampHcon obtained in Example 1 was determined after subcloning by sequencing (primer in Table 1) and was used for projection of primer pairs (C C; D D '; Table 2). The DNA fragments rescanned from the V segments are cut with appropriate restriction nucleases (summarized in Table 2) and then ligated into the expression vectors. The vectors (Figures 2 and 3) contain human light and heavy constant region genes. Thus, insertion of the cut and enlarged V segments reconstitutes the original genetic organization of the lg genes in the vectors without altering any amino acid in the V regions.
[000241] The parental vector for the light chain contains the VJ region of the mouse light chain and the C region of the kapa de ser gene. Restriction sites were introduced at the required locations (Xhol and Spel) in order to replace the Xhol-Spel light chain fragment with the appropriate light chain VJ fragment of BV10 or 4G8 monoclonal antibodies or any other monoclonal antibody. The relevant region for fragment exchange is shown enlarged in Figure 2. The fragment to be exchanged contains part of the second exon of the leader sequence, an appropriate site (Xhol) for a frame fusion, the VJ region and part of the second intron with restriction site Spel.
[000242] The original vector for the heavy chain contains the human y1 isotype 1g heavy chain. Restriction sites were introduced at the positions required in intron I and II to exchange the Aatll-Clal fragment with the VDJ fragment of the BV10 or 4G8 monoclonal antibody heavy chain or any other monoclonal antibody. The region relevant to the cloning of the VDJ fragment is shown enlarged in figure 3a. The fragment to be exchanged contains parts of the first intron with an Aatll restriction site, the second exon of the leader sequence, the VDJ region and part of the second intron with the Clal restriction site. For the replacement of all exons in the constant region, restriction sites were introduced at the required position in intron II (Mlul) and intron VI (Spel). The M-Spel fragment to be exchanged (shown enlarged in Figure 3 b) contains the entire human y2 heavy chain constant region and two amino acid modifications in the CH2 domain as indicated (Ser239-Asp; Ile332-Glu)
[000243] Furthermore, with the expression vectors used, it is possible to exchange the entire constant region of the human Igy1 isotype (Mlul-Spel fragment; see Figure 3) or against constant regions of all other antibody isotypes or against Fc parts with optimized or reduced effector function. In the case of antibodies optimized for triggering ADCC to S239D and exchanging I332E (amino acid position according to the Kabat nomenclature), they were introduced into the CH2 domain of the y1 constant region of human beings. This was done according to the publication by Lazar and others (Lazar GA, Dang W, Karki S, Vafa O, Peng JS, Hyun L, Chan C, Chung HS, Ei- vazi A, Yoder SC, Vielmetter J, Carmichael DF, Hayes RJ, Dahiyat Bl Engineered antibody Fc variants with enhanced effector function (Proc. Natl. Acad. Sci. USA 103: 4005-4010, 2006). Table 2: Oligonucleotides used to enlarge VJ and VDJ segments obtained by iPCR for insertion into expression vectors
D 4G8-L-for (Xhol) 5'-act cga gga gat art gtg eta act cag tct cca gee acc ctg-3 '(SEQ ID NO: 61) D' 4G8-L-rev (Spel) 5'-tac tag tac tta cgt ttt art tec age ttg gtc ccc cct cc-3 '(SEQ ID NO: 62) D BV10-L-for (Xhol) 5'-act cga gga gac att gtg atg aca cag tct cca tec tecc-3 '(SEQ ID NO: 63) D' BV10-L-rev (Spel) 5'-act agt act tac gtt tea get cca get tgg tec cag cac cga acg tg-3 '(SEQ ID NO: 64)
[000244] Restriction sites are shown in bold and indicated by the letters in parentheses.
[000245] Thus, chimeric antibodies 4G8 and BV10 and the optimized Fc variants SDIE 4G8 and SDIE BV10 were obtained. These comprise the following amino acid and nucleotide sequences.
[000246] Chimeric antibody 4G8: light chain amino acid sequence as indicated in SEQ ID NO: 23 and as encoded by the nucleotide sequence indicated in SEQ ID NO: 24, heavy chain amino acid sequence as indicated in SEQ ID NO: 25 and as encoded by the nucleotide sequence indicated in SEQ ID NO: 26.
[000247] SDIE 4G8 (Fc-optimized antibody, chimeric): light chain amino acid sequence indicated in SEQ ID NO: 23 and encoded by the nucleotide sequence indicated in SEQ ID NO: 24, heavy chain amino acid sequence indicated in SEQ ID NO: 27 and encoded by the nucleotide sequence indicated in SEQ ID NO: 28.
[000248] Chimeric antibody BV10: light chain amino acid sequence as indicated in SEQ ID NO: 39 and as encoded by the nucleotide sequence indicated in SEQ ID NO: 40, heavy chain amino acid sequence as indicated in SEQ ID NO: 41 and as encoded by the nucleotide sequence indicated in SEQ ID NO: 42.
[000249] SDIE BV10 (Fc-optimized antibody, chimeric): light chain amino acid sequence indicated in SEQ ID NO: 3 and encoded by the nucleotide sequence indicated in SEQ ID NO: 40, heavy chain amino acid sequence indicated in SEQ ID NO: 43 and encoded by the nucleotide sequence indicated in SEQ ID NO: 44. Example 3: Expression and purification of anti-FLT3 antibodies
[000250] Cotransfection of the expression vectors encoding the chimeric light and heavy chain (IgGI / kappa) or modified heavy chains in the Sp2 / 0 non-lg production myeloma cell line provided stable transfectomas that secrete chimeric monoclonal antibodies that are capable of specifically binding to FLT3 in human REH cells, and FLT3 transfectants (Sp2 / 0).
[000251] Chimeric antibodies were purified from cell culture supernatant by protein A affinity chromatography. Example 4: ADCC effector function of anti-FLT3 antibodies
[000252] The ADCC effector function of the anti-FLT3 chimeric antibodies, optimized by 4Gc-SDIE and BV10-SDIE Fc, compared to the corresponding chimeric antibodies without Fc optimization (figures 4A and B) as well as an antibody of chimeric anti-NG2 comprising the same Fc modification (figure 4C) has been demonstrated using chromium release assays. Furthermore, the cell killing activity of 4G8-SDIE and unstimulated PBMCs compared to the 4G8 parental mouse antibody has been shown for immature AML precursor cells isolated from a human patient with acute myelogenous leukemia (Figure 5). The target cells used were:
[000253] NALM16: an acute lymphoblastic leukemia cell line (ALL), supplier: Department of Pediatric Oncology, University of Tiibingeno, original characterization: Minowada J and others J Cancer Res Clin Oncol 101: 91-100 (1981) .
[000254] SK-Mel63: human melanoma cell line, original supplier: Dr. A. Knuth, Nordwestkrankenhaus Frank-furt / Maina, Germany.
[000255] SG3: leukemic cells, isolated from the peripheral blood of a patient with acute myelogenous leukemia (AML) by gradient centrifugation density; provided by Dr. H. Salih, Department of Medical Oncology, University of Tübingen.
[000256] The effector cells used were peripheral blood mononuclear cells (PBMCs) isolated from the blood of healthy normal donors.
[000257] The chromium release assay was performed as follows: 106 target cells were labeled with sodium chromate (51Cr, 150 pCi / ml) for 1 hr, washed and laminated in 96 well microtiter plates (10,000 cells per well). PBMC and antibodies were then added at the indicated concentrations. After 4 and 20 hrs respectively supernatant was collected and counted in a MicroBeta counter. Cytotoxicity was determined according to the standard formula:% specific 51 Cr release = (experimental release - spontaneous release): (total release - spontaneous release) x 100. Spontaneous release and total release were determined by incubation of target cells in the medium with or without 2% Triton-X100, respectively.
[000258] The results shown in Figures 4 and 5 clearly show that the introduction of the F23 modifications of S239D and I332E into the CH2 domain of the anti-FLT3 4G8 and BV10 chimeric antibody heavy chain could induce significant cell killing activity in both the antibodies. In contrast to these results, introducing the same modifications into a chimeric anti-NG2 antibody had no such effect. Correspondingly, there is generally no principle that two modifications are used to confer cell killing activity on any given antibody, but they certainly have to be carefully selected for each individual monoclonal antibody. Example 5: Production and purification of Fc-optimized antibodies and recombinants
[000259] The BV10 and 4G8 murine antibody mRNA (both IgGI kappa) was isolated from hybridomas with the RNeasy kit (Qiageno, Hildemann, Germany). Unknown heavy-chain (VDJ) and light-chain (VJ) variable regions were identified by sequencing the reverse PCR amplicons generated as previously described (Herrmann T, Grosse-Hovest L, Otz T, Krammer PH, Ramrnensee HG, Jung G. Construction of bispecific antibodies optimized for selective activation of the CD95 death receptor. Cancer Res. 2008; 68 (4): 1221-1227), using specific primers for light chain mouse constant genes (Ck- para (SEQ ID NO: 47); Ck-back (SEQ ID NO: 48)) and heavy chain (gamal-for (SEQ ID NO: 45); gamai-back (SEQ IS NO: 46). The cloning of the variable regions of the hybridoma 9.2.27 (GenBank: # AJ459796; # AJ459797), production of an IgG2a / K CSPG4 antibody has also been described previously (Grosse-Hovest L, Hartlapp I, Marwan W, Brem G, Ramrnensee HG, Jung G. An antibody of recombinant bispecific single strand induces stimulation of CD28 of targeted supra-agonistic and tumor cell death. Eur J Immunol. 2003; 33 (5): 1334-1340). For the generation of chimerized and optimized antibodies, the elements of VJ and VDJ were re-amplified using the oligonucleotides listed in table 2 and were cloned into eukaryotic expression vectors as shown in figures 2 and 3. In addition to the amino acid exchanges at S239D and I332E, the optimized Gl Fc part contains an M C-terminal tag (PTHVNVSVVM AEEQKLISEEDLLR; SEQ ID NO: 66, which was derived from the PTHVNVSVVMAE amino acid sequences (amino acid # 455- 466 of the Igalfal de ser end piece human) (SEQ ID NO: 67) and the c-myc epitope EQKLISEEDLLR (SEQ ID NO: 68) (Evan Gl, Lewis GK, Ramsay G, Bishop JM. Isolation of monoclonal antibodies specific for c-proto-oncogene product -myc from human. Mol Cell Biol. 1985; 5 (12): 3610-3616). Recombinant antibodies, as well as parental mouse 4G8 and BV10, were purified from the culture supernatant of transfectants and hybridoma cells. , respectively, using protein affinity chromatography a A (GE Healthcare, Munich, Germany). In the case of 4G8SDIEM, a large load of the antibody (15 g) was produced in clean GMP-compatible chambers using disposable technology including a 100 L bio-wave reactor (Sartorius; Goettingeno, Germany) for fermentation and a system Akta Ready for purification by hydrophobic interaction chromatography, ion exchange and protein A (MabSelect Sure and CaptoAdhere columns, GE Healthcare, Munich, Germany). Example 6: Avidity and specificity of antibody binding FLT3
[000260] Parental mouse antibodies of 4G8 and BV10 were originally described and were characterized as recognition of the FLT3 protein (Rappold I, Ziegler BL, Kohler I, and others. Functional or phenotypic characterization of bone marrow and cord blood subsets which express FLT3 receptor tyrosine kinase (CD135). Blood. 1997; 90 (1): 111-125). Figure 7A shows that both antibodies modified by SDIEM specifically bind to that protein in the transfected mouse Sp2 / 0 cells. In Figure 7B the binding of the two FLT3 positive antibodies to human NALM1-6 cells is assessed by flow cytometry. Antibodies do not cross-block (data not shown) and thus recognize two spatially separate epitopes of the FLT3 protein. Both antibodies to FLT3 molecules saturated in NALM16 cells at low concentrations of 1 ng / ml. The binding of the chimeric 4G8 antibody was stronger than that of BV10. This is not due to chimerization or optimization since a similar difference was observed when linking the parental mouse versions of 4G8 and BV10 was compared (Figure 7C). No difference in binding between the chimeric versions modified by SDIEM and chimeric antibodies was detected (Figure 7B). The SDIE-modified antibody, called 9.2.27SDIE, directed against a melanoma-associated surface antigen, did not bind to NALM16 cells and was used as a negative control in this and several subsequent experiments. Example 7: Competition with FLT3 ligand binding (FLT3L)
[000261] In general, interference with natural ligand binding can contribute to the therapeutic activity of an antibody. Figure 8 A shows that recombinant FLT3L partially inhibits binding of 4G8SDIEM, but not BV10SDIEM to NALM16 cells, indicating that the 4G8 antibody binding site is in close proximity to that of the FLT3 ligand. Therefore, the effect of 4G8SDIEM on the spontaneous proliferation of immature leukemic precursor cells from the three different patients was evaluated in vitro using the non-binding SDIE modified 9.2.27 antibody as a control. While spontaneous proliferation of primary AML cells varied substantially, significant effects of antibodies on cell proliferation were not observed (Figure 8B). Example 8: Antibody-dependent cellular cytotoxicity
[000262] Figure 9 shows that ADCC activity of PBMCs against NALM16 cells is markedly increased in the presence of modified SDIEM antibodies when compared to that of unmodified chimeric antibody versions. In various experiments, the concentrations required to achieve comparable lysis by modified and unmodified antibodies differentiated by a factor of at least 100. Death by the 4G8SDIEM antibody will be significantly better than that achieved by BV10SDIEM, in particular at low concentrations. This corresponds to the moderately low binding avidity of BV10 (Figure 7).
[000263] In figure 10A the ADCC activity of 4G8SDIEM is shown using PBMCs from three different healthy donors (# 1-3). In these experiments, SDIE-modified mAb 9.2.27 was used as a negative control. The cytolytic activity in the presence of this reagent did not exceed that of NK cells in the absence of antibodies that varied between 0 and 20%. In Figure 10B the ADCC activity of PBMCs from a healthy donor (# 2) against immature leukemic precursor cells from the three different patients is shown. ADCC activity measured against these immature precursor cells (AML # 1, AML # 2, AML # 7), which carry 4000, 4500 and 3200 FLT3 molecules per cell, respectively, was less pronounced than that against cultured NALM 16 cells . It required 8 instead of 4 hours to become clearly detectable. In general, ADCC as well as NK activity against NALM 16 cells and immature leukemic precursor cells continue to appear after 8 hours. However, using primary immature precursor cells, it was difficult to further extend the test time due to the release of spontaneous growing corm.
[000264] The next ADCC activity of PBMCs isolated from the blood of AML patients against autologous immature precursor cells was evaluated. To that end, immature leukemic precursor cells from BMC preparations were depleted and depleted PBMCs were used as effector cells against positively selected immature precursor cells (see Materials and Methods). Under these conditions, significant lysis in 2 (AML # 1, # 15) of every 5 independent experiments with immature precursor cells and autologous PBMCs of the respective patients (Figure IOC) was detected. Example 9: Antigen change
[000265] Modulation of target antigen expression on antibody binding is a phenomenon frequently observed during antibody therapy. In particular, a sustained and complete loss has been reported in the treatment of patients with AML with a saturation dose of the CD33 antibody. Lintuzumab (Feldman EJ, Brandwein J, Stone R, and others. Randomized Phase III multi-center study of a humanized anti-CD33 monoclonal antibody, lintuzumab, in combination with chemotherapy, versus chemotherapy alone in patients with acute myeloid leukemia of first relapse or contumaz J Clin Oncol. 2005; 23 (18): 4110-4116). Figure 11A shows the antigen change induced after incubation of NALM cells or primary immature leukemic precursor cells from two patients (AML # 1 and # 2) with various concentrations of 4G8SDIEM for 48 h. In all of these cells a moderate change in antigen was observed that was already complete after 24 h of incubation (data not shown). Example 10: Binding to leukemic and normal cells
[000266] Figures 11B and 11C show binding of a parent mouse 4G8 and 4G8SDIEM antibody, respectively, to a panel of leukemic cells obtained from patients suffering from the indicated AML subtypes. Controlled CD33 + CD45dim or CD34 + CD45dim cells were analyzed. FLT3 was detected in all 15 patient samples. The number of molecules per well by indirect immunofluorescence and quantitative flow cytometry varied from 500 to 6000, whereas in NALM16 cells from 6000 to 9000 (Figure 11B). In Figure 11C, 4G8SDIEM-PE instead of 4G8 mouse was used for staining. In that case, an SFI value was calculated to quantify antibody binding. For immature precursor cells from 4 out of 15 donors, this index was not determined because of the high non-specific reactivity with the control antibody of 9.2.27SDIE. As expected, SFI values from the samples that can be evaluated closely match the numbers of molecules determined by quantitative FACS (Figure 11C).
[000267] Figures 12A-C show that the binding of 4G8 mouse to CDIIc positive mDCs and purified CD303 positive pDCs from normal PBMCs was marginal at best. The numbers of FLT3 molecules expressed in these cells were below 100 / cell. In addition, DCs from normal PBMCs were generated. Although these cells express large amounts of CD80, CD86 and CD 123 associated DC markers, binding of 4G8 antibodies was again only detectable (data not shown). Next, binding of 4G8 positive cells to CD34 in normal bone marrow was assessed. Again, antibody binding to bone marrow cells from three different donors was marginal with less than 300 molecules per cell (Figure 12D). In summary, the binding of FLT3 antibodies to normal DC cells and bone marrow cells was significantly lower than to all the FLT3 expression leukemic cells examined. In addition, binding of FLT3 antibodies to PBMCs, thrombocytes, erythrocytes and granulocytes was observed (data not shown). Example 11: In vitro toxicity
[000268] Despite relatively low levels of 4G8SDIEM binding to normal bone marrow precursor cells and DCs, the potential toxicity of that antibody to such cells has been assessed. To that end, we incubate bone marrow cells with saturation concentrations of 4G8SDIEM and 9.2.27SDIE and determine the influence of these antibodies on the ability of bone marrow cells to originate colonies (CFlIs) in semi-solid medium. CFU formation capacity was detected in two experiments with bone marrow cells from different healthy donors (Figure 13 A). Likewise, human DCs were incubated with autologous PBMCs as effector cells. While 4G8SDIEM-mediated effective ADCC against NALM16 cells, used as a positive control, no autologous DC deaths were observed (figure 13B). Example 12: Clinical application of 4G8-SDIEM
[000269] A 30-year-old man diagnosed in 2008 with AML (FAB M0, 45XY, complex karyotype including inv (3) (q21q26), -7) was treated with 4G8-SDIEM. The patient fails to achieve complete remission (CR) after two different induction therapy regimens. He subsequently received allogeneic SCT (stem cell transplantation) from a donor paired with HLA, relapsed, received haploidentical SCT from his sister and relapsed again. 4G8-SDIEM treatment was considered and pre-clinical testing was performed. FACS analysis of patients' immature precursor cells (CD34 +) revealed homogeneous expression of FLT3 to about 4000 cell molecules (figure 14A and data not shown). Effective ADCC induced by 4G8-SDIEM, in vitro of the patient's peripheral blood mononuclear cells (PBMC) against NALM16 leukemia cells and - to a lesser extent, - against autologous immature precursor cells (figure 14B, C). The patient was then treated with staggered doses of 4G8-SDIEM ranging from 10 pig to 10 mg. Several hours after the first 10 mg dose, 5x108 PBMCs from a CD3 / CD19-depleted donor from her sister were adopted. Serum concentration of 4G8-SDIEM reached 0.8 pig / ml I h after the first 10 mg dose and subsequently decreased to 0.3 pig / ml at 24 h (figure 15A). During treatment (i) an almost complete saturation of leukemic cells in the bone marrow (BM) (figure 15B), (ii) a marked increase in activated NK cells in peripheral blood (PB) (figure 16A) and BM (figure 16B ) that was associated with an increase in serum levels of the cytokine TNF index (figure 16C), and (iii) a marked reduction in immature leukemic precursor cells in BP (figure 16A) was observed. While the decline in immature PB precursor cells was transient, but almost complete, reduction in BM was less pronounced (figure 16B). This is most likely due to the different ratios of NKJeukemia cells in two compartments: In PB the cell ratio of CD56 + NK and immature precursor cells was about 1 while in BM it was only I / 7, as determined by FACS ( data not shown). Side effects were moderate and consisted of subfebrile temperature (max. 38.2 ° C) and a transient exacerbation of a pre-existing transient acneiform skin rash.
[000270] Despite the merely transient response to antibody treatment, the patient unexpectedly remained in good clinical condition for several months with a slow increase in immature precursor cell counts under better supportive care and hydroxyurea. Therefore, a second haploidentical SCT from a different donor was performed. After recovery, the patient achieved a CR without minimal detectable residual disease (MRD). We then applied 45.5 mg of 4G8-SDIEM in scaled doses. At that time, neither the release of the relevant cytokine nor the activation of the entire NK cell (figure 16D) was observed, and side effects were totally absent.
[000271] The invention has been described widely and generically here. Each of the narrowest species and subgeneric groupings that fall within the generic revelation are also part of the invention. This includes the generic description of the invention with the negative condition or limitation of removal of any material object of the species, regardless of whether or not the excised material is specifically reported here. Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of the Markush groups, those skilled in the art will recognize that the invention is also described in terms of any individual member or subgroup of members of the Markush group.
[000272] One skilled in the art will readily appreciate that the present invention is well adapted to achieve the objectives and will obtain the mentioned purposes and advantages, as well as those inherent here. In addition, it will be apparent to one skilled in the art that variation of substitutions and modifications can be made to the invention disclosed here without departing from the scope and spirit of the invention. The specific compositions, methods, procedures, treatments, molecules and compounds described herein are presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes here and other uses will occur for those skilled who are included within the spirit of the invention are defined by the scope of the claims. The listing and discussion of a document previously published in this report would not necessarily be taken as an acknowledgment that the document is part of the state of the art or is common knowledge.
[000273] The invention illustratively described here can suitably be practiced in the absence of any element or elements, limitations or limitations, not specifically disclosed here. Thus, for example, the terms "comprising", "including," containing ", etc. will be read extensively and without limitation. The word" understand "or variations such as" comprises "or" comprising "accordingly will be understood to imply inclusion of an integer or groups of integers stated but not excluding any other integer or group of integers. Additionally, the terms and expressions used here were used as terms of description and not of limitation, not of intention to use of such terms and expressions to the exclusion of any equivalents of the features shown and described or their portions, but it is recognized that several modifications are possible within the scope of the claimed invention, so it would be understood that although the present invention was specifically disclosed by exemplary embodiments and optional features, modification and variation of the inventions expressed herein can be used by those those skilled in the art, and that such modifications and variations are considered to be within the scope of that invention.
[000274] The contents of all documents and patent documents cited here are incorporated by reference in their entirety.

权利要求:
Claims (14)
[0001]
1. Antibody that binds to the human tyrosine kinase receptor FLT3, characterized by the fact that it comprises a heavy chain and a light chain, the light chain comprising a VL CDR1 that comprises or consists of the amino acid sequence indicated in SEQ ID NO: 1; a VL CDR2 that comprises or consists of the amino acid sequence indicated in SEQ ID NO: 2; and a VL CDR3 comprising or consisting of the amino acid sequence indicated in SEQ ID NO: 3, and the heavy chain comprising a VH CDR1 comprising or consisting of the amino acid sequence indicated in SEQ ID NO: 4; a VH CDR2 that comprises or consists of the amino acid sequence indicated in SEQ ID NO: 5; and a VH CDR3 comprising or consisting of the amino acid sequence indicated in SEQ ID NO: 6, and the amino acid substitutions S239D and I332E in the constant region relative to a parental anti-FLT3 antibody, where the positioning is in accordance with the EU index.
[0002]
2. Antibody that binds to the human tyrosine kinase receptor FLT3, characterized by the fact that it comprises a heavy chain and a light chain, the light chain comprising a VL CDR1 that comprises or consists of the amino acid sequence indicated in SEQ ID NO: 7; a VL CDR2 that comprises or consists of the amino acid sequence indicated in SEQ ID NO: 8; and a VL CDR3 comprising or consisting of the amino acid sequence indicated in SEQ ID NO: 9, and the heavy chain comprising a VH CDR1 comprising or consisting of the amino acid sequence indicated in SEQ ID NO: 10; a VH CDR2 that comprises or consists of the amino acid sequence indicated in SEQ ID NO: 11; and a VH CDR3 comprising or consisting of the amino acid sequence indicated in SEQ ID NO: 12, and the amino acid substitutions S239D and I332E in the constant region relative to a parental anti-FLT3 antibody, where the position numbering is in accordance with the EU index.
[0003]
3. Antibody according to claim 1, characterized in that the heavy chain comprises a VH domain that comprises or consists of the amino acid sequence indicated in SEQ ID NO: 14 and the light chain comprises a VL domain that comprises or consists of the amino acid sequence indicated in SEQ ID NO: 13.
[0004]
4. Antibody according to claim 2, characterized in that the heavy chain comprises a VH domain that comprises or consists of the amino acid sequence indicated in SEQ ID NO: 30 and the light chain comprises a VL domain that comprises or consists of the amino acid sequence indicated in SEQ ID NO: 29.
[0005]
5. Antibody according to claim 1, characterized in that the antibody is a chimeric antibody and comprises a heavy chain having the amino acid sequence indicated in SEQ ID NO: 27 and a light chain having the amino acid sequence indicated in SEQ ID NO: 23.
[0006]
6. Antibody according to claim 2, characterized in that the antibody is a chimeric antibody and comprises a heavy chain having the amino acid sequence indicated in SEQ ID NO: 43 and a light chain having the amino acid sequence indicated in SEQ ID NO: 39.
[0007]
Antibody according to any one of claims 1 to 6, characterized by the fact that said antibody binds with increased affinity to the FcyRllla receptor or has increased ADCC effector function when compared to the parent antibody.
[0008]
Nucleic acid molecule, characterized by the fact that it encodes a light and heavy chain of the antibody, as defined in any one of claims 1, 3 or 5, wherein the nucleic acid molecule comprises a sequence of nucleotides that encodes the domain light chain variable indicated in SEQ ID NO: 17 and where the nucleic acid molecule comprises a nucleotide sequence encoding the heavy chain variable domain indicated in SEQ ID NO: 18 or where the nucleic acid molecule encoding antibody light chain has a nucleotide sequence indicated in SEQ ID NO: 24 and the nucleic acid molecule encoding the antibody heavy chain has a nucleotide sequence indicated in SEQ ID NO: 28.
[0009]
Nucleic acid molecule, characterized by the fact that it encodes a light and heavy chain of the antibody, as defined in any one of claims 2, 4 or 6, wherein the nucleic acid molecule comprises a sequence of nucleotides that encodes the domain light chain variable indicated in SEQ ID NO: 33 and where the nucleic acid molecule comprises a nucleotide sequence encoding the heavy chain variable domain indicated in SEQ ID NO: 34 or where the nucleic acid molecule encoding antibody light chain has a nucleotide sequence indicated in SEQ ID NO: 40 and the nucleic acid molecule encoding the antibody heavy chain has a nucleotide sequence indicated in SEQ ID NO: 44.
[0010]
10. Use of an antibody, as defined in any of claims 1 to 7, characterized by the fact that it is for the preparation of a pharmaceutical composition for the treatment of lymphoma or leukemia in mammals.
[0011]
11. Use according to claim 10, characterized by the fact that lymphoma or leukemia is in the stage of minimal residual disease (MRD).
[0012]
12. Use according to claim 10 or 11, characterized by the fact that lymphoma or leukemia is selected from the group consisting of: non-Hodgkin's lymphomas (NHL), chronic lymphocytic leukemia (CLL), leukemia / acute B-cell lymphoblastic lymphoma (B-ALL), mantle cell lymphoma (MCL), hair cell leukemia (HCL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), and multiple myeloma (MM ).
[0013]
13. Use according to any one of claims 10 to 12, characterized in that said antibody is administered in combination with at least one agent selected from the group consisting of a cytotoxic agent, a chemotherapeutic agent, a cytokine, a growth inhibitory agent, anti-hormonal agent, kinase inhibitor, antiangiogenic agent, cardioprotective agent, immunostimulatory agent, immunosuppressive agent, angiogenesis inhibitor, protein tyrosine kinase inhibitor and second antibody .
[0014]
14. Pharmaceutical composition, characterized in that it comprises an antibody, as defined in any of claims 1 to 7, and a pharmaceutically acceptable carrier.

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law|

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2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|

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2019-05-21| B07E| Notice of approval relating to section 229 industrial property law|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-06-25| B06T| Formal requirements before examination|

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2019-10-15| B07A| Technical examination (opinion): publication of technical examination (opinion)|

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2020-05-05| B09A| Decision: intention to grant|

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2020-09-29| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 23/12/2010, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US28952909P| true| 2009-12-23|2009-12-23|

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US61/289,529|2009-12-23|

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PCT/EP2010/070659|WO2011076922A1|2009-12-23|2010-12-23|Anti-flt3 antibodies and methods of using the same|

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