WO2022148853A1 - Immunoconjugates - Google Patents
Immunoconjugates Download PDFInfo
- Publication number
- WO2022148853A1 WO2022148853A1 PCT/EP2022/050310 EP2022050310W WO2022148853A1 WO 2022148853 A1 WO2022148853 A1 WO 2022148853A1 EP 2022050310 W EP2022050310 W EP 2022050310W WO 2022148853 A1 WO2022148853 A1 WO 2022148853A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- amino acid
- seq
- acid sequence
- domain
- immunoconjugate
- Prior art date
Links
- 229940127121 immunoconjugate Drugs 0.000 title claims abstract description 272
- 108010002350 Interleukin-2 Proteins 0.000 claims abstract description 350
- 102000000588 Interleukin-2 Human genes 0.000 claims abstract description 349
- 101001109501 Homo sapiens NKG2-D type II integral membrane protein Proteins 0.000 claims abstract description 228
- 102100022680 NKG2-D type II integral membrane protein Human genes 0.000 claims abstract description 222
- 238000000034 method Methods 0.000 claims abstract description 115
- 102000040430 polynucleotide Human genes 0.000 claims abstract description 46
- 108091033319 polynucleotide Proteins 0.000 claims abstract description 46
- 239000002157 polynucleotide Substances 0.000 claims abstract description 46
- 239000013598 vector Substances 0.000 claims abstract description 36
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 19
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 427
- 235000001014 amino acid Nutrition 0.000 claims description 234
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 164
- 241000282414 Homo sapiens Species 0.000 claims description 145
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 139
- 229920001184 polypeptide Polymers 0.000 claims description 134
- 230000027455 binding Effects 0.000 claims description 120
- 238000006467 substitution reaction Methods 0.000 claims description 101
- 101001002657 Homo sapiens Interleukin-2 Proteins 0.000 claims description 56
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 56
- 108060003951 Immunoglobulin Proteins 0.000 claims description 52
- 102000018358 immunoglobulin Human genes 0.000 claims description 52
- 239000000203 mixture Substances 0.000 claims description 51
- 201000010099 disease Diseases 0.000 claims description 47
- 238000011282 treatment Methods 0.000 claims description 45
- 206010028980 Neoplasm Diseases 0.000 claims description 43
- 108010087819 Fc receptors Proteins 0.000 claims description 41
- 102000009109 Fc receptors Human genes 0.000 claims description 41
- 239000012636 effector Substances 0.000 claims description 41
- 230000014509 gene expression Effects 0.000 claims description 37
- 108010047041 Complementarity Determining Regions Proteins 0.000 claims description 33
- 239000003814 drug Substances 0.000 claims description 32
- 238000012986 modification Methods 0.000 claims description 31
- 230000004048 modification Effects 0.000 claims description 30
- 102000005962 receptors Human genes 0.000 claims description 30
- 108020003175 receptors Proteins 0.000 claims description 30
- 125000000539 amino acid group Chemical group 0.000 claims description 29
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 claims description 27
- 108700004922 F42A Proteins 0.000 claims description 22
- 102220505697 Palmitoyl-protein thioesterase 1_Y45F_mutation Human genes 0.000 claims description 21
- 102220610852 Thialysine N-epsilon-acetyltransferase_L72G_mutation Human genes 0.000 claims description 21
- 201000011510 cancer Diseases 0.000 claims description 18
- 238000004519 manufacturing process Methods 0.000 claims description 18
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 claims description 18
- 230000000295 complement effect Effects 0.000 claims description 17
- 210000000987 immune system Anatomy 0.000 claims description 14
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 claims description 14
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 claims description 13
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 claims description 13
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 claims description 13
- 125000001909 leucine group Chemical group [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 12
- 230000001737 promoting effect Effects 0.000 claims description 11
- 239000003937 drug carrier Substances 0.000 claims description 9
- 239000013604 expression vector Substances 0.000 claims description 9
- 125000000430 tryptophan group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12 0.000 claims description 9
- 125000002987 valine group Chemical group [H]N([H])C([H])(C(*)=O)C([H])(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 9
- 102200134447 rs41295338 Human genes 0.000 claims description 8
- 230000004936 stimulating effect Effects 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims description 3
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 claims description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 2
- 102220495631 Putative uncharacterized protein LOC645739_F42A_mutation Human genes 0.000 claims 1
- 210000004027 cell Anatomy 0.000 description 222
- 229940024606 amino acid Drugs 0.000 description 137
- 210000001744 T-lymphocyte Anatomy 0.000 description 132
- 150000001413 amino acids Chemical class 0.000 description 114
- 230000035772 mutation Effects 0.000 description 92
- 210000000822 natural killer cell Anatomy 0.000 description 79
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 description 73
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 67
- 230000000694 effects Effects 0.000 description 61
- 239000000427 antigen Substances 0.000 description 59
- 108010038453 Interleukin-2 Receptors Proteins 0.000 description 57
- 102000010789 Interleukin-2 Receptors Human genes 0.000 description 57
- 102000036639 antigens Human genes 0.000 description 57
- 108091007433 antigens Proteins 0.000 description 57
- 230000002829 reductive effect Effects 0.000 description 56
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 description 53
- 108090000623 proteins and genes Proteins 0.000 description 47
- 239000000872 buffer Substances 0.000 description 44
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 43
- 230000035755 proliferation Effects 0.000 description 41
- 230000004913 activation Effects 0.000 description 36
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 36
- 230000006870 function Effects 0.000 description 36
- 229940027941 immunoglobulin g Drugs 0.000 description 36
- 235000018102 proteins Nutrition 0.000 description 36
- 102000004169 proteins and genes Human genes 0.000 description 36
- 150000007523 nucleic acids Chemical class 0.000 description 26
- 102220619344 RNA polymerase I-specific transcription initiation factor RRN3_F42D_mutation Human genes 0.000 description 25
- 108020001507 fusion proteins Proteins 0.000 description 25
- 102000037865 fusion proteins Human genes 0.000 description 25
- 102000039446 nucleic acids Human genes 0.000 description 23
- 108020004707 nucleic acids Proteins 0.000 description 23
- 210000003289 regulatory T cell Anatomy 0.000 description 22
- 230000008685 targeting Effects 0.000 description 20
- 238000013459 approach Methods 0.000 description 18
- 239000002953 phosphate buffered saline Substances 0.000 description 18
- 241001465754 Metazoa Species 0.000 description 17
- 229940124597 therapeutic agent Drugs 0.000 description 16
- 102000004127 Cytokines Human genes 0.000 description 15
- 108090000695 Cytokines Proteins 0.000 description 15
- 239000003795 chemical substances by application Substances 0.000 description 15
- 238000001727 in vivo Methods 0.000 description 15
- 239000002609 medium Substances 0.000 description 15
- 239000000243 solution Substances 0.000 description 15
- 238000000684 flow cytometry Methods 0.000 description 14
- 239000012634 fragment Substances 0.000 description 14
- 238000002360 preparation method Methods 0.000 description 14
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 14
- 102000001712 STAT5 Transcription Factor Human genes 0.000 description 13
- 108010029477 STAT5 Transcription Factor Proteins 0.000 description 13
- 238000005734 heterodimerization reaction Methods 0.000 description 13
- 230000001225 therapeutic effect Effects 0.000 description 13
- 102000053602 DNA Human genes 0.000 description 12
- 108020004414 DNA Proteins 0.000 description 12
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 12
- 230000006786 activation induced cell death Effects 0.000 description 12
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 12
- 238000005516 engineering process Methods 0.000 description 12
- 230000026731 phosphorylation Effects 0.000 description 12
- 238000006366 phosphorylation reaction Methods 0.000 description 12
- -1 tripeptides Proteins 0.000 description 12
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 11
- 241000124008 Mammalia Species 0.000 description 11
- 238000003556 assay Methods 0.000 description 11
- 230000003993 interaction Effects 0.000 description 11
- 239000000047 product Substances 0.000 description 11
- 239000006228 supernatant Substances 0.000 description 11
- 108091026890 Coding region Proteins 0.000 description 10
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 10
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 10
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 10
- 108010076504 Protein Sorting Signals Proteins 0.000 description 10
- 235000004279 alanine Nutrition 0.000 description 10
- 210000004899 c-terminal region Anatomy 0.000 description 10
- 238000004113 cell culture Methods 0.000 description 10
- 210000002865 immune cell Anatomy 0.000 description 10
- 238000009169 immunotherapy Methods 0.000 description 10
- 241000196324 Embryophyta Species 0.000 description 9
- 102100026120 IgG receptor FcRn large subunit p51 Human genes 0.000 description 9
- 239000004480 active ingredient Substances 0.000 description 9
- 230000015572 biosynthetic process Effects 0.000 description 9
- 230000000903 blocking effect Effects 0.000 description 9
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 9
- 230000004540 complement-dependent cytotoxicity Effects 0.000 description 9
- 238000012217 deletion Methods 0.000 description 9
- 230000037430 deletion Effects 0.000 description 9
- 208000035475 disorder Diseases 0.000 description 9
- 239000012737 fresh medium Substances 0.000 description 9
- 210000004408 hybridoma Anatomy 0.000 description 9
- 238000002372 labelling Methods 0.000 description 9
- 210000004698 lymphocyte Anatomy 0.000 description 9
- 238000001542 size-exclusion chromatography Methods 0.000 description 9
- 230000001988 toxicity Effects 0.000 description 9
- 231100000419 toxicity Toxicity 0.000 description 9
- 230000009261 transgenic effect Effects 0.000 description 9
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 8
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 8
- 230000003213 activating effect Effects 0.000 description 8
- 238000001042 affinity chromatography Methods 0.000 description 8
- 210000003719 b-lymphocyte Anatomy 0.000 description 8
- 230000004927 fusion Effects 0.000 description 8
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 8
- 238000011534 incubation Methods 0.000 description 8
- 230000006698 induction Effects 0.000 description 8
- 238000002347 injection Methods 0.000 description 8
- 239000007924 injection Substances 0.000 description 8
- 230000001404 mediated effect Effects 0.000 description 8
- 108020004999 messenger RNA Proteins 0.000 description 8
- 125000003729 nucleotide group Chemical group 0.000 description 8
- 238000012552 review Methods 0.000 description 8
- 229920002477 rna polymer Polymers 0.000 description 8
- 102200013599 rs452472 Human genes 0.000 description 8
- 150000003839 salts Chemical class 0.000 description 8
- 230000028327 secretion Effects 0.000 description 8
- 230000011664 signaling Effects 0.000 description 8
- NFGXHKASABOEEW-UHFFFAOYSA-N 1-methylethyl 11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate Chemical compound COC(C)(C)CCCC(C)CC=CC(C)=CC(=O)OC(C)C NFGXHKASABOEEW-UHFFFAOYSA-N 0.000 description 7
- 241000282412 Homo Species 0.000 description 7
- 101710177940 IgG receptor FcRn large subunit p51 Proteins 0.000 description 7
- 102000006496 Immunoglobulin Heavy Chains Human genes 0.000 description 7
- 108010019476 Immunoglobulin Heavy Chains Proteins 0.000 description 7
- 241000699666 Mus <mouse, genus> Species 0.000 description 7
- 239000012980 RPMI-1640 medium Substances 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- 239000002585 base Substances 0.000 description 7
- 230000008901 benefit Effects 0.000 description 7
- 230000003915 cell function Effects 0.000 description 7
- 239000006285 cell suspension Substances 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 230000001419 dependent effect Effects 0.000 description 7
- 230000002068 genetic effect Effects 0.000 description 7
- 230000013595 glycosylation Effects 0.000 description 7
- 238000006206 glycosylation reaction Methods 0.000 description 7
- 239000012642 immune effector Substances 0.000 description 7
- 229940121354 immunomodulator Drugs 0.000 description 7
- 238000003780 insertion Methods 0.000 description 7
- 230000037431 insertion Effects 0.000 description 7
- 239000002773 nucleotide Substances 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 239000000725 suspension Substances 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- 238000002560 therapeutic procedure Methods 0.000 description 7
- 230000003827 upregulation Effects 0.000 description 7
- 239000004471 Glycine Substances 0.000 description 6
- 241000238631 Hexapoda Species 0.000 description 6
- 239000004698 Polyethylene Substances 0.000 description 6
- 241000700159 Rattus Species 0.000 description 6
- 230000005888 antibody-dependent cellular phagocytosis Effects 0.000 description 6
- 230000006907 apoptotic process Effects 0.000 description 6
- 230000004069 differentiation Effects 0.000 description 6
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 102000044042 human KLRK1 Human genes 0.000 description 6
- 230000001900 immune effect Effects 0.000 description 6
- 230000001976 improved effect Effects 0.000 description 6
- 230000001939 inductive effect Effects 0.000 description 6
- 210000004962 mammalian cell Anatomy 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 238000003752 polymerase chain reaction Methods 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 230000009467 reduction Effects 0.000 description 6
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 6
- 230000001105 regulatory effect Effects 0.000 description 6
- 230000000284 resting effect Effects 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 210000004881 tumor cell Anatomy 0.000 description 6
- 101000861452 Homo sapiens Forkhead box protein P3 Proteins 0.000 description 5
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 description 5
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 5
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 5
- 239000004472 Lysine Substances 0.000 description 5
- 241000288906 Primates Species 0.000 description 5
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 5
- 230000004071 biological effect Effects 0.000 description 5
- 231100000433 cytotoxic Toxicity 0.000 description 5
- 230000001472 cytotoxic effect Effects 0.000 description 5
- 238000009472 formulation Methods 0.000 description 5
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 5
- 230000036541 health Effects 0.000 description 5
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 5
- 230000001506 immunosuppresive effect Effects 0.000 description 5
- 210000003292 kidney cell Anatomy 0.000 description 5
- 230000007246 mechanism Effects 0.000 description 5
- 230000002093 peripheral effect Effects 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 230000005180 public health Effects 0.000 description 5
- 230000004044 response Effects 0.000 description 5
- 241000894007 species Species 0.000 description 5
- 230000000638 stimulation Effects 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 238000013518 transcription Methods 0.000 description 5
- 230000035897 transcription Effects 0.000 description 5
- STMRGLKPBJVVEG-UHFFFAOYSA-N 2-(2-oxopropyl)isoindole-1,3-dione Chemical compound C1=CC=C2C(=O)N(CC(=O)C)C(=O)C2=C1 STMRGLKPBJVVEG-UHFFFAOYSA-N 0.000 description 4
- 239000004475 Arginine Substances 0.000 description 4
- 102100031491 Arylsulfatase B Human genes 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 4
- 102100027581 Forkhead box protein P3 Human genes 0.000 description 4
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 4
- 241001529936 Murinae Species 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- 230000004989 O-glycosylation Effects 0.000 description 4
- 108700026244 Open Reading Frames Proteins 0.000 description 4
- 206010035226 Plasma cell myeloma Diseases 0.000 description 4
- 239000002202 Polyethylene glycol Substances 0.000 description 4
- 241000283984 Rodentia Species 0.000 description 4
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 4
- 230000002411 adverse Effects 0.000 description 4
- 238000010171 animal model Methods 0.000 description 4
- 239000002246 antineoplastic agent Substances 0.000 description 4
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 4
- 235000003704 aspartic acid Nutrition 0.000 description 4
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 238000013368 capillary electrophoresis sodium dodecyl sulfate analysis Methods 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 210000004748 cultured cell Anatomy 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 150000002148 esters Chemical class 0.000 description 4
- 210000003527 eukaryotic cell Anatomy 0.000 description 4
- 230000002349 favourable effect Effects 0.000 description 4
- IKAIKUBBJHFNBZ-UHFFFAOYSA-N glycyl-lysine Chemical group NCCCCC(C(O)=O)NC(=O)CN IKAIKUBBJHFNBZ-UHFFFAOYSA-N 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 229940072221 immunoglobulins Drugs 0.000 description 4
- 239000004615 ingredient Substances 0.000 description 4
- 239000003094 microcapsule Substances 0.000 description 4
- 201000000050 myeloid neoplasm Diseases 0.000 description 4
- 229920001223 polyethylene glycol Polymers 0.000 description 4
- 229920000642 polymer Polymers 0.000 description 4
- 230000003389 potentiating effect Effects 0.000 description 4
- 239000003755 preservative agent Substances 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 230000002062 proliferating effect Effects 0.000 description 4
- 238000000159 protein binding assay Methods 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 238000002741 site-directed mutagenesis Methods 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 239000003381 stabilizer Substances 0.000 description 4
- 239000011550 stock solution Substances 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 230000014616 translation Effects 0.000 description 4
- 102000003390 tumor necrosis factor Human genes 0.000 description 4
- 101710117290 Aldo-keto reductase family 1 member C4 Proteins 0.000 description 3
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 3
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 description 3
- 206010006187 Breast cancer Diseases 0.000 description 3
- 241000282693 Cercopithecidae Species 0.000 description 3
- 206010009944 Colon cancer Diseases 0.000 description 3
- 108020004635 Complementary DNA Proteins 0.000 description 3
- 102220497066 DNA dC->dU-editing enzyme APOBEC-3C_L72D_mutation Human genes 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 3
- 206010062016 Immunosuppression Diseases 0.000 description 3
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 3
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 3
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 3
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 description 3
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 102220509039 Platelet-activating factor acetylhydrolase IB subunit beta_L72A_mutation Human genes 0.000 description 3
- 102220509020 Platelet-activating factor acetylhydrolase IB subunit beta_L72E_mutation Human genes 0.000 description 3
- 102220477050 Protein C-ets-1_F42N_mutation Human genes 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 3
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 3
- 239000004473 Threonine Substances 0.000 description 3
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 3
- 102220618307 YLP motif-containing protein 1_F42E_mutation Human genes 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 230000001270 agonistic effect Effects 0.000 description 3
- 230000004075 alteration Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000000890 antigenic effect Effects 0.000 description 3
- 239000003963 antioxidant agent Substances 0.000 description 3
- 235000006708 antioxidants Nutrition 0.000 description 3
- 235000009582 asparagine Nutrition 0.000 description 3
- 229960001230 asparagine Drugs 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 238000010804 cDNA synthesis Methods 0.000 description 3
- 238000007385 chemical modification Methods 0.000 description 3
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 230000021615 conjugation Effects 0.000 description 3
- 239000003431 cross linking reagent Substances 0.000 description 3
- 239000003405 delayed action preparation Substances 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 238000010494 dissociation reaction Methods 0.000 description 3
- 230000005593 dissociations Effects 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003623 enhancer Substances 0.000 description 3
- 238000001502 gel electrophoresis Methods 0.000 description 3
- 235000013922 glutamic acid Nutrition 0.000 description 3
- 239000004220 glutamic acid Substances 0.000 description 3
- 239000000710 homodimer Substances 0.000 description 3
- 230000036737 immune function Effects 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 230000002163 immunogen Effects 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 238000001802 infusion Methods 0.000 description 3
- 238000001990 intravenous administration Methods 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- 210000001672 ovary Anatomy 0.000 description 3
- 238000010647 peptide synthesis reaction Methods 0.000 description 3
- 238000002823 phage display Methods 0.000 description 3
- 239000000546 pharmaceutical excipient Substances 0.000 description 3
- COLNVLDHVKWLRT-QMMMGPOBSA-N phenylalanine group Chemical group N[C@@H](CC1=CC=CC=C1)C(=O)O COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 3
- 230000036470 plasma concentration Effects 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 230000002035 prolonged effect Effects 0.000 description 3
- 238000003259 recombinant expression Methods 0.000 description 3
- 230000004043 responsiveness Effects 0.000 description 3
- 102200150061 rs1553765909 Human genes 0.000 description 3
- 102220000379 rs397514441 Human genes 0.000 description 3
- 102220344309 rs397514441 Human genes 0.000 description 3
- 102220339220 rs771012029 Human genes 0.000 description 3
- 230000003248 secreting effect Effects 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 230000009870 specific binding Effects 0.000 description 3
- 235000000346 sugar Nutrition 0.000 description 3
- 231100000440 toxicity profile Toxicity 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 210000005253 yeast cell Anatomy 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Natural products CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 2
- 102220540040 Alkaline phosphatase, placental type_R38A_mutation Human genes 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 206010005003 Bladder cancer Diseases 0.000 description 2
- 208000003174 Brain Neoplasms Diseases 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 2
- 241000699802 Cricetulus griseus Species 0.000 description 2
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 2
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 2
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical class C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 208000008839 Kidney Neoplasms Diseases 0.000 description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 2
- 241000282567 Macaca fascicularis Species 0.000 description 2
- 241000829100 Macaca mulatta polyomavirus 1 Species 0.000 description 2
- 206010027476 Metastases Diseases 0.000 description 2
- 241000699660 Mus musculus Species 0.000 description 2
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Natural products OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 2
- 108091007960 PI3Ks Proteins 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Natural products OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 108090000430 Phosphatidylinositol 3-kinases Proteins 0.000 description 2
- 102000003993 Phosphatidylinositol 3-kinases Human genes 0.000 description 2
- 206010060862 Prostate cancer Diseases 0.000 description 2
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 2
- 102220472091 Protein ENL_D20T_mutation Human genes 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 206010038389 Renal cancer Diseases 0.000 description 2
- 208000000453 Skin Neoplasms Diseases 0.000 description 2
- 230000024932 T cell mediated immunity Effects 0.000 description 2
- 108091008874 T cell receptors Proteins 0.000 description 2
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 2
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 2
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 210000000612 antigen-presenting cell Anatomy 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 239000008228 bacteriostatic water for injection Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 239000011230 binding agent Substances 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 238000002619 cancer immunotherapy Methods 0.000 description 2
- 238000005251 capillar electrophoresis Methods 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical compound OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 description 2
- 238000001516 cell proliferation assay Methods 0.000 description 2
- 238000012054 celltiter-glo Methods 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000002648 combination therapy Methods 0.000 description 2
- 230000001447 compensatory effect Effects 0.000 description 2
- 238000004590 computer program Methods 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 102000003675 cytokine receptors Human genes 0.000 description 2
- 108010057085 cytokine receptors Proteins 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 238000002784 cytotoxicity assay Methods 0.000 description 2
- 231100000263 cytotoxicity test Toxicity 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 229940039227 diagnostic agent Drugs 0.000 description 2
- 239000000032 diagnostic agent Substances 0.000 description 2
- 239000000539 dimer Substances 0.000 description 2
- 238000006471 dimerization reaction Methods 0.000 description 2
- 239000002612 dispersion medium Substances 0.000 description 2
- 230000003828 downregulation Effects 0.000 description 2
- 230000009977 dual effect Effects 0.000 description 2
- 210000003162 effector t lymphocyte Anatomy 0.000 description 2
- 230000008030 elimination Effects 0.000 description 2
- 238000003379 elimination reaction Methods 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 210000002744 extracellular matrix Anatomy 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 238000002523 gelfiltration Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 229940093915 gynecological organic acid Drugs 0.000 description 2
- 201000010536 head and neck cancer Diseases 0.000 description 2
- 208000014829 head and neck neoplasm Diseases 0.000 description 2
- 102000049018 human NCAM1 Human genes 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 238000000099 in vitro assay Methods 0.000 description 2
- 230000036512 infertility Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 239000002050 international nonproprietary name Substances 0.000 description 2
- 238000001361 intraarterial administration Methods 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 238000001155 isoelectric focusing Methods 0.000 description 2
- YWXYYJSYQOXTPL-SLPGGIOYSA-N isosorbide mononitrate Chemical compound [O-][N+](=O)O[C@@H]1CO[C@@H]2[C@@H](O)CO[C@@H]21 YWXYYJSYQOXTPL-SLPGGIOYSA-N 0.000 description 2
- 201000010982 kidney cancer Diseases 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 210000005229 liver cell Anatomy 0.000 description 2
- 201000005202 lung cancer Diseases 0.000 description 2
- 208000020816 lung neoplasm Diseases 0.000 description 2
- RLSSMJSEOOYNOY-UHFFFAOYSA-N m-cresol Chemical compound CC1=CC=CC(O)=C1 RLSSMJSEOOYNOY-UHFFFAOYSA-N 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 238000007726 management method Methods 0.000 description 2
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 2
- 229930182817 methionine Natural products 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 108010068617 neonatal Fc receptor Proteins 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 150000007524 organic acids Chemical class 0.000 description 2
- 235000005985 organic acids Nutrition 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- AQIXEPGDORPWBJ-UHFFFAOYSA-N pentan-3-ol Chemical compound CCC(O)CC AQIXEPGDORPWBJ-UHFFFAOYSA-N 0.000 description 2
- 230000000144 pharmacologic effect Effects 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- 229920001451 polypropylene glycol Polymers 0.000 description 2
- 230000004481 post-translational protein modification Effects 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 230000000770 proinflammatory effect Effects 0.000 description 2
- 125000001500 prolyl group Chemical group [H]N1C([H])(C(=O)[*])C([H])([H])C([H])([H])C1([H])[H] 0.000 description 2
- 230000000069 prophylactic effect Effects 0.000 description 2
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 2
- 230000006337 proteolytic cleavage Effects 0.000 description 2
- 230000002685 pulmonary effect Effects 0.000 description 2
- 230000002285 radioactive effect Effects 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- GHMLBKRAJCXXBS-UHFFFAOYSA-N resorcinol Chemical compound OC1=CC=CC(O)=C1 GHMLBKRAJCXXBS-UHFFFAOYSA-N 0.000 description 2
- 238000002702 ribosome display Methods 0.000 description 2
- 102200001994 rs121908462 Human genes 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 201000000849 skin cancer Diseases 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000010532 solid phase synthesis reaction Methods 0.000 description 2
- 239000000600 sorbitol Substances 0.000 description 2
- 230000000087 stabilizing effect Effects 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000007910 systemic administration Methods 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 231100001274 therapeutic index Toxicity 0.000 description 2
- 239000004308 thiabendazole Substances 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 230000002103 transcriptional effect Effects 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 201000005112 urinary bladder cancer Diseases 0.000 description 2
- 239000004474 valine Substances 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- QBYIENPQHBMVBV-HFEGYEGKSA-N (2R)-2-hydroxy-2-phenylacetic acid Chemical compound O[C@@H](C(O)=O)c1ccccc1.O[C@@H](C(O)=O)c1ccccc1 QBYIENPQHBMVBV-HFEGYEGKSA-N 0.000 description 1
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- UKAUYVFTDYCKQA-UHFFFAOYSA-N -2-Amino-4-hydroxybutanoic acid Natural products OC(=O)C(N)CCO UKAUYVFTDYCKQA-UHFFFAOYSA-N 0.000 description 1
- ASJSAQIRZKANQN-UHFFFAOYSA-N 2-deoxypentose Chemical compound OCC(O)C(O)CC=O ASJSAQIRZKANQN-UHFFFAOYSA-N 0.000 description 1
- BRMWTNUJHUMWMS-UHFFFAOYSA-N 3-Methylhistidine Natural products CN1C=NC(CC(N)C(O)=O)=C1 BRMWTNUJHUMWMS-UHFFFAOYSA-N 0.000 description 1
- 229940117976 5-hydroxylysine Drugs 0.000 description 1
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 108091008875 B cell receptors Proteins 0.000 description 1
- 230000003844 B-cell-activation Effects 0.000 description 1
- 206010005949 Bone cancer Diseases 0.000 description 1
- 208000018084 Bone neoplasm Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 102100033640 Bromodomain-containing protein 1 Human genes 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 101800005309 Carboxy-terminal peptide Proteins 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 241000282552 Chlorocebus aethiops Species 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 239000012625 DNA intercalator Substances 0.000 description 1
- 108010049207 Death Domain Receptors Proteins 0.000 description 1
- 102000009058 Death Domain Receptors Human genes 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 108010016626 Dipeptides Proteins 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 206010014733 Endometrial cancer Diseases 0.000 description 1
- 206010014759 Endometrial neoplasm Diseases 0.000 description 1
- 102000005593 Endopeptidases Human genes 0.000 description 1
- 108010059378 Endopeptidases Proteins 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 1
- 206010015866 Extravasation Diseases 0.000 description 1
- 101150064015 FAS gene Proteins 0.000 description 1
- 102000015212 Fas Ligand Protein Human genes 0.000 description 1
- 108010039471 Fas Ligand Protein Proteins 0.000 description 1
- 102100036089 Fascin Human genes 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 102220630762 Fibrinogen alpha chain_R38N_mutation Human genes 0.000 description 1
- 102220630764 Fibrinogen alpha chain_R38S_mutation Human genes 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- 102000002812 Heat-Shock Proteins Human genes 0.000 description 1
- 108010004889 Heat-Shock Proteins Proteins 0.000 description 1
- 206010019837 Hepatocellular injury Diseases 0.000 description 1
- 102100026122 High affinity immunoglobulin gamma Fc receptor I Human genes 0.000 description 1
- 101000690301 Homo sapiens Aldo-keto reductase family 1 member C4 Proteins 0.000 description 1
- 101000869050 Homo sapiens Caveolae-associated protein 2 Proteins 0.000 description 1
- 101000935587 Homo sapiens Flavin reductase (NADPH) Proteins 0.000 description 1
- 101000913074 Homo sapiens High affinity immunoglobulin gamma Fc receptor I Proteins 0.000 description 1
- 101000878602 Homo sapiens Immunoglobulin alpha Fc receptor Proteins 0.000 description 1
- 101000878605 Homo sapiens Low affinity immunoglobulin epsilon Fc receptor Proteins 0.000 description 1
- 101000917826 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor II-a Proteins 0.000 description 1
- 101000917824 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor II-b Proteins 0.000 description 1
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 description 1
- 101001116548 Homo sapiens Protein CBFA2T1 Proteins 0.000 description 1
- 101000611023 Homo sapiens Tumor necrosis factor receptor superfamily member 6 Proteins 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 102000013463 Immunoglobulin Light Chains Human genes 0.000 description 1
- 108010065825 Immunoglobulin Light Chains Proteins 0.000 description 1
- 102100038005 Immunoglobulin alpha Fc receptor Human genes 0.000 description 1
- 238000012695 Interfacial polymerization Methods 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- UKAUYVFTDYCKQA-VKHMYHEASA-N L-homoserine Chemical compound OC(=O)[C@@H](N)CCO UKAUYVFTDYCKQA-VKHMYHEASA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 1
- 102100038007 Low affinity immunoglobulin epsilon Fc receptor Human genes 0.000 description 1
- 102100029204 Low affinity immunoglobulin gamma Fc region receptor II-a Human genes 0.000 description 1
- 102100029193 Low affinity immunoglobulin gamma Fc region receptor III-A Human genes 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 239000012515 MabSelect SuRe Substances 0.000 description 1
- 239000004907 Macro-emulsion Substances 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 206010027480 Metastatic malignant melanoma Diseases 0.000 description 1
- 206010050513 Metastatic renal cell carcinoma Diseases 0.000 description 1
- 102000029749 Microtubule Human genes 0.000 description 1
- 108091022875 Microtubule Proteins 0.000 description 1
- 102220555203 Myoblast determination protein 1_R38E_mutation Human genes 0.000 description 1
- JDHILDINMRGULE-LURJTMIESA-N N(pros)-methyl-L-histidine Chemical compound CN1C=NC=C1C[C@H](N)C(O)=O JDHILDINMRGULE-LURJTMIESA-N 0.000 description 1
- 230000004988 N-glycosylation Effects 0.000 description 1
- 108010001657 NK Cell Lectin-Like Receptor Subfamily K Proteins 0.000 description 1
- 102000000812 NK Cell Lectin-Like Receptor Subfamily K Human genes 0.000 description 1
- 102220537072 NKG2-D type II integral membrane protein_K35E_mutation Human genes 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 1
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000282577 Pan troglodytes Species 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 208000006994 Precancerous Conditions Diseases 0.000 description 1
- 102100040678 Programmed cell death protein 1 Human genes 0.000 description 1
- 101710089372 Programmed cell death protein 1 Proteins 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 206010037423 Pulmonary oedema Diseases 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- IWYDHOAUDWTVEP-UHFFFAOYSA-N R-2-phenyl-2-hydroxyacetic acid Natural products OC(=O)C(O)C1=CC=CC=C1 IWYDHOAUDWTVEP-UHFFFAOYSA-N 0.000 description 1
- 241000700157 Rattus norvegicus Species 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 208000015634 Rectal Neoplasms Diseases 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- 241000714474 Rous sarcoma virus Species 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 1
- 241000256251 Spodoptera frugiperda Species 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- 230000020385 T cell costimulation Effects 0.000 description 1
- 230000037453 T cell priming Effects 0.000 description 1
- 230000006052 T cell proliferation Effects 0.000 description 1
- 102220500923 Telomerase reverse transcriptase_K35D_mutation Human genes 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 102000003978 Tissue Plasminogen Activator Human genes 0.000 description 1
- 108090000373 Tissue Plasminogen Activator Proteins 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 108060008683 Tumor Necrosis Factor Receptor Proteins 0.000 description 1
- 102100040247 Tumor necrosis factor Human genes 0.000 description 1
- 102100040403 Tumor necrosis factor receptor superfamily member 6 Human genes 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 208000002495 Uterine Neoplasms Diseases 0.000 description 1
- 101150117115 V gene Proteins 0.000 description 1
- 244000000188 Vaccinium ovalifolium Species 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 239000003070 absorption delaying agent Substances 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 102000035181 adaptor proteins Human genes 0.000 description 1
- 108091005764 adaptor proteins Proteins 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 230000001919 adrenal effect Effects 0.000 description 1
- 108700025316 aldesleukin Proteins 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 239000002168 alkylating agent Substances 0.000 description 1
- 229940100198 alkylating agent Drugs 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 230000009435 amidation Effects 0.000 description 1
- 238000007112 amidation reaction Methods 0.000 description 1
- 150000003862 amino acid derivatives Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 239000004037 angiogenesis inhibitor Substances 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000340 anti-metabolite Effects 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 230000005809 anti-tumor immunity Effects 0.000 description 1
- 230000009830 antibody antigen interaction Effects 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 229940100197 antimetabolite Drugs 0.000 description 1
- 239000002256 antimetabolite Substances 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 238000003782 apoptosis assay Methods 0.000 description 1
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 238000003705 background correction Methods 0.000 description 1
- 229960000686 benzalkonium chloride Drugs 0.000 description 1
- 229960001950 benzethonium chloride Drugs 0.000 description 1
- UREZNYTWGJKWBI-UHFFFAOYSA-M benzethonium chloride Chemical compound [Cl-].C1=CC(C(C)(C)CC(C)(C)C)=CC=C1OCCOCC[N+](C)(C)CC1=CC=CC=C1 UREZNYTWGJKWBI-UHFFFAOYSA-M 0.000 description 1
- 235000019445 benzyl alcohol Nutrition 0.000 description 1
- CADWTSSKOVRVJC-UHFFFAOYSA-N benzyl(dimethyl)azanium;chloride Chemical compound [Cl-].C[NH+](C)CC1=CC=CC=C1 CADWTSSKOVRVJC-UHFFFAOYSA-N 0.000 description 1
- 238000013357 binding ELISA Methods 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- HUTDDBSSHVOYJR-UHFFFAOYSA-H bis[(2-oxo-1,3,2$l^{5},4$l^{2}-dioxaphosphaplumbetan-2-yl)oxy]lead Chemical compound [Pb+2].[Pb+2].[Pb+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O HUTDDBSSHVOYJR-UHFFFAOYSA-H 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 108010006025 bovine growth hormone Proteins 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- LRHPLDYGYMQRHN-UHFFFAOYSA-N butyl alcohol Substances CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 208000035269 cancer or benign tumor Diseases 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 208000019065 cervical carcinoma Diseases 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 230000006690 co-activation Effects 0.000 description 1
- 230000004186 co-expression Effects 0.000 description 1
- 238000005354 coacervation Methods 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 230000024203 complement activation Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 239000012050 conventional carrier Substances 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- HPXRVTGHNJAIIH-UHFFFAOYSA-N cyclohexanol Chemical compound OC1CCCCC1 HPXRVTGHNJAIIH-UHFFFAOYSA-N 0.000 description 1
- 230000001461 cytolytic effect Effects 0.000 description 1
- 210000005220 cytoplasmic tail Anatomy 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 239000000824 cytostatic agent Substances 0.000 description 1
- 239000002254 cytotoxic agent Substances 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 231100000599 cytotoxic agent Toxicity 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- YSMODUONRAFBET-UHFFFAOYSA-N delta-DL-hydroxylysine Natural products NCC(O)CCC(N)C(O)=O YSMODUONRAFBET-UHFFFAOYSA-N 0.000 description 1
- 230000004041 dendritic cell maturation Effects 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- UGMCXQCYOVCMTB-UHFFFAOYSA-K dihydroxy(stearato)aluminium Chemical compound CCCCCCCCCCCCCCCCCC(=O)O[Al](O)O UGMCXQCYOVCMTB-UHFFFAOYSA-K 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 1
- 239000003534 dna topoisomerase inhibitor Substances 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 230000002222 downregulating effect Effects 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 230000036267 drug metabolism Effects 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 210000003372 endocrine gland Anatomy 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 239000006167 equilibration buffer Substances 0.000 description 1
- YSMODUONRAFBET-UHNVWZDZSA-N erythro-5-hydroxy-L-lysine Chemical compound NC[C@H](O)CC[C@H](N)C(O)=O YSMODUONRAFBET-UHNVWZDZSA-N 0.000 description 1
- 201000004101 esophageal cancer Diseases 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 230000036251 extravasation Effects 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 239000010685 fatty oil Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 239000012458 free base Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 210000004475 gamma-delta t lymphocyte Anatomy 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 201000005787 hematologic cancer Diseases 0.000 description 1
- 230000002489 hematologic effect Effects 0.000 description 1
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 125000005842 heteroatom Chemical group 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 238000001794 hormone therapy Methods 0.000 description 1
- 230000005745 host immune response Effects 0.000 description 1
- 102000054751 human RUNX1T1 Human genes 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 235000011167 hydrochloric acid Nutrition 0.000 description 1
- 229920001477 hydrophilic polymer Polymers 0.000 description 1
- 229920001600 hydrophobic polymer Polymers 0.000 description 1
- 150000004679 hydroxides Chemical class 0.000 description 1
- 229920003063 hydroxymethyl cellulose Polymers 0.000 description 1
- 229940031574 hydroxymethyl cellulose Drugs 0.000 description 1
- 229960002591 hydroxyproline Drugs 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 230000008076 immune mechanism Effects 0.000 description 1
- 230000006058 immune tolerance Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 239000002955 immunomodulating agent Substances 0.000 description 1
- 230000001024 immunotherapeutic effect Effects 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 239000007972 injectable composition Substances 0.000 description 1
- 150000007529 inorganic bases Chemical class 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 238000010212 intracellular staining Methods 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 229940065638 intron a Drugs 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- FZWBNHMXJMCXLU-BLAUPYHCSA-N isomaltotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)O1 FZWBNHMXJMCXLU-BLAUPYHCSA-N 0.000 description 1
- JJWLVOIRVHMVIS-UHFFFAOYSA-N isopropylamine Chemical compound CC(C)N JJWLVOIRVHMVIS-UHFFFAOYSA-N 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- 229940043355 kinase inhibitor Drugs 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 231100000849 liver cell damage Toxicity 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000005265 lung cell Anatomy 0.000 description 1
- 210000004324 lymphatic system Anatomy 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 238000002824 mRNA display Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 229960002510 mandelic acid Drugs 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Chemical class 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 208000037819 metastatic cancer Diseases 0.000 description 1
- 208000011575 metastatic malignant neoplasm Diseases 0.000 description 1
- 208000021039 metastatic melanoma Diseases 0.000 description 1
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 description 1
- 239000004292 methyl p-hydroxybenzoate Substances 0.000 description 1
- 229960002216 methylparaben Drugs 0.000 description 1
- 239000004530 micro-emulsion Substances 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 210000004688 microtubule Anatomy 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 239000002088 nanocapsule Substances 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 210000000581 natural killer T-cell Anatomy 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 230000000926 neurological effect Effects 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000004768 organ dysfunction Effects 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 229960003104 ornithine Drugs 0.000 description 1
- LXCFILQKKLGQFO-UHFFFAOYSA-N p-hydroxybenzoic acid methyl ester Natural products COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 230000000849 parathyroid Effects 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 description 1
- 210000004303 peritoneum Anatomy 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 235000011007 phosphoric acid Nutrition 0.000 description 1
- 150000003016 phosphoric acids Chemical class 0.000 description 1
- 238000003566 phosphorylation assay Methods 0.000 description 1
- 239000003757 phosphotransferase inhibitor Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 230000001817 pituitary effect Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 239000001608 potassium adipate Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- MFDFERRIHVXMIY-UHFFFAOYSA-N procaine Chemical compound CCN(CC)CCOC(=O)C1=CC=C(N)C=C1 MFDFERRIHVXMIY-UHFFFAOYSA-N 0.000 description 1
- 229960004919 procaine Drugs 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000005522 programmed cell death Effects 0.000 description 1
- 229940087463 proleukin Drugs 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 235000010232 propyl p-hydroxybenzoate Nutrition 0.000 description 1
- 239000004405 propyl p-hydroxybenzoate Substances 0.000 description 1
- 229960003415 propylparaben Drugs 0.000 description 1
- 108020001775 protein parts Proteins 0.000 description 1
- 230000004850 protein–protein interaction Effects 0.000 description 1
- 239000003586 protic polar solvent Substances 0.000 description 1
- 208000005333 pulmonary edema Diseases 0.000 description 1
- 239000002510 pyrogen Substances 0.000 description 1
- 238000007420 radioactive assay Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 239000002464 receptor antagonist Substances 0.000 description 1
- 229940044551 receptor antagonist Drugs 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 206010038038 rectal cancer Diseases 0.000 description 1
- 201000001275 rectum cancer Diseases 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 210000003935 rough endoplasmic reticulum Anatomy 0.000 description 1
- 102200042537 rs121909608 Human genes 0.000 description 1
- 102220013601 rs397516689 Human genes 0.000 description 1
- 102220057404 rs730881766 Human genes 0.000 description 1
- 102220198260 rs772110575 Human genes 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 210000000717 sertoli cell Anatomy 0.000 description 1
- 239000008159 sesame oil Substances 0.000 description 1
- 235000011803 sesame oil Nutrition 0.000 description 1
- 230000007781 signaling event Effects 0.000 description 1
- 229940018944 simlukafusp alfa Drugs 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 239000001601 sodium adipate Substances 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 206010041823 squamous cell carcinoma Diseases 0.000 description 1
- SFVFIFLLYFPGHH-UHFFFAOYSA-M stearalkonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1 SFVFIFLLYFPGHH-UHFFFAOYSA-M 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 238000011146 sterile filtration Methods 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 238000011287 therapeutic dose Methods 0.000 description 1
- 210000000115 thoracic cavity Anatomy 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 210000001685 thyroid gland Anatomy 0.000 description 1
- 229960000187 tissue plasminogen activator Drugs 0.000 description 1
- 229940044693 topoisomerase inhibitor Drugs 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 102000003298 tumor necrosis factor receptor Human genes 0.000 description 1
- 210000003171 tumor-infiltrating lymphocyte Anatomy 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 210000002229 urogenital system Anatomy 0.000 description 1
- 206010046766 uterine cancer Diseases 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 230000008728 vascular permeability Effects 0.000 description 1
- 210000005166 vasculature Anatomy 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- C07K14/55—IL-2
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/74—Inducing cell proliferation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
- C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
Definitions
- FIG. 6A-F Proliferation and activation of CD8 T cells, NK cells and CD4 T cells within PBMCs upon treatment for 5 days with 3 different NKG2D-IL2v constructs was determined by flow cytometry. Proliferation of CD4 T cells (Fig.6A), NK cells (Fig.6B), CD8 T cells (Fig.6C), and activation by CD25 expression of CD4 T cells (Fig.6D), NK cells (Fig.6E), and CD8 T cells (Fig.6F) upon treatment with NKG2D-IL2v (5C5), NKG2D-IL2v (13C6), NKG2D-IL2v (395 cl.80), and FAP-IL2v are shown.
- NKG2D-IL2v 5C5
- NKG2D-IL2v 13C6
- NKG2D-IL2v 395 cl.80
- FAP-IL2v FAP-IL2v
- a “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to a subject.
- a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
- package insert is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
- a mutant IL-2 polypeptide shares a carboxy-terminal peptide bond with a first antibody, particularly a first Fab molecule, and further shares an amino- terminal peptide bond with a second antibody, particularly a second Fab molecule.
- a first antibody, particularly a first Fab molecule shares a carboxy-terminal peptide bond with a mutant IL-2 polypeptide, and further shares an amino-terminal peptide bond with a second antibody, particularly a second Fab molecule.
- the antibody comprises a heavy chain complementary determining region (HCDR) 1 comprising the amino acid sequence of SEQ ID NO:83, a HCDR 2 comprising the amino acid sequence of SEQ ID NO:84, and a HCDR 3 comprising the amino acid sequence of SEQ ID NO:85; and a light chain complementarity determining region (LCDR) 1 comprising the amino acid sequence of SEQ ID NO:86, a LCDR 2 comprising the amino acid sequence of SEQ ID NO:87, and a LCDR 3 comprising the amino acid sequence of SEQ ID NO:88.
- HCDR heavy chain complementary determining region
- LCDR light chain complementarity determining region
- the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to NKG2D, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising the amino acid substitutions T3A, F42A, Y45A, L72G and C125A (numbering relative to the human IL-2 sequence SEQ ID NO: 41); and wherein the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:7, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:8.
- VH heavy chain variable region
- VL light chain variable region
- the invention provides an immunoconjugate comprising a polypeptide comprising an amino acid sequence of SEQ ID NO:44, a polypeptide comprising an amino acid sequence of SEQ ID NO:45, and a polypeptide comprising an amino acid sequence of SEQ ID NO:46.
- the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to NKG2D, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising the amino acid substitutions T3A, F42A, Y45A, L72G and C125A (numbering relative to the human IL-2 sequence SEQ ID NO: 41); and wherein the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:39, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:40.
- VH heavy chain variable region
- VL light chain variable region
- compositions are lyophilized formulations or aqueous solutions.
- pharmaceutically acceptable carrier includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g.
- the invention provides a method for treating a disease in an individual.
- the method comprises administering to an individual having such disease a therapeutically effective amount of an immunoconjugate of the invention.
- a composition is administered to said invididual, comprising the immunoconjugate of the invention in a pharmaceutically acceptable form.
- the disease to be treated is a proliferative disorder.
- the disease is cancer.
- the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer.
Abstract
The present invention generally relates to immunoconjugates, particularly immunoconjugates comprising a mutant interleukin-2 polypeptide and an antibody that binds to NKG2D. In addition, the invention relates to polynucleotide molecules encoding the immunoconjugates, and vectors and host cells comprising such polynucleotide molecules. The invention further relates to methods for producing the mutant immunoconjugates, pharmaceutical compositions comprising the same, and uses thereof.
Description
Immunoconjugates
FIELD OF THE INVENTION
The present invention generally relates to immunoconjugates, particularly immunoconjugates comprising a mutant interleukin-2 polypeptide and an antibody that binds to NKG2D. In addition, the invention relates to polynucleotide molecules encoding the immunoconjugates, and vectors and host cells comprising such polynucleotide molecules. The invention further relates to methods for producing the mutant immunoconjugates, pharmaceutical compositions comprising the same, and uses thereof.
BACKGROUND Interleukin-2 (IL-2), also known as T cell growth factor (TCGF), is a 15.5 kDa globular glycoprotein playing a central role in lymphocyte generation, survival and homeostasis. It has a length of 133 amino acids and consists of four antiparallel, amphiphatic a-helices that form a quaternary structure indispensable of its function (Smith, Science 240, 1169-76 (1988); Bazan, Science 257, 410-413 (1992)). Sequences of IL-2 from different species are found under NCBI RefSeq Nos. NP000577 (human), NP032392 (mouse), NP446288 (rat) or NP517425 (chimpanzee).
IL-2 mediates its action by binding to IL-2 receptors (IL-2R), which consist of up to three individual subunits, the different association of which can produce receptor forms that differ in their affinity to IL-2. Association of the a (CD25), b (CD122), and g (yc, CD132) subunits results in a trimeric, high-affinity receptor for IL-2. Dimeric IL-2 receptor consisting of the b and g subunits is termed intermediate-affinity IL-2R. The a subunit forms the monomeric low affinity IL-2 receptor. Although the dimeric intermediate-affinity IL-2 receptor binds IL-2 with approximately 100-fold lower affinity than the trimeric high-affinity receptor, both the dimeric and the trimeric IL-2 receptor variants are able to transmit signal upon IL-2 binding (Minami et ah, Annu Rev Immunol 11, 245-268 (1993)). Hence, the a-subunit, CD25, is not essential for IL- 2 signalling. It confers high-affinity binding to its receptor, whereas the b subunit, CD 122, and
the g-subunit are crucial for signal transduction (Krieg et al., Proc Natl Acad Sci 107, 11906-11 (2010)). Trimeric IL-2 receptors including CD25 are expressed by (resting) CD4+ forkhead box P3 (FoxP3)+ regulatory T (Treg) cells. They are also transiently induced on conventional activated T cells, whereas in the resting state these cells express only dimeric IL-2 receptors. Treg cells consistently express the highest level of CD25 in vivo (Fontenot et al., Nature Immunol 6, 1142- 51 (2005)).
IL-2 is synthesized mainly by activated T-cells, in particular CD4+ helper T cells. It stimulates the proliferation and differentiation of T cells, induces the generation of cytotoxic T lymphocytes (CTLs) and the differentiation of peripheral blood lymphocytes to cytotoxic cells and lymphokine-activated killer (LAK) cells, promotes cytokine and cytolytic molecule expression by T cells, facilitates the proliferation and differentiation of B-cells and the synthesis of immunoglobulin by B-cells, and stimulates the generation, proliferation and activation of natural killer (NK) cells (reviewed e.g. in Waldmann, Nat Rev Immunol 6, 595-601 (2009); Olejniczak and Kasprzak, Med Sci Monit 14, RA179-89 (2008); Malek, Annu Rev Immunol 26, 453-79 (2008)).
Its ability to expand lymphocyte populations in vivo and to increase the effector functions of these cells confers antitumor effects to IL-2, making IL-2 immunotherapy an attractive treatment option for certain metastatic cancers. Consequently, high-dose IL-2 treatment has been approved for use in patients with metastatic renal-cell carcinoma and malignant melanoma.
However, IL-2 has a dual function in the immune response in that it not only mediates expansion and activity of effector cells, but also is crucially involved in maintaining peripheral immune tolerance.
A major mechanism underlying peripheral self-tolerance is IL-2 induced activation-induced cell death (AICD) in T cells. AICD is a process by which fully activated T cells undergo programmed cell death through engagement of cell surface-expressed death receptors such as CD95 (also known as Fas) or the TNF receptor. When antigen-activated T cells expressing a high-affinity IL-2 receptor (after previous exposure to IL-2) during proliferation are re stimulated with antigen via the T cell receptor (TCR)/CD3 complex, the expression of Fas ligand (FasL) and/or tumor necrosis factor (TNF) is induced, making the cells susceptible for Fas- mediated apoptosis. This process is IL-2 dependent (Lenardo, Nature 353, 858-61 (1991)) and mediated via STAT5. By the process of AICD in T lymphocytes tolerance can not only be
established to self-antigens, but also to persistent antigens that are clearly not part of the host’s makeup, such as tumor antigens.
Moreover, IL-2 is also involved in the maintenance of peripheral CD4+ CD25+ regulatory T (Treg) cells (Fontenot et al., Nature Immunol 6, 1142-51 (2005); D’Cruz and Klein, Nature Immunol 6, 1152-59 (2005); Maloy and Powrie, Nature Immunol 6, 1171-72 (2005), which are also known as suppressor T cells. They suppress effector T cells from destroying their (self-)target, either through cell-cell contact by inhibiting T cell help and activation, or through release of immunosuppressive cytokines such as IL-10 or TGF-b. Depletion of Treg cells was shown to enhance IL-2 induced anti-tumor immunity (Imai et al., Cancer Sci 98, 416-23 (2007)).
Therefore, IL-2 is not optimal for inhibiting tumor growth, because in the presence of IL-2 either the CTLs generated might recognize the tumor as self and undergo AICD or the immune response might be inhibited by IL-2 dependent Treg cells.
A further concern in relation to IL-2 immunotherapy are the side effects produced by recombinant human IL-2 treatment. Patients receiving high-dose IL-2 treatment frequently experience severe cardiovascular, pulmonary, renal, hepatic, gastrointestinal, neurological, cutaneous, haematological and systemic adverse events, which require intensive monitoring and in-patient management. The majority of these side effects can be explained by the development of so-called vascular (or capillary) leak syndrome (VLS), a pathological increase in vascular permeability leading to fluid extravasation in multiple organs (causing e.g. pulmonary and cutaneous edema and liver cell damage) and intravascular fluid depletion (causing a drop in blood pressure and compensatory increase in heart rate). There is no treatment of VLS other than withdrawal of IL-2. Low-dose IL-2 regimens have been tested in patients to avoid VLS, however, at the expense of suboptimal therapeutic results. VLS was believed to be caused by the release of proinflammatory cytokines, such as tumor necrosis factor (TNF)-a from IL-2-activated NK cells, however it has recently been shown that IL-2-induced pulmonary edema resulted from direct binding of IL-2 to lung endothelial cells, which expressed low to intermediate levels of functional abg IL-2 receptors (Krieg et al., Proc Nat Acad Sci USA 107, 11906-11 (2010)).
Several approaches have been taken to overcome these problems associated with IL-2 immunotherapy. For example, it has been found that the combination of IL-2 with certain anti- IL-2 monoclonal antibodies enhances treatment effects of IL-2 in vivo (Kamimura et al., J Immunol 177, 306-14 (2006); Boyman et al., Science 311, 1924-27 (2006)). In an alternative
approach, IL-2 has been mutated in various ways to reduce its toxicity and/or increase its efficacy. Hu et al. (Blood 101, 4853-4861 (2003), US Pat. Publ. No. 2003/0124678) have substituted the arginine residue in position 38 of IL-2 by tryptophan to eliminate IL-2’s vasopermeability activity. Shanafelt et al. (Nature Biotechnol 18, 1197-1202 (2000)) have mutated asparagine 88 to arginine to enhance selectivity for T cells over NK cells. Heaton et al. (Cancer Res 53, 2597-602 (1993); US Pat. No. 5,229,109) have introduced two mutations, Arg38Ala and Phe42Lys, to reduce the secretion of proinflammatory cytokines from NK cells. Gillies et al. (US Pat. Publ. No. 2007/0036752) have substituted three residues of IL-2 (Asp20Thr, Asn88Arg, and Glnl26Asp) that contribute to affinity for the intermediate-affinity IL-2 receptor to reduce VLS. Gillies et al. (WO 2008/0034473) have also mutated the interface of IL-2 with CD25 by amino acid substitution Arg38Trp and Phe42Lys to reduce interaction with CD25 and activation of Treg cells for enhancing efficacy. To the same aim, Wittrup et al. (WO 2009/061853) have produced IL-2 mutants that have enhanced affinity to CD25, but do not activate the receptor, thus act as antagonists. The mutations introduced were aimed at disrupting the interaction with the b- and/or g-subunit of the receptor.
A particular mutant IL-2 polypeptide, designed to overcome the above-mentioned problems associated with IL-2 immunotherapy (toxicity caused by the induction of VLS, tumor tolerance caused by the induction of AICD, and immunosuppression caused by activation of Treg cells), is described in WO 2012/107417. Substitution of the phenylalanine residue at position 42 by alanine, the tyrosine residue at position 45 by alanine and the leucine residue at position 72 of IL-2 by glycine essentially abolishes binding of this mutant IL-2 polypeptide to the a-subunit of the IL-2 receptor (CD25).
Further to the above-mentioned approaches, IL-2 immunotherapy may be improved by selectively targeting IL-2 to tumors, e.g. in the form of immunoconjugates comprising an antibody that binds to an antigen expressed on tumor cells. Several such immunoconjugates have been described (see e.g. Ko et al., J Immunother (2004) 27, 232-239; Klein et al., Oncoimmunology (2017) 6(3), el277306).
Tumors may be able, however, to escape such targeting by shedding, mutating or downregulating the target antigen of the antibody. Moreover, tumor-targeted IL-2 may not come into optimal contact with effector cells such as cytotoxic T lymphocytes (CTLs), in tumor microenvironments that actively exclude lymphocytes.
Thus there remains a need to further improve IL-2 immunotherapy. An approach, which may circumvent the problems of tumor-targeting, is to target IL-2 directly to effector cells, in particular CTLs.
NKG2D is an activating receptor expressed on cytotoxic effector cells described for the first time in 1991 (Houchnins et al. (1991) J Exp Med 173, 1017-1020). It has no own signaling motif in the cytoplasmic tail but associates via charged amino acids with the adapter protein DNAX activating protein of 10 kDa (DAPIO). DAPIO has a cytoplasmic YxxM motif, which recruits phosphatidylinositol 3-kinase (PI3K) after phosphorylation at its tyrosine residue eventually resulting in the activation of NK cells, cytotoxicity and CD8 T cell co-stimulation. NKG2D is constitutively expressed on almost all NK cells, CD8 T cells, gd T cells and on a subset of NKT cells but not in normal tissues (Bauer et al. (1999) Science 285, 727-729). NKG2D expression can be modulated by different cytokines; IL-2 and IL-15 induce upregulation whereas TGFP and IL-21 were shown to down-modulate NKG2D. Also on tumor infiltrating lymphocytes NKG2D can be detected.
NKG2D serves as a sensor for transformed cells via the upregulation of NKG2D ligands (NKG2DL). Many viruses and tumors have developed mechanisms to evade the sensing via NKG2D, suggesting that this receptor plays an important role in the immunosurveillance of tumors and virus infections and making it a compelling target for cancer immunotherapy.
An anti-NKG2D antibody with dual antagonistic and agonistic activity, KYK-2.0, has been reported by Kwong et al. (Kwong et al. (2008) J Mol Biol 384, 1143-1156; WO 2010/017103). A bispecific antibody derived therefrom has been reported in WO 2016/134371. Trispecific antibodies targeting NKG2D, CD 16 and a tumor-associated antigen have been reported e.g. in WO 2018/148445.
Ghasemi et al. have described a fusion protein of IL-2 and an NKG2D binding protein (Ghashemi et al., Nat Comm (2016) 7, 12878), for targeting IL-2 to NKG2D-bearing cells such as natural killer (NK) cells.
There remains a need, however, for antibodies and immunoconjugates targeting NKG2D with improved efficacy and/or safety, e.g. for use in cancer immunotherapy.
SUMMARY OF THE INVENTION
The present invention provides a novel approach of targeting a mutant form of IL-2 with advantageous properties for immunotherapy directly to immune effector cells, such as cytotoxic T lymphocytes, rather than tumor cells. Targeting to immune effector cells is achieved by recombinant fusion of the mutant IL-2 molecule to an antibody that binds to NKG2D.
The IL-2 mutant used in the present invention has been designed to overcome the problems associated with IL-2 immunotherapy, in particular toxicity caused by the induction of VLS, tumor tolerance caused by the induction of AICD, and immunosuppression caused by activation of Treg cells. In addition to circumventing escape of tumors from tumor-targeting as mentioned above, targeting of the IL-2 mutant to immune effector cells may further increase the preferential activation of CTLs over immunosuppressive Treg cells. By using an antibody that binds to NKG2D, specific ‘in cis’ targeting of IL2 to cytotoxic T lymphocytes, NK cells, and other cytotoxic immune effector cells is achieved, thus further enhancing the immune response.
In a first aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to NKG2D, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising the amino acid substitutions F42A, Y45A and L72G (numbering relative to the human IL-2 sequence SEQ ID NO: 41).
In some embodiments the immunoconjugate accordint to the invention, the antibody comprises (i) a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 1, a HCDR 2 of SEQ ID NO: 2, and a HCDR 3 of SEQ ID NO: 3, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 4, a LCDR 2 of SEQ ID NO: 5 and a LCDR 3 of SEQ ID NO: 6; (ii) a VH comprising a HCDR 1 of SEQ ID NO: 9, a HCDR 2 of SEQ ID NO: 10, and a HCDR 3 of SEQ ID NO: 11, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 12, a LCDR 2 of SEQ ID NO: 13 and a LCDR 3 of SEQ ID NO: 14; (iii) a VH comprising a HCDR 1 of SEQ ID NO: 17, a HCDR 2 of SEQ ID NO: 18, and a HCDR 3 of SEQ ID NO: 19, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 20, a LCDR 2 of SEQ ID NO: 21 and a LCDR 3 of SEQ ID NO: 22; (iv) a VH comprising a HCDR 1 of SEQ ID NO: 25, a HCDR 2 of SEQ ID NO: 26, and a HCDR 3 of SEQ ID NO: 27, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 28, a LCDR 2 of SEQ ID NO: 29 and a LCDR 3 of SEQ ID NO: 30;
(v) a VH comprising a HCDR 1 of SEQ ID NO: 33, a HCDR 2 of SEQ ID NO: 34, and a HCDR 3 of SEQ ID NO: 35, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 36, a LCDR 2 of SEQ ID NO: 37 and a LCDR 3 of SEQ ID NO: 38; or (vi) a VH comprising a HCDR 1 of SEQ ID NO: 83, a HCDR 2 of SEQ ID NO: 84, and a HCDR 3 of SEQ ID NO: 85, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 86, a LCDR 2 of SEQ ID NO: 87 and a LCDR 3 of SEQ ID NO: 88.
In some embodiments of the immunoconjugate according to the invention, the antibody comprises (i) (a) a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:7, and (b) a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:8; (ii) (a) a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 15, and (b) a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16; (iii) (a) a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:23, and (b) a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:24; (iv) (a) a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:31, and (b) a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:32; (v) (a) a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:39, and (b) a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:40; or (vi) (a) a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:89, and (b) a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:90. In some embodiments of the immunoconjugate according to the invention, the mutant IL-2 polypeptide further comprises the amino acid substitution T3A and/or the amino acid substitution C125A. In some embodiments, the mutant IL-2 polypeptide comprises the sequence of SEQ ID NO: 42. In some embodiments
the the immunoconjugate comprises not more than one mutant IL-2 polypeptide. In some emebodiments, the antibody comprises an Fc domain composed of a first and a second subunit. In some emebodiments, the Fc domain is an IgG class, particularly an IgGl subclass, Fc domain. In some embodiments, the Fc domain is a human Fc domain. In some embodiments, the antibody is an IgG class, particularly an IgGl subclass immunoglobulin.
In some emebodiments, the Fc domain comprises a modification promoting the association of the first and the second subunit of the Fc domain. In some emebodiments, the CH3 domain of the first subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable. In some emebodiments, the first subunit of the Fc domain the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the second subunit of the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V) and optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numberings according to Kabat EU index). In some emebodiments, in the first subunit of the Fc domain additionally the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C), and in the second subunit of the Fc domain additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (numberings according to Kabat EU index). In some emebodiments, the mutant IL-2 polypeptide is fused at its amino-terminal amino acid to the carboxy -terminal amino acid of one of the subunits of the Fc domain, particularly the first subunit of the Fc domain, optionally through a linker peptide. In some emebodiments, the linker peptide has the amino acid sequence of SEQ ID NO:43.
In some emebodiments, wherein the immunoconjugate comprises an Fc domain, the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor, particularly an Fey receptor, and/or effector function, particularly antibody-dependent cell- mediated cytotoxicity (ADCC). In some such embodiments, said one or more amino acid substitution is at one or more position selected from the group of L234, L235, and P329 (Kabat
EU index numbering). In some embodiments, each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering).
In some embodiments, the immunoconjugate according to the invention comprises a polypeptide (i) comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:44, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:45, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:46; (ii) comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:47, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:48, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:49; (iii) comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:50, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:51, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:52; (iv) comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:53, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:54, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:55; (v) comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:56, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:57, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:58; or (vi) comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:91, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to
the sequence of SEQ ID NO:92, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:93. In some embodiemnts, the immunoconjugate essentially consists of a mutant IL- 2 polypeptide and an IgGl immunoglobulin molecule, joined by a linker sequence.
The invention further provides one or more isolated polynucleotide encoding an immunoconjugate of the invention, one or more vector, particularly expression vector, comprising said polynucleotides, and a host cell comprising said polynucleotide(s) or said vector(s).
Also provided by the invention is a method of producing an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to NKG2D, comprising (a) culturing the host cell of the invention under conditions suitable for the expression of the immunoconjugate, and optionally (b) recovering the immunoconjugate. Also provided by the invention is an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to NKG2D, produced by said method.
The invention further provides a pharmaceutical composition comprising an immunoconjugate of the invention and a pharmaceutically acceptable carrier, and methods of using an immunoconjugate of the invention.
In particular, the invention encompasses an immunoconjugate according to the invention for use as a medicament, and for use in the treatment of a disease. In a particular embodiment, said disease is cancer.
Also encompassed by the invention is the use of an immunoconjugate according to the invention in the manufacture of a medicament for the treatment of a disease. In a particular embodiment, said disease is cancer.
Further provided is a method of treating disease in an individual, comprising administering to said individual a therapeutically effective amount of a composition comprising an immunoconjugate according to the invention in a pharmaceutically acceptable form. In a particular embodiment, said disease is cancer.
Also provided is a method of stimulating the immune system of an individual, comprising administering to said individual an effective amount of a composition comprising an immunoconjugate according to the invention in a pharmaceutically acceptable form.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Schematic representation of the IgG-IL-2 immunoconjugate format, comprising mutant IL-2 polypeptide (NKG2D-targeted IgG PG LALA with IL2v fused to the C-terminus of the Fc knob chain).
Figure 2A-B. Binding of NKG2D-IL2v fusion proteins, the respective IgG controls and CEA- IL2v to NK92 cells was determined by flow cytometry. Figure 2A compares NKG2D(320)-IL2v, NKG2D(320) IgG and CEA-IL2v. Figure 2B compares NKG2D(13C6)-IL2v, NKG2DQ3C6) IgG and CEA-IL2v.
Figure 3A-F. Proliferation and activation of CD8 T cells, NK cells and CD4 T cells within PBMCs upon treatment for 5 days with NKG2D-IL2v and CEA-IL2v was determined by flow cytometry. Proliferation of NK cells (Fig.3 A), CD8 T cells (Fig.3B) and CD4 T cells (Fig.3C), and activation by CD25 expression of NK cells (Fig. 3D), CD8 T cells (Fig.3E) and CD4 T cells (Fig.3F) upon treatment with NKG2D(320)-IL2v, NK62D(13C6)-IL2v and CEA-IL2v are shown.
Figure 4A-L. Proliferation and activation of CD8 T cells, NK cells and CD4 T cells within PBMCs upon treatment for 5 days with NKG2D-IL2v alone (Fig.4A-F) or in the presence of a blocking NKG2D antibody was determined by flow cytometry (Fig.4A-F) and compared to CEA-IL2v alone or int presence of blocking NKG2D IgG (Fig.4G-L). Proliferation of CD8 T cells (Fig.4A), NK cells (Fig.4B) and CD4 T cells (Fig.4C), and activation by CD25 expression of CD8 T cells (Fig. 4D), NK cells (Fig.4E) and CD4 T cells (Fig.4F) upon treatment with NKG2D(320)-IL2v and the combination of NKG2D(320)-IL2v and blocking NKG2D IgG are shown. Proliferation of CD8 T cells (Fig.4G), NK cells (Fig.4H) and CD4 T cells (Fig.41), and activation by CD25 expression of CD8 T cells (Fig. 4J), NK cells (Fig.4K) and CD4 T cells (Fig.4L) upon treatment with CEA-IL2v and the combination of CEA-IL2v and blocking NKG2D IgG are shown.
Figure 5A-H. Proliferation and activation of CD8 T cells, NK cells and CD4 T cells within PBMCs upon treatment for 5 days with 4 different NKG2D-IL2v constructs alone (Fig.5A-F) or proliferation and activation of CD8 T cells in the presence of recombinant NKG2D (Fig.5G-H) was determined by flow cytometry. Proliferation of CD8 T cells (Fig.5A), NK cells (Fig.5B) and CD4 T cells (Fig.5C), and activation by CD25 expression of CD8 T cells (Fig.5D), NK cells (Fig.5E) and CD4 T cells (Fig.5F) upon treatment with NKG2D(13C6)-IL2v, NKG2D(320)- IL2v, NKG2D(014)-IL2v and NKG2D(C5C)-IL2v are shown. Proliferation of CD8 T cells
(Fig.5G) and activation by CD25 expression of CD8 T cells (Fig.5H) upon treatment with NKG2D( 13 C6)-IL2v, NKG2D(320)-IL2v, NKG2D(014)-IL2v and NKG2D(C5C)-IL2v in the presence of soluble (recombinant) NKG2D are shown.
Figure 6A-F. Proliferation and activation of CD8 T cells, NK cells and CD4 T cells within PBMCs upon treatment for 5 days with 3 different NKG2D-IL2v constructs was determined by flow cytometry. Proliferation of CD4 T cells (Fig.6A), NK cells (Fig.6B), CD8 T cells (Fig.6C), and activation by CD25 expression of CD4 T cells (Fig.6D), NK cells (Fig.6E), and CD8 T cells (Fig.6F) upon treatment with NKG2D-IL2v (5C5), NKG2D-IL2v (13C6), NKG2D-IL2v (395 cl.80), and FAP-IL2v are shown.
Figure 7A-B. STAT5 phosphorylation in CD8 T cells (Fig.7A) and NK cells (Fig.7B) upon treatment of resting PBMCs with NKG2D-IL2v and FAP-IL2v was determined by flow cytometry.
Figure 8A-C. STAT5 phosphorylation in CD8 T cells (Fig.8A), CD4 T cells (Fig.8C), and NK cells (Fig.8B) upon treatment of resting PBMCs with NKG2D(5C5)-IL2v and FAP-IL2v was determined by flow cytometry.
Figure 9A-D. STAT5 phosphorylation in CD4 T cells (Fig.9A), CD8 T cells (Fig.9B) Tregs (Fig.9C), NK cells (Fig.9D) and upon treatment of resting PBMCs with NKG2D-IL2v (5C5), NKG2D-IL2v (13C6), NKG2D-IL2v (395 cl.80), and FAP-IL2v was determined by flow cytometry.
Figure 10A-C. Binding of NKG2D(5C5)-IL2v and FAP-IL2v to CD8 T cells (Fig.lOA), NK cells (Fig.lOB) and CD4 T cells (Fig. IOC) was determined by flow cytometry.
Figure 11A-C. Binding of NKG2D-IL2v (5C5), NKG2D-IL2v (13C6), NKG2D-IL2v (395 cl.80), and FAP-IL2v on PBMCs (CD4 T cells (Fig.11 A), CD8 T cells (Fig.1 IB), NK cells (Fig.11C) was determined by flow cytometry.
Figure 12A-C. Activation of CD8 T cells (Fig.9A), NK cells (Fig.9B) and CD4 T cells (Fig.9C) within PBMCs upon treatment for 5 days with NKG2D(5C5)-IL2v and NKG2D(296)-IL2v was determined by flow cytometry.
Figure 13. Binding of the NKG2D IgGs 296 and 5C5 to NKG2D on NK92 cells was determined by flow cytometry.
Figure 14. Proliferation of NK92 upon treatment for 4 days with NKG2D-IL2v (5C5), NKG2D- IL2v (13C6), NKG2D-IL2v (395 cl.80), and FAP-IL2v was determined by flow cytometry
DETAILED DESCRIPTION OF THE INVENTION
I. DEFINITIONS
Terms are used herein as generally used in the art, unless otherwise defined in the following.
The term “interleukin-2” or “IL-2” as used herein, refers to any native IL-2 from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses unprocessed IL-2 as well as any form of IL-2 that results from processing in the cell. The term also encompasses naturally occurring variants of IL-2, e.g. splice variants or allelic variants. The amino acid sequence of an exemplary human IL-2 is shown in SEQ ID NO: 41. Unprocessed human IL-2 additionally comprises an N- terminal 20 amino acid signal peptide having the sequence of SEQ ID NO: 59, which is absent in the mature IL-2 molecule.
The term "IL-2 mutant" or "mutant IL-2 polypeptide" as used herein is intended to encompass any mutant forms of various forms of the IL-2 molecule including full-length IL-2, truncated forms of IL-2 and forms where IL-2 is linked to another molecule such as by fusion or chemical conjugation. "Full-length" when used in reference to IL-2 is intended to mean the mature, natural length IL-2 molecule. For example, full-length human IL-2 refers to a molecule that has 133 amino acids (see e.g. SEQ ID NO: 41). The various forms of IL-2 mutants are characterized in having a at least one amino acid mutation affecting the interaction of IL-2 with CD25. This mutation may involve substitution, deletion, truncation or modification of the wild-type amino acid residue normally located at that position. Mutants obtained by amino acid substitution are preferred. Unless otherwise indicated, an IL-2 mutant may be referred to herein as a mutant IL-2 peptide sequence, a mutant IL-2 polypeptide, a mutant IL-2 protein or a mutant IL-2 analog.
Designation of various forms of IL-2 is herein made with respect to the sequence shown in SEQ ID NO: 41. Various designations may be used herein to indicate the same mutation. For example a mutation from phenylalanine at position 42 to alanine can be indicated as 42A, A42, A42, F42A, or Phe42Ala.
By a “human IL-2 molecule” as used herein is meant an IL-2 molecule comprising an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95% or at least about 96% identical to the human IL-2 sequence of SEQ ID NO:41. Particularly, the sequence identity is at least about 95%, more particularly at
least about 96%. In particular embodiments, the human IL-2 molecule is a full-length IL-2 molecule.
The term “amino acid mutation” as used herein is meant to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitution, deletion, insertion, and modification can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g. reduced binding to CD25. Amino acid sequence deletions and insertions include amino- and/or carboxy-terminal deletions and insertions of amino acids. An example of a terminal deletion is the deletion of the alanine residue in position 1 of full-length human IL-2. Preferred amino acid mutations are amino acid substitutions. For the purpose of altering e.g. the binding characteristics of an IL-2 polypeptide, non-conservative amino acid substitutions, i.e. replacing one amino acid with another amino acid having different structural and/or chemical properties, are particularly preferred. Preferred amino acid substitions include replacing a hydrophobic by a hydrophilic amino acid. Amino acid substitutions include replacement by non-naturally occurring amino acids or by naturally occurring amino acid derivatives of the twenty standard amino acids (e.g. 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5 -hydroxy lysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis and the like. It is contemplated that methods of altering the side chain group of an amino acid by methods other than genetic engineering, such as chemical modification, may also be useful. Various designations may be used herein to indicate the same amino acid mutation. For example, a substitution from proline at position 329 of the Fc domain to glycine can be indicated as 329G, G329, G329, P329G, or Pro329Gly.
As used herein, a “wild-type” form of IL-2 is a form of IL-2 that is otherwise the same as the mutant IL-2 polypeptide except that the wild-type form has a wild-type amino acid at each amino acid position of the mutant IL-2 polypeptide. For example, if the IL-2 mutant is the full-length IL-2 (i.e. IL-2 not fused or conjugated to any other molecule), the wild-type form of this mutant is full-length native IL-2. If the IL-2 mutant is a fusion between IL-2 and another polypeptide encoded downstream of IL-2 (e.g. an antibody chain) the wild-type form of this IL-2 mutant is IL-2 with a wild-type amino acid sequence, fused to the same downstream polypeptide. Furthermore, if the IL-2 mutant is a truncated form of IL-2 (the mutated or modified sequence within the non-truncated portion of IL-2) then the wild-type form of this IL-2 mutant is a similarly truncated IL-2 that has a wild-type sequence. For the purpose of comparing IL-2 receptor binding affinity or biological activity of various forms of IL-2 mutants to the
corresponding wild-type form of IL-2, the term wild-type encompasses forms of IL-2 comprising one or more amino acid mutation that does not affect IL-2 receptor binding compared to the naturally occurring, native IL-2, such as e.g. a substitution of cysteine at a position corresponding to residue 125 of human IL-2 to alanine. In some embodiments wild-type IL-2 for the purpose of the present invention comprises the amino acid substitution C125A (see SEQ ID NO: 60). In certain embodiments according to the invention the wild-type IL-2 polypeptide to which the mutant IL-2 polypeptide is compared comprises the amino acid sequence of SEQ ID NO: 41. In other embodiments the wild-type IL-2 polypeptide to which the mutant IL-2 polypeptide is compared comprises the amino acid sequence of SEQ ID NO: 60.
The term “CD25” or “a-subunit of the IL-2 receptor” as used herein, refers to any native CD25 from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length”, unprocessed CD25 as well as any form of CD25 that results from processing in the cell. The term also encompasses naturally occurring variants of CD25, e.g. splice variants or allelic variants. In certain embodiments CD25 is human CD25. The amino acid sequence of human CD25 is found e.g. in UniProt entry no. P01589 (version 185).
The term “high-affinity IL-2 receptor” as used herein refers to the heterotrimeric form of the IL- 2 receptor, consisting of the receptor g-subunit (also known as common cytokine receptor g- subunit, yc, or CD132, see UniProt entry no. P14784 (version 192)), the receptor b-subunit (also known as CD122 or p70, see UniProt entry no. P31785 (version 197)) and the receptor a-subunit (also known as CD25 or p55, see UniProt entry no. P01589 (version 185)). The term “intermediate-affinity IL-2 receptor” by contrast refers to the IL-2 receptor including only the g- subunit and the b-subunit, without the a-subunit (for a review see e.g. Olejniczak and Kasprzak, Med Sci Monit 14, RA179-189 (2008)).
The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen binding activity.
The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies comprised in the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody
preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.
An "isolated" antibody is one which has been separated from a component of its natural environment. In some aspects, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC, affinity chromatography, size exclusion chromatography) methods. For review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007). In some aspects, the antibodies provided by the present invention are isolated antibodies.
The term “chimeric” antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure.
An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab’-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g. scFv), and single-domain antibodies. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).
The term “immunoglobulin molecule” refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric
glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or a heavy chain variable region, followed by three constant domains (CHI, CH2, and CH3), also called a heavy chain constant region. Similarly, from N- to C-terminus, each light chain has a variable domain (VL), also called a variable light domain or a light chain variable region, followed by a constant light (CL) domain, also called a light chain constant region. The heavy chain of an immunoglobulin may be assigned to one of five types, called a (IgA), d (IgD), e (IgE), g (IgG), or m (IgM), some of which may be further divided into subtypes, e.g. gΐ (IgGl), g2 (IgG2), g3 (IgG3), g4 (IgG4), al (IgAl) and a2 (IgA2). The light chain of an immunoglobulin may be assigned to one of two types, called kappa (K) and lambda (l), based on the amino acid sequence of its constant domain. An immunoglobulin essentially consists of two Fab molecules and an Fc domain, linked via the immunoglobulin hinge region.
The term "antigen binding domain" refers to the part of an antibody that comprises the area which specifically binds to and is complementary to part or all of an antigen. An antigen binding domain may be provided by, for example, one or more antibody variable domains (also called antibody variable regions). Particularly, an antigen binding domain comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).
An “antigen binding site” refers to the site, i.e. one or more amino acid residues, of an antigen binding molecule which provides interaction with the antigen. For example, the antigen binding site of an antibody comprises amino acid residues from the complementarity determining regions (CDRs). A native immunoglobulin molecule typically has two antigen binding sites, a Fab molecule typically has a single antigen binding site.
As used herein, the term "antigenic determinant" or "antigen" refers to a site (e.g. a contiguous stretch of amino acids or a conformational configuration made up of different regions of non contiguous amino acids) on a polypeptide macromolecule to which an antigen binding domain binds, forming an antigen binding domain-antigen complex. Useful antigenic determinants can be found, for example, on the surfaces of tumor cells, on the surfaces of virus-infected cells, on the surfaces of other diseased cells, on the surface of immune cells, free in blood serum, and/or in the extracellular matrix (ECM). In a preferred aspect, the antigen is a human protein.
The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar
structures, with each domain comprising four conserved framework regions (FRs) and complementarity determining regions (CDRs). See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman & Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991). As used herein in connection with variable region sequences, "Rabat numbering" refers to the numbering system set forth by Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).
As used herein, the amino acid positions of all constant regions and domains of the heavy and light chain are numbered according to the Rabat numbering system described in Rabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), referred to as “numbering according to Rabat” or “Rabat numbering” herein. Specifically the Rabat numbering system (see pages 647-660 of Rabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)) is used for the light chain constant domain CL of kappa and lambda isotype and the Rabat EU index numbering system (see pages 661-723) is used for the heavy chain constant domains (CHI, hinge, CH2 and CH3), which is herein further clarified by referring to “numbering according to Rabat EU index” or “Rabat EU index numbering” in this case.
The term “hypervariable region” or “HVR”, as used herein, refers to each of the regions of an antibody variable domain which are hypervariable in sequence and which determine antigen binding specificity, for example “complementarity determining regions” (“CDRs”). Generally, antibodies comprise six CDRs; three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1, LCDR2, LCDR3). Exemplary CDRs herein include:
(a) hypervariable loops occurring at amino acid residues 26-32 (LI), 50-52 (L2), 91-96 (L3), 26- 32 (HI), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));
(b) CDRs occurring at amino acid residues 24-34 (LI), 50-56 (L2), 89-97 (L3), 31-35b (HI), 50- 65 (H2), and 95-102 (H3) (Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and
(c) antigen contacts occurring at amino acid residues 27c-36 (LI), 46-55 (L2), 89-96 (L3), 30- 35b (HI), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)).
Unless otherwise indicated, the CDRs are determined according to Kabat et al., supra. One of skill in the art will understand that the CDR designations can also be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.
"Framework" or "FR" refers to variable domain residues other than complementarity determining regions (CDRs). The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following order in VH (or VL): FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3- HCDR3 (LCDR3 )-FR4.
Unless otherwise indicated, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.
A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non human CDRs and amino acid residues from human FRs. In certain aspects, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. Such variable domains are referred to herein as “humanized variable region”. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. In some aspects, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity. A “humanized form” of an antibody, e.g. of a non-human antibody, refers to an antibody that has undergone humanization.
An “acceptor human framework” for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some aspects, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some aspects, the VL acceptor human framework is identical in
sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.
A “human consensus framework” is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et ah, Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3.
A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. In certain embodiments, a human antibody is derived from a non human transgenic mammal, for example a mouse, a rat, or a rabbit. In certain embodiments, a human antibody is derived from a hybridoma cell line. Antibodies or antibody fragments isolated from human antibody libraries are also considered human antibodies or human antibody fragments herein.
The “class” of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, d, e, g, and m, respectively.
A “Fab molecule” refers to a protein consisting of the VH and CHI domain of the heavy chain (the “Fab heavy chain”) and the VL and CL domain of the light chain (the “Fab light chain”) of an immunoglobulin.
The term “Fc domain” or “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain might vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain.
Therefore an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain (also referred to herein as a “cleaved variant heavy chain”). This may be the case where the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to Kabat EU index). Therefore, the C- terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (K447), of the Fc region may or may not be present. Amino acid sequences of heavy chains including Fc domains (or a subunit of an Fc domain as defined herein) are denoted herein without C-terminal glycine-lysine dipeptide if not indicated otherwise. In one embodiment of the invention, a heavy chain including a subunit of an Fc domain as specified herein, comprised in an immunoconjugate according to the invention, comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to EU index of Kabat). In one embodiment of the invention, a heavy chain including a subunit of an Fc domain as specified herein, comprised in an immunoconjuate according to the invention, comprises an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat). Compositions of the invention, such as the pharmaceutical compositions described herein, comprise a population of immunoconjugates of the invention. The population of immunoconjugates may comprise molecules having a full- length heavy chain and molecules having a cleaved variant heavy chain. The population of immunoconjugates may consist of a mixture of molecules having a full-length heavy chain and molecules having a cleaved variant heavy chain, wherein at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the immunoconjugates have a cleaved variant heavy chain. In one embodiment of the invention, a composition comprising a population of immunoconjugates of the invention comprises an immunoconjugate comprising a heavy chain including a subunit of an Fc domain as specified herein with an additional C-terminal glycine- lysine dipeptide (G446 and K447, numbering according to EU index of Kabat). In one embodiment of the invention, a composition comprising a population of immunoconjugates of the invention comprises an immunoconjugate comprising a heavy chain including a subunit of an Fc domain as specified herein with an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat). In one embodiment of the invention, such a composition comprises a population of immunoconjugates comprised of molecules comprising a heavy chain including a subunit of an Fc domain as specified herein; molecules comprising a heavy chain including a subunit of a Fc domain as specified herein with an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat); and molecules comprising a heavy
chain including a subunit of an Fc domain as specified herein with an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to EU index of Kabat). Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also above). A “subunit” of an Fc domain as used herein refers to one of the two polypeptides forming the dimeric Fc domain, i.e. a polypeptide comprising C-terminal constant regions of an immunoglobulin heavy chain, capable of stable self-association. For example, a subunit of an IgG Fc domain comprises an IgG CH2 and an IgG CH3 constant domain.
A “modification promoting the association of the first and the second subunit of the Fc domain” is a manipulation of the peptide backbone or the post-translational modifications of an Fc domain subunit that reduces or prevents the association of a polypeptide comprising the Fc domain subunit with an identical polypeptide to form a homodimer. A modification promoting association as used herein particularly includes separate modifications made to each of the two Fc domain subunits desired to associate (i.e. the first and the second subunit of the Fc domain), wherein the modifications are complementary to each other so as to promote association of the two Fc domain subunits. For example, a modification promoting association may alter the structure or charge of one or both of the Fc domain subunits so as to make their association sterically or electrostatically favorable, respectively. Thus, (hetero)dimerization occurs between a polypeptide comprising the first Fc domain subunit and a polypeptide comprising the second Fc domain subunit, which might be non-identical in the sense that further components fused to each of the subunits (e.g. antigen binding moieties) are not the same. In some embodiments the modification promoting association comprises an amino acid mutation in the Fc domain, specifically an amino acid substitution. In a particular embodiment, the modification promoting association comprises a separate amino acid mutation, specifically an amino acid substitution, in each of the two subunits of the Fc domain.
The term “effector functions” when used in reference to antibodies refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune
complex-mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g. B cell receptor), and B cell activation.
Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism leading to the lysis of antibody-coated target cells by immune effector cells. The target cells are cells to which antibodies or derivatives thereof comprising an Fc region specifically bind, generally via the protein part that is N-terminal to the Fc region. As used herein, the term “reduced ADCC” is defined as either a reduction in the number of target cells that are lysed in a given time, at a given concentration of antibody in the medium surrounding the target cells, by the mechanism of ADCC defined above, and/or an increase in the concentration of antibody in the medium surrounding the target cells, required to achieve the lysis of a given number of target cells in a given time, by the mechanism of ADCC. The reduction in ADCC is relative to the ADCC mediated by the same antibody produced by the same type of host cells, using the same standard production, purification, formulation and storage methods (which are known to those skilled in the art), but that has not been engineered. For example the reduction in ADCC mediated by an antibody comprising in its Fc domain an amino acid substitution that reduces ADCC, is relative to the ADCC mediated by the same antibody without this amino acid substitution in the Fc domain. Suitable assays to measure ADCC are well known in the art (see e.g. PCT publication no. WO 2006/082515 or PCT publication no. WO 2012/130831).
An “activating Fc receptor” is an Fc receptor that following engagement by an Fc domain of an antibody elicits signaling events that stimulate the receptor-bearing cell to perform effector functions. Human activating Fc receptors include FcyRIIIa (CD16a), FcyRI (CD64), FcyRIIa (CD32), and FcaRI (CD89).
As used herein, the terms “engineer, engineered, engineering”, are considered to include any manipulation of the peptide backbone or the post-translational modifications of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modifications of the amino acid sequence, of the glycosylation pattern, or of the side chain group of individual amino acids, as well as combinations of these approaches.
“Reduced binding”, for example reduced binding to an Fc receptor or CD25, refers to a decrease in affinity for the respective interaction, as measured for example by SPR. For clarity, the term includes also reduction of the affinity to zero (or below the detection limit of the analytic method), i.e. complete abolishment of the interaction. Conversely, “increased binding” refers to an increase in binding affinity for the respective interaction.
As used herein, the term "immunoconjugate" refers to a polypeptide molecule that includes at least one IL-2 molecule and at least one antibody. The IL-2 molecule can be joined to the antibody by a variety of interactions and in a variety of configurations as described herein. In particular embodiments, the IL-2 molecule is fused to the antibody via a peptide linker. Particular immunoconjugates according to the invention essentially consist of one IL-2 molecule and an antibody joined by one or more linker sequences.
By “fused” is meant that the components (e.g. an antibody and an IL-2 molecule) are linked by peptide bonds, either directly or via one or more peptide linkers.
As used herein, the terms "first" and "second" with respect to Fc domain subunits etc., are used for convenience of distinguishing when there is more than one of each type of moiety. Use of these terms is not intended to confer a specific order or orientation of the immunoconjugate unless explicitly so stated.
“Affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by well-established methods known in the art, including those described herein. A preferred method for measuring affinity is Surface Plasmon Resonance (SPR).
An “affinity matured” antibody refers to an antibody with one or more alterations in one or more complementary determining regions (CDRs), compared to a parent antibody, which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.
The affinity of the mutant or wild-type IL-2 polypeptide for various forms of the IL-2 receptor can be determined in accordance with the method set forth in the WO 2012/107417 by surface plasmon resonance (SPR), using standard instrumentation such as a BIAcore instrument (GE Healthcare) and receptor subunits such as may be obtained by recombinant expression (see e.g. Shanafelt et ah, Nature Biotechnol 18, 1197-1202 (2000)). Alternatively, binding affinity of IL-2 mutants for different forms of the IL-2 receptor may be evaluated using cell lines known to express one or the other such form of the receptor. Specific illustrative and exemplary embodiments for measuring binding affinity are described hereinafter.
By “regulatory T cell” or “Treg cell” is meant a specialized type of CD4+ T cell that can suppress the responses of other T cells. Treg cells are characterized by expression of the a- subunit of the IL-2 receptor (CD25) and the transcription factor forkhead box P3 (FOXP3) (Sakaguchi, Annu Rev Immunol 22, 531-62 (2004)) and play a critical role in the induction and maintenance of peripheral self-tolerance to antigens, including those expressed by tumors. Treg cells require IL-2 for their function and development and induction of their suppressive characteristics.
As used herein, the term “effector cells” refers to a population of lymphocytes that mediate the cytotoxic effects of IL-2. Effector cells include effector T cells such as CD8+cytotoxic T cells, NK cells, lymphokine-activated killer (LAK) cells and macrophages/monocytes.
“NKG2D” refers to any native NKG2D from any vertebrate source, including mammals such as primates (e.g. humans), non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed NKG2D as well as any form of NKG2D that results from processing in the cell. The term also encompasses naturally occurring variants of NKG2D, e.g., splice variants or allelic variants. In one aspect, NKG2D is human NKG2D, particularly the extracellular domain (ECD) of human NKG2D. The amino acid sequence of human NKG2D and its ECD are shown in SEQ ID NO: 65 and SEQ ID NO: 66, respectively. See also UniProt (www.uniprot.org) entry P26718 (version 176). In another aspect, NKG2D is cynomolgus ( Macaca fascicularis) NKG2D, particularly the ECD of cynomolgus NKG2D. The amino acid sequence of cynomolgus NKG2D and its ECD are shown in SEQ ID NO: 67 and SEQ ID NO: 68, respectively. See also UniProt entry P61252 (version 71). In another aspect, NKG2D is murine ( Mus musculus ) NKG2D, particularly the ECD of murine NKG2D. The amino acid sequence of murine NKG2D and its ECD are shown in SEQ ID NO: 69 and SEQ ID NO: 70, respectively. See also UniProt entry 054709 (version 151). In certain aspects the antibody of the invention binds to an epitope of NKG2D that is conserved among the NKG2D antigens from different species, particularly human and cynomolgus NKG2D. In preferred aspects, the antibody binds to human NKG2D. In one aspect the first antigen binding domain is cross-reactive for (i.e. binds to) human and cynomolgus NKG2D.
The terms “anti-NKG2D antibody” and “an antibody that binds to NKG2D” refer to an antibody that is capable of binding NKG2D with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting NKG2D. In one aspect, the extent of binding of an anti-NKG2D antibody to an unrelated, non-NKG2D protein is less than about 10% of the binding of the antibody to NKG2D as measured, e.g., by surface plasmon resonance (SPR). In
certain aspects, an antibody that binds to NKG2D has a dissociation constant (KD) of < 1 mM, < 500 nM, < 200 nM, or < 100 nM. An antibody is said to “specifically bind” to NKG2D when the antibody has a KD of 1 mM or less, as measured, e.g., by SPR. In certain aspects, an anti- NKG2D antibody binds to an epitope of NKG2D that is conserved among NKG2D from different species.
Conversely, an antibody that “does not bind” to a certain antigen is not capable of binding said antigen with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting said antigen. In certain aspects, an antibody that does not bind to a certain antigen has a dissociation constant (KD) of > 1 pM to said antigen.
By "specific binding" is meant that the binding is selective for the antigen and can be discriminated from unwanted or non-specific interactions. The ability of an antibody to bind to a specific antigen (e.g. NKG2D) can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g. surface plasmon resonance (SPR) technique (analyzed e.g. on a BIAcore instrument) (Liljeblad et ah, Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of binding of an antibody to an unrelated protein is less than about 10% of the binding of the antibody to the antigen as measured, e.g., by SPR. The antibody comprised in the immunoconjugate described herein specifically binds to NKG2D.
As used herein, term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein", "amino acid chain", or any other term used to refer to a chain of two or more amino acids, are included within the definition of "polypeptide", and the term "polypeptide" may be used instead of, or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis. Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides which do not possess a defined three-
dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded.
By an "isolated" polypeptide or a variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.
“Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Alternatively, the percent identity values can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087 and is described in WO 2001/007611.
Unless otherwise indicated, for purposes herein, % amino acid sequence identity values are generated using the ggsearch program of the FASTA package version 36.3.8c or later with a BLOSUM50 comparison matrix. The FASTA program package was authored by W. R. Pearson and D. J. Lipman (“Improved Tools for Biological Sequence Analysis”, PNAS 85 (1988) 2444- 2448), W. R. Pearson (“Effective protein sequence comparison” Meth. Enzymol. 266 (1996) 227- 258), and Pearson et. al. (Genomics 46 (1997) 24-36) and is publicly available from www.fasta.bioch.virginia.edu/fasta_www2/fasta_down.shtml or www.ebi.ac.uk/Tools/sss/fasta. Alternatively, a public server accessible at fasta.bioch.virginia.edu/fasta_www2/index.cgi can be used to compare the sequences, using the ggsearch (global protein: protein) program and default
options (BLOSUM50; open: -10; ext: -2; Ktup = 2) to ensure a global, rather than local, alignment is performed. Percent amino acid identity is given in the output alignment header.
The term “polynucleotide” or “nucleic acid molecule” includes any compound and/or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine- or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose), and a phosphate group. Often, the nucleic acid molecule is described by the sequence of bases, whereby said bases represent the primary structure (linear structure) of a nucleic acid molecule. The sequence of bases is typically represented from 5’ to 3’. Herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) including e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. The nucleic acid molecule may be linear or circular. In addition, the term nucleic acid molecule includes both, sense and antisense strands, as well as single stranded and double stranded forms. Moreover, the herein described nucleic acid molecule can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugars or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules which are suitable as a vector for direct expression of an antibody of the invention in vitro and/or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors, can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and/or expression of the encoded molecule so that mRNA can be injected into a subject to generate the antibody in vivo (see e.g., Stadler et al. (2017) Nature Medicine 23:815-817, or EP 2 101 823 Bl).
“Isolated polynucleotide (or nucleic acid) encoding an antibody” refers to one or more polynucleotide molecules encoding antibody heavy and light chains (or fragments thereof), including such polynucleotide molecule(s) in a single vector or separate vectors, and such polynucleotide molecule(s) present at one or more locations in a host cell.
The term “vector”, as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors”.
The terms "host cell", "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. A host cell is any type of cellular system that can be used to generate the antibodies of the present invention. Host cells include cultured cells, e.g. mammalian cultured cells, such as HEK cells, CHO cells, BHK cells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue. In one aspect, the host cell of the invention is a eukaryotic cell, particularly a mammalian cell. In one aspect, the host cell is not a cell within a human body.
An “effective amount” of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
A "therapeutically effective amount" of an agent, e.g. a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent for example eliminates, decreases, delays, minimizes or prevents adverse effects of a disease.
An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g. cows, sheep, cats, dogs, and horses), primates (e.g. humans and non human primates such as monkeys), rabbits, and rodents (e.g. mice and rats). Particularly, the individual or subject is a human.
The term "pharmaceutical composition" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the composition would be administered.
A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, immunoconjugates of the invention are used to delay development of a disease or to slow the progression of a disease.
II. COMPOSITIONS AND METHODS A. MUTANT INTERLEUKIN-2 POLYPEPTIDES
The immunoconjugates according to the present invention comprise a mutant IL-2 polypeptide having advantageous properties for immunotherapy. In particular, pharmacological properties of IL-2 that contribute to toxicity but are not essential for efficacy of IL-2 are eliminated in the mutant IL-2 polypeptide. Such mutant IL-2 polypeptides are described in detail in WO 2012/107417, which is incorporated herein by reference in its entirety. As discussed above, different forms of the IL-2 receptor consist of different subunits and exhibit different affinities for IL-2. The intermediate-affinity IL-2 receptor, consisting of the b and g receptor subunits, is expressed on resting effector cells and is sufficient for IL-2 signaling. The high-affinity IL-2 receptor, additionally comprising the a-subunit of the receptor, is mainly expressed on regulatory T (Treg) cells as well as on activated effector cells where its engagement by IL-2 can promote Treg cell-mediated immunosuppression or activation-induced cell death (AICD), respectively. Thus, without wishing to be bound by theory, reducing or abolishing the affinity of IL-2 to the a- subunit of the IL-2 receptor should reduce IL-2 induced downregulation of effector cell function by regulatory T cells and development of tumor tolerance by the process of AICD. On the other
hand, maintaining the affinity to the intermediate-affinity IL-2 receptor should preserve the induction of proliferation and activation of effector cells like NK and T cells by IL-2.
The mutant interleukin-2 (IL-2) polypeptide comprised in the immunoconjugate according to the invention comprises at least one amino acid mutation that abolishes or reduces affinity of the mutant IL-2 polypeptide to the a-subunit of the IL-2 receptor and preserves affinity of the mutant IL-2 polypeptide to the intermediate-affinity IL-2 receptor each compared to a wild-type IL-2 polypeptide.
Mutants of human IL-2 (hIL-2) with decreased affinity to CD25 may for example be generated by amino acid substitution at amino acid position 35, 38, 42, 43, 45 or 72 or combinations thereof (numbering relative to the human IL-2 sequence SEQ ID NO: 41). Exemplary amino acid substitutions include K35E, K35A, R38A, R38E, R38N, R38F, R38S, R38L, R38G, R38Y, R38W, F42L, F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, K43E, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K. Particular IL-2 mutants useful in the immunoconjugates of the invention comprise an amino acid mutation at an amino acid position corresponding to residue 42, 45, or 72 of human IL-2, or a combination thereof. In one embodiment said amino acid mutation is an amino acid substitution selected from the group of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K, more specifically an amino acid substitution selected from the group of F42A, Y45A and L72G. These mutants exhibit substantially similar binding affinity to the intermediate-affinity IL-2 receptor, and have substantially reduced affinity to the a-subunit of the IL-2 receptor and the high-affinity IL-2 receptor compared to a wild-type form of the IL-2 mutant.
Other characteristics of useful mutants may include the ability to induce proliferation of IL-2 receptor-bearing T and/or NK cells, the ability to induce IL-2 signaling in IL-2 receptor-bearing T and/or NK cells, the ability to generate interferon (åFN)-y as a secondary cytokine by NK cells, a reduced ability to induce elaboration of secondary cytokines - particularly IL-10 and TNF-a - by peripheral blood mononuclear cells (PBMCs), a reduced ability to activate regulatory T cells, a reduced ability to induce apoptosis in T cells, and a reduced toxicity profile in vivo.
Particular mutant IL-2 polypeptides useful in the invention comprise three amino acid mutations that abolish or reduce affinity of the mutant IL-2 polypeptide to the a-subunit of the IL-2
receptor but preserve affinity of the mutant IL-2 polypeptide to the intermediate affinity IL-2 receptor. In one embodiment said three amino acid mutations are at positions corresponding to residue 42, 45 and 72 of human IL-2. In one embodiment said three amino acid mutations are amino acid substitutions. In one embodiment said three amino acid mutations are amino acid substitutions selected from the group of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K. In a specific embodiment said three amino acid mutations are amino acid substitutions F42A, Y45A and L72G (numbering relative to the human IL-2 sequence of SEQ ID NO: 41).
In certain embodiments said amino acid mutation reduces the affinity of the mutant IL-2 polypeptide to the a-subunit of the IL-2 receptor by at least 5 -fold, specifically at least 10-fold, more specifically at least 25-fold. In embodiments where there is more than one amino acid mutation that reduces the affinity of the mutant IL-2 polypeptide to the a-subunit of the IL-2 receptor, the combination of these amino acid mutations may reduce the affinity of the mutant IL-2 polypeptide to the a-subunit of the IL-2 receptor by at least 30-fold, at least 50-fold, or even at least 100-fold. In one embodiment said amino acid mutation or combination of amino acid mutations abolishes the affinity of the mutant IL-2 polypeptide to the a-subunit of the IL-2 receptor so that no binding is detectable by surface plasmon resonance.
Substantially similar binding to the intermediate-affinity receptor, i.e. preservation of the affinity of the mutant IL-2 polypeptide to said receptor, is achieved when the IL-2 mutant exhibits greater than about 70% of the affinity of a wild-type form of the IL-2 mutant to the intermediate- affinity IL-2 receptor. IL-2 mutants of the invention may exhibit greater than about 80% and even greater than about 90% of such affinity.
Reduction of the affinity of IL-2 for the a-subunit of the IL-2 receptor in combination with elimination of the O-glycosylation of IL-2 results in an IL-2 protein with improved properties. For example, elimination of the O-glycosylation site results in a more homogenous product when the mutant IL-2 polypeptide is expressed in mammalian cells such as CHO or HEK cells.
Thus, in certain embodiments the mutant IL-2 polypeptide comprises an additional amino acid mutation which eliminates the O-glycosylation site of IL-2 at a position corresponding to residue 3 of human IL-2. In one embodiment said additional amino acid mutation which eliminates the O-glycosylation site of IL-2 at a position corresponding to residue 3 of human IL-2 is an amino acid substitution. Exemplary amino acid substitutions include T3A, T3G, T3Q, T3E, T3N, T3D,
T3R, T3K, and T3P. In a specific embodiment, said additional amino acid mutation is the amino acid substitution T3A.
In certain embodiments the mutant IL-2 polypeptide is essentially a full-length IL-2 molecule. In certain embodiments the mutant IL-2 polypeptide is a human IL-2 molecule. In one embodiment the mutant IL-2 polypeptide comprises the sequence of SEQ ID NO: 41 with at least one amino acid mutation that abolishes or reduces affinity of the mutant IL-2 polypeptide to the a-subunit of the IL-2 receptor but preserve affinity of the mutant IL-2 polypeptide to the intermediate affinity IL-2 receptor, compared to an IL-2 polypeptide comprising SEQ ID NO: 19 without said mutation. In another embodiment, the mutant IL-2 polypeptide comprises the sequence of SEQ ID NO: 60 with at least one amino acid mutation that abolishes or reduces affinity of the mutant IL-2 polypeptide to the a-subunit of the IL-2 receptor but preserve affinity of the mutant IL-2 polypeptide to the intermediate affinity IL-2 receptor, compared to an IL-2 polypeptide comprising SEQ ID NO: 60 without said mutation.
In a specific embodiment, the mutant IL-2 polypeptide can elicit one or more of the cellular responses selected from the group consisting of: proliferation in an activated T lymphocyte cell, differentiation in an activated T lymphocyte cell, cytotoxic T cell (CTL) activity, proliferation in an activated B cell, differentiation in an activated B cell, proliferation in a natural killer (NK) cell, differentiation in a NK cell, cytokine secretion by an activated T cell or an NK cell, and NK/lymphocyte activated killer (LAK) antitumor cytotoxicity.
In one embodiment the mutant IL-2 polypeptide has a reduced ability to induce IL-2 signaling in regulatory T cells, compared to a wild-type IL-2 polypeptide. In one embodiment the mutant IL- 2 polypeptide induces less activation-induced cell death (AICD) in T cells, compared to a wild- type IL-2 polypeptide. In one embodiment the mutant IL-2 polypeptide has a reduced toxicity profile in vivo, compared to a wild-type IL-2 polypeptide. In one embodiment the mutant IL-2 polypeptide has a prolonged serum half-life, compared to a wild-type IL-2 polypeptide.
A particular mutant IL-2 polypeptide useful in the invention comprises four amino acid substitutions at positions corresponding to residues 3, 42, 45 and 72 of human IL-2. Specific amino acid substitutions are T3A, F42A, Y45A and L72G. As demonstrated in WO 2012/107417, said quadruple mutant IL-2 polypeptide exhibits no detectable binding to CD25, reduced ability to induce apoptosis in T cells, reduced ability to induce IL-2 signaling in Treg cells, and a reduced toxicity profile in vivo. However, it retains ability to activate IL-2 signaling
in effector cells, to induce proliferation of effector cells, and to generate IFN-g as a secondary cytokine by NK cells.
Moreover, said mutant IL-2 polypeptide has further advantageous properties, such as reduced surface hydrophobicity, good stability, and good expression yield, as described in WO 2012/107417. Unexpectedly, said mutant IL-2 polypeptide also provides a prolonged serum half- life, compared to wild-type IL-2.
IL-2 mutants useful in the invention, in addition to having mutations in the region of IL-2 that forms the interface of IL-2 with CD25 or the glycosylation site, also may have one or more mutations in the amino acid sequence outside these regions. Such additional mutations in human IL-2 may provide additional advantages such as increased expression or stability. For example, the cysteine at position 125 may be replaced with a neutral amino acid such as serine, alanine, threonine or valine, yielding C125S IL-2, C125A IL-2, C125T IL-2 or C125V IL-2 respectively, as described in U.S. Patent no. 4,518,584. As described therein, one may also delete the N- terminal alanine residue of IL-2 yielding such mutants as des-Al C125S or des-Al C125A. Alternatively or conjunctively, the IL-2 mutant may include a mutation whereby methionine normally occurring at position 104 of wild-type human IL-2 is replaced by a neutral amino acid such as alanine (see U.S. Patent no. 5,206,344). The resulting mutants, e. g., des-Al M104A IL- 2, des-Al Ml 04 A C125S IL-2, M104A IL-2, M104A C125A IL-2, des-Al M104A C125A IL-2, or M104A C125S IL-2 (these and other mutants may be found in U.S. Patent No. 5,116,943 and in Weiger et al., Eur J Biochem 180, 295-300 (1989)) may be used in conjunction with the particular IL-2 mutations of the invention.
Thus, in certain embodiments the mutant IL-2 polypeptide comprises an additional amino acid mutation at a position corresponding to residue 125 of human IL-2. In one embodiment said additional amino acid mutation is the amino acid substitution C125A.
The skilled person will be able to determine which additional mutations may provide additional advantages for the purpose of the invention. For example, he will appreciate that amino acid mutations in the IL-2 sequence that reduce or abolish the affinity of IL-2 to the intermediate- affinity IL-2 receptor, such as D20T, N88R or Q126D (see e.g. US 2007/0036752), may not be suitable to include in the mutant IL-2 polypeptide according to the invention.
In one embodiment, the mutant IL-2 polypeptide comprises no more than 12, no more than 11, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, or no more than 5 amino acid mutations as compared to the corresponding wild-type IL-2 sequence, e.g. the
human IL-2 sequence of SEQ ID NO: 41. In a particular embodiment, the mutant IL-2 polypeptide comprises no more than 5 amino acid mutations as compared to the corresponding wild-type IL-2 sequence, e.g. the human IL-2 sequence of SEQ ID NO: 41.
In one embodiment the mutant IL-2 polypeptide comprises the sequence of SEQ ID NO: 42. In one embodiment the mutant IL-2 polypeptide consists of the sequence of SEQ ID NO: 42.
B. IMMUNOCONJUGATES
Immunoconjugates as described herein comprise an IL-molecule and an antibody. Such immunoconjugates significantly increase the efficacy of IL-2 therapy by directly targeting IL-2 e.g. into a tumor microenvironment. According to the invention, an antibody comprised in the immunoconjugate can be a whole antibody or immunoglobulin, or a portion or variant thereof that has a biological function such as antigen specific binding affinity.
The general benefits of immunoconjugate therapy are readily apparent. For example, an antibody comprised in an immunoconjugate recognizes a tumor-specific epitope and results in targeting of the immunoconjugate molecule to the tumor site. Therefore, high concentrations of IL-2 can be delivered into the tumor microenvironment, thereby resulting in activation and proliferation of a variety of immune effector cells mentioned herein using a much lower dose of the immunoconjugate than would be required for unconjugated IL-2. Moreover, since application of IL-2 in form of immunoconjugates allows lower doses of the cytokine itself, the potential for undesirable side effects of IL-2 is restricted, and targeting the IL-2 to a specific site in the body by means of an immunoconjugate may also result in a reduction of systemic exposure and thus less side effects than obtained with unconjugated IL-2. In addition, the increased circulating half- life of an immunoconjugate compared to unconjugated IL-2 contributes to the efficacy of the immunoconjugate. However, this characteristic of IL-2 immunoconjugates may again aggravate potential side effects of the IL-2 molecule: Because of the significantly longer circulating half- life of IL-2 immunoconjugate in the bloodstream relative to unconjugated IL-2, the probability for IL-2 or other portions of the fusion protein molecule to activate components generally present in the vasculature is increased. The same concern applies to other fusion proteins that contain IL-2 fused to another moiety such as Fc or albumin, resulting in an extended half-life of IL-2 in the circulation. Therefore an immunoconjugate comprising a mutant IL-2 polypeptide as described herein and in WO 2012/107417, with reduced toxicity compared to wild-type forms of IL-2, is particularly advantageous.
As described hereinabove, targeting IL-2 directly to immune effector cells rather than tumor cells may be advantageous for IL-2 immunotherapy.
Accordingly, the invention provides a mutant IL-2 polypeptide as described hereinbefore, and an antibody that binds to NKG2D. In one embodiment the mutant IL-2 polypeptide and the antibody form a fusion protein, i.e. the mutant IL-2 polypeptide shares a peptide bond with the antibody. In some embodiments, the antibody comprises an Fc domain composed of a first and a second subunit. In a specific embodiment the mutant IL-2 polypeptide is fused at its amino- terminal amino acid to the carboxy -terminal amino acid of one of the subunits of the Fc domain, optionally through a linker peptide. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an immunoglobulin molecule, particularly an IgG class immunoglobulin molecule, more particularly an IgGl subclass immunoglobulin molecule. In one such embodiment, the mutant IL-2 polypeptide shares an amino-terminal peptide bond with one of the immunoglobulin heavy chains. In certain embodiments the antibody is an antibody fragment. In some embodiments the antibody is a Fab molecule or a scFv molecule. In one embodiment the antibody is a Fab molecule. In another embodiment the antibody is a scFv molecule. The immunoconjugate may also comprise more than one antibody. Where more than one antibody is comprised in the immunoconjugate, e.g. a first and a second antibody, each antibody can be independently selected from various forms of antibodies and antibody fragments. For example, the first antibody can be a Fab molecule and the second antibody can be a scFv molecule. In a specific embodiment each of said first and said second antibodies is a scFv molecule or each of said first and said second antibodies is a Fab molecule. In a particular embodiment each of said first and said second antibodies is a Fab molecule. In one embodiment each of said first and said second antibodies binds to NKG2D.
1. IMMUNOCONJUGATE FORMATS
Exemplary immunoconjugate formats are described in PCT publication no. WO 2011/020783, which is incorporated herein by reference in its entirety. These immunoconjugates comprise at least two antibodies. Thus, in one embodiment, the immunoconjugate according to the present invention comprises a mutant IL-2 polypeptide as described herein, and at least a first and a second antibody. In a particular embodiment, said first and second antibody are independently selected from the group consisting of an Fv molecule, particularly a scFv molecule, and a Fab molecule. In a specific embodiment, said mutant IL-2 polypeptide shares an amino- or carboxy-
terminal peptide bond with said first antibody and said second antibody shares an amino- or carboxy-terminal peptide bond with either i) the mutant IL-2 polypeptide or ii) the first antibody. In a particular embodiment, the immunoconjugate consists essentially of a mutant IL-2 polypeptide and first and second antibodies, particularly Fab molecules, joined by one or more linker sequences. Such formats have the advantage that they bind with high affinity to the target antigen (NKG2D), but provide only monomeric binding to the IL-2 receptor, thus avoiding targeting the immunoconjugate to IL-2 receptor bearing immune cells at other locations than the target site. In a particular embodiment, a mutant IL-2 polypeptide shares a carboxy-terminal peptide bond with a first antibody, particularly a first Fab molecule, and further shares an amino- terminal peptide bond with a second antibody, particularly a second Fab molecule. In another embodiment, a first antibody, particularly a first Fab molecule, shares a carboxy-terminal peptide bond with a mutant IL-2 polypeptide, and further shares an amino-terminal peptide bond with a second antibody, particularly a second Fab molecule. In another embodiment, a first antibody, particularly a first Fab molecule, shares an amino-terminal peptide bond with a first mutant IL-2 polypeptide, and further shares a carboxy-terminal peptide with a second antibody, particularly a second Fab molecule. In a particular embodiment, a mutant IL-2 polypeptide shares a carboxy- terminal peptide bond with a first heavy chain variable region and further shares an amino- terminal peptide bond with a second heavy chain variable region. In another embodiment a mutant IL-2 polypeptide shares a carboxy-terminal peptide bond with a first light chain variable region and further shares an amino-terminal peptide bond with a second light chain variable region. In another embodiment, a first heavy or light chain variable region is joined by a carboxy-terminal peptide bond to a mutant IL-2 polypeptide and is further joined by an amino- terminal peptide bond to a second heavy or light chain variable region. In another embodiment, a first heavy or light chain variable region is joined by an amino-terminal peptide bond to a mutant IL-2 polypeptide and is further joined by a carboxy-terminal peptide bond to a second heavy or light chain variable region. In one embodiment, a mutant IL-2 polypeptide shares a carboxy- terminal peptide bond with a first Fab heavy or light chain and further shares an amino-terminal peptide bond with a second Fab heavy or light chain. In another embodiment, a first Fab heavy or light chain shares a carboxy-terminal peptide bond with a mutant IL-2 polypeptide and further shares an amino-terminal peptide bond with a second Fab heavy or light chain. In other embodiments, a first Fab heavy or light chain shares an amino-terminal peptide bond with a mutant IL-2 polypeptide and further shares a carboxy-terminal peptide bond with a second Fab heavy or light chain. In one embodiment, the immunoconjugate comprises a mutant IL-2
polypeptide sharing an amino-terminal peptide bond with one or more scFv molecules and further sharing a carboxy-terminal peptide bond with one or more scFv molecules.
Particularly suitable formats for the immunoconjugates according to the present invention, however comprise an immunoglobulin molecule as antibody. Such immunoconjugate formats are described in WO 2012/146628, which is incorporated herein by reference in its entirety.
Accordingly, in particular embodiments, the immunoconjugate comprises a mutant IL-2 polypeptide as described herein and an immunoglobulin molecule that binds to NKG2D, particularly an IgG molecule, more particularly an IgGl molecule. In one embodiment the immunoconjugate comprises not more than one mutant IL-2 polypeptide. In one embodiment the immunoglobulin molecule is human. In one embodiment, the immunoglobulin molecule comprises a human constant region, e.g. a human CHI, CH2, CH3 and/or CL domain. In one embodiment, the immunoglobulin comprises a human Fc domain, particularly a human IgGl Fc domain. In one embodiment the mutant IL-2 polypeptide shares an amino- or carboxy-terminal peptide bond with the immunoglobulin molecule. In one embodiment, the immunoconjugate essentially consists of a mutant IL-2 polypeptide and an immunoglobulin molecule, particularly an IgG molecule, more particularly an IgGl molecule, joined by one or more linker sequences. In a specific embodiment the mutant IL-2 polypeptide is fused at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the immunoglobulin heavy chains, optionally through a linker peptide.
The mutant IL-2 polypeptide may be fused to the antibody directly or through a linker peptide, comprising one or more amino acids, typically about 2-20 amino acids. Linker peptides are known in the art and are described herein. Suitable, non-immunogenic linker peptides include, for example, (G4S)n, (SG4)n, (G4S)n or G4(SG4)n linker peptides “n” is generally an integer from 1 to 10, typically from 2 to 4. In one embodiment the linker peptide has a length of at least 5 amino acids, in one embodiment a length of 5 to 100, in a further embodiment of 10 to 50 amino acids. In a particular embodiment, the linker peptide has a length of 15 amino acids. In one embodiment the linker peptide is (GxS)n or (GxS)nGm with G=glycine, S=serine, and (x=3, n= 3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3, 4 or 5 and m= 0, 1, 2 or 3), in one embodiment x=4 and n=2 or 3, in a further embodiment x=4 and n=3. In a particular embodiment the linker peptide is (G4S)3 (SEQ ID NO: 43). In one embodiment, the linker peptide has (or consists of) the amino acid sequence of SEQ ID NO: 43.
In a particular embodiment, the immunoconjugate comprises a mutant IL-2 molecule and an immunoglobulin molecule, particularly an IgGl subclass immunoglobulin molecule, that binds to NKG2D, wherein the mutant IL-2 molecule is fused at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the immunoglobulin heavy chains through the linker peptide of SEQ ID NO: 43.
In a particular embodiment, the immunoconjugate comprises a mutant IL-2 molecule and an antibody that binds to NKG2D, wherein the antibody comprises an Fc domain, particularly a human IgGl Fc domain, composed of a first and a second subunit, and the mutant IL-2 molecule is fused at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the subunits of the Fc domain through the linker peptide of SEQ ID NO: 43.
2. NKG2D ANTIBODIES
The antibody comprised in the immunoconjugate of the invention binds to NKG2D, particularly human NKG2D, and is able to direct the mutant IL-2 polypeptide to a target site where NKG2D is expressed, particularly to a T cell that expresses NKG2D, for example associated with a tumor.
Suitable NKG2D antibodies that may be used in the immunoconjugate of the invention are described in PCT patent application no. PCT/EP2020/069813, which is incorporated herein by reference in its entirety.
The immunoconjugate of the invention may comprise two or more antibodies, which may bind to the same or to different antigens. In particular embodiments, however, each of these antibodies binds to NKG2D. In one embodiment, the antibody comprised in the immunoconjugate of the invention is monospecific. In a particular embodiment, the immunoconjugate comprises a single, monospecific antibody, particularly a monospecific immunoglobulin molecule.
The antibody can be any type of antibody or fragment thereof that retains specific binding to NKG2D, particularly human NKG2D. Antibody fragments include, but are not limited to, Fv molecules, scFv molecule, Fab molecule, and F(ab')2 molecules. In particular embodiments, however, the antibody is a full-length antibody. In some embodiments, the antibody comprises an Fc domain, composed of a first and a second subunit. In some embodiments, the antibody is an immunoglobulin, particularly an IgG class, more particularly an IgGl subclass immunoglobulin.
In some embodiments, the antibody is a monoclonal antibody.
In some embodiments, the antibody comprises a heavy chain complementary determining region (HCDR) 1 comprising the amino acid sequence of SEQ ID NO:l, a HCDR 2 comprising the amino acid sequence of SEQ ID NO:2, and a HCDR 3 comprising the amino acid sequence of SEQ ID NO:3; and a light chain complementarity determining region (LCDR) 1 comprising the amino acid sequence of SEQ ID NO:4, a LCDR 2 comprising the amino acid sequence of SEQ ID NO:5, and a LCDR 3 comprising the amino acid sequence of SEQ ID NO:6.
In some embodiments, the antibody comprises (a) a heavy chain variable region (VH) comprising a HCDR 1 comprising the amino acid sequence of SEQ ID NO:l, a HCDR 2 comprising the amino acid sequence of SEQ ID NO:2, and a HCDR 3 comprising the amino acid sequence of SEQ ID NO:3, and (b) a light chain variable region (VL) comprising a LCDR 1 comprising the amino acid sequence of SEQ ID NO:4, a LCDR 2 comprising the amino acid sequence of SEQ ID NO:5, and a LCDR 3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the heavy and/or light chain variable region is a humanized variable region. In some embodiments, the heavy and/or light chain variable region comprises human framework regions (FR).
In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:7. In some embodiments, the antibody comprises a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence of SEQ ID NO: 8.
In a particular embodiment, the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 7, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 8.
In some embodiments, the antibody comprises a heavy chain complementary determining region (HCDR) 1 comprising the amino acid sequence of SEQ ID NO:9, a HCDR 2 comprising the amino acid sequence of SEQ ID NO: 10, and a HCDR 3 comprising the amino acid sequence of SEQ ID NO: 11; and a light chain complementarity determining region (LCDR) 1 comprising the amino acid sequence of SEQ ID NO: 12, a LCDR 2 comprising the amino acid sequence of SEQ ID NO: 13, and a LCDR 3 comprising the amino acid sequence of SEQ ID NO: 14.
In some embodiments, the antibody comprises (a) a heavy chain variable region (VH) comprising a HCDR 1 comprising the amino acid sequence of SEQ ID NO:9, a HCDR 2
comprising the amino acid sequence of SEQ ID NO: 10, and a HCDR 3 comprising the amino acid sequence of SEQ ID NO: 11, and (b) a light chain variable region (VL) comprising a LCDR 1 comprising the amino acid sequence of SEQ ID NO: 12, a LCDR 2 comprising the amino acid sequence of SEQ ID NO: 13, and a LCDR 3 comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the heavy and/or light chain variable region is a humanized variable region. In some embodiments, the heavy and/or light chain variable region comprises human framework regions (FR).
In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the antibody comprises a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence of SEQ ID NO: 16.
In a particular embodiment, the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16.
In some embodiments, the antibody comprises a heavy chain complementary determining region (HCDR) 1 comprising the amino acid sequence of SEQ ID NO: 17, a HCDR 2 comprising the amino acid sequence of SEQ ID NO: 18, and a HCDR 3 comprising the amino acid sequence of SEQ ID NO: 19; and a light chain complementarity determining region (LCDR) 1 comprising the amino acid sequence of SEQ ID NO:20, a LCDR 2 comprising the amino acid sequence of SEQ ID NO:21, and a LCDR 3 comprising the amino acid sequence of SEQ ID NO:22.
In some embodiments, the antibody comprises (a) a heavy chain variable region (VH) comprising a HCDR 1 comprising the amino acid sequence of SEQ ID NO: 17, a HCDR 2 comprising the amino acid sequence of SEQ ID NO: 18, and a HCDR 3 comprising the amino acid sequence of SEQ ID NO: 19, and (b) a light chain variable region (VL) comprising a LCDR 1 comprising the amino acid sequence of SEQ ID NO:20, a LCDR 2 comprising the amino acid sequence of SEQ ID NO:21, and a LCDR 3 comprising the amino acid sequence of SEQ ID NO:22. In some embodiments, the heavy and/or light chain variable region is a humanized variable region. In some embodiments, the heavy and/or light chain variable region comprises human framework regions (FR).
In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the
amino acid sequence of SEQ ID NO:23. In some embodiments, the antibody comprises a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence of SEQ ID NO:24.
In a particular embodiment, the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 23, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 24.
In some embodiments, the antibody comprises a heavy chain complementary determining region (HCDR) 1 comprising the amino acid sequence of SEQ ID NO:25, a HCDR 2 comprising the amino acid sequence of SEQ ID NO:26, and a HCDR 3 comprising the amino acid sequence of SEQ ID NO:27; and a light chain complementarity determining region (LCDR) 1 comprising the amino acid sequence of SEQ ID NO:28, a LCDR 2 comprising the amino acid sequence of SEQ ID NO:29, and a LCDR 3 comprising the amino acid sequence of SEQ ID NO:30.
In some embodiments, the antibody comprises (a) a heavy chain variable region (VH) comprising a HCDR 1 comprising the amino acid sequence of SEQ ID NO:25, a HCDR 2 comprising the amino acid sequence of SEQ ID NO:26, and a HCDR 3 comprising the amino acid sequence of SEQ ID NO:27, and (b) a light chain variable region (VL) comprising a LCDR 1 comprising the amino acid sequence of SEQ ID NO:28, a LCDR 2 comprising the amino acid sequence of SEQ ID NO:29, and a LCDR 3 comprising the amino acid sequence of SEQ ID NO:30. In some embodiments, the heavy and/or light chain variable region is a humanized variable region. In some embodiments, the heavy and/or light chain variable region comprises human framework regions (FR).
In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:31. In some embodiments, the antibody comprises a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence of SEQ ID NO:32.
In a particular embodiment, the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 31, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 32.
In some embodiments, the antibody comprises a heavy chain complementary determining region (HCDR) 1 comprising the amino acid sequence of SEQ ID NO:33, a HCDR 2 comprising the amino acid sequence of SEQ ID NO:34, and a HCDR 3 comprising the amino acid sequence of
SEQ ID NO:35; and a light chain complementarity determining region (LCDR) 1 comprising the amino acid sequence of SEQ ID NO:36, a LCDR 2 comprising the amino acid sequence of SEQ ID NO:37, and a LCDR 3 comprising the amino acid sequence of SEQ ID NO:38.
In some embodiments, the antibody comprises (a) a heavy chain variable region (VH) comprising a HCDR 1 comprising the amino acid sequence of SEQ ID NO:33, a HCDR 2 comprising the amino acid sequence of SEQ ID NO:34, and a HCDR 3 comprising the amino acid sequence of SEQ ID NO:35, and (b) a light chain variable region (VL) comprising a LCDR 1 comprising the amino acid sequence of SEQ ID NO:36, a LCDR 2 comprising the amino acid sequence of SEQ ID NO:37, and a LCDR 3 comprising the amino acid sequence of SEQ ID NO:38. In some embodiments, the heavy and/or light chain variable region is a humanized variable region. In some embodiments, the heavy and/or light chain variable region comprises human framework regions (FR).
In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:39. In some embodiments, the antibody comprises a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence of SEQ ID NO:40.
In a particular embodiment, the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 39, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 40.
In some embodiments, the antibody comprises a heavy chain complementary determining region (HCDR) 1 comprising the amino acid sequence of SEQ ID NO:83, a HCDR 2 comprising the amino acid sequence of SEQ ID NO:84, and a HCDR 3 comprising the amino acid sequence of SEQ ID NO:85; and a light chain complementarity determining region (LCDR) 1 comprising the amino acid sequence of SEQ ID NO:86, a LCDR 2 comprising the amino acid sequence of SEQ ID NO:87, and a LCDR 3 comprising the amino acid sequence of SEQ ID NO:88.
In some embodiments, the antibody comprises (a) a heavy chain variable region (VH) comprising a HCDR 1 comprising the amino acid sequence of SEQ ID NO:83, a HCDR 2 comprising the amino acid sequence of SEQ ID NO:84, and a HCDR 3 comprising the amino acid sequence of SEQ ID NO:85, and (b) a light chain variable region (VL) comprising a LCDR 1 comprising the amino acid sequence of SEQ ID NO:86, a LCDR 2 comprising the amino acid sequence of SEQ ID NO:87, and a LCDR 3 comprising the amino acid sequence of SEQ ID
NO:88. In some embodiments, the heavy and/or light chain variable region is a humanized variable region. In some embodiments, the heavy and/or light chain variable region comprises human framework regions (FR).
In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:89. In some embodiments, the antibody comprises a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence of SEQ ID NO:90.
In a particular embodiment, the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 89, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 90.
In some embodiments, the antibody is a humanized antibody. In one embodiment, the antibody is an immunoglobulin molecule comprising a human constant region, particularly an IgG class immunoglobulin molecule comprising a human CHI, CH2, CH3 and/or CL domain. Exemplary sequences of human constant domains are given in SEQ ID NOs 62 and 63 (human kappa and lambda CL domains, respectively) and SEQ ID NO: 64 (human IgGl heavy chain constant domains CH1-CH2-CH3). In some embodiments, the antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 62 or SEQ ID NO: 63, particularly the amino acid sequence of SEQ ID NO: 62. In some embodiments, the antibody comprises a heavy chain constant region comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 64. Particularly, the heavy chain constant region may comprise amino acid mutations in the Fc domain as described herein.
NKG2D antibodies are disclosed in PCT application PCT/EP2020/069813, which is incorporated by reference in its entirety.
3. FC DOMAIN
In particular embodiments, the antibody comprised in the immunconjugates according to the invention comprises an Fc domain, composed of a first and a second subunit. The Fc domain of an antibody consists of a pair of polypeptide chains comprising heavy chain domains of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule
is a dimer, each subunit of which comprises the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain are capable of stable association with each other. In one embodiment the immunoconjugate of the invention comprises not more than one Fc domain.
In one embodiment the Fc domain of the antibody comprised in the immunoconjugate is an IgG Fc domain. In a particular embodiment the Fc domain is an IgGl Fc domain. In another embodiment the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (Kabat EU index numbering), particularly the amino acid substitution S228P. This amino acid substitution reduces in vivo Fab arm exchange of IgG4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In a further particular embodiment the Fc domain is a human Fc domain. In an even more particular embodiment, the Fc domain is a human IgGl Fc domain. An exemplary sequence of a human IgGl Fc region is given in SEQ ID NO: 61.
Immunoconjugate s according to the invention comprise a mutant IL-2 polypeptide, particularly a single (not more than one) mutant IL-2 polypeptide, fused to one or the other of the two subunits of the Fc domain, thus the two subunits of the Fc domain are typically comprised in two non identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization leads to several possible combinations of the two polypeptides. To improve the yield and purity of the immunoconjugate in recombinant production, it will thus be advantageous to introduce in the Fc domain of the antibody a modification promoting the association of the desired polypeptides.
Accordingly, in particular embodiments, the Fc domain of the antibody comprised in the immunoconjugate according to the invention comprises a modification promoting the association of the first and the second subunit of the Fc domain. The site of most extensive protein-protein interaction between the two subunits of a human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one embodiment said modification is in the CH3 domain of the Fc domain.
There exist several approaches for modifications in the CH3 domain of the Fc domain in order to enforce heterodimerization, which are well described e.g. in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, in all such approaches the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are both engineered in a complementary manner so that each CH3 domain (or the heavy chain comprising it) can no longer homodimerize
with itself but is forced to heterodimerize with the complementarily engineered other CH3 domain (so that the first and second CH3 domain heterodimerize and no homodimers between the two first or the two second CH3 domains are formed).
In a specific embodiment said modification promoting the association of the first and the second subunit of the Fc domain is a so-called “knob-into-hole” modification, comprising a “knob” modification in one of the two subunits of the Fc domain and a “hole” modification in the other one of the two subunits of the Fc domain.
The knob-into-hole technology is described e.g. in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine).
Accordingly, in a particular embodiment, in the CH3 domain of the first subunit of the Fc domain of the antibody comprised in the immunoconjugate an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable.
Preferably said amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).
Preferably said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).
The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis.
In a specific embodiment, in the CH3 domain of the first subunit of the Fc domain (the “knobs” subunit) the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain (the “hole” subunit) the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain additionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numberings according to Kabat EU index).
In yet a further embodiment, in the first subunit of the Fc domain additionally the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C) (particularly the serine residue at position 354 is replaced with a cysteine residue), and in the second subunit of the Fc domain additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (numberings according to Kabat EU index). Introduction of these two cysteine residues results in formation of a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).
In a particular embodiment, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to Kabat EU index).
In some embodiments, the second subunit of the Fc domain additionally comprises the amino acid substitutions H435R and Y436F (numbering according to Kabat EU index).
In a particular embodiment the mutant IL-2 polypeptide is fused (optionally through a linker peptide) to the first subunit of the Fc domain (comprising the “knob” modification). Without wishing to be bound by theory, fusion of the mutant IL-2 polypeptide to the knob-containing subunit of the Fc domain will (further) minimize the generation of immunoconjugates comprising two mutant IL-2 polypeptides (steric clash of two knob-containing polypeptides).
Other techniques of CH3 -modification for enforcing the heterodimerization are contemplated as alternatives according to the invention and are described e.g. in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO 2013/096291.
In one embodiment the heterodimerization approach described in EP 1870459, is used alternatively. This approach is based on the introduction of charged amino acids with opposite charges at specific amino acid positions in the CH3/CH3 domain interface between the two
subunits of the Fc domain. One preferred embodiment for the antibody comprised in the immunoconjugate of the invention are amino acid mutations R409D; K370E in one of the two CH3 domains (of the Fc domain) and amino acid mutations D399K; E357K in the other one of the CH3 domains of the Fc domain (numbering according to Kabat EU index).
In another embodiment, the antibody comprised in the immunoconjugate of the invention comprises amino acid mutation T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and additionally amino acid mutations R409D; K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutations D399K; E357K in the CH3 domain of the second subunit of the Fc domain (numberings according to Kabat EU index).
In another embodiment, the antibody comprised in the immunoconjugate of the invention comprises amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, or said antibody comprises amino acid mutations Y349C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations S354C, T366S, L368A, Y407V in the CH3 domains of the second subunit of the Fc domain and additionally amino acid mutations R409D; K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutations D399K; E357K in the CH3 domain of the second subunit of the Fc domain (all numberings according to Kabat EU index).
In one embodiment, the heterodimerization approach described in WO 2013/157953 is used alternatively. In one embodiment, a first CH3 domain comprises amino acid mutation T366K and a second CH3 domain comprises amino acid mutation L351D (numberings according to Kabat EU index). In a further embodiment, the first CH3 domain comprises further amino acid mutation L351K. In a further embodiment, the second CH3 domain comprises further an amino acid mutation selected from Y349E, Y349D and L368E (preferably L368E) (numberings according to Kabat EU index).
In one embodiment, the heterodimerization approach described in WO 2012/058768 is used alternatively. In one embodiment, a first CH3 domain comprises amino acid mutations L351Y, Y407A and a second CH3 domain comprises amino acid mutations T366A, K409F. In a further embodiment, the second CH3 domain comprises a further amino acid mutation at position T411, D399, S400, F405, N390, or K392, e.g. selected from a) T411N, T411R, T411Q, T411K, T411D, T411E or T411W, b) D399R, D399W, D399Y or D399K, c) S400E, S400D, S400R, or
S400K, d) F405I, F405M, F405T, F405S, F405V or F405W, e) N390R, N390K or N390D, f) K392V, K392M, K392R, K392L, K392F or K392E (numberings according to Rabat EU index). In a further embodiment, a first CH3 domain comprises amino acid mutations L351Y, Y407A and a second CH3 domain comprises amino acid mutations T366V, K409F. In a further embodiment a first CH3 domain comprises amino acid mutation Y407A and a second CH3 domain comprises amino acid mutations T366A, K409F. In a further embodiment, the second CH3 domain further comprises amino acid mutations K392E, T411E, D399R and S400R (numberings according to Rabat EU index).
In one embodiment the heterodimerization approach described in WO 2011/143545 is used alternatively, e.g. with the amino acid modification at a position selected from the group consisting of 368 and 409 (numbering according to Rabat EU index).
In one embodiment, the heterodimerization approach described in WO 2011/090762, which also uses the knobs-into-holes technology described above, is used alternatively. In one embodiment, a first CH3 domain comprises amino acid mutation T366W and a second CH3 domain comprises amino acid mutation Y407A. In one embodiment, a first CH3 domain comprises amino acid mutation T366Y and a second CH3 domain comprises amino acid mutation Y407T (numberings according to Rabat EU index).
In one embodiment, the antibody comprised in the immunoconjugate or its Fc domain is of IgG2 subclass and the heterodimerization approach described in WO 2010/129304 is used alternatively.
In an alternative embodiment, a modification promoting association of the first and the second subunit of the Fc domain comprises a modification mediating electrostatic steering effects, e.g. as described in PCT publication WO 2009/089004. Generally, this method involves replacement of one or more amino acid residues at the interface of the two Fc domain subunits by charged amino acid residues so that homodimer formation becomes electrostatically unfavorable but heterodimerization electrostatically favorable. In one such embodiment, a first CH3 domain comprises amino acid substitution of R392 or N392 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D), preferably R392D or N392D) and a second CH3 domain comprises amino acid substitution of D399, E356, D356, or E357 with a positively charged amino acid (e.g. lysine (R) or arginine (R), preferably D399R, E356R, D356R, or E357R, and more preferably D399R and E356R). In a further embodiment, the first CH3 domain further comprises amino acid substitution of R409 or R409 with a negatively charged amino acid (e.g.
glutamic acid (E), or aspartic acid (D), preferably K409D or R409D). In a further embodiment, the first CH3 domain further or alternatively comprises amino acid substitution of K439 and/or K370 with a negatively charged amino acid (e.g. glutamic acid (E), or aspartic acid (D)) (all numberings according to Kabat EU index).
In yet a further embodiment, the heterodimerization approach described in WO 2007/147901 is used alternatively. In one embodiment, a first CH3 domain comprises amino acid mutations K253E, D282K, and K322D and a second CH3 domain comprises amino acid mutations D239K, E240K, and K292D (numberings according to Kabat EU index).
In still another embodiment, the heterodimerization approach described in WO 2007/110205 can be used alternatively.
In one embodiment, the first subunit of the Fc domain comprises amino acid substitutions K392D and K409D, and the second subunit of the Fc domain comprises amino acid substitutions D356K and D399K (numbering according to Kabat EU index).
The Fc domain confers to the immunoconjugate favorable pharmacokinetic properties, including a long serum half-life which contributes to good accumulation in the target tissue and a favorable tissue-blood distribution ratio. At the same time it may, however, lead to undesirable targeting of the immunoconjugate to cells expressing Fc receptors rather than to the preferred antigen bearing cells. Moreover, the co-activation of Fc receptor signaling pathways may lead to cytokine release which, in combination with the IL-2 polypeptide and the long half-life of the immunoconjugate, results in excessive activation of cytokine receptors and severe side effects upon systemic administration. In line with this, conventional IgG-IL-2 immunoconjugates have been described to be associated with infusion reactions (see e.g. King et ak, J Clin Oncol 22, 4463-4473 (2004)).
Accordingly, in particular embodiments, the Fc domain of the antibody comprised in the immunoconjugate according to the invention exhibits reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgGl Fc domain. In one such embodiment the Fc domain (or the antibody comprising said Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the binding affinity to an Fc receptor, as compared to a native IgGl Fc domain (or an antibody comprising a native IgGl Fc domain), and/or less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the effector function, as compared to a native IgGl Fc domain domain (or an antibody comprising a native IgGl Fc domain). In one
embodiment, the Fc domain domain (or an antibody comprising said Fc domain) does not substantially bind to an Fc receptor and/or induce effector function. In a particular embodiment the Fc receptor is an Fey receptor. In one embodiment the Fc receptor is a human Fc receptor. In one embodiment the Fc receptor is an activating Fc receptor. In a specific embodiment the Fc receptor is an activating human Fey receptor, more specifically human FcyRIIIa, FcyRI or FcyRIIa, most specifically human FcyRIIIa. In one embodiment the effector function is one or more selected from the group of CDC, ADCC, ADCP, and cytokine secretion. In a particular embodiment the effector function is ADCC. In one embodiment the Fc domain domain exhibits substantially similar binding affinity to neonatal Fc receptor (FcRn), as compared to a native IgGl Fc domain domain. Substantially similar binding to FcRn is achieved when the Fc domain (or an antibody comprising said Fc domain) exhibits greater than about 70%, particularly greater than about 80%, more particularly greater than about 90% of the binding affinity of a native IgGl Fc domain (or an antibody comprising a native IgGl Fc domain) to FcRn.
In certain embodiments the Fc domain is engineered to have reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a non-engineered Fc domain. In particular embodiments, the Fc domain of the antibody comprised in the immunoconjugate comprises one or more amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and/or effector function. Typically, the same one or more amino acid mutation is present in each of the two subunits of the Fc domain. In one embodiment the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor. In one embodiment the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor by at least 2- fold, at least 5-fold, or at least 10-fold. In embodiments where there is more than one amino acid mutation that reduces the binding affinity of the Fc domain to the Fc receptor, the combination of these amino acid mutations may reduce the binding affinity of the Fc domain to an Fc receptor by at least 10-fold, at least 20-fold, or even at least 50-fold. In one embodiment the antibody comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, more particularly less than 5% of the binding affinity to an Fc receptor as compared to an antibody comprising a non-engineered Fc domain. In a particular embodiment the Fc receptor is an Fey receptor. In some embodiments the Fc receptor is a human Fc receptor. In some embodiments the Fc receptor is an activating Fc receptor. In a specific embodiment the Fc receptor is an activating human Fey receptor, more specifically human FcyRIIIa, FcyRI or FcyRIIa, most specifically human FcyRIIIa. Preferably, binding to each of these receptors is reduced. In some embodiments binding affinity to a complement component, specifically binding affinity to Clq,
is also reduced. In one embodiment binding affinity to neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e. preservation of the binding affinity of the Fc domain to said receptor, is achieved when the Fc domain (or an antibody comprising said Fc domain) exhibits greater than about 70% of the binding affinity of a non-engineered form of the Fc domain (or an antibody comprising said non-engineered form of the Fc domain) to FcRn. The Fc domain, or antibody comprised in the immunoconjugate of the invention comprising said Fc domain, may exhibit greater than about 80% and even greater than about 90% of such affinity. In certain embodiments the Fc domain of the antibody comprised in the immunoconjugate is engineered to have reduced effector function, as compared to a non-engineered Fc domain. The reduced effector function can include, but is not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NR cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling inducing apoptosis, reduced crosslinking of target-bound antibodies, reduced dendritic cell maturation, or reduced T cell priming. In one embodiment the reduced effector function is one or more selected from the group of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a particular embodiment the reduced effector function is reduced ADCC. In one embodiment the reduced ADCC is less than 20% of the ADCC induced by a non-engineered Fc domain (or an antibody comprising a non-engineered Fc domain).
In one embodiment the amino acid mutation that reduces the binding affinity of the Fc domain to an Fc receptor and/or effector function is an amino acid substitution. In one embodiment the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329 (numberings according to Rabat EU index). In a more specific embodiment the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235 and P329 (numberings according to Rabat EU index). In some embodiments the Fc domain comprises the amino acid substitutions L234A and L235A (numberings according to Rabat EU index). In one such embodiment, the Fc domain is an IgGl Fc domain, particularly a human IgGl Fc domain. In one embodiment the Fc domain comprises an amino acid substitution at position P329. In a more specific embodiment the amino acid substitution is P329A or P329G, particularly P329G (numberings according to Rabat EU index). In one embodiment the Fc domain comprises an amino acid substitution at position P329 and a
further amino acid substitution at a position selected from E233, L234, L235, N297 and P331 (numberings according to Kabat EU index). In a more specific embodiment the further amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In particular embodiments the Fc domain comprises amino acid substitutions at positions P329, L234 and L235 (numberings according to Kabat EU index). In more particular embodiments the Fc domain comprises the amino acid mutations L234A, L235A and P329G (“P329G LALA”, “PGLALA” or “LALAPG”). Specifically, in particular embodiments, each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering), i.e. in each of the first and the second subunit of the Fc domain the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A) and the proline residue at position 329 is replaced by a glycine residue (P329G) (numbering according to Kabat EU index). In one such embodiment, the Fc domain is an IgGl Fc domain, particularly a human IgGl Fc domain. The “P329G LALA” combination of amino acid substitutions almost completely abolishes Fey receptor (as well as complement) binding of a human IgGl Fc domain, as described in PCT publication no. WO 2012/130831, which is incorporated herein by reference in its entirety. WO 2012/130831 also describes methods of preparing such mutant Fc domains and methods for determining its properties such as Fc receptor binding or effector functions.
IgG4 antibodies exhibit reduced binding affinity to Fc receptors and reduced effector functions as compared to IgGl antibodies. Hence, in some embodiments the Fc domain of the antibody comprised in the immunoconjugate of the invention is an IgG4 Fc domain, particularly a human IgG4 Fc domain. In one embodiment the IgG4 Fc domain comprises amino acid substitutions at position S228, specifically the amino acid substitution S228P (numberings according to Kabat EU index). To further reduce its binding affinity to an Fc receptor and/or its effector function, in one embodiment the IgG4 Fc domain comprises an amino acid substitution at position L235, specifically the amino acid substitution L235E (numberings according to Kabat EU index). In another embodiment, the IgG4 Fc domain comprises an amino acid substitution at position P329, specifically the amino acid substitution P329G (numberings according to Kabat EU index). In a particular embodiment, the IgG4 Fc domain comprises amino acid substitutions at positions S228, L235 and P329, specifically amino acid substitutions S228P, L235E and P329G (numberings according to Kabat EU index). Such IgG4 Fc domain mutants and their Fey receptor binding properties are described in PCT publication no. WO 2012/130831, incorporated herein by reference in its entirety.
In a particular embodiment, the Fc domain exhibiting reduced binding affinity to an Fc receptor and/or reduced effector function, as compared to a native IgGl Fc domain, is a human IgGl Fc domain comprising the amino acid substitutions L234A, L235A and optionally P329G, or a human IgG4 Fc domain comprising the amino acid substitutions S228P, L235E and optionally P329G (numberings according to Kabat EU index).
In certain embodiments N-glycosylation of the Fc domain has been eliminated. In one such embodiment, the Fc domain comprises an amino acid mutation at position N297, particularly an amino acid substitution replacing asparagine by alanine (N297A) or aspartic acid (N297D) (numberings according to Kabat EU index).
In addition to the Fc domains described hereinabove and in PCT publication no. WO 2012/130831, Fc domains with reduced Fc receptor binding and/or effector function also include those with substitution of one or more of Fc domain residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056) (numberings according to Kabat EU index). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).
Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide changes can be verified for example by sequencing.
Binding to Fc receptors can be easily determined e.g. by ELISA, or by Surface Plasmon Resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare), and Fc receptors such as may be obtained by recombinant expression. Alternatively, binding affinity of Fc domains or antibodies comprising an Fc domain for Fc receptors may be evaluated using cell lines known to express particular Fc receptors, such as human NK cells expressing Fcyllla receptor.
Effector function of an Fc domain, or an antibody comprising an Fc domain, can be measured by methods known in the art. Examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Patent No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non radioactive assays methods may be employed (see, for example, ACTI™ non-radioactive
cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g. in a animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).
In some embodiments, binding of the Fc domain to a complement component, specifically to Clq, is reduced. Accordingly, in some embodiments wherein the Fc domain is engineered to have reduced effector function, said reduced effector function includes reduced CDC. Clq binding assays may be carried out to determine whether the Fc domain, or antibody comprising the Fc domain, is able to bind Clq and hence has CDC activity. See e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano- Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738- 2743 (2004)).
FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int’l. Immunol. 18(12): 1759-1769 (2006); WO 2013/120929).
4. PARTICULAR ASPECTS OF THE INVENTION
In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to NKG2D, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising the amino acid substitutions F42A, Y45A and L72G (numbering relative to the human IL-2 sequence SEQ ID NO: 41); and wherein the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 7, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:8.
In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to NKG2D, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising the amino acid substitutions T3A, F42A, Y45A, L72G and C125A (numbering relative to the human IL-2 sequence SEQ ID NO: 41); and wherein the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ
ID NO:7, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:8.
In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to NKG2D, wherein the mutant IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 42; and wherein the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:7, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:8.
In one embodiment according to any of the above aspects of the invention, the antibody is an IgG class immunoglobulin, comprising a human IgGl Fc domain composed of a first and a second subunit, wherein in the first subunit of the Fc domain the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the second subunit of the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V) and optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numberings according to Kabat EU index), and wherein further each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering).
In this embodiment, the mutant IL-2 polypeptide may be fused at its amino-terminal amino acid to the carboxy -terminal amino acid of the first subunit of the Fc domain, through a linker peptide of SEQ ID NO: 43.
In one aspect, the invention provides an immunoconjugate comprising a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:44, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:45, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:46.
In one aspect, the invention provides an immunoconjugate comprising a polypeptide comprising an amino acid sequence of SEQ ID NO:44, a polypeptide comprising an amino acid sequence of SEQ ID NO:45, and a polypeptide comprising an amino acid sequence of SEQ ID NO:46.
In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to NKG2D, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising the amino acid substitutions F42A, Y45A and L72G (numbering relative to
the human IL-2 sequence SEQ ID NO: 41); and wherein the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16.
In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to NKG2D, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising the amino acid substitutions T3A, F42A, Y45A, L72G and C125A (numbering relative to the human IL-2 sequence SEQ ID NO: 41); and wherein the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16.
In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to NKG2D, wherein the mutant IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 42; and wherein the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 15, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 16.
In one embodiment according to any of the above aspects of the invention, the antibody is an IgG class immunoglobulin, comprising a human IgGl Fc domain composed of a first and a second subunit, wherein in the first subunit of the Fc domain the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the second subunit of the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V) and optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numberings according to Kabat EU index), and wherein further each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering).
In this embodiment, the mutant IL-2 polypeptide may be fused at its amino-terminal amino acid to the carboxy -terminal amino acid of the first subunit of the Fc domain, through a linker peptide of SEQ ID NO: 43.
In one aspect, the invention provides an immunoconjugate comprising a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:47, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:48, and a polypeptide comprising an amino acid sequence that is at
least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:49.
In one aspect, the invention provides an immunoconjugate comprising a polypeptide comprising an amino acid sequence of SEQ ID NO:47, a polypeptide comprising an amino acid sequence of SEQ ID NO:48, and a polypeptide comprising an amino acid sequence of SEQ ID NO:49.
In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to NKG2D, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising the amino acid substitutions F42A, Y45A and L72G (numbering relative to the human IL-2 sequence SEQ ID NO: 41); and wherein the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:23, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:24.
In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to NKG2D, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising the amino acid substitutions T3A, F42A, Y45A, L72G and C125A (numbering relative to the human IL-2 sequence SEQ ID NO: 41); and wherein the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:23, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:24.
In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to NKG2D, wherein the mutant IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 42; and wherein the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:23, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:24.
In one embodiment according to any of the above aspects of the invention, the antibody is an IgG class immunoglobulin, comprising a human IgGl Fc domain composed of a first and a second subunit, wherein in the first subunit of the Fc domain the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the second subunit of the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V) and optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numberings according to Kabat EU index), and wherein further each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering).
In this embodiment, the mutant IL-2 polypeptide may be fused at its amino-terminal amino acid to the carboxy -terminal amino acid of the first subunit of the Fc domain, through a linker peptide of SEQ ID NO: 43.
In one aspect, the invention provides an immunoconjugate comprising a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:50, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:51, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:52.
In one aspect, the invention provides an immunoconjugate comprising a polypeptide comprising an amino acid sequence of SEQ ID NO:50, a polypeptide comprising an amino acid sequence of SEQ ID NO:51, and a polypeptide comprising an amino acid sequence of SEQ ID NO:52.
In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to NKG2D, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising the amino acid substitutions F42A, Y45A and L72G (numbering relative to the human IL-2 sequence SEQ ID NO: 41); and wherein the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:31, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:32.
In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to NKG2D, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising the amino acid substitutions T3A, F42A, Y45A, L72G and C125A (numbering relative to the human IL-2 sequence SEQ ID NO: 41); and wherein the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:31, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:32.
In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to NKG2D, wherein the mutant IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 42; and wherein the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:31, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:32.
In one embodiment according to any of the above aspects of the invention, the antibody is an IgG class immunoglobulin, comprising a human IgGl Fc domain composed of a first and a second subunit, wherein in the first subunit of the Fc domain the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the second subunit of the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V) and optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numberings according to Kabat EU index), and wherein further each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering).
In this embodiment, the mutant IL-2 polypeptide may be fused at its amino-terminal amino acid to the carboxy -terminal amino acid of the first subunit of the Fc domain, through a linker peptide of SEQ ID NO: 43.
In one aspect, the invention provides an immunoconjugate comprising a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:53, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:54, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:55.
In one aspect, the invention provides an immunoconjugate comprising a polypeptide comprising an amino acid sequence of SEQ ID NO:53, a polypeptide comprising an amino acid sequence of SEQ ID NO:54, and a polypeptide comprising an amino acid sequence of SEQ ID NO:55.
In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to NKG2D, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising the amino acid substitutions F42A, Y45A and L72G (numbering relative to the human IL-2 sequence SEQ ID NO: 41); and wherein the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:39, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:40.
In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to NKG2D, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising the amino acid substitutions T3A, F42A, Y45A, L72G and C125A (numbering relative to the human IL-2 sequence SEQ ID NO: 41); and wherein the antibody
comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:39, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:40.
In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to NKG2D, wherein the mutant IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 42; and wherein the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:39, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:40.
In one embodiment according to any of the above aspects of the invention, the antibody is an IgG class immunoglobulin, comprising a human IgGl Fc domain composed of a first and a second subunit, wherein in the first subunit of the Fc domain the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the second subunit of the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V) and optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numberings according to Kabat EU index), and wherein further each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering).
In this embodiment, the mutant IL-2 polypeptide may be fused at its amino-terminal amino acid to the carboxy -terminal amino acid of the first subunit of the Fc domain, through a linker peptide of SEQ ID NO: 43.
In one aspect, the invention provides an immunoconjugate comprising a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:56, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:57, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:58.
In one aspect, the invention provides an immunoconjugate comprising a polypeptide comprising an amino acid sequence of SEQ ID NO:56, a polypeptide comprising an amino acid sequence of SEQ ID NO:57, and a polypeptide comprising an amino acid sequence of SEQ ID NO:58.
In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to NKG2D, wherein the mutant IL-2 polypeptide is a human IL-2
molecule comprising the amino acid substitutions F42A, Y45A and L72G (numbering relative to the human IL-2 sequence SEQ ID NO: 41); and wherein the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:89, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:90.
In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to NKG2D, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising the amino acid substitutions T3A, F42A, Y45A, L72G and C125A (numbering relative to the human IL-2 sequence SEQ ID NO: 41); and wherein the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:89, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:90.
In one aspect, the invention provides an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to NKG2D, wherein the mutant IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 42; and wherein the antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:89, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:90.
In one embodiment according to any of the above aspects of the invention, the antibody is an IgG class immunoglobulin, comprising a human IgGl Fc domain composed of a first and a second subunit, wherein in the first subunit of the Fc domain the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the second subunit of the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V) and optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numberings according to Kabat EU index), and wherein further each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering).
In this embodiment, the mutant IL-2 polypeptide may be fused at its amino-terminal amino acid to the carboxy -terminal amino acid of the first subunit of the Fc domain, through a linker peptide of SEQ ID NO: 43.
In one aspect, the invention provides an immunoconjugate comprising a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:91, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to
the sequence of SEQ ID NO:92, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 93.
In one aspect, the invention provides an immunoconjugate comprising a polypeptide comprising an amino acid sequence of SEQ ID NO:91, a polypeptide comprising an amino acid sequence of SEQ ID NO:92, and a polypeptide comprising an amino acid sequence of SEQ ID NO:93.
5. POLYNUCLEOTIDES
The invention further provides isolated polynucleotides encoding an immunoconjugate as described herein or a fragment thereof. In some embodiments, said fragment is an antigen binding fragment.
The polynucleotides encoding immunoconjugates of the invention may be expressed as a single polynucleotide that encodes the entire immunoconjugate or as multiple (e.g., two or more) polynucleotides that are co-expressed. Polypeptides encoded by polynucleotides that are co expressed may associate through, e.g., disulfide bonds or other means to form a functional immunoconjugate. For example, the light chain portion of an antibody may be encoded by a separate polynucleotide from the portion of the immunoconjugate comprising the heavy chain portion of the antibody and the mutant IL-2 polypeptide. When co-expressed, the heavy chain polypeptides will associate with the light chain polypeptides to form the immunoconjugate. In another example, the portion of the immunoconjugate comprising one of the two Fc domain subunits and the mutant IL-2 polypeptide could be encoded by a separate polynucleotide from the portion of the immunoconjugate comprising the the other of the two Fc domain subunits. When co-expressed, the Fc domain subunits will associate to form the Fc domain.
In some embodiments, the isolated polynucleotide encodes the entire immunoconjugate according to the invention as described herein. In other embodiments, the isolated polynucleotide encodes a polypeptide comprised in the immunoconjugate according to the invention as described herein.
In one embodiment, an isolated polynucleotide of the invention encodes the heavy chain of the antibody comprised in the immunoconjugate (e.g. an immunoglobulin heavy chain), and the mutant IL-2 polypeptide. In another embodiment, an isolated polynucleotide of the invention encodes the light chain of the antibody comprised in the immunoconjugate.
In certain embodiments the polynucleotide or nucleic acid is DNA. In other embodiments, a polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA). RNA of the present invention may be single stranded or double stranded.
6. RECOMBINANT METHODS
Mutant IL-2 polypeptides useful in the invention can be prepared by deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide changes can be verified for example by sequencing. In this regard, the nucleotide sequence of native IL-2 has been described by Taniguchi et al. (Nature 302, 305-10 (1983)) and nucleic acid encoding human IL-2 is available from public depositories such as the American Type Culture Collection (Rockville MD). The sequence of native human IL-2 is shown in SEQ ID NO: 41. Substitution or insertion may involve natural as well as non-natural amino acid residues. Amino acid modification includes well known methods of chemical modification such as the addition of glycosylation sites or carbohydrate attachments, and the like.
Immunoconjugates of the invention may be obtained, for example, by solid-state peptide synthesis (e.g. Merrifield solid phase synthesis) or recombinant production. For recombinant production one or more polynucleotide encoding the immunoconjugate (fragment), e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such polynucleotide may be readily isolated and sequenced using conventional procedures. In one embodiment a vector, preferably an expression vector, comprising one or more of the polynucleotides of the invention is provided. Methods which are well known to those skilled in the art can be used to construct expression vectors containing the coding sequence of an immunoconjugate (fragment) along with appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See, for example, the techniques described in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y (1989). The expression vector can be part of a plasmid, virus, or may be a nucleic acid fragment. The expression vector includes an expression cassette into which the polynucleotide encoding the immunoconjugate
(fragment) (i.e. the coding region) is cloned in operable association with a promoter and/or other transcription or translation control elements. As used herein, a "coding region" is a portion of nucleic acid which consists of codons translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, if present, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, 5' and 3' untranslated regions, and the like, are not part of a coding region. Two or more coding regions can be present in a single polynucleotide construct, e.g. on a single vector, or in separate polynucleotide constructs, e.g. on separate (different) vectors. Furthermore, any vector may contain a single coding region, or may comprise two or more coding regions, e.g. a vector of the present invention may encode one or more polypeptides, which are post- or co-translationally separated into the final proteins via proteolytic cleavage. In addition, a vector, polynucleotide, or nucleic acid of the invention may encode heterologous coding regions, either fused or unfused to a polynucleotide encoding the immunoconjugate of the invention, or variant or derivative thereof. Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain. An operable association is when a coding region for a gene product, e.g. a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated" if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein. A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions, which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (e.g. the immediate early promoter, in conjunction with
intron-A), simian virus 40 (e.g. the early promoter), and retroviruses (such as, e.g. Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit b-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as inducible promoters (e.g. promoters inducible tetracyclins). Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence). The expression cassette may also include other features such as an origin of replication, and/or chromosome integration elements such as retroviral long terminal repeats (LTRs), or adeno- associated viral (AAV) inverted terminal repeats (ITRs).
Polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce a secreted or "mature" form of the polypeptide. For example, human IL-2 is translated with a 20 amino acid signal sequence at the N-terminus of the polypeptide, which is subsequently cleaved off to produce the mature, 133 amino acid human IL-2. In certain embodiments, the native signal peptide, e.g. the IL-2 signal peptide or an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it. Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, may be used. For example, the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TP A) or mouse b-glucuronidase.
DNA encoding a short protein sequence that could be used to facilitate later purification (e.g. a histidine tag) or assist in labeling the immunoconjugate may be included within or at the ends of the immunoconjugate (fragment) encoding polynucleotide.
In a further embodiment, a host cell comprising one or more polynucleotides of the invention is provided. In certain embodiments a host cell comprising one or more vectors of the invention is provided. The polynucleotides and vectors may incorporate any of the features, singly or in combination, described herein in relation to polynucleotides and vectors, respectively. In one such embodiment a host cell comprises (e.g. has been transformed or transfected with) one or more vector comprising one or more polynucleotide that encodes the immunoconjugate of the invention. As used herein, the term "host cell" refers to any kind of cellular system which can be engineered to generate the immunoconjugates of the invention or fragments thereof. Host cells suitable for replicating and for supporting expression of immunoconjugates are well known in the art. Such cells may be transfected or transduced as appropriate with the particular expression vector and large quantities of vector containing cells can be grown for seeding large scale fermenters to obtain sufficient quantities of the immunoconjugate for clinical applications. Suitable host cells include prokaryotic microorganisms, such as E. coli , or various eukaryotic cells, such as Chinese hamster ovary cells (CHO), insect cells, or the like. For example, polypeptides may be produced in bacteria in particular when glycosylation is not needed. After expression, the polypeptide may be isolated from the bacterial cell paste in a soluble fraction and can be further purified. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized”, resulting in the production of a polypeptide with a partially or fully human glycosylation pattern. See Gemgross, Nat Biotech 22, 1409-1414 (2004), and Li et ah, Nat Biotech 24, 210-215 (2006). Suitable host cells for the expression of (glycosylated) polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be utilized as hosts. See e.g. US Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIESTM technology for producing antibodies in transgenic plants). Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells as described, e.g., in Graham et ak, J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells
(VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3 A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (as described, e.g., in Mather et al., Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr- CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63 and Sp2/0. For a review of certain mammalian host cell lines suitable for protein production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). Host cells include cultured cells, e.g., mammalian cultured cells, yeast cells, insect cells, bacterial cells and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, a human embryonic kidney (HEK) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell).
Standard technologies are known in the art to express foreign genes in these systems. Cells expressing a mutant-IL-2 polypeptide fused to either the heavy or the light chain of an antibody may be engineered so as to also express the other of the antibody chains such that the expressed mutant IL-2 fusion product is an antibody that has both a heavy and a light chain.
In one embodiment, a method of producing an immunoconjugate according to the invention is provided, wherein the method comprises culturing a host cell comprising one or more polynucleotide encoding the immunoconjugate, as provided herein, under conditions suitable for expression of the immunoconjugate, and optionally recovering the immunoconjugate from the host cell (or host cell culture medium).
In the immunoconjugate of the invention, the mutant IL-2 polypeptide may be genetically fused to the antibody, or may be chemically conjugated to the antibody. Genetic fusion of the IL-2 polypeptide to the antibody can be designed such that the IL-2 sequence is fused directly to the polypeptide or indirectly through a linker sequence. The composition and length of the linker may be determined in accordance with methods well known in the art and may be tested for efficacy. Particular linker peptides are described herein. Additional sequences may also be included to incorporate a cleavage site to separate the individual components of the fusion if desired, for example an endopeptidase recognition sequence. In addition, an IL-2 fusion protein may also be synthesized chemically using methods of polypeptide synthesis as is well known in the art (e.g. Merrifield solid phase synthesis). Mutant IL-2 polypeptides may be chemically conjugated to other molecules, e.g. antibodies, using well known chemical conjugation methods.
Bi-functional cross-linking reagents such as homofunctional and heterofunctional cross-linking reagents well known in the art can be used for this purpose. The type of cross-linking reagent to use depends on the nature of the molecule to be coupled to IL-2 and can readily be identified by those skilled in the art. Alternatively, or in addition, mutant IL-2 and/or the molecule to which it is intended to be conjugated may be chemically derivatized such that the two can be conjugated in a separate reaction as is also well known in the art.
The immunoconjugates of the invention comprise an antibody. Methods to produce antibodies are well known in the art (see e.g. Harlow and Lane, "Antibodies, a laboratory manual", Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodies can be constructed using solid phase-peptide synthesis, can be produced recombinantly (e.g. as described in U.S. patent No. 4,186,567) or can be obtained, for example, by screening combinatorial libraries comprising variable heavy chains and variable light chains (see e.g. U.S. Patent. No. 5,969,108 to McCafferty). Immunoconjugates, antibodies, and methods for producing the same are also described in detail e.g. in PCT publication nos. WO 2011/020783, WO 2012/107417, and WO 2012/146628, each of which are incorporated herein by reference in their entirety.
Any animal species of antibody may be used in the immunoconjugates of the invention. Non limiting antibodies useful in the present invention can be of murine, primate, or human origin. If the immunoconjugate is intended for human use, a chimeric form of antibody may be used wherein the constant regions of the antibody are from a human. A humanized or fully human form of the antibody can also be prepared in accordance with methods well known in the art (see e. g. U.S. Patent No. 5,565,332 to Winter). Humanization may be achieved by various methods including, but not limited to (a) grafting the non-human (e.g., donor antibody) CDRs onto human (e.g. recipient antibody) framework and constant regions with or without retention of critical framework residues (e.g. those that are important for retaining good antigen binding affinity or antibody functions), (b) grafting only the non-human specificity-determining regions (SDRs or a-CDRs; the residues critical for the antibody-antigen interaction) onto human framework and constant regions, or (c) transplanting the entire non-human variable domains, but "cloaking" them with a human-like section by replacement of surface residues. Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619- 1633 (2008), and are further described, e.g., in Riechmann et ak, Nature 332:323-329 (1988); Queen et ak, Proc. NatT Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5, 821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et ak, Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991)
(describing “resurfacing”); Dall’Acqua et al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the “guided selection” approach to FR shuffling). Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et ak, J. Biol. Chem. 271:22611-22618 (1996)).
Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr Opin Pharmacol 5, 368-74 (2001) and Lonberg, Curr Opin Immunol 20, 450-459 (2008). Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal’s chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSETM technology; U.S. Patent No. 5,770,429 describing HuMab® technology; U.S. Patent No. 7,041,870 describing K-M MOUSE® technology, and U.S. Patent Application Publication No. US 2007/0061900, describing VelociMouse® technology). Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.
Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse- human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boemer et al., J. Immunol., 147: 86 (1991).) Human antibodies generated via human
B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) andNi, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-91 (2005).
Human antibodies may also be generated by isolation from human antibody libraries, as described herein.
Antibodies useful in the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. Methods for screening combinatorial libraries are reviewed, e.g., in Lerner et al. in Nature Reviews 16:498-508 (2016). For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Frenzel et al. in mAbs 8:1177-1194 (2016); Bazan et al. in Human Vaccines and Immunotherapeutics 8:1817-1828 (2012) and Zhao et al. in Critical Reviews in Biotechnology 36:276-289 (2016) as well as in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O’Brien et al., ed., Human Press, Totowa, NJ, 2001) and in Marks and Bradbury in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003).
In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al. in Annual Review of Immunology 12: 433-455 (1994). Phage typically display antibody fragments, either as single chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high- affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al. in EMBO Journal 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter in Journal of Molecular Biology 227: 381-388 (1992). Patent publications describing human antibody phage
libraries include, for example: US Patent Nos. 5,750,373; 7,985,840; 7,785,903 and 8,679,490 as well as US Patent Publication Nos. 2005/0079574, 2007/0117126, 2007/0237764 and 2007/0292936. Further examples of methods known in the art for screening combinatorial libraries for antibodies with a desired activity or activities include ribosome and mRNA display, as well as methods for antibody display and selection on bacteria, mammalian cells, insect cells or yeast cells. Methods for yeast surface display are reviewed, e.g., in Scholler et al. in Methods in Molecular Biology 503:135-56 (2012) and in Cherf et al. in Methods in Molecular biology 1319:155-175 (2015) as well as in the Zhao et al. in Methods in Molecular Biology 889:73-84 (2012). Methods for ribosome display are described, e.g., in He et al. in Nucleic Acids Research 25:5132-5134 (1997) and in Hanes et al. in PNAS 94:4937-4942 (1997).
Further chemical modification of the immunoconjugate of the invention may be desirable. For example, problems of immunogenicity and short half-life may be improved by conjugation to substantially straight chain polymers such as polyethylene glycol (PEG) or polypropylene glycol (PPG) (see e.g. WO 87/00056).
Immunoconjugates prepared as described herein may be purified by art-known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity etc., and will be apparent to those having skill in the art. For affinity chromatography purification an antibody, ligand, receptor or antigen can be used to which the immunoconjugate binds. For example, an antibody which specifically binds the mutant IL-2 polypeptide may be used. For affinity chromatography purification of immunoconjugates of the invention, a matrix with protein A or protein G may be used. For example, sequential Protein A or G affinity chromatography and size exclusion chromatography can be used to isolate an immunoconjugate essentially as described in the Examples. The purity of the immunoconjugate can be determined by any of a variety of well known analytical methods including gel electrophoresis, high pressure liquid chromatography, and the like.
7. COMPOSITIONS, FORMULATIONS AND ROUTES OF ADMINISTRATION
In a further aspect, the invention provides pharmaceutical compositions comprising an immunoconjugate as described herein, e.g., for use in any of the below therapeutic methods. In one embodiment, a pharmaceutical composition comprises any of the immunoconjugates
provided herein and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical composition comprises any of the immunoconjugates provided herein and at least one additional therapeutic agent, e.g., as described below.
Further provided is a method of producing an immunoconjugate of the invention in a form suitable for administration in vivo, the method comprising (a) obtaining an immunoconjugate according to the invention, and (b) formulating the immunoconjugate with at least one pharmaceutically acceptable carrier, whereby a preparation of immunoconjugate is formulated for administration in vivo.
Pharmaceutical compositions of the present invention comprise a therapeutically effective amount of immunoconjugate dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that are generally non-toxic to recipients at the dosages and concentrations employed, i.e. do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains immunoconjugate and optionally an additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards or corresponding authorities in other countries. Preferred compositions are lyophilized formulations or aqueous solutions. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g. antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.
An immunoconjugate of the invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular,
intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
Parenteral compositions include those designed for administration by injection, e.g. subcutaneous, intradermal, intralesional, intravenous, intraarterial intramuscular, intrathecal or intraperitoneal injection. For injection, the immunoconjugates of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. The solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the immunoconjugates may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. Sterile injectable solutions are prepared by incorporating the immunoconjugates of the invention in the required amount in the appropriate solvent with various of the other ingredients enumerated below, as required. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein. Suitable pharmaceutically acceptable carriers include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or
dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, or the like. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl cleats or triglycerides, or liposomes.
Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin- microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g. films, or microcapsules. In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.
In addition to the compositions described previously, the immunoconjugates may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the immunoconjugates may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
Pharmaceutical compositions comprising the immunoconjugates of the invention may be manufactured by means of conventional mixing, dissolving, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients
or auxiliaries which facilitate processing of the proteins into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
The immunoconjugates may be formulated into a composition in a free acid or base, neutral or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or base. These include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than are the corresponding free base forms.
8. THERAPEUTIC METHODS AND COMPOSITIONS
Any of the immunoconjugates provided herein may be used in therapeutic methods. Immunoconjugates of the invention may be used as immunotherapeutic agents, for example in the treatment of cancers.
For use in therapeutic methods, immunoconjugates of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
Immunoconjugates of the invention may be particularly useful in treating disease states where stimulation of the immune system of the host is beneficial, in particular conditions where an enhanced cellular immune response is desirable. These may include disease states where the host immune response is insufficient or deficient. Disease states for which the immunoconjugates of the invention may be administered comprise, for example, a tumor or infection where a cellular immune response would be a critical mechanism for specific immunity. The immunoconjugates of the invention may be administered per se or in any suitable pharmaceutical composition.
In one aspect, immunoconjugates of the invention for use as a medicament are provided. In further aspects, immunoconjugates of the invention for use in treating a disease are provided. In certain embodiments, immunoconjugates of the invention for use in a method of treatment are provided. In one embodiment, the invention provides an immunoconjugate as described herein for use in the treatment of a disease in an individual in need thereof. In certain embodiments, the invention provides an immunoconjugate for use in a method of treating an individual having a disease comprising administering to the individual a therapeutically effective amount of the immunoconjugate. In certain embodiments the disease to be treated is a proliferative disorder. In a particular embodiment the disease is cancer. In certain embodiments the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer. In further embodiments, the invention provides an immunoconjugate for use in stimulating the immune system. In certain embodiments, the invention provides an immunoconjugate for use in a method of stimulating the immune system in an individual comprising administering to the individual an effective amount of the immunoconjugate to stimulate the immune system. An “individual” according to any of the above embodiments is a mammal, preferably a human. “Stimulation of the immune system” according to any of the above embodiments may include any one or more of a general increase in immune function, an increase in T cell function, an increase in B cell function, a restoration of lymphocyte function, an increase in the expression of IL-2 receptors, an increase in T cell responsiveness, an increase in natural killer cell activity or lymphokine-activated killer (LAK) cell activity, and the like.
In a further aspect, the invention provides for the use of an immunconjugate of the invention in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of a disease in an individual in need thereof. In one embodiment, the medicament is for use in a method of treating a disease comprising administering to an individual having the disease a therapeutically effective amount of the medicament. In certain embodiments the disease to be treated is a proliferative disorder. In a particular embodiment the disease is cancer. In one embodiment, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer. In a further embodiment, the medicament is for stimulating the immune system. In a further embodiment, the medicament is for use in a method of stimulating the immune system in an individual comprising administering to the individual an effective amount of the medicament to stimulate the immune system. An “individual” according
to any of the above embodiments may be a mammal, preferably a human. “Stimulation of the immune system” according to any of the above embodiments may include any one or more of a general increase in immune function, an increase in T cell function, an increase in B cell function, a restoration of lymphocyte function, an increase in the expression of IL-2 receptors, an increase in T cell responsiveness, an increase in natural killer cell activity or lymphokine- activated killer (LAK) cell activity, and the like.
In a further aspect, the invention provides a method for treating a disease in an individual. In one embodiment, the method comprises administering to an individual having such disease a therapeutically effective amount of an immunoconjugate of the invention. In one embodiment a composition is administered to said invididual, comprising the immunoconjugate of the invention in a pharmaceutically acceptable form. In certain embodiments the disease to be treated is a proliferative disorder. In a particular embodiment the disease is cancer. In certain embodiments the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, e.g., an anti-cancer agent if the disease to be treated is cancer. In a further aspect, the invention provides a method for stimulating the immune system in an individual, comprising administering to the individual an effective amount of an immunoconjugate to stimulate the immune system. An “individual” according to any of the above embodiments may be a mammal, preferably a human. “Stimulation of the immune system” according to any of the above embodiments may include any one or more of a general increase in immune function, an increase in T cell function, an increase in B cell function, a restoration of lymphocyte function, an increase in the expression of IL-2 receptors, an increase in T cell responsiveness, an increase in natural killer cell activity or lymphokine-activated killer (LAK) cell activity, and the like.
In certain embodiments the disease to be treated is a proliferative disorder, particularly cancer. Non-limiting examples of cancers include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. Other cell proliferation disorders that may be treated using an immunoconjugate of the present invention include, but are not limited to neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic region, and urogenital
system. Also included are pre-cancerous conditions or lesions and cancer metastases. In certain embodiments the cancer is chosen from the group consisting of kidney cancer, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer, prostate cancer and bladder cancer. A skilled artisan readily recognizes that in many cases the immunoconjugates may not provide a cure but may only provide partial benefit. In some embodiments, a physiological change having some benefit is also considered therapeutically beneficial. Thus, in some embodiments, an amount of immunoconjugate that provides a physiological change is considered an "effective amount" or a "therapeutically effective amount". The subject, patient, or individual in need of treatment is typically a mammal, more specifically a human.
In some embodiments, an effective amount of an immunoconjugate of the invention is administered to a cell. In other embodiments, a therapeutically effective amount of an immunoconjugates of the invention is administered to an individual for the treatment of disease.
For the prevention or treatment of disease, the appropriate dosage of an immunoconjugate of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the route of administration, the body weight of the patient, the type of molecule (e.g. comprising an Fc domain or not), the severity and course of the disease, whether the immunoconjugate is administered for preventive or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's clinical history and response to the immunoconjugate, and the discretion of the attending physician.. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.
The immunoconjugates of the invention will generally be used in an amount effective to achieve the intended purpose. For use to treat or prevent a disease condition, the immunoconjugates of the invention, or pharmaceutical compositions thereof, are administered or applied in a therapeutically effective amount. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.
For systemic administration, a therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays. A dose can then be formulated in animal models to
achieve a circulating concentration range that includes the IC50 as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art. One having ordinary skill in the art could readily optimize administration to humans based on animal data.
Dosage amount and interval may be adjusted individually to provide plasma levels of the immunoconjugates which are sufficient to maintain therapeutic effect. Usual patient dosages for administration by injection range from about 0.1 to 50 mg/kg/day, typically from about 0.5 to 1 mg/kg/day. Therapeutically effective plasma levels may be achieved by administering multiple doses each day. Levels in plasma may be measured, for example, by HPLC.
In cases of local administration or selective uptake, the effective local concentration of the immunoconjugates may not be related to plasma concentration. One having skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation.
A therapeutically effective dose of the immunoconjugates described herein will generally provide therapeutic benefit without causing substantial toxicity. Toxicity and therapeutic efficacy of an immunoconjugate can be determined by standard pharmaceutical procedures in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine the LD50 (the dose lethal to 50% of a population) and the ED50 (the dose therapeutically effective in 50% of a population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50/ED50. Immunoconjugates that exhibit large therapeutic indices are preferred. In one embodiment, the immunoconjugate according to the present invention exhibits a high therapeutic index. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosages suitable for use in humans. The dosage lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon a variety of factors, e.g., the dosage form employed, the route of administration utilized, the condition of the subject, and the like. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1, incorporated herein by reference in its entirety).
The attending physician for patients treated with immunoconjugates of the invention would know how and when to terminate, interrupt, or adjust administration due to toxicity, organ
dysfunction, and the like. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administered dose in the management of the disorder of interest will vary with the severity of the condition to be treated, with the route of administration, and the like. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency will also vary according to the age, body weight, and response of the individual patient.
The maximum therapeutic dose of an immunoconjugate comprising a mutant IL-2 polypeptide as described herein may be increased from those used for an immunoconjugate comprising wild- type IL-2.
9. FURTHER AGENTS AND TREATMENTS
The immunoconjugates according to the invention may be administered in combination with one or more other agents in therapy. For instance, an immunoconjugate of the invention may be co administered with at least one additional therapeutic agent. The term "therapeutic agent” encompasses any agent administered to treat a symptom or disease in an individual in need of such treatment. Such additional therapeutic agent may comprise any active ingredients suitable for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. In certain embodiments, an additional therapeutic agent is an immunomodulatory agent, a cytostatic agent, an inhibitor of cell adhesion, a cytotoxic agent, an activator of cell apoptosis, or an agent that increases the sensitivity of cells to apoptotic inducers. In a particular embodiment, the additional therapeutic agent is an anti-cancer agent, for example a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormonal therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an antiangiogenic agent.
Such other agents are suitably present in combination in amounts that are effective for the purpose intended. The effective amount of such other agents depends on the amount of immunoconjugate used, the type of disorder or treatment, and other factors discussed above. The immunoconjugates are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.
Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate compositions), and separate administration, in which case, administration of the immunoconjugate of the invention can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant. Immunoconjugates of the invention may also be used in combination with radiation therapy.
10. ARTICLES OF MANUFACURE
In another aspect of the invention, an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an immunoconjugate of the invention. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an immunoconjugate of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided above.
Example 1
Example 1 A. Preparation of NKG2D-IL2v fusion proteins
The expression cassette for the antibody heavy chain - interleukin-2 (IL-2) fusion protein [heavy chain variable region of anti-NKG2D antibody, human IgGl heavy chain (bearing mutations L234A, L235A and P329G (EU numbering) for removal of effector functions, and mutations S354C and T366W (EU numbering) for heterodimerization (“knob”)), (G4S)3 linker, and human IL-2v (bearing the mutations T3A, F42A, Y45A, L72G and C125A)], the expression cassette for the antibody heavy chain [heavy chain variable region of anti-human NKG2D antibody, and human IgGl heavy chain (bearing mutations L234A, L235A and P329G (EU numbering) for removal of effector functions, mutations Y349C, T366S, L368A and Y410V (EU numbering) for heterodimerization (“hole”), and optionally mutations H435R and Y436F (EU numbering)] and the expression cassette for the antibody light chain [light chain variable region of anti-human PD-1 antibody, and human Ckappa constant region] and was produced by gene-synthesis.
They were each cloned via Hindlll and Nhel digestion into an expression vector under the control of the CMV-promoter followed by IntronA and terminated by BGH-poly A signal. The vector further contained a bacterial ampicillin resistance gene and an origin of replication from E.coli.
The NKG2D(5C5)-IL-2v fusion protein (SEQ ID NOs 44, 45 and 46) was generated by cotransfection of HEK293F cells (Invitrogen) with the above-described vectors in the ratio of 1:1:1 in shaking flasks. After one week, supernatant was harvested and filtrated through sterile filters.
The NKG2D(13C6)-IL-2v fusion protein (SEQ ID NOs 47, 48 and 49) was generated by cotransfection of HEK293F cells (Invitrogen) with the above-described vectors in the ratio of 1:1:1 in shaking flasks. After one week, supernatant was harvested and filtrated through sterile filters.
The NKG2D(014)-IL-2v fusion protein (SEQ ID NOs 50, 51 and 52) was generated by cotransfection of HEK293F cells (Invitrogen) with the above-described vectors in the ratio of
1:1:1 in shaking flasks. After one week, supernatant was harvested and filtrated through sterile filters.
The NKG2D(296)-IL-2v fusion protein (SEQ ID NOs 53, 54 and 55) was generated by cotransfection of HEK293F cells (Invitrogen) with the above-described vectors in the ratio of 1:1:1 in shaking flasks. After one week, supernatant was harvested and filtrated through sterile filters.
The NKG2D(320)-IL-2v fusion protein (SEQ ID NOs 56, 57 and 58) was generated by cotransfection of HEK293F cells (Invitrogen) with the above-described vectors in the ratio of 1:1:1 in shaking flasks. After one week, supernatant was harvested and filtrated through sterile filters.
The NKG2D(395 cl.80)-IL-2v fusion protein (SEQ ID NOs 91, 92 and 93) was generated by cotransfection of HEK293F cells (Invitrogen) with the above-described vectors in the ratio of 1:1:1 in shaking flasks. After one week supernatant was harvested and filtrated through sterile filters. The fusion proteins were purified from the supernatant by a combination of Protein A affinity chromatography and size exclusion chromatography. The obtained product was characterized for identity by mass spectrometry and analytical properties such as purity by capillary electrophoresis (CE-SDS), monomer content and stability. The fusion proteins could be produced in good yields and are stable.
The immunoconjugates prepared in this Example were further used in the following Examples.
Example IB. Preparation of NKG2D-IgGs
The antibody molecules were generated in transiently transfected HEK293 cells (human embryonic kidney cell line 293-derived) cultivated in F17 Medium (Invitrogen Corp.). For transfection "293-Free" Transfection Reagent (Novagen) was used. The respective antibody heavy- and light chains were expressed from individual expression plasmids. Transfections were performed as specified in the manufacturer’s instructions. Immunoglobulin-containing cell culture supernatants were harvested three to seven (3-7) days after transfection and frozen at - 80°C until purification. General information regarding the recombinant expression of human immunoglobulins in e.g. HEK293 cells is given in: Meissner, P. et al., (2001), Transient gene expression: recombinant protein production with suspension-adapted HEK293-EBNA cells. Biotechnol. Bioeng. 75, 197-203.
The following antibodiy molecules were generated: NKG2D(5C5)-IgG (SEQ ID NOs 71 and 72); NKG2D( 13 C6)-IgG (SEQ ID NOs 73 and 74); NKG2D(014)-IgG (SEQ ID NOs 75 and 76); NKG2D(296)-IgG (SEQ ID NOs 77 and 78); NKG2D(320)-IgG (SEQ ID NOs 79 and 80); and NKG2D(395 cl.80)-IgG (SEQ ID NOs 81 and 82).
The recombinant antibodies were purified from the supernatant in two steps by affinity chromatography using protein A-SepharoseTM affinity chromatography (GE Healthcare, Sweden) and Superdex200 (GE Healthcare, Sweden) size exclusion chromatography. Briefly, the antibody-containing clarified culture supernatants were loaded onto a MabSelectSuRe Protein A (5-50 ml) column equilibrated with PBS buffer (10 mM Na2HP04, 1 mM KH2P04, 137 mM NaCl and 2.7 mM KC1, pH 7.4). Unbound proteins were washed out with equilibration buffer. The antibodies were eluted with 100 mM citrate buffer, pH 2.8. The protein-containing fractions were neutralized with 1/10 eluate volume of 2 M Tris buffer, pH 9.0. In a subsequent step, the eluted protein fractions were pooled and processed according to one of the three options: a) concentrated with an Amicon Ultra centrifugal filter device (MWCO: 30 K, Millipore) and loaded on a Superdex200 HiLoad 16/60 gel filtration column (GE Healthcare, Sweden) equilibrated with 20 mM histidine, 140 mM NaCl, at pH 6.0, or b) loaded onto a Superdex200 HiLoad 16/60 gel filtration column (GE Healthcare, Sweden) equilibrated with 20 mM histidine, 140 mM NaCl, at pH 6.0, or c) dialysed with 10K Slide-A-Lyzer (Thermo Fisher Scientific). Monomeric antibody fractions were pooled. The protein concentration of purified antibodies and derivatives was determined by determining the optical density (OD) at 280 nm with the OD at 320 nm as background correction, using the molar extinction coefficient calculated on the basis of the amino acid sequence according to Pace et. al., Protein Science 4 (1995) 2411-2423. Antibody samples were snap-frozen and stored at -80 °C.
The homogeneity of the antibodies was confirmed by CE-SDS LabChip GX (PerkinElmer) in the presence or absence of a reducing agent. CE-SDS is based on traditional gel electrophoresis principles. The chip is designed to sieve proteins by size as run through the chip matrix by means of electrophoresis, similar to using agarose or polyacrylamide gels. Under reducing conditions, light and heavy chain polypeptide chains of the IgGs were identified after CE-SDS at apparent molecular sizes analogous to the calculated molecular weights.
The quality of the antibodies was confirmed by analytical SEC (size-exclusion chromatography) using a BioSuite High Resolution SEC, 250A, 5pm run on an UltiMate 3000 HPLC system (Thermo Fisher Scientific). Elution from the chromatography material was performed by
applying 200 mM K2HP04/KH2P04, 250 mM KC1, pH 6.2. Main peak of analytical SEC resulted in >91% for all analyzed samples.
Example 2
Binding of NKG2D-IL2v constructs to NK cells
Two agonistic NKG2D antibodies, namely clone 320 and clone 13C6, where chosen and converted into a NKG2D-IL2v fusion protein to build a immune cell targeted IL2v. Clone 320 and clone 13C6 are abbreviated as “320” and “13C6” herein, respectively. NKG2D(320)-IL2v comprises the polypeptides with the amino acid sequence of SEQ ID NOs 56, 57 and 58. NKG2D(13C6)-IL2v comprises the polypeptides with the amino acid sequence of SEQ ID NOs 47, 48 and 49.
NK92 cells (DSMZ ACC 488) is a human natural killer lymphoma cell line. The cells were cultivated in RPMI1640, 10% FCS, 1% Glutamine and 10 ng/ml Proleukin (Novartis). NK92 cells were harvested, washed twice with PBS and resuspended in FACS buffer (PBS, 2% FBS, 5 mM EDTA, 0.025% NaN3) to a final concentration of 1 million cells per ml. 100 mΐ of the cell suspension was seeded in each well of a 96well round bottom plate. The cells were stained with 40 mΐ of the antibody dilutions for 30 min at 4°C. The cells were washed twice with FACS buffer to remove unbound antibodies. Then 30 mΐ of the diluted PE anti -human Fc specific secondary antibody (1:50 dilution, 109-116-170, Jackson ImmunoResearch) was added to the cells. After 30 min incubation at 4°C the unbound antibodies were removed by washing twice with FACS buffer. Finally, the cells were fixed with 1% PFA in FACS buffer. Before measuring, the cells were resuspended in FACS buffer and measured using a BD low cytometer.
Binding of these NKG2D-IL2v constructs to the NKG2D and IL2 receptor positive NK cell line NK92 was tested and binding to CEA-IL2v and the respective NKG2D IgGs comprising clone 13C6 and 320 was compared. NKG2D(320)-IL2v showed a stronger binding to NK92 cells as the respective IgG and CEA-IL2v. NKG2D(13C6)-IL2v showed a similar binding to NK92 cells as the respective IgG. Binding of 13C6 to NK92 cells was stronger than binding of CEA-IL2v whereas binding of 320 was weaker than binding of CEA-IL2v (Figure 2A and 2B). In summary, for both tested constructs more NKG2D-IL2v constructs could bind to NK92 cells than the non- immune cell targeted CEA-IL2v. This might help to induce a stronger IL2v induced activation of NKG2D expressing immune cells.
Example 3
Proliferation and activation of PBMCs upon NKG2D-IL2v treatment
To test if targeting of IL2v to NKG2D expressed on immune cells can enhance the activity of IL2v, the two IL2v fusion proteins NKG2D(320)-IL2v and NKG2D(13C6)-IL2v were compared to CEA-IL2v. CEA-IL2v as used herein is disclosed in PCT-application WO 2012/146628 Al, and comprises the SEQ ID NOs 277, 281 and 283 of said application. We addressed their capacity to induce proliferation and activation of NK cells, CD8 T cells and CD4 T cells within PBMCs.
Freshly isolated PBMCs from healthy donors were labeled with CFSE (5(6)-Carboxyfluorescein diacetate N-succinimidyl ester, 21888, Sigma- Aldrich). Briefly, PBMCs were washed once with PBS. In parallel, the CSFE stock solution (2 mM in DMSO) was diluted 1:20 in PBS. PBMCs were resuspended in prewarmed PBS to 1 Mio/ml, 1 mΐ of the CFSE solution was added to 1 ml cell suspension and the cells were mixed immediately. For an optimal labeling, the cells were incubated for 15 min at 37°C. Then 10 ml prewarmed medium (RPMI1640, 10% FCS, 1 %
Glutamine) were added to stop the labeling reaction. The cells were spun down for 10 min at 400g and resuspended in ml fresh medium to 1 Mio/ml and incubated for additional 30 min at 37°C. Finally, the cells were washed once with medium and resuspended in fresh medium and used directly or stored overnight in the incubator. The labeled PBMCs were seeded in a 96 well round bottom plate (100Ό00 cells per well) and treated for 5 days with the indicated molecules. After the incubation the cells were washed once with FACS buffer and stained with 20 pi of a mixture of anti-human CD3 APC-Cy7 (300318, BioLegend), anti-human CD8 BV421 (301036, BioLegend), anti-human CD56 APC (318310 , BioLegend) and CD25 PE (356104, BioLegend) in FACS buffer for 30 min at 4°C. Afterwards PBMCs were washed twice with FACS buffer before fixing them with 2% PFA in FACS buffer and measuring the fluorescence with a BD Fortessa. Proliferation was determined by measuring CFSE dilution of CD8 T cells (CD3+CD8+), CD4 T cells (CD3+CD8-) and NK cells (CD3-CD56+) and activation was determined by CD25 upregulation on CD8 T cell, CD4 T cells and NK cells.
NK cells and CD8 T cells express NKG2D and the IL2 receptor, CD4 T cells express only the IL2 receptor but not NKG2D and serve therefore as negative control. As expected, all three tested IL2v fusion molecules had the same activity on CD4 T cells to induce proliferation and CD25 upregulation. On NK cells and CD8 T cells that are NKG2D positive, NKG2D(320)-IL2v was more potent compared to CEA-IL2v whereas NKG2D(13C6)-IL2v had the same activity as
CEA-IL2v (Figure 3A-F). NKG2D(320)-IL2v was about 13-fold more potent on CD8 T cells and about 6-fold more potent on NK cells compared to CEA-IL2v (as indicated in Table 1) indicating that cis targeting of IL2v via NKG2D to immune cells increased the potency of the IL2v fusion protein and that this effect was dependent on the chosen NKG2D antibody. Table 1. EC50 values proliferation
Example 4
NKG2D dependent proliferation of NK cells and CD8 T cells To prove that the increased proliferation and activation of NK cells and CD8 T cells detected with NKG2D(320)-IL2v is dependent on binding of the construct to NKG2D, the proliferation and activation assay was performed in the presence of an NKG2D antibody that blocks binding of clone 320 to NKG2D. As control that the blocking antibody had no direct effect on the immune cells, the activity of CEA-IL2v was also tested in the presence and absence of the NKG2D antibody.
Freshly isolated PBMCs from healthy donors were labeled with CFSE (5(6)-Carboxyfluorescein diacetate N-succinimidyl ester, 21888, Sigma- Aldrich). Briefly, PBMCs were washed once with PBS. In parallel, the CSFE stock solution (2 mM in DMSO) was diluted 1:20 in PBS. PBMCs were resuspended in prewarmed PBS to 1 Mio/ml, 1 mΐ of the CFSE solution was added to 1 ml cell suspension and the cells were mixed immediately. For an optimal labeling, the cells were incubated for 15 min at 37°C. Then 10 ml prewarmed medium (RPMI1640, 10% FCS, 1 %
Glutamine) were added to stop the labeling reaction. The cells were spun down for 10 min at 400g and resuspended in ml fresh medium to 1 Mio/ml and incubated for additional 30 min at 37°C. Finally, the cells were washed once with medium and resuspended in fresh medium and used directly or stored overnight in the incubator. The labeled PBMCs were seeded in a 96 well round bottom plate (100Ό00 cells per well), pre-treated for 30 min at 37°C with 200 nM of the blocking NKG2D antibody before adding the IL2v fusion proteins. PBMCs were treated for 5 days with the indicated molecules. After the incubation the cells were washed once with FACS buffer and stained with 20 mΐ of a mixture of anti-human CD3 APC-Cy7 (300318, BioLegend), anti-human CD8 BV421 (301036, BioLegend), anti-human CD56 APC (318310 , BioLegend) and CD25 PE (356104, BioLegend) in FACS buffer for 30 min at 4°C. Afterwards PBMCs were washed twice with FACS buffer before fixing them with 1% PFA in FACS buffer and measuring the fluorescence with a BD Fortessa. Proliferation was determined by measuring CFSE dilution of CD8 T cells (CD3+CD8+), CD4 T cells (CD3+CD8-) and NK cells (CD3-CD56+) and activation was determined by CD25 upregulation on CD8 T cell, CD4 T cells and NK cells.
The presence of the blocking NKG2D antibody (NKG2D(13C6)-IgG) strongly reduced the activity of NKG2D(320)-IL2v on NK cells and CD8 T cells but not on CD4 T cells (Figure 4A- F). The presence of the blocking NKG2D antibody had no effect on the activity of CEA-IL2v on NK cells, CD8 T cells and CD4 T cells (Figure 4G-L). These results supported the concept that the increased activity observed with NKG2D(320)-IL2v was dependent on binding in cis of the fusion protein to NKG2D and the IL2 receptor on NK cells and CD8 T cells.
Example 5
Evaluation of additional NKG2D antibodies
Because it was observed that the activity of the NKG2D-IL2v fusion protein strongly depends on the selected NKG2D antibody, additional NKG2D antibodies were tested as IL2v fusion protein in a proliferation and activation assay with PBMCs. In addition to the NKG2D antibodies 13C6
and 320 tested in Examples 2 to 4, the agonistic clones 014, 5C5 and 395 cl.80 were selected for testing. Clone 014 and clone 5C5 are abbreviated as “014” and “5C5” herein, respectively. NKG2D(014)-IL2v comprises the polypeptides with the amino acid sequence of SEQ ID NOs 50, 51 and 52. NKG2D(5C5)-IL2v comprises the polypeptides with the amino acid sequence of SEQ ID NOs 44, 45 and 46. NKG2D(395 cl.80)-IL2v comprises polypeptides with the amino acid sequence of SEQ ID NOs 91, 92 and 93.
Freshly isolated PBMCs from healthy donors were labeled with CFSE (5(6)-Carboxyfluorescein diacetate N-succinimidyl ester, 21888, Sigma- Aldrich). Briefly, PBMCs were washed once with PBS. In parallel, the CSFE stock solution (2 mM in DMSO) was diluted 1:20 in PBS. PBMCs were resuspended in prewarmed PBS to 1 Mio/ml, 1 mΐ of the CFSE solution was added to 1 ml cell suspension and the cells were mixed immediately. For an optimal labeling, the cells were incubated for 15 min at 37°C. Then 10 ml prewarmed medium (RPMI1640, 10% FCS, 1 %
Glutamine) were added to stop the labeling reaction. The cells were spun down for 10 min at 400g and resuspended in ml fresh medium to 1 Mio/ml and incubated for additional 30 min at 37°C. Finally, the cells were washed once with medium and resuspended in fresh medium and used directly or stored overnight in the incubator. The labeled PBMCs were seeded in a 96 well round bottom plate (100Ό00 cells per well) and the IL2v fusion proteins were pre-incubated with the recombinant NKG2D protein for 30 min at 37°C. The IL2v fusion proteins with or without NKG2D were added to the PBMCs and incubated for 5 days. After the incubation the cells were washed once with FACS buffer and stained with 20 mΐ of a mixture of anti-human CD3 APC-Cy7 (300318, BioLegend), anti-human CD8 BV421 (301036, BioLegend), anti human CD56 APC (318310 , BioLegend) and CD25 PE (356104, BioLegend) in FACS buffer for 30 min at 4°C. Afterwards PBMCs were washed twice with FACS buffer before fixing them with 1% PFA in FACS buffer and measuring the fluorescence with a BD Fortessa. Proliferation was determined by measuring CFSE dilution of CD8 T cells (CD3+CD8+), CD4 T cells (CD3+CD8-) and NK cells (CD3-CD56+) and activation was determined by CD25 upregulation on CD8 T cell, CD4 T cells and NK cells.
Again all tested molecules had the same activity on CD4 T cells that were included as negative control. On NK cells and CD8 T cells NKG2D(5C5)-IL2v had the highest activity in inducing proliferation and activation, the second best molecule was NKG2D(320)-IL2v. NKG2D(13C6)- IL2v and NKG2D(014)-IL2v had the same activity which was lower compared to the other tested constructs (Figure 5A-5F). As control, we did the same experiment in the presence of
recombinant NKG2D to block binding of the NKG2D-IL2v constructs to NKG2D on the immune cells and analyzed CD8 T cells proliferation and activation. In the presence of recombinant NKG2D, all four tested NKG2D-IL2v constructs had the same activity (Figure 5G- 5H). This indicated that the two NKG2D antibodies 320 and 5C5 increased the activity of IL2v by binding in cis to NKG2D on NK cells and CD8 T cells. The other two NKG2D antibodies 13C6 and 014 did not increased the activity of IL2v upon cis targeting. In a separate assay NKG2D(5C5)-IL2v, NKG2D(13C6)-IL2v, NKG2D(395 cl.80)-IL2v and FAP-IL2v were compared (Figure 6A-F). As seen before, the tested molecules had the same activity on CD4 T cells except for NKG2D(395 cl.80)-IL2v which had a higher activity. On NK cells and CD8 T cells, NKG2D(5C5)-IL2v had the highest activity in inducing proliferation and activation. NKG2D(13C6)-IL2v, NKG2D(395 cl.80)-IL2v and FAP-IL2v had the same activity in inducing proliferation and activation of CD8 T cells and NK cells.
Example 6
STAT5 phosphorylation upon treatment with NKG2D-IL2v
Next, the five previously tested NKG2D-IL2v molecules (Example 1-5) were compared in their ability to induce phosphorylation of STAT5 in CD8 T cells and NK cells to FAP-IL2v (Figure 7A-7B). FAP-IL2v as used herein relates to the International Nonproprietary Name (INN) simlukafusp alfa. FAP-IL2v is disclosed in the PCT-application WO 2012/107417 Al.
Freshly isolated PBMCs from healthy donors were seeded in warm medium (RPMI1640, 10% FCS, 2 mM Glutamine) into a 96 well round bottom plate (200Ό00 cells/well). The plates were centrifuged at 300 g for 10 min and the supernatant was removed. The cells were re-suspended in
100 pi medium containing the IL2v molecules and stimulated for 20 min at 37°C. To preserve the phosphorylation status, the cells were immediately fixed after stimulation with equal amount of pre-warmed Cytofix buffer (554655, BD Bioscience) for 10 min at 37°C. Afterwards the plates were centrifuged for 5 min at 350 g and the supernatant was removed. To allow intracellular staining, the cells were permeabilized in 100 mΐ Phosflow Perm buffer III (558050, BD Bioscience) for 30 min at 4°C. Then the cells were washed twice with 150 mΐ cold FACS buffer and split in two 96 well round bottom plates and stained each with 20 mΐ of the antibody mix I or II for 60 min in the fridge. Antibody mix I was used to stain pSTAT5 in CD4 T cells and regulatory T cells and antibody mix II was used to stain pSTAT5 in CD8 T cells and NK cells. Afterwards the cells were washed twice with FACS buffer and re-suspended in 200 mΐ
FACS buffer containing 2 % PFA per well. The analysis was performed using a BD flow cytometer gating on CD8 T cells (CD3+CD8+), NK cells (CD3-CD56+, CD4 T cells (CD4+) and Tregs (CD4+CD25+FoxP3+).
As seen in the proliferation assay shown in Figures 7A and 7B, NKG2D(5C5)-IL2v had the highest activity followed by NKG2D(13C6)-IL2v and NKG2D(320)-IL2v which had an intermediate activity. The increased activity of NKG2D(13C6)-IL2v to phosphorylate STAT5 did not increase proliferation of CD8 T cells and NK cells. NKG2D(014)-IL2v had the same activity as FAP-IL2v to induce STAT5 phosphorylation which is in line with the results from the proliferation assay. NKG2D(5C5)-IL2v was tested in another STAT5 phosphorylation assay on CD8 T cells, NK cells and CD4 T cells (Figure 8A-8C). As seen before, NKG2D(5C5)-IL2v had a higher activity to induce phosphorylation of STAT5 in NK cells and CD8 T cells compared to FAP-IL2v. In contrast, there was no difference in the activity on CD4 T cells between NKG2D(5C5)-IL2v and FAP-IL2v. This showed that the binding of the constructs to NKG2D on the same cell was necessary for the increased activity. In a separate assay, NKG2D(5C5)-IL2v, NKG2D(13C6)- IL2v and NKG2D(395 cl.80)-IL2v and FAP-IL2v were tested in STAT5 phopshorylation assay on CD4 T cells, CD8 T cells, regulatory T cells and NK cells (Figure 9A-D). As seen before, NKG2D(5C5)-IL2v had a higher activity to induce phosphorylation of STAT5 in NK cells and CD8 T cells. The second best molecule was NKG2D(395 cl.80)-IL2v and the NKG2D(13C6)- IL2v showed lower STAT5 phosphorylation of CD8 T cells and NK cells. There was no difference in the activity on NKG2D negative CD4 T cells and regulatory T cells between NKG2D(5C5)-IL2v, NKG2D(13C6)-IL2v, NKG2D(395 cl.80)-IL2v and FAP-IL2v
Table 2. FACS antibody mix I (CD4 T cells and regulatory T cells)
Table 3. FACS antibody mix II (CD8 T cells and NK cells)
Example 7
Binding of NKG2D(5C5)-IL2v to PBMCs The construct with the highest activity to activate NK cells and CD8 T cells, NKG2D(5C5)- IL2v), was tested in binding assays and compared to FAP-IL2v (Figure 10A-C and Figure 11A- C).
Freshly isolated PBMCs from healthy donors were counted and transferred into a 96 well round bottom plate (100Ό00 cells per well). The cells were washed with FACS buffer (PBS, 2% FBS, 5 mM EDTA, 0.025% NaN3) and stained with 30 pi of the indicated molecules in FACS buffer for 30 min at 4°C. The cells were washed twice with FACS buffer to remove unbound molecules. Then 20 mΐ of the diluted APC anti-human Fc specific secondary antibody (1:50 dilution, 109-136-098, Jackson ImmunoResearch) together with CD3 BUV395 antibody (563548, BD Bioscience), CD4 PE antibody (300508, BioLegend), CD56 BV421 (318328, BioLegend) and CD8 FITC antibody (344704, BioLegend) was added to the cells. After 30 min incubation at 4°C the unbound antibodies were removed by washing twice with FACS buffer. Finally, the cells were resuspended in FACS buffer and measured using a BD flow cytometer gating on CD3+CD8+ cells (CD 8 T cells), CD3+CD4+ cells (CD4 T cells) and CD3-CD56+ cells (NK cells). Figure 10A-10C show that at the same concentration more NKG2D(5C5)-IL2v constructs bound to NK cells and CD8 T cells compared to FAP-IL2v. On NKG2D negative CD4 T cells there was no difference detectable between binding of NKG2D(5C5)-IL2v and FAP-IL2v. In a separate binding assay, NKG2D(5C5)-IL2v, NKG2D(13C6)-IL2v and NKG2D(395 cl.80)-IL2v and FAP-IL2v were compared (Figure 11A-11C). As seen before, NKG2D(5C5)-IL2v binds
better to CD8 T cells and NK cells compared to FAP-IL2v. NKG2D(13C6)-IL2v and NKG2D(395 cl.80)-IL2v show intermediate binding to CD8 T cells and NK cells. On NKG2D negative CD4 T cells, there was no difference detectable between binding of NKG2D(5C5)- IL2v, NKG2D( 13 C6)-IL2v and NKG2D(395 cl.80)-IL2v and FAP-IL2v.
Example 8
Activation of PBMCs with a high affinity NKG2D-IL2v construct
Next, the NKG2D(296)-IL2v construct was compared to NKG2D(5C5)-IL2v (Figure 12A-12C).
Binding to the human NK cell line NK92 : Viability of NK92 cells was checked and cells were re-suspended and adjusted to a density of 1 Mio cells / ml. 100 mΐ of this cell suspension (containing 0.1 Mio cells) were seeded into a 96 well round bottom plate. The plate was centrifuged for 4 min at 400xg and the supernatant was removed. Then 40 mΐ of the diluted antibodies or FACS buffer were added to the cells and incubated for 30 min at 4°C. After the incubation the cells were washed twice with 150 mΐ FACS buffer per well. Then 20 mΐ of the diluted secondary APC anti-human Fc specific secondary antibody (109-116-170, Jackson ImmunoResearch) was added to the cells. The cells were incubated for an additional 30 min at 4°C. To remove unbound antibody the cells were washed again twice with 150 mΐ per well FACS buffer. To fix the cells 100 mΐ of FACS buffer containing 1% PFA were added to the wells. Before measuring the cells were re-suspended in 150 mΐ FACS buffer. The fluorescence was measured using a BD CantoII flow cytometer.
Activation of PBMCs: Freshly isolated PBMCs from healthy donors were labeled with CFSE (5(6)-Carboxyfluorescein diacetate N-succinimidyl ester, 21888, Sigma- Aldrich). Briefly, PBMCs were washed once with PBS. In parallel, the CSFE stock solution (2 mM in DMSO) was diluted 1:20 in PBS. PBMCs were resuspended in prewarmed PBS to 1 Mio/ml, 1 mΐ of the CFSE solution was added to 1 ml cell suspension and the cells were mixed immediately. For an optimal labeling, the cells were incubated for 15 min at 37°C. Then 10 ml prewarmed medium
(RPMI1640, 10% FCS, 1 % Glutamine) were added to stop the labeling reaction. The cells were spun down for 10 min at 400g and resuspended in ml fresh medium to 1 Mio/ml and incubated for additional 30 min at 37°C. Finally, the cells were washed once with medium and resuspended in fresh medium and used directly or stored overnight in the incubator. The labeled PBMCs were seeded in a 96 well round bottom plate (100Ό00 cells per well) and treated for 5 days with the indicated molecules. After the incubation the cells were washed once with FACS buffer and
stained with 20 mΐ of a mixture of anti-human CD3 APC/Cy7 antibody (300318, BioLegend), anti-human CD8 APC (344722, BioLegend), anti-human CD4 PE (300508, BioLegend), anti human CD56 BV711 (318336, BioLegend) and anti-human CD25 BV421 (356114, BioLegend) in FACS buffer for 30 min at 4°C. Afterwards PBMCs were washed twice with FACS buffer before fixing them with 1% PFA in FACS buffer and measuring the fluorescence with a BD Fortessa. Activaiton was determined by measuring CD25 upregulation on CD8 T cells (CD3+CD8+), CD4 T cells (CD3+CD4+) and NK cells (CD3-CD56+).
The NKG2D binder clone 296 had a higher overall binding to NKG2D as IgG compared to the clone 5C5 (Figure 13). However, the higher binding did not translate into increased activity. NKG2D(5C5)-IL2v had among all tested NKG2D-IL2v constructs the highest activity on NK cells and CD8 T cells, as shown in Figure 5A, 5B, 5D, 5E, Figure 6B, 6C, 6E, 6F, Figure 7A-7B, Figure 9A-9D, Figure 12A-12C and Figure 14. This indicates that the NKG2D antibody 5C5 recognizes a specific epitope that enables superior cis-targeting of the IL2v in NKG2D(5C5)- IL2v to NKG2D positive immune cells, namely NK cells and CD8 T cells translating to increased activity on these cells.
Example 9
Example Proliferation of NK92 cells upon treatment with NKG2D-IL2v
Proliferation of the human NK cell line NK92 was assessed upon treatment for 4 days with NKG2D-IL2v (5C5), NKG2D-IL2v (13C6) and NKG2D-IL2v (359 cl.80) and compared to FAP-IL2v.
NK92 cells were harvested, counted and assessed for viability. Cells were washed three times with PBS to remove residual IL2. The washed NK92 cells were re-suspended in fresh medium (advanced RPMI1640, 2% FCS, 1% Glutamine) without IL2 to 160Ό00 cells per ml and 12.5 mΐ of the cell suspension was transferred in a 384-well cell culture treated flat bottom plate. The plate was incubated for 4 days in the incubator.
After 4 days, the CellTiter-Glo (Promega) reagents and the cell culture plate were equilibrated to room temperature. The CellTiter-Glo solution was prepared as described in the manufacturer’s instructions and 25 mΐ of the solution were added to each well. After 10 min of incubation, remaining aggregates were re-suspended by pipetting and 40 mΐ of the mixture were transferred to a white flat bottom plate. The luminescence was measured with a Tecan Spark 10M multimode reader.
The NKG2D-IL2v (13C6) and NKG2D-IL2v (395 cl.80) showed comparable activity to induce proliferation on NK92 cells to the FAP-IL2v. The NKG2D-IL2v (5C5) was more potent compared to the other tested constructs (Figure 14). z * * *
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference. 0
Claims
1. An immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to NKG2D, wherein the mutant IL-2 polypeptide is a human IL-2 molecule comprising the amino acid substitutions F42A, Y45A and L72G (numbering relative to the human IL-2 sequence SEQ ID NO: 41).
2. The immunoconjugate according to claim 1, wherein the antibody comprises
(i) a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 1, a HCDR 2 of SEQ ID NO: 2, and a HCDR 3 of SEQ ID NO: 3, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 4, a LCDR 2 of SEQ ID NO: 5 and a LCDR 3 of SEQ ID NO: 6;
(ii) a VH comprising a HCDR 1 of SEQ ID NO: 9, a HCDR 2 of SEQ ID NO: 10, and a HCDR 3 of SEQ ID NO: 11, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 12, a LCDR 2 of SEQ ID NO: 13 and a LCDR 3 of SEQ ID NO: 14;
(iii) a VH comprising a HCDR 1 of SEQ ID NO: 17, a HCDR 2 of SEQ ID NO: 18, and a HCDR 3 of SEQ ID NO: 19, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 20, a LCDR 2 of SEQ ID NO: 21 and a LCDR 3 of SEQ ID NO: 22;
(iv) a VH comprising a HCDR 1 of SEQ ID NO: 25, a HCDR 2 of SEQ ID NO: 26, and a HCDR 3 of SEQ ID NO: 27, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 28, a LCDR 2 of SEQ ID NO: 29 and a LCDR 3 of SEQ ID NO: 30;
(v) a VH comprising a HCDR 1 of SEQ ID NO: 33, a HCDR 2 of SEQ ID NO: 34, and a HCDR 3 of SEQ ID NO: 35, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 36, a LCDR 2 of SEQ ID NO: 37 and a LCDR 3 of SEQ ID NO: 38; or
(vi) a VH comprising a HCDR 1 of SEQ ID NO: 83, a HCDR 2 of SEQ ID NO: 84, and a HCDR 3 of SEQ ID NO: 85, and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 86, a LCDR 2 of SEQ ID NO: 87 and a LCDR 3 of SEQ ID NO: 88.
3. The immunoconjugate according to claim 1 or 2, wherein the antibody comprises
(i) (a) a heavy chain variable region (VH) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:7, and (b) a light chain variable region (VL) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:8;
(ii) (a) a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 15, and (b) a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 16;
(iii) (a) a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:23, and (b) a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:24;
(iv) (a) a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:31, and (b) a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:32;
(v) (a) a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:39, and (b) a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:40; or
(vi) (a) a VH comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:89, and (b) a VL comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:90.
4. The immunoconjugate of any one of claims 1 to 3, wherein the mutant IL-2 polypeptide further comprises the amino acid substitution T3A and/or the amino acid substitution C125A.
5. The immunoconjugate of any one of claims 1 to 4, wherein the mutant IL-2 polypeptide comprises the sequence of SEQ ID NO: 42.
6. The immunoconjugate of any one of claims 1 to 5, wherein the immunoconjugate comprises not more than one mutant IL-2 polypeptide.
7. The immunoconjugate of any one of claims 1 to 6, wherein the antibody comprises an Fc domain composed of a first and a second subunit.
8. The immunoconjugate of claim 7, wherein the Fc domain is an IgG class, particularly an IgGl subclass, Fc domain.
9. The immunoconjugate of claim 7 or 8, wherein the Fc domain is a human Fc domain.
10. The immunoconjugate of any one of claims 1 to 9, wherein the antibody is an IgG class, particularly an IgGl subclass immunoglobulin.
11. The immunoconjugate of any one of claims 7 to 10, wherein the Fc domain comprises a modification promoting the association of the first and the second subunit of the Fc domain.
12. The immunoconjugate of any one of claims 7 to 11, wherein in the CH3 domain of the first subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in
the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable.
13. The immunoconjugate of any one of claims 7 to 12, wherein in the first subunit of the Fc domain the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the second subunit of the Fc domain the tyrosine residue at position 407 is replaced with a valine residue (Y407V) and optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numberings according to Kabat EU index).
14. The immunoconjugate of claim 13, wherein in the first subunit of the Fc domain additionally the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C), and in the second subunit of the Fc domain additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (numberings according to Kabat EU index).
15. The immunoconjugate of any one of claims 7 to 14, wherein the mutant IL-2 polypeptide is fused at its amino-terminal amino acid to the carboxy-terminal amino acid of one of the subunits of the Fc domain, particularly the first subunit of the Fc domain, optionally through a linker peptide.
16. The immunoconjugate of claim 15, wherein the linker peptide has the amino acid sequence of SEQ ID NO:43.
17. The immunoconjugate of any one of claims 7 to 15, wherein the Fc domain comprises one or more amino acid substitution that reduces binding to an Fc receptor, particularly an Fey receptor, and/or effector function, particularly antibody-dependent cell-mediated cytotoxicity (ADCC).
18. The immunoconjugate of claim 17, wherein said one or more amino acid substitution is at one or more position selected from the group of L234, L235, and P329 (Kabat EU index numbering).
19. The immunoconjugate of any one of claims 7 to 18 wherein each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (Kabat EU index numbering).
20. The immunoconjugate of any one of claims 1 to 19, comprising a polypeptide
(i) comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:44, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:45, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:46;
(ii) comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:47, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:48, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:49;
(iii) comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:50, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:51, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 52;
(iv) comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:53, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:54, and a polypeptide comprising an amino acid
sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 55;
(v) comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:56, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:57, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:58; or
(vi) comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:91, a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:92, and a polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 93.
21. The immunoconjugate of any one of claims 1 to 20, essentially consisting of a mutant IL-2 polypeptide and an IgGl immunoglobulin molecule, joined by a linker sequence.
22. One or more isolated polynucleotide encoding the immunoconjugate of any one of claims 1 to 21.
23. One or more vector, particularly expression vector, comprising the polynucleotide(s) of claim 22
24. A host cell comprising the polynucleotide(s) of claim 22 or the vector(s) of claim 23.
25. A method of producing an immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to NKG2D, comprising (a) culturing the host cell of claim 24 under conditions suitable for the expression of the immunoconjugate, and optionally (b) recovering the immunoconjugate.
26. An immunoconjugate comprising a mutant IL-2 polypeptide and an antibody that binds to NKG2D, produced by the method of claim 25.
27. A pharmaceutical composition comprising the immunoconjugate of any one of claims 1 to 21 or 26 and a pharmaceutically acceptable carrier.
28. The immunoconjugate of any one of claims 1 to 21 or 26 for use as a medicament.
29. The immunoconjugate of any one of claims 1 to 21 or 26 for use in the treatment of a disease.
30. The immunoconjugate for use in the treatment of a disease of claim 29, wherein said disease is cancer.
31. Use of the immunoconjugate of any one of claims 1 to 21 or 26 in the manufacture of a medicament for the treatment of a disease.
32. The use of claim 31, wherein said disease is cancer.
33. A method of treating a disease in an individual, comprising administering to said individual a therapeutically effective amount of a composition comprising the immunoconjugate of any one of claims 1 to 21 or 26 in a pharmaceutically acceptable form.
34. The method of claim 33, wherein said disease is cancer.
35. A method of stimulating the immune system of an individual, comprising administering to said individual an effective amount of a composition comprising the immunoconjugate of any one of claims 1 to 21 or 26 in a pharmaceutically acceptable form.
36. The invention as described hereinbefore.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP21150846 | 2021-01-11 | ||
EP21150846.0 | 2021-01-11 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2022148853A1 true WO2022148853A1 (en) | 2022-07-14 |
Family
ID=74130061
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2022/050310 WO2022148853A1 (en) | 2021-01-11 | 2022-01-10 | Immunoconjugates |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2022148853A1 (en) |
Citations (63)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4186567A (en) | 1977-04-18 | 1980-02-05 | Hitachi Metals, Ltd. | Ornament utilizing rare earth-cobalt magnet |
US4518584A (en) | 1983-04-15 | 1985-05-21 | Cetus Corporation | Human recombinant interleukin-2 muteins |
WO1987000056A1 (en) | 1985-06-26 | 1987-01-15 | Cetus Corporation | Solubilization of proteins for pharmaceutical compositions using polymer conjugation |
US5116943A (en) | 1985-01-18 | 1992-05-26 | Cetus Corporation | Oxidation-resistant muteins of Il-2 and other protein |
US5206344A (en) | 1985-06-26 | 1993-04-27 | Cetus Oncology Corporation | Interleukin-2 muteins and polymer conjugation thereof |
US5229109A (en) | 1992-04-14 | 1993-07-20 | Board Of Regents, The University Of Texas System | Low toxicity interleukin-2 analogues for use in immunotherapy |
US5500362A (en) | 1987-01-08 | 1996-03-19 | Xoma Corporation | Chimeric antibody with specificity to human B cell surface antigen |
WO1996027011A1 (en) | 1995-03-01 | 1996-09-06 | Genentech, Inc. | A method for making heteromultimeric polypeptides |
US5565332A (en) | 1991-09-23 | 1996-10-15 | Medical Research Council | Production of chimeric antibodies - a combinatorial approach |
US5750373A (en) | 1990-12-03 | 1998-05-12 | Genentech, Inc. | Enrichment method for variant proteins having altered binding properties, M13 phagemids, and growth hormone variants |
US5770429A (en) | 1990-08-29 | 1998-06-23 | Genpharm International, Inc. | Transgenic non-human animals capable of producing heterologous antibodies |
US5821337A (en) | 1991-06-14 | 1998-10-13 | Genentech, Inc. | Immunoglobulin variants |
WO1998050431A2 (en) | 1997-05-02 | 1998-11-12 | Genentech, Inc. | A method for making multispecific antibodies having heteromultimeric and common components |
US5959177A (en) | 1989-10-27 | 1999-09-28 | The Scripps Research Institute | Transgenic plants expressing assembled secretory antibodies |
US5969108A (en) | 1990-07-10 | 1999-10-19 | Medical Research Council | Methods for producing members of specific binding pairs |
US6040498A (en) | 1998-08-11 | 2000-03-21 | North Caroline State University | Genetically engineered duckweed |
US6075181A (en) | 1990-01-12 | 2000-06-13 | Abgenix, Inc. | Human antibodies derived from immunized xenomice |
US6150584A (en) | 1990-01-12 | 2000-11-21 | Abgenix, Inc. | Human antibodies derived from immunized xenomice |
WO2001007611A2 (en) | 1999-07-26 | 2001-02-01 | Genentech, Inc. | Novel polynucleotides and method for the use thereof |
US6420548B1 (en) | 1999-10-04 | 2002-07-16 | Medicago Inc. | Method for regulating transcription of foreign genes |
US20030124678A1 (en) | 2001-08-13 | 2003-07-03 | University Of Southern California | Interleukin-2 mutants with reduced toxicity |
US6737056B1 (en) | 1999-01-15 | 2004-05-18 | Genentech, Inc. | Polypeptide variants with altered effector function |
US20050079574A1 (en) | 2003-01-16 | 2005-04-14 | Genentech, Inc. | Synthetic antibody phage libraries |
WO2005100402A1 (en) | 2004-04-13 | 2005-10-27 | F.Hoffmann-La Roche Ag | Anti-p-selectin antibodies |
US6982321B2 (en) | 1986-03-27 | 2006-01-03 | Medical Research Council | Altered antibodies |
WO2006029879A2 (en) | 2004-09-17 | 2006-03-23 | F.Hoffmann-La Roche Ag | Anti-ox40l antibodies |
US7041870B2 (en) | 2000-11-30 | 2006-05-09 | Medarex, Inc. | Transgenic transchromosomal rodents for making human antibodies |
US7087409B2 (en) | 1997-12-05 | 2006-08-08 | The Scripps Research Institute | Humanization of murine antibody |
WO2006082515A2 (en) | 2005-02-07 | 2006-08-10 | Glycart Biotechnology Ag | Antigen binding molecules that bind egfr, vectors encoding same, and uses thereof |
US7125978B1 (en) | 1999-10-04 | 2006-10-24 | Medicago Inc. | Promoter for regulating expression of foreign genes |
US20070036752A1 (en) | 2001-12-04 | 2007-02-15 | Emd Lexigen Research Center Corp. | IL-2 fusion proteins with modulated selectivity |
US7189826B2 (en) | 1997-11-24 | 2007-03-13 | Institute For Human Genetics And Biochemistry | Monoclonal human natural antibodies |
US20070061900A1 (en) | 2000-10-31 | 2007-03-15 | Murphy Andrew J | Methods of modifying eukaryotic cells |
US20070117126A1 (en) | 1999-12-15 | 2007-05-24 | Genentech, Inc. | Shotgun scanning |
WO2007110205A2 (en) | 2006-03-24 | 2007-10-04 | Merck Patent Gmbh | Engineered heterodimeric protein domains |
US20070237764A1 (en) | 2005-12-02 | 2007-10-11 | Genentech, Inc. | Binding polypeptides with restricted diversity sequences |
US20070292936A1 (en) | 2006-05-09 | 2007-12-20 | Genentech, Inc. | Binding polypeptides with optimized scaffolds |
EP1870459A1 (en) | 2005-03-31 | 2007-12-26 | Chugai Seiyaku Kabushiki Kaisha | Methods for producing polypeptides by regulating polypeptide association |
WO2007147901A1 (en) | 2006-06-22 | 2007-12-27 | Novo Nordisk A/S | Production of bispecific antibodies |
WO2008034473A1 (en) | 2006-09-20 | 2008-03-27 | Dge Dr.-Ing. Günther Engineering Gmbh | Method and device for separating methane and carbon dioxide from biogas |
US7527791B2 (en) | 2004-03-31 | 2009-05-05 | Genentech, Inc. | Humanized anti-TGF-beta antibodies |
WO2009061853A2 (en) | 2007-11-05 | 2009-05-14 | Massachusetts Institute Of Technology | Mutant interleukin-2 (il-2) polypeptides |
WO2009089004A1 (en) | 2008-01-07 | 2009-07-16 | Amgen Inc. | Method for making antibody fc-heterodimeric molecules using electrostatic steering effects |
EP2101823A2 (en) | 2007-01-09 | 2009-09-23 | Curevac GmbH | Rna-coded antibody |
WO2010017103A2 (en) | 2008-08-04 | 2010-02-11 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Servic | Fully human anti-human nkg2d monoclonal antibodies |
US7785903B2 (en) | 2004-04-09 | 2010-08-31 | Genentech, Inc. | Variable domain library and uses |
WO2010129304A2 (en) | 2009-04-27 | 2010-11-11 | Oncomed Pharmaceuticals, Inc. | Method for making heteromultimeric molecules |
WO2011020783A2 (en) | 2009-08-17 | 2011-02-24 | Roche Glycart Ag | Targeted immunoconjugates |
US7985840B2 (en) | 2002-06-03 | 2011-07-26 | Genentech, Inc | Synthetic antibody phage libraries |
WO2011090762A1 (en) | 2009-12-29 | 2011-07-28 | Emergent Product Development Seattle, Llc | Heterodimer binding proteins and uses thereof |
WO2011143545A1 (en) | 2010-05-14 | 2011-11-17 | Rinat Neuroscience Corporation | Heterodimeric proteins and methods for producing and purifying them |
WO2012058768A1 (en) | 2010-11-05 | 2012-05-10 | Zymeworks Inc. | Stable heterodimeric antibody design with mutations in the fc domain |
WO2012107417A1 (en) | 2011-02-10 | 2012-08-16 | Roche Glycart Ag | Mutant interleukin-2 polypeptides |
WO2012130831A1 (en) | 2011-03-29 | 2012-10-04 | Roche Glycart Ag | Antibody fc variants |
WO2012146628A1 (en) | 2011-04-29 | 2012-11-01 | Roche Glycart Ag | Novel immunoconjugates |
WO2013096291A2 (en) | 2011-12-20 | 2013-06-27 | Medimmune, Llc | Modified polypeptides for bispecific antibody scaffolds |
WO2013120929A1 (en) | 2012-02-15 | 2013-08-22 | F. Hoffmann-La Roche Ag | Fc-receptor based affinity chromatography |
WO2013157954A1 (en) | 2012-04-20 | 2013-10-24 | Merus B.V. | Methods and means for the production of ig-like molecules |
US8679490B2 (en) | 2005-11-07 | 2014-03-25 | Genentech, Inc. | Binding polypeptides with diversified and consensus VH/VL hypervariable sequences |
WO2016134371A2 (en) | 2015-02-20 | 2016-08-25 | Ohio State Innovation Foundation | Bivalent antibody directed against nkg2d and tumor associated antigens |
WO2018148445A1 (en) | 2017-02-08 | 2018-08-16 | Adimab, Llc | Multi-specific binding proteins for activation of natural killer cells and therapeutic uses thereof to treat cancer |
US20180326010A1 (en) * | 2017-04-03 | 2018-11-15 | Hoffmann-La Roche Inc. | Immunoconjugates |
US20190119345A1 (en) * | 2016-02-05 | 2019-04-25 | Washington University | Compositions and methods for targeted cytokine delivery |
-
2022
- 2022-01-10 WO PCT/EP2022/050310 patent/WO2022148853A1/en active Application Filing
Patent Citations (69)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4186567A (en) | 1977-04-18 | 1980-02-05 | Hitachi Metals, Ltd. | Ornament utilizing rare earth-cobalt magnet |
US4518584A (en) | 1983-04-15 | 1985-05-21 | Cetus Corporation | Human recombinant interleukin-2 muteins |
US5116943A (en) | 1985-01-18 | 1992-05-26 | Cetus Corporation | Oxidation-resistant muteins of Il-2 and other protein |
US5206344A (en) | 1985-06-26 | 1993-04-27 | Cetus Oncology Corporation | Interleukin-2 muteins and polymer conjugation thereof |
WO1987000056A1 (en) | 1985-06-26 | 1987-01-15 | Cetus Corporation | Solubilization of proteins for pharmaceutical compositions using polymer conjugation |
US6982321B2 (en) | 1986-03-27 | 2006-01-03 | Medical Research Council | Altered antibodies |
US5500362A (en) | 1987-01-08 | 1996-03-19 | Xoma Corporation | Chimeric antibody with specificity to human B cell surface antigen |
US5959177A (en) | 1989-10-27 | 1999-09-28 | The Scripps Research Institute | Transgenic plants expressing assembled secretory antibodies |
US6417429B1 (en) | 1989-10-27 | 2002-07-09 | The Scripps Research Institute | Transgenic plants expressing assembled secretory antibodies |
US6150584A (en) | 1990-01-12 | 2000-11-21 | Abgenix, Inc. | Human antibodies derived from immunized xenomice |
US6075181A (en) | 1990-01-12 | 2000-06-13 | Abgenix, Inc. | Human antibodies derived from immunized xenomice |
US5969108A (en) | 1990-07-10 | 1999-10-19 | Medical Research Council | Methods for producing members of specific binding pairs |
US5770429A (en) | 1990-08-29 | 1998-06-23 | Genpharm International, Inc. | Transgenic non-human animals capable of producing heterologous antibodies |
US5750373A (en) | 1990-12-03 | 1998-05-12 | Genentech, Inc. | Enrichment method for variant proteins having altered binding properties, M13 phagemids, and growth hormone variants |
US5821337A (en) | 1991-06-14 | 1998-10-13 | Genentech, Inc. | Immunoglobulin variants |
US5565332A (en) | 1991-09-23 | 1996-10-15 | Medical Research Council | Production of chimeric antibodies - a combinatorial approach |
US5229109A (en) | 1992-04-14 | 1993-07-20 | Board Of Regents, The University Of Texas System | Low toxicity interleukin-2 analogues for use in immunotherapy |
WO1996027011A1 (en) | 1995-03-01 | 1996-09-06 | Genentech, Inc. | A method for making heteromultimeric polypeptides |
US7695936B2 (en) | 1995-03-01 | 2010-04-13 | Genentech, Inc. | Knobs and holes heteromeric polypeptides |
US5731168A (en) | 1995-03-01 | 1998-03-24 | Genentech, Inc. | Method for making heteromultimeric polypeptides |
WO1998050431A2 (en) | 1997-05-02 | 1998-11-12 | Genentech, Inc. | A method for making multispecific antibodies having heteromultimeric and common components |
US7189826B2 (en) | 1997-11-24 | 2007-03-13 | Institute For Human Genetics And Biochemistry | Monoclonal human natural antibodies |
US7087409B2 (en) | 1997-12-05 | 2006-08-08 | The Scripps Research Institute | Humanization of murine antibody |
US6040498A (en) | 1998-08-11 | 2000-03-21 | North Caroline State University | Genetically engineered duckweed |
US6737056B1 (en) | 1999-01-15 | 2004-05-18 | Genentech, Inc. | Polypeptide variants with altered effector function |
US7332581B2 (en) | 1999-01-15 | 2008-02-19 | Genentech, Inc. | Polypeptide variants with altered effector function |
WO2001007611A2 (en) | 1999-07-26 | 2001-02-01 | Genentech, Inc. | Novel polynucleotides and method for the use thereof |
US6420548B1 (en) | 1999-10-04 | 2002-07-16 | Medicago Inc. | Method for regulating transcription of foreign genes |
US7125978B1 (en) | 1999-10-04 | 2006-10-24 | Medicago Inc. | Promoter for regulating expression of foreign genes |
US20070117126A1 (en) | 1999-12-15 | 2007-05-24 | Genentech, Inc. | Shotgun scanning |
US20070061900A1 (en) | 2000-10-31 | 2007-03-15 | Murphy Andrew J | Methods of modifying eukaryotic cells |
US7041870B2 (en) | 2000-11-30 | 2006-05-09 | Medarex, Inc. | Transgenic transchromosomal rodents for making human antibodies |
US20030124678A1 (en) | 2001-08-13 | 2003-07-03 | University Of Southern California | Interleukin-2 mutants with reduced toxicity |
US20070036752A1 (en) | 2001-12-04 | 2007-02-15 | Emd Lexigen Research Center Corp. | IL-2 fusion proteins with modulated selectivity |
US7985840B2 (en) | 2002-06-03 | 2011-07-26 | Genentech, Inc | Synthetic antibody phage libraries |
US20050079574A1 (en) | 2003-01-16 | 2005-04-14 | Genentech, Inc. | Synthetic antibody phage libraries |
US7527791B2 (en) | 2004-03-31 | 2009-05-05 | Genentech, Inc. | Humanized anti-TGF-beta antibodies |
US7785903B2 (en) | 2004-04-09 | 2010-08-31 | Genentech, Inc. | Variable domain library and uses |
WO2005100402A1 (en) | 2004-04-13 | 2005-10-27 | F.Hoffmann-La Roche Ag | Anti-p-selectin antibodies |
WO2006029879A2 (en) | 2004-09-17 | 2006-03-23 | F.Hoffmann-La Roche Ag | Anti-ox40l antibodies |
WO2006082515A2 (en) | 2005-02-07 | 2006-08-10 | Glycart Biotechnology Ag | Antigen binding molecules that bind egfr, vectors encoding same, and uses thereof |
EP1870459A1 (en) | 2005-03-31 | 2007-12-26 | Chugai Seiyaku Kabushiki Kaisha | Methods for producing polypeptides by regulating polypeptide association |
US8679490B2 (en) | 2005-11-07 | 2014-03-25 | Genentech, Inc. | Binding polypeptides with diversified and consensus VH/VL hypervariable sequences |
US20070237764A1 (en) | 2005-12-02 | 2007-10-11 | Genentech, Inc. | Binding polypeptides with restricted diversity sequences |
WO2007110205A2 (en) | 2006-03-24 | 2007-10-04 | Merck Patent Gmbh | Engineered heterodimeric protein domains |
US20070292936A1 (en) | 2006-05-09 | 2007-12-20 | Genentech, Inc. | Binding polypeptides with optimized scaffolds |
WO2007147901A1 (en) | 2006-06-22 | 2007-12-27 | Novo Nordisk A/S | Production of bispecific antibodies |
WO2008034473A1 (en) | 2006-09-20 | 2008-03-27 | Dge Dr.-Ing. Günther Engineering Gmbh | Method and device for separating methane and carbon dioxide from biogas |
EP2101823A2 (en) | 2007-01-09 | 2009-09-23 | Curevac GmbH | Rna-coded antibody |
WO2009061853A2 (en) | 2007-11-05 | 2009-05-14 | Massachusetts Institute Of Technology | Mutant interleukin-2 (il-2) polypeptides |
WO2009089004A1 (en) | 2008-01-07 | 2009-07-16 | Amgen Inc. | Method for making antibody fc-heterodimeric molecules using electrostatic steering effects |
WO2010017103A2 (en) | 2008-08-04 | 2010-02-11 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Servic | Fully human anti-human nkg2d monoclonal antibodies |
WO2010129304A2 (en) | 2009-04-27 | 2010-11-11 | Oncomed Pharmaceuticals, Inc. | Method for making heteromultimeric molecules |
WO2011020783A2 (en) | 2009-08-17 | 2011-02-24 | Roche Glycart Ag | Targeted immunoconjugates |
WO2011090754A1 (en) | 2009-12-29 | 2011-07-28 | Emergent Product Development Seattle, Llc | Polypeptide heterodimers and uses thereof |
WO2011090762A1 (en) | 2009-12-29 | 2011-07-28 | Emergent Product Development Seattle, Llc | Heterodimer binding proteins and uses thereof |
WO2011143545A1 (en) | 2010-05-14 | 2011-11-17 | Rinat Neuroscience Corporation | Heterodimeric proteins and methods for producing and purifying them |
WO2012058768A1 (en) | 2010-11-05 | 2012-05-10 | Zymeworks Inc. | Stable heterodimeric antibody design with mutations in the fc domain |
WO2012107417A1 (en) | 2011-02-10 | 2012-08-16 | Roche Glycart Ag | Mutant interleukin-2 polypeptides |
WO2012130831A1 (en) | 2011-03-29 | 2012-10-04 | Roche Glycart Ag | Antibody fc variants |
WO2012146628A1 (en) | 2011-04-29 | 2012-11-01 | Roche Glycart Ag | Novel immunoconjugates |
WO2013096291A2 (en) | 2011-12-20 | 2013-06-27 | Medimmune, Llc | Modified polypeptides for bispecific antibody scaffolds |
WO2013120929A1 (en) | 2012-02-15 | 2013-08-22 | F. Hoffmann-La Roche Ag | Fc-receptor based affinity chromatography |
WO2013157954A1 (en) | 2012-04-20 | 2013-10-24 | Merus B.V. | Methods and means for the production of ig-like molecules |
WO2013157953A1 (en) | 2012-04-20 | 2013-10-24 | Merus B.V. | Methods and means for the production of ig-like molecules |
WO2016134371A2 (en) | 2015-02-20 | 2016-08-25 | Ohio State Innovation Foundation | Bivalent antibody directed against nkg2d and tumor associated antigens |
US20190119345A1 (en) * | 2016-02-05 | 2019-04-25 | Washington University | Compositions and methods for targeted cytokine delivery |
WO2018148445A1 (en) | 2017-02-08 | 2018-08-16 | Adimab, Llc | Multi-specific binding proteins for activation of natural killer cells and therapeutic uses thereof to treat cancer |
US20180326010A1 (en) * | 2017-04-03 | 2018-11-15 | Hoffmann-La Roche Inc. | Immunoconjugates |
Non-Patent Citations (90)
Title |
---|
"NCBI", Database accession no. NP517425 |
"Remington's Pharmaceutical Sciences", 1990, MACK PRINTING COMPANY, pages: 1289 - 1329 |
ALMAGROFRANSSON, FRONT. BIOSCI., vol. 13, 2008, pages 1619 - 1633 |
BACA ET AL., J. BIOL. CHEM., vol. 272, 1997, pages 10678 - 10684 |
BAUER ET AL., SCIENCE, vol. 285, 1999, pages 727 - 729 |
BAZAN ET AL., HUMAN VACCINES AND IMMUNOTHERAPEUTICS, vol. 8, 2012, pages 1817 - 1828 |
BAZAN, SCIENCE, vol. 257, 1992, pages 410 - 413 |
BOERNER ET AL., J. IMMUNOL., vol. 147, 1991, pages 86 |
BOYMAN ET AL., SCIENCE, vol. 311, 2006, pages 1924 - 27 |
BRODEUR ET AL.: "Monoclonal Antibody Production Techniques and Applications", 1987, MARCEL DEKKER, INC., pages: 51 - 63 |
BRUGGEMANN ET AL., J EXP MED, vol. 166, 1987, pages 1351 - 1361 |
CARTER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 4285 |
CARTER, J IMMUNOL METH, vol. 248, 2001, pages 7 - 15 |
CARTER, J IMMUNOL METHODS, vol. 248, 2001, pages 7 - 15 |
CHERF ET AL., METHODS IN MOLECULAR BIOLOGY, vol. 1319, 2015, pages 155 - 175 |
CHOTHIALESK, J. MOL. BIOL., vol. 196, 1987, pages 901 - 917 |
CLARKSON ET AL., NATURE, vol. 352, 1991, pages 624 - 628 |
CLYNES ET AL., PROC NATL ACAD SCI USA, vol. 95, 1998, pages 652 - 656 |
CRAGG ET AL., BLOOD, vol. 101, 2003, pages 1045 - 1052 |
CRAGGGLENNIE, BLOOD, vol. 103, 2004, pages 2738 - 2743 |
DALL'ACQUA ET AL., METHODS, vol. 36, 2005, pages 61 - 68 |
FLATMAN ET AL., J. CHROMATOGR. B, vol. 848, 2007, pages 79 - 87 |
FONTENOT ET AL., NATURE IMMUNOL, vol. 6, 2005, pages 1171 - 72 |
FRENZEL ET AL., MABS, vol. 8, 2016, pages 1177 - 1194 |
GAZZANO-SANTORO ET AL., J IMMUNOL METHODS, vol. 202, 1996, pages 163 |
GERNGROSS, NAT BIOTECH, vol. 22, 2004, pages 1409 - 1414 |
GHASHEMI ET AL., NAT COMM, vol. 7, 2016, pages 12878 |
GRAHAM ET AL., J GEN VIROL, vol. 36, 1977, pages 59 |
GRIFFITHS ET AL., EMBO JOURNAL, vol. 12, 1993, pages 725 - 734 |
HANES ET AL., PNAS, vol. 94, 1997, pages 4937 - 4942 |
HE ET AL., NUCLEIC ACIDS RESEARCH, vol. 25, 1997, pages 5132 - 5134 |
HEATON, CANCER RES, vol. 53, 1993, pages 2597 - 602 |
HEELEY, ENDOCR RES, vol. 28, 2002, pages 217 - 229 |
HELLSTROM ET AL., PROC NATL ACAD SCI USA, vol. 82, 1985, pages 1499 - 1502 |
HELLSTROM ET AL., PROC NATL ACAD SCI USA, vol. 83, 1986, pages 7059 - 7063 |
HOLLIGERHUDSON, NATURE BIOTECHNOLOGY, vol. 23, 2005, pages 1126 - 1136 |
HOOGENBOOM ET AL.: "Methods in Molecular Biology", vol. 248, 2003, HUMANA PRESS, pages: 161 - 175 |
HOOGENBOOMWINTER, JOURNAL OF MOLECULAR BIOLOGY, vol. 227, 1992, pages 381 - 388 |
HOUCHNINS ET AL., J EXP MED, vol. 173, 1991, pages 1017 - 1020 |
IMAI ET AL., CANCER SCI, vol. 98, 2007, pages 416 - 23 |
KAMIMURA ET AL., J IMMUNOL, vol. 177, 2006, pages 306 - 14 |
KINDT ET AL.: "Kuby Immunology", 2007, W.H. FREEMAN & CO., pages: 91 |
KING ET AL., J CLIN ONCOL, vol. 22, 2004, pages 4463 - 4473 |
KLEIN ET AL., ONCOIMMUNOLOGY, vol. 6, no. 3, 2017, pages el277306 |
KLIMKA ET AL., BR. J. CANCER, vol. 83, 2000, pages 252 - 260 |
KO ET AL., J IMMUNOTHER, vol. 27, 2004, pages 232 - 239 |
KOZBOR, J. IMMUNOL., vol. 133, 1984, pages 3001 |
KRIEG ET AL., PROC NAT ACAD SCI USA, vol. 107, 2010, pages 11906 - 11 |
KRIEG ET AL., PROC NATL ACAD SCI, vol. 107, 2010, pages 11906 - 11 |
KWONG ET AL., J MOL BIOL, vol. 384, 2008, pages 1143 - 1156 |
LERNER ET AL., NATURE REVIEWS, vol. 16, 2016, pages 498 - 508 |
LI ET AL., NAT BIOTECH, vol. 24, 2006, pages 210 - 215 |
LI ET AL., PROC. NATL. ACAD. SCI. USA, vol. 103, 2006, pages 3557 - 3562 |
LILJEBLAD ET AL., GLYCO J, vol. 17, 2000, pages 323 - 329 |
LONBERG, CURR OPIN IMMUNOL, vol. 20, 2008, pages 450 - 459 |
LONBERG, NAT. BIOTECH., vol. 23, 2005, pages 1117 - 1125 |
MACCALLUM ET AL., J. MOL. BIOL., vol. 262, 1996, pages 732 - 745 |
MALEK, ANNU REV IMMUNOL, vol. 26, 2008, pages 453 - 79 |
MATHER ET AL., NNALS N.Y. ACAD SCI, vol. 383, 1982, pages 44 - 68 |
MATHER, BIOL REPROD, vol. 23, 1980, pages 243 - 251 |
MEISSNER, P. ET AL.: "Transient gene expression: recombinant protein production with suspension-adapted HEK293-EBNA cells", BIOTECHNOL. BIOENG., vol. 75, 2001, pages 197 - 203, XP002283991, DOI: 10.1002/bit.1179 |
MINAMI ET AL., ANNU REV IMMUNOL, vol. 11, 1993, pages 245 - 268 |
NI, XIANDAI MIANYIXUE, vol. 26, no. 4, 2006, pages 265 - 268 |
OLEJNICZAKKASPRZAK, MED SCI MONIT, vol. 14, 2008, pages RA179 - 89 |
PACE, PROTEIN SCIENCE, vol. 4, 1995, pages 2411 - 2423 |
PADLAN, MOL. IMMUNOL., vol. 1-3, 1991, pages 489 - 498 |
PEARSON, GENOMICS, vol. 46, 1997, pages 24 - 36 |
PETKOVA, S.B. ET AL., INT'L. IMMUNOL., vol. 18, no. 12, 2006, pages 1759 - 1769 |
PORTOLANO ET AL., J. IMMUNOL., vol. 151, 1993, pages 2623 - 887 |
QUEEN ET AL., PROC. NAT'L ACAD. SCI. USA, vol. 86, 1989, pages 10029 - 10033 |
RIDGWA ET AL., PROT ENG, vol. 9, 1996, pages 617 - 621 |
RIECHMANN ET AL., NATURE, vol. 332, 1988, pages 323 - 329 |
ROSOK ET AL., J. BIOL. CHEM., vol. 271, 1996, pages 22611 - 22618 |
SAKAGUCHI, ANNU REV IMMUNOL, vol. 22, 2004, pages 531 - 62 |
SCHOLLER ET AL., METHODS IN MOLECULAR BIOLOGY, vol. 889, 2012, pages 135 - 84 |
SHANAFELT ET AL., NATURE BIOTECHNOL, vol. 18, 2000, pages 1197 - 1202 |
SMITH, SCIENCE, vol. 240, 1988, pages 1169 - 76 |
STADLER ET AL., NATURE MEDICINE, vol. 23, 2017, pages 815 - 817 |
STUBENRAUCH ET AL., DRUG METABOLISM AND DISPOSITION, vol. 38, 2010, pages 84 - 91 |
TANIGUCHI ET AL., NATURE, vol. 302, 1983, pages 305 - 10 |
URLAUB ET AL., PROC NATL ACAD SCI USA, vol. 77, 1980, pages 4216 |
VAN DIJKVAN DE WINKEL, CURR OPIN PHARMACOL, vol. 5, 2001, pages 368 - 74 |
VOLLMERSBRANDLEIN, HISTOLOGY AND HISTOPATHOLOGY, vol. 20, no. 3, 2005, pages 927 - 937 |
VOLLMERSBRANDLEIN, METHODS AND FINDINGS IN EXPERIMENTAL AND CLINICAL PHARMACOLOGY, vol. 27, no. 3, 2005, pages 185 - 91 |
W. R. PEARSON: "Effective protein sequence comparison", METH. ENZYMOL., vol. 266, 1996, pages 227 - 258 |
W. R. PEARSOND. J. LIPMAN: "Improved Tools for Biological Sequence Analysis", PNAS, vol. 85, 1988, pages 2444 - 2448 |
WALDMANN, NAT REV IMMUNOL, vol. 6, 2009, pages 595 - 601 |
WEIGER ET AL., EUR J BIOCHEM, vol. 180, 1989, pages 295 - 300 |
WINTER ET AL., ANNUAL REVIEW OF IMMUNOLOGY, vol. 12, 1994, pages 433 - 455 |
ZHAO ET AL., CRITICAL REVIEWS IN BIOTECHNOLOGY, vol. 36, 2016, pages 276 - 289 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20230071733A1 (en) | Immunoconjugates | |
US20230134606A1 (en) | Immunoconjugates | |
WO2021001289A1 (en) | Immunoconjugates comprising a mutant interleukin-2 and an anti-cd8 antibody | |
US20230192795A1 (en) | Immunoconjugates | |
JP2023509952A (en) | Novel 4-1BBL trimer-containing antigen-binding molecule | |
IL296225A (en) | Immune activating fc domain binding molecules | |
WO2022148853A1 (en) | Immunoconjugates | |
WO2023062048A1 (en) | Alternative pd1-il7v immunoconjugates for the treatment of cancer | |
WO2023062050A1 (en) | New interleukin-7 immunoconjugates |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 22700051 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 22700051 Country of ref document: EP Kind code of ref document: A1 |