US20230272028A1 - Separation moieties and methods of use thereof - Google Patents
Separation moieties and methods of use thereof Download PDFInfo
- Publication number
- US20230272028A1 US20230272028A1 US18/315,927 US202318315927A US2023272028A1 US 20230272028 A1 US20230272028 A1 US 20230272028A1 US 202318315927 A US202318315927 A US 202318315927A US 2023272028 A1 US2023272028 A1 US 2023272028A1
- Authority
- US
- United States
- Prior art keywords
- polypeptide
- seq
- cytokine
- protease
- domain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000926 separation method Methods 0.000 title abstract description 143
- 238000000034 method Methods 0.000 title description 31
- 230000004071 biological effect Effects 0.000 claims abstract description 25
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 265
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 245
- 229920001184 polypeptide Polymers 0.000 claims description 230
- 108090000695 Cytokines Proteins 0.000 claims description 209
- 102000004127 Cytokines Human genes 0.000 claims description 207
- 230000027455 binding Effects 0.000 claims description 190
- 239000000427 antigen Substances 0.000 claims description 157
- 108091007433 antigens Proteins 0.000 claims description 156
- 102000036639 antigens Human genes 0.000 claims description 156
- 239000004365 Protease Substances 0.000 claims description 136
- 108091005804 Peptidases Proteins 0.000 claims description 133
- 230000000903 blocking effect Effects 0.000 claims description 125
- 238000003776 cleavage reaction Methods 0.000 claims description 94
- 230000007017 scission Effects 0.000 claims description 88
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 79
- 239000012634 fragment Substances 0.000 claims description 66
- 102000005962 receptors Human genes 0.000 claims description 61
- 108020003175 receptors Proteins 0.000 claims description 60
- 108090000623 proteins and genes Proteins 0.000 claims description 56
- 150000001413 amino acids Chemical class 0.000 claims description 54
- 102000004169 proteins and genes Human genes 0.000 claims description 53
- 239000000758 substrate Substances 0.000 claims description 48
- 108091006905 Human Serum Albumin Proteins 0.000 claims description 41
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 41
- 150000007523 nucleic acids Chemical class 0.000 claims description 31
- 102000039446 nucleic acids Human genes 0.000 claims description 28
- 108020004707 nucleic acids Proteins 0.000 claims description 28
- 102000019034 Chemokines Human genes 0.000 claims description 27
- 108010012236 Chemokines Proteins 0.000 claims description 27
- 108060003951 Immunoglobulin Proteins 0.000 claims description 23
- 102000018358 immunoglobulin Human genes 0.000 claims description 23
- 108091008874 T cell receptors Proteins 0.000 claims description 19
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 claims description 19
- 239000000203 mixture Substances 0.000 claims description 18
- 239000003446 ligand Substances 0.000 claims description 17
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 claims description 15
- 230000002238 attenuated effect Effects 0.000 claims description 12
- 239000013598 vector Substances 0.000 claims description 10
- 239000003102 growth factor Substances 0.000 claims description 7
- 230000003834 intracellular effect Effects 0.000 claims description 5
- 102000000844 Cell Surface Receptors Human genes 0.000 claims description 4
- 108010001857 Cell Surface Receptors Proteins 0.000 claims description 4
- 102000006306 Antigen Receptors Human genes 0.000 claims description 2
- 108010083359 Antigen Receptors Proteins 0.000 claims description 2
- 239000013603 viral vector Substances 0.000 claims description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims 4
- 230000001225 therapeutic effect Effects 0.000 abstract description 23
- 230000002829 reductive effect Effects 0.000 abstract description 10
- 229920002521 macromolecule Polymers 0.000 abstract 2
- 125000005647 linker group Chemical group 0.000 description 145
- 102000000588 Interleukin-2 Human genes 0.000 description 139
- 108010002350 Interleukin-2 Proteins 0.000 description 139
- 102000035195 Peptidases Human genes 0.000 description 129
- 102000037865 fusion proteins Human genes 0.000 description 110
- 108020001507 fusion proteins Proteins 0.000 description 110
- 230000000694 effects Effects 0.000 description 108
- 235000019419 proteases Nutrition 0.000 description 98
- 206010028980 Neoplasm Diseases 0.000 description 95
- 235000001014 amino acid Nutrition 0.000 description 83
- 210000004027 cell Anatomy 0.000 description 59
- 210000002966 serum Anatomy 0.000 description 59
- 229940024606 amino acid Drugs 0.000 description 55
- 102000003812 Interleukin-15 Human genes 0.000 description 48
- 108090000172 Interleukin-15 Proteins 0.000 description 48
- 235000018102 proteins Nutrition 0.000 description 46
- 102000013462 Interleukin-12 Human genes 0.000 description 41
- 108010065805 Interleukin-12 Proteins 0.000 description 41
- -1 but not limited to Proteins 0.000 description 38
- 230000008685 targeting Effects 0.000 description 38
- 241000282414 Homo sapiens Species 0.000 description 37
- 210000001744 T-lymphocyte Anatomy 0.000 description 36
- 238000006467 substitution reaction Methods 0.000 description 36
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 34
- 238000003556 assay Methods 0.000 description 30
- 230000004927 fusion Effects 0.000 description 30
- 102000025171 antigen binding proteins Human genes 0.000 description 29
- 108091000831 antigen binding proteins Proteins 0.000 description 29
- 229940117681 interleukin-12 Drugs 0.000 description 28
- 210000003289 regulatory T cell Anatomy 0.000 description 27
- 201000010099 disease Diseases 0.000 description 26
- 241000699670 Mus sp. Species 0.000 description 24
- 230000006870 function Effects 0.000 description 24
- 239000003814 drug Substances 0.000 description 23
- 238000011282 treatment Methods 0.000 description 23
- 102000000704 Interleukin-7 Human genes 0.000 description 22
- 108010002586 Interleukin-7 Proteins 0.000 description 22
- 229940100994 interleukin-7 Drugs 0.000 description 22
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 20
- 201000011510 cancer Diseases 0.000 description 20
- 238000002560 therapeutic procedure Methods 0.000 description 20
- 108010038453 Interleukin-2 Receptors Proteins 0.000 description 19
- 102000010789 Interleukin-2 Receptors Human genes 0.000 description 19
- 102000015696 Interleukins Human genes 0.000 description 19
- 108010063738 Interleukins Proteins 0.000 description 19
- 230000004913 activation Effects 0.000 description 19
- 108090000624 Cathepsin L Proteins 0.000 description 18
- 102000004172 Cathepsin L Human genes 0.000 description 18
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 18
- 102000002274 Matrix Metalloproteinases Human genes 0.000 description 18
- 108010000684 Matrix Metalloproteinases Proteins 0.000 description 18
- 230000015556 catabolic process Effects 0.000 description 18
- 238000006731 degradation reaction Methods 0.000 description 18
- 229920001223 polyethylene glycol Polymers 0.000 description 18
- 102220211407 rs762135776 Human genes 0.000 description 18
- 239000002202 Polyethylene glycol Substances 0.000 description 16
- 102200053439 rs72466487 Human genes 0.000 description 16
- 102000004190 Enzymes Human genes 0.000 description 15
- 108090000790 Enzymes Proteins 0.000 description 15
- 241001465754 Metazoa Species 0.000 description 15
- 229940079593 drug Drugs 0.000 description 15
- 229940088598 enzyme Drugs 0.000 description 15
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 14
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 14
- 206010061218 Inflammation Diseases 0.000 description 13
- 108010076557 Matrix Metalloproteinase 14 Proteins 0.000 description 13
- 102100030216 Matrix metalloproteinase-14 Human genes 0.000 description 13
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 13
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 13
- 239000008194 pharmaceutical composition Substances 0.000 description 13
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 12
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 12
- 102000012479 Serine Proteases Human genes 0.000 description 12
- 108010022999 Serine Proteases Proteins 0.000 description 12
- 101000915480 Streptococcus pneumoniae serotype 4 (strain ATCC BAA-334 / TIGR4) Zinc metalloprotease ZmpC Proteins 0.000 description 12
- 108060008683 Tumor Necrosis Factor Receptor Proteins 0.000 description 12
- 238000013459 approach Methods 0.000 description 12
- 230000004054 inflammatory process Effects 0.000 description 12
- 230000014759 maintenance of location Effects 0.000 description 12
- 210000004881 tumor cell Anatomy 0.000 description 12
- 102000003298 tumor necrosis factor receptor Human genes 0.000 description 12
- 239000003981 vehicle Substances 0.000 description 12
- 102000005600 Cathepsins Human genes 0.000 description 11
- 108010084457 Cathepsins Proteins 0.000 description 11
- 101000851370 Homo sapiens Tumor necrosis factor receptor superfamily member 9 Proteins 0.000 description 11
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 11
- 108090000190 Thrombin Proteins 0.000 description 11
- 102100036856 Tumor necrosis factor receptor superfamily member 9 Human genes 0.000 description 11
- 230000009471 action Effects 0.000 description 11
- 239000000562 conjugate Substances 0.000 description 11
- 230000002757 inflammatory effect Effects 0.000 description 11
- 210000000822 natural killer cell Anatomy 0.000 description 11
- 102000009027 Albumins Human genes 0.000 description 10
- 108010088751 Albumins Proteins 0.000 description 10
- 239000004475 Arginine Substances 0.000 description 10
- 239000004471 Glycine Substances 0.000 description 10
- 101000716102 Homo sapiens T-cell surface glycoprotein CD4 Proteins 0.000 description 10
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 10
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 10
- 239000004472 Lysine Substances 0.000 description 10
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 description 10
- 239000000556 agonist Substances 0.000 description 10
- 238000004458 analytical method Methods 0.000 description 10
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 10
- 230000003247 decreasing effect Effects 0.000 description 10
- 230000001965 increasing effect Effects 0.000 description 10
- 230000001939 inductive effect Effects 0.000 description 10
- 230000003993 interaction Effects 0.000 description 10
- 229960003136 leucine Drugs 0.000 description 10
- 235000018977 lysine Nutrition 0.000 description 10
- 230000011664 signaling Effects 0.000 description 10
- 229960004072 thrombin Drugs 0.000 description 10
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 9
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 9
- 102000003810 Interleukin-18 Human genes 0.000 description 9
- 108090000171 Interleukin-18 Proteins 0.000 description 9
- 102100030703 Interleukin-22 Human genes 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 9
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 9
- 102000007562 Serum Albumin Human genes 0.000 description 9
- 108010071390 Serum Albumin Proteins 0.000 description 9
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 9
- 235000004279 alanine Nutrition 0.000 description 9
- 235000009582 asparagine Nutrition 0.000 description 9
- 229960001230 asparagine Drugs 0.000 description 9
- 230000001419 dependent effect Effects 0.000 description 9
- 238000001727 in vivo Methods 0.000 description 9
- 235000004400 serine Nutrition 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 9
- 102100026802 72 kDa type IV collagenase Human genes 0.000 description 8
- 208000023275 Autoimmune disease Diseases 0.000 description 8
- 102100025248 C-X-C motif chemokine 10 Human genes 0.000 description 8
- 102100036705 Interleukin-23 subunit alpha Human genes 0.000 description 8
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 8
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 8
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 8
- 102000004083 Lymphotoxin-alpha Human genes 0.000 description 8
- 108090000542 Lymphotoxin-alpha Proteins 0.000 description 8
- 229940049595 antibody-drug conjugate Drugs 0.000 description 8
- 239000003795 chemical substances by application Substances 0.000 description 8
- 231100000433 cytotoxic Toxicity 0.000 description 8
- 230000001472 cytotoxic effect Effects 0.000 description 8
- 208000035475 disorder Diseases 0.000 description 8
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 8
- 235000004554 glutamine Nutrition 0.000 description 8
- 210000000987 immune system Anatomy 0.000 description 8
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Chemical group OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 8
- COLNVLDHVKWLRT-QMMMGPOBSA-N phenylalanine group Chemical group N[C@@H](CC1=CC=CC=C1)C(=O)O COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 8
- LKDMKWNDBAVNQZ-UHFFFAOYSA-N 4-[[1-[[1-[2-[[1-(4-nitroanilino)-1-oxo-3-phenylpropan-2-yl]carbamoyl]pyrrolidin-1-yl]-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-4-oxobutanoic acid Chemical compound OC(=O)CCC(=O)NC(C)C(=O)NC(C)C(=O)N1CCCC1C(=O)NC(C(=O)NC=1C=CC(=CC=1)[N+]([O-])=O)CC1=CC=CC=C1 LKDMKWNDBAVNQZ-UHFFFAOYSA-N 0.000 description 7
- 102100039160 Amiloride-sensitive amine oxidase [copper-containing] Human genes 0.000 description 7
- 108090000663 Annexin A1 Proteins 0.000 description 7
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 7
- 101100506090 Caenorhabditis elegans hil-2 gene Proteins 0.000 description 7
- 102000004173 Cathepsin G Human genes 0.000 description 7
- 108090000617 Cathepsin G Proteins 0.000 description 7
- 108090000625 Cathepsin K Proteins 0.000 description 7
- 102000004171 Cathepsin K Human genes 0.000 description 7
- 108700022150 Designed Ankyrin Repeat Proteins Proteins 0.000 description 7
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 7
- 102000001398 Granzyme Human genes 0.000 description 7
- 102000014150 Interferons Human genes 0.000 description 7
- 108010050904 Interferons Proteins 0.000 description 7
- 102100030698 Interleukin-12 subunit alpha Human genes 0.000 description 7
- 102000004890 Interleukin-8 Human genes 0.000 description 7
- 108090001007 Interleukin-8 Proteins 0.000 description 7
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 7
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 7
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 7
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 7
- 108010016165 Matrix Metalloproteinase 2 Proteins 0.000 description 7
- 241001529936 Murinae Species 0.000 description 7
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 7
- 108010072866 Prostate-Specific Antigen Proteins 0.000 description 7
- 102100038358 Prostate-specific antigen Human genes 0.000 description 7
- 108010089417 Sex Hormone-Binding Globulin Proteins 0.000 description 7
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 description 7
- 239000000611 antibody drug conjugate Substances 0.000 description 7
- 210000003719 b-lymphocyte Anatomy 0.000 description 7
- 230000003197 catalytic effect Effects 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 229940127089 cytotoxic agent Drugs 0.000 description 7
- 239000002254 cytotoxic agent Substances 0.000 description 7
- 210000004443 dendritic cell Anatomy 0.000 description 7
- 239000000499 gel Substances 0.000 description 7
- 235000013922 glutamic acid Nutrition 0.000 description 7
- 239000004220 glutamic acid Substances 0.000 description 7
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 7
- 235000014304 histidine Nutrition 0.000 description 7
- 210000002865 immune cell Anatomy 0.000 description 7
- 230000028993 immune response Effects 0.000 description 7
- 108010074108 interleukin-21 Proteins 0.000 description 7
- 239000000543 intermediate Substances 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 208000024891 symptom Diseases 0.000 description 7
- 230000001988 toxicity Effects 0.000 description 7
- 231100000419 toxicity Toxicity 0.000 description 7
- 108091007505 ADAM17 Proteins 0.000 description 6
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 6
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 6
- 102100027207 CD27 antigen Human genes 0.000 description 6
- 108090000712 Cathepsin B Proteins 0.000 description 6
- 102000004225 Cathepsin B Human genes 0.000 description 6
- 102000003908 Cathepsin D Human genes 0.000 description 6
- 108090000258 Cathepsin D Proteins 0.000 description 6
- 102100031111 Disintegrin and metalloproteinase domain-containing protein 17 Human genes 0.000 description 6
- 102100037362 Fibronectin Human genes 0.000 description 6
- 108010067306 Fibronectins Proteins 0.000 description 6
- 102100027581 Forkhead box protein P3 Human genes 0.000 description 6
- 101000858088 Homo sapiens C-X-C motif chemokine 10 Proteins 0.000 description 6
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 description 6
- 101000861452 Homo sapiens Forkhead box protein P3 Proteins 0.000 description 6
- 101001002657 Homo sapiens Interleukin-2 Proteins 0.000 description 6
- 108010065637 Interleukin-23 Proteins 0.000 description 6
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 6
- 102000019298 Lipocalin Human genes 0.000 description 6
- 108050006654 Lipocalin Proteins 0.000 description 6
- 102100033732 Tumor necrosis factor receptor superfamily member 1A Human genes 0.000 description 6
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 6
- 230000006907 apoptotic process Effects 0.000 description 6
- 235000003704 aspartic acid Nutrition 0.000 description 6
- 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 6
- 239000011230 binding agent Substances 0.000 description 6
- 238000001516 cell proliferation assay Methods 0.000 description 6
- 230000021615 conjugation Effects 0.000 description 6
- 239000000409 cytokine receptor agonist Substances 0.000 description 6
- 102000003675 cytokine receptors Human genes 0.000 description 6
- 108010057085 cytokine receptors Proteins 0.000 description 6
- 238000011161 development Methods 0.000 description 6
- 230000018109 developmental process Effects 0.000 description 6
- 230000004069 differentiation Effects 0.000 description 6
- 108040006849 interleukin-2 receptor activity proteins Proteins 0.000 description 6
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 6
- 229960000310 isoleucine Drugs 0.000 description 6
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 6
- 230000035755 proliferation Effects 0.000 description 6
- 238000011002 quantification Methods 0.000 description 6
- 102220044643 rs587781450 Human genes 0.000 description 6
- 230000004083 survival effect Effects 0.000 description 6
- 239000004474 valine Substances 0.000 description 6
- 235000014393 valine Nutrition 0.000 description 6
- 102100027398 A disintegrin and metalloproteinase with thrombospondin motifs 1 Human genes 0.000 description 5
- 108091007504 ADAM10 Proteins 0.000 description 5
- 108091007507 ADAM12 Proteins 0.000 description 5
- 108091005660 ADAMTS1 Proteins 0.000 description 5
- 102000004506 Blood Proteins Human genes 0.000 description 5
- 108010017384 Blood Proteins Proteins 0.000 description 5
- 102100036842 C-C motif chemokine 19 Human genes 0.000 description 5
- 102100036848 C-C motif chemokine 20 Human genes 0.000 description 5
- 102100036846 C-C motif chemokine 21 Human genes 0.000 description 5
- 102000003902 Cathepsin C Human genes 0.000 description 5
- 108090000267 Cathepsin C Proteins 0.000 description 5
- 102000004178 Cathepsin E Human genes 0.000 description 5
- 108090000611 Cathepsin E Proteins 0.000 description 5
- 102100039673 Disintegrin and metalloproteinase domain-containing protein 10 Human genes 0.000 description 5
- 102100031112 Disintegrin and metalloproteinase domain-containing protein 12 Human genes 0.000 description 5
- 102100024364 Disintegrin and metalloproteinase domain-containing protein 8 Human genes 0.000 description 5
- 101710116123 Disintegrin and metalloproteinase domain-containing protein 8 Proteins 0.000 description 5
- 102100024361 Disintegrin and metalloproteinase domain-containing protein 9 Human genes 0.000 description 5
- 101710116121 Disintegrin and metalloproteinase domain-containing protein 9 Proteins 0.000 description 5
- 108010074860 Factor Xa Proteins 0.000 description 5
- 208000009329 Graft vs Host Disease Diseases 0.000 description 5
- 101000694288 Homo sapiens 40S ribosomal protein SA Proteins 0.000 description 5
- 101000713106 Homo sapiens C-C motif chemokine 19 Proteins 0.000 description 5
- 101000713099 Homo sapiens C-C motif chemokine 20 Proteins 0.000 description 5
- 101000713085 Homo sapiens C-C motif chemokine 21 Proteins 0.000 description 5
- 101001090483 Homo sapiens Glutathione S-transferase LANCL1 Proteins 0.000 description 5
- 101000617830 Homo sapiens Sterol O-acyltransferase 1 Proteins 0.000 description 5
- 102000004889 Interleukin-6 Human genes 0.000 description 5
- 108090001005 Interleukin-6 Proteins 0.000 description 5
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 5
- 102100021993 Sterol O-acyltransferase 1 Human genes 0.000 description 5
- 101000697584 Streptomyces lavendulae Streptothricin acetyltransferase Proteins 0.000 description 5
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 5
- 239000004473 Threonine Substances 0.000 description 5
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 5
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 5
- 230000003213 activating effect Effects 0.000 description 5
- 108700025316 aldesleukin Proteins 0.000 description 5
- 125000000539 amino acid group Chemical group 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 210000004899 c-terminal region Anatomy 0.000 description 5
- 239000013592 cell lysate Substances 0.000 description 5
- 230000004087 circulation Effects 0.000 description 5
- 231100000599 cytotoxic agent Toxicity 0.000 description 5
- 238000012217 deletion Methods 0.000 description 5
- 230000037430 deletion Effects 0.000 description 5
- 239000010432 diamond Substances 0.000 description 5
- 210000002889 endothelial cell Anatomy 0.000 description 5
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 5
- 208000024908 graft versus host disease Diseases 0.000 description 5
- 229940047122 interleukins Drugs 0.000 description 5
- 210000004072 lung Anatomy 0.000 description 5
- 230000003278 mimic effect Effects 0.000 description 5
- 108020001580 protein domains Proteins 0.000 description 5
- 230000009467 reduction Effects 0.000 description 5
- 230000019491 signal transduction Effects 0.000 description 5
- 230000009885 systemic effect Effects 0.000 description 5
- 229940124597 therapeutic agent Drugs 0.000 description 5
- 235000008521 threonine Nutrition 0.000 description 5
- 102100038222 60 kDa heat shock protein, mitochondrial Human genes 0.000 description 4
- 101150013553 CD40 gene Proteins 0.000 description 4
- 108090000397 Caspase 3 Proteins 0.000 description 4
- 102100029855 Caspase-3 Human genes 0.000 description 4
- 102000011727 Caspases Human genes 0.000 description 4
- 108010076667 Caspases Proteins 0.000 description 4
- 108010058432 Chaperonin 60 Proteins 0.000 description 4
- 108090000227 Chymases Proteins 0.000 description 4
- 102000003858 Chymases Human genes 0.000 description 4
- 102000005927 Cysteine Proteases Human genes 0.000 description 4
- 108010005843 Cysteine Proteases Proteins 0.000 description 4
- 102100039498 Cytotoxic T-lymphocyte protein 4 Human genes 0.000 description 4
- 102100035308 Fibroblast growth factor 17 Human genes 0.000 description 4
- 108060005986 Granzyme Proteins 0.000 description 4
- 102000004989 Hepsin Human genes 0.000 description 4
- 108090001101 Hepsin Proteins 0.000 description 4
- 241000282412 Homo Species 0.000 description 4
- 101000914324 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 5 Proteins 0.000 description 4
- 101000914321 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 7 Proteins 0.000 description 4
- 101000889276 Homo sapiens Cytotoxic T-lymphocyte protein 4 Proteins 0.000 description 4
- 101001013150 Homo sapiens Interstitial collagenase Proteins 0.000 description 4
- 102100026120 IgG receptor FcRn large subunit p51 Human genes 0.000 description 4
- 101710177940 IgG receptor FcRn large subunit p51 Proteins 0.000 description 4
- 102000001399 Kallikrein Human genes 0.000 description 4
- 108060005987 Kallikrein Proteins 0.000 description 4
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 4
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 4
- 102000000380 Matrix Metalloproteinase 1 Human genes 0.000 description 4
- 206010027480 Metastatic malignant melanoma Diseases 0.000 description 4
- 102000016387 Pancreatic elastase Human genes 0.000 description 4
- 108010067372 Pancreatic elastase Proteins 0.000 description 4
- 206010037423 Pulmonary oedema Diseases 0.000 description 4
- 102000004338 Transferrin Human genes 0.000 description 4
- 102000009618 Transforming Growth Factors Human genes 0.000 description 4
- 108010009583 Transforming Growth Factors Proteins 0.000 description 4
- 102100033598 Triosephosphate isomerase Human genes 0.000 description 4
- 101710187743 Tumor necrosis factor receptor superfamily member 1A Proteins 0.000 description 4
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 description 4
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 description 4
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 description 4
- 239000005557 antagonist Substances 0.000 description 4
- 238000003570 cell viability assay Methods 0.000 description 4
- 238000012054 celltiter-glo Methods 0.000 description 4
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 4
- 231100000673 dose–response relationship Toxicity 0.000 description 4
- 239000012636 effector Substances 0.000 description 4
- 230000008030 elimination Effects 0.000 description 4
- 238000003379 elimination reaction Methods 0.000 description 4
- 230000013632 homeostatic process Effects 0.000 description 4
- 102000055277 human IL2 Human genes 0.000 description 4
- 230000002209 hydrophobic effect Effects 0.000 description 4
- 230000001976 improved effect Effects 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 230000028709 inflammatory response Effects 0.000 description 4
- 229940047124 interferons Drugs 0.000 description 4
- 210000003734 kidney Anatomy 0.000 description 4
- 238000004020 luminiscence type Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 210000003071 memory t lymphocyte Anatomy 0.000 description 4
- 208000021039 metastatic melanoma Diseases 0.000 description 4
- 239000000178 monomer Substances 0.000 description 4
- 229940012957 plasmin Drugs 0.000 description 4
- 108010094020 polyglycine Proteins 0.000 description 4
- 229940087463 proleukin Drugs 0.000 description 4
- 208000005333 pulmonary edema Diseases 0.000 description 4
- 239000000018 receptor agonist Substances 0.000 description 4
- 229940044601 receptor agonist Drugs 0.000 description 4
- 102220050130 rs187015338 Human genes 0.000 description 4
- 108091006024 signal transducing proteins Proteins 0.000 description 4
- 102000034285 signal transducing proteins Human genes 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 108090000250 sortase A Proteins 0.000 description 4
- 239000012581 transferrin Substances 0.000 description 4
- 230000004614 tumor growth Effects 0.000 description 4
- 102000003390 tumor necrosis factor Human genes 0.000 description 4
- 238000012447 xenograft mouse model Methods 0.000 description 4
- 108010032088 Calpain Proteins 0.000 description 3
- 102000007590 Calpain Human genes 0.000 description 3
- 108010080937 Carboxypeptidases A Proteins 0.000 description 3
- 102000000496 Carboxypeptidases A Human genes 0.000 description 3
- 102100025012 Dipeptidyl peptidase 4 Human genes 0.000 description 3
- 101150064015 FAS gene Proteins 0.000 description 3
- 108010049003 Fibrinogen Proteins 0.000 description 3
- 102000008946 Fibrinogen Human genes 0.000 description 3
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 3
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 3
- 108050003624 Granzyme M Proteins 0.000 description 3
- 101000959820 Homo sapiens Interferon alpha-1/13 Proteins 0.000 description 3
- 101000801234 Homo sapiens Tumor necrosis factor receptor superfamily member 18 Proteins 0.000 description 3
- 101000851376 Homo sapiens Tumor necrosis factor receptor superfamily member 8 Proteins 0.000 description 3
- 102100040019 Interferon alpha-1/13 Human genes 0.000 description 3
- 102100037850 Interferon gamma Human genes 0.000 description 3
- 108090000467 Interferon-beta Proteins 0.000 description 3
- 108010074328 Interferon-gamma Proteins 0.000 description 3
- 102220470904 Interferon-induced protein with tetratricopeptide repeats 5_K48E_mutation Human genes 0.000 description 3
- 102000003814 Interleukin-10 Human genes 0.000 description 3
- 108090000174 Interleukin-10 Proteins 0.000 description 3
- 108090000177 Interleukin-11 Proteins 0.000 description 3
- 102000003815 Interleukin-11 Human genes 0.000 description 3
- 102000004560 Interleukin-12 Receptors Human genes 0.000 description 3
- 108010017515 Interleukin-12 Receptors Proteins 0.000 description 3
- 102000014154 Interleukin-12 Subunit p35 Human genes 0.000 description 3
- 108010011301 Interleukin-12 Subunit p35 Proteins 0.000 description 3
- 102000003816 Interleukin-13 Human genes 0.000 description 3
- 108090000176 Interleukin-13 Proteins 0.000 description 3
- 102000004388 Interleukin-4 Human genes 0.000 description 3
- 108090000978 Interleukin-4 Proteins 0.000 description 3
- 108010002616 Interleukin-5 Proteins 0.000 description 3
- 102000000743 Interleukin-5 Human genes 0.000 description 3
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical group OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 3
- 101710180643 Leishmanolysin Proteins 0.000 description 3
- 108010061593 Member 14 Tumor Necrosis Factor Receptors Proteins 0.000 description 3
- 102000005741 Metalloproteases Human genes 0.000 description 3
- 108010006035 Metalloproteases Proteins 0.000 description 3
- 101001044384 Mus musculus Interferon gamma Proteins 0.000 description 3
- 102100034681 Myeloblastin Human genes 0.000 description 3
- 108090000973 Myeloblastin Proteins 0.000 description 3
- 102000001938 Plasminogen Activators Human genes 0.000 description 3
- 108010001014 Plasminogen Activators Proteins 0.000 description 3
- 241000700159 Rattus Species 0.000 description 3
- 108010029477 STAT5 Transcription Factor Proteins 0.000 description 3
- 102100024481 Signal transducer and activator of transcription 5A Human genes 0.000 description 3
- 230000006052 T cell proliferation Effects 0.000 description 3
- 102100031294 Thymic stromal lymphopoietin Human genes 0.000 description 3
- 108090000901 Transferrin Proteins 0.000 description 3
- 102000046299 Transforming Growth Factor beta1 Human genes 0.000 description 3
- 101800002279 Transforming growth factor beta-1 Proteins 0.000 description 3
- 108060005989 Tryptase Proteins 0.000 description 3
- 102000001400 Tryptase Human genes 0.000 description 3
- 102100028785 Tumor necrosis factor receptor superfamily member 14 Human genes 0.000 description 3
- 102100033728 Tumor necrosis factor receptor superfamily member 18 Human genes 0.000 description 3
- 102100033733 Tumor necrosis factor receptor superfamily member 1B Human genes 0.000 description 3
- 101710187830 Tumor necrosis factor receptor superfamily member 1B Proteins 0.000 description 3
- 102100036857 Tumor necrosis factor receptor superfamily member 8 Human genes 0.000 description 3
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 3
- 102220348979 c.262A>G Human genes 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 230000011712 cell development Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000002950 deficient Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 210000003162 effector t lymphocyte Anatomy 0.000 description 3
- 210000002919 epithelial cell Anatomy 0.000 description 3
- 229940012952 fibrinogen Drugs 0.000 description 3
- 238000002866 fluorescence resonance energy transfer Methods 0.000 description 3
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 3
- 239000000833 heterodimer Substances 0.000 description 3
- 210000003630 histaminocyte Anatomy 0.000 description 3
- 238000009169 immunotherapy Methods 0.000 description 3
- 208000027866 inflammatory disease Diseases 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 229940079322 interferon Drugs 0.000 description 3
- 230000033001 locomotion Effects 0.000 description 3
- 230000002132 lysosomal effect Effects 0.000 description 3
- 210000002540 macrophage Anatomy 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 210000001616 monocyte Anatomy 0.000 description 3
- 239000004031 partial agonist Substances 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 230000003836 peripheral circulation Effects 0.000 description 3
- 229940127126 plasminogen activator Drugs 0.000 description 3
- 230000004983 pleiotropic effect Effects 0.000 description 3
- 230000003389 potentiating effect Effects 0.000 description 3
- 229940002612 prodrug Drugs 0.000 description 3
- 239000000651 prodrug Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000000770 proinflammatory effect Effects 0.000 description 3
- 125000001500 prolyl group Chemical group [H]N1C([H])(C(=O)[*])C([H])([H])C([H])([H])C1([H])[H] 0.000 description 3
- 230000007026 protein scission Effects 0.000 description 3
- 102220008330 rs199476310 Human genes 0.000 description 3
- 102200002393 rs3816873 Human genes 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 230000004936 stimulating effect Effects 0.000 description 3
- 238000007920 subcutaneous administration Methods 0.000 description 3
- 231100000057 systemic toxicity Toxicity 0.000 description 3
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 3
- 231100001274 therapeutic index Toxicity 0.000 description 3
- 108010029307 thymic stromal lymphopoietin Proteins 0.000 description 3
- 229920003169 water-soluble polymer Polymers 0.000 description 3
- CVOFKRWYWCSDMA-UHFFFAOYSA-N 2-chloro-n-(2,6-diethylphenyl)-n-(methoxymethyl)acetamide;2,6-dinitro-n,n-dipropyl-4-(trifluoromethyl)aniline Chemical compound CCC1=CC=CC(CC)=C1N(COC)C(=O)CCl.CCCN(CCC)C1=C([N+]([O-])=O)C=C(C(F)(F)F)C=C1[N+]([O-])=O CVOFKRWYWCSDMA-UHFFFAOYSA-N 0.000 description 2
- 108091005508 Acid proteases Proteins 0.000 description 2
- 108010032595 Antibody Binding Sites Proteins 0.000 description 2
- 102100029470 Apolipoprotein E Human genes 0.000 description 2
- 101710095339 Apolipoprotein E Proteins 0.000 description 2
- 102100021943 C-C motif chemokine 2 Human genes 0.000 description 2
- 101710155857 C-C motif chemokine 2 Proteins 0.000 description 2
- 101710098275 C-X-C motif chemokine 10 Proteins 0.000 description 2
- 102100036150 C-X-C motif chemokine 5 Human genes 0.000 description 2
- 102000002110 C2 domains Human genes 0.000 description 2
- 108050009459 C2 domains Proteins 0.000 description 2
- 102100032912 CD44 antigen Human genes 0.000 description 2
- 210000001239 CD8-positive, alpha-beta cytotoxic T lymphocyte Anatomy 0.000 description 2
- 102000000905 Cadherin Human genes 0.000 description 2
- 108050007957 Cadherin Proteins 0.000 description 2
- 102000014914 Carrier Proteins Human genes 0.000 description 2
- 108090000426 Caspase-1 Proteins 0.000 description 2
- 102100021809 Chorionic somatomammotropin hormone 1 Human genes 0.000 description 2
- 108010005939 Ciliary Neurotrophic Factor Proteins 0.000 description 2
- 102100031614 Ciliary neurotrophic factor Human genes 0.000 description 2
- 102000029816 Collagenase Human genes 0.000 description 2
- 108060005980 Collagenase Proteins 0.000 description 2
- 241000701022 Cytomegalovirus Species 0.000 description 2
- 101001027406 Danio rerio Fibroblast growth factor 8b Proteins 0.000 description 2
- 102100029727 Enteropeptidase Human genes 0.000 description 2
- 108010013369 Enteropeptidase Proteins 0.000 description 2
- 108010092408 Eosinophil Peroxidase Proteins 0.000 description 2
- 102100028471 Eosinophil peroxidase Human genes 0.000 description 2
- 108010066687 Epithelial Cell Adhesion Molecule Proteins 0.000 description 2
- 102000018651 Epithelial Cell Adhesion Molecule Human genes 0.000 description 2
- 102000008857 Ferritin Human genes 0.000 description 2
- 108050000784 Ferritin Proteins 0.000 description 2
- 238000008416 Ferritin Methods 0.000 description 2
- 108090000386 Fibroblast Growth Factor 1 Proteins 0.000 description 2
- 102000003971 Fibroblast Growth Factor 1 Human genes 0.000 description 2
- 108090000569 Fibroblast Growth Factor-23 Proteins 0.000 description 2
- 102100028412 Fibroblast growth factor 10 Human genes 0.000 description 2
- 108090001047 Fibroblast growth factor 10 Proteins 0.000 description 2
- 108050003237 Fibroblast growth factor 11 Proteins 0.000 description 2
- 102100028413 Fibroblast growth factor 11 Human genes 0.000 description 2
- 108050003239 Fibroblast growth factor 12 Proteins 0.000 description 2
- 102100028417 Fibroblast growth factor 12 Human genes 0.000 description 2
- 102000003685 Fibroblast growth factor 14 Human genes 0.000 description 2
- 108090000046 Fibroblast growth factor 14 Proteins 0.000 description 2
- 108050002072 Fibroblast growth factor 16 Proteins 0.000 description 2
- 102100035307 Fibroblast growth factor 16 Human genes 0.000 description 2
- 108050002074 Fibroblast growth factor 17 Proteins 0.000 description 2
- 102100031734 Fibroblast growth factor 19 Human genes 0.000 description 2
- 108050002085 Fibroblast growth factor 20 Proteins 0.000 description 2
- 102100031361 Fibroblast growth factor 20 Human genes 0.000 description 2
- 108090000376 Fibroblast growth factor 21 Proteins 0.000 description 2
- 102000003973 Fibroblast growth factor 21 Human genes 0.000 description 2
- 108050002062 Fibroblast growth factor 22 Proteins 0.000 description 2
- 102100024804 Fibroblast growth factor 22 Human genes 0.000 description 2
- 102100024802 Fibroblast growth factor 23 Human genes 0.000 description 2
- 108090000378 Fibroblast growth factor 3 Proteins 0.000 description 2
- 102100028043 Fibroblast growth factor 3 Human genes 0.000 description 2
- 102100028072 Fibroblast growth factor 4 Human genes 0.000 description 2
- 108090000381 Fibroblast growth factor 4 Proteins 0.000 description 2
- 108090000380 Fibroblast growth factor 5 Proteins 0.000 description 2
- 102100028073 Fibroblast growth factor 5 Human genes 0.000 description 2
- 108090000382 Fibroblast growth factor 6 Proteins 0.000 description 2
- 102100028075 Fibroblast growth factor 6 Human genes 0.000 description 2
- 102100028071 Fibroblast growth factor 7 Human genes 0.000 description 2
- 108090000385 Fibroblast growth factor 7 Proteins 0.000 description 2
- 102100037665 Fibroblast growth factor 9 Human genes 0.000 description 2
- 108090000367 Fibroblast growth factor 9 Proteins 0.000 description 2
- 108010001498 Galectin 1 Proteins 0.000 description 2
- 108010001517 Galectin 3 Proteins 0.000 description 2
- 102100021736 Galectin-1 Human genes 0.000 description 2
- 102100039558 Galectin-3 Human genes 0.000 description 2
- 102000006395 Globulins Human genes 0.000 description 2
- 108010044091 Globulins Proteins 0.000 description 2
- 102100039619 Granulocyte colony-stimulating factor Human genes 0.000 description 2
- 108010051696 Growth Hormone Proteins 0.000 description 2
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 2
- 102100022893 Histone acetyltransferase KAT5 Human genes 0.000 description 2
- 101000947186 Homo sapiens C-X-C motif chemokine 5 Proteins 0.000 description 2
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 2
- 101000846394 Homo sapiens Fibroblast growth factor 19 Proteins 0.000 description 2
- 101000746367 Homo sapiens Granulocyte colony-stimulating factor Proteins 0.000 description 2
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 2
- 101000599048 Homo sapiens Interleukin-6 receptor subunit alpha Proteins 0.000 description 2
- 101000990912 Homo sapiens Matrilysin Proteins 0.000 description 2
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 2
- 101000577877 Homo sapiens Stromelysin-3 Proteins 0.000 description 2
- 101000801228 Homo sapiens Tumor necrosis factor receptor superfamily member 1A Proteins 0.000 description 2
- 101000611023 Homo sapiens Tumor necrosis factor receptor superfamily member 6 Proteins 0.000 description 2
- 101000997832 Homo sapiens Tyrosine-protein kinase JAK2 Proteins 0.000 description 2
- 229940099539 IL-36 receptor antagonist Drugs 0.000 description 2
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 2
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 2
- 102100026720 Interferon beta Human genes 0.000 description 2
- 108010047761 Interferon-alpha Proteins 0.000 description 2
- 102000006992 Interferon-alpha Human genes 0.000 description 2
- 108010066719 Interleukin Receptor Common gamma Subunit Proteins 0.000 description 2
- 102000018682 Interleukin Receptor Common gamma Subunit Human genes 0.000 description 2
- 102000000589 Interleukin-1 Human genes 0.000 description 2
- 108010002352 Interleukin-1 Proteins 0.000 description 2
- 102100020790 Interleukin-12 receptor subunit beta-1 Human genes 0.000 description 2
- 101710103841 Interleukin-12 receptor subunit beta-1 Proteins 0.000 description 2
- 102100020792 Interleukin-12 receptor subunit beta-2 Human genes 0.000 description 2
- 101710103840 Interleukin-12 receptor subunit beta-2 Proteins 0.000 description 2
- 108010076561 Interleukin-23 Subunit p19 Proteins 0.000 description 2
- 108010067003 Interleukin-33 Proteins 0.000 description 2
- 102000017761 Interleukin-33 Human genes 0.000 description 2
- 102100021150 Interleukin-36 receptor antagonist protein Human genes 0.000 description 2
- 101710089409 Interleukin-36 receptor antagonist protein Proteins 0.000 description 2
- 102100037792 Interleukin-6 receptor subunit alpha Human genes 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
- 102000016267 Leptin Human genes 0.000 description 2
- 108010092277 Leptin Proteins 0.000 description 2
- 108090000581 Leukemia inhibitory factor Proteins 0.000 description 2
- 102100032352 Leukemia inhibitory factor Human genes 0.000 description 2
- 108020002496 Lysophospholipase Proteins 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 102100030417 Matrilysin Human genes 0.000 description 2
- 108010032605 Nerve Growth Factor Receptors Proteins 0.000 description 2
- 108090000630 Oncostatin M Proteins 0.000 description 2
- 102000004140 Oncostatin M Human genes 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 102000000470 PDZ domains Human genes 0.000 description 2
- 108050008994 PDZ domains Proteins 0.000 description 2
- 102220536899 PRKC apoptosis WT1 regulator protein_E68D_mutation Human genes 0.000 description 2
- 108090000526 Papain Proteins 0.000 description 2
- 108090000284 Pepsin A Proteins 0.000 description 2
- 102000057297 Pepsin A Human genes 0.000 description 2
- 108010067902 Peptide Library Proteins 0.000 description 2
- 108090000279 Peptidyltransferases Proteins 0.000 description 2
- 206010057249 Phagocytosis Diseases 0.000 description 2
- 108010003044 Placental Lactogen Proteins 0.000 description 2
- 239000000381 Placental Lactogen Substances 0.000 description 2
- 108010057464 Prolactin Proteins 0.000 description 2
- 102100024819 Prolactin Human genes 0.000 description 2
- 102100023832 Prolyl endopeptidase FAP Human genes 0.000 description 2
- 102000003923 Protein Kinase C Human genes 0.000 description 2
- 108090000315 Protein Kinase C Proteins 0.000 description 2
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 2
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 2
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 108010017324 STAT3 Transcription Factor Proteins 0.000 description 2
- 102000005886 STAT4 Transcription Factor Human genes 0.000 description 2
- 108010019992 STAT4 Transcription Factor Proteins 0.000 description 2
- 102100024040 Signal transducer and activator of transcription 3 Human genes 0.000 description 2
- 102220494399 Signal transducer and activator of transcription 5A_Y31H_mutation Human genes 0.000 description 2
- 102100038803 Somatotropin Human genes 0.000 description 2
- 102100028847 Stromelysin-3 Human genes 0.000 description 2
- 102220572243 Toll-interacting protein_N29S_mutation Human genes 0.000 description 2
- 108010033576 Transferrin Receptors Proteins 0.000 description 2
- 102100026144 Transferrin receptor protein 1 Human genes 0.000 description 2
- 108700015934 Triose-phosphate isomerases Proteins 0.000 description 2
- 102100033725 Tumor necrosis factor receptor superfamily member 16 Human genes 0.000 description 2
- 102100040403 Tumor necrosis factor receptor superfamily member 6 Human genes 0.000 description 2
- 102100033444 Tyrosine-protein kinase JAK2 Human genes 0.000 description 2
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 2
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000006786 activation induced cell death Effects 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 230000004721 adaptive immunity Effects 0.000 description 2
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 2
- 229960005310 aldesleukin Drugs 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 230000005756 apoptotic signaling Effects 0.000 description 2
- 230000001363 autoimmune Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 102220402176 c.236A>G Human genes 0.000 description 2
- 102220367675 c.268A>C Human genes 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000002975 chemoattractant Substances 0.000 description 2
- 230000035605 chemotaxis Effects 0.000 description 2
- 229960002424 collagenase Drugs 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 239000003636 conditioned culture medium Substances 0.000 description 2
- 238000011393 cytotoxic chemotherapy Methods 0.000 description 2
- 239000000539 dimer Substances 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000003366 endpoint assay Methods 0.000 description 2
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 2
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 108090000047 fibroblast growth factor 13 Proteins 0.000 description 2
- 108090000370 fibroblast growth factor 18 Proteins 0.000 description 2
- 102000003977 fibroblast growth factor 18 Human genes 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 239000000122 growth hormone Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 231100000086 high toxicity Toxicity 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000015788 innate immune response Effects 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 102000006495 integrins Human genes 0.000 description 2
- 108010044426 integrins Proteins 0.000 description 2
- 229960003130 interferon gamma Drugs 0.000 description 2
- 108010074109 interleukin-22 Proteins 0.000 description 2
- 230000002601 intratumoral effect Effects 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 125000000741 isoleucyl group Chemical group [H]N([H])C(C(C([H])([H])[H])C([H])([H])C([H])([H])[H])C(=O)O* 0.000 description 2
- 238000005304 joining Methods 0.000 description 2
- 238000011813 knockout mouse model Methods 0.000 description 2
- NRYBAZVQPHGZNS-ZSOCWYAHSA-N leptin Chemical compound O=C([C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CC(C)C)CCSC)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CS)C(O)=O NRYBAZVQPHGZNS-ZSOCWYAHSA-N 0.000 description 2
- 229940039781 leptin Drugs 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 210000001165 lymph node Anatomy 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 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 2
- 210000003712 lysosome Anatomy 0.000 description 2
- 230000001868 lysosomic effect Effects 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 230000035800 maturation Effects 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 210000000107 myocyte Anatomy 0.000 description 2
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 description 2
- 210000000440 neutrophil Anatomy 0.000 description 2
- 230000000269 nucleophilic effect Effects 0.000 description 2
- 238000012261 overproduction Methods 0.000 description 2
- 229940055729 papain Drugs 0.000 description 2
- 235000019834 papain Nutrition 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 229940111202 pepsin Drugs 0.000 description 2
- 230000008782 phagocytosis Effects 0.000 description 2
- 230000000144 pharmacologic effect Effects 0.000 description 2
- 229940097325 prolactin Drugs 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 230000012743 protein tagging Effects 0.000 description 2
- 230000002685 pulmonary effect Effects 0.000 description 2
- 238000010188 recombinant method Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 102220209560 rs1057521321 Human genes 0.000 description 2
- 102200097961 rs11539202 Human genes 0.000 description 2
- 102200038856 rs150565592 Human genes 0.000 description 2
- 102220024713 rs397515486 Human genes 0.000 description 2
- 102220058220 rs730881937 Human genes 0.000 description 2
- 102220097954 rs876658140 Human genes 0.000 description 2
- 108010073863 saruplase Proteins 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 210000005212 secondary lymphoid organ Anatomy 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 102000003137 synaptotagmin Human genes 0.000 description 2
- 108060008004 synaptotagmin Proteins 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 2
- 229940126585 therapeutic drug Drugs 0.000 description 2
- 230000002992 thymic effect Effects 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000001052 transient effect Effects 0.000 description 2
- 230000001810 trypsinlike Effects 0.000 description 2
- 238000013414 tumor xenograft model Methods 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
- 235000002374 tyrosine Nutrition 0.000 description 2
- 125000002987 valine group Chemical group [H]N([H])C([H])(C(*)=O)C([H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- 230000003442 weekly effect Effects 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- AOUOVFRSCMDPFA-QSDJMHMYSA-N (2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-amino-3-carboxypropanoyl]amino]-4-carboxybutanoyl]amino]-3-methylbutanoyl]amino]butanedioic acid Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC(O)=O AOUOVFRSCMDPFA-QSDJMHMYSA-N 0.000 description 1
- KQRHTCDQWJLLME-XUXIUFHCSA-N (2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-aminopropanoyl]amino]-4-methylpentanoyl]amino]propanoyl]amino]-4-methylpentanoic acid Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)N KQRHTCDQWJLLME-XUXIUFHCSA-N 0.000 description 1
- GHKCSRZBNZQHKW-UHFFFAOYSA-N 1-sulfanylethanol Chemical compound CC(O)S GHKCSRZBNZQHKW-UHFFFAOYSA-N 0.000 description 1
- 101710151806 72 kDa type IV collagenase Proteins 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 102000054930 Agouti-Related Human genes 0.000 description 1
- 102400000344 Angiotensin-1 Human genes 0.000 description 1
- 101800000734 Angiotensin-1 Proteins 0.000 description 1
- 108010049777 Ankyrins Proteins 0.000 description 1
- 102000008102 Ankyrins Human genes 0.000 description 1
- 108091005504 Asparagine peptide lyases Proteins 0.000 description 1
- 102000030431 Asparaginyl endopeptidase Human genes 0.000 description 1
- 238000012935 Averaging Methods 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 108091007065 BIRCs Proteins 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 101000984722 Bos taurus Pancreatic trypsin inhibitor Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 101000773693 Brassica napus Acyl carrier protein, chloroplastic Proteins 0.000 description 1
- 108010004032 Bromelains Proteins 0.000 description 1
- 101710149863 C-C chemokine receptor type 4 Proteins 0.000 description 1
- 102100032367 C-C motif chemokine 5 Human genes 0.000 description 1
- 102100032976 CCR4-NOT transcription complex subunit 6 Human genes 0.000 description 1
- 102100035793 CD83 antigen Human genes 0.000 description 1
- 102220615759 CD83 antigen_E67G_mutation Human genes 0.000 description 1
- 206010006895 Cachexia Diseases 0.000 description 1
- 101000898643 Candida albicans Vacuolar aspartic protease Proteins 0.000 description 1
- 101000898783 Candida tropicalis Candidapepsin Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 102100026548 Caspase-8 Human genes 0.000 description 1
- 108090000538 Caspase-8 Proteins 0.000 description 1
- 102220557289 Cathepsin G_N71A_mutation Human genes 0.000 description 1
- 241000700199 Cavia porcellus Species 0.000 description 1
- 108010055166 Chemokine CCL5 Proteins 0.000 description 1
- 108090000746 Chymosin Proteins 0.000 description 1
- 102100027995 Collagenase 3 Human genes 0.000 description 1
- 206010010099 Combined immunodeficiency Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 102000002585 Contractile Proteins Human genes 0.000 description 1
- 108010068426 Contractile Proteins Proteins 0.000 description 1
- 241000938605 Crocodylia Species 0.000 description 1
- 101000898784 Cryphonectria parasitica Endothiapepsin Proteins 0.000 description 1
- ONIBWKKTOPOVIA-SCSAIBSYSA-N D-Proline Chemical group OC(=O)[C@H]1CCCN1 ONIBWKKTOPOVIA-SCSAIBSYSA-N 0.000 description 1
- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-N 0.000 description 1
- COLNVLDHVKWLRT-MRVPVSSYSA-N D-phenylalanine Chemical group OC(=O)[C@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-MRVPVSSYSA-N 0.000 description 1
- 108091008102 DNA aptamers Proteins 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 108090000194 Dipeptidyl-peptidases and tripeptidyl-peptidases Proteins 0.000 description 1
- 102000003779 Dipeptidyl-peptidases and tripeptidyl-peptidases Human genes 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 108700033317 EC 3.4.23.12 Proteins 0.000 description 1
- 101150084967 EPCAM gene Proteins 0.000 description 1
- 101000609473 Ecballium elaterium Trypsin inhibitor 2 Proteins 0.000 description 1
- 102100029722 Ectonucleoside triphosphate diphosphohydrolase 1 Human genes 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 206010015150 Erythema Diseases 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 108050001049 Extracellular proteins Proteins 0.000 description 1
- 108010071289 Factor XIII Proteins 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 102000002090 Fibronectin type III Human genes 0.000 description 1
- 108050009401 Fibronectin type III Proteins 0.000 description 1
- 239000004606 Fillers/Extenders Substances 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 101000766307 Gallus gallus Ovotransferrin Proteins 0.000 description 1
- 102100032518 Gamma-crystallin B Human genes 0.000 description 1
- 101710092798 Gamma-crystallin B Proteins 0.000 description 1
- 101000930822 Giardia intestinalis Dipeptidyl-peptidase 4 Proteins 0.000 description 1
- 102100038393 Granzyme H Human genes 0.000 description 1
- 101710113220 Granzyme H Proteins 0.000 description 1
- 108010010369 HIV Protease Proteins 0.000 description 1
- 102220541629 HLA class II histocompatibility antigen, DQ beta 1 chain_M46L_mutation Human genes 0.000 description 1
- 102400001369 Heparin-binding EGF-like growth factor Human genes 0.000 description 1
- 101800001649 Heparin-binding EGF-like growth factor Proteins 0.000 description 1
- 206010019837 Hepatocellular injury Diseases 0.000 description 1
- 101000946856 Homo sapiens CD83 antigen Proteins 0.000 description 1
- 101000577887 Homo sapiens Collagenase 3 Proteins 0.000 description 1
- 101000908391 Homo sapiens Dipeptidyl peptidase 4 Proteins 0.000 description 1
- 101001012447 Homo sapiens Ectonucleoside triphosphate diphosphohydrolase 1 Proteins 0.000 description 1
- 101001055157 Homo sapiens Interleukin-15 Proteins 0.000 description 1
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 1
- 101000853002 Homo sapiens Interleukin-25 Proteins 0.000 description 1
- 101001128431 Homo sapiens Myeloid-derived growth factor Proteins 0.000 description 1
- 101000990908 Homo sapiens Neutrophil collagenase Proteins 0.000 description 1
- 101000617725 Homo sapiens Pregnancy-specific beta-1-glycoprotein 2 Proteins 0.000 description 1
- 101000684208 Homo sapiens Prolyl endopeptidase FAP Proteins 0.000 description 1
- 101000990915 Homo sapiens Stromelysin-1 Proteins 0.000 description 1
- 101100369992 Homo sapiens TNFSF10 gene Proteins 0.000 description 1
- 101000997835 Homo sapiens Tyrosine-protein kinase JAK1 Proteins 0.000 description 1
- 101000934996 Homo sapiens Tyrosine-protein kinase JAK3 Proteins 0.000 description 1
- 241000725303 Human immunodeficiency virus Species 0.000 description 1
- 108010016183 Human immunodeficiency virus 1 p16 protease Proteins 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 208000001953 Hypotension Diseases 0.000 description 1
- 101150029684 IL2RA gene Proteins 0.000 description 1
- 102000013463 Immunoglobulin Light Chains Human genes 0.000 description 1
- 108010065825 Immunoglobulin Light Chains Proteins 0.000 description 1
- 102000012745 Immunoglobulin Subunits Human genes 0.000 description 1
- 108010079585 Immunoglobulin Subunits Proteins 0.000 description 1
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 1
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 1
- 102000055031 Inhibitor of Apoptosis Proteins Human genes 0.000 description 1
- 102100022469 Interferon kappa Human genes 0.000 description 1
- 102000003996 Interferon-beta Human genes 0.000 description 1
- 102000019223 Interleukin-1 receptor Human genes 0.000 description 1
- 108050006617 Interleukin-1 receptor Proteins 0.000 description 1
- 102000014158 Interleukin-12 Subunit p40 Human genes 0.000 description 1
- 108010011429 Interleukin-12 Subunit p40 Proteins 0.000 description 1
- 108010017535 Interleukin-15 Receptors Proteins 0.000 description 1
- 102000004556 Interleukin-15 Receptors Human genes 0.000 description 1
- 101800003050 Interleukin-16 Proteins 0.000 description 1
- 102000049772 Interleukin-16 Human genes 0.000 description 1
- 108050003558 Interleukin-17 Proteins 0.000 description 1
- 102000013691 Interleukin-17 Human genes 0.000 description 1
- 102100026878 Interleukin-2 receptor subunit alpha Human genes 0.000 description 1
- 102000000646 Interleukin-3 Human genes 0.000 description 1
- 108010002386 Interleukin-3 Proteins 0.000 description 1
- 108010038501 Interleukin-6 Receptors Proteins 0.000 description 1
- 102000010781 Interleukin-6 Receptors Human genes 0.000 description 1
- 108010002335 Interleukin-9 Proteins 0.000 description 1
- 102000000585 Interleukin-9 Human genes 0.000 description 1
- 101150009057 JAK2 gene Proteins 0.000 description 1
- 101150069380 JAK3 gene Proteins 0.000 description 1
- 108010024121 Janus Kinases Proteins 0.000 description 1
- 102000015617 Janus Kinases Human genes 0.000 description 1
- 102100027612 Kallikrein-11 Human genes 0.000 description 1
- 102100038356 Kallikrein-2 Human genes 0.000 description 1
- 101710176220 Kallikrein-2 Proteins 0.000 description 1
- 208000008839 Kidney Neoplasms Diseases 0.000 description 1
- 102100035792 Kininogen-1 Human genes 0.000 description 1
- 108010077861 Kininogens Proteins 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 101100385641 Locusta migratoria ACP21 gene Proteins 0.000 description 1
- 101000680845 Luffa aegyptiaca Ribosome-inactivating protein luffin P1 Proteins 0.000 description 1
- 101150113681 MALT1 gene Proteins 0.000 description 1
- 102000019149 MAP kinase activity proteins Human genes 0.000 description 1
- 108040008097 MAP kinase activity proteins Proteins 0.000 description 1
- 102000043136 MAP kinase family Human genes 0.000 description 1
- 108091054455 MAP kinase family Proteins 0.000 description 1
- 102000043131 MHC class II family Human genes 0.000 description 1
- 108091054438 MHC class II family Proteins 0.000 description 1
- 102100030612 Mast cell carboxypeptidase A Human genes 0.000 description 1
- 101710119290 Mast cell carboxypeptidase A Proteins 0.000 description 1
- 108010091175 Matriptase Proteins 0.000 description 1
- 102000000422 Matrix Metalloproteinase 3 Human genes 0.000 description 1
- 102100030412 Matrix metalloproteinase-9 Human genes 0.000 description 1
- 108010015302 Matrix metalloproteinase-9 Proteins 0.000 description 1
- 102000003735 Mesothelin Human genes 0.000 description 1
- 108090000015 Mesothelin Proteins 0.000 description 1
- 102000003843 Metalloendopeptidases Human genes 0.000 description 1
- 108090000131 Metalloendopeptidases Proteins 0.000 description 1
- 108010045057 Metalloexopeptidases Proteins 0.000 description 1
- 102000005612 Metalloexopeptidases Human genes 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 206010050513 Metastatic renal cell carcinoma Diseases 0.000 description 1
- 102100023482 Mitogen-activated protein kinase 14 Human genes 0.000 description 1
- 102220485631 Mitogen-activated protein kinase 14_K54R_mutation Human genes 0.000 description 1
- 108010006519 Molecular Chaperones Proteins 0.000 description 1
- 102000005431 Molecular Chaperones Human genes 0.000 description 1
- 108700026676 Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Proteins 0.000 description 1
- 102100038732 Mucosa-associated lymphoid tissue lymphoma translocation protein 1 Human genes 0.000 description 1
- 101000959789 Mus musculus Interferon alpha-1 Proteins 0.000 description 1
- 206010028289 Muscle atrophy Diseases 0.000 description 1
- 102100031789 Myeloid-derived growth factor Human genes 0.000 description 1
- 108010057466 NF-kappa B Proteins 0.000 description 1
- 102000003945 NF-kappa B Human genes 0.000 description 1
- 206010028851 Necrosis Diseases 0.000 description 1
- 102100030411 Neutrophil collagenase Human genes 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 102100040557 Osteopontin Human genes 0.000 description 1
- 108010081689 Osteopontin Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 108010079855 Peptide Aptamers Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- KHGNFPUMBJSZSM-UHFFFAOYSA-N Perforine Natural products COC1=C2CCC(O)C(CCC(C)(C)O)(OC)C2=NC2=C1C=CO2 KHGNFPUMBJSZSM-UHFFFAOYSA-N 0.000 description 1
- 241000009328 Perro Species 0.000 description 1
- 108010022233 Plasminogen Activator Inhibitor 1 Proteins 0.000 description 1
- 102100039418 Plasminogen activator inhibitor 1 Human genes 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 102220557308 Proliferating cell nuclear antigen_I128A_mutation Human genes 0.000 description 1
- 101710129873 Prolyl endopeptidase FAP Proteins 0.000 description 1
- 102100038946 Proprotein convertase subtilisin/kexin type 6 Human genes 0.000 description 1
- 101000774655 Protobothrops mucrosquamatus Snake venom metalloproteinase TM-1 Proteins 0.000 description 1
- 108091008103 RNA aptamers Proteins 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 206010038389 Renal cancer Diseases 0.000 description 1
- 208000006265 Renal cell carcinoma Diseases 0.000 description 1
- 108090000783 Renin Proteins 0.000 description 1
- 102100028255 Renin Human genes 0.000 description 1
- 101000933133 Rhizopus niveus Rhizopuspepsin-1 Proteins 0.000 description 1
- 101000910082 Rhizopus niveus Rhizopuspepsin-2 Proteins 0.000 description 1
- 101000910079 Rhizopus niveus Rhizopuspepsin-3 Proteins 0.000 description 1
- 101000910086 Rhizopus niveus Rhizopuspepsin-4 Proteins 0.000 description 1
- 101000910088 Rhizopus niveus Rhizopuspepsin-5 Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 101000898773 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) Saccharopepsin Proteins 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- 102100030416 Stromelysin-1 Human genes 0.000 description 1
- 101710135785 Subtilisin-like protease Proteins 0.000 description 1
- 102100037942 Suppressor of tumorigenicity 14 protein Human genes 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 102000000551 Syk Kinase Human genes 0.000 description 1
- 108010016672 Syk Kinase Proteins 0.000 description 1
- 201000001322 T cell deficiency Diseases 0.000 description 1
- 230000024932 T cell mediated immunity Effects 0.000 description 1
- 210000000662 T-lymphocyte subset Anatomy 0.000 description 1
- 108700012411 TNFSF10 Proteins 0.000 description 1
- 102220532327 Testis-expressed protein 10_K76R_mutation Human genes 0.000 description 1
- 210000000447 Th1 cell Anatomy 0.000 description 1
- 102100036407 Thioredoxin Human genes 0.000 description 1
- 108091005501 Threonine proteases Proteins 0.000 description 1
- 102000035100 Threonine proteases Human genes 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- 101800004564 Transforming growth factor alpha Proteins 0.000 description 1
- 102400001320 Transforming growth factor alpha Human genes 0.000 description 1
- 101710152431 Trypsin-like protease Proteins 0.000 description 1
- 102100040247 Tumor necrosis factor Human genes 0.000 description 1
- 102100024598 Tumor necrosis factor ligand superfamily member 10 Human genes 0.000 description 1
- 102100022153 Tumor necrosis factor receptor superfamily member 4 Human genes 0.000 description 1
- 101710165473 Tumor necrosis factor receptor superfamily member 4 Proteins 0.000 description 1
- 102100033438 Tyrosine-protein kinase JAK1 Human genes 0.000 description 1
- 102100025387 Tyrosine-protein kinase JAK3 Human genes 0.000 description 1
- 108090000848 Ubiquitin Proteins 0.000 description 1
- 102000044159 Ubiquitin Human genes 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 238000002679 ablation Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 108090000350 actinidain Proteins 0.000 description 1
- 210000001789 adipocyte Anatomy 0.000 description 1
- 238000011467 adoptive cell therapy Methods 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000009824 affinity maturation Effects 0.000 description 1
- 230000008484 agonism Effects 0.000 description 1
- 108010054982 alanyl-leucyl-alanyl-leucine Proteins 0.000 description 1
- 125000003545 alkoxy group Chemical group 0.000 description 1
- 208000030961 allergic reaction Diseases 0.000 description 1
- 229920005603 alternating copolymer Polymers 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000001195 anabolic effect Effects 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 239000002870 angiogenesis inducing agent Substances 0.000 description 1
- ORWYRWWVDCYOMK-HBZPZAIKSA-N angiotensin I Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C1=CC=C(O)C=C1 ORWYRWWVDCYOMK-HBZPZAIKSA-N 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000002424 anti-apoptotic effect Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000005875 antibody response Effects 0.000 description 1
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 description 1
- 108010055066 asparaginylendopeptidase Proteins 0.000 description 1
- 229940009098 aspartate Drugs 0.000 description 1
- 208000037979 autoimmune inflammatory disease Diseases 0.000 description 1
- 201000004988 autoimmune vasculitis Diseases 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 229920001400 block copolymer Polymers 0.000 description 1
- 239000002981 blocking agent Substances 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 235000019835 bromelain Nutrition 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000010001 cellular homeostasis Effects 0.000 description 1
- 150000005829 chemical entities Chemical class 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 229940080701 chymosin Drugs 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 239000013256 coordination polymer Substances 0.000 description 1
- GICLSALZHXCILJ-UHFFFAOYSA-N ctk5a5089 Chemical compound NCC(O)=O.NCC(O)=O GICLSALZHXCILJ-UHFFFAOYSA-N 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 229960003067 cystine Drugs 0.000 description 1
- 230000001461 cytolytic effect Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 230000004041 dendritic cell maturation Effects 0.000 description 1
- 238000012938 design process Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000000032 diagnostic agent Substances 0.000 description 1
- 229940039227 diagnostic agent Drugs 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000003292 diminished effect Effects 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000002901 elastaselike Effects 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000003979 eosinophil Anatomy 0.000 description 1
- 231100000321 erythema Toxicity 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000005713 exacerbation Effects 0.000 description 1
- 230000003090 exacerbative effect Effects 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 210000001723 extracellular space Anatomy 0.000 description 1
- 229940012444 factor xiii Drugs 0.000 description 1
- 231100001264 fatal toxicity Toxicity 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 108010072257 fibroblast activation protein alpha Proteins 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 102000006815 folate receptor Human genes 0.000 description 1
- 108020005243 folate receptor Proteins 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 238000009650 gentamicin protection assay Methods 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 description 1
- 230000002489 hematologic effect Effects 0.000 description 1
- 230000002008 hemorrhagic effect Effects 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 231100000753 hepatic injury Toxicity 0.000 description 1
- 238000005734 heterodimerization reaction Methods 0.000 description 1
- 230000003284 homeostatic effect Effects 0.000 description 1
- 239000000710 homodimer Substances 0.000 description 1
- 229920001519 homopolymer Polymers 0.000 description 1
- 102000056003 human IL15 Human genes 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000036543 hypotension Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 230000005934 immune activation Effects 0.000 description 1
- 239000012651 immune agonist Substances 0.000 description 1
- 229940044680 immune agonist Drugs 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 239000002955 immunomodulating agent Substances 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 230000002584 immunomodulator Effects 0.000 description 1
- 230000004957 immunoregulator effect Effects 0.000 description 1
- 230000003308 immunostimulating effect Effects 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 230000001024 immunotherapeutic effect Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 108010080375 interferon kappa Proteins 0.000 description 1
- 108010045648 interferon omega 1 Proteins 0.000 description 1
- 229960001388 interferon-beta Drugs 0.000 description 1
- 229940076264 interleukin-3 Drugs 0.000 description 1
- 210000005025 intestinal intraepithelial lymphocyte Anatomy 0.000 description 1
- 230000004068 intracellular signaling Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 210000002510 keratinocyte Anatomy 0.000 description 1
- 201000010982 kidney cancer Diseases 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 125000001909 leucine group Chemical group [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 108020001756 ligand binding domains Proteins 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 150000002632 lipids Chemical group 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 231100000849 liver cell damage Toxicity 0.000 description 1
- 230000005923 long-lasting effect Effects 0.000 description 1
- 210000005265 lung cell Anatomy 0.000 description 1
- 210000003563 lymphoid tissue Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 108091007169 meprins Proteins 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 230000002025 microglial effect Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000000663 muscle cell Anatomy 0.000 description 1
- 201000000585 muscular atrophy Diseases 0.000 description 1
- 210000004985 myeloid-derived suppressor cell Anatomy 0.000 description 1
- 210000000651 myofibroblast Anatomy 0.000 description 1
- 210000001087 myotubule Anatomy 0.000 description 1
- GNOLWGAJQVLBSM-UHFFFAOYSA-N n,n,5,7-tetramethyl-1,2,3,4-tetrahydronaphthalen-1-amine Chemical compound C1=C(C)C=C2C(N(C)C)CCCC2=C1C GNOLWGAJQVLBSM-UHFFFAOYSA-N 0.000 description 1
- DAZSWUUAFHBCGE-KRWDZBQOSA-N n-[(2s)-3-methyl-1-oxo-1-pyrrolidin-1-ylbutan-2-yl]-3-phenylpropanamide Chemical compound N([C@@H](C(C)C)C(=O)N1CCCC1)C(=O)CCC1=CC=CC=C1 DAZSWUUAFHBCGE-KRWDZBQOSA-N 0.000 description 1
- OHDXDNUPVVYWOV-UHFFFAOYSA-N n-methyl-1-(2-naphthalen-1-ylsulfanylphenyl)methanamine Chemical compound CNCC1=CC=CC=C1SC1=CC=CC2=CC=CC=C12 OHDXDNUPVVYWOV-UHFFFAOYSA-N 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 210000003061 neural cell Anatomy 0.000 description 1
- 230000009438 off-target cleavage Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000005305 organ development Effects 0.000 description 1
- 108010068338 p38 Mitogen-Activated Protein Kinases Proteins 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 229940023041 peptide vaccine Drugs 0.000 description 1
- 229930192851 perforin Natural products 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 108010020708 plasmepsin Proteins 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920000232 polyglycine polymer Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical group 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 210000001948 pro-b lymphocyte Anatomy 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 229920005604 random copolymer Polymers 0.000 description 1
- 230000009711 regulatory function Effects 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 208000015347 renal cell adenocarcinoma Diseases 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 102200005671 rs1013940 Human genes 0.000 description 1
- 102220196890 rs1057519097 Human genes 0.000 description 1
- 102220227469 rs1064794097 Human genes 0.000 description 1
- 102200139755 rs11575292 Human genes 0.000 description 1
- 102220001328 rs121434258 Human genes 0.000 description 1
- 102220289727 rs1253463092 Human genes 0.000 description 1
- 102220277069 rs1553408375 Human genes 0.000 description 1
- 102220269398 rs1555403432 Human genes 0.000 description 1
- 102220164875 rs199968672 Human genes 0.000 description 1
- 102200082906 rs33932981 Human genes 0.000 description 1
- 102220044801 rs587781587 Human genes 0.000 description 1
- 102220044167 rs73334764 Human genes 0.000 description 1
- 102220328481 rs768061768 Human genes 0.000 description 1
- 102220060547 rs786203080 Human genes 0.000 description 1
- 102220020885 rs80356880 Human genes 0.000 description 1
- 102220097508 rs876658707 Human genes 0.000 description 1
- 102220114396 rs886039195 Human genes 0.000 description 1
- 102220119381 rs886042300 Human genes 0.000 description 1
- 102200061173 rs886054247 Human genes 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000007781 signaling event Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 210000000329 smooth muscle myocyte Anatomy 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 210000002536 stromal cell Anatomy 0.000 description 1
- 108091007196 stromelysin Proteins 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000007910 systemic administration Methods 0.000 description 1
- 102220482199 tRNA pseudouridine synthase A_K49E_mutation Human genes 0.000 description 1
- 238000011287 therapeutic dose Methods 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 108060008226 thioredoxin Proteins 0.000 description 1
- 229940094937 thioredoxin Drugs 0.000 description 1
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 206010043554 thrombocytopenia Diseases 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 231100000732 tissue residue Toxicity 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 208000030218 transient fever Diseases 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 239000013638 trimer Substances 0.000 description 1
- 239000000439 tumor marker Substances 0.000 description 1
- 108010072415 tumor necrosis factor precursor Proteins 0.000 description 1
- 230000001173 tumoral effect Effects 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 230000006444 vascular growth Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Images
Classifications
-
- 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
-
- 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
- C07K14/555—Interferons [IFN]
- C07K14/57—IFN-gamma
-
- 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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
- C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
-
- 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
- C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/22—Cysteine endopeptidases (3.4.22)
- C12Y304/22015—Cathepsin L (3.4.22.15)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/642—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
-
- 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
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
-
- 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
- C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/50—Fusion polypeptide containing protease site
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
Definitions
- Recombinant fusion proteins containing two or more functional polypeptides have utility in many fields including, for use in protein purification, imaging, as therapeutics, and drug delivery.
- protein drugs can be fused to Fc domains of antibodies or to carrier proteins (i.e., human serum albumin) for targeting, to extend their plasma half-lives and/or to achieve therapeutic effects. Chen et al., (2013), Adv Drug Deliv Rev. 65(10):1357-1369.
- linker sequences such as length, hydrophobicity, amino acid composition, secondary structures, and the overall folding can affect linker suitability and need to be taken into consideration in designing and selecting an appropriate linker.
- linkers that can be cleaved under selected conditions or in selected biological locations (e.g., in tumor microenvironment) to deliver active therapeutic agents (e.g., therapeutic polypeptides) can provide targeted pharmacological activity of the therapeutic agents and reduce unwanted systemic effects. This introduces further complexity into designing suitable linkers.
- novel separation moieties or linkers are disclosed herein.
- the separation moieties or linkers disclosed herein can be utilized, for example to specifically deliver prodrugs such as conditionally active and/or targeted cytokines to target sites where the linkers are processed to activate bioactivity.
- compositions and methods to generate and use high efficiency separation moieties and/or linkers can confer site-selectivity with regards to biological activity of the attached payload or payloads.
- the separation moieties and/or linkers are used in conjugation with therapeutic proteins to treat a disease or disorder, such as proliferative disease, a tumorous disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, graft-versus-host disease and the like.
- recombinant polypeptides comprising a separation moiety, wherein the separation moiety comprises an amino acid sequence is a substrate for an enzyme, specifically a protease.
- the protease can be selected from the group consisting of Fibroblast activation protein alpha (FAP ⁇ , also known as prolyl endopeptidase FAP), Cathepsin L (CTSL1), an ADAM selected from ADAM 8, ADAM 9, ADAM 10, ADAM12 ADAM17, and ADAMTS1, and an MMP selected from MMP1, MMP2, MMP9 or MMP14.
- the cleavable moiety can also be a substrate for a cathepsin, such as cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G, cathepsin K and/or cathepsin L.
- the cathepsin is Cathepsin L.
- the protease is MMP14 or cathepsin L.
- the cleavable moiety can comprise an amino acid sequence that is a substrate for at least two proteases.
- the separation moiety comprises two or more cleavable moieties, each of which is a substrate for a protease.
- the separation moiety can comprise a first cleavable moiety comprising a first amino acid sequence that is a substrate for a first protease and a second cleavable moiety comprising a second amino acid sequence that is a substrate for a second protease.
- the disclosure related to recombinant polypeptides that comprise a separation moiety that contains a protease cleavage motif as disclosed herein.
- the recombinant polypeptide can comprise a separation moiety that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 195-220, or an amino acid sequence that has at least 90% identity to SEQ ID NOs:195-220.
- Preferred separation moieties comprise the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198).
- the disclosure also relates to functional variants of separation moieties comprising SEQ IND NOs: 195-220.
- the functional variants of of SEQ ID NO: 195 can comprise any of SEQ ID NOS: 258-331.
- the functional variants of SEQ ID NO: 198 can comprise of SEQ ID NO:198 comprises SEQ ID NOS: 199 or any of SEQ ID NOS: 332-408.
- the separation moieties disclosed herein can comprise Formula I: [D1]-[L1]-[D2]. D1 is a first domain of interest.
- L1 is a separation moiety that connects or links D1 to D2, wherein the separation moiety comprises an amino acid sequence selected from SEQ ID NOs: 195-220 or an amino acid sequence that has at least about 90% identity to SEQ ID NOs:195-220.
- D2 is a second domain of interest.
- the recombinant polypeptide can further comprise a non-cleavable linker sequence.
- the recombinant polypeptide can include a therapeutic protein, such as a cytokine, chemokine, growth factor, soluble receptor, antigen-binding portion of an antibody (e.g., scFV, dAb) and the like.
- the recombinant polypeptide comprises a cytokine, chemokine, growth factor, a soluble receptor, or any combination thereof.
- the recombinant polypeptide comprises at least one of an extracellular domain, a transmembrane domain, and an intracellular domain.
- the recombinant polypeptide comprises a cell surface receptor, a chimeric antigen receptor (CAR), or a T Cell Receptor (TCR) subunit. In one embodiment, the recombinant polypeptide comprises an antigen-binding polypeptide, an antibody or an antigen-binding portion thereof.
- the cleavable moiety is cleaved with either (a) greater catalytic efficiency or (b) greater specificity or (c) both (a) and (b), by one or more proteases than a reference polypeptide sequence.
- the one or more proteases are selected from the group consisting of FAP ⁇ , CTSL1, an ADAM selected from ADAM 8, ADAM 9, ADAM 10, ADAM12 ADAM17, and ADAMTS1, and an MMP selected from MMP1, MMP2, MMP9 and MMP14.
- the one or more proteases can also include a protease selected from cathepsins, such as cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G, cathepsin K and/or cathepsin L.
- cathepsins such as cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G, cathepsin K and/or cathepsin L.
- the reference polypeptide sequence is present in a naturally occurring polypeptide substrate for FAP ⁇ , CTSL1, an ADAM selected from ADAM 8, ADAM 9, ADAM 10, ADAM12 ADAM17, and ADAMTS1, and an MMP selected from MMP1, MMP2, MMP9 and MMP14, a cathepsin, such as cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G, cathepsin K and/or cathepsin L or a combination thereof.
- the cleavable moiety is cleaved with reduced catalytic efficiency by one or more proteases than a reference polypeptide sequence.
- the cleavable moiety is cleaved with reduced catalytic efficiency by one or more serum proteases. In another embodiment, the cleavable moiety is cleaved with reduced catalytic efficiency by one or more hepatic proteases. In another embodiment, the cleavable moiety is cleaved with reduced catalytic efficiency by one or more Factor Xa, hepsin, or thrombin.
- the recombinant polypeptide comprises two or more separation moieties.
- the recombinant polypeptide is operably linked to a moiety selected from the group consisting of a polypeptide moiety, a lipid moiety, a nucleic acid moiety, a detectable moiety, and a small molecule.
- a recombinant pro-protein comprising: a recombinant polypeptide comprising a cleavable moiety that is a substrate for a protease, wherein the cleavable moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 195-220; and a polypeptide with biological activity, wherein the pro-protein has attenuated biological activity and wherein cleavage of the cleavable moiety by the protease produces a polypeptide with biological activity that is not attenuated.
- the polypeptide with biological activity comprises a cytokine, chemokine, growth factor, a soluble receptor or a combination thereof.
- the linkers are components of fusion proteins with therapeutic utility, and the linkers are not cleaved or are cleaved with low efficiency in the peripheral circulation but are cleaved with higher efficiency at a desired location in the body, such as a tumor microenvironment or site of inflammation.
- the polypeptide with biological activity comprises at least one of an extracellular domain, a transmembrane domain, and an intracellular domain.
- the polypeptide with biological activity comprises a cell surface receptor, a chimeric antigen receptor (CAR), or a T Cell Receptor (TCR) subunit.
- the polypeptide with biological activity comprises an antigen-binding polypeptide, an antibody or an antigen-binding portion thereof.
- the disclosure also relates to a fusion protein comprising: a. a signaling protein or molecule; and b. a blocking moiety selected from a steric blocking moiety, a specific blocking moiety, and the combination thereof; and c. a peptide linker that comprises a cleavable moiety, e.g., a cleavable moiety disclosed herein, having at least one protease-cleavable sequence.
- the fusion protein wherein (a) and (b) are operably linked by (c).
- the fusion protein, wherein the peptide linker comprises two or more copies of the same cleavable moiety.
- the fusion protein wherein the steric blocking moiety comprises human serum albumin (HSA) or an anti-HSA antibody.
- the fusion protein wherein the signaling protein is an interleukin-2 amino acid sequence comprising: (i) a non-native N terminus and/or (ii) a non-native C terminus.
- the fusion protein wherein the signaling protein is an interleukin-2 amino acid sequence.
- the fusion protein further comprising one or more half-life extension domains that are not also a specific blocker.
- the fusion protein wherein the non-native N and/or C termini are generated by circular permutation.
- the fusion polypeptide provided herein can a first polypeptide fusion partner linked to a ligand by a protease cleavable linker, wherein the cleavable linker has been optimized for catalytic efficiency, and wherein the ligand has been optionally modified, wherein the first polypeptide fusion partner is a blocking moiety which prevents binding of the modified ligand to a target receptor or a subunit of a target receptor until cleavage of the protease cleavable linker.
- the fusion polypeptide provided herein can also a fusion polypeptide comprising a first polypeptide fusion partner linked to a ligand by a protease cleavable linker, wherein the cleavable linker has been optimized for catalytic efficiency, and wherein the ligand has been optionally modified, including by circularly permutation to create a non-native N-terminus and a new C-terminus as compared to a native ligand, and wherein at least one of the new N-terminus or the new C-terminus of the modified ligand is operably linked to a first polypeptide fusion partner to form a fusion polypeptide wherein the first polypeptide fusion partner is a blocking moiety which prevents binding of the modified ligand to a target receptor or a subunit of a target receptor until cleavage of the protease cleavable linker.
- the first polypeptide fusion partner is selected from the group consisting of an antibody, an antibody fragment, and an albumin molecule.
- the first polypeptide fusion partner further comprising a second polypeptide fusion partner comprising a second blocking moiety.
- the second polypeptide fusion partner is a different kind of blocking moiety than the first polypeptide fusion partner.
- the first polypeptide fusion partner is albumin and the second polypeptide fusion partner is a domain comprising a complementary amino acid sequence that blocks activity of the cytokine.
- the first polypeptide fusion partner is a steric blocker, such as albumin
- the second polypeptide is a specific blocker, such as a cytokine receptor, portion of a cytokine receptor, a de novo affinity peptide specific for the cytokine, or an antibody or antibody fragment that specifically binds the cytokine of the fusion polypeptide.
- the second polypeptide fusion partner is the same kind of blocking moiety as the first polypeptide fusion partner.
- the fusion protein further comprises a tumor antigen binding component.
- the fusion protein further comprises a serum half-life extension domain.
- the ligand is selected from the group consisting of helix bundle proteins and cytokines (including, but not limited to, growth hormone, IL-2, IL-4, IL-5, IL-6, IL-10, IL-22, IL-23p19, IL-11, IL-13, IL-15, IL-12p35, IL-21, IL-30 (IL27p28), IL-34, IL-35, IL-35p35, IFN- ⁇ , IFN- ⁇ , IFN ⁇ , LIF, CNTF, oncostatin M, CLCF-1, GCSF, GM-CSF, EPO, ferritin, leptin, placental lactogen, prolactin, apolipoprotein e), b-trefoil proteins (including, but not limited to, IL-1 ⁇ , IL-1 ⁇ , IL
- the ligand comprises IL-2 polypeptide or a fragment or fragments thereof.
- the protease-cleavable linker polypeptide comprises a sequence that is capable of being cleaved by at least one protease selected from the group consisting of a kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin G, an elastase, a FAP, an ADAM selected from ADAM 8, ADAM 9, ADAM 10, ADAM12 ADAM17, and ADAMTS1, PR-3, granzyme M, a calpain, a matrix metalloproteinase (MMP), a plasminogen activator, a cathepsin, a caspase, a tryptase, and a tumor cell surface protease.
- the cytokine or fragment or mutein thereof is substantially dissociated from the cytokine blocking moiety after the proteas
- fusion polypeptide comprising at least one of each of: a cytokine polypeptide or functional fragment or mutein thereof [A]; a cytokine blocking moiety [B]; and an optimized protease-cleavable polypeptide linker [L]; wherein the blocking moiety is selected from the group consisting of an antibody, an antibody fragment, and an albumin, and wherein the cytokine comprises a circularly permuted cytokine.
- the fusion protein further comprises a tumor antigen binding component and/or a serum half-life extension domain.
- the fusion polypeptide wherein the cytokine peptide or functional fragment or mutein thereof is selected from the group consisting of helix bundle proteins and cytokines (including, but not limited to, growth hormone, IL-2, IL-4, IL-5, IL-6, IL-10, IL-22, IL-23p19, IL-11, IL-13, IL-15, IL-12p35, IL-21, IL-30 (IL27p28), IL-34, IL-35, IL-35p35, IFN- ⁇ , IFN ⁇ , LIF, CNTF, oncostatin M, CLCF-1, GCSF, GM-CSF, EPO, ferritin, leptin, placental lactogen, prolactin, apolipoprotein e), a FAP (e.g., Fap ⁇ ), an ADAM selected from ADAM 8, ADAM 9, ADAM 10, ADAM12 ADAM17, and ADAMTS1, b-trefoil proteins (e.g
- the cytokine peptide or functional fragment or mutein thereof comprises IL-2.
- the cytokine blocking moiety comprises a ligand binding domain or fragment or mutein of a cognate receptor for the cytokine, a single domain antibody or scFv that binds the cytokine polypeptide or functional fragment or mutein thereof, or an antibody or antibody fragment that binds a receptor of the cytokine.
- antibody is a single domain antibody or scFv.
- the blocking moiety extends the serum half-life of the cytokine or fragment thereof.
- fusion polypeptides comprising a protease cleavable moiety, wherein the sequence is catalytically optimized for cleavage by certain proteases and wherein protease cleavage renders the composition inducible in a tumor microenvironment.
- the fusion protein further comprises a biologically inactive polypeptide, wherein cleavage of the cleavable moiety by the protease converts the biologically inactive polypeptide to a biologically active polypeptide.
- the biologically inactive polypeptide comprises a cytokine, chemokine, growth factor, or soluble receptor.
- the biologically inactive polypeptide comprises at least one of an extracellular domain, a transmembrane domain, and an intracellular domain.
- the biologically inactive polypeptide comprises a cell surface receptor, a chimeric antigen receptor (CAR), or a T Cell Receptor (TCR) subunit.
- the biologically inactive polypeptide comprises an antigen-binding polypeptide, an antibody or an antigen-binding portion thereof.
- the disclosure further relates to a nucleic acid encoding any of the polypeptides disclosed herein, a vector comprising any of the nucleic acids encoding any of the polypeptides disclosed herein, and a host cell comprising said vector.
- Methods of making a pharmaceutical composition comprising culturing the host cell comprising a vector comprising a nucleic acid encoding any of the polypeptides disclosed herein, under suitable conditions for expression and collection of desired polypeptides are provided herein.
- Method of using any of the polypeptides disclosed herein comprising administering an effective amount of a pharmaceutical composition comprising such polypeptides to a subject in need thereof are described. For example, use for treating a subject with a disease or disorder disclosed herein.
- compositions comprising an effective amount of any of the recombinant polypeptides, any of the pro-proteins, any of the fusion proteins, any of the fusion polypeptides, any of the nucleic acids, any of the vectors, or any of the host cells comprising such vectors disclosed herein.
- FIG. 1 A is a schematic illustrating a protease-activated cytokine or chemokine that includes a blocking moiety.
- the blocking moiety may optionally function as a serum half-life extending domain.
- a cytokine is connected to a blocking moiety via a protease-cleavable linker, thus blocking its ability to bind to its receptor.
- the drawing shows that in an inflammatory or tumor environment a protease cleaves at a protease-cleavage site on the linker, releasing the blocking moiety and allowing the cytokine to bind to its receptor.
- FIG. 1 B is a schematic illustrating a protease-activated cytokine or chemokine wherein HSA (blocking moiety) is directly bound to the cytokine or chemokine of interest, with a protease cleavage site between the HSA and a cytokine or chemokine of interest.
- HSA blocking moiety
- FIG. 1 B shows that a cytokine is connected to a blocking moiety via a protease-cleavable linker, thus blocking its ability to bind to its receptor.
- the drawing shows that in an inflammatory or tumor environment, the protease cleaves at a protease-cleavage site on linker, releasing the blocking moiety and allowing the cytokine to bind to its receptor.
- FIG. 1 C is a schematic illustrating a protease-activated cytokine or chemokine wherein more than one HSA (blocking moiety) is bound directly to the molecule of interest.
- HSA blocking moiety
- one or more of the HSA can be bonded to the cytokine or chemokine through a linker, such as a linker that contains a protease cleavage site.
- linker such as a linker that contains a protease cleavage site.
- the drawing shows that a cytokine is connected to a blocking moiety via a protease-cleavable linker, thus blocking its ability to bind to its receptor.
- the drawing shows that in an inflammatory or tumor environment, protease cleaves at protease-cleavage site on linker, releasing the blocking moiety and allowing cytokine to bind receptor.
- the cytokine now has similar pK properties as compared to the native cytokine (e.g., has a short half-life).
- FIG. 1 D is a schematic illustrating a protease-activated cytokine or chemokine comprising more than one cytokine, of the same type or different type, each of which is bonded to a binding domain through a protease-cleavable linker.
- a cytokine is connected to a blocking moiety via a protease-cleavable linker, thus blocking its ability to bind to its receptor.
- the drawing shows that in an inflammatory or tumor environment a protease cleaves at a protease cleavage site on linker, releasing the blocking moiety and allowing the cytokine to bind to its receptor.
- FIG. 2 is a schematic illustrating a protease-activated cytokine or chemokine comprising a cytokine or chemokine polypeptide, a blocking moiety, and a serum half-life extending domain connected by at least one protease-cleavable linker.
- a cytokine is connected to a blocking moiety via protease-cleavable linkers, thus blocking its ability to bind to its receptor. It is also bound to a separate half-life extension element, which extends half-life in serum.
- the drawing shows that in an inflammatory or tumor environment a protease cleaves at a protease-cleavage site on linker, thus releasing the serum half-life extension element and the blocking moiety and allowing the cytokine to bind to its receptor.
- the cytokine now has similar pK properties as compared to the native cytokine (e.g., a short half-life).
- FIG. 3 is a schematic illustrating a protease-activated cytokine or chemokine comprising a cytokine or chemokine polypeptide, a blocking moiety, and a targeting domain connected by at least one protease-cleavable linker.
- a cytokine is connected to a blocking moiety and a targeting domain via a protease-cleavable linker, thus blocking its ability to bind to its receptor.
- the drawing shows that in an inflammatory or tumor microenvironment a protease cleaves at the protease cleavage site in the linker, releasing the targeting domain and the blocking moiety and allowing the cytokine to bind to its receptor.
- FIG. 4 A is a schematic illustrating a protease-activated cytokine or chemokine comprising a cytokine or chemokine polypeptide, a blocking moiety, a targeting domain, and a serum half-life extending domain connected by at least one protease-cleavable linker, wherein the cytokine polypeptide and the targeting domain are connected by a protease-cleavable linker.
- the drawing shows that a cytokine or chemokine is connected to targeting domain, blocking moiety, and half-life extension element via protease-cleavable linker(s), thus blocking its ability to bind to its receptor.
- the drawing shows that in an inflammatory or tumor environment, the protease cleaves at a protease-cleavage site on linker(s), releasing the half-life extension element, the targeting domain, and the blocking moiety, and allowing the cytokine to bind to its receptor.
- the cytokine now has similar pK properties as compared to the native cytokine (e.g., short half-life).
- FIG. 4 B is a schematic illustrating a protease-activated cytokine or chemokine comprising a cytokine or chemokine polypeptide, a blocking moiety, a targeting domain, and a serum half-life extending domain connected by at least one protease-cleavable linker.
- a cytokine is connected to targeting domain, a blocking moiety, and a half-life extension element via protease-cleavable linker(s), thus blocking its ability to bind to its receptor.
- the drawing shows that in an inflammatory or tumor environment, the protease cleaves at a protease-cleavage site on linker(s), releasing the half-life extension element and the blocking moiety and allowing the cytokine to bind to the receptor.
- the targeting moiety remains bound, keeping the cytokine in the tumor microenvironment.
- the cytokine now has similar pK properties as compared to the native cytokine (e.g., a short half-life).
- FIG. 5 depicts a graph showing that Linkers-2 (GPAGLYAQ, SEQ ID NO: 195) and Linkers-3 (ALFKSSFP, SEQ ID NO: 198) are minimally cleaved in lung, kidney, and liver cells.
- FIGS. 6 A- 6 B show graphs that polypeptides containing recombinant human IL-2 and the sequence for Linker-1 (GPAGMKGL, SEQ ID NO: 196), Linker-2 (GPAGLYAQ, SEQ ID NO: 195), or Linker-3 (ALFKSSFP, SEQ ID NO: 198) are not processed by healthy lung fibroblasts.
- FIGS. 7 A- 7 H is a series of graphs showing activity of exemplary IL-2 fusion proteins in IL-2 dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows results of the IL-2 proliferation assay as quantified by CellTiter-Glo® (Promega) luminescence-based cell viability assay. Each proliferation assay was performed with HSA ( FIGS. 7 B, 7 D, 7 F, 7 H ) or without ( FIGS. 7 A, 7 C, 7 E, 7 G ). Each fusion protein comprises an anti-HSA binder, and both uncleaved and MMP9 protease cleaved versions of the fusion protein were used in each assay.
- FIGS. 8 A- 8 F is a series of graphs showing activity of exemplary IL-2 fusion proteins in IL-2 dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows results of the IL-2 proliferation assay as quantified by CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease cleaved versions of the fusion protein were used in each assay.
- FIGS. 9 A- 9 Z is a series of graphs showing activity of exemplary IL-2 fusion proteins in IL-2 dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows results of the IL-2 proliferation assay as quantified by CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease cleaved versions of the fusion protein were used in each assay.
- FIG. 10 shows results of protein cleavage assay. Fusion protein ACP16 was run on an SDS-PAGE gel in both cleaved and uncleaved form. As can be seen in the gel, cleavage was complete.
- FIGS. 11 A- 11 B are a series of graphs depicting results from a HEK-Blue IL-12 reporter assay performed on human p40/murine p35 IL12 fusion proteins and recombinant human IL12 (Rec hIL-12). Analysis was performed based on quantification of Secreted Alkaline Phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). Results confirm that IL12 protein fusion proteins are active.
- SEAP Secreted Alkaline Phosphatase
- FIGS. 12 A- 12 F show a series of graphs depicting the results of HEK-blue assay of four IL-12 fusion proteins, before and after cleavage by MMP9. Analysis was performed based on quantification of Secreted Alkaline Phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). The data show greater activity in the cleaved IL12 than in the full fusion protein. Constructs tested were ACP06 ( FIG. 12 A ), ACP07 ( FIG. 12 C ), ACP08 ( FIG. 12 B ), ACP09 ( FIG. 12 D ), ACP10 ( FIG. 12 E ), ACP11 ( FIG. 12 F )
- FIG. 13 shows results of protein cleavage assay. Fusion protein ACP11 was run on an SDS-PAGE gel in both cleaved and uncleaved form. As can be seen in the gel, cleavage was complete.
- FIG. 14 is a schematic which depicts a non-limiting example of an inducible cytokine protein, wherein the construct is activated upon protease cleavage of a linker attached between two subunits of the cytokine.
- FIGS. 15 A- 15 D are graphs depicting results from a HEK-Blue assay performed on human p40/murine p35 IL12 fusion proteins and recombinant human IL12 (Rec hIL-12). Results confirm that IL12 protein fusion proteins are active. Each proliferation assay was performed with HSA or without HSA.
- FIGS. 16 A- 16 F are a series of graphs showing activity of exemplary IFN ⁇ fusion proteins compared to activity of mouse IFN ⁇ control using WEHI 279 cell survival assay. Each assay was performed with medium containing HSA (+HSA) or not containing HSA ( ⁇ HSA). Each fusion protein comprises an anti-HSA binder, and both uncleaved and MMP9 protease cleaved versions of the fusion protein were used in each assay.
- FIGS. 17 A- 17 F are a series of graphs showing activity of exemplary IFN ⁇ fusion proteins compared to activity of mouse IFN ⁇ control using B16 reporter assay. Each assay was performed with medium containing HSA (+HSA) or not containing HSA ( ⁇ HSA). Each fusion protein comprises an anti-HSA binder, and both uncleaved and MMP9 protease cleaved versions of the fusion protein were used in each assay.
- FIGS. 18 A- 18 B shows results of protein cleavage assay.
- FIGS. 19 A- 19 B are a series of graphs showing activity of exemplary IFN ⁇ fusion proteins compared to activity of mouse IFN ⁇ control using B16 reporter assay.
- Each assay was performed with culture medium containing HSA, and each fusion protein comprises an anti-HSA binder. Both uncleaved and MMP9 protease cleaved versions of the fusion protein were used in each assay.
- FIGS. 20 A- 20 B are a series of graphs showing activity of exemplary IFN ⁇ fusion proteins compared to activity of mouse IFNalphaA control using a B16 reporter assay.
- Each assay was performed with medium containing HSA, and each fusion protein comprises an anti-HSA binder. Both uncleaved and MMP9 protease cleaved versions of the fusion protein were used in each assay.
- FIGS. 21 A- 21 D are a series of graphs depicting the results of tumor growth studies using the MC38 cell line.
- FIG. 21 D shows the effect of intratumoral (IT) injection of IFN ⁇ and IL-2 on tumor growth.
- FIGS. 22 A- 22 B are a series of graphs showing activity of exemplary IFN ⁇ fusion proteins (ACP51 and ACP52) cleaved by MMP9 protease compared to activity of uncleaved fusion proteins using B16 reporter assay.
- Each fusion protein comprises an anti-HSA binder and a tumor targeting domain.
- FIGS. 23 A- 23 B are a series of graphs showing activity of exemplary IFN ⁇ fusion proteins (ACP53 and ACP54) cleaved by MMP9 protease compared to activity of uncleaved fusion proteins using B16 reporter assay.
- Each fusion protein comprises IFN ⁇ directly fused to albumin.
- FIGS. 24 A- 24 B are two graphs showing the stability of IL-2 fusion proteins containing Linker-1 (GPAGMKGL, SEQ ID NO: 196), Linker-2 (GPAGLYAQ, SEQ ID NO: 195), or Linker-3 (ALFKSSFP, SEQ ID NO: 198) in human serum in normal patient and a cancer patient.
- FIG. 24 A depicts the stability of the IL-2 fusion proteins containing Linker-1 (GPAGMKGL, SEQ ID NO: 196), Linker-2 (GPAGLYAQ, SEQ ID NO: 195), or Linker-3 (ALFKSSFP, SEQ ID NO: 198) at 24 hours.
- FIG. 24 A depicts the stability of the IL-2 fusion proteins containing Linker-1 (GPAGMKGL, SEQ ID NO: 196), Linker-2 (GPAGLYAQ, SEQ ID NO: 195), or Linker-3 (ALFKSSFP, SEQ ID NO: 198) at 24 hours.
- FIG. 24 A depicts
- 24 B depicts the stability of the IL-2 fusion proteins containing Linker-1 (GPAGMKGL, SEQ ID NO: 196), Linker-2 (GPAGLYAQ, SEQ ID NO: 195), or Linker-3 (ALFKSSFP, SEQ ID NO: 198) at 72 hours.
- Linker-1 GAAGMKGL, SEQ ID NO: 196
- Linker-2 GPAGLYAQ, SEQ ID NO: 195
- Linker-3 ALFKSSFP, SEQ ID NO: 198
- FIGS. 25 A and 25 B are two graphs showing analysis of ACP16 ( FIG. 25 A ) and ACP124 ( FIG. 25 B ) in a HEKBlue IL-2 reporter assay in the presence of HSA. Circles depict the activity of the uncut polypeptide, squares depict activity of the cut polypeptide, and triangles depict IL-2 alone as a control.
- FIG. 25 C is a graph showing results of a CTLL-2 proliferation assay.
- CTLL2 cells ATCC
- HSA human serum albumin
- FIGS. 26 A- 26 C are a series of graphs showing activity of fusion proteins in an HEKBlue IL-12 reporter assay.
- FIG. 26 A is a graph showing activity of cut and uncut ACP11 (a human p40/murine p35 IL12 fusion protein).
- FIG. 26 B is a graph showing analysis of ACP91 (a chimeric IL-12 fusion protein). Squares depict activity of the uncut ACP91 polypeptide, and triangles depict the activity of the cut polypeptide (ACP91+MMP9). EC50 values for each are shown in the table.
- FIG. 26 C is a graph showing analysis of ACP136 (a chimeric IL-12 fusion protein). Squares depict activity of the uncut ACP136 polypeptide, and triangles depict the activity of the cut polypeptide (ACP136+MMP9). EC50 values for each are shown in the table insert.
- FIGS. 27 A- 27 F are a series of graphs showing that cleaved IL-12 polypeptides are active in a HEKBlue IL2 reporter assay. Fusion proteins are evaluated both uncut (circles) and cut (squares) form, and wild type IL2 is used as a control +HSA for FIGS. 27 A-C ; ACP131 is used as a control (triangles) for FIGS. 27 D- 27 F . Shown are data for APC31+HSA ( FIG. 27 A ), ACP125+HSA ( FIG. 27 B ), ACP126+HSA ( FIG. 27 C ), ACP127 ( FIG. 27 D ), ACP128 ( FIG. 27 E ), and ACP129 ( FIG. 27 F ). The EC50 values for each are shown in the table below each graph.
- FIGS. 28 A- 28 N are a series of graphs depicting the activity of APC56 ( FIG. 28 A ), APC57 ( FIG. 28 B ) APC58 ( FIG. 28 C ), APC59 ( FIG. 28 D ), APC60 ( FIG. 28 E ), APC61+HSA ( FIG. 28 F ), ACP30+HSA ( FIG. 28 G ), ACP73 ( FIG. 28 H ), ACP70+HSA ( FIG. 28 I ), ACP71 ( FIG. 28 J ), ACP72 ( FIG. 28 K ), ACP 73 ( FIG. 28 L ), ACP74 ( FIG. 28 M ), and ACP75 ( FIG. 28 N ) in a HEKBlue IFN ⁇ reporter assay. Each fusion was tested for its activity when cut (squares) and uncut (circles). Analysis of murine IFN ⁇ is included in each graph as a comparator.
- FIGS. 29 A- 29 B is two graphs showing results of analyzing ACP31 (mouse IFN ⁇ 1 fusion protein) and ACP11 (a human p40/murine p35 IL12 fusion protein) in a tumor xenograft model.
- FIG. 29 A shows tumor volume over time in mice treated with 33 ⁇ g ACP31 (circles), 110 ⁇ g ACP31 (triangles), 330 ⁇ g ACP31 (diamonds), and as controls 1 ⁇ g murine wild type IFN ⁇ 1 (dashed line, squares) and 10 ⁇ g mIFN ⁇ 1 (dashed line, small circles). Vehicle alone is indicated by large open circles.
- FIG. 29 B shows tumor volume over time in mice treated with 17.5 ⁇ g ACP11 (squares), 175 ⁇ g ACP11 (triangles), 525 ⁇ g ACP11 (circles), and as controls 2 ⁇ g ACP04 (dashed line, triangles) and 10 ⁇ g ACP04 (dashed line, diamonds). Vehicle alone is indicated by large open circles.
- the data show tumor volume decreasing over time in a dose-dependent manner in mice treated with both ACP11 and ACP04 (a human p40/murine p35 IL12 fusion protein).
- FIGS. 30 A- 30 F are a series of spaghetti plots showing tumor volume over time in a mouse xenograft tumor model in mice each treated with vehicle alone ( FIG. 30 A ), 2 ⁇ g ACP04 ( FIG. 30 B ), 10 ⁇ g ACP04 ( FIG. 30 C ), 17.5 ⁇ g ACP11 ( FIG. 30 D ), 175 ⁇ g ACP11 ( FIG. 30 E ), and 525 ⁇ g ACP11 ( FIG. 30 F ). Each line represents a single mouse.
- FIGS. 31 A- 31 C depicts three graphs showing results of analyzing ACP16 and ACP124 in a tumor xenograft model.
- FIG. 31 A shows tumor volume over time in mice treated with 4.4 ⁇ g ACP16 (squares), 17 ⁇ g ACP16 (triangles), 70 ⁇ g ACP16 (downward triangles), 232 ⁇ g ACP16 (dark circles), and as a comparator 12 ⁇ g wild type IL-2 (dashed line, triangles) and 36 ⁇ g wild type IL-2 (dashed line, diamonds). Vehicle alone is indicated by large open circles.
- the data show tumor volume decreasing over time in a dose-dependent manner in mice treated with ACP16 at higher concentrations.
- FIG. 31 A shows tumor volume over time in mice treated with 4.4 ⁇ g ACP16 (squares), 17 ⁇ g ACP16 (triangles), 70 ⁇ g ACP16 (downward triangles), 232 ⁇ g ACP16 (dark circles), and as a comparator
- FIG. 31 B shows tumor volume over time in mice treated with 17 ⁇ g ACP124 (squares), 70 ⁇ g ACP124 (triangles), 230 ⁇ g ACP124 (downward triangles), and 700 ⁇ g ACP124 (diamonds). Vehicle alone is indicated by large open circles.
- FIG. 31 C shows tumor volume over time in mice treated with 17 ⁇ g ACP16 (dashed line, triangles), 70 ⁇ g ACP16 (circles), 232 ⁇ g ACP16 (dark circles), and as a comparator 17 ⁇ g ACP124 (downward triangles) 70 ⁇ g ACP124 (dashed line, diamonds), 230 ⁇ g ACP124 (dashed line, stars). Vehicle alone is indicated by dark downward triangles. The data show tumor volume decreasing over time in a dose-dependent manner in mice treated with ACP16, but not ACP124.
- FIGS. 32 A- 32 U are a series of spaghetti plots showing activity of fusion proteins in an MC38 mouse xenograft model. Each line in the plots is a single mouse.
- FIG. 33 is a graph showing tumor volume over time in a mouse xenograft model showing tumor growth in control mice (open circles) and AP16-treated mice (squares).
- FIGS. 34 A- 34 D are a series of survival plots showing survival of mice over time after treatment with cleavable fusion proteins.
- FIG. 34 A shows data for mice treated with vehicle alone (dotted line), 17 ⁇ g ACP16 (dark line), and 17 ⁇ g ACP124 (dashed line).
- FIG. 34 B shows data for mice treated with vehicle alone (dotted line), 70 ⁇ g ACP16 (dark line), and 70 ⁇ g ACP124 (dashed line).
- FIG. 34 C shows data for mice treated with vehicle alone (dotted line), 232 ⁇ g ACP16 (dark line), and 230 ⁇ g ACP124 (dashed line).
- FIG. 34 D shows data for mice treated with vehicle alone (dotted line), 232 ⁇ g ACP16 (dark line), and 700 ⁇ g ACP124 (dashed line).
- FIGS. 35 A- 35 M are a series of spaghetti plots showing activity of fusion proteins in an MC38 mouse xenograft model. All mouse groups were given four doses total except for the highest three doses of APC132, wherein fatal toxicity was detected after 1 week/2 doses.
- FIG. 35 A shows tumor volume over time for mice treated with vehicle.
- FIGS. 35 B- 3 E shows tumor volume over tume in mice treated with ACP16 at 17 ⁇ g/animal ( FIG. 35 B ), 55 ⁇ g/animal ( FIG. 35 C ), 70 ⁇ g/animal ( FIG. 35 C ), and 230 ⁇ g/animal 35E.
- FIGS. 35 A shows tumor volume over time for mice treated with vehicle.
- FIGS. 35 B- 3 E shows tumor volume over tume in mice treated with ACP16 at 17 ⁇ g/animal ( FIG. 35 B ), 55 ⁇ g/animal ( FIG. 35 C ), 70 ⁇ g/animal ( FIG. 35 C ),
- 35 F- 35 I shows tumor volume over time for mice treated with ACP132 at 9 ⁇ g/animal ( FIG. 35 F ), 28 ⁇ g/animal ( FIG. 35 G ). 36 ⁇ g/animal ( FIG. 35 H ), and 119 ⁇ g/animal ( FIG. 35 I ).
- FIGS. 35 J- 35 M shows tumor volume over time for mice treated with ACP21 at 13 ⁇ g/animal ( FIG. 35 J ), 42 ⁇ g/animal ( FIG. 35 K ), 54 ⁇ g/animal ( FIG. 35 L ), and 177 ⁇ g/animal ( FIG. 35 M ). Each line in the plots represents an individual animal.
- FIG. 36 is a schematic illustrating substrate cleavage activity in conditioned complete (+FBS) media by FRET endpoint assay across four cell lines. The ratio of tumor vs control activity was approximated by averaging the three tumor cell lines and comparing to the control myofibroblast cell line where signal was detectable.
- FIG. 36 discloses SEQ ID NOs: 201, 198, 197, 196 and 195, respectively, in order of appearance.
- FIG. 37 is a schematic illustrating ADAM17_2 substrate kinetics in cell culture conditioned media.
- FIG. 37 discloses SEQ ID NO: 235.
- FIG. 38 is a schematic illustrating FAP ⁇ _1 substrate kinetics in conditioned media.
- FIG. 38 discloses SEQ ID NO: 197.
- FIG. 39 is a schematic illustrating FAP ⁇ _1 substrate kinetics in cell lysates.
- FIG. 39 discloses SEQ ID NO: 197.
- FIG. 40 is a schematic illustrating MMP9_1 substrate kinetics in cell lysates.
- FIG. 40 discloses SEQ ID NO: 196.
- FIG. 41 is a schematic illustrating a substrate cleavage activity in cell lysates by FRET endpoint assay.
- FIG. 41 discloses SEQ ID NOS 198 and 197, respectively, in order of appearance.
- FIG. 42 is a schematic illustrating CTSL1_1 substrate kinetics in cell lysates.
- FIG. 42 discloses SEQ ID NO: 198.
- FIG. 43 is a schematic illustrating MMP14_1 substrate kinetics in cell lysates.
- FIG. 43 discloses SEQ ID NO: 195.
- FIG. 44 is a schematic illustrating calculated concentration of enzyme equivalents per cell culture-derived sample.
- FIG. 44 discloses SEQ ID NOS 201, 198, 197, 197, 196 and 195, respectively, in order of appearance.
- FIG. 45 is a schematic illustrating an enzyme progress curve for CTSL1 cleavage of CSTL1_2 vs CTSL1_1.
- FIG. 45 discloses SEQ ID NOS 198, 199 and 236, respectively, in order of appearance.
- FIG. 46 is a schematic illustrating 30-mer cleavage of CTSL1_1 (ALFKSSFP, SEQ ID NO: 198) vs CTSL1_2 (ALFFSSPP, SEQ ID NO: 199).
- FIG. 47 is a schematic illustrating susceptibility of the CTSL1 FRET substrates to CTSK cleavage. Rates of product formation were measured as a specific activity in units pmol min ⁇ 1 ⁇ g ⁇ 1 .
- the threshold value for the reference substrate, Z-LR-AMC is shown as a dashed line.
- FIG. 47 discloses SEQ ID NOS 198 and 199, respectively, in order of appearance.
- FIG. 48 is a schematic illustrating 30-mer Substrate Degradation by MMP9 Ranking of the substrates by relative rates of degradation are shown with “+”; uncleaved substrates are indicated as “ ⁇ ”.
- FIG. 48 discloses SEQ ID NOS 204, 205, 214, 216, 202, 217, 203, 211, 219, 207, 215, 212, 213, 206, 208, 209, 210, 218 and 220, respectively, in order of appearance.
- FIG. 49 is a schematic illustrating tandem MMP14_1 Motif degradation by MMP9. Top: substrate degradation traces, modeled with first-order kinetics. Bottom: product formation traces showing complex kinetics. FIG. 49 discloses SEQ ID NOS 202-205, respectively, in order of appearance.
- FIG. 50 is a schematic illustrating 30-mer Substrate Degradation by FAP ⁇ Ranking of the substrates by relative rates of degradation are shown with “+”; uncleaved substrates are indicated as “ ⁇ ”.
- FIG. 50 discloses SEQ ID NOS 205, 204, 206, 217, 203, 218, 219, 213, 216, 207, 214, 210, 202, 211, 208, 209, 212, 215 and 220, respectively, in order of appearance.
- FIG. 51 is a schematic illustrating 30-mer Substrate Degradation by CTSL1 Ranking of the substrates by relative rates of degradation are shown with “+”; uncleaved substrates are indicated as “ ⁇ ”.
- FIG. 51 discloses SEQ ID NOS 207, 208, 202, 218, 219, 212, 215, 217, 211, 209, 214, 206, 213, 210, 216, 203, 204, 205 and 220, respectively, in order of appearance.
- FIG. 52 is a schematic illustrating 30-mer Substrate Degradation by ADAM17 Ranking of the substrates by relative rates of degradation are shown with “+”; uncleaved substrates are indicated as “ ⁇ ”.
- FIG. 52 discloses SEQ ID NOS 208, 209, 211, 214, 217, 219, 213, 218, 215, 210, 212, 216, 207, 206, 202, 203, 204, 205 and 220, respectively, in order of appearance.
- FIG. 53 is a schematic illustrating 30-mer Substrate Degradation by Factor Xa Ranking of the substrates by relative rates of degradation are shown with “+”; uncleaved substrates are indicated as “ ⁇ ”.
- FIG. 53 discloses SEQ ID NOS 220, 206, 202, 214, 208, 209, 215, 210, 218, 217, 207, 213, 216, 211, 212, 219, 203, 204 and 205, respectively, in order of appearance.
- FIG. 54 is a schematic illustrating 30-mer Substrate Degradation by Thrombin Ranking of the substrates by relative rates of degradation are shown with “+”; uncleaved substrates are indicated as “ ⁇ ”.
- FIG. 54 discloses SEQ ID NOS 220, 204, 202, 207, 205, 211, 212, 215, 209, 218, 219, 217, 210, 213, 216, 214, 208, 206 and 203, respectively, in order of appearance.
- FIG. 55 is a schematic illustrating 30-mer Substrate Degradation by hepsin Ranking of the substrates by relative rates of degradation are shown with “+”; uncleaved substrates are indicated as “ ⁇ ”.
- FIG. 55 discloses SEQ ID NOS 220, 209, 216, 215, 210, 213, 206, 214, 212, 207, 217, 208, 211, 218, 219, 202, 203, 204 and 205, respectively, in order of appearance.
- FIGS. 56 A- 56 C show western blots probed with an IL-2 antibody demonstrating the stability of ACP16 ( FIG. 56 A ), ACP153 ( FIG. 56 B ), and ACP157 ( FIG. 56 C ) in 90% serum.
- FIG. 57 A- 57 D show western blots probed with an IL-2 antibody demonstrating the stability of ACP153, ACP155, ACP156, ACP16, and ACP372 in 90% serum.
- FIGS. 57 A- 57 B shows the result using serum from a human donor and FIGS. 57 C- 57 D shows the result using serum from a mouse donor.
- FIGS. 58 A- 58 D show a series of spaghetti plots showing activity of fusion proteins in an MC38 mouse xenograft model. Shown are vehicle alone ( FIG. 58 A , top), 17, 55, and 230 ⁇ g ACP16 ( FIG. 58 A ), 55 and 230 ⁇ g ACP153 ( FIG. 58 B ), 55 and 230 ⁇ g ACP155 ( FIG. 58 C ), and 55 and 230 ⁇ g ACP156 ( FIG. 58 D ). Each line in the plots represents an individual animal.
- FIG. 59 shows a graph depicting results from a STAT activation reporter assay performed on IL-2 fusion proteins and recombinant human IL2 (Rec hIL-2). Analysis was performed based on quantification of Secreted Alkaline Phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen).
- SEAP Secreted Alkaline Phosphatase
- FIG. 60 shows a graph depicting results from a STAT activation reporter assay performed on IL-2 fusion proteins and recombinant human IL2 (Rec hIL-2). Analysis was performed based on quantification of Secreted Alkaline Phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen).
- SEAP Secreted Alkaline Phosphatase
- FIG. 61 shows a graph depicting results from a STAT activation reporter assay performed on IL-2 fusion proteins and recombinant human IL2 (Rec hIL-2). Analysis was performed based on quantification of Secreted Alkaline Phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen).
- SEAP Secreted Alkaline Phosphatase
- FIG. 62 B shows a graph depicting results from a STAT activation reporter assay performed on IL-2 fusion proteins and recombinant human IL2 (Rec hIL-2). Analysis was performed based on quantification of Secreted Alkaline Phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen).
- FIG. 62 A shows an SDS-PAGE gel of construct cleavage by MMP9 protease.
- FIGS. 63 A- 63 B shows tables reporting engineered cleavage substrates described herein and the extent of cleavage observed using relevant proteases Flanking sequences are lower case, the first cleavable sequence is underlined, the second is in bold font, and the third cleavable sequence is in italics. In some cases, there is overlap between cleavable sequences, which are indicated accordingly.
- FIGS. 63 A- 63 B discloses SEQ ID NOS 202-220, respectively, in order of appearance.
- FIG. 64 is a schematic of an inducible tetravalent antibody format.
- FIGS. 65 A- 65 B show that multivalent 4-1BB antibodies are able to inducibly agonize 4-1BB.
- linkers are preferably protease cleavable and link a first amino acid sequence of interest (e.g. a first domain of interest) to a second amino acid sequence of interest (e.g. a second domain of interest).
- the disclosed separation moieties confer site-selectivity with regard to the action of the attached payload or payloads.
- the payload can be a therapeutic agent, a half-life extender, a blocking agent and the like, or any combination thereof.
- the separation moieties may be used to attach any payload of interest, including e.g. cytokines, antibodies, cell-based therapies, etc.
- the separation moieties may be used individually or be used in tandem, triplicate, quadruplicate, and so forth, as long as the separation moiety is smaller than about 100 amino acids. Individual separation moieties may be directly joined to each other, or may be interspersed with non-cleavable linkers, whichever promotes high efficiency and site-specificity.
- Cytokine is a well-known term of art that refers to any of a class of immunoregulatory proteins (such as interleukin or interferon) that are secreted by cells especially of the immune system and that are modulators of the immune system.
- immunoregulatory proteins such as interleukin or interferon
- Cytokine polypeptides that can be used in the fusion proteins disclosed herein include, but are not limited to transforming growth factors, such as TGF- ⁇ and TGF- ⁇ (e.g., TGFbeta1, TGFbeta2, TGFbeta3); interferons, such as interferon- ⁇ , interferon- ⁇ , interferon- ⁇ , interferon-kappa and interferon-omega; interleukins, such as IL-1, IL-1 ⁇ , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21 and IL-25; tumor necrosis factors, such as tumor necrosis factor alpha and lymphotoxin; transforming growth factor beta (TGFbeta) family proteins, chemokines (e.g., C—X—C motif
- Cytokines are well-known to have short serum half-lives that frequently are only a few minutes. Even forms of cytokines that have altered amino acid sequences intended to extend the serum half-life yet retain receptor agonist activity typically also have short serum half-lives.
- a “short-half-life cytokine” refers to a cytokine that has a substantially brief half-life circulating in the serum of a subject, such as a serum half-life that is less than 10, less than 15, less than 30, less than 60, less than 90, less than 120, less than 240, or less than 480 minutes.
- a short half-life cytokine includes cytokines which have not been modified in their sequence to achieve a longer than usual half-life in the body of a subject and polypeptides that have altered amino acid sequences intended to extend the serum half-life yet retain receptor agonist activity. This latter case is not meant to include the addition of heterologous protein domains, such as a bona fide half-life extension element, such as serum albumin.
- a “conservative” amino acid substitution generally refers to substitution of one amino acid residue with another amino acid residue from within a recognized group which can change the structure of the peptide but biological activity of the peptide is substantially retained.
- Conservative substitutions of amino acids are known to those skilled in the art. Conservative substitutions of amino acids can include, but not limited to, substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.
- Sortases are transpeptidases that modify proteins by recognizing and cleaving a carboxyl-terminal sorting signal embedded in or terminally attached to a target protein or peptide.
- Sortase A catalyzes the cleavage of the LPXTG motif (where X is any standard amino acid) (SEQ ID NO: 237) between the Thr and Gly residue on the target protein, with transient attachment of the Thr residue to the active site Cys residue on the enzyme, forming an enzyme-thioacyl intermediate.
- a biomolecule with an N-terminal nucleophilic group typically an oligoglycine motif, attacks the intermediate, displacing Sortase A and joining the two molecules.
- steric blocker refers to a polypeptide or polypeptide moiety that can be covalently bonded to a cytokine polypeptide directly or indirectly through other moieties such as linkers, for example in the form of a chimeric polypeptide (fusion protein), but otherwise does not covalently bond to the cytokine polypeptide.
- a steric blocker can non-covalently bond to the cytokine polypeptide, for example though electrostatic, hydrophobic, ionic or hydrogen bonding.
- a steric blocker typically inhibits or blocks the activity of the cytokine moiety due to its proximity to the cytokine moiety and comparative size.
- a “half-life extension element” is a part of the chimeric polypeptide that increases the serum half-life and improve pK, for example, by altering its size (e.g., to be above the kidney filtration cutoff), shape, hydrodynamic radius, charge, or parameters of absorption, biodistribution, metabolism, and elimination.
- separation moiety refers to an amino acid sequence typically less than about 100 amino acids that connects or links a first amino acid sequence of interest (e.g., an amino acid sequence that folds to form a first protein domain) to a second amino acid sequence of interest (e.g., an amino acid sequence that folds to form a second protein domain) in a contiguous polypeptide chain.
- the separation moiety or linker typically include one or more protease cleavage sites and thus is protease cleavable.
- a “tandem linker” refers to a linker that comprises two or more protease cleavages sites which can be cleaved by the same or different proteases, and which can be arranged in any desired orientation, such as one cleavage site adjacent to another cleavage site, one cleavage site overlapping another cleavage site, one cleavage site following by another cleavage site with intervening amino acids between the two cleavage sites.
- the terms “activatable,” “activate,” “induce,” and “inducible” refer to the ability of a protein, i.e. a cytokine, that is part of a conjugate, to bind its receptor and effectuate activity upon cleavage of additional elements from the conjugate.
- Plasmids or “viral vectors” are agents that transport the disclosed nucleic acids into the cell without degradation and include a promoter yielding expression of the nucleic acid molecule and/or polypeptide in the cells into which it is delivered.
- peptide As used herein, the terms “peptide”, “polypeptide”, or “protein” are used broadly to mean two or more amino acids linked by a peptide bond. Protein, peptide, and polypeptide are also used herein interchangeably to refer to amino acid sequences. It should be recognized that the term polypeptide is not used herein to suggest a particular size or number of amino acids comprising the molecule and that a peptide of the invention can contain up to several amino acid residues or more.
- subject can be a vertebrate, more specifically a mammal (e g a human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig), birds, reptiles, amphibians, fish, and any other animal.
- a mammal e g a human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig
- the term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered.
- patient or “subject” may be used interchangeably and can refer to a subject with a disease or disorder (e.g. cancer).
- a disease or disorder e.g. cancer
- patient or subject includes human and veterinary subjects.
- treatment refers to a method of reducing the effects of a disease or condition or symptom of the disease or condition.
- treatment can refer to at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or substantially complete reduction in the severity of an established disease or condition or symptom of the disease or condition.
- a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject as compared to a control.
- the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels.
- treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition. 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.
- the terms “prevent”, “preventing”, and “prevention” of a disease or disorder refers to an action, for example, administration of the chimeric polypeptide or nucleic acid sequence encoding the chimeric polypeptide, that occurs before or at about the same time a subject begins to show one or more symptoms of the disease or disorder, which inhibits or delays onset or exacerbation of one or more symptoms of the disease or disorder.
- references to “decreasing”, “reducing”, or “inhibiting” include a change of at least about 10%, of at least about 20%, of at least about 30%, of at least about 40%, of at least about 50%, of at least about 60%, of at least about 70%, of at least about 80%, of at least about 90% or greater as compared to a suitable control level.
- Such terms can include but do not necessarily include complete elimination of a function or property, such as agonist activity.
- an “attenuated cytokine receptor agonist” is a cytokine receptor agonist that has decreased receptor agonist activity as compared to the cytokine receptor's naturally occurring agonist.
- An attenuated cytokine agonist may have at least about 10 ⁇ , at least about 50 ⁇ , at least about 100 ⁇ , at least about 250 ⁇ , at least about 500 ⁇ , at least about 1000 ⁇ or less agonist activity as compared to the receptor's naturally occurring agonist.
- a fusion protein that contains a cytokine polypeptide as described herein is described as “attenuated” or having “attenuated activity”, it is meant that the fusion protein is an attenuated cytokine receptor agonist.
- an “intact fusion protein” is a fusion protein in which no domain has been removed from the fusion protein, for example by protease cleavage.
- a domain may be removable by protease cleavage or other enzymatic activity, but when the fusion protein is “intact”, this has not occurred.
- moiety refers to a portion of a molecule that has a distinct function within that molecule, and that function may be performed by that moiety in the context of another molecule.
- a moiety may be a chemical entity with a particular function, or a portion of a biological molecule with a particular function.
- a “blocking moiety” within a fusion protein is a portion of the fusion protein which is capable of blocking the activity of some or all of the fusion polypeptide. This may be a protein domain, such as serum albumin.
- the disclosure relates to novel protease cleavable separation moieties.
- the protease cleavable separation moieties were designed so that the separation moieties are cleaved with high efficiency by proteases at a desired location (e.g., proteases that are selectively expressed or expressed at high levels in the tumor microenvironment) but are stable and not cleaved or cleaved with low efficiency in other locations (e.g., in the periphery, for example healthy tissue or serum).
- the protease cleavable separation moieties were designed using a process that included prioritizing proteases that would be suitable for cleaving the separation moieties based on expression in target indications, such as, expression in particular types of tumors (e.g., colon, lung, breast, melanoma). Multiple data sources for increased expression or specific expression of proteases in the target indications were used, including mRNA, proteomics and tissue staining data. The proteases were also prioritized based on their specific activity as well as intrinsic specificity, with high specific activity and high intrinsic activity preferred.
- target indications such as, expression in particular types of tumors (e.g., colon, lung, breast, melanoma).
- Multiple data sources for increased expression or specific expression of proteases in the target indications were used, including mRNA, proteomics and tissue staining data.
- the proteases were also prioritized based on their specific activity as well as intrinsic specificity, with high specific activity and high intrinsic activity preferred.
- Stability in the serum is an important design consideration, and to avoid potential off-target cleavage of the separation moieties by serum proteases, proteases that are not dependent on arginine in their substrate were selected, since many off-target enzymes are active towards arginine residues.
- This process yielded peptides containing sequence motifs that are cleaved by certain tumor-associated proteases (e.g., Matrix Metaloprotease 9 (MMP9), MMP14 and/or Cathepsin L) with high efficiency, but are stable (not cleaved or cleaved with low efficiency) in the serum or normal healthy tissue (e.g., by thrombin, Factor Xa, hepsin and the like).
- MMP9 Matrix Metaloprotease 9
- MMP14 Matrix Metaloprotease 9
- Cathepsin L e.g., Cathepsin L
- the separation moieties disclosed herein are efficient cleavage by human tumors and minimal cleavage by normal tissues or serum.
- This disclosure relates to separation moieties or linkers that connect a first amino acid sequence of interest (e.g. a first domain of interest) to a second amino acid sequence of interest (e.g. a second domain of interest).
- first amino acid sequence of interest and the second amino acid sequence of interest are not found together in a naturally occurring protein.
- the separation moiety can connect or link a first domain of interest to a second domain of interest in a fusion protein.
- the separation moiety is an amino acid sequence that can be of any suitable length, and preferably can be cleaved by a protease.
- the separation moieties disclosed herein can confer functionality, including flexibility as well as the ability to be cleaved. Flexible linkers are usually applied when joined domains requires a certain degree of movement or interaction. Cleavable linkers are introduced to release free and functional domains in vivo at a target site.
- the separation moieties disclosed herein serve to connect at least two domains of interest.
- the separation moieties can maintain cooperative inter-domain interactions or preserving biological activity.
- the separation moieties can join functional domains (e.g., a payload and half-life extension element) that are released from the separation moiety at a target site (e.g. a tumor microenvironment).
- the separation moiety is cleavable by a cleaving agent, e.g., an enzyme.
- the separation moiety comprises a protease cleavage site.
- the separation moiety comprises one or more cleavage sites.
- the separation moiety can comprise a single protease cleavage site.
- the separation moiety can also comprise 2 or more protease cleavage sites. For example, 2 cleavage sites, 3 cleavage sites, 4, cleavage sites, 5 cleavage sites, or more.
- the separation moiety comprises 2 or more protease cleavage sites
- the cleavage sites can be cleaved by the same protease or different proteases.
- a separation moiety comprising two or more cleavage sites is referred to as a “tandem linker.”
- the two or more cleavage sites can be arranged in any desired orientation, including, but not limited tom one cleavage site adjacent to another cleavage site, one cleavage site overlapping another cleavage site, or one cleavage site following by another cleavage site with intervening amino acids between the two cleavage sites.
- protease-cleavable linkers are disease specific protease-cleavable linkers. Also preferred are protease-cleavable linkers that are preferentially cleaved at a desired location in the body, such as the tumor microenvironment, relative to the peripheral circulation.
- the rate at which the protease-cleavable linker is cleaved in the tumor microenvironment can be at least about 10 times, at least about 100 times, at least about 1000 times or at least about 10,000 times faster in the desired location in the body, e.g., the tumor microenvironment, in comparison to in the peripheral circulation (e.g., in plasma).
- Proteases known to be associated with diseased cells or tissues include but are not limited to serine proteases, cysteine proteases, aspartate proteases, threonine proteases, glutamic acid proteases, metalloproteases, asparagine peptide lyases, serum proteases, cathepsins, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin G, Cathepsin K, Cathepsin L, kallikreins, hK1, hK10, hK15, plasmin, collagenase, Type IV collagenase, stromelysin, Factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidain, bromelain, calpain, caspase
- Proteases capable of cleaving linker amino acid sequences can, for example, be selected from the group consisting of a prostate specific antigen (PSA), a matrix metalloproteinase (MMP), an A Disintigrin and a Metalloproteinase (ADAM), a plasminogen activator, a cathepsin, a caspase, a tumor cell surface protease, and an elastase.
- the MMP can, for example, be matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), matrix metalloproteinase 14 (MMP14).
- the linker can be cleaved by a cathepsin, such as, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin G, Cathepsin K and/or Cathepsin L.
- a cathepsin such as, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin G, Cathepsin K and/or Cathepsin L.
- the linker can be cleaved by MMP14 or Cathepsin L.
- Granzyme B Cleaves after Asp Type of serine protease; strongly residues (asp-ase) implicated in inducing perforin-dependent target cell apoptosis
- Granzyme A grA
- grH Granzyme H
- grH Unknown substrate Type of serine protease
- specificity granzymes are also secreted by killer T cells, but not all are present in humans
- Protease Cleavage Domain Sequence SEQ ID NO: MMP7 KRALGLPG 3 MMP7 (DE) 8 RPLALWRS(DR) 8 4 MMP9 PR(S/T)(L/I)(S/T) 5 MMP9 LEATA 6 MMP11 GGAANLVRGG 7 MMP14 SGRIGFLRTA 8 MMP PLGLAG 9 MMP PLGLAX 10 MMP PLGC(me)AG 11 MMP ESPAYYTA 12 MMP RLQLKL 13 MMP RLQLKAC 14 MMP2, MMP9, MMP14 EP(Cit)G(Hof)YL 15 Urokinase plasminogen activator (uPA) SGRSA 16 Urokinase plasminogen activator (uPA) DAFK 17 Urokinase plasminogen activator (uPA) GGGRR 18 Lysosomal Enzyme GFLG 19 Lysosomal Enzyme
- Exemplary protease linkers include, but are not limited to kallikrein cleavable linkers, thrombin cleavable linkers, chymase cleavable linkers, carboxypeptidase A cleavable linkers, cathepsin cleavable linkers, elastase cleavable linkers, FAP cleavable linkers, ADAM cleavable linkers, PR-3 cleavable linkers, granzyme M cleavable linkers, a calpain cleavable linkers, a matrix metalloproteinase (MMP) cleavable linkers, a plasminogen activator cleavable linkers, a caspase cleavable linkers, a tryptase cleavable linkers, or a tumor cell surface protease.
- MMP matrix metalloproteinase
- MMP9 cleavable linkers Specifically, MMP9 cleavable linkers, ADAM cleavable linkers, CTSL1 cleavable linkers, FAP ⁇ cleavable linkers, and cathepsin cleavable linkers.
- Some preferred protease-cleavable linkers are cleaved by a MMP and/or a cathepsin.
- the separation moieties disclosed herein are typically less than 100 amino acids. Such separation moieties can be of different lengths, such as from 1 amino acid (e.g., Gly) to 30 amino acids, from 1 amino acid to 40 amino acids, from 1 amino acid to 50 amino acids, from 1 amino acid to 60 amino acids, from 1 to 70 amino acids, from 1 to 80 amino acids, from 1 to 90 amino acids, and from 1 to 100 amino acids.
- the linker is at least about 1, about 2, about 3, about 4, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 amino acids in length.
- Preferred linkers are typically from about 5 amino acids to about 30 amino acids.
- the lengths of linkers vary from 2 to 30 amino acids, optimized for each condition so that the linker does not impose any constraints on the conformation or interactions of the linked domains.
- the separation moiety comprises the sequence GPAGLYAQ (SEQ ID NO: 195); GPAGMKGL (SEQ ID NO: 196); PGGPAGIG (SEQ ID NO: 197); ALFKSSFP (SEQ ID NO: 198); ALFFSSPP (SEQ ID NO: 199); LAQRLRSS (SEQ ID NO: 200); LAQKLKSS (SEQ ID NO; 201); GALFKSSFPSGGGPAGLYAQGGSGKGGSGK (SEQ ID NO: 202); RGSGGGPAGLYAQGSGGGPAGLYAQGGSGK (SEQ ID NO: 203); KGGGPAGLYAQGPAGLYAQGPAGLYAQGSR (SEQ ID NO: 204); RGGPAGLYAQGGPAGLYAQGGGPAGLYAQK (SEQ ID NO: 205); KGGALFKSSFPGGPAGIGPLAQKLKSSGGS (SEQ ID NO: 206); SGGPGGPAGIGALFKSSFPLAQKLKSSGGG (SEQ ID NO:
- Certain preferred separation moieties comprises the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198).
- the separation moieties disclosed herein can comprise one or more cleavage motif or functional variants that are the same or different.
- the separation moieties can comprise 1, 2, 3, 4, 5, or more cleavage motifs or functional variants.
- Separation moieties comprising 30 amino acids can contain 2 cleavage motifs or functional variants, 3 cleavage motifs or functional variants or more.
- a “functional variant” of a separation moiety retains the ability to be cleaved with high efficiency at a target site (e.g., a tumor microenvironment that expresses high levels of the protease) and are not cleaved or cleaved with low efficiency in the periphery (e.g., serum).
- a target site e.g., a tumor microenvironment that expresses high levels of the protease
- the functional variants retain at least about 50%, about 55%, about 60%, about 70%, about 80%, about 85%, about 95% or more of the cleavage efficiency of a separation moiety comprising any one of SEQ ID NOs. 195-220.
- the separation moieties comprising more than one cleavage motif can be selected from SEQ ID NOs: 195-201 and combinations thereof.
- Preferred separation moieties comprising more than one cleavage motif comprise the amino acids selected from SEQ ID NO: 202-220.
- the separation moiety can comprise both ALFKSSFP (SEQ ID NO: 198) and GPAGLYAQ (SEQ ID NO: 195).
- the separation moiety can comprise two cleavage motifs that each have the sequence GPAGLYAQ (SEQ ID NO: 195).
- the separation moiety can comprise two cleavage motifs that each have the sequence ALFKSSFP (SEQ ID NO: 198).
- the separation moiety can comprise a third cleavage motif that is the same or different.
- the separation moiety comprises an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99% identical to SEQ ID NOs: 195 to SEQ ID NO: 220 over the full length of SEQ ID NO: 195-220.
- the functional variants can contain at least one or more amino acid substitutions, deletions, or insertions relative to the separation moieties comprising SEQ ID NOs. 195-220.
- the functional variant can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations comparted to the separation moieties comprising SEQ ID NOs. 195-220.
- the functional variant differs from the separation moiety comprising SEQ ID NOs. 195-220 by less than 10, less, than 8, less than 5, less than 4, less than 3, less than 2, or one amino acid alterations, e.g., amino acid substitutions or deletions.
- the functional variant may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to SEQ ID NOs. 195-220.
- the amino acid substitution can be a conservative substitution or a non-conservative substitution, but preferably is a conservative substitution.
- the functional variants of the separation moieties may comprise 1, 2, 3, 4, or 5 or more non-conservative amino acid substitutions compared the separation moieties comprising SEQ ID NOs: 195-220.
- Non-conservative amino acid substitutions could be recognized by one of skill in the art.
- the functional variant of the separation moiety preferably contains no more than 1, 2, 3, 4, or 5 amino acid deletions.
- separation moieties comprising 8 amino acid protease substrates (e.g., SEQ ID Nos: 195-201) contain amino acid at positions P4, P3, P2, P1, P1′, P2′, P3′, P4′, wherein the sissile bond is between P1 and P1′.
- amino acid positions for the separation moiety comprising the sequence GPAGLYAQ (SEQ ID NO: 195) can be described as follows:
- Amino acids positions for the separation moiety comprising the sequence ALFKSSFP (SEQ ID NO: 198) can be described as follows:
- amino acids surrounding the cleavage site e.g., positions P1 and P1′ for SEQ ID NOs: 195-201 are not substituted.
- the separation moiety comprises the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) or a functional variant of SEQ ID NO: 195 or a function variant of SEQ ID NO: 198.
- a functional variant of PAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) can comprise one or more amino acid substitutions, and substantially retain their ability to be cleaved by a protease.
- the functional variants of GPAGLYAQ (SEQ ID NO: 195) is cleaved by MMP14, and the functional variant of ALFKSSFP (SEQ ID NO: 198) is cleaved by Capthepsin L (CTSL1).
- the functional variants also retain their ability to be cleaved with high efficiency at a target site (e.g., a tumor microenvironment that expresses high levels of the protease).
- the functional variants of GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) retain at least about 50%, about 55%, about 60%, about 70%, about 80%, about 85%, about 95% or more of the cleavage efficiency of a separation moiety comprising amino acid sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198), respectively.
- the functional variant of GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) comprise no more than 1, 2, 3, 4, or 5 conservative amino acid substitutions compared to GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198).
- the amino acids at position P1 and P1′ are not substituted.
- the amino acids at positions P1 and P1′ in SEQ ID NO: 195 are G and L
- the amino acids at positions P1 and P1′ in SEQ ID NO: 198 are K and S.
- the functional variant of GPAGLYAQ can preferably comprise one or more of the following: a) an arginine amino acid substitution at position P4, b) a leucine, valine, asparagine, or proline amino acid substitution at position P3, c) a asparagine amino acid substitution at position P2, d) a histidine, asparagine, or glycine amino acid substitution at position P1, e) a asparagine, isoleucine, or leucine amino acid substitution at position P1′, f) a tyrosine or arginine amino acid substitution at position P2′, g) a glycine, arginine, or alanine amino acid substitution at position P3′, h) or a serine, glutamine, or lysine amino acid substitution at position P4′.
- GPAGLYAQ The following amino acid substitutions are disfavored in functional variants of GPAGLYAQ (SEQ ID NO: 195): a) arginine or isoleucine at position P3, b) alanine at position P2, c) valine at position P1, d) arginine, glycine, asparagine, or threonine at position P1′, e) aspartic acid or glutamic acid at position P2′, f) isoleucine at position P3′, g) valine at position P4′.
- the functional variant of GPAGLYAQ does not comprise an amino acid substitution at position P1 and/or P1′.
- the amino acid substitution of the functional variant of GPAGLYAQ preferably comprises an amino acid substitution at position P4 and/or P4′.
- the functional variant of GPAGLYAQ (SEQ ID NO: 195) can comprises a leucine at position P4, or serine, glutamine, lysine, or phenylalanine at position P4.
- the functional variant of GPAGLYAQ (SEQ ID NO: 195) can comprises a glycine, phenylalanine, or a proline at position P4′.
- amino acid substitutions at position P2 or P2′ of GPAGLYAQ are not preferred.
- the functional variants of LFKSSFP preferably comprises hydrophobic amino acid substitutions.
- the functional variant of LFKSSFP can preferably comprise one or more of the following: (a) lysine, histidine, serine, glutamine, leucine, proline, or phenylalanine at position P4; (b) lysine, histidine, glycine, proline, asparagine, phenylalanine at position P3; (c) arginine, leucine, alanine, glutamine, or histatine at position P2; (d) phenylalanine, histidine, threonine, alanine, or glutamine at position P1; (e) histidine, leucine, lysine, alanine, isoleucine, arginine, phenylalanine, asparagine, glutamic acid, or glycine at position P1′,
- aspartic acid and/or glutamic acid are generally disfavored and avoided.
- the following amino acid substitutions are also disfavored in functional variants of LFKSSFP (SEQ ID NO: 198): (a) alanine, serine, or glutamic acid at position P3; (b) proline, threonine, glycine, or aspartic acid at position P2; (c) proline at position P1; (d) proline at position P1′; (e) glycine at position P2′; (f) lysine or glutamic acid at position P3′; (g) aspartic acid at position P4′.
- the amino acid substitution of the functional variant of LFKSSFP preferably comprises an amino acid substitution at position P4 and/or P1. In some embodiments, an amino acid substitution of the functional variant of LFKSSFP (SEQ ID NO: 198) at position P4′ is not preferred.
- the functional variant of LFKSSFP comprises the amino acid sequence selected from SEQ ID NOs: 332-408.
- Specific functional variants of LFKSSFP include ALFFSSPP (SEQ ID NO: 199), ALFKSFPP (SEQ ID NO: 381), ALFKSLPP (SEQ ID NO: 382); ALFKHSPP (SEQ ID NO: 370); ALFKSIPP (SEQ ID NO: 383); ALFKSSLP (SEQ ID NO: 390); or SPFRSSRQ (SEQ ID NO: 333).
- the separation moieties disclosed herein can form a stable complex under physiological conditions with the amino acid sequences (e.g. domains) that they link, while being capable of being cleaved by a protease.
- the separation moiety is stable (e.g., not cleaved or cleaved with low efficiency) in the circulation and cleaved with higher efficiency at a target site (i.e. a tumor microenvironment).
- fusion polypeptides that include the linkers disclosed herein can, if desired, have a prolonged circulation half-life and/or lower biological activity in the circulation in comparison to the components of the fusion polypeptide as separate molecular entities.
- the linkers when in the desired location (e.g., tumor microenvironment) the linkers can be efficiently cleaved to release the components that are joined together by the linker and restoring or nearly restoring the half-life and biological activity of the components as separate molecular entities.
- the separation moiety desirably remains stable in the circulation for at least 2 hours, at least 5, hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 24 hours, at least 30 hours, at least 35 hours, at least 40 hours, at least 45 hours, at least 50 hours, at least 60 hours, at least 65 hours, at least 70 hours, at least 80 hours, at least 90 hours, or longer.
- the separation moiety is cleaved by less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 20%, 5%, or 1% in the circulation as compared to the target location.
- the separation moiety is also stable in the absence of an enzyme capable of cleaving the linker. However, upon expose to a suitable enzyme (i.e., a protease), the separation moiety is cleaved resulting in separation of the linked domain.
- the separation moieties disclosed herein can be used in a wide range of applications. Without limitation, they are suitable for us in fusion proteins. As further described herein, the separation moieties are particularly useful for preparing therapeutic fusion proteins in which the therapeutic of biological activity of the fusion protein is attenuated and the attenuation is removed upon cleavage of the separation moiety.
- the separation moieties can also be used to conjugate a variety of payloads such as therapeutic and/or diagnostic agents to carriers or targeting agents (e.g., antibodies and fragments of antibodies, nanoparticles). Suitable methods for prepare such conjugates are well-known in the art, see for example, Bioconjugate Techniques, Third Ed., G. T. Hermanson (Ed.) Academic Press 2013.
- Exemplary payloads include, but are not limited to cytokines, antibodies, cell based-therapies, antibiotics, cytotoxic drugs, or other recombinant polypeptide complexes.
- separation moieties that are suitable for use in conjugation with payloads that target or are targeted to tumor microenvironments.
- This disclosure relates recombinant polypeptides in which a separation moiety as disclosed herein links a first amino acid sequence of interest (e.g. a first domain of interest) to a second amino acid sequence of interest (e.g. a second domain of interest).
- a separation moiety as disclosed herein links a first amino acid sequence of interest (e.g. a first domain of interest) to a second amino acid sequence of interest (e.g. a second domain of interest).
- the first amino acid sequence of interest and the second amino acid sequence of interest are not found together in a naturally occurring protein.
- Preferred linkers are SEQ ID NOS:195-220.
- at least one of the first amino acid sequence of interest and the second amino acid sequence of interest is the amino acid sequence of a therapeutic polypeptide.
- the other amino acid sequence of interest can be the amino acid sequence of a targeting polypeptide, a half-life extending polypeptide and/or a blocking polypeptide.
- polypeptides that contain a separation moiety can be represented by Formula I: [D1]-[L1]-[D2], wherein D1 is a first amino acid sequence of interest (e.g., a domain of interest), L1 is a separation moiety that connects or links D1 to D2; and D2 is a second amino acid sequence of interest (e.g., a second domain of interest).
- D1 is a protease-cleavable separation moiety, and more preferably L1 comprises or consists of any of SEQ ID NOS: 195-220.
- the polypeptides can also be represented by Formula II: [D1]-[L1]-[D2]-[L2]-[D3], wherein D1 is a first amino acid of interest (e.g., a domain of interest), L1 and L2 are each, independently, a linker; D2 is a second amino acid sequence of interest (e.g., a domain of interest), and D3 is a third amino acid of interest (e.g., a domain of interest), wherein at least one of L1 and L2 is a protease-cleavable separation moiety, and preferably at least one of L1 and L2 comprises or consists of any of SEQ ID NOS: 195-220.
- D1 is a first amino acid of interest (e.g., a domain of interest)
- L1 and L2 are each, independently, a linker
- D2 is a second amino acid sequence of interest (e.g., a domain of interest)
- D3 is a third amino acid of
- the separation moieties as described herein can be used to attach a therapeutic drug-moiety.
- a therapeutic drug moiety is attached to the separation moiety to make a therapeutic drug moiety complex.
- the separate drug moiety complex can be a prodrug that is inactive until the target protease cleaves the prodrug, releasing the free drug.
- ADCs antibody-drug conjugates
- ADCs typically are an antibody that is linked to a cytotoxic moiety, such as a cytotoxic drug.
- the ADCs discriminate between the healthy and diseased cells and provide targeted delivery of drug (e.g. cytotoxic drug) to diseased cells.
- ADCs typically comprise an antibody that targets a tumor marker that is specific to tumor cells, whereupon the antibody attaches itself to the tumor cell, causing the ADC to be absorbed into the cell, which enables the cytotoxic component to be released to kill the tumor cell.
- a key aspect of ADCs is the provision of a stable linker between the antibody component and the cytotoxic agent.
- linkers may be cleavable or non-cleavable.
- the antibody, linker and cytotoxic unit is incorporated into the tumor cell.
- the nature of the linker typically determines the release profile of the cytotoxic agent.
- cleavable linkers between the antibody and the cytotoxic agent are typically catalyzed by enzymes in the tumor cell or in the tumor microenvironment, wherein the antibody and the cytotoxic agents are cleaved to release the cytotoxic agent.
- the separation moiety disclosed herein links or connects a drug moiety to an antibody moiety.
- the separation moieties disclosed herein are suitable for use in peptide-drug conjugates. These compounds typically comprise a cytotoxic payload and linker; however, instead of antibodies, peptide-drug conjugates are equipped with peptides that have the ability to penetrate tumors, thus allowing the cargo to be delivered inside the tumor.
- the separation moiety links or connects a cytotoxic payload with a peptide. The peptide-drug conjugate remains stable and has no biological activity until the target protease cleaves the separation moiety.
- the separation moieties disclosed herein are suitable for use in constructs engineered for use in adoptive cell transfer (ACT) therapies.
- the field of adoptive cell transfer (ACT) is currently comprised of chimeric antigen receptor (CAR) engineered T cells (and next generation therapies) that target T cells to cell surface expressed targets (e.g., tumor cells expressing surface targets), and T cell receptor (TCR) engineered T cells that can target intracellular antigens.
- CAR chimeric antigen receptor
- TCR T cell receptor
- the separation moiety is used to tether a targeting moiety to a CAR construct.
- a CAR replaces the endogenous TCR complex with a new receptor that uses a fragment of a human or mouse antibody to bind to targets outside of a cancer cell.
- the antibody fragment is linked to various signaling proteins inside T-cell which mediate receptor activation when the CAR binds to its target.
- the separation moiety is used to tether a targeting moiety to a TCR construct.
- a TCR is based on the gene for the protein receptor that is already naturally present in T-cells. The gene for a desired TCR can be discovered in a single patient, e.g., a patient that is able to mount an effective immune response against a type of cancer. This gene can then be introduced into other patients by incorporating it into TCR T cell constructs or reengineered to improve the binding interaction with its MHC target.
- engineered T cells whether autologous or allogenic, comprise engineered T cell receptor components that comprise a targeting agent such as an isolated human or humanized antibody.
- a targeting agent such as an isolated human or humanized antibody.
- the binding affinity of the targeting moiety may be affected by the steric, chemical, or flexibility properties of the separation moiety tethering the targeting moiety to the rest of the construct and the T cell.
- the separation moieties disclosed herein are suitable for use with engineered constructs for making CAR T and TCR T cells.
- the separation moieties disclosed herein are suitable for use in antigen-binding proteins.
- An “antigen-binding protein” (ABP) is a protein comprising one or more antigen-binding domains that specifically bind to an antigen or epitope.
- the antigen-binding domain binds the antigen or epitope with specificity and affinity similar to that of naturally occurring antibodies.
- the separation moieties link a polypeptide that blocks the antigen-binding site of the ABP from binding to its cognate antigen. But when the separation moiety is cleaved the blocking polypeptide can diffuse away from the ABP antigen binding site and the ABP can bind to its cognate antigen.
- Exemplary binding polypeptides that can block the antigen binding site of an ABP include steric blocker such as human serum albumin, and peptides that interact with one or more of the complementarity determining regions (CDRs) in the antigen binding site of the ABP.
- Such blocking peptides can be obtained by screening libraries or by screening peptide fragments of the cognate antigen of an ABP of interest.
- the separation moiety and blocker will be bonded to the amino terminus of the antibody light chain, or the amino terminus of the antibody heavy chain, so that the blocker is tethered close to and will readily block the antigen binding site.
- the ABP contains an alternative scaffold for the binding site.
- the blocker-separation moiety is typically bonded to the amino terminus near the antigen binding site.
- the ABP comprises an antibody binding site that comprises a VH and a VL, and the blocker-separation moiety is bonded to the amino terminus of the VL.
- the ABP can be an antibody (e.g., the first and second antigen binding domains are in the form of an antibody).
- at least one antigen binding domain of the ABP is in the form of an antibody.
- a first or second antigen binding domain is in the form of an antibody, and a first or second antigen binding domain is in the form of an antigen-binding fragment (e.g., the first antigen binding domain is an antibody and the second antigen binding domain is an antigen-binding fragment.
- the first antigen binding domain is an antigen binding fragment and the second antigen binding domain is an antibody).
- the ABP consists of an antibody. In some embodiments, the ABP consists essentially of an antibody. In some embodiments, the ABP comprises an alternative scaffold. In some embodiments, the ABP consists of an alternative scaffold. In some embodiments, the ABP consists essentially of an alternative scaffold. In some embodiments, the ABP comprises an antibody fragment. In some embodiments, the ABP consists of an antibody fragment. In some embodiments, the ABP consists essentially of an antibody fragment.
- the separation moieties disclosed herein are suitable for use in conjunction with an antibody.
- antibody is used herein in its broadest sense and includes certain types of immunoglobulin molecules comprising one or more antigen-binding domains that specifically bind to an antigen or epitope.
- An antibody specifically includes intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multi-specific antibodies.
- An antibody is one type of ABP.
- the separation moieties disclosed herein are suitable for use in conjunction with an antigen binding protein comprising an alternative scaffold.
- alternative scaffold refers to a molecule in which one or more regions may be diversified to produce one or more antigen-binding domains that specifically bind to an antigen or epitope.
- the antigen-binding domain binds the antigen or epitope with specificity and affinity similar to that of an antibody.
- Exemplary alternative scaffolds include those derived from fibronectin (e.g., AdnectinsTM), the ⁇ -sandwich (e.g., iMab), lipocalin (e.g., Anticalins®), EETI-II/AGRP, BPTI/LACI-D1/ITI-D2 (e.g., Kunitz domains), thioredoxin peptide aptamers, protein A (e.g., Affibody®), ankyrin repeats (e.g., DARPins), gamma-B-crystallin/ubiquitin (e.g., Affilins), CTLD3 (e.g., Tetranectins), Fynomers, and (LDLR-A module) (e.g., Avimers).
- fibronectin e.g., Adnectin
- An alternative scaffold is one type of ABP.
- the separation moieties disclosed herein are suitable for use in conjunction with an antibody fragment.
- An “antibody fragment” comprises a portion of an intact antibody, such as the antigen-binding or variable region of an intact antibody.
- Antibody fragments include, for example, Fv fragments, Fab fragments, F(ab′)2 fragments, Fab′ fragments, scFv (sFv) fragments, and scFv-Fc fragments.
- the separation moieties disclosed herein are suitable for use in conjunction with one or more Fv, Fab, or F(ab′)2 fragments.
- Fv fragments comprise a non-covalently-linked dimer of one heavy chain variable domain and one light chain variable domain.
- Fab fragments comprise, in addition to the heavy and light chain variable domains, the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments may be generated, for example, by recombinant methods or by papain digestion of a full-length antibody.
- “F(ab′)2” fragments contain two Fab′ fragments joined, near the hinge region, by disulfide bonds. F(ab′)2 fragments may be generated, for example, by recombinant methods or by pepsin digestion of an intact antibody. The F(ab′) fragments can be dissociated, for example, by treatment with 1-mercaptoethanol.
- the separation moieties disclosed herein are suitable for use in conjunction with an scFv or scFv-Fc.
- Single-chain Fv” or “sFv” or “scFv” antibody fragments comprise a VH domain and a VL domain in a single polypeptide chain.
- the VH and VL are generally linked by a peptide linker. See Plückthun A. (1994). Any suitable linker may be used.
- scFv-Fc” fragments comprise an scFv attached to an Fc domain. For example, an Fc domain may be attached to the C-terminal of the scFv.
- the Fc domain may follow the VH or VL, depending on the orientation of the variable domains in the scFv (i.e., VH-VL or VL-VH). Any suitable Fc domain known in the art or described herein may be used. In some cases, the Fc domain comprises an IgG4 Fc domain.
- the separation moieties disclosed herein are suitable for use in conjunction with a single domain antibody.
- the term “single domain antibody” refers to a molecule in which one variable domain of an antibody specifically binds to an antigen without the presence of another variable domain.
- Single domain antibodies, and fragments thereof, are described in Arabi Ghahroudi et al., FEBS Letters, 1998, 414:521-526 and Muyldermans et al., Trends in Biochem. Sci., 2001, 26:230-245, each of which is incorporated by reference in its entirety.
- Single domain antibodies are also known as sdAbs or nanobodies.
- the separation moieties disclosed herein are suitable for use in conjunction with a monospecific ABP.
- a “monospecific ABP” is an ABP that comprises one or more binding sites that specifically bind to the same epitope.
- An example of a monospecific ABP is a naturally occurring IgG molecule which, while divalent (i.e., having two antigen-binding domains), recognizes the same epitope at each of the two antigen-binding domains.
- the binding specificity may be present in any suitable valency.
- the separation moieties disclosed herein are suitable for use in conjunction with a monoclonal antibody.
- the term “monoclonal antibody” refers to an antibody from a population of substantially homogeneous antibodies.
- a population of substantially homogeneous antibodies comprises antibodies that are substantially similar and that bind the same epitope(s), except for variants that may normally arise during production of the monoclonal antibody. Such variants are generally present in only minor amounts.
- a monoclonal antibody is typically obtained by a process that includes the selection of a single antibody from a plurality of antibodies.
- the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones.
- the selected antibody can be further altered, for example, to improve affinity for the TNFR superfamily member proteins (“affinity maturation”), to humanize the antibody, to improve its production in cell culture, and/or to reduce its immunogenicity in a subject.
- affinity maturation affinity maturation
- the separation moieties disclosed herein are suitable for use in conjunction with a chimeric antibody.
- 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 separation moieties disclosed herein are suitable for use in conjunction with a humanized antibody.
- “Humanized” forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody.
- a humanized antibody is generally a human antibody (recipient antibody) in which residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody).
- the donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody having a desired specificity, affinity, or biological effect.
- selected framework region residues of the recipient antibody are replaced by the corresponding framework region residues from the donor antibody.
- Humanized antibodies may also comprise residues that are not found in either the recipient antibody or the donor antibody. Such modifications may be made to further refine antibody function. For further details, see Jones et al., Nature, 1986, 321:522-525; Riechmann et al., Nature, 1988, 332:323-329; and Presta, Curr. Op. Struct. Biol., 1992, 2:593-596, each of which is incorporated by reference in its entirety.
- the separation moieties disclosed herein are suitable for use in conjunction with a human antibody.
- a “human antibody” is one which possesses an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or derived from a non-human source that utilizes a human antibody repertoire or human antibody-encoding sequences (e.g., obtained from human sources or designed de novo). Human antibodies specifically exclude humanized antibodies.
- the ABPs provided herein specifically bind to the extracellular domain of a TNFR Superfamily Protein.
- the TNFR superfamily protein is CD27, CD137, CD40, GITR, LT-betaR, CD30, HVEM, TNFR1, TNFR2, or OX-40.
- the TNFR superfamily protein may be expressed on the surface of any suitable target cell.
- the target cell is a T cell.
- the target cell is an effector T cell.
- the target cell is a regulatory T cell.
- the target cell is a natural killer (NK) cell.
- the target cell is a natural killer T (NKT) cell.
- the target cell is a B cell. In some embodiments, the target cell is a myeloid derived cell. In some embodiments, the target cell is a myeloid derived suppressor cell. In some embodiments, the target cell is a dendritic cell.
- an ABP provided herein is an antibody. In some embodiments, an ABP provided herein is an antibody fragment. In some embodiments, an ABP provided herein is an alternative scaffold.
- the ABPs provided herein comprise an immunoglobulin molecule. In some embodiments, the ABPs provided herein consist of an immunoglobulin molecule. In some embodiments, the ABPs provided herein consist essentially of an immunoglobulin molecule. In some aspects, the immunoglobulin molecule comprises an antibody. In some aspects, the immunoglobulin molecule consists of an antibody. In some aspects, the immunoglobulin molecule consists essentially of an antibody.
- the ABPs provided herein comprise a light chain.
- the light chain is a kappa light chain.
- the light chain is a lambda light chain.
- the ABPs provided herein comprise a heavy chain.
- the heavy chain is an IgA.
- the heavy chain is an IgD.
- the heavy chain is an IgE.
- the heavy chain is an IgG.
- the heavy chain is an IgM.
- the heavy chain is an IgG1.
- the heavy chain is an IgG2.
- the heavy chain is an IgG3.
- the heavy chain is an IgG4.
- the heavy chain is an IgA1. In some aspects, the heavy chain is an IgA2.
- the separation moieties connect to a further blocking moiety.
- the separation moieties are positioned such that movement of the antigen-binding protein subunits is restricted.
- the separation moieties disclosed herein are suitable for use in conjunction with a multi-specific antigen binding protein (ABP).
- Multispecific ABP's provided herein bind more than one antigen.
- a multispecific antibody can bind 2 antigens, 3 antigens, binds 4 antigens, 5 antigens, or more.
- the multispecific ABP can bind 2 or more different epitopes.
- the two or more different epitopes may be epitopes on the same antigen (e.g., a single TNFR superfamily protein molecule expressed by a cell) or on different antigens (e.g., different TNFR superfamily member protein molecules expressed by the same cell, or a TNFR superfamily member protein molecule and a non-TNFR Superfamily member protein molecule).
- a multi-specific ABP binds two different epitopes (i.e., a “bispecific ABP”).
- a multi-specific ABP binds three different epitopes (i.e., a “trispecific ABP”).
- a multi-specific ABP binds four different epitopes (i.e., a “quadspecific ABP”). In some aspects, a multi-specific ABP binds five different epitopes (i.e., a “quintspecific ABP”). In some aspects, a multi-specific ABP binds 6, 7, 8, or more different epitopes. Each binding specificity may be present in any suitable valency.
- the antigen binding protein comprises a blocking domain.
- the separation moiety disclosed herein links the blocking domain to a first antigen binding domain and consequently the blocking domain inhibits (a) binding affinity or avidity of the antigen binding protein on the epitope and/or (b) agonist or antagonist activity of the antigen binding protein on the epitope.
- the separation moiety comprises a cleavage site.
- a disease-specific enzyme i.e., a protease
- the antigen binding protein comprises an additional separation moiety that links or connects an additional blocking domain to an antigen binding domain.
- the additional separation moiety comprises a cleavage site.
- the antigen binding protein comprises an additional blocking domain (e.g., a second, third, or fourth blocking domain) that is/are operably linked to an antigen binding domain (e.g., the second, third, or fourth antigen binding domain) by a cleavable separation moiety as disclosed herein.
- the blocking domain can be a steric blocker or a specific blocker.
- the steric blocker is independently selected from the group consisting of a fragment of the extracellular portion of the antibody binding protein, serum albumin, a fragment of serum albumin, and an antibody or antigen binding fragment thereof that binds serum albumin.
- the specific blocker is independently selected from a fragment of an antibody or antigen-binding fragment thereof that binds to the first, second, third or fourth antigen binding domain of the antigen binding protein described herein.
- the blocking domain is serum albumin or a fragment thereof, or an antibody or antigen binding fragment thereof that binds serum albumin.
- the antigen binding domain may further comprise a third antigen binding domain and a fourth antigen binding domain each with binding specificity for a target antigen.
- TNFR superfamily target antigens are well known in the art and are encompassed by this disclosure. Exemplary TNFR superfamily members include, but are not limited to CD27, CD30, CD137 (4-1BB), TNFR1 (CD120a), TNFR2 (CD120b), CD40, CD95 (Fas/Apo-1), HVEM, LT-betaR, GITR, nerve growth factor receptor or OX-40 (CD34). Preferred TNFR superfamily target antigens are CD27, CD137, and OX40.
- the antigen binding domains with specificity for an antigen can have binding specificity for the same epitope or for a different epitope on the same antigen.
- the first, second and third antigen binding domains can have binding specificity for the same epitope, for a different epitope, two of the antigen binding domains may binding specificity for the same epitope, or three of the antigen binding domains may have binding specificity for the same epitope.
- the antigen binding protein may further comprise a fifth antigen binding domain with specificity for a tumor antigen.
- the antigen binding polypeptide comprises an Fc region.
- two antigen binding domains can be located on each end of the Fc region.
- Another format comprises two antibody arms attached to the N terminus of the Fc region, each arm comprising 2 antigen binding domains.
- the antigen binding domain can also comprise a half-life extension domain.
- the half-life extension domain can be albumin, an antigen binding domain that recruits albumin, or an immunoglobulin Fc, or a fragment thereof.
- the half-life extension domain is operably linked to the antigen binding polypeptide by a cleavable linker.
- the disclosure also relates to an antigen binding protein comprising at least a first polypeptide and a second polypeptide.
- the first polypeptide comprises at least a portion of an antibody heavy chain constant region and an antibody heavy chain variable region (VH).
- the second polypeptide comprises at least a portion of an antibody light chain constant region and an antibody light chain variable region (VL).
- At least one of the first and second polypeptide further comprises a blocking domain that is operably linked to the VH or VL through a protease cleavable linker.
- the first polypeptide associates with the second polypeptide, and VH and VL form an antigen binding site with binding specificity for a target antigen (e.g., 4-1BB or CS27).
- a target antigen e.g., 4-1BB or CS27
- the blocking domain inhibits binding of the antigen binding site to the target antigen (e.g., 4-1BB or CD27).
- the first polypeptide further comprises a second VH, and the first polypeptide associates with two of said second polypeptides to form two VH/VL antigen binding sites that each have specificity for human 4-1BB.
- the antigen binding protein is a dimer of the first polypeptide with the associated second polypeptides.
- the antigen binding protein can further comprise a third, fourth, fifth, or a sixth polypeptide.
- the ABP is monospecific multivalent ABP.
- Such formats can have a variety of structures and be prepared using suitable antibody engineering techniques. For example, a dual Fab antibody that contains a heavy chain with the structure VH—CH1-noncleavable liner-VH—CH1-CH2-CH3 can be prepare. Such a heavy chain can be expressed and can pair with two light chains. The heavy chain can dimerize through conventional interchain disulfide bonds to form an antibody format that contains 4 Fab antigen binding sites.
- the separation moieties described herein can be bonded to the amino terminus of the light chain polypeptides to link a blocker to each of the Fab antigen binding sites.
- Suitable monospecific, multivalent ABP formats can be readily envisioned by those of skill in the art.
- a heavy chain is prepared that has the structure VH—CH1-CH2-CH3-VH—CH1.
- the separation moieties described herein can be bonded to the amino terminus of the light chain polypeptides to link a blocker to each of the Fab antigen binding sites.
- binding activity of such monospecific tetravalent ABPs is masked by the blocking domain, and the mask is removed upon cleavage of the separation domain (e.g., in a tumor microenvironment) and the ABP is able to bind its cognate antigen.
- the monospecific multivalent ABP formats can have binding specificity for any desired antigen.
- the monospecific multivalent ABP formats specifically bind to the extracellular domain of a TNFR Superfamily Protein, such as CD27, CD30, CD137 (4-1BB), TNFR1 (CD120a), TNFR2 (CD120b), CD40, CD95 (Fas/Apo-1), HVEM, LT-betaR, GITR, nerve growth factor receptor or OX-40 (CD34).
- a TNFR Superfamily Protein such as CD27, CD30, CD137 (4-1BB), TNFR1 (CD120a), TNFR2 (CD120b), CD40, CD95 (Fas/Apo-1), HVEM, LT-betaR, GITR, nerve growth factor receptor or OX-40 (CD34).
- the multivalent antigen binding protein can comprise a first antigen binding site with specificity for a target antigen (e.g., CD27 or TNFR1), second antigen binding site with specificity for the target antigen, a blocking polypeptide, at least one protease cleavable linker, and an optional half-life extension element.
- a first blocking polypeptide is operably linked to the first antigen binding site by a protease cleavable linker and, optionally, a second blocking polypeptide is operably linked to the second antigen binding site by a protease cleavable linker.
- the blocking polypeptide is operably linked to the second antigen binding site by a protease cleavable linker.
- the blocking polypeptide inhibits (a) binding affinity or avidity of the antigen binding protein on the target antigen and/or (b) agonist or antagonist activity of the antigen binding protein on the target antigen and upon cleavage of the protease cleavable linker (a) the binding affinity or avidity of the antigen binding protein on the target antigen and/or (b) agonist activity of the antigen binding protein on the target antigen is increased.
- the optional half-life extension element can be operably linked to the first antigen binding site and/or second antigen binding site through a optionally protease cleavable linker.
- the first and second binding sites can have specificity for the same epitope or different epitopes.
- the antigen binding protein further comprises a third antigen binding site with specificity for the same target antigen.
- the antigen binding protein can further comprise a fourth antigen binding site with specificity for the same target antigen as the first and second and third antigen binding sites.
- the third and fourth antigen binding sites can each further comprise a blocking polypeptide that is operable linked to the antigen-binding sight through a protease clevable linker.
- This disclosure also relates to a tetravalent antigen binding protein that can comprise a first polypeptide that comprises at least a portion of an antibody heavy chain constant region and an antibody heavy chain variable region (VH) and a second polypeptide that comprises at least a portion of an antibody light chain constant region, an antibody light chain variable region (VL).
- the first polypeptide associates with the second polypeptide and the VH and VL form an antigen binding site with binding specificity for a target antigen.
- the tetravalent antigen binding protein further comprises a blocking polypeptide that is operably linked to the VH or VL through a protease cleavable linker. The blocking polypeptide inhibits binding of the antigen binding site to the target antigen.
- the first polypeptide can further comprise a second VH. The first polypeptide associates with two of the second polypeptides to form two VH/VL antigen binding site that each have specificity for the target antigen.
- the tetravalent antigen binding protein can further comprise a third polypeptide, a fourth polypeptide, a fifth polypeptide, a sixth polypeptide, a seventh polypeptide, and an eight polypeptide.
- the third polypeptide can comprise at least a portion of an antibody heavy chain constant region and an antibody heavy chain variable region (VH).
- the fourth polypeptide can comprise at least a portion of an antibody light chain constant region, an antibody light chain variable region (VL).
- the firth polypeptide can comprise at least portion of an antibody heavy chain constant region and an antibody heavy chain variable region (VH).
- the sixth polypeptide can comprise at least a portion of an antibody light chain constant region, an antibody light chain variable region (VL).
- the seventh polypeptide can comprise at least portion of an antibody heavy chain constant region and an antibody heavy chain variable region (VH).
- the eighth polypeptide can comprise at least a portion of an antibody light chain constant region, an antibody light chain variable region (VL).
- At least one of the third and fourth polypeptide further comprises a blocking polypeptide that is operably linked to the VH or VL through a protease cleavable linker.
- the third polypeptide associates with the fourth polypeptide and VH and VL form an antigen binding site with binding specificity for a target antigen, and the blocking domain inhibits binding of the antigen binding site to the target antigen.
- the fifth and sixth polypeptide further comprises a blocking polypeptide that is operably linked to the VH or VL through a protease cleavable linker.
- the fifth polypeptide associates with the sixth polypeptide and VH and VL form an antigen binding site with binding specificity for a target antigen, and the blocking domain inhibits binding of the antigen binding site to the target antigen.
- the seventh and eighth polypeptide further comprises a blocking polypeptide that is operably linked to the VH or VL through a protease cleavable linker.
- the seventh polypeptide associates with the eighth polypeptide and VH and VL form an antigen binding site with binding specificity for a target antigen, and the blocking domain inhibits binding of the antigen binding site to the target antigen.
- Cytokines are potent immune agonists, which lead to them being considered promising therapeutic agents for oncology.
- cytokines have a very narrow therapeutic window.
- Cytokines have short serum half-lives and are also considered to be highly potent. Consequently, therapeutic administration of cytokines produces undesirable systemic effects and toxicities. These were exacerbated by the need to administer large quantities of cytokine in order to achieve the desired levels of cytokine at the intended site of cytokine action (e.g., a tumor).
- cytokines due to the biology of cytokines and inability to effectively target and control their activity, cytokines have not achieved the hoped-for clinical advantages in the treatment of tumors.
- the fusion proteins contain cytokine polypeptides that have receptor agonist activity. But in the context of the fusion protein, the cytokine receptor agonist activity is attenuated, and the circulating half-life is extended.
- the fusion proteins include protease cleave sites, which are cleaved by proteases that are associated with a desired site of cytokine activity (e.g., a tumor), and are typically enriched or selectively present at the site of desired activity.
- the fusion proteins are preferentially (or selectively) and efficiently cleaved at the desired site of activity to limit cytokine activity substantially to the desired site of activity, such as the tumor microenvironment.
- Protease cleavage at the desired site of activity such as in a tumor microenvironment, releases a form of the cytokine from the fusion protein that is much more active as a cytokine receptor agonist than the fusion protein (typically at least about 100 ⁇ more active than the fusion protein).
- the form of the cytokine that is released upon cleavage of the fusion protein typically has a short half-life, which is often substantially similar to the half-life of the naturally occurring cytokine, further restricting cytokine activity to the tumor microenvironment.
- the fusion proteins described herein enable the administration of an effective therapeutic dose of a cytokine to treat tumors with the activity of the cytokine substantially limited to the tumor microenvironment, and dramatically reduces or eliminates unwanted systemic effects and toxicity of the cytokine.
- cytokines In general, the therapeutic use of cytokines is strongly limited by their systemic toxicity. TNF, for example, was originally discovered for its capacity of inducing the hemorrhagic necrosis of some tumors, and for its in vitro cytotoxic effect on different tumoral lines, but it subsequently proved to have strong pro-inflammatory activity, which can, in case of overproduction conditions, dangerously affect the human body.
- systemic toxicity is a fundamental problem with the use of pharmacologically active amounts of cytokines in humans, novel derivatives and therapeutic strategies are now under evaluation, aimed at reducing the toxic effects of this class of biological effectors while keeping their therapeutic efficacy.
- IL-2 exerts both stimulatory and regulatory functions in the immune system and is, along with other members of the common ⁇ chain ( ⁇ c) cytokine family, central to immune homeostasis.
- IL-2 mediates its action by binding to IL-2 receptors (IL-2R), consisting of either trimeric receptors made of IL-2R ⁇ (CD25), IL-2R ⁇ (CD122), and IL-2R ⁇ ( ⁇ c, CD132) chains or dimeric ⁇ IL-2Rs (1, 3). Both IL-2R variants are able to transmit signal upon IL-2 binding.
- IL-2R IL-2 receptors
- Trimeric ⁇ IL-2Rs have a roughly 10-100 times higher affinity for IL-2 than dimeric ⁇ IL-2Rs (3), implicating that CD25 confers high-affinity binding of IL-2 to its receptor but is not crucial for signal transduction.
- Trimeric IL-2Rs are found on activated T cells and CD4+ forkhead box P3 (FoxP3)+ T regulatory cells (Treg), which are sensitive to IL-2 in vitro and in vivo.
- antigen-experienced (memory) CD8+, CD44 high memory-phenotype (MP) CD8+, and natural killer (NK) cells are endowed with high levels of dimeric ⁇ IL-2Rs, and these cells also respond vigorously to IL-2 in vitro and in vivo.
- IL-2R ⁇ expression is absent on naive and memory T cells but is induced after antigen activation.
- IL-2R ⁇ is constitutively expressed by NK, NKT, and memory CD8+ T cells but is also induced on naive T cells after antigen activation.
- ⁇ c is much less stringently regulated and is constitutively expressed by all lymphoid cells.
- IL-2R signaling upregulates the expression of IL-2R ⁇ in part through Stat5-dependent regulation of Il2ra transcription (Kim et al., 2001). This process represents a mechanism to maintain expression of the high-affinity IL-2R and sustain IL-2 signaling while there remains a source of IL-2.
- IL-2 is captured by IL-2R ⁇ through a large hydrophobic binding surface surrounded by a polar periphery that results in a relatively weak interaction (Kd 10-8 M) with rapid on-off binding kinetics.
- Kd 10-8 M relatively weak interaction
- the IL-2R ⁇ -IL-2 binary complex leads to a very small conformational change in IL-2 that promotes association with IL-2R ⁇ through a distinct polar interaction between IL-2 and IL-2R ⁇ .
- the pseudo-high affinity of the IL2/ ⁇ / ⁇ trimeric complex i.e. Kd ⁇ 300 pM
- the IL2/ ⁇ / ⁇ trimer then recruits the ⁇ chain into the quaternary complex capable of signaling, which is facilitated by the large composite binding site on the IL2-bound ⁇ chain for the ⁇ chain.
- the ternary IL-2R ⁇ -IL-2R ⁇ -IL-2 complex then recruits ⁇ c through a weak interaction with IL-2 and a stronger interaction with IL-2R ⁇ to produce a stable quaternary high-affinity IL-2R (Kd 10-11 M which is 10 pM).
- Kd 10-11 M which is 10 pM.
- the formation of the high-affinity quaternary IL-2-IL-2R complex leads to signal transduction through the tyrosine kinases Jak1 and Jak3, which are associated with IL-2R ⁇ and ⁇ c, respectively (Nelson and Willerford, 1998).
- IL-2-IL-2R complex The quaternary IL-2-IL-2R complex is rapidly internalized, where IL-2, IL-2R ⁇ , and ⁇ c are rapidly degraded, but IL-2R ⁇ is recycled to the cell surface (Hémar et al., 1995; Yu and Malek, 2001).
- those functional activities that require sustained IL-2R signaling require a continued source of IL-2 to engage IL-2R ⁇ and form additional IL-2-IL-2R signaling complexes.
- Interleukin-15 (IL-15), another member of the 4-alpha-helix bundle family of cytokines, has also emerged as an immunomodulator for the treatment of cancer.
- IL-15 is initially captured via IL-15R ⁇ , which is expressed on antigen-presenting dendritic cells, monocytes and macrophages.
- IL-15 exhibits broad activity and induces the differentiation and proliferation of T, B and natural killer (NK) cells via signaling through the IL-15/IL-2-R- ⁇ (CD122) and the common ⁇ chain (CD132). It also enhances cytolytic activity of CD8 + T cells and induces long-lasting antigen-experienced CD8 + CD44 memory T cells.
- IL-15 stimulates differentiation and immunoglobulin synthesis by B cells and induces maturation of dendritic cells. It does not stimulate immunosuppressive T regulatory cells (Tregs). Thus, boosting IL-15 activity selectively in the tumor micro-environment could enhance innate and specific immunity and fight tumors (Waldmann et al., 2012).
- IL-15 was initially identified for its ability to stimulate T cell proliferation in an IL-2-like manner through common receptor components (IL-2R/15R ⁇ - ⁇ c) and signaling through JAK1/JAK3 and STAT3/STAT5.
- IL-15 has been shown to stimulate proliferation of activated CD4 ⁇ CD8 ⁇ , CD4+CD8+, CD4+ and CD8+ T cells as well as facilitate the induction of cytotoxic T-lymphocytes, and the generation, proliferation and activation of NK cells (Waldmann et al., 1999).
- IL-2 which is required to maintain forkhead box P3 (FOXP3)-expressing CD4+CD25+ Treg cells and for the retention of these cells in the periphery
- IL-15 has little effect on Tregs (Berger et al., 2009). This is important as FOXP3-expressing CD4+CD25+ Tregs inhibit effector T cells, thereby inhibiting immune responses including those directed against the tumor.
- IL-2 also has a crucial role in initiating activation induced cell death (AICD), a process that leads to the elimination of self-reactive T cells, whereas IL-15 is an anti-apoptotic factor for T cells (Marks-Konczalik et al., 2000).
- IL-15 co-delivered with HIV peptide vaccines has been shown to overcome CD4+ T cell deficiency by promoting longevity of antigen-specific CD8+ T cells and blocking TRAIL-mediated apoptosis (Oh et al., 2008).
- IL-15 promotes the long-term maintenance of CD8+CD44hi memory T cells (Kanegane et al., 1996).
- IL-15 and IL-15R ⁇ are further highlighted by the phenotype of IL-15R ⁇ ⁇ / ⁇ and IL-15 ⁇ / ⁇ mice.
- Knockout mice demonstrate decreased numbers of total CD8+ T cells, and are deficient in memory-phenotype CD8+ T cells, NK cells, NK/T cells and some subsets of intestinal intraepithelial lymphocytes, indicating that IL-15 provides essential positive homeostatic functions for these subsets of cells (Lodolce et al., 1996; Kennedy et al., 1998).
- the similarities in the phenotypes of these two strains of knockout mice suggest the importance of IL-15R ⁇ in maintaining physiologically relevant IL-15 signals.
- IL-15 is presented in trans by the IL-15 receptor alpha-chain to the IL-15R ⁇ c complex displayed on the surface of T cells and natural killer (NK) cells (Han et al., 2011).
- the IL-15Ra-chain plays a role of chaperone protein, stabilizes, and increases IL-15 activity (Desbois et al., 2016).
- exogenous IL-15 may have a limited impact on patients with cancer due to its dependency on IL-15Ra frequently downregulated in cancer patients. Therefore, the fusion protein RLI, composed of the sushi+ domain of IL15Ra coupled via a linker to IL-15, has been suggested as an alternative approach to IL15 therapy (Bessard et al., 2009). It was found that administration of soluble IL-15/IL-15R ⁇ complexes greatly enhanced IL-15 serum half-life and bioavailability in vivo (Stoklasek et al., 2010).
- IL-15 In addition to the effects on T and NK cells, IL-15 also has several effects on other components of the immune system. IL-15 protects neutrophils from apoptosis, modulates phagocytosis and stimulates the secretion of IL-8 and IL-1R antagonist. It functions through the activation of JAK2, p38 and ERK1/2 MAPK, Syk kinase and the NF-kB transcriptional factor (Pelletier et al., 2002). In mast cells, IL-15 can act as a growth factor and an inhibitor of apoptosis. In these cells IL-15 activates the JAK2/STAT5 pathway without the requirement of ⁇ c binding (Tagaya et al., 1996).
- IL-15 also induces B lymphocyte proliferation and differentiation, and increases immunoglobulin secretion (Armitage et al., 1995). It also prevents Fas-mediated apoptosis and allows induction of antibody responses partially independent of CD4-help (Demerci et al., 2004; Steel et al., 2010). Monocytes, macrophages and dendritic cells effectively transcribe and translate IL-15. They also respond to IL-15 stimulation. Macrophages respond by increasing phagocytosis, inducing IL-8, IL-12 and MCP-1 expression, and secreting IL-6, IL-8 and TNF ⁇ (Budagian et al., 2006).
- Dendritic cells incubated with IL-15 demonstrate maturation with increased CD83, CD86, CD40, and MHC class II expression, are also resistant to apoptosis, and show enhanced interferon- ⁇ secretion (Anguille et al., 2009).
- IL-15 has also been shown to have effects on non-hematological cells including myocytes, adipocytes, endothelial and neural cells.
- IL-15 has an anabolic effect on muscle and may support muscle cell differentiation (Quinn et al., 1995). It stimulates myocytes and muscle fibers to accumulate contractile protein and is able to slow muscle wasting in rats with cancer-related cachexia (Figueras et al., 2004).
- IL-15 has also been shown to stimulate angiogenesis (Angiolillo et al., 1997) and induce microglial growth and survival (Hanisch et al., 1997).
- Interleukin-7 also of the IL-2/IL-15 family, is a well-characterized pleiotropic cytokine, and is expressed by stromal cells, epithelial cells, endothelial cells, fibroblasts, smooth muscle cells and keratinocytes, and following activation, by dendritic cells (Alpdogan et al., 2005). Although it was originally described as a growth and differentiation factor for precursor B lymphocytes, subsequent studies have shown that IL-7 is critically involved in T-lymphocyte development and differentiation.
- Interleukin-7 signaling is essential for optimal CD8 T-cell function, homeostasis and establishment of memory (Schluns et al., 2000); it is required for the survival of most T-cell subsets, and its expression has been proposed to be important for regulating T-cell numbers.
- IL-7 binds to a dimeric receptor, including IL-7R ⁇ and ⁇ c to form a ternary complex that plays fundamental roles in extracellular matrix remodeling, development, and homeostasis of T and B cells (Mazzucchelli and Durum, 2007).
- IL-7R ⁇ also cross-reacts to form a ternary complex with thymic stromal lymphopoietin (TSLP) and its receptor (TSLPR), and activates the TSLP pathway, resulting in T and dendritic cell proliferation in humans and further B cell development in mice (Leonard, 2002). Tight regulation of the signaling cascades activated by the complexes are therefore crucial to normal cellular function.
- IL-7 has a potential role in enhancing immune reconstitution in cancer patients following cytotoxic chemotherapy.
- IL-7 therapy enhances immune reconstitution and can augment even limited thymic function by facilitating peripheral expansion of even small numbers of recent thymic emigrants. Therefore, IL-7 therapy could potentially repair the immune system of patients who have been depleted by cytotoxic chemotherapy (Capitini et al., 2010).
- Interleukin-12 is a disulfide-linked heterodimer of two separately encoded subunits (p35 and p40), which are linked covalently to give rise to the so-called bioactive heterodimeric (p70) molecule (Lieschke et al., 1997; Jana et al., 2014).
- the p40 subunit is also secreted as a monomer (p40) and a homodimer (p40 2 ). It is known in the art that synthesis of the heterodimer as a single chain with a linker connecting the p35 to the p40 subunit preserves the full biological activity of the heterodimer.
- IL-12 plays a critical role in the early inflammatory response to infection and in the generation of Th1 cells, which favor cell-mediated immunity. It has been found that overproduction of IL-12 can be dangerous to the host because it is involved in the pathogenesis of a number of autoimmune inflammatory diseases (e.g. MS, arthritis, type 1 diabetes).
- autoimmune inflammatory diseases e.g. MS, arthritis, type 1 diabetes.
- the IL-12 receptor is a heterodimeric complex consisting of IL-12R ⁇ 1 and IL-12R ⁇ 2 chains expressed on the surface of activated T-cells and natural killer cells (Trinchieri et al., 2003).
- the IL-12R ⁇ 1 chain binds to the IL-12p40 subunit, whereas IL-12p35 in association with IL-12R ⁇ 2 confers an intracellular signaling ability (Benson et al., 2011).
- IL-12R Signal transduction through IL-12R induces phosphorylation of Janus kinase (Jak2) and tyrosine kinase (Tyk2), that phosphorylate and activate signal transducer and activator of transcription (STAT)1, STAT3, STAT4, and STAT5.
- the specific cellular effects of IL-12 are due mainly to activation of STAT4.
- IL-12 induces natural killer and T-cells to produce cytokines, in particular interferon (IFN) ⁇ , that mediate many of the proinflammatory activities of IL-12, including CD4+ T-cell differentiation toward the Th1 phenotype (Montepaone et al., 2014).
- IFN interferon
- Treg cells actively suppress activation of the immune system and prevent pathological self-reactivity and consequent autoimmune disease. Developing drugs and methods to selectively activate regulatory T cells for the treatment of autoimmune disease is the subject of intense research and, until the development of the present invention, which can selectively deliver active interleukins at the site of inflammation, has been largely unsuccessful.
- Treg are a class of CD4+CD25+ T cells that suppress the activity of other immune cells. Treg are central to immune system homeostasis and play a major role in maintaining tolerance to self-antigens and in modulating the immune response to foreign antigens.
- T1D Type 1 Diabetes
- SLE Systemic Lupus Erythematosus
- GVHD Graft-versus-Host Disease
- Treg cells that boost the numbers and/or function of Treg cells.
- One treatment approach for autoimmune diseases being investigated is the transplantation of autologous, ex vivo-expanded Treg cells (Tang, Q., et al, 2013, Cold Spring Harb. Perspect. Med., 3:1-15). While this approach has shown promise in treating animal models of disease and in several early stage human clinical trials, it requires personalized treatment with the patient's own T cells, is invasive, and is technically complex.
- Another approach is treatment with low dose Interleukin-2 (IL-2).
- IL-2 Interleukin-2
- Treg cells characteristically express high constitutive levels of the high affinity IL-2 receptor, IL2R ⁇ , which is composed of the subunits IL2R ⁇ (CD25), IL2R ⁇ (CD122), and IL2R ⁇ (CD132), and Treg cell growth has been shown to be dependent on IL-2 (Malek, T. R., et al., 2010, Immunity, 33:153-65).
- IL2R ⁇ which is composed of the subunits IL2R ⁇ (CD25), IL2R ⁇ (CD122), and IL2R ⁇ (CD132)
- IL-2 recombinant IL-2
- Proleukin® recombinant IL-2
- High-dose IL-2 is used for the treatment of patients with metastatic melanoma and metastatic renal cell carcinoma with a long-term impact on overall survival.
- Aldesleukin (marketed as Proleukin® by Prometheus Laboratories, San Diego, Calif.), the recombinant form of IL-2 used in these trials, is associated with high toxicity. Aldesleukin is approved for the treatment of metastatic melanoma and metastatic renal cancer, but its side effects are so severe that its use is only recommended in a hospital setting with access to intensive care (Web address: www.proleukin com/assets/pdf/proleukin.pdf).
- IL-2 receptors are expressed on a variety of different immune cell types, including T cells, NK cells, eosinophils, and monocytes, and this broad expression pattern likely contributes to its pleiotropic effect on the immune system and high systemic toxicity.
- activated T effector cells express IL2R ⁇ , as do pulmonary epithelial cells. But, activating T effector cells runs directly counter to the goal of down-modulating and controlling an immune response, and activating pulmonary epithelial cells leads to known dose-limiting side effects of IL-2 including pulmonary edema.
- VLS vascular leak syndrome
- VLS is believed to be caused by the release of proinflammatory cytokines from IL-2-activated NK cells.
- pulmonary edema results from direct binding of IL-2 to lung endothelial cells, which expressed low to intermediate levels of functional ⁇ IL-2Rs.
- the pulmonary edema associated with interaction of IL-2 with lung endothelial cells was abrogated by blocking binding to CD25 with an anti-CD25 monoclonal antibody (mAb), in CD25-deficient host mice, or by the use of CD122-specific IL-2/anti-IL-2 mAb (IL-2/mAb) complexes, thus preventing VLS.
- mAb monoclonal antibody
- IL-15 displays immune cell stimulatory activity similar to that of IL-2 but without the same inhibitory effects, thus making it a promising immunotherapeutic candidate.
- Clinical trials of recombinant human IL-15 for the treatment of metastatic malignant melanoma or renal cell cancer demonstrated appreciable changes in immune cell distribution, proliferation, and activation and suggested potential antitumor activity (Conlon et. al., 2014).
- IL-15 is currently in clinical trials to treat various forms of cancer.
- IL-15 therapy is known to be associated with undesired and toxic effects, such as exacerbating certain leukemias, graft-versus-host disease, hypotension, thrombocytopenia, and liver injury.
- IL-7 promotes lymphocyte development in the thymus and maintains survival of naive and memory T cell homeostasis in the periphery. Moreover, it is important for the organogenesis of lymph nodes (LN) and for the maintenance of activated T cells recruited into the secondary lymphoid organs (SLOs) (Gao et. al., 2015). In clinical trials of IL-7, patients receiving IL-7 showed increases in both CD4+ and CD8+ T cells, with no significant increase in regulatory T cell numbers as monitored by FoxP3 expression (Sportes et al., 2008).
- IL-12 is a pleiotropic cytokine, the actions of which create an interconnection between the innate and adaptive immunity.
- IL-12 was first described as a factor secreted from PMA-induced EBV-transformed B-cell lines. Based on its actions, IL-12 has been designated as cytotoxic lymphocyte maturation factor and natural killer cell stimulatory factor. Due to bridging the innate and adaptive immunity and potently stimulating the production of IFN ⁇ , a cytokine coordinating natural mechanisms of anticancer defense, IL-12 seemed ideal candidate for tumor immunotherapy in humans.
- compositions and methods comprising IL-2 and IL-15 and other cytokines, functional fragments and muteins of cytokines as well as conditionally active cytokines designed to address these risks and provide needed immunomodulatory therapeutics.
- the present invention is designed to address the shortcomings of direct IL-2 therapy and therapy using other cytokines, for example using cytokine blocking moieties, e.g. steric blocking polypeptides, serum half-life extending polypeptides, targeting polypeptides, linking polypeptides, including protease cleavable linkers, and combinations thereof.
- cytokine blocking moieties e.g. steric blocking polypeptides, serum half-life extending polypeptides, targeting polypeptides, linking polypeptides, including protease cleavable linkers, and combinations thereof.
- Cytokines including interleukins (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-21 IL-23), interferons (IFNs, including IFN ⁇ , IFN ⁇ and IFN ⁇ ), tumor necrosis factors (e.g., TNF ⁇ , lymphotoxin), transforming growth factors (e.g., TGF ⁇ 1, TGF ⁇ 2, TGF ⁇ 3), chemokines (C—X—C motif chemokine 10 (CXCL10), CCL19, CCL20, CCL21), and granulocyte macrophage-colony stimulating factor (GM-CS) are highly potent when administered to patients.
- interleukins e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-21 IL-23
- IFNs interferons
- tumor necrosis factors e.g., TNF ⁇ , lymphotoxin
- chemokine means a family of small cytokines with the ability to induce directed chemotaxis in nearby responsive cells Cytokines can provide powerful therapy but are accompanied by undesired effects that are difficult to control clinically and which have limited the clinical use of cytokines.
- This disclosure relates to new forms of cytokines that can be used in patients with reduced or eliminated undesired effects.
- pharmaceutical compositions including chimeric polypeptides (fusion proteins), nucleic acids encoding fusion proteins and pharmaceutical formulations of the foregoing that contain cytokines or active fragments or muteins of cytokines that have decreased cytokine receptor activating activity in comparison to the corresponding cytokine.
- the chimeric polypeptides activate their cognate receptors, often with the same or higher potency as the corresponding naturally occurring cytokine.
- this is typically achieved using a cytokine blocking moiety that blocks or inhibits the receptor activating function of the cytokine, active fragment or mutein thereof under general conditions but not under selected conditions, such as those present at the desired site of cytokine activity (e.g., an inflammatory site or a tumor).
- the chimeric polypeptides and nucleic acids encoding the chimeric polypeptides can be made using any suitable method.
- nucleic acids encoding a chimeric polypeptide can be made using recombinant DNA techniques, synthetic chemistry or combinations of these techniques, and expressed in a suitable expression system, such as in CHO cells.
- Chimeric polypeptides can similarly be made, for example by expression of a suitable nucleic acid, using synthetic or semi-synthetic chemical techniques, and the like.
- the blocking moiety can be attached to the cytokine polypeptide via sortase-mediated conjugation.
- Sortases are transpeptidases that modify proteins by recognizing and cleaving a carboxyl-terminal sorting signal embedded in or terminally attached to a target protein or peptide.
- Sortase A catalyzes the cleavage of the LPXTG motif (where X is any standard amino acid) (SEQ ID NO: 237) between the Thr and Gly residue on the target protein, with transient attachment of the Thr residue to the active site Cys residue on the enzyme, forming an enzyme-thioacyl intermediate.
- a biomolecule with an N-terminal nucleophilic group typically an oligoglycine motif, attacks the intermediate, displacing Sortase A and joining the two molecules.
- the cytokine polypeptide is first tagged at the N-terminus with a polyglycine sequence, or alternatively, with at the C-terminus with a LPXTG (SEQ ID NO: 237) motif.
- the blocking moiety or other element has respective peptides attached that serve as acceptor sites for the tagged polypeptides.
- the polypeptide will be tagged with an N-terminal poly-glycine stretch.
- the polypeptide For conjugation to domain carrying a poly-glycine peptide attached via its C-terminus, the polypeptide will be tagged at its C-terminus with a LPXTG (SEQ ID NO: 237) sortase recognition sequence. Recognizing poly-glycine and LPXTG (SEQ ID NO: 237) sequences, sortase will form a peptide bond between polymer-peptide and tagged polypeptides. The sortase reaction cleaves off glycine residues as intermediates and occurs at room temperature.
- the pharmaceutical compositions can include a cytokine moiety and a blocking moiety, e.g. a steric blocking moiety, with a protease cleavable linker comprising a protease cleavage site located between the cytokine and cytokine blocking moiety or within the cytokine blocking moiety.
- a blocking moiety e.g. a steric blocking moiety
- a protease cleavable linker comprising a protease cleavage site located between the cytokine and cytokine blocking moiety or within the cytokine blocking moiety.
- the linker can comprise glycine-glycine, a sortase-recognition motif, or a sortase-recognition motif and a peptide sequence (Gly 4 Ser) n (SEQ ID NO: 238) or (Gly 3 Ser) n (SEQ ID NO: 239), wherein n is 1, 2, 3, 4 or 5.
- the sortase-recognition motif comprises a peptide sequence LPXTG (SEQ ID NO: 237), where X is any amino acid.
- the covalent linkage is between a reactive lysine residue attached to the C-terminal of the cytokine polypeptide and a reactive aspartic acid attached to the N-terminal of the blocker or other domain. In other embodiments, the covalent linkage is between a reactive aspartic acid residue attached to the N-terminal of the cytokine polypeptide and a reactive lysine residue attached to the C-terminal of the blocker or another domain.
- the cytokine blocking moieties used can be steric blockers.
- a “steric blocker” refers to a polypeptide or polypeptide moiety that can be covalently bonded to a cytokine polypeptide directly or indirectly through other moieties such as linkers, for example in the form of a chimeric polypeptide (fusion protein), but otherwise does not covalently bond to the cytokine polypeptide.
- a steric blocker can non-covalently bond to the cytokine polypeptide, for example though electrostatic, hydrophobic, ionic or hydrogen bonding.
- a steric blocker typically inhibits or blocks the activity of the cytokine moiety due to its proximity to the cytokine moiety and comparative size.
- the steric inhibition of the cytokine moiety can be removed by spatially separating the cytokine moiety from the steric blocker, such as by enzymatically cleaving a fusion protein that contains a steric blocker and a cytokine polypeptide at a site between the steric blocker and the cytokine polypeptide.
- the blocking function can be combined with or due to the presence of additional functional components in the pharmaceutical composition, such as a targeting domain, a serum half-life extension element, and protease-cleavable linking polypeptides.
- a serum half-life extending polypeptide can also be a steric blocker.
- cytokine IL-2 as an exemplary cytokine.
- the invention and this disclosure are not limited to IL-2.
- this disclosure including the disclosed methods, polypeptides and nucleic acids, adequately describes and enables the use of other cytokines, fragments and muteins, such as IL-2, IL-7, IL-12, IL-15, IL-18, IL-21 IL-23, IFN ⁇ , IFN ⁇ , IFN ⁇ , TNF ⁇ , lymphotoxin, TGF- ⁇ 1, TGF ⁇ 2, TGF ⁇ 3, GM-CSF, CXCL10, CCL19, CCL20, CCL21 and functional fragments or muteins of any of the foregoing.
- Various elements ensure the delivery and activity of IL-2 preferentially at the site of desired IL-2 activity and to severely limit systemic exposure to the interleukin via a blocking and/or a targeting strategy preferentially linked to a serum half-life extension strategy.
- a blocking and/or a targeting strategy preferentially linked to a serum half-life extension strategy.
- the blocked version of interleukin circulates for extended times (preferentially 1-2 or more weeks) but the activated version has the typical serum half-life of the interleukin.
- IL-2 has a very short plasma half-life of 85 minutes for intravenous administration and 3.3 hours subcutaneous administration (Kirchner, G. I., et al., 1998, Br J Clin Pharmacol. 46:5-10).
- the half-life extension element is linked to the interleukin via a linker which is cleaved at the site of action (e.g. by inflammation-specific proteases) releasing the interleukin's full activity at the desired site and also separating it from the half-life extension of the uncleaved version.
- the fully active and free interleukin would have very different pharmacokinetic (pK) properties—a half-life of hours instead of weeks.
- exposure to active cytokine is limited to the site of desired cytokine activity (e.g., an inflammatory site or tumor) and systemic exposure to active cytokine, and associated toxicity and side effects, are reduced.
- cytokines envisioned in this invention have similar pharmacology (e.g. IL-15 as reported by Blood 2011 117:4787-4795; doi: doi.org/10.1182/blood-2010-10-311456) as IL-2 and accordingly, the designs of this invention address the shortcomings of using these agents directly, and provide chimeric polypeptides that can have extended half-life and/or be targeted to a site of desired activity (e.g., a site of inflammation or a tumor).
- a site of desired activity e.g., a site of inflammation or a tumor.
- IL-2 can be engineered to bind the IL-2R complex generally or one of the three IL-2R subunits specifically with an affinity that differs from that of the corresponding wild-type IL-2, for example to selectively activate Tregs or Teff (Effector T Cell).
- IL-2 polypeptides that are said to have higher affinity for the trimeric form of the IL-2 receptor relative to the dimeric beta/gamma form of the 11-2 receptor in comparison to wild type IL-2 can have an amino acid sequence that includes one of the following sets of mutations with respect to SEQ ID NO: 1 (a mature IL-2 protein comprising amino acids 21-153 of human IL-2 having the UniProt Accession No.
- P60568-1) (a) K64R, V69A, and Q74P; (b) V69A, Q74P, and T101A; (c) V69A, Q74P, and I128T; (d) N30D, V69A, Q74P, and F103S; (e) K49E, V69A, A73V, and K76E; (f) V69A, Q74P, T101A, and T133N; (g) N30S, V69A, Q74P, and I128A; (h) V69A, Q74P, N88D, and S99P; (i) N30S, V69A, Q74P, and I128T; (j) K9T, Q11R, K35R, V69A, and Q74P; (k) A1T, M46L, K49R, E61D, V69A, and H79R; (1) K48E, E68D, N71T, N90H, F103S, and 1114V
- muteins of other cytokines including interleukins (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-23), interferons (IFNs, including IFNalpha, IFNbeta and IFNgamma), tumor necrosis factors (e.g., TNFalpha, lymphotoxin), transforming growth factors (e.g., TGFbeta1, TGFbeta2, TGFbeta3) and granulocyte macrophage-colony stimulating factor (GM-CS).
- interleukins e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-23
- IFNs interferons
- tumor necrosis factors e.g., TNFalpha, lymphotoxin
- transforming growth factors e.g., TGFbeta1, TGFbeta2, TGFbeta3
- GM-CS granulocyte macrophage-
- any of the mutant IL-2 polypeptides disclosed herein can include the sequences described; they can also be limited to the sequences described and otherwise identical to SEQ ID NO:1. Moreover, any of the mutant IL-2 polypeptides disclosed herein can optionally include a substitution of the cysteine residue at position 125 with another residue (e.g., serine) and/or can optionally include a deletion of the alanine residue at position 1 of SEQ ID NO:1.
- IL-2 IL-2 stimulation of human T cell proliferation in vitro required a minimum of 5-6 hours exposure to effective concentrations of IL-2 (Cantrell, D. A., et. al., 1984, Science, 224: 1312-1316).
- IL-2 has a very short plasma half-life of 85 minutes for intravenous administration and 3.3 hours subcutaneous administration (Kirchner, G. I., et al., 1998, Br J Clin Pharmacol. 46:5-10).
- IL-2 peak levels can activate IL2R ⁇ receptors and have other unintended or adverse effects, for example VLS as noted above.
- An IL-2 analog, or a multifunctional protein with IL-2 attached to a domain that enables binding to the FcRn receptor, with a longer circulating half-life than IL-2 can achieve a target drug concentration for a specified period of time at a lower dose than IL-2, and with lower peak levels.
- IL-2 analog will therefore require either lower doses or less frequent administration than IL-2 to effectively stimulate Treg cells. Less frequent subcutaneous administration of an IL-2 drug will also be more tolerable for patients. A therapeutic with these characteristics will translate clinically into improved pharmacological efficacy, reduced toxicity, and improved patient compliance with therapy.
- IL-2 or muteins of IL-2 can be selectively targeted to the intended site of action (e.g. sites of inflammation). This targeting can be achieved by one of several strategies, including the addition of domains to the administered agent that comprise blockers of the IL-2 (or muteins) that are cleaved away or by targeting domains or a combination of the two.
- IL-2* partial agonists can be tailored to bind with higher or lower affinity depending on the desired target; for example, an IL-2* can be engineered to bind with enhanced affinity to one of the receptor subunits and not the others.
- These types of partial agonists unlike full agonists or complete antagonists, offer the ability to tune the signaling properties to an amplitude that elicits desired functional properties while not meeting thresholds for undesired properties.
- a repertoire of IL-2 variants could be engineered to exhibit an even finer degree of distinctive signaling activities, ranging from almost full to partial agonism to complete antagonism.
- the IL-2* has altered affinity for IL-2R ⁇ . In some embodiments, the IL-2* has a higher affinity for IL-2R ⁇ than wild-type IL-2. In other embodiments, the IL-2* has altered affinity for IL-2R ⁇ . In one embodiment, IL-2* has enhanced binding affinity for IL-2R ⁇ , e.g., the N-terminus of IL-2R ⁇ , that eliminates the functional requirement for IL-2R ⁇ . In another embodiment, an IL-2* is generated that has increased binding affinity for IL-2R ⁇ but that exhibited decreased binding to IL-2R ⁇ , and thereby is defective IL-2R ⁇ heterodimerization and signaling.
- Blocking moieties can also be used to favor binding to or activation of one or more receptors.
- blocking moieties are added such that IL-2R ⁇ binding or activation is blocked but IL-2R ⁇ binding or activation is not changed.
- blocking moieties are added such that IL-2R ⁇ binding or activation is diminished.
- blocking moieties are added such that binding to and or activation of all three receptors is inhibited. This blocking may be relievable by removal of the blocking moieties in a particular environment, for example by proteolytic cleavage of a linker linking one or more blocking moieties to the cytokine.
- the pharmacokinetics and/or pharmacodynamics of the cytokine can be tailored to maximally activate effector cells (e.g., effect T cells, NK cells) and/or cytotoxic immune response promoting cells (e.g., induce dendritic cell maturation) at a site of desired activity, such as in a tumor, but preferably not systemically.
- effector cells e.g., effect T cells, NK cells
- cytotoxic immune response promoting cells e.g., induce dendritic cell maturation
- compositions comprising at least one cytokine polypeptide, such as interleukins (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23), interferons (IFNs, including IFN ⁇ , IFN ⁇ and IFN ⁇ ), tumor necrosis factors (e.g., TNF ⁇ , lymphotoxin), transforming growth factors (e.g., TGF- ⁇ 1, TGF ⁇ 2, TGF ⁇ 3), chemokines (e.g. CXCL10, CCL19, CCL20, CCL21) and granulocyte macrophage-colony stimulating factor (GM-CS) or a functional fragment or mutein of any of the foregoing.
- interleukins e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23
- IFNs interferons
- tumor necrosis factors e.g., TNF ⁇ , lymphot
- the polypeptide typically also includes at least one linker amino acid sequence, wherein the amino acid sequence is in certain embodiments capable of being cleaved by an endogenous protease.
- the linker comprises an amino acid sequence comprising HSSKLQ (SEQ ID NO: 25), GPLGVRG (SEQ ID NO: 221), IPVSLRSG (SEQ ID NO: 222), VPLSLYSG (SEQ ID NO: 223), or SGESPAYYTA (SEQ ID NO: 224).
- the chimeric polypeptide further contains a blocking moiety, e.g. a steric blocking polypeptide moiety, capable of blocking the activity of the interleukin polypeptide.
- the blocking moiety for example, can comprise a human serum albumin (HSA) binding domain or an optionally branched or multi-armed polyethylene glycol (PEG).
- the pharmaceutical composition comprises a first cytokine polypeptide or a fragment thereof, and blocking moiety, e.g. a steric blocking polypeptide moiety, wherein the blocking moiety blocks the activity of the cytokine polypeptide on the cytokine receptor, and wherein the blocking moiety in certain embodiments comprises a protease cleavable domain.
- blockade and reduction of cytokine activity is achieved simply by attaching additional domains with very short linkers to the N or C terminus of the interleukin domain.
- the blockade is relieved by protease digestion of the blocking moiety or of the short linker that tethers the blocker to the interleukin. Once the domain is clipped or is released, it will no longer be able to achieve blockade of cytokine activity.
- the pharmaceutical composition e.g., chimeric polypeptide can comprise two or more cytokines, which can be the same cytokine polypeptide or different cytokine polypeptides.
- the two or more different types of cytokines have complementary functions.
- a first cytokine is IL-2 and a second cytokine is IL-12.
- each of the two or more different types of cytokine polypeptides have activities that modulate the activity of the other cytokine polypeptides.
- a first cytokine polypeptide is T-cell activating, and a second cytokine polypeptide is non-T-cell-activating.
- a first cytokine is a chemoattractant, e.g., CXCL10, and a second cytokine is an immune cell activator.
- the cytokine polypeptides (including functional fragments) that are included in the fusion proteins disclosed herein are not mutated or engineered to alter the properties of the naturally occurring cytokine, including receptor binding affinity and specificity or serum half-life.
- changes in amino acid sequence from naturally occurring (including wild type) cytokine are acceptable to facilitate cloning and to achieve desired expression levels, for example.
- the blocking moiety can be any moiety that inhibits the ability of the cytokine to bind and/or activate its receptor.
- the blocking moiety can inhibit the ability of the cytokine to bind and/or activate its receptor sterically blocking and/or by noncovalently binding to the cytokine.
- suitable blocking moieties include the full length or a cytokine-binding fragment or mutein of the cognate receptor of the cytokine.
- Antibodies and fragments thereof including, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody a single chain variable fragment (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain of camelid-type nanobody (VHH), a sdAb and the like that bind the cytokine can also be used.
- scFv single chain variable fragment
- VH heavy chain variable domain
- VL light chain variable domain
- VHH camelid-type nanobody
- Suitable antigen-binding domain that bind the cytokine can also be used, include non-immunoglobulin proteins that mimic antibody binding and/or structure such as, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin and CTLA4 scaffolds.
- suitable blocking polypeptides include polypeptides that sterically inhibit or block binding of the cytokine to its cognate receptor.
- such moieties can also function as half-life extending elements.
- a peptide that is modified by conjugation to a water-soluble polymer, such as PEG can sterically inhibit or prevent binding of the cytokine to its receptor.
- Polypeptides, or fragments thereof, that have long serum half-lives can also be used, such as serum albumin (human serum albumin), immunoglobulin Fc, transferring and the like, as well as fragments and muteins of such polypeptides.
- antigen-binding polypeptides include a single chain variable fragment (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain of camelid-type nanobody (VHH), a sdAb and the like.
- antigen-binding domain that bind the cytokine can also be used, include non-immunoglobulin proteins that mimic antibody binding and/or structure such as, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin and CTLA4 scaffolds.
- non-immunoglobulin proteins that mimic antibody binding and/or structure such as, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocal
- the blocking moiety when IL-2 is the cytokine in the chimeric polypeptide, can be the full length or fragment or mutein of the alpha chain of IL-2 receptor (IL-2R ⁇ ) or beta (IL-2R ⁇ ) or gamma chain of IL-2 receptor (IL-2R ⁇ ), an anti-IL-2 single-domain antibody (sdAb) or scFv, an anti-CD25 antibody or fragment thereof, and anti-HSA sdAb or scFv, and the like.
- IL-2R ⁇ alpha chain of IL-2 receptor
- beta IL-2R ⁇
- IL-2R ⁇ gamma chain of IL-2 receptor
- sdAb anti-IL-2 single-domain antibody
- scFv an anti-CD25 antibody or fragment thereof
- anti-HSA sdAb or scFv and the like.
- the chimeric polypeptides comprise an in vivo half-life extension element.
- a “half-life extension element” is a part of the chimeric polypeptide that increases the in vivo half-life and improve pK, for example, by altering its size (e.g., to be above the kidney filtration cutoff), shape, hydrodynamic radius, charge, or parameters of absorption, biodistribution, metabolism, and elimination.
- An exemplary way to improve the pK of a polypeptide is by expression of an element in the polypeptide chain that binds to receptors that are recycled to the plasma membrane of cells rather than degraded in the lysosomes, such as the FcRn receptor on endothelial cells and transferrin receptor.
- an element in the polypeptide chain that binds to receptors that are recycled to the plasma membrane of cells rather than degraded in the lysosomes, such as the FcRn receptor on endothelial cells and transferrin receptor.
- Three types of proteins, e.g., human IgGs, HSA (or fragments), and transferrin persist for much longer in human serum than would be predicted just by their size, which is a function of their ability to bind to receptors that are recycled rather than degraded in the lysosome.
- These proteins, or fragments of them that retain the FcRn binding are routinely linked to other polypeptides to extend their serum half-life.
- the half-life extension element is a human serum albumin (HSA) binding domain.
- HSA serum albumin (SEQ ID NO:2) may also be directly bound to the pharmaceutical compositions or bound via a short linker. Fragments of HSA may also be used. HSA and fragments thereof can function as both a blocking moiety and a half-life extension element. Human IgGs can also carry out a similar function.
- the serum half-life extension element can also be antigen-binding polypeptide that binds to a protein with a long serum half-life such as serum albumin, transferrin and the like.
- polypeptides include antibodies and fragments thereof including, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody a single chain variable fragment (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain of camelid-type nanobody (VHH), a sdAb and the like.
- antigen-binding domain include non-immunoglobulin proteins that mimic antibody binding and/or structure such as, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin and CTLA4 scaffolds.
- non-immunoglobulin proteins that mimic antibody binding and/or structure such as, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin and CTLA4 scaffolds.
- antigen-binding polypeptides include a ligand for a desired receptor, a ligand-binding portion of a receptor, a lectin, and peptides that binds to or associates with one or more target antigens.
- Some preferred serum half-life extension elements are polypeptides that comprise complementarity determining regions (CDRs), and optionally non-CDR loops.
- CDRs complementarity determining regions
- serum half-life extension elements can extend the serum half-life of the cytokine, and also function as inhibitors of the cytokine (e.g., via steric blocking, non-covalent interaction or combination thereof) and/or as targeting domains.
- the serum half-life extension elements are domains derived from an immunoglobulin molecule (Ig molecule) or engineered protein scaffolds that mimic antibody structure and/or binding activity.
- the Ig may be of any class or subclass (IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM etc.).
- a polypeptide chain of an Ig molecule folds into a series of parallel beta strands linked by loops.
- three of the loops constitute the “complementarity determining regions” (CDRs) which determine the antigen binding specificity of the molecule.
- An IgG molecule comprises at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding fragment thereof.
- Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
- the heavy chain constant region is comprised of three domains, CH1, CH2 and CH3.
- Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
- the light chain constant region is comprised of one domain, CL.
- CL The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs) with are hypervariable in sequence and/or involved in antigen recognition and/or usually form structurally defined loops, interspersed with regions that are more conserved, termed framework regions (FR).
- CDRs complementarity determining regions
- FR framework regions
- Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
- variable domains of an immunoglobulin molecule has several beta strands that are arranged in two sheets.
- the variable domains of both light and heavy immunoglobulin chains contain three hypervariable loops, or complementarity-determining regions (CDRs).
- CDR1, CDR2, CDR3 cluster at one end of the beta barrel.
- the CDRs are the loops that connect beta strands B-C, C′-C′′, and F-G of the immunoglobulin fold, whereas the bottom loops that connect beta strands AB, CC′, C′′-D and E-F of the immunoglobulin fold, and the top loop that connects the D-E strands of the immunoglobulin fold are the non-CDR loops.
- at least some amino acid residues of a constant domain, CH1, CH2, or CH3 are part of the “non-CDR loop” of the binding moieties described herein.
- Non-CDR loops comprise, in some embodiments, one or more of AB, CD, EF, and DE loops of a C1-set domain of an Ig or an Ig-like molecule; AB, CC′, EF, FG, BC, and EC′ loops of a C2-set domain of an Ig or an Ig-like molecule; DE, BD, GF, A(A1A2)B, and EF loops of I(Intermediate)-set domain of an Ig or Ig-like molecule.
- the CDRs are believed to be responsible for antigen recognition and binding, while the FR residues are considered a scaffold for the CDRs.
- some of the FR residues play an important role in antigen recognition and binding.
- Framework region residues that affect Ag binding are divided into two categories. The first are FR residues that contact the antigen, thus are part of the binding-site, and some of these residues are close in sequence to the CDRs. Other residues are those that are far from the CDRs in sequence but are in close proximity to it in the 3-D structure of the molecule, e.g., a loop in heavy chain.
- the binding moieties are any kinds of polypeptides.
- the binding moieties are natural peptides, synthetic peptides, or fibronectin scaffolds, or engineered bulk serum proteins.
- the bulk serum protein comprises, for example, albumin, fibrinogen, or a globulin.
- the binding moieties are engineered scaffolds.
- Engineered scaffolds comprise, for example, a sdAb, a scFv, a Fab, a VHH, a fibronectin type III domain, immunoglobulin-like scaffold (as suggested in Halaby et al., 1999. Prot Eng 12(7):563-571), DARPin, cystine knot peptide, lipocalin, three-helix bundle scaffold, protein G-related albumin-binding module, or a DNA or RNA aptamer scaffold.
- the serum half-life extending element comprises a binding site for a bulk serum protein.
- the CDRs provide the binding site for the bulk serum protein.
- the bulk serum protein is, in some examples, a globulin, albumin, transferrin, IgG1, IgG2, IgG4, IgG3, IgA monomer, Factor XIII, Fibrinogen, IgE, or pentameric IgM.
- the CDR form a binding site for an immunoglobulin light chain, such as an Ig ⁇ free light chain or an Ig ⁇ free light chain.
- the serum half-life extension element can be any type of binding domain, including but not limited to, domains from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody.
- the binding moiety is a single chain variable fragment (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of camelid derived nanobody.
- the binding moieties are non-Ig binding domains, i.e., antibody mimetic, such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, and monobodies.
- antibody mimetic such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, and monobodies.
- the serum half-life extension element can be a water-soluble polymer or a peptide that is conjugated to a water-soluble polymer, such as PEG.
- PEG polyethylene glycol
- PEG poly(ethylene glycol)
- PEG also means a polymer that contains a majority, that is to say, greater than 50%, of —OCH 2 CH 2 — repeating subunits.
- the PEG can take any number of a variety of molecular weights, as well as structures or geometries such as “branched,” “linear,” “forked,” “multifunctional,” and the like, to be described in greater detail below.
- the PEG is not limited to a particular structure and can be linear (e.g., an end capped, e.g., alkoxy PEG or a bifunctional PEG), branched or multi-armed (e.g., forked PEG or PEG attached to a polyol core), a dendritic (or star) architecture, each with or without one or more degradable linkages.
- the internal structure of the PEG can be organized in any number of different repeat patterns and can be selected from the group consisting of homopolymer, alternating copolymer, random copolymer, block copolymer, alternating tripolymer, random tripolymer, and block tripolymer.
- PEGs can be conjugated to polypeptide and peptides through any suitable method.
- a reactive PEG derivative such as N-hydroxysuccinamidyl ester PEG
- a peptide or polypeptide that includes amino acids with a side chain that contains an amine, sulfhydryl, carboxylic acid or hydroxyl functional group, such as cysteine, lysine, asparagine, glutamine, threonine, tyrosine, serine, aspartic acid, and glutamic acid.
- the construct may be desirable to maximize the amount of time the construct is present in its desired location in the body.
- This can be achieved by including one further domain in the chimeric polypeptide (fusion protein) to influence its movements within the body.
- the chimeric nucleic acids can encode a domain that directs the polypeptide to a location in the body, e.g., tumor cells or a site of inflammation; this domain is termed a “targeting domain” and/or encode a domain that retains the polypeptide in a location in the body, e.g., tumor cells or a site of inflammation; this domain is termed a “retention domain”
- a domain can function as both a targeting and a retention domain.
- the targeting domain and/or retention domain are specific to a protease-rich environment.
- the encoded targeting domain and/or retention domain are specific for regulatory T cells (Tregs), for example targeting the CCR4 or CD39 receptors.
- Tregs regulatory T cells
- Other suitable targeting and/or retention domains comprise those that have a cognate ligand that is overexpressed in inflamed tissues, e.g., the IL-1 receptor, or the IL-6 receptor.
- the suitable targeting and/or retention domains comprise those who have a cognate ligand that is overexpressed in tumor tissue, e.g., Epcam, CEA or mesothelin.
- the targeting domain is linked to the interleukin via a linker which is cleaved at the site of action (e.g. by inflammation or cancer specific proteases) releasing the interleukin full activity at the desired site.
- the targeting and/or retention domain is linked to the interleukin via a linker which is not cleaved at the site of action (e.g. by inflammation or cancer specific proteases), causing the cytokine to remain at the desired site.
- Antigens of choice are expressed on the surface of a diseased cell or tissue, for example a tumor or a cancer cell.
- Antigens useful for tumor targeting and retention include but are not limited to EpCAM, EGFR, HER-2, HER-3, c-Met, FoIR, and CEA.
- Pharmaceutical compositions disclosed herein also include proteins comprising two targeting and/or retention domains that bind to two different target antigens known to be expressed on a diseased cell or tissue. Exemplary pairs of antigen binding domains include but are not limited to EGFR/CEA, EpCAM/CEA, and HER-2/HER-3.
- Suitable targeting and/or retention domains include antigen-binding domains, such as antibodies and fragments thereof including, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody a single chain variable fragment (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain of camelid-type nanobody (VHH), a sdAb and the like.
- antigen-binding domains such as antibodies and fragments thereof including, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody a single chain variable fragment (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain of camelid-type nanobody (VHH), a sdAb and the like.
- antigen-binding domain include non-immunoglobulin proteins that mimic antibody binding and/or structure such as, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin and CTLA4 scaffolds.
- non-immunoglobulin proteins that mimic antibody binding and/or structure such as, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin and CTLA4 scaffolds.
- antigen-binding polypeptides include a ligand for a desired receptor, a ligand-binding portion of a receptor, a lectin, and peptides that binds to or associates with one or more target antigens.
- the targeting and/or retention domains specifically bind to a cell surface molecule. In some embodiments, the targeting and/or retention domains specifically bind to a tumor antigen. In some embodiments, the targeting polypeptides specifically and independently bind to a tumor antigen selected from at least one of EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FoIR. In some embodiments, the targeting polypeptides specifically and independently bind to two different antigens, wherein at least one of the antigens is a tumor antigen selected from EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FoIR.
- the targeting and/or retention antigen can be a tumor antigen expressed on a tumor cell.
- Tumor antigens are well known in the art and include, for example, EpCAM, EGFR, HER-2, HER-3, c-Met, FolR, PSMA, CD38, BCMA, and CEA.
- the targeting and/or retention antigen can be an immune checkpoint protein.
- immune checkpoint proteins include but are not limited to CD27, CD137, 2B4, TIGIT, CD155, ICOS, HVEM, CD40L, LIGHT, TIM-1, OX40, DNAM-1, PD-L1, PD1, PD-L2, CTLA-4, CD8, CD40, CEACAM1, CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA, IDO1, IDO2, TDO, KIR, LAG-3, TIM-3, or VISTA.
- the targeting and/or retention antigen can be a cell surface molecule such as a protein, lipid or polysaccharide.
- a targeting and/or retention antigen is a on a tumor cell, virally infected cell, bacterially infected cell, damaged red blood cell, arterial plaque cell, inflamed or fibrotic tissue cell.
- the targeting and/or retention antigen can comprise an immune response modulator.
- immune response modulator examples include but are not limited to granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 12 (IL-12), interleukin 15 (IL-15), B7-1 (CD80), B7-2 (CD86), GITRL, CD3, or GITR.
- GM-CSF granulocyte-macrophage colony stimulating factor
- M-CSF macrophage colony stimulating factor
- G-CSF granulocyte colony stimulating factor
- interleukin 2 IL-2
- IL-3 interleukin 3
- IL-12 interleukin 12
- IL-15 interleukin 15
- B7-1 CD80
- B7-2 CD86
- GITRL GITRL
- the targeting and/or retention antigen can be a cytokine receptor.
- cytokine receptors include but are not limited to Type I cytokine receptors, such as GM-CSF receptor, G-CSF receptor, Type I IL receptors, Epo receptor, LIF receptor, CNTF receptor, TPO receptor; Type II Cytokine receptors, such as IFN-alpha receptor (IFNAR1, IFNAR2), IFB-beta receptor, IFN-gamma receptor (IFNGR1, IFNGR2), Type II IL receptors; chemokine receptors, such as CC chemokine receptors, CXC chemokine receptors, CX3C chemokine receptors, XC chemokine receptors; tumor necrosis receptor superfamily receptors, such as TNFRSF5/CD40, TNFRSF8/CD30, TNFRSF7/CD27, TNFRSF1A/TNFR1/CD120a, TNFRSF1B/TNFR2/CD120b
- the pharmaceutical compositions comprise one or more linker sequences.
- a linker sequence serves to provide flexibility between polypeptides, such that, for example, the blocking moiety is capable of inhibiting the activity of the cytokine polypeptide.
- the linker sequence can be located between any or all of the cytokine polypeptide, the serum half-life extension element, and/or the blocking moiety.
- at least one of the linkers is protease cleavable, and contains a (one or more) cleavage site for a (one or more) desired protease.
- the desired protease is enriched or selectively expressed at the desired site of cytokine activity (e.g., the tumor microenvironment).
- the fusion protein is preferentially or selectively cleaved at the site of desired cytokine activity.
- a blocking moiety can be located C-terminally or N-terminally to a cytokine polypeptide.
- compositions comprising polypeptide sequences.
- polypeptide sequences As with all peptides, polypeptides, and proteins, including fragments thereof, it is understood that additional modifications in the amino acid sequence of the chimeric polypeptides (amino acid sequence variants) can occur that do not alter the nature or function of the peptides, polypeptides, or proteins. Such modifications include conservative amino acid substitutions and are discussed in greater detail below.
- compositions provided herein have a desired function.
- the compositions are comprised of at least a cytokine polypeptide, such as IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, or IFN ⁇ , or a chemokine, such as CXCL10, CCL19, CCL20, CCL21, a blocking moiety, e.g. a steric blocking polypeptide, and an optional serum half-life extension element, and an optional targeting polypeptide, with one or more linkers connecting each polypeptide in the composition.
- the first polypeptide e.g., an IL-2 mutein, is provided to be an active agent.
- the blocking moiety is provided to block the activity of the interleukin.
- the linker polypeptide e.g., a protease cleavable polypeptide
- the blocking moiety blocks the activity of the first polypeptide by binding the interleukin polypeptide.
- the blocking moiety e.g. a steric blocking peptide
- the linker comprises glycine-glycine, a sortase-recognition motif, or a sortase-recognition motif and a peptide sequence (Gly 4 Ser) n (SEQ ID NO: 238) or (Gly 3 Ser) n (SEQ ID NO: 239), wherein n is 1, 2, 3, 4 or 5.
- the sortase-recognition motif comprises a peptide sequence LPXTG, where X is any amino acid (SEQ ID NO: 237).
- the covalent linkage is between a reactive lysine residue attached to the C-terminal of the cytokine polypeptide and a reactive aspartic acid attached to the N-terminal of the blocking or other moiety. In one embodiment, the covalent linkage is between a reactive aspartic acid residue attached to the N-terminal of the cytokine polypeptide and a reactive lysine residue attached to the C-terminal of the blocking or other moiety.
- the activity of the cytokine polypeptide the context of the fusion protein is attenuated, and protease cleavage at the desired site of activity, such as in a tumor microenvironment, releases a form of the cytokine from the fusion protein that is much more active as a cytokine receptor agonist than the fusion protein.
- the cytokine-receptor activating (agonist) activity of the fusion polypeptide can be at least about 10 ⁇ , at least about 50 ⁇ , at least about 100 ⁇ , at least about 250 ⁇ , at least about 500 ⁇ , or at least about 1000 ⁇ less than the cytokine receptor activating activity of the cytokine polypeptide as a separate molecular entity.
- the cytokine polypeptide that is part of the fusion protein exists as a separate molecular entity when it contains an amino acid that is substantially identical to the cytokine polypeptide and does not substantially include additional amino acids and is not associated (by covalent or non-covalent bonds) with other molecules. If necessary, a cytokine polypeptide as a separate molecular entity may include some additional amino acid sequences, such as a tag or short sequence to aid in expression and/or purification.
- the cytokine-receptor activating (agonist) activity of the fusion polypeptide is at least about 10 ⁇ , at least about 50 ⁇ , at least about 100 ⁇ , at least about 250 ⁇ , at least about 500 ⁇ , or about 1000 ⁇ less than the cytokine receptor activating activity of the polypeptide that contains the cytokine polypeptide that is produced by cleavage of the protease cleavable linker in the fusion protein.
- the cytokine receptor activating (agonist) activity of the polypeptide that contains the cytokine polypeptide that is produced by cleavage of the protease cleavable linker in the fusion protein is at least about 10 ⁇ , at least about 50 ⁇ , at least about 100 ⁇ , at least about 250 ⁇ , at least about 500 ⁇ , or at least about 1000 ⁇ greater than the cytokine receptor activating activity of the fusion protein.
- a recombinant polypeptide is in conjugation with a cleavable moiety wherein the cleavable moiety is cleaved with reduced catalytic efficiency by one or more proteases than a reference polypeptide sequence.
- the cleavable moiety is resistant to proteolytic cleavage by one or more proteases.
- a cleavable moiety is resistant to a protease if the sequence comprises a binding site that is altered from the canonical cleavable motif sequence for the specific protease.
- a binding site is altered compared to a reference sequence by making one or more substitutions in the protease cleavage motif sequence.
- the protease cathepsin S cleaves the sequence GAVVRGA (SEQ ID NO: 240); a sequence is made to substitution can be made to substitute the arginine (R) with a glutamine (Q), thus changing a charged residue to a shorter, polar residue, reducing the ability of cathepsin S to bind and cleave the sequence.
- GAVVRGA SEQ ID NO: 240
- Q glutamine
- Such semi-conservative amino acid substitutions in protease target sequence motifs can lead to reduced binding ability, and such altered sequence motifs are therefore protease resistant.
- An uncleavable moiety can be made by inserting a disruptive amino acid into the protease target sequence motif, such as a proline (causes curves in the secondary structure of the peptide) or histidine (causes steric interference with other amino acid side chains).
- a “protease-resistant” peptide linker is one with reduced or undetectable cleavage by one or more specified proteases. Exemplary protease-resistant peptide linkers can be tested, e.g., in vitro by incubation with a specific protease followed by analysis of the digestion products by western blot.
- polypeptides described herein can include components (e.g., the cytokine, the blocking moiety) that have the same amino acid sequence of the corresponding naturally occurring protein (e.g., IL-2, IL-15, HSA) or can have an amino acid sequence that differs from the naturally occurring protein so long as the desired function is maintained. It is understood that one way to define any known modifications and derivatives or those that might arise, of the disclosed proteins and nucleic acids that encode them is through defining the sequence variants in terms of identity to specific known reference sequences.
- polypeptides and nucleic acids which have at least, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent identity to the chimeric polypeptides provided herein.
- polypeptides or nucleic acids that have at least, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent identity to the sequence of any of the nucleic acids or polypeptides described herein.
- polypeptides or nucleic acids that have at least, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent identity to the sequence of the cleavable linkers provided herein.
- This also includes variants of the linker or inducible polypeptides that comprise 1, 2, 3, 4, 5, or 6 variants from the cleavage domain sequences.
- the identity can be calculated after aligning the two sequences so that the identity is at its highest level.
- Optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman Adv. Appl. Math. 2:482 (1981), by the identity alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection.
- Protein modifications include amino acid sequence modifications. Modifications in amino acid sequence may arise naturally as allelic variations (e.g., due to genetic polymorphism), may arise due to environmental influence (e.g., by exposure to ultraviolet light), or may be produced by human intervention (e.g., by mutagenesis of cloned DNA sequences), such as induced point, deletion, insertion and substitution mutants. These modifications can result in changes in the amino acid sequence, provide silent mutations, modify a restriction site, or provide other specific mutations. Amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional or deletional modifications. Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues.
- Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues.
- Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about from 2 to 6 residues are deleted at any one site within the protein molecule Amino acid substitutions are typically of single residues but can occur at a number of different locations at once; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues.
- Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues.
- substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct.
- the mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure.
- Substitutional modifications are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Table 2 and are referred to as conservative substitutions.
- Modifications including the specific amino acid substitutions, are made by known methods. For example, modifications are made by site specific mutagenesis of nucleotides in the DNA encoding the polypeptide, thereby producing DNA encoding the modification, and thereafter expressing the DNA in recombinant cell culture. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example M13 primer mutagenesis and PCR mutagenesis.
- Modifications can be selected to optimize binding.
- affinity maturation techniques can be used to alter binding of the scFv by introducing random mutations inside the complementarity determining regions (CDRs).
- CDRs complementarity determining regions
- Such random mutations can be introduced using a variety of techniques, including radiation, chemical mutagens or error-prone PCR. Multiple rounds of mutation and selection can be performed using, for example, phage display.
- the disclosure also relates to nucleic acids that encode the chimeric polypeptides described herein, and to the use of such nucleic acids to produce the chimeric polypeptides and for therapeutic purposes.
- the invention includes DNA and RNA molecules (e.g., mRNA, self-replicating RNA) that encode a chimeric polypeptide and to the therapeutic use of such DNA and RNA molecules.
- Exemplary fusion proteins of the invention combine the above described elements in a variety of orientations.
- the orientations described in this section are meant as examples and are not to be considered limiting.
- the fusion protein comprises a cytokine, a blocking moiety and a half-life extension element.
- the cytokine is positioned between the half-life extension element and the blocking moiety.
- the cytokine is N-terminal to the blocking moiety and the half-life extension element.
- the cytokine is proximal to the blocking moiety; in some such embodiments, the cytokine is proximal to the half-life extension element.
- At least one protease-cleavable linker must be included in all embodiments, such that the cytokine may be active upon cleavage.
- the cytokine is C-terminal to the blocking moiety and the half-life extension element. Additional elements may be attached to one another by a cleavable linker, a non-cleavable linker, or by direct fusion.
- the blocking domains used are capable of extending half-life, and the cytokine is positioned between two such blocking domains. In some embodiments, the cytokine is positioned between two blocking domains, one of which is capable of extending half-life.
- two cytokines are included in the same construct.
- the cytokines are connected to two blocking domains each (three in total in one molecule), with a blocking domain between the two cytokine domains.
- one or more additional half-life extension domains may be included to optimize pharmacokinetic properties.
- three cytokines are included in the same construct.
- the third cytokine may function to block the other two in place of a blocking domain between the two cytokines.
- Preferred half-life extension elements for use in the fusion proteins are human serum albumin (HSA), an antibody or antibody fragment (e.g., scFV, dAb) which binds serum albumin, a human or humanized IgG, or a fragment of any of the foregoing.
- the blocking moiety is human serum albumin (HSA), or an antibody or antibody fragment which binds serum albumin, an antibody which binds the cytokine and prevents activation of binding or activation of the cytokine receptor, another cytokine, or a fragment of any of the foregoing.
- the targeting domain is an antibody which binds a cell surface protein which is enriched on the surface of cancer cells, such as EpCAM, FOLR1, and Fibronectin.
- the separation moieties disclosed herein can be used for antibody-antibiotic conjugates.
- the separation moiety disclosed herein connects or links an antimicrobial antibiotic to an antibody specific for a bacterial strain (e.g., Staphylococcus aureus Ab).
- the antibody-antibiotic conjugate does not display antibacterial activity when the antibody is linked to the antibiotic.
- the separation moiety is cleaved by proteases releasing the free antibiotic. The free antibiotic kills the intracellular bacteria.
- the separation moieties disclosed herein can be used for applications in chemical probes use for detection and isolation of proteins.
- Chemical probes are designed based on small molecule interaction with proteins.
- the probes typically comprise a covalent binding motif in order for the probe to interact with the target protein, a detection/purification tag for visualization/purification of the protein target and a linker group.
- the separation moieties described herein can be incorporated to enable the target protein to be detected and isolated.
- a disease or disorder such as proliferative disease, a tumorous disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, or graft-versus-host disease.
- the methods administering to a subject in need thereof an effective amount of a fusion protein as disclosed herein that is typically administered as a pharmaceutical composition.
- the method further comprises selecting a subject with or at risk of developing such a disease or disorder.
- the pharmaceutical composition preferably comprises a blocked cytokine, fragment or mutein thereof that is activated at a site of inflammation.
- the chimeric polypeptide comprises a cytokine polypeptide, fragment or mutein thereof and a serum half-life extension element.
- the chimeric polypeptide comprises a cytokine polypeptide, fragment or mutein thereof and a blocking moiety, e.g. a steric blocking polypeptide, wherein the steric blocking polypeptide is capable of sterically blocking the activity of the cytokine polypeptide, fragment or mutein thereof.
- the chimeric polypeptide comprises a cytokine polypeptide, fragment or mutein thereof, a blocking moiety, and a serum half-life extension element.
- Inflammation is part of the complex biological response of body tissues to harmful stimuli, such as pathogens, damaged cells, or irritants, and is a protective response involving immune cells, blood vessels, and molecular mediators.
- the function of inflammation is to eliminate the initial cause of cell injury, clear out necrotic cells and tissues damaged from the original insult and the inflammatory process, and to initiate tissue repair.
- Inflammation can occur from infection, as a symptom or a disease, e.g., cancer, atherosclerosis, allergies, myopathies, HIV, obesity, or an autoimmune disease.
- An autoimmune disease is a chronic condition arising from an abnormal immune response to a self-antigen.
- Autoimmune diseases that may be treated with the polypeptides disclosed herein include but are not limited to lupus, celiac disease, diabetes mellitus type 1, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.
- the pharmaceutical composition can comprise one or more protease-cleavable linker sequences.
- the linker sequence serves to provide flexibility between polypeptides, such that each polypeptide is capable of inhibiting the activity of the first polypeptide.
- the linker sequence can be located between any or all of the cytokine polypeptide, fragment or mutein thereof, the blocking moiety, and serum half-life extension element.
- the composition comprises, two, three, four, or five linker sequences.
- the linker sequence, two, three, or four linker sequences can be the same or different linker sequences.
- the linker sequence comprises GGGGS (SEQ ID NO: 232), GSGSGS (SEQ ID NO: 233), or G(SGGG) 2 SGGT (SEQ ID NO: 234).
- the linker comprises a protease-cleavable sequence selected from group consisting of HSSKLQ (SEQ ID NO: 25), GPLGVRG (SEQ ID NO:221), IPVSLRSG (SEQ ID NO: 222), VPLSLYSG (SEQ ID NO: 223), or SGESPAYYTA (SEQ ID NO: 224).
- the linker is cleaved by a protease selected from the group consisting of a kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin G, an elastase, PR-3, granzyme M, a calpain, a matrix metalloproteinase (MMP), a plasminogen activator, a cathepsin, a caspase, a tryptase, or a tumor cell surface protease.
- a protease selected from the group consisting of a kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin G, an elastase, PR-3, granzyme M, a calpain, a matrix metalloproteinase (MMP), a plasminogen activator, a cathepsin, a caspase, a tryptase
- the methods comprise administering to the subject in need thereof an effective amount of a chimeric polypeptide (a fusion protein) as disclosed herein that is typically administered as a pharmaceutical composition.
- the method further comprises selecting a subject with or at risk of developing cancer.
- the pharmaceutical composition preferably comprises a blocked cytokine, fragment or mutein thereof that is activated at a tumor site.
- the tumor is a solid tumor.
- the cancer may be a colon cancer, a lung cancer, a melanoma, a sarcoma, a renal cell carcinoma, a breast cancer,
- the method can further involve the administration of one or more additional agents to treat cancer, such as chemotherapeutic agents (e.g., Adriamycin, Cerubidine, Bleomycin, Alkeran, Velban, Oncovin, Fluorouracil, Thiotepa, Methotrexate, Bisantrene, Noantrone, Thiguanine, Cytaribine, Procarabizine), immuno-oncology agents (e.g., anti-PD-L1, anti-CTLA4, anti-PD-1, anti-CD47, anti-GD2), cellular therapies (e.g, CAR-T, T-cell therapy), oncolytic viruses and the like.
- chemotherapeutic agents e.g., Adriamycin, Cerubidine, Bleomycin, Alkeran, Velban, Oncovin, Fluorouracil, Thiotepa, Methotrexate, Bisantrene, Noantrone, Thiguanine, Cytaribine, Procarabizine
- immuno-oncology agents e.g
- compositions are suitable for administration in vitro or in vivo.
- pharmaceutically acceptable carrier is meant a material that is not biologically or otherwise undesirable, i.e., the material is administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical formulation or composition in which it is contained.
- the carrier is selected to minimize degradation of the active ingredient and to minimize adverse side effects in the subject.
- Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 21 st Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005).
- an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic, although the formulate can be hypertonic or hypotonic if desired.
- the pharmaceutically acceptable carriers include, but are not limited to, sterile water, saline, buffered solutions like Ringer's solution, and dextrose solution. The pH of the solution is generally about 5 to about 8 or from about 7 to 7.5.
- Other carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the immunogenic polypeptides.
- Matrices are in the form of shaped articles, e.g., films, liposomes, or microparticles. Certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. Carriers are those suitable for administration of the chimeric polypeptides or nucleic acid sequences encoding the chimeric polypeptides to humans or other subjects.
- compositions are administered in a number of ways depending on whether local or systemic treatment is desired and, on the area to be treated.
- the compositions are administered via any of several routes of administration, including topically, orally, parenterally, intravenously, intra-articularly, intraperitoneally, intramuscularly, subcutaneously, intracavity, transdermally, intrahepatically, intracranially, nebulization/inhalation, or by installation via bronchoscopy.
- the compositions are administered locally (non-systemically), including intratumorally, intra-articularly, intrathecally, etc.
- Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
- non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
- Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
- Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
- Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives are optionally present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
- Formulations for topical administration include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders.
- Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners and the like are optionally necessary or desirable.
- compositions for oral administration include powders or granules, suspension or solutions in water or non-aqueous media, capsules, sachets, or tables. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders are optionally desirable.
- the chimeric polypeptides or nucleic acid sequences encoding the chimeric polypeptides are administered by a vector.
- a vector There are a number of compositions and methods which can be used to deliver the nucleic acid molecules and/or polypeptides to cells, either in vitro or in vivo via, for example, expression vectors. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non-viral based delivery systems. Such methods are well known in the art and readily adaptable for use with the compositions and methods described herein.
- compositions and methods can be used to transfect or transduce cells in vitro or in vivo, for example, to produce cell lines that express and preferably secrete the encoded chimeric polypeptide or to therapeutically deliver nucleic acids to a subject.
- the components of the chimeric nucleic acids disclosed herein typically are operably linked in frame to encode a fusion protein.
- plasmid or viral vectors are agents that transport the disclosed nucleic acids into the cell without degradation and include a promoter yielding expression of the nucleic acid molecule and/or polypeptide in the cells into which it is delivered.
- Viral vectors are, for example, Adenovirus, Adeno-associated virus, herpes virus, Vaccinia virus, Polio virus, Sindbis, and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviral vectors, in general are described by Coffin et al., Retroviruses, Cold Spring Harbor Laboratory Press (1997), which is incorporated by reference herein for the vectors and methods of making them.
- replication-defective adenoviruses has been described (Berkner et al., J. Virol. 61:1213-20 (1987); Massie et al., Mol. Cell. Biol. 6:2872-83 (1986); Haj-Ahmad et al., J. Virol. 57:267-74 (1986); Davidson et al., J. Virol. 61:1226-39 (1987); Zhang et al., BioTechniques 15:868-72 (1993)).
- the benefit and the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell types, since they can replicate within an initial infected cell, but are unable to form new infectious viral particles.
- Recombinant adenoviruses have been shown to achieve high efficiency after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma, and a number of other tissue sites.
- Other useful systems include, for example, replicating and host-restricted non-replicating vaccinia virus vectors.
- VLPs Virus like particles
- VLPs consist of viral protein(s) derived from the structural proteins of a virus. Methods for making and using virus like particles are described in, for example, Garcea and Gissmann, Current Opinion in Biotechnology 15:513-7 (2004).
- the provided polypeptides can be delivered by subviral dense bodies (DBs).
- DBs transport proteins into target cells by membrane fusion.
- Methods for making and using DBs are described in, for example, Pepperl-Klindworth et al., Gene Therapy 10:278-84 (2003).
- the provided polypeptides can be delivered by tegument aggregates. Methods for making and using tegument aggregates are described in International Publication No. WO 2006/110728.
- Non-viral based delivery methods can include expression vectors comprising nucleic acid molecules and nucleic acid sequences encoding polypeptides, wherein the nucleic acids are operably linked to an expression control sequence.
- Suitable vector backbones include, for example, those routinely used in the art such as plasmids, artificial chromosomes, BACs, YACs, or PACs. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, Wis.), Clonetech (Pal Alto, Calif.), Stratagene (La Jolla, Calif.), and Invitrogen/Life Technologies (Carlsbad, Calif.). Vectors typically contain one or more regulatory regions.
- Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5′ and 3′ untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, and introns.
- a suitable host cell such as CHO cells.
- Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis B virus, and most preferably cytomegalovirus (CMV), or from heterologous mammalian promoters, e.g. ⁇ -actin promoter or EF1 ⁇ promoter, or from hybrid or chimeric promoters (e.g., CMV promoter fused to the ⁇ -actin promoter).
- viruses such as polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis B virus, and most preferably cytomegalovirus (CMV), or from heterologous mammalian promoters, e.g. ⁇ -actin promoter or EF1 ⁇ promoter, or from hybrid or chimeric promoters (e.g., CMV promoter fuse
- Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5′ or 3′ to the transcription unit. Furthermore, enhancers can be within an intron as well as within the coding sequence itself. They are usually between 10 and 300 base pairs (bp) in length, and they function in cis. Enhancers usually function to increase transcription from nearby promoters. Enhancers can also contain response elements that mediate the regulation of transcription. While many enhancer sequences are known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
- the promoter and/or the enhancer can be inducible (e.g. chemically or physically regulated).
- a chemically regulated promoter and/or enhancer can, for example, be regulated by the presence of alcohol, tetracycline, a steroid, or a metal.
- a physically regulated promoter and/or enhancer can, for example, be regulated by environmental factors, such as temperature and light.
- the promoter and/or enhancer region can act as a constitutive promoter and/or enhancer to maximize the expression of the region of the transcription unit to be transcribed.
- the promoter and/or enhancer region can be active in a cell type specific manner.
- the promoter and/or enhancer region can be active in all eukaryotic cells, independent of cell type.
- Preferred promoters of this type are the CMV promoter, the SV40 promoter, the ⁇ -actin promoter, the EF1 ⁇ promoter, and the retroviral long terminal repeat (LTR).
- the vectors also can include, for example, origins of replication and/or markers.
- a marker gene can confer a selectable phenotype, e.g., antibiotic resistance, on a cell.
- the marker product is used to determine if the vector has been delivered to the cell and once delivered is being expressed.
- selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, puromycin, and blasticidin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. Examples of other markers include, for example, the E.
- an expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide.
- Tag sequences such as GFP, glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or FLAGTM tag (Kodak; New Haven, Conn.) sequences typically are expressed as a fusion with the encoded polypeptide.
- GFP glutathione S-transferase
- GST glutathione S-transferase
- polyhistidine polyhistidine
- c-myc hemagglutinin
- FLAGTM tag FLAGTM tag
- peptide, polypeptide, or protein are used broadly to mean two or more amino acids linked by a peptide bond. Protein, peptide, and polypeptide are also used herein interchangeably to refer to amino acid sequences. It should be recognized that the term polypeptide is not used herein to suggest a particular size or number of amino acids comprising the molecule and that a peptide of the invention can contain up to several amino acid residues or more.
- subject can be a vertebrate, more specifically a mammal (e g a human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig), birds, reptiles, amphibians, fish, and any other animal.
- patient or subject may be used interchangeably and can refer to a subject with a disease or disorder (e.g. cancer).
- a disease or disorder e.g. cancer
- patient or subject includes human and veterinary subjects.
- a subject at risk of developing a disease or disorder can be genetically predisposed to the disease or disorder, e.g., have a family history or have a mutation in a gene that causes the disease or disorder, or show early signs or symptoms of the disease or disorder.
- a subject currently with a disease or disorder has one or more than one symptom of the disease or disorder and may have been diagnosed with the disease or disorder.
- a therapeutically effective amount of the chimeric polypeptides or chimeric nucleic acid sequences encoding the chimeric polypeptides described herein are administered to a subject prior to onset (e.g., before obvious signs of cancer or inflammation) or during early onset (e.g., upon initial signs and symptoms of cancer or inflammation).
- Prophylactic administration can occur for several days to years prior to the manifestation of symptoms of cancer or inflammation.
- Prophylactic administration can be used, for example, in the preventative treatment of subjects diagnosed with a genetic predisposition to cancer.
- Therapeutic treatment involves administering to a subject a therapeutically effective amount of the chimeric polypeptides or nucleic acid sequences encoding the chimeric polypeptides described herein after diagnosis or development of cancer or inflammation (e.g., an autoimmune disease). Prophylactic use may also apply when a patient is undergoing a treatment, e.g., a chemotherapy, in which inflammation is expected.
- a treatment e.g., a chemotherapy
- the subject is administered an effective amount of the agent (e.g., a chimeric polypeptide).
- effective amount and effective dosage are used interchangeably.
- the term effective amount is defined as any amount necessary to produce a desired physiologic response.
- Effective amounts and schedules for administering the agent may be determined empirically, and making such determinations is within the skill in the art.
- the dosage ranges for administration are those large enough to produce the desired effect in which one or more symptoms of the disease or disorder are affected (e.g., reduced or delayed). The dosage should not be so large as to cause substantial adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
- the dosage will vary with the age, condition, sex, type of disease, the extent of the disease or disorder, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art.
- the dosage can be adjusted by the individual physician in the event of any contraindications. Dosages can vary and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.
- treatment refers to a method of reducing the effects of a disease or condition or symptom of the disease or condition.
- treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or condition or symptom of the disease or condition.
- a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject as compared to a control.
- the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.
- the terms prevent, preventing, and prevention of a disease or disorder refers to an action, for example, administration of the chimeric polypeptide or nucleic acid sequence encoding the chimeric polypeptide, that occurs before or at about the same time a subject begins to show one or more symptoms of the disease or disorder, which inhibits or delays onset or exacerbation of one or more symptoms of the disease or disorder.
- references to decreasing, reducing, or inhibiting include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater as compared to a control level. Such terms can include but do not necessarily include complete elimination.
- IL-2 variants have been developed that are selective for IL2R ⁇ relative to IL2R ⁇ (Shanafelt, A. B., et al., 2000, Nat Biotechnol. 18:1197-202; Cassell, D. J., et. al., 2002, Curr Pharm Des., 8:2171-83). These variants have amino acid substitutions which reduce their affinity for IL2RB. Because IL-2 has undetectable affinity for IL2RG, these variants consequently have reduced affinity for the IL2R ⁇ receptor complex and reduced ability to activate IL2R ⁇ -expressing cells but retain the ability to bind IL2RA and the ability to bind and activate the IL2R ⁇ receptor complex.
- IL2/N88R Boy 50-4798
- Bay 50-4798 was shown to selectively stimulate the proliferation of activated T cells relative to NK cells, and was evaluated in phase I/II clinical trials in cancer patients (Margolin, K., et. al., 2007, Clin Cancer Res., 13:3312-9) and HIV patients (Davey, R. T., et. al., 2008, J Interferon Cytokine Res., 28:89-100).
- mutants can be made that selectively alter the affinity for the CD25 chain relative to native 11-2.
- IL-2 can be engineered to produce mutants that bind the IL-2R complex generally or the IL-2R ⁇ subunit specifically with an affinity that differs from that of the corresponding wild-type IL-2 or of a presently available mutant (referred to as C125 S, as the cysteine residue at position 125 is replaced with a serine residue).
- the present invention features mutant interleukin-2 (IL-2*) polypeptides that include an amino acid sequence that is at least 80% identical to wild-type IL-2 (e.g., 85, 87, 90, 95, 97, 98, or 99% identical) and that bind, as compared to WT IL-2, with higher to the IL-2 trimeric receptor relative to the dimeric IL-2 receptor.
- the muteins will also bind an IL-2 receptor a subunit (IL-2R ⁇ ) with an affinity that is greater than the affinity with which wild type IL-2 binds the IL-2R ⁇ .
- mutant IL-2 polypeptides can vary from SEQ ID NO:1 (UniProtKB accession number P60568) by virtue of containing (or only containing) one or more amino acid substitutions, which may be considered conservative or non-conservative substitutions. Non-naturally occurring amino acids can also be incorporated.
- amino acid sequence can vary from SEQ ID NO:1 (which may be considered the “reference” sequence) by virtue of containing and addition and/or deletion of one or more amino acid residues.
- the amino acid sequence can differ from that of SEQ ID NO:1 by virtue of a mutation at least one of the following positions of SEQ ID NO:1: 1, 4, 8, 9, 10, 11, 13, 15, 26, 29, 30, 31, 35, 37, 46, 48, 49, 54, 61, 64, 67, 68, 69, 71, 73, 74, 75, 76, 79, 88, 89, 90, 92, 99, 101, 103, 114, 125, 128, or 133 (or combinations thereof). As noted, as few as one of these positions may be altered, as may two, three, four, five, six, seven, eight, nine, ten, or 11 or more (including up to all) of the positions.
- amino acid sequence can differ from SEQ ID NO:1 at positions 69 and 74 and further at one or more of positions 30, 35, and 128.
- the amino acid sequence can also differ from SEQ ID NO:2 (as disclosed in U.S. Pat. No.
- the amino acid sequence of the mutant IL-2 polypeptide can otherwise be identical to SEQ ID NO:1.
- the amino acid sequence can differ from SEQ ID NO:1 by virtue of having one or more of the following mutations: A1T, S4P, K8R, K9T, T10A, Q11R, Q13R, E15K, N26D, N29S, N30S, N30D, N30T, Y31H, Y31C, K35R, T37A, T37R, M46L, K48E, K49R, K49E, K54R, E61D, K64R, E67G, E68D, V69A, N71T, N71A, N71R, A73V, Q74P, 575P, K76E, K76R, H79R, N88D, I89V, N90H, I92T, S99P, T101A, F103S, I114V, I128T, I1
- mutant polypeptides within the scope of the invention include those that include a mutant of SEQ ID NO:2 having substitutions at V69 (e.g. A) and Q74 (e.g., P).
- the amino acid sequence can include one of the following sets of mutations with respect to SEQ ID NO:2: (a) K64R, V69A, and Q74P; (b) V69A, Q74P, and T101A; (c) V69A, Q74P, and I128T; (d) N30D, V69A, Q74P, and F103S; (e) K49E, V69A, A73V, and K76E; (f) V69A, Q74P, T101A, and T133N; (g) N30S, V69A, Q74P, and I128A; (h) V69A, Q74P, N88D, and S99P; (i) N30S, V69A, Q74P, and I128T; (j) K9T, Q11R, K35R, V69A, and Q74P; (k) A1T, M46L, K49R, E61D, V69A, and H79R; (1) K48E,
- any of the mutant IL-2 polypeptides disclosed herein can include the sequences described; they can also be limited to the sequences described and otherwise identical to SEQ ID NO:1. Moreover, any of the mutant IL-2 polypeptides described herein can optionally include a substitution of the cysteine residue at position 125 with another residue (e.g., serine) and/or can optionally include a deletion of the alanine residue at position 1 of SEQ ID NO:1.
- the mutant IL-2 polypeptides disclosed herein can bind to the IL-2R ⁇ subunit with a K d of less than about 28 nM (e.g., less than about 25 nM; less than about 5 nM; about 1 nM; less than about 500 pM; or less than about 100 pM). More specifically, a mutant IL-2 polypeptide can have an affinity equilibrium constant less than 1.0 nM (e.g., about 0.8, 0.6, 0.4, or 0.2 nM). Affinity can also be expressed as a relative rate of dissociation from an IL-2R ⁇ subunit or from an IL-2 receptor complex (e.g., a complex expressed on the surface of a cell or otherwise membrane bound).
- the mutant IL-2 polypeptides can dissociate from, e.g., IL-2R ⁇ , at a decreased rate relative to a wild-type polypeptide or to an IL-2 based therapeutic, e.g., IL-2*.
- affinity can be characterized as the time, or average time, an IL-2* polypeptide persists on, for example, the surface of a cell expressing an IL-2R.
- an IL-2*polypeptide can persist on the receptor for at least about 2, 5, 10, 50, 100, or 250 times (or more).
- any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.
- Example 1 Detection of IL-2, IL-2 Mutein, IL-2R ⁇ and IL-2R ⁇ in Fusion Proteins by ELISA
- IL-2 mutein is detected with a commercially available antibody, e.g., the anti-IL-2 monoclonal (JES6-1A12) (BD Pharmingen; San Jose, Calif.). A positive control is used to show whether the monoclonal antibody recognizes the cytokine or mutein. Antibodies against IL-2R ⁇ and IL-2Ry chain are also used. Wells of a 96-well plate are coated with an antibody (2.5 ⁇ g/ml) in PBS. Wells are blocked with 5% non-fat milk in PBS with 0.2% Tween®20 (PBS-M-Tw) and fusion proteins are added for 1-2 hours at 37° C.
- JES6-1A12 anti-IL-2 monoclonal
- BD Pharmingen San Jose, Calif.
- a positive control is used to show whether the monoclonal antibody recognizes the cytokine or mutein.
- Antibodies against IL-2R ⁇ and IL-2Ry chain are also used. Wells of
- an anti-IL-2 biotin-labeled antibody e.g., JES5H4 (BD Pharmingen) is added and binding is detected using Streptavidin HRP (Southern Biotechnology Associates; Birmingham, Ala.).
- the ELISA plate is developed by adding 50 ⁇ l O-phenylenediamine (OPD) (Sigma-Aldrich) in 0.1M Citrate pH 4.5 and 0.04% H 2 O 2 , stopped by adding 50 ⁇ l/well 2N H 2 SO 4 and the absorbance was read at 490 nm.
- OPD O-phenylenediamine
- CTLL2 cells were plated in suspension at a concentration of 500,000 cells/well in culture media with or without 40 mg/ml human serum albumin (HSA) and stimulated with a dilution series of recombinant hIL2 or activatable hIL2 for 72 hours at 37° C. and 5% CO 2 .
- HSA human serum albumin
- Activity of uncleaved and cleaved activatable hIL2 was tested.
- Cleaved activatable hIL2 was generated by incubation with active MMP9.
- Cell activity was assessed using a CellTiter-Glo® (Promega) luminescence-based cell viability assay. Results are shown in FIGS. 8 A- 8 F , FIGS. 9 A- 9 Z , FIG. 25 C .
- the IL-2 mutein fusion proteins are biochemically characterized before and after cleavage with a protease, e.g., PSA Immunoblot analyses will show that the fusion proteins can be cleaved by PSA and that there is an increase in intensity of the predicted low molecular weight cleavage product of approximately 20 kDa reactive with an anti-IL-2 antibody after treatment of the samples with PSA.
- the degree of cleavage is dependent upon the amount of PSA as well as the time of incubation.
- the fusion protein is analyzed before and after PSA treatment by ELISA, it was found that the apparent amount of IL-2 is increased after PSA cleavage.
- the amount of IL-2 mutein increase will suggest that after PSA cleavage there is an increase in the predicted low molecular weight cleavage fragment of approximately 20 kDa reactive with an anti-IL-2 antibody, an increase in antibody accessibility, and most importantly, an increase in the amount of biologically active IL-2 mutein.
- the chimeric polypeptide is examined to determine if it could have biological effects in vivo.
- a system is used in which tumor cells injected intraperitoneally rapidly and preferentially attach and grow initially on the milky spots, a series of organized immune aggregates found on the omentum (Gerber et al., Am. J. Pathol. 169:1739-52 (2006)).
- This system offers a convenient way to examine the effects of fusion protein treatment on tumor growth since fusion proteins can be delivered intraperitoneally multiple times and tumor growth can be analyzed by examining the dissociated omental cells.
- the Colon 38 cell line a rapidly growing tumor cell line that expresses both MMP2 and MMP9 in vitro, may be used.
- the omental tissue normally expresses a relatively small amount of MMP2 and MMP9, but, when Colon 38 tumor is present on the omentum, MMP levels increase.
- MMP2 and MMP9 the ability of IL-2 mutein fusion proteins to affect tumor growth is examined Colon 38 cells are injected intraperitoneally, allowed to attach and grow for 1 day, and then treated daily with fusion protein intraperitoneally. At day 7, the animals are sacrificed and the omenta examined for tumor growth using flow cytometry and by a colony-forming assay.
- Activatable interleukin proteins are tested for their binding affinities to human CD20 + cells and cynomolgus CD20 + cells.
- CD20 + cells are incubated with 100 ⁇ L of serial dilutions of the activatable interleukin proteins and at least one protease. After washing three times with FACS buffer the cells are incubated with 0.1 mL of 10 ⁇ g/mL mouse monoclonal anti-idiotype antibody in the same buffer for 45 min on ice. After a second washing cycle, the cells are incubated with 0.1 mL of 15 ⁇ g/mL FITC-conjugated goat anti-mouse IgG antibodies under the same conditions as before. As a control, cells are incubated with the anti-His IgG followed by the FITC-conjugated goat anti-mouse IgG antibodies without the activatable IL2 proteins.
- the cells were then washed again and resuspended in 0.2 mL of FACS buffer containing 2 ⁇ g/mL propidium iodide (PI) in order to exclude dead cells.
- the fluorescence of 1 ⁇ 10 4 living cells is measured using a Beckman-Coulter FC500 MPL flow cytometer using the MXP software (Beckman-Coulter, Krefeld, Germany) or a Millipore Guava EasyCyte flow cytometer using the Incyte software (Merck Millipore, Schwalbach, Germany).
- Mean fluorescence intensities of the cell samples are calculated using CXP software (Beckman-Coulter, Krefeld, Germany) or Incyte software (Merck Millipore, Schwalbach, Germany).
- CD20 binding and crossreactivity are assessed on the human CD20 + tumor cell lines.
- the K D ratio of crossreactivity is calculated using the K D values determined on the CHO cell lines expressing either recombinant human or recombinant cynomolgus antigens.
- Activatable interleukin protein is evaluated in vitro on its mediation of immune response to CD20 + target cells.
- Fluorescence labeled CD20 + REC-1 cells (a Mantle cell lymphoma cell line, ATCC CRL-3004) are incubated with isolated PBMC of random donors or CB15 T-cells (standardized T-cell line) as effector cells in the presence of the activatable IL2 protein and at least one protease. After incubation for 4 h at 37° C. in a humidified incubator, the release of the fluorescent dye from the target cells into the supernatant is determined in a spectro-fluorimeter. Target cells incubated without the activatable IL2 protein and target cells totally lysed by the addition of saponin at the end of the incubation serve as negative and positive controls, respectively.
- the percentage of specific cell lysis is calculated according to the following formula: [1 ⁇ (number of living targets (sample) /number of living targets (spontaneous) ] ⁇ 100%.
- Sigmoidal dose response curves and EC 50 values are calculated by non-linear regression/4-parameter logistic fit using the GraphPad Software.
- the lysis values obtained for a given antibody concentration are used to calculate sigmoidal dose-response curves by 4 parameter logistic fit analysis using the Prism® GraphPad® software.
- Activatable interleukin protein is evaluated for half-time elimination in animal studies.
- the activatable IL2 protein is administered to cynomolgus monkeys as a 0.5 mg/kg bolus injection into the saphenous vein.
- Another cynomolgus monkey group receives a comparable IL2 construct in size but lacking a serum half-life extension element.
- a third and fourth group receive an IL2 construct with serum half-life extension element and a cytokine with CD20 and serum half-life extension elements respectively, and both comparable in size to the activatable interleukin protein.
- Each test group consists of 5 monkeys. Serum samples are taken at indicated time points, serially diluted, and the concentration of the proteins is determined using a binding ELISA to CD20.
- Pharmacokinetic analysis is performed using the test article plasma concentrations.
- Group mean plasma data for each test article conforms to a multi-exponential profile when plotted against the time post-dosing.
- the data are fit by a standard two-compartment model with bolus input and first-order rate constants for distribution and elimination phases.
- the ⁇ -phase is the initial phase of the clearance and reflects distribution of the protein into all extracellular fluid of the animal, whereas the second or ⁇ -phase portion of the decay curve represents true plasma clearance.
- the activatable interleukin protein has improved pharmacokinetic parameters such as an increase in elimination half-time as compared to proteins lacking a serum half-life extension element.
- Activatable IL2 protein is evaluated in a xenograft model.
- mice Female immune-deficient NOD/scid mice are sub-lethally irradiated (2 Gy) and subcutaneously inoculated with 4 ⁇ 10 6 Ramos RA1 cells into the right dorsal flank. When tumors reach 100 to 200 mm 3 , animals are allocated into 3 treatment groups. Groups 2 and 3 (8 animals each) are intraperitoneally injected with 1.5 ⁇ 10 7 activated human T-cells. Three days later, animals from Group 3 are subsequently treated with a total of 9 intravenous doses of 50 ⁇ g activatable interleukin protein. Groups 1 and 2 are only treated with vehicle. Body weight and tumor volume are determined for 30 days.
- WEHI279 cells were plated in suspension at a concentration of 25,000 cells/well in culture media with or without 1.5% human serum albumin (HSA) and stimulated with a dilution series of recombinant mIFN ⁇ or inducible mIFN ⁇ for 72 hours at 37° C. and 5% CO 2 .
- Activity of uncleaved and cleaved inducible mIFN ⁇ was tested.
- Cleaved inducible mIFN ⁇ was generated by incubation with active MMP9.
- Cell survival was assessed using a CellTiter-Glo (Promega) luminescence-based cell viability assay.
- the EC50 values for cleaved inducible mIFN ⁇ molecules were at least 100 ⁇ more potent than un-cleaved inducible mIFN ⁇ molecules. As shown in FIGS. 16 A- 16 , greater inducibility was seen in assays wherein the culture medium contained human serum albumin.
- B16-Blue IFN ⁇ cells (InvivoGen) were plated at a concentration of 75,000 cells/well in culture media with or without 1.5% human serum albumin (HSA) and stimulated with a dilution series of recombinant mIFN ⁇ or inducible mIFN ⁇ for 24 hours at 37° C. and 5% CO 2 .
- Activity of uncleaved and cleaved inducible mIFN ⁇ was tested.
- Cleaved inducible mIFN ⁇ was generated by incubation with active MMP9. Supernatants were harvested, and SEAP activation was assessed by adding QUANTI-Blue Reagent (InvivoGen), incubating at 37° C. for 2 hours, and measuring absorbance at 620 nm.
- the EC50 values for cleaved inducible mIFN ⁇ molecules were at least 100 ⁇ more potent than un-cleaved inducible mIFN ⁇ molecules. Results are shown in e.g., FIG. 19 A- 19 B , FIGS. 22 A- 22 B , FIGS. 23 A- 23 B . This experiment was repeated with for IFN ⁇ conjugates using B16-Blue IFN ⁇ / ⁇ cells. The EC50 values for cleaved inducible mIFN ⁇ molecules were at least 100 ⁇ more potent than un-cleaved inducible mIFN ⁇ molecules. See FIGS. 20 A- 20 B .
- the chimeric polypeptide is examined to determine if it could have biological effects in vivo.
- a system is used in which tumor cells injected intraperitoneally rapidly and preferentially attach and grow initially on the milky spots, a series of organized immune aggregates found on the omentum (Gerber et al., Am. J. Pathol. 169:1739-52 (2006)).
- This system offers a convenient way to examine the effects of fusion protein treatment on tumor growth since fusion proteins can be delivered intraperitoneally multiple times and tumor growth can be analyzed by examining the dissociated omental cells.
- the Colon 38 cell line a rapidly growing tumor cell line that expresses both MMP2 and MMP9 in vitro, may be used.
- the omental tissue normally expresses a relatively small amount of MMP2 and MMP9, but, when Colon 38 tumor is present on the omentum, MMP levels increase.
- Colon 38 cells are injected intraperitoneally, allowed to attach and grow for 1 day, and then treated daily with fusion protein intraperitoneally.
- the animals are sacrificed and the omenta examined for tumor growth using flow cytometry and by a colony-forming assay.
- the MC38 cell line a rapidly growing colon adenocarcinoma cell line that expresses MMP9 in vitro, was used. Using this tumor model, the ability of IFN ⁇ fusion proteins to affect tumor growth was examined MC38 cells were injected subcutaneously, allowed to grow for 10-14 days, and then treated with fusion protein twice weekly intraperitoneally for a total of four doses. As a comparator, wild type mIFN ⁇ was administered at the dose levels indicated, twice daily for 2 weeks on a 5 day on/2 day off schedule (10 total doses). Tumor growth and body weight were monitored approximately twice per week for two weeks.
- An IFN ⁇ polypeptide capable of binding to CD20 polypeptide present in a tumor or on a tumor cell is produced as follows.
- a nucleic acid is produced that contains nucleic acid sequences: (1) encoding an IFN ⁇ polypeptide sequence and (2) one or more polypeptide linkers.
- Activatable IFN ⁇ plasmid constructs can have optional Flag, His or other affinity tags, and are electroporated into HEK293 or other suitable human or mammalian cell lines and purified.
- Validation assays include T cell activation assays using T cells responsive to IFN ⁇ stimulation in the presence of a protease.
- CD20 is one of the cell surface proteins present on B-lymphocytes.
- CD20 antigen is found in normal and malignant pre-B and mature B lymphocytes, including those in over 90% of B-cell non-Hodgkin's lymphomas (NHL).
- the antigen is absent in hematopoietic stem cells, activated B lymphocytes (plasma cells) and normal tissue.
- hematopoietic stem cells activated B lymphocytes (plasma cells) and normal tissue.
- Human or humanized anti-CD20 antibodies are therefore used to generate scFv sequences for CD20 binding domains of an activatable IFN ⁇ protein.
- DNA sequences coding for human or humanized VL and VH domains are obtained, and the codons for the constructs are, optionally, optimized for expression in cells from Homo sapiens .
- the order in which the VL and VH domains appear in the scFv is varied (i.e., VL-VH, or VH-VL orientation), and three copies of the “G4S” (SEQ ID NO: 241) or “G 4 S” (SEQ ID NO: 241) subunit (G 4 S) 3 (SEQ ID NO: 242) connect the variable domains to create the scFv domain.
- Anti-CD20 scFv plasmid constructs can have optional Flag, His or other affinity tags, and are electroporated into HEK293 or other suitable human or mammalian cell lines and purified.
- Validation assays include binding analysis by FACS, kinetic analysis using Proteon, and staining of CD20-expressing cells.
- the activatable IFN ⁇ construct with protease cleavage site domains is used to construct an activatable IFN ⁇ protein in combination with an anti-CD20 scFv domain and a serum half-life extension element (e.g., a HSA binding peptide or VH domain).
- a serum half-life extension element e.g., a HSA binding peptide or VH domain.
- coding sequences of all protein domains are cloned into a mammalian expression vector system.
- gene sequences encoding the activatable IFN ⁇ domain, serum half-life extension element, and CD20 binding domain along with peptide linkers L1 and L2 are separately synthesized and subcloned.
- the resulting constructs are then ligated together in the order of CD20 binding domain-L1-IFN ⁇ subunit 1-L2-protease cleavage domain-L3-IFN ⁇ subunit2-L4-anti-CD20 scFv-L5-serum half-life extension element to yield a final construct.
- All expression constructs are designed to contain coding sequences for an N-terminal signal peptide and a C-terminal hexahistidine (6 ⁇ His)-tag (SEQ ID NO: 243) to facilitate protein secretion and purification, respectively.
- a CHO cell expression system (Flp-In®, Life Technologies), a derivative of CHO—K1 Chinese Hamster ovary cells (ATCC, CCL-61) (Kao and Puck, Proc. Natl. Acad. Sci. USA 1968; 60(4):1275-81), is used. Adherent cells are subcultured according to standard cell culture protocols provided by Life Technologies.
- cells are detached from tissue culture flasks and placed in serum-free medium. Suspension-adapted cells are cryopreserved in medium with 10% DMSO.
- Recombinant CHO cell lines stably expressing secreted activatable IFN ⁇ proteins are generated by transfection of suspension-adapted cells. During selection with the antibiotic Hygromycin B viable cell densities are measured twice a week, and cells are centrifuged and resuspended in fresh selection medium at a maximal density of 0.1 ⁇ 10 6 viable cells/mL. Cell pools stably expressing activatable IFN ⁇ proteins are recovered after 2-3 weeks of selection at which point cells are transferred to standard culture medium in shake flasks. Expression of recombinant secreted proteins is confirmed by performing protein gel electrophoresis or flow cytometry. Stable cell pools are cryopreserved in DMSO containing medium.
- Activatable IFN ⁇ proteins are produced in 10-day fed-batch cultures of stably transfected CHO cell lines by secretion into the cell culture supernatant.
- Cell culture supernatants are harvested after 10 days at culture viabilities of typically >75%. Samples are collected from the production cultures every other day and cell density and viability are assessed. On day of harvest, cell culture supernatants are cleared by centrifugation and vacuum filtration before further use.
- Protein expression titers and product integrity in cell culture supernatants are analyzed by SDS-PAGE.
- Activatable IFN ⁇ proteins are purified from CHO cell culture supernatants in a two-step procedure.
- the constructs are subjected to affinity chromatography in a first step followed by preparative size exclusion chromatography (SEC) on Superdex 200 in a second step.
- SEC preparative size exclusion chromatography
- Samples are buffer-exchanged and concentrated by ultrafiltration to a typical concentration of >1 mg/mL.
- Purity and homogeneity typically >90%) of final samples are assessed by SDS PAGE under reducing and non-reducing conditions, followed by immunoblotting using an anti-HSA or anti idiotype antibody as well as by analytical SEC, respectively.
- Purified proteins are stored at aliquots at ⁇ 80° C. until use.
- the activatable IFN ⁇ proteins are tested for their binding affinities to human CD20 + cells and cynomolgus CD20 + cells.
- CD20 + cells are incubated with 100 ⁇ L of serial dilutions of the activatable IFN ⁇ proteins and at least one protease. After washing three times with FACS buffer the cells are incubated with 0.1 mL of 10 ⁇ g/mL mouse monoclonal anti-idiotype antibody in the same buffer for 45 min on ice. After a second washing cycle, the cells are incubated with 0.1 mL of 15 ⁇ g/mL FITC-conjugated goat anti-mouse IgG antibodies under the same conditions as before. As a control, cells are incubated with the anti-His IgG followed by the FITC-conjugated goat anti-mouse IgG antibodies without the activatable IFN ⁇ proteins.
- the cells were then washed again and resuspended in 0.2 mL of FACS buffer containing 2 ⁇ g/mL propidium iodide (PI) in order to exclude dead cells.
- the fluorescence of 1 ⁇ 10 4 living cells is measured using a Beckman-Coulter FC500 MPL flow cytometer using the MXP software (Beckman-Coulter, Krefeld, Germany) or a Millipore Guava EasyCyte flow cytometer using the Incyte software (Merck Millipore, Schwalbach, Germany).
- Mean fluorescence intensities of the cell samples are calculated using CXP software (Beckman-Coulter, Krefeld, Germany) or Incyte software (Merck Millipore, Schwalbach, Germany).
- CD20 binding and cross-reactivity are assessed on the human CD20 + tumor cell lines.
- the K D ratio of cross-reactivity is calculated using the K D values determined on the CHO cell lines expressing either recombinant human or recombinant cynomolgus antigens.
- the activatable IFN ⁇ protein is evaluated in vitro on its mediation of immune response to CD20 + target cells.
- Fluorescence labeled CD20 + REC-1 cells (a Mantle cell lymphoma cell line, ATCC CRL-3004) are incubated with isolated PBMC of random donors or CB15 T-cells (standardized T-cell line) as effector cells in the presence of the activatable IFN ⁇ protein and at least one protease. After incubation for 4 h at 37° C. in a humidified incubator, the release of the fluorescent dye from the target cells into the supernatant is determined in a spectrofluorometer. Target cells incubated without the activatable IFN ⁇ protein and target cells totally lysed by the addition of saponin at the end of the incubation serve as negative and positive controls, respectively.
- the percentage of specific cell lysis is calculated according to the following formula: [1 ⁇ (number of living targets (sample) /number of living targets (spontaneous) ] ⁇ 100%.
- Sigmoidal dose response curves and EC 50 values are calculated by non-linear regression/4-parameter logistic fit using the GraphPad Software. The lysis values obtained for a given antibody concentration are used to calculate sigmoidal dose-response curves by 4 parameter logistic fit analysis using the Prism software.
- the activatable IFN ⁇ protein is evaluated for half-time elimination in animal studies.
- the activatable IFN ⁇ protein is administered to cynomolgus monkeys as a 0.5 mg/kg bolus injection into the saphenous vein.
- Another cynomolgus monkey group receives a comparable cytokine in size but lacking a serum half-life extension element.
- a third and fourth group receive a cytokine with serum half-life extension elements and a cytokine with CD20 and serum half-life extension elements respectively, and both comparable in size to the activatable IFN ⁇ protein.
- Each test group consists of 5 monkeys. Serum samples are taken at indicated time points, serially diluted, and the concentration of the proteins is determined using a binding ELISA to CD20.
- Pharmacokinetic analysis is performed using the test article plasma concentrations.
- Group mean plasma data for each test article conforms to a multi-exponential profile when plotted against the time post-dosing.
- the data are fit by a standard two-compartment model with bolus input and first-order rate constants for distribution and elimination phases.
- the ⁇ -phase is the initial phase of the clearance and reflects distribution of the protein into all extracellular fluid of the animal, whereas the second or ⁇ -phase portion of the decay curve represents true plasma clearance.
- the activatable IFN ⁇ protein has improved pharmacokinetic parameters such as an increase in elimination half-time as compared to proteins lacking a serum half-life extension element.
- the activatable IFN ⁇ protein is evaluated in a xenograft model.
- mice Female immune-deficient NOD/scid mice are sub-lethally irradiated (2 Gy) and subcutaneously inoculated with 4 ⁇ 10 ⁇ Ramos RA1 cells into the right dorsal flank. When tumors reach 100 to 200 mm3, animals are allocated into 3 treatment groups. Groups 2 and 3 (8 animals each) are intraperitoneally injected with 1.5 ⁇ 10 7 activated human T-cells. Three days later, animals from Group 3 are subsequently treated with a total of 9 intravenous doses of 50 ⁇ g activatable IFN ⁇ protein. Groups 1 and 2 are only treated with vehicle. Body weight and tumor volume are determined for 30 days.
- HEK-Blue IL12 cells (InvivoGen) were plated in suspension at a concentration of 250,000 cells/well in culture media with or without 40 mg/ml human serum albumin (HSA) and stimulated with a dilution series of recombinant hIL12, chimeric IL12 (mouse p35/human p40) or activatable hIL12 for 24 hours at 37° C. and 5% CO 2 .
- Activity of uncleaved and cleaved activatable hIL12 was tested.
- Cleaved inducible hIL12 was generated by incubation with active MMP9.
- IL12 activity was assessed by quantification of Secreted Alkaline Phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen), a colorimetric based assay.
- SEAP Secreted Alkaline Phosphatase
- HEK-Blue IL2 cells (InvivoGen) were plated in suspension at a concentration of 50,000 cells/well in culture media with or without 15-40 mg/ml human serum albumin (HSA) and stimulated with a dilution series of recombinant hIL2 or activatable hIL2 for 24 hours at 37° C. and 5% CO 2 .
- HSA human serum albumin
- Activity of uncleaved and cleaved activatable hIL2 was tested.
- Cleaved inducible hIL2 was generated by incubation with active MMP9 or another protease.
- IL2 activity was assessed by quantification of Secreted Alkaline Phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen), a colorimetric based assay. Results are shown in FIGS. 59 - 62 .
- SEAP Secreted Alkaline Phosphatase
- T-Blasts were induced from murine splenocytes with a 6-day incubation with PHA and a 24 hr incubation with recombinant hIL12.
- T-blasts were then plated in suspension at a concentration of 200,000 cells/well in culture media with or without 40 mg/ml human serum albumin (HSA) and stimulated with a dilution series of recombinant hIL12 or chimeric IL12 (mouse p35/human p40) or mouse IL12 for 72 hours at 37° C. and 5% CO 2 .
- HSA human serum albumin
- HSA human serum albumin
- Cleaved inducible hIL12 was generated by incubation with active MMP9.
- IL12 activity was assessed by downstream quantification of IFN ⁇ production using a mIFN ⁇ alphaLISA.
- Example 22 In Vivo Delivery of a Protease Activated Fusion Protein Results in Decreased Tumor Growth
- the chimeric polypeptide is examined to determine if it could have biological effects in vivo.
- a system is used in which tumor cells injected intraperitoneally rapidly and preferentially attach and grow initially on the milky spots, a series of organized immune aggregates found on the omentum (Gerber et al., Am. J. Pathol. 169:1739-52 (2006)).
- This system offers a convenient way to examine the effects of fusion protein treatment on tumor growth since fusion proteins can be delivered intraperitoneally multiple times and tumor growth can be analyzed by examining the dissociated omental cells.
- the Colon 38 cell line a rapidly growing tumor cell line that expresses both MMP2 and MMP9 in vitro, may be used.
- the omental tissue normally expresses a relatively small amount of MMP2 and MMP9, but, when Colon 38 tumor is present on the omentum, MMP levels increase.
- MMP2 and MMP9 the ability of IL-2 mutein fusion proteins to affect tumor growth is examined Colon 38 cells are injected intraperitoneally, allowed to attach and grow for 1 day, and then treated daily with fusion protein intraperitoneally. At day 7, the animals are sacrificed and the omenta examined for tumor growth using flow cytometry and by a colony-forming assay.
- Example 23A Construction of an Exemplary Activatable Interleukin Protein TargetingCD20
- the human IL-12p35 chain canonical sequence is UniProt Accession No. P29459.
- the human IL-12p40 chain canonical sequence is UniProt Accession No. P29460.
- IL-12p35 and IL-12p40 are cloned into an expression construct.
- a protease cleavage site is included between the IL-12p35 and IL-12p40 domains.
- An IL-12 polypeptide capable of binding to CD20 polypeptide present in a tumor or on a tumor cell is produced as follows.
- a nucleic acid is produced that contains nucleic acid sequences: (1) encoding an IFN ⁇ polypeptide sequence and (2) one or more polypeptide linkers.
- Activatable interleukin plasmid constructs can have optional Flag, His or other affinity tags, and are electroporated into HEK293 or other suitable human or mammalian cell lines and purified.
- Validation assays include T cell activation assays using T cells responsive to IL-12 stimulation in the presence of a protease.
- CD20 is one of the cell surface proteins present on B-lymphocytes.
- CD20 antigen is found in normal and malignant pre-B and mature B lymphocytes, including those in over 90% of B-cell non-Hodgkin's lymphomas (NHL).
- the antigen is absent in hematopoietic stem cells, activated B lymphocytes (plasma cells) and normal tissue.
- hematopoietic stem cells activated B lymphocytes (plasma cells) and normal tissue.
- Human or humanized anti-CD20 antibodies are therefore used to generate scFv sequences for CD20 binding domains of an activatable interleukin protein.
- DNA sequences coding for human or humanized VL and VH domains are obtained, and the codons for the constructs are, optionally, optimized for expression in cells from Homo sapiens .
- the order in which the VL and VH domains appear in the scFv is varied (i.e., VL-VH, or VH-VL orientation), and three copies of the “G4S” (SEQ ID NO: 241) or “G 4 S” (SEQ ID NO: 241) subunit (G 4 S) 3 (SEQ ID NO: 242) connect the variable domains to create the scFv domain.
- Anti-CD20 scFv plasmid constructs can have optional Flag, His or other affinity tags, and are electroporated into HEK293 or other suitable human or mammalian cell lines and purified.
- Validation assays include binding analysis by FACS, kinetic analysis using Proteon, and staining of CD20-expressing cells.
- the activatable interleukin construct with protease cleavage site domains are used to construct an activatable interleukin protein in combination with an anti-CD20 scFv domain and a serum half-life extension element (e.g., a HSA binding peptide or VH domain).
- a serum half-life extension element e.g., a HSA binding peptide or VH domain.
- coding sequences of all protein domains are cloned into a mammalian expression vector system.
- gene sequences encoding the activatable interleukin domain, serum half-life extension element, and CD20 binding domain along with peptide linkers L1 and L2 are separately synthesized and subcloned.
- the resulting constructs are then ligated together in the order of CD20 binding domain-L1-IL-12p35-L2-protease cleavage domain-L3-IL-12p40-L4-anti-CD20 scFv-L5-serum half-life extension element to yield a final construct.
- All expression constructs are designed to contain coding sequences for an N-terminal signal peptide and a C-terminal hexahistidine (6 ⁇ His)-tag (SEQ ID NO: 243) to facilitate protein secretion and purification, respectively.
- a CHO cell expression system (Flp-In®, Life Technologies), a derivative of CHO—K1 Chinese Hamster ovary cells (ATCC, CCL-61) (Kao and Puck, Proc. Natl. Acad Sci USA 1968; 60(4):1275-81), is used. Adherent cells are subcultured according to standard cell culture protocols provided by Life Technologies.
- cells are detached from tissue culture flasks and placed in serum-free medium. Suspension-adapted cells are cryopreserved in medium with 10% DMSO.
- Recombinant CHO cell lines stably expressing secreted activatable interleukin proteins are generated by transfection of suspension-adapted cells. During selection with the antibiotic Hygromycin B viable cell densities are measured twice a week, and cells are centrifuged and resuspended in fresh selection medium at a maximal density of 0.1 ⁇ 10 6 viable cells/mL. Cell pools stably expressing activatable interleukin proteins are recovered after 2-3 weeks of selection at which point cells are transferred to standard culture medium in shake flasks. Expression of recombinant secreted proteins is confirmed by performing protein gel electrophoresis or flow cytometry. Stable cell pools are cryopreserved in DMSO containing medium.
- Activatable interleukin proteins are produced in 10-day fed-batch cultures of stably transfected CHO cell lines by secretion into the cell culture supernatant.
- Cell culture supernatants are harvested after 10 days at culture viabilities of typically >75%. Samples are collected from the production cultures every other day and cell density and viability are assessed. On day of harvest, cell culture supernatants are cleared by centrifugation and vacuum filtration before further use.
- Protein expression titers and product integrity in cell culture supernatants are analyzed by SDS-PAGE.
- Activatable interleukin proteins are purified from CHO cell culture supernatants in a two-step procedure.
- the constructs are subjected to affinity chromatography in a first step followed by preparative size exclusion chromatography (SEC) on Superdex 200 in a second step.
- SEC preparative size exclusion chromatography
- Samples are buffer-exchanged and concentrated by ultrafiltration to a typical concentration of >1 mg/mL.
- Purity and homogeneity typically >90%) of final samples are assessed by SDS PAGE under reducing and non-reducing conditions, followed by immunoblotting using an anti-HSA or anti idiotype antibody as well as by analytical SEC, respectively.
- Purified proteins are stored at aliquots at ⁇ 80° C. until use.
- Activatable interleukin proteins are tested for their binding affinities to human CD20 + cells and cynomolgus CD20 + cells.
- CD20 + cells are incubated with 100 ⁇ L of serial dilutions of the activatable interleukin proteins and at least one protease. After washing three times with FACS buffer the cells are incubated with 0.1 mL of 10 ⁇ g/mL mouse monoclonal anti-idiotype antibody in the same buffer for 45 min on ice. After a second washing cycle, the cells are incubated with 0.1 mL of 15 ⁇ g/mL FITC-conjugated goat anti-mouse IgG antibodies under the same conditions as before. As a control, cells are incubated with the anti-His IgG followed by the FITC-conjugated goat anti-mouse IgG antibodies without the activatable interleukin proteins.
- the cells were then washed again and resuspended in 0.2 mL of FACS buffer containing 2 ⁇ g/mL propidium iodide (PI) in order to exclude dead cells.
- the fluorescence of 1 ⁇ 10 4 living cells is measured using a Beckman-Coulter FC500 MPL flow cytometer using the MXP software (Beckman-Coulter, Krefeld, Germany) or a Millipore Guava EasyCyte flow cytometer using the Incyte software (Merck Millipore, Schwalbach, Germany).
- Mean fluorescence intensities of the cell samples are calculated using CXP software (Beckman-Coulter, Krefeld, Germany) or Incyte software (Merck Millipore, Schwalbach, Germany).
- CD20 binding and cross-reactivity are assessed on the human CD20 + tumor cell lines.
- the K D ratio of cross-reactivity is calculated using the K D values determined on the CHO cell lines expressing either recombinant human or recombinant cynomolgus antigens.
- the activatable interleukin protein is evaluated in vitro on its mediation of immune response to CD20 + target cells.
- Fluorescence labeled CD20 + REC-1 cells (a Mantle cell lymphoma cell line, ATCC CRL-3004) are incubated with isolated PBMC of random donors or CB15 T-cells (standardized T-cell line) as effector cells in the presence of the activatable interleukin protein and at least one protease. After incubation for 4 h at 37° C. in a humidified incubator, the release of the fluorescent dye from the target cells into the supernatant is determined in a spectrofluorometer. Target cells incubated without the activatable interleukin protein and target cells totally lysed by the addition of saponin at the end of the incubation serve as negative and positive controls, respectively.
- the percentage of specific cell lysis is calculated according to the following formula: [1 ⁇ (number of living targets (sample) /number of living targets (spontaneous) ] ⁇ 100%.
- Sigmoidal dose response curves and EC 50 values are calculated by non-linear regression/4-parameter logistic fit using the GraphPad Software. The lysis values obtained for a given antibody concentration are used to calculate sigmoidal dose-response curves by 4 parameter logistic fit analysis using the Prism software.
- the activatable interleukin protein is evaluated for half-time elimination in animal studies.
- the activatable interleukin protein is administered to cynomolgus monkeys as a 0.5 mg/kg bolus injection into the saphenous vein.
- Another cynomolgus monkey group receives a comparable cytokine in size but lacking a serum half-life extension element.
- a third and fourth group receive a cytokine with serum half-life extension elements and a cytokine with CD20 and serum half-life extension elements respectively, and both comparable in size to the activatable interleukin protein.
- Each test group consists of 5 monkeys. Serum samples are taken at indicated time points, serially diluted, and the concentration of the proteins is determined using a binding ELISA to CD20.
- Pharmacokinetic analysis is performed using the test article plasma concentrations.
- Group mean plasma data for each test article conforms to a multi-exponential profile when plotted against the time post-dosing.
- the data are fit by a standard two-compartment model with bolus input and first-order rate constants for distribution and elimination phases.
- the ⁇ -phase is the initial phase of the clearance and reflects distribution of the protein into all extracellular fluid of the animal, whereas the second or ⁇ -phase portion of the decay curve represents true plasma clearance.
- the activatable interleukin protein has improved pharmacokinetic parameters such as an increase in elimination half-time as compared to proteins lacking a serum half-life extension element.
- Activatable interleukin protein is evaluated in a xenograft model.
- mice Female immune-deficient NOD/scid mice are sub-lethally irradiated (2 Gy) and subcutaneously inoculated with 4 ⁇ 10 6 Ramos RA1 cells into the right dorsal flank. When tumors reach 100 to 200 mm 3 , animals are allocated into 3 treatment groups. Groups 2 and 3 (8 animals each) are intraperitoneally injected with 1.5 ⁇ 10 7 activated human T-cells. Three days later, animals from Group 3 are subsequently treated with a total of 9 intravenous doses of 50 ⁇ g activatable interleukin protein. Groups 1 and 2 are only treated with vehicle. Body weight and tumor volume are determined for 30 days.
- the MC38 cell line a rapidly growing colon adenocarcinoma cell line that expresses MMP9 in vitro, was used. Using this tumor model, the ability of fusion proteins to affect tumor growth was examined.
- Example 27A MC38 IL-2POC
- Example 27B MC38 IL-2
- mice were anaesthetized with isoflurane for implant of cells to reduce the ulcerations.
- 308 CR female C57BL/6 mice were set up with 5 ⁇ 105 MC38 tumor cells in 0% Matrigel sc in flank.
- Cell Injection Volume was 0.1 mL/mouse.
- Mouse age at start date was 8 to 12 weeks. Pair matches were performed when tumors reach an average size of 100-150 mm 3 and begin treatment. Body weights were taken at initiation and then biweekly to the end. Caliper measurements were taken biweekly to the end. Any adverse reactions were to be reported immediately.
- Dosing volume was 0.2 mL/mouse for IL-2-WTI; 0.3 mL for ACP16, ACP130; 0.5 mL for ACP124. Do not adjust for body weight.
- ACP16 current amount of required compound—13.45 mg
- ACP130 current amount of required compound ⁇ 25.83 mg
- ACP124 current amount of required compound ⁇ 42.31 mg
- IL-2-WTI current amount of required compound ⁇ 9.98 mg
- Necropsy was to be performed in case of unexpected toxicity
- Example 27C MC38 IFN ⁇ and IL-12
- mice were anaesthetized with isoflurane for implant of cells to reduce the ulcerations.
- 308 CR female C57BL/6 mice were set up with 5 ⁇ 10 5 MC38 tumor cells in 0% Matrigel sc in flank.
- Cell Injection Volume was 0.1 mL/mouse.
- Mouse age at start date was 8 to 12 weeks. Pair matches were performed when tumors reach an average size of 100-150 mm 3 and begin treatment. Body weights were taken at initiation and then biweekly to the end. Caliper measurements were taken biweekly to the end. Any adverse reactions were to be reported immediately.
- ACP11 For ACP11, ACP31, ACP131, the amount needed per week was calculated, aliquoted accordingly, and stored at ⁇ 20 C. For each week of dosing, one aliquot was thawed and stored at 4 C, the required amount was diluted with PBS right before each injection.
- PBS was used as the vehicle for all tests.
- Example 27D MC38 Rechallenge
- mice were anaesthetized with isoflurane for implant of cells to reduce the ulcerations. This portion of the study began on the day of implant (Day 1).
- Group 1 consisted of 33 CR female C57BL/6 mice set up with 5 ⁇ 105 MC38 tumor cells in 0% Matrigel subcutaneously in the flank.
- Groups 2-6 consisted of 33 CR female C57BL/6 mice set up with 5 ⁇ 105 MC38 tumor cells in 0% Matrigel sc in the left flank.
- the tumors from the previous MC38 experiment (example 25 ⁇ ) were implanted in the right flank of each animal Cell Injection Volume was 0.1 mL/mouse. Age of control mice at initiation was 14 to 17 weeks.
- mice were age matched to mice from the previous MC38 experiment (example 25 ⁇ ). No dosing of active agent occurred during rechallenge. Body Weights were taken biweekly until end, as were caliper measurements. Any adverse reactions or death were reported immediately. Any individual animal with a single observation of >than 30% body weight loss or three consecutive measurements of >25% body weight loss was euthanized Endpoint was tumor growth delay (TGD). Animals were monitored individually. The endpoint of the experiment was a tumor volume of 1000 mm3 or 45 days, whichever comes first. Responders were followed longer when possible. When the endpoint is reached, the animals were euthanized.
- TGD tumor growth delay
- Example 27E Treatment with ACP16, APC153, ACP155, and ACP156 (see FIG. 58 )
- mice were anaesthetized with isoflurane for implant of cells to reduce the ulcerations.
- CR female C57BL/6 mice were set up with 5 ⁇ 10 5 MC38 tumor cells in 0% Matrigel sc in flank.
- Cell Injection Volume was 0.1 mL/mouse.
- Mouse age at start date was 8 to 12 weeks. Pair matches were performed when tumors reach an average size of 100-150 mm 3 and begin treatment.
- ACP16 was dosed at 17, 55 or 230 ⁇ g/animal;
- ACP153, ACP155 and ACP156 were dosed at 55 or 230 ⁇ g/animal
- Body weights were taken at initiation and then biweekly to the end. Caliper measurements were taken biweekly to the end. Any adverse reactions were to be reported immediately.
- FRET fluorescent resonance energy transfer
- Protease specificity screening was performed using Multiplexed Substrate Profiling by Mass Spectrometry (MSP-MS), the method described in, e.g. O'Donoghue A. J. et al., Nat Methods., 2012; 9(11): 1095-1100.
- MSP-MS Multiplexed Substrate Profiling by Mass Spectrometry
- This method employs a physico-chemically diverse peptide library as substrates for proteases, and reactions are monitored over time with mass spectrometric detection of cleaved products. The resulting cleavages are assessed for specific cleavage in the enzyme-treated sample by comparison with results from a no-enzyme control incubation. Sequence logos for motif analysis are generated with iceLogo software (v.1.2) (iomics.ugent.be/icelogoserver/).
- Recombinant human enzymes were sourced from R&D Systems (Bio-techne): CTSL1 (#952-CY), ADAM17 (a.k.a. TACE, TNF-alpha Converting Enzyme) (#930-ADB), FAP-alpha (#930-ADB-010), MMP-14 (#918-MP-010), furin (#1503-SE-010), MMP-9 (#911-MP-010), thrombin (#2196-SE-200), thermolysin (#3097-ZN), Factor Xa (#1063-SE-010), hepsin (#4776-SE) and ADAM-TS1 (#2197-AD). Enzymes were activated and assayed following the manufacturer's recommendations.
- MSP-MS reactions were then started with the mixing of enzyme and substrate, at final CTSL1 concentration of 0.04 ng/ ⁇ l (0.77 nM), and peptide substrate concentration at 500 nM.
- ADAM17 activity was monitored following manufacturer's recommended conditions using the FRET substrate Mca-PLAQAV-Dpa-RSSSR—NH2 (SEQ ID NO: 244), in the recommended assay buffer: 25 mM TRIS, 2.5 ⁇ M ZnCl 2 at the optimal pH 9.0, and also at physiological pH 7.4.
- ADAM17 enzyme specificity was profiled in the MSP-MS experiment at pH 9.0 using 10 nM enzyme, and at pH 7.4 using 50 nM enzyme.
- MMP-14 was activated following manufacturer's recommended conditions by pre-incubation with the enzyme furin, at a molar ratio of 1:100 furin to MMP-14, at 37° C. and pH 9.0 for 1.5 hours. FRET assays were performed using 20 nM MMP-14 at 37° C. in activation buffer: 50 mM TRIS HCl, 3 mM CaCl 2 , 1 ⁇ M ZnCl 2 at pH 8.5.
- MMP9 was activated by incubation with 1 mM p-amino phenyl mercuric acetate (prepared from a stock at 100 mM in DMSO) for 24h at 37° C. in activation buffer: 50 mM TRIS HCl (pH 7.5), 10 mM CaCl 2 , 150 mM NaCl, 0.05% Brij35.
- activation buffer 50 mM TRIS HCl (pH 7.5), 10 mM CaCl 2 , 150 mM NaCl, 0.05% Brij35.
- the final enzyme concentration used for fluorescence assays was 2.5 nM.
- FAP ⁇ activity was tested with fluorescence detection using a generic Z-GP-AMC substrate.
- the final enzyme concentration was 5 nM and the buffer was 50 mM TRIS HCl, 1 M NaCl, 1 mg/mL BSA, pH 7.5.
- Thrombin was activated following the manufacturer's recommended conditions by pre-incubation with the enzyme with thermolysin for 15 min, and then the thermolysin was quenched with 1,10 phenanthroline treatment.
- Activated thrombin was assayed at 1.2 nM in activation buffer: 50 mM TRIS HCl, 10 mM CaCl 2 , 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5.
- Factor Xa was assayed at 4.7 nM enzyme, at 37° C. in the manufacturer recommended buffer: 50 mM TRIS, 10 mM CaCl 2 , 150 mM NaCl, pH 7.5.
- Hepsin was pre-activated overnight at 37° C. in the manufacturer recommended buffer: 100 mM TRIS, 10 mM CaCl 2 , 150 mM NaCl, 0.05% Brij-35, pH 8.0. Activity was measured with 2.4 or 24 nM enzyme in the buffer: 50 mM TRIS HCl at pH 7.4.
- ADAM-TS1 was assayed at 5 ⁇ M enzyme concentration in the manufacturer recommended buffer: 50 mM TRIS, 10 mM CaCl2, 150 mM NaCl at pH 7.5.
- Conditioned media and cell lysate samples were received from Charles River Laboratories for the murine colon cancer cell lines: MC38 (epithelial), CT26 (fibroblast), and CT26 transduced with a lentivirus for doxycyclin-inducible expression of MMP9 (CT26 pLVX MMP9-5T4 +dox).
- An immortalized mouse intestinal myofibroblast cell line (ABM Good #T0565) was used as a control stromal cell line, grown under manufacturer recommended conditions.
- Conditioned media and cell lysate samples were also received for screening purposes from ABM Good for the immortalized mouse colonic epithelial cell line YAMC (ABM Good #T0567).
- Conditioned media samples were prepared according to the standard protocol using logarithmic phase cells. Briefly, cells were grown for 16h in either serum-free media or in complete media containing 10% fetal bovine serum (FBS). After conditioned media collection, the adherent cells were washed then lysed on plate with non-denaturing lysis buffer to produce a lysed “cell pellet” to capture any activity that remained associated with the cells. All cell culture-derived conditioned media samples, cell lysates from Charles River Laboratories, and cell lysates derived internally were processed with identical methods, to allow comparison between samples.
- FBS fetal bovine serum
- the resulting conditioned media was buffer exchanged with PBS and concentrated to 10 ⁇ the original titer as a stock solution.
- FBS fetal bovine serum
- K cat is the rate of product formation under steady-state conditions in units (s ⁇ 1 )
- K M is the Michaelis constant that gives the substrate concentration in molar units (M) at which half maximum velocity of the reaction is produced for a given enzyme
- [E 0 ] is the enzyme concentration
- [S] the substrate concentration in (M).
- the Michaelis-Menten parameters are as classically defined above.
- the effective titer of conditioned media per assay was adjusted to approximately a 2 ⁇ concentrate of the original unprocessed media samples to assure that the detection range matched the signal produced.
- the enzyme component in the calculation is redefined as a mixture of possible proteases that contribute to the cumulative observed activity, each with intrinsic reactivity toward a substrate. In this case, it is more appropriate to consider k cat and K M as macroscopic constants that represent the overall efficiency of catalysis, and E 0 is an enzyme equivalent, expressed in units of concentration.
- k cat /K M can be fixed at that of the recombinant enzyme and enzyme equivalents per cell culture volume can be output.
- the effective titer of enzyme equivalents in this case can be used to compare the activity produced by the different cell lines.
- the ratios of enzyme equivalents between tumor and control cell lines may be a contributing factor to therapeutic index in a pro-drug employing protease-cleavable linkers.
- the ADAM17 substrate with the sequence LAQKLKSS (ADAM17_2) (SEQ ID NO: 201), based on the results in the end-point screen experiments, had approximately 3-fold greater activity in the conditioned media produced from the three tumor cell lines, compared to the myofibroblast cell line ( FIG. 36 , black bars).
- the activity of recombinant ADAM17 at 12 nM is shown in the same assay format.
- the steady state kinetic curves for ADAM17 substrate are shown in FIG. 37 . There was essentially undetectable ADAM17_2 background activity in the myofibroblast sample.
- CTSL1_1 The CTSL1 substrate, ALFKSSFP (SEQ ID NO: 198) (CTSL1_1), based on the results in the end-point screen experiments, had undetectable activity in the control myofibroblast cell line, as well had low activity in the MC38 and CT26 parental cell lines, and had undetectable activity in the CT26-MMP9+ cell line ( FIG. 36 ).
- This assay was benchmarked with CTSL1 at 1.5 nM, showing that the extracellular concentration of secreted CTSL1 activity was below this effective concentration across the four cell lines.
- Re-analysis of conditioned media with the CTSL1_1 substrate using the steady-state kinetics analysis yielded no measurable activity above background.
- the secreted activity for FAP ⁇ was very low, but was still detected in all three tumor cell lines ( FIG. 36 , blue bars).
- the FAP ⁇ _1 activity was benchmarked with 5 nM FAP ⁇ enzyme. This enzyme is also found in a plasma membrane-associated form, therefore FAP ⁇ activity was also tested in the cell pellet fraction that was collected after the media conditioning procedure.
- the MMP substrates MMP9_1 and MMP14_1 all had high activity in the end point screen, and the activity was significantly higher in the tumor cell lines than in the myofibroblast control cells.
- MMP9_1 (second column from right, FIG. 38 ) had approximately 4-fold greater activity in the tumor lines than the myofibroblast control; and MMP14_1 had approximately 57-fold greater activity in the tumor lines.
- the MMP14_1 substrate had the lowest activity in the myofibroblast cells, thus contributing to the higher ratio of tumor vs control in this assay. Secreted activity was measured for the MMP9_1 substrate in the conditioned media for each of the tumor cell lines using the steady-state kinetics analysis ( FIG. 36 ).
- the myofibroblast cell line had an artefact in analysis of MMP9_1 secreted activity ( FIG. 36 ); this will be addressed with a repeat analysis at higher enzyme titer.
- the MMP14_1 substrate had significant measurable activity in the conditioned media from the tumor cells but not in the myofibroblast cell line.
- CTSL1_1 cleavage in tumor vs control cell lines in this screening format was ⁇ 1 ⁇ (grey bars, left-hand bar of each pair, FIG. 41 ).
- FAP ⁇ fibroblast activating protein-alpha
- CT26 fibroblast
- myofibroblast cell lines an additional non-fibroblast, epithelial cell line was obtained as an alternate negative control cell line, comparable to the MC38 epithelial cell line.
- YAMC Immortalized Mouse Colonic Epithelial Cell Line
- YAMC cell line generally had more background activity than did the myofibroblast cell line with all of the substrates.
- This cell line required non-standard cell culture conditions, including growth with murine IFN ⁇ , and at low temperature to maintain, and may not be fully representative of epithelial cell lines.
- Example 33 Cleavage of CTSL1_2 Motif in the Context of an 8-Mer FRET Peptide and in the Context of an Extended Tandem Linker
- CTSL1_2 motif has the sequence ALFFSSPP (SEQ ID NO: 199).
- CTSL1 cleaves the FRET substrate bearing the CTSL1_1 motif highly efficiently ( FIG. 46 ).
- initial experiments with the CTSL1_2 FRET substrate were inconclusive, possibly due to artifacts in the FRET assay; therefore, a new FRET peptide construct was designed with the 9-mer sequence ALFFSSPPS (SEQ ID NO: 236). This 9-mer FRET substrate bearing the CTSL1_2 motif produced comparable catalytic efficiency to the CTSL1_1 motif.
- CTSL1_1 (SEQ ID NO: 207) SGGPGGPAGIGALFKSSFPLAQKLKSSGGG (CTSL1_2) (SEQ ID NO: 219) KSGPGGPAGIGALFFSSPPLAQKLKSSGGR
- CTSL1_1 had a ⁇ 1 site product at ALF
- CTSK Cathepsin K
- the substrate CTSL1_2 although lacking a basic P1 residue (ALFFSSPP (SEQ ID NO: 199)) was measurably cleaved by CTSK but at a 15 ⁇ lower level than the CTSL1_1 substrate (ALFKSSFP (SEQ ID NO: 198)) FIG. 47 .
- the specific activity for reference substrates such as Z-LR-AMC and Z-FR-AMC is 15,000-25,000 pmol/min/ ⁇ g, with the average value indicated by a dashed line in FIG. 47 .
- the library of tandem linker sequences was designed to incorporate three individual protease motifs within the context of a longer, 30-mer peptide sequence.
- Table 11 lists the sequences and motif arrangement of the 30-mer peptides.
- motifs A, B, and C were arranged in all permutations.
- the combination of motifs A, B, and C could be arranged as: A-B-C, C-A-B, B-C-A, B-A-C, A-C-B, and C-B-A.
- MMP14_1, ADAM17_2, and CTSL1_1 were tested for the tandem combinations of MMP14_1, ADAM17_2, and CTSL1_1 as a broad set of motifs susceptible to matrix metalloprotease (MMP) activity (ALU30-11 to -16), and for FAPa_1 with ADAM17_2, and CTSL1_1 as a set with lower MMP sensitivity (ALU30-5 to -10).
- MMP matrix metalloprotease
- ADAM17 and CTSL1 Two additional peptides were designed, swapping the ADAM17_2 motif with ADAM17_1 (in ALU30-17), or CTSL1_1 with CTSL1_2 (in ALU30-18), demonstrating a strategy for testing further tandem linker designs with new individual protease motifs.
- Catalytic efficiency (k cat /K M ) estimations were used to rank the top cleaved substrates in the MSP-MS reaction.
- Peptides were quantified by mass spectrometric label-free quantitation from the MS1 precursor ion peak areas for each peptide.
- Enzyme progress curves were modeled from six-time point measurements in GraphPad Prism. Data were fitted using non-linear least squares fitting to the first order kinetics equation:
- the observed rate is a function of the enzyme concentration [E 0 ], and an observed catalytic efficiency (k cat /K M ) in units M ⁇ 1 s ⁇ 1
- the substrate degradation traces for the 30-mer library peptides are shown from MMP9-treatment in FIG. 55 .
- the most efficient MMP9 substrates were those that bore three-repeats of the MMP14_1 motif.
- the next most efficient substrates contained the pair of motifs where MMP14_1 was followed directly by ADAM17_2.
- the single or double MMP14_1 motif-bearing peptides were next in the order.
- An unanticipated result was that the FAPa_1 motif was also cleaved within the peptides bearing FAPa_1 directly followed by the CTSL1_1 motif at PGGPAG
- the FAPa_1 motif was not cleaved by MMP9, however, when followed either by the ADAM17_2 motif or the spacing residues GG.
- the slowest peptides bear the MMP14_1 motif near the C-terminus of the 30-mer peptide. From this experiment, a trend emerged that MMP9 has additional sequence preferences in the downstream prime-side positions, and that the combination of MMP14_1 upstream of ADAM17_2 is most efficient.
- FAP ⁇ was able to cleave both FAP ⁇ _1 and MMP14_1 motifs, at PGGP
- Degradation of the 30-mer peptides by FAP ⁇ showed highest cleavage activity toward the peptides bearing tandem MMP14_1 motifs ( FIG. 50 , Alu30-4 or Alu30-3).
- the peptide bearing two MMP14_1 motifs (Alu30-2) was also efficiently cleaved.
- FAP ⁇ _1 motifs were cleaved more efficiently when followed by the ADAM17_1 or ADAM17_2 motifs than when followed by CTSL1_1 or CTSL1_2.
- the Lys-bearing ADAM17_2 or CTSL1_1 motifs were lower efficiency than the new motifs tested in this experiment, ADAM17_1 and CTSL1_2.
- FAP ⁇ _1 motif Another trend was a preference for cleavage of the FAP ⁇ _1 motif in the first or second position relative to the N-terminus of these peptides.
- FAP ⁇ _1 motif When the FAP ⁇ _1 motif was in the third position closest to the C-terminus, these peptides were not cleaved (Alu30-7 or Alu30-8).
- the MMP14_1 motif was readily cleaved in the third position closest to the C-terminus, but this was most evident in the tandem MMP14_1 motif-bearing peptides, where N-terminal cleavages assist in shortening the 30-mer.
- FAP ⁇ could serve as a secondary-cleaving enzyme after activation with a first enzyme elsewhere in a hybrid linker design.
- CTSL1 treatment of the 30-mer peptide library produced cleavages in all peptides bearing a CTSL1_1 or CTSL1_2 motif ( FIG. 51 ).
- the most efficiently cleaved peptides were those containing the FAPa_1, CTSL1_1 and ADAM17_2 motifs, in multiple orders.
- Peptides bearing the MMP14_1 motif with CTSL1_1 and ADAM17_2 were slightly lower efficiency.
- CTSL1 motifs in the middle of the 30-mer peptides were also favorable, thus CTSL1 may be able to serve as a first cleavage in the hybrid motifs.
- ADAM17 produced cleavages in all peptides bearing an ADAM17_1 or ADAM17_2 motif as well ( FIG. 52 ).
- the most efficient peptide cleavages occurred in the peptides containing ADAM17_2, CTSL1_1 and FAPa_1 motifs. Slightly lower efficiency cleavages were obtained in peptides containing the MMP14_1 with ADAM17_2 and CTSL1_1.
- Peptides bearing only the MMP14_1 motif were not cleaved.
- the library peptide Alu30-19 was designed to include authentic sites for each enzyme, for comparison of overall cleavage efficiency as well as cleavage site specificity within each motif.
- the substrate designed for Factor Xa has the motif FNPR
- the substrate motif for Thrombin was FPR
- the substrate motif for hepsin was RKRR
- FIG. 53 Shown in FIG. 53 are the results of Factor Xa cleavage.
- the authentic cleavage site motif was Peptide Alu30-19 with this motif was completely degraded before the 5 min time point. Products of the Alu30-19 cleavage were formed from all three sites in this peptide. In comparison, the only library peptide with significant cleavage was Alu30-5, cleaved at the CTSL1_1 motif ALFKS
- CTSL1_1 motif all other peptides bearing a CTSL1_1 motif were cleaved by Factor Xa at either ALF
- the ADAM17 motifs were also cleaved at KLK
- the peptide with the motif CTSL1_2 was cleaved at the lowest efficiency.
- Thrombin showed higher activity than Factor Xa, and it cleaved each of the 30-mer peptides.
- the MMP14_1 motifs were cleaved at GPAG
- the two alternate ADAM17 motifs were cleaved with similar efficiency (peptides Alu30-17 vs Alu30-9). However, the CTSL1_2 motif had lower cleavage activity than did the CTSL1_1 motif (peptides Alu30-18 vs Alu30-6).
- the peptides Alu30-1 and Alu30-3 were the most rapidly cleaved in the library, and Alu30-4 was also an efficient substrate, showing the susceptibility of MMP14_1 motifs in this experiment.
- the peptide Alu30-2, bearing two MMP14_1 motifs was less susceptible to thrombin, however, potentially due to altered spacing with additional glycine residues between motifs.
- peptides Alu30-12, Alu30-13 and Alu30-15, with MMP141 motifs in combination with ADAM17_2 and CTSL1_1, were cleaved at lower efficiency by thrombin.
- increased spacing between the motifs or specific arrangements may rescue a thrombin-susceptible linker design ( FIG. 54 ).
- the favored arrangements for reducing thrombin susceptibility include using ADAM17_2 upstream of CTSL1_1, and CTSL1_1 upstream of FAPa_1.
- the library peptides were all cleaved at P1 Lys sites, QKLK
- the ADAM17_1 motif was less efficiently cleaved than ADAM172, and the alternate CTSL1_2 motif was also cleaved at a much slower rate than CTSL1_1.
- tandem linker analysis with mass spectrometric detection revealed motif order preferences and unexpected side-reactions.
- the tandem linker with the arrangement of ADAM17_2-MMP14_1-CTSL1_1 in the peptide Alu30-16 had generally high activity toward the full set of five targeted enzymes, as well as the lowest susceptibility to thrombin, Factor Xa, and hepsin.
- the next best configuration was MMP14_1-ADAM17_2-CTSL1_1 in Alu30-15 which, although it had slightly higher hepsin susceptibility and lower CTSL1 activity may be rescued by the replacement with the CTSL1_2 motif.
- Replacement of the ADAM17_2 motif with ADAM17_1 may also enhance activity toward FAP-alpha and CTSL1, even if ADAM17 activity enhancement is minor, and hepsin susceptibility would be predicted to be reduced.
- IL2 fusion proteins The stability of IL2 fusion proteins in human serum (normal and cancer patient) was measured using capillary electrophoresis-based immunoassays (Jess instrument, Protein Simple). Fusion proteins were concentrated to 10 mg/ml and incubated with serum (90%). A time zero sample was immediately stored on ice while 24 hour and 72 hour samples were placed at 37° C. Post incubation, samples were diluted 1:1000 with 0.1 ⁇ sample buffer (Protein Simple) and loaded on the Jess cartridge per the manufacturer's protocol.
- Jess instrument Protein Simple
- the primary antibody was a monoclonal human IL2 antibody (R&D Systems, cat #AF-202-NA, stock concentration 0.2 mg/ml, working concentration 1:100), and the secondary antibody was a peroxidase-conjugated AffiiniPure Bovine Anti Goat IgG (H+L) (Jackson Immuno Research, cat #805-035-18, reconstituted at 0.8 mg/ml, working concentration 1:5000 dilution). All antibodies were diluted in milk free diluent (Protein Simple). Quantitation of IL2 containing species was quantitated using Compass software (Protein Simple) to determine the extent of cleavage (i.e. amount of IL2 containing species smaller than the input fusion protein). Results are shown in FIGS. 24 A- 24 B .
- Primary human lung epithelial cells and renal epithelial cells were obtained from ATCC. Primary hepatocytes were obtained from Lonza and Sigma. Cells were thawed, counted, and plated at 1e4 cells per well in a 96 well round bottom plate in their respective growth medias. Polypeptides containing recombinant human IL-2 and the sequence for Linker-2 and Linker-3 were incubated with cells or media alone for 24 and 72 hours at a concentration 5 ⁇ g/mL. Cell culture supernatants were collected, and cells were discarded. Protein cleavage was measured by western blot for IL-2 using the protein simple JESS system and Compass software. Results are shown in FIG. 5 .
- Primary human lung fibroblasts were obtained from ATCC. Prior to the processing assay, human PBMCs were stimulated with 5 ug/mL PHA for 72 hours to generate T blasts, which were then frozen and used to measure polypeptide processing. Primary human fibroblasts were thawed, counted, and plated at 1e5 cells per well in a 96 well round bottom plate in X-Vivo 15 media.
- Polypeptides containing recombinant human IL-2 and the sequence for Linker-1 (GPAGMKGL, SEQ ID NO: 196), Linker-2 (GPAGLYAQ, SEQ ID NO: 195), or Linker-3 (ALFKSSFP, SEQ ID NO: 198), or a non-cleavable sequence were incubated with or without fibroblasts cells for 48 hours at a concentration of either A) 3.3 nM or B) 0.33 nM.
- As a positive control some wells were also incubated with a polypeptide that was pre-cut in vitro overnight with 1 ⁇ g pre-activated MMP9 enzyme at 37° C. After 48 hours, cell culture supernatants were collected and healthy fibroblasts were discarded.
- Polypeptide processing was measured either by A) western blot for IL-2 using the protein simple JESS system, or by B) measuring the capacity of the cell culture supernatants to stimulate T blast proliferation. Briefly, T blasts were incubated for 72 hours with cell culture supernatants containing polypeptides previously exposed to healthy human fibroblasts, before T blast proliferation was measured by Cell Titer Glow analysis. Results are shown in FIG. 6 A- 6 B .
- Cleavage efficiency of Linker-2 (GPAGLYAQ, SEQ ID NO: 195) was evaluated in tumor tissue derived from human samples. A total of 66 samples were analyzed from seven solid tumor types. The tumor types are shown in Table 5 below.
- DTC frozen dissociated tumor cells
- IL-2 cytokine containing Linker 1 (a cleavable linker that was not designed using the processes described herein) or Linker-2 (GPAGLYAQ, SEQ ID NO: 195).
- Linker 1 a cleavable linker that was not designed using the processes described herein
- Linker-2 GPAGLYAQ, SEQ ID NO: 195.
- IL-2 proteins have no or minimal IL-2 biological activity when the linker is intact, and IL-2 biological activity is induced when the linker is cleaved.
- Cell culture supernatants were collected and frozen before analysis for IL-2 biological activity using T-Blast proliferation and Jess protein assays.
- the minimum fold change in IL-2 activity is 1.00, and the maximum is 2.50.
- a 1.3 fold change is indicative of a significant increase in biological activity relative to the uncleaved control.
- Table 5 shows the average fold change for Linker-2 (GPAGLYAQ, SEQ ID NO: 195) in comparison to an uncleaved control, a cleaved control, and Linker-1.
- the symbol “ ⁇ ” indicate essentially no increase in activity; “+/ ⁇ ” indicates some increase in activity but not significant; “+,” “++,” and “+++” indicate the relative significant increase in activity compared to the uncleavable control.
- Linker-2 is sufficiently cleaved in human tumor types to induce IL-2 biological activity.
- Linker-3 (ALFKSSFP, SEQ ID NO: 198) was evaluated in tumor tissue derived from human samples. A total of 66 samples were analyzed from seven solid tumor types. The tumor types are shown in Table 6 below.
- the minimum fold change in IL-2 activity is 1.00, and the maximum is 2.50.
- a 1.3 fold change is indicative of a significant increase in biological activity relative to the uncleaved control.
- Table 6 shows the average fold change for Linker-3 (ALFKSSFP, SEQ ID NO: 198) in comparison to an uncleaved control, a cleaved control, and Linker 1.
- the symbol “ ⁇ ” indicate essentially no increase in activity; “+/ ⁇ ” indicates some increase in activity but not significant; “+,” “++,” and “+++” indicate the relative significant increase in activity compared to the uncleavable control.
- Linker-3 (ALFKSSFP, SEQ ID NO: 198) is sufficiently cleaved in human tumor types to induce IL-2 biological activity.
- a stable HT-1080 cell line expressing human 4-1BB was established. Agonism of human-4-1BB in these cells resulted in increased secretion of IL-8. Tetravalent monospecific antibodies, inducible format tetravalent monospecific antibodies (protease cleaved or uncleaved), or trimeric ligands were tested for ability to agonize 4-1BB.
- Exemplary polypeptide constructs are detailed herein in Appendix A. While the exemplary polypeptides that contain Linker-2 (GPAGLYAQ, SEQ ID NO: 195), or Linker-3 (ALFKSSFP, SEQ ID NO: 198) or other cleavable linkers are disclosed in Appendix A, for each construct, the disclosed linker can be replaced with either Linker-2 (GPAGLYAQ, SEQ ID NO: 195), or Linker-3 (ALFKSSFP, SEQ ID NO: 198) or other cleavable linkers disclosed herein. For example construct
- ACP355 (IgG4_Fc(S228P)-X-CD25ecd_C213S-LX-IL2-LX
- L refers to a linker.
- X refers to a cleavable linker.
- L refers a linker that is optionally cleavable. When L is the only linker in a polypeptide, L is cleavable.
- LX or “XL” each refer to a cleavable linker with an extended non-cleavable sequence adjacent to it. Cleav. Lin. Also refers to a cleavable linker.
- Other abbreviations used include: “mIFNg” for mouse interferon gamma (IFNg); (5) “hAlbumin” for human serum albumin (HSA); and (6) “mAlbumin” indicates mouse serum albumin.
Abstract
Description
- The present application claims the benefit of U.S. Provisional Application No. 62/847,914 filed on May 14, 2019 and U.S. Provisional Application No. 62/938,786 filed on Nov. 21, 2019, each of which are incorporated herein by reference in their entireties.
- The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 14, 2020, is named 761146_000140_SL.txt and is 896,815 bytes in size.
- Recombinant fusion proteins containing two or more functional polypeptides have utility in many fields including, for use in protein purification, imaging, as therapeutics, and drug delivery. For example, protein drugs can be fused to Fc domains of antibodies or to carrier proteins (i.e., human serum albumin) for targeting, to extend their plasma half-lives and/or to achieve therapeutic effects. Chen et al., (2013), Adv Drug Deliv Rev. 65(10):1357-1369.
- Direct fusion of functional polypeptide or domains without a linker may lead to many undesirable outcomes, including misfolding of the fusion proteins (Zhao et al., (2008), Protein Expr. Purif., 61:73-77), low yield in protein production (Amet et al., (2009), Pharm. Res. 26:523-528), or impaired bioactivity (Bai et al., (2006) Proc. Natl Acad. Sci. USA, 102:7292-7296). One approach to overcome these difficulties is to use linker sequences between the component polypeptides or domains in a fusion protein. However, the selection of a suitable linker to join protein domains together can be complicated and is often neglected in the design of fusion proteins. Chen et al., (2013), Adv Drug Deliv Rev. 65(10):1357-1369. The properties of linker sequences, such as length, hydrophobicity, amino acid composition, secondary structures, and the overall folding can affect linker suitability and need to be taken into consideration in designing and selecting an appropriate linker. In addition, linkers that can be cleaved under selected conditions or in selected biological locations (e.g., in tumor microenvironment) to deliver active therapeutic agents (e.g., therapeutic polypeptides) can provide targeted pharmacological activity of the therapeutic agents and reduce unwanted systemic effects. This introduces further complexity into designing suitable linkers.
- There is a need for improved liner sequences that can be used to prepare stable fusion proteins, including linkers that can be cleaved under selected conditions. Accordingly, novel separation moieties or linkers are disclosed herein. The separation moieties or linkers disclosed herein can be utilized, for example to specifically deliver prodrugs such as conditionally active and/or targeted cytokines to target sites where the linkers are processed to activate bioactivity.
- Provided herein are compositions and methods to generate and use high efficiency separation moieties and/or linkers. The linkers can confer site-selectivity with regards to biological activity of the attached payload or payloads. In some embodiments, the separation moieties and/or linkers are used in conjugation with therapeutic proteins to treat a disease or disorder, such as proliferative disease, a tumorous disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, graft-versus-host disease and the like.
- Disclosed herein are recombinant polypeptides comprising a separation moiety, wherein the separation moiety comprises an amino acid sequence is a substrate for an enzyme, specifically a protease. The protease can be selected from the group consisting of Fibroblast activation protein alpha (FAPα, also known as prolyl endopeptidase FAP), Cathepsin L (CTSL1), an ADAM selected from
ADAM 8, ADAM 9, ADAM 10, ADAM12 ADAM17, and ADAMTS1, and an MMP selected from MMP1, MMP2, MMP9 or MMP14. The cleavable moiety can also be a substrate for a cathepsin, such as cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G, cathepsin K and/or cathepsin L. Preferably, the cathepsin is Cathepsin L. Preferably, the protease is MMP14 or cathepsin L. The cleavable moiety can comprise an amino acid sequence that is a substrate for at least two proteases. In another embodiment, the separation moiety comprises two or more cleavable moieties, each of which is a substrate for a protease. The separation moiety can comprise a first cleavable moiety comprising a first amino acid sequence that is a substrate for a first protease and a second cleavable moiety comprising a second amino acid sequence that is a substrate for a second protease. In embodiments, the disclosure related to recombinant polypeptides that comprise a separation moiety that contains a protease cleavage motif as disclosed herein. The recombinant polypeptide can comprise a separation moiety that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 195-220, or an amino acid sequence that has at least 90% identity to SEQ ID NOs:195-220. Preferred separation moieties comprise the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198). The disclosure also relates to functional variants of separation moieties comprising SEQ IND NOs: 195-220. The functional variants of of SEQ ID NO: 195 can comprise any of SEQ ID NOS: 258-331. The functional variants of SEQ ID NO: 198 can comprise of SEQ ID NO:198 comprises SEQ ID NOS: 199 or any of SEQ ID NOS: 332-408. The separation moieties disclosed herein can comprise Formula I: [D1]-[L1]-[D2]. D1 is a first domain of interest. L1 is a separation moiety that connects or links D1 to D2, wherein the separation moiety comprises an amino acid sequence selected from SEQ ID NOs: 195-220 or an amino acid sequence that has at least about 90% identity to SEQ ID NOs:195-220. D2 is a second domain of interest. - In one embodiment, the recombinant polypeptide can further comprise a non-cleavable linker sequence. The recombinant polypeptide can include a therapeutic protein, such as a cytokine, chemokine, growth factor, soluble receptor, antigen-binding portion of an antibody (e.g., scFV, dAb) and the like. In another embodiment, the recombinant polypeptide comprises a cytokine, chemokine, growth factor, a soluble receptor, or any combination thereof. In another embodiment, the recombinant polypeptide comprises at least one of an extracellular domain, a transmembrane domain, and an intracellular domain. In one embodiment, the recombinant polypeptide comprises a cell surface receptor, a chimeric antigen receptor (CAR), or a T Cell Receptor (TCR) subunit. In one embodiment, the recombinant polypeptide comprises an antigen-binding polypeptide, an antibody or an antigen-binding portion thereof.
- In one embodiment, the cleavable moiety is cleaved with either (a) greater catalytic efficiency or (b) greater specificity or (c) both (a) and (b), by one or more proteases than a reference polypeptide sequence. In another embodiment, the one or more proteases are selected from the group consisting of FAPα, CTSL1, an ADAM selected from
ADAM 8, ADAM 9, ADAM 10, ADAM12 ADAM17, and ADAMTS1, and an MMP selected from MMP1, MMP2, MMP9 and MMP14. The one or more proteases can also include a protease selected from cathepsins, such as cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G, cathepsin K and/or cathepsin L. In another embodiment, the reference polypeptide sequence is present in a naturally occurring polypeptide substrate for FAPα, CTSL1, an ADAM selected fromADAM 8,ADAM 9, ADAM 10, ADAM12 ADAM17, and ADAMTS1, and an MMP selected from MMP1, MMP2, MMP9 and MMP14, a cathepsin, such as cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin G, cathepsin K and/or cathepsin L or a combination thereof. In another embodiment, the cleavable moiety is cleaved with reduced catalytic efficiency by one or more proteases than a reference polypeptide sequence. In another embodiment the cleavable moiety is cleaved with reduced catalytic efficiency by one or more serum proteases. In another embodiment, the cleavable moiety is cleaved with reduced catalytic efficiency by one or more hepatic proteases. In another embodiment, the cleavable moiety is cleaved with reduced catalytic efficiency by one or more Factor Xa, hepsin, or thrombin. - In one embodiment, the recombinant polypeptide comprises two or more separation moieties. In one embodiment, the recombinant polypeptide is operably linked to a moiety selected from the group consisting of a polypeptide moiety, a lipid moiety, a nucleic acid moiety, a detectable moiety, and a small molecule.
- Provided herein is a recombinant pro-protein comprising: a recombinant polypeptide comprising a cleavable moiety that is a substrate for a protease, wherein the cleavable moiety comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 195-220; and a polypeptide with biological activity, wherein the pro-protein has attenuated biological activity and wherein cleavage of the cleavable moiety by the protease produces a polypeptide with biological activity that is not attenuated. In one embodiment, the polypeptide with biological activity comprises a cytokine, chemokine, growth factor, a soluble receptor or a combination thereof. In some preferred aspects, the linkers are components of fusion proteins with therapeutic utility, and the linkers are not cleaved or are cleaved with low efficiency in the peripheral circulation but are cleaved with higher efficiency at a desired location in the body, such as a tumor microenvironment or site of inflammation. In another embodiment, the polypeptide with biological activity comprises at least one of an extracellular domain, a transmembrane domain, and an intracellular domain. In another embodiment, the polypeptide with biological activity comprises a cell surface receptor, a chimeric antigen receptor (CAR), or a T Cell Receptor (TCR) subunit. In another embodiment, the polypeptide with biological activity comprises an antigen-binding polypeptide, an antibody or an antigen-binding portion thereof.
- The disclosure also relates to a fusion protein comprising: a. a signaling protein or molecule; and b. a blocking moiety selected from a steric blocking moiety, a specific blocking moiety, and the combination thereof; and c. a peptide linker that comprises a cleavable moiety, e.g., a cleavable moiety disclosed herein, having at least one protease-cleavable sequence. In one embodiment, the fusion protein, wherein (a) and (b) are operably linked by (c). In another embodiment, the fusion protein, wherein the peptide linker comprises two or more copies of the same cleavable moiety. In another embodiment, the fusion protein wherein the steric blocking moiety comprises human serum albumin (HSA) or an anti-HSA antibody. In another embodiment, the fusion protein wherein the signaling protein is an interleukin-2 amino acid sequence comprising: (i) a non-native N terminus and/or (ii) a non-native C terminus. In another embodiment, the fusion protein wherein the signaling protein is an interleukin-2 amino acid sequence. In another embodiment, the fusion protein further comprising one or more half-life extension domains that are not also a specific blocker. In another embodiment, the fusion protein wherein the non-native N and/or C termini are generated by circular permutation.
- The fusion polypeptide provided herein can a first polypeptide fusion partner linked to a ligand by a protease cleavable linker, wherein the cleavable linker has been optimized for catalytic efficiency, and wherein the ligand has been optionally modified, wherein the first polypeptide fusion partner is a blocking moiety which prevents binding of the modified ligand to a target receptor or a subunit of a target receptor until cleavage of the protease cleavable linker.
- The fusion polypeptide provided herein can also a fusion polypeptide comprising a first polypeptide fusion partner linked to a ligand by a protease cleavable linker, wherein the cleavable linker has been optimized for catalytic efficiency, and wherein the ligand has been optionally modified, including by circularly permutation to create a non-native N-terminus and a new C-terminus as compared to a native ligand, and wherein at least one of the new N-terminus or the new C-terminus of the modified ligand is operably linked to a first polypeptide fusion partner to form a fusion polypeptide wherein the first polypeptide fusion partner is a blocking moiety which prevents binding of the modified ligand to a target receptor or a subunit of a target receptor until cleavage of the protease cleavable linker.
- In one embodiment, the first polypeptide fusion partner is selected from the group consisting of an antibody, an antibody fragment, and an albumin molecule. In another embodiment, the first polypeptide fusion partner further comprising a second polypeptide fusion partner comprising a second blocking moiety. In another embodiment, the second polypeptide fusion partner is a different kind of blocking moiety than the first polypeptide fusion partner. In another embodiment, the first polypeptide fusion partner is albumin and the second polypeptide fusion partner is a domain comprising a complementary amino acid sequence that blocks activity of the cytokine. In another embodiment, the first polypeptide fusion partner is a steric blocker, such as albumin, and the second polypeptide is a specific blocker, such as a cytokine receptor, portion of a cytokine receptor, a de novo affinity peptide specific for the cytokine, or an antibody or antibody fragment that specifically binds the cytokine of the fusion polypeptide. In another embodiment, the second polypeptide fusion partner is the same kind of blocking moiety as the first polypeptide fusion partner.
- In one embodiment, the fusion protein further comprises a tumor antigen binding component. In another embodiment, the fusion protein further comprises a serum half-life extension domain. In another embodiment, the ligand is selected from the group consisting of helix bundle proteins and cytokines (including, but not limited to, growth hormone, IL-2, IL-4, IL-5, IL-6, IL-10, IL-22, IL-23p19, IL-11, IL-13, IL-15, IL-12p35, IL-21, IL-30 (IL27p28), IL-34, IL-35, IL-35p35, IFN-α, IFN-β, IFNγ, LIF, CNTF, oncostatin M, CLCF-1, GCSF, GM-CSF, EPO, ferritin, leptin, placental lactogen, prolactin, apolipoprotein e), b-trefoil proteins (including, but not limited to, IL-1α, IL-1β, IL-1Ra, IL18, IL-33, IL-36Ra, IL-36a, IL-36b, IL-36g, IL-37, IL-38, IL1Hy2, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8a, FGF-8b, FGF-8e, FGF-8f, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23), α/β (TIM) barrel proteins (including, but not limited to, triosephosphate isomerase), beta sandwich proteins (including, but not limited to, galectin-1, galectin-3, TNF-beta, seven β-propeller proteins, class 1 MHC α1α2 domain, integrin I domain, GYF domain, C1 domain, C2 domain (for example, from cPLA2, PKC, synaptotagmin), PDZ domains, C3d, C5a. In one embodiment, wherein the ligand comprises IL-2 polypeptide or a fragment or fragments thereof. In another embodiment, the protease-cleavable linker polypeptide comprises a sequence that is capable of being cleaved by at least one protease selected from the group consisting of a kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin G, an elastase, a FAP, an ADAM selected from
ADAM 8,ADAM 9,ADAM 10, ADAM12 ADAM17, and ADAMTS1, PR-3, granzyme M, a calpain, a matrix metalloproteinase (MMP), a plasminogen activator, a cathepsin, a caspase, a tryptase, and a tumor cell surface protease. In another embodiment, the cytokine or fragment or mutein thereof is substantially dissociated from the cytokine blocking moiety after the protease-cleavable polypeptide linker is cleaved by a protease. - Disclosed herein are fusion polypeptide comprising at least one of each of: a cytokine polypeptide or functional fragment or mutein thereof [A]; a cytokine blocking moiety [B]; and an optimized protease-cleavable polypeptide linker [L]; wherein the blocking moiety is selected from the group consisting of an antibody, an antibody fragment, and an albumin, and wherein the cytokine comprises a circularly permuted cytokine. In some embodiments, the fusion protein further comprises a tumor antigen binding component and/or a serum half-life extension domain. In some embodiment, the fusion polypeptide wherein the cytokine peptide or functional fragment or mutein thereof is selected from the group consisting of helix bundle proteins and cytokines (including, but not limited to, growth hormone, IL-2, IL-4, IL-5, IL-6, IL-10, IL-22, IL-23p19, IL-11, IL-13, IL-15, IL-12p35, IL-21, IL-30 (IL27p28), IL-34, IL-35, IL-35p35, IFN-β, IFNγ, LIF, CNTF, oncostatin M, CLCF-1, GCSF, GM-CSF, EPO, ferritin, leptin, placental lactogen, prolactin, apolipoprotein e), a FAP (e.g., Fapα), an ADAM selected from ADAM 8, ADAM 9, ADAM 10, ADAM12 ADAM17, and ADAMTS1, b-trefoil proteins (including, but not limited to, IL-1α, IL-1β, IL-1Ra, IL18, IL-33, IL-36Ra, IL-36a, IL-36b, IL-36g, IL-37, IL-38, IL1Hy2, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8a, FGF-8b, FGF-8e, FGF-8f, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23), α/β (TIM) barrel proteins (including, but not limited to, triosephosphate isomerase), beta sandwich proteins (including, but not limited to, galectin-1, galectin-3, TNF-β, seven β-propeller proteins, class 1 MHC α1α2 domain, integrin I domain, GYF domain, C1 domain, C2 domain (for example, from cPLA2, PKC, synaptotagmin), PDZ domains, C3d, C5a. In one embodiment, the cytokine peptide or functional fragment or mutein thereof comprises IL-2. In another embodiment, the cytokine blocking moiety comprises a ligand binding domain or fragment or mutein of a cognate receptor for the cytokine, a single domain antibody or scFv that binds the cytokine polypeptide or functional fragment or mutein thereof, or an antibody or antibody fragment that binds a receptor of the cytokine. In another embodiment, antibody is a single domain antibody or scFv. In another embodiment, the blocking moiety extends the serum half-life of the cytokine or fragment thereof.
- Disclosed herein are fusion polypeptides comprising a protease cleavable moiety, wherein the sequence is catalytically optimized for cleavage by certain proteases and wherein protease cleavage renders the composition inducible in a tumor microenvironment. In one embodiment, the fusion protein further comprises a biologically inactive polypeptide, wherein cleavage of the cleavable moiety by the protease converts the biologically inactive polypeptide to a biologically active polypeptide. In one embodiment, the biologically inactive polypeptide comprises a cytokine, chemokine, growth factor, or soluble receptor. In another embodiment, the biologically inactive polypeptide comprises at least one of an extracellular domain, a transmembrane domain, and an intracellular domain. In another embodiment, the biologically inactive polypeptide comprises a cell surface receptor, a chimeric antigen receptor (CAR), or a T Cell Receptor (TCR) subunit. In another embodiment, the biologically inactive polypeptide comprises an antigen-binding polypeptide, an antibody or an antigen-binding portion thereof.
- The disclosure further relates to a nucleic acid encoding any of the polypeptides disclosed herein, a vector comprising any of the nucleic acids encoding any of the polypeptides disclosed herein, and a host cell comprising said vector.
- Methods of making a pharmaceutical composition, comprising culturing the host cell comprising a vector comprising a nucleic acid encoding any of the polypeptides disclosed herein, under suitable conditions for expression and collection of desired polypeptides are provided herein. Method of using any of the polypeptides disclosed herein comprising administering an effective amount of a pharmaceutical composition comprising such polypeptides to a subject in need thereof are described. For example, use for treating a subject with a disease or disorder disclosed herein.
- Provided herein are pharmaceutical compositions comprising an effective amount of any of the recombinant polypeptides, any of the pro-proteins, any of the fusion proteins, any of the fusion polypeptides, any of the nucleic acids, any of the vectors, or any of the host cells comprising such vectors disclosed herein. For example, a pharmaceutical composition for treating a subject with a disease or disorder disclosed herein.
- Also disclosed is the use of the recombinant polypeptides, pro-proteins, fusion proteins, fusion polypeptides, nucleic acids, vectors, or host cells comprising such vectors disclosed herein for the manufacture of a medicament for treating a disease or disorder disclosed herein.
-
FIG. 1A is a schematic illustrating a protease-activated cytokine or chemokine that includes a blocking moiety. The blocking moiety may optionally function as a serum half-life extending domain. To the left of the arrow the drawing shows that a cytokine is connected to a blocking moiety via a protease-cleavable linker, thus blocking its ability to bind to its receptor. To the right of the arrow the drawing shows that in an inflammatory or tumor environment a protease cleaves at a protease-cleavage site on the linker, releasing the blocking moiety and allowing the cytokine to bind to its receptor. -
FIG. 1B is a schematic illustrating a protease-activated cytokine or chemokine wherein HSA (blocking moiety) is directly bound to the cytokine or chemokine of interest, with a protease cleavage site between the HSA and a cytokine or chemokine of interest. To the left of the arrow the drawing shows that a cytokine is connected to a blocking moiety via a protease-cleavable linker, thus blocking its ability to bind to its receptor. To the right of the arrow the drawing shows that in an inflammatory or tumor environment, the protease cleaves at a protease-cleavage site on linker, releasing the blocking moiety and allowing the cytokine to bind to its receptor. -
FIG. 1C is a schematic illustrating a protease-activated cytokine or chemokine wherein more than one HSA (blocking moiety) is bound directly to the molecule of interest. If desired, one or more of the HSA can be bonded to the cytokine or chemokine through a linker, such as a linker that contains a protease cleavage site. To the left of the arrow the drawing shows that a cytokine is connected to a blocking moiety via a protease-cleavable linker, thus blocking its ability to bind to its receptor. To the right of the arrow the drawing shows that in an inflammatory or tumor environment, protease cleaves at protease-cleavage site on linker, releasing the blocking moiety and allowing cytokine to bind receptor. The cytokine now has similar pK properties as compared to the native cytokine (e.g., has a short half-life). -
FIG. 1D is a schematic illustrating a protease-activated cytokine or chemokine comprising more than one cytokine, of the same type or different type, each of which is bonded to a binding domain through a protease-cleavable linker. To the left of the arrow the drawing shows that a cytokine is connected to a blocking moiety via a protease-cleavable linker, thus blocking its ability to bind to its receptor. To the right of the arrow the drawing shows that in an inflammatory or tumor environment a protease cleaves at a protease cleavage site on linker, releasing the blocking moiety and allowing the cytokine to bind to its receptor. -
FIG. 2 is a schematic illustrating a protease-activated cytokine or chemokine comprising a cytokine or chemokine polypeptide, a blocking moiety, and a serum half-life extending domain connected by at least one protease-cleavable linker. To the left of the arrow the drawing shows that a cytokine is connected to a blocking moiety via protease-cleavable linkers, thus blocking its ability to bind to its receptor. It is also bound to a separate half-life extension element, which extends half-life in serum. To the right of the arrow the drawing shows that in an inflammatory or tumor environment a protease cleaves at a protease-cleavage site on linker, thus releasing the serum half-life extension element and the blocking moiety and allowing the cytokine to bind to its receptor. The cytokine now has similar pK properties as compared to the native cytokine (e.g., a short half-life). -
FIG. 3 is a schematic illustrating a protease-activated cytokine or chemokine comprising a cytokine or chemokine polypeptide, a blocking moiety, and a targeting domain connected by at least one protease-cleavable linker. To the left of the arrow the drawing shows that a cytokine is connected to a blocking moiety and a targeting domain via a protease-cleavable linker, thus blocking its ability to bind to its receptor. To the right of the arrow the drawing shows that in an inflammatory or tumor microenvironment a protease cleaves at the protease cleavage site in the linker, releasing the targeting domain and the blocking moiety and allowing the cytokine to bind to its receptor. -
FIG. 4A is a schematic illustrating a protease-activated cytokine or chemokine comprising a cytokine or chemokine polypeptide, a blocking moiety, a targeting domain, and a serum half-life extending domain connected by at least one protease-cleavable linker, wherein the cytokine polypeptide and the targeting domain are connected by a protease-cleavable linker. To the left of the arrow, the drawing shows that a cytokine or chemokine is connected to targeting domain, blocking moiety, and half-life extension element via protease-cleavable linker(s), thus blocking its ability to bind to its receptor. To the right of the arrow the drawing shows that in an inflammatory or tumor environment, the protease cleaves at a protease-cleavage site on linker(s), releasing the half-life extension element, the targeting domain, and the blocking moiety, and allowing the cytokine to bind to its receptor. The cytokine now has similar pK properties as compared to the native cytokine (e.g., short half-life). -
FIG. 4B is a schematic illustrating a protease-activated cytokine or chemokine comprising a cytokine or chemokine polypeptide, a blocking moiety, a targeting domain, and a serum half-life extending domain connected by at least one protease-cleavable linker. To the left of the arrow, the drawing shows that a cytokine is connected to targeting domain, a blocking moiety, and a half-life extension element via protease-cleavable linker(s), thus blocking its ability to bind to its receptor. To the right of the arrow the drawing shows that in an inflammatory or tumor environment, the protease cleaves at a protease-cleavage site on linker(s), releasing the half-life extension element and the blocking moiety and allowing the cytokine to bind to the receptor. The targeting moiety remains bound, keeping the cytokine in the tumor microenvironment. The cytokine now has similar pK properties as compared to the native cytokine (e.g., a short half-life). -
FIG. 5 depicts a graph showing that Linkers-2 (GPAGLYAQ, SEQ ID NO: 195) and Linkers-3 (ALFKSSFP, SEQ ID NO: 198) are minimally cleaved in lung, kidney, and liver cells. -
FIGS. 6A-6B show graphs that polypeptides containing recombinant human IL-2 and the sequence for Linker-1 (GPAGMKGL, SEQ ID NO: 196), Linker-2 (GPAGLYAQ, SEQ ID NO: 195), or Linker-3 (ALFKSSFP, SEQ ID NO: 198) are not processed by healthy lung fibroblasts. -
FIGS. 7A-7H is a series of graphs showing activity of exemplary IL-2 fusion proteins in IL-2 dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows results of the IL-2 proliferation assay as quantified by CellTiter-Glo® (Promega) luminescence-based cell viability assay. Each proliferation assay was performed with HSA (FIGS. 7B, 7D, 7F, 7H ) or without (FIGS. 7A, 7C, 7E, 7G ). Each fusion protein comprises an anti-HSA binder, and both uncleaved and MMP9 protease cleaved versions of the fusion protein were used in each assay. -
FIGS. 8A-8F is a series of graphs showing activity of exemplary IL-2 fusion proteins in IL-2 dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows results of the IL-2 proliferation assay as quantified by CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease cleaved versions of the fusion protein were used in each assay. -
FIGS. 9A-9Z is a series of graphs showing activity of exemplary IL-2 fusion proteins in IL-2 dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows results of the IL-2 proliferation assay as quantified by CellTiter-Glo (Promega) luminescence-based cell viability assay. Both uncleaved and MMP9 protease cleaved versions of the fusion protein were used in each assay. -
FIG. 10 shows results of protein cleavage assay. Fusion protein ACP16 was run on an SDS-PAGE gel in both cleaved and uncleaved form. As can be seen in the gel, cleavage was complete. -
FIGS. 11A-11B are a series of graphs depicting results from a HEK-Blue IL-12 reporter assay performed on human p40/murine p35 IL12 fusion proteins and recombinant human IL12 (Rec hIL-12). Analysis was performed based on quantification of Secreted Alkaline Phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). Results confirm that IL12 protein fusion proteins are active. -
FIGS. 12A-12F show a series of graphs depicting the results of HEK-blue assay of four IL-12 fusion proteins, before and after cleavage by MMP9. Analysis was performed based on quantification of Secreted Alkaline Phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). The data show greater activity in the cleaved IL12 than in the full fusion protein. Constructs tested were ACP06 (FIG. 12A ), ACP07 (FIG. 12C ), ACP08 (FIG. 12B ), ACP09 (FIG. 12D ), ACP10 (FIG. 12E ), ACP11 (FIG. 12F ) -
FIG. 13 shows results of protein cleavage assay. Fusion protein ACP11 was run on an SDS-PAGE gel in both cleaved and uncleaved form. As can be seen in the gel, cleavage was complete. -
FIG. 14 is a schematic which depicts a non-limiting example of an inducible cytokine protein, wherein the construct is activated upon protease cleavage of a linker attached between two subunits of the cytokine. -
FIGS. 15A-15D are graphs depicting results from a HEK-Blue assay performed on human p40/murine p35 IL12 fusion proteins and recombinant human IL12 (Rec hIL-12). Results confirm that IL12 protein fusion proteins are active. Each proliferation assay was performed with HSA or without HSA. -
FIGS. 16A-16F are a series of graphs showing activity of exemplary IFNγ fusion proteins compared to activity of mouse IFNγ control using WEHI 279 cell survival assay. Each assay was performed with medium containing HSA (+HSA) or not containing HSA (−HSA). Each fusion protein comprises an anti-HSA binder, and both uncleaved and MMP9 protease cleaved versions of the fusion protein were used in each assay. -
FIGS. 17A-17F are a series of graphs showing activity of exemplary IFNγ fusion proteins compared to activity of mouse IFNγ control using B16 reporter assay. Each assay was performed with medium containing HSA (+HSA) or not containing HSA (−HSA). Each fusion protein comprises an anti-HSA binder, and both uncleaved and MMP9 protease cleaved versions of the fusion protein were used in each assay. -
FIGS. 18A-18B shows results of protein cleavage assay. Two constructs, ACP31 (IFN-a fusion protein;FIG. 18A ) and ACP55 (IFN-γ fusion protein;FIG. 18B ), were run on an SDS-PAGE gel in both cleaved and uncleaved form. As can be seen in the gel, cleavage was complete. -
FIGS. 19A-19B are a series of graphs showing activity of exemplary IFNγ fusion proteins compared to activity of mouse IFNγ control using B16 reporter assay. Each assay was performed with culture medium containing HSA, and each fusion protein comprises an anti-HSA binder. Both uncleaved and MMP9 protease cleaved versions of the fusion protein were used in each assay. -
FIGS. 20A-20B are a series of graphs showing activity of exemplary IFNα fusion proteins compared to activity of mouse IFNalphaA control using a B16 reporter assay. Each assay was performed with medium containing HSA, and each fusion protein comprises an anti-HSA binder. Both uncleaved and MMP9 protease cleaved versions of the fusion protein were used in each assay. -
FIGS. 21A-21D are a series of graphs depicting the results of tumor growth studies using the MC38 cell line.FIG. 21A-21C show the effect of IFNγ and IFNγ fusion proteins on tumor growth when injected intraperitoneally (IP) using different dosing levels and schedules (μg=micrograms, BID=twice daily, BIW=twice weekly, QW=weekly).FIG. 21D shows the effect of intratumoral (IT) injection of IFNγ and IL-2 on tumor growth. -
FIGS. 22A-22B are a series of graphs showing activity of exemplary IFNγ fusion proteins (ACP51 and ACP52) cleaved by MMP9 protease compared to activity of uncleaved fusion proteins using B16 reporter assay. Each fusion protein comprises an anti-HSA binder and a tumor targeting domain. -
FIGS. 23A-23B are a series of graphs showing activity of exemplary IFNγ fusion proteins (ACP53 and ACP54) cleaved by MMP9 protease compared to activity of uncleaved fusion proteins using B16 reporter assay. Each fusion protein comprises IFNγ directly fused to albumin. -
FIGS. 24A-24B are two graphs showing the stability of IL-2 fusion proteins containing Linker-1 (GPAGMKGL, SEQ ID NO: 196), Linker-2 (GPAGLYAQ, SEQ ID NO: 195), or Linker-3 (ALFKSSFP, SEQ ID NO: 198) in human serum in normal patient and a cancer patient.FIG. 24A depicts the stability of the IL-2 fusion proteins containing Linker-1 (GPAGMKGL, SEQ ID NO: 196), Linker-2 (GPAGLYAQ, SEQ ID NO: 195), or Linker-3 (ALFKSSFP, SEQ ID NO: 198) at 24 hours.FIG. 24B depicts the stability of the IL-2 fusion proteins containing Linker-1 (GPAGMKGL, SEQ ID NO: 196), Linker-2 (GPAGLYAQ, SEQ ID NO: 195), or Linker-3 (ALFKSSFP, SEQ ID NO: 198) at 72 hours. -
FIGS. 25A and 25B are two graphs showing analysis of ACP16 (FIG. 25A ) and ACP124 (FIG. 25B ) in a HEKBlue IL-2 reporter assay in the presence of HSA. Circles depict the activity of the uncut polypeptide, squares depict activity of the cut polypeptide, and triangles depict IL-2 alone as a control.FIG. 25C is a graph showing results of a CTLL-2 proliferation assay. CTLL2 cells (ATCC) were plated in suspension at a concentration of 500,000 cells/well in culture media with or without 40 mg/ml human serum albumin (HSA) and stimulated with a dilution series of recombinant hIL2 or activatable hIL2 for 72 hours at 37° C. and 5% CO2. Activity of uncleaved and cleaved activatable ACP16 was tested. Cleaved activatable hIL2 was generated by incubation with active MMP9 Cell activity was assessed using a CellTiter-Glo (Promega) luminescence-based cell viability assay. Triangles show wile-type cytokine, circles depict intact fusion protein, and squares depict protease-cleaved fusion protein. -
FIGS. 26A-26C are a series of graphs showing activity of fusion proteins in an HEKBlue IL-12 reporter assay.FIG. 26A is a graph showing activity of cut and uncut ACP11 (a human p40/murine p35 IL12 fusion protein).FIG. 26B is a graph showing analysis of ACP91 (a chimeric IL-12 fusion protein). Squares depict activity of the uncut ACP91 polypeptide, and triangles depict the activity of the cut polypeptide (ACP91+MMP9). EC50 values for each are shown in the table.FIG. 26C is a graph showing analysis of ACP136 (a chimeric IL-12 fusion protein). Squares depict activity of the uncut ACP136 polypeptide, and triangles depict the activity of the cut polypeptide (ACP136+MMP9). EC50 values for each are shown in the table insert. -
FIGS. 27A-27F are a series of graphs showing that cleaved IL-12 polypeptides are active in a HEKBlue IL2 reporter assay. Fusion proteins are evaluated both uncut (circles) and cut (squares) form, and wild type IL2 is used as a control +HSA forFIGS. 27A-C ; ACP131 is used as a control (triangles) forFIGS. 27D-27F . Shown are data for APC31+HSA (FIG. 27A ), ACP125+HSA (FIG. 27B ), ACP126+HSA (FIG. 27C ), ACP127 (FIG. 27D ), ACP128 (FIG. 27E ), and ACP129 (FIG. 27F ). The EC50 values for each are shown in the table below each graph. -
FIGS. 28A-28N are a series of graphs depicting the activity of APC56 (FIG. 28A ), APC57 (FIG. 28B ) APC58 (FIG. 28C ), APC59 (FIG. 28D ), APC60 (FIG. 28E ), APC61+HSA (FIG. 28F ), ACP30+HSA (FIG. 28G ), ACP73 (FIG. 28H ), ACP70+HSA (FIG. 28I ), ACP71 (FIG. 28J ), ACP72 (FIG. 28K ), ACP 73 (FIG. 28L ), ACP74 (FIG. 28M ), and ACP75 (FIG. 28N ) in a HEKBlue IFNα reporter assay. Each fusion was tested for its activity when cut (squares) and uncut (circles). Analysis of murine IFNγ is included in each graph as a comparator. -
FIGS. 29A-29B is two graphs showing results of analyzing ACP31 (mouse IFNα1 fusion protein) and ACP11 (a human p40/murine p35 IL12 fusion protein) in a tumor xenograft model.FIG. 29A shows tumor volume over time in mice treated with 33 μg ACP31 (circles), 110 μg ACP31 (triangles), 330 μg ACP31 (diamonds), and ascontrols 1 μg murine wild type IFNα1 (dashed line, squares) and 10 μg mIFNα1 (dashed line, small circles). Vehicle alone is indicated by large open circles. The data show tumor volume decreasing over time in a dose-dependent manner in mice treated with ACP31.FIG. 29B shows tumor volume over time in mice treated with 17.5 μg ACP11 (squares), 175 μg ACP11 (triangles), 525 μg ACP11 (circles), and ascontrols 2 μg ACP04 (dashed line, triangles) and 10 μg ACP04 (dashed line, diamonds). Vehicle alone is indicated by large open circles. The data show tumor volume decreasing over time in a dose-dependent manner in mice treated with both ACP11 and ACP04 (a human p40/murine p35 IL12 fusion protein). -
FIGS. 30A-30F are a series of spaghetti plots showing tumor volume over time in a mouse xenograft tumor model in mice each treated with vehicle alone (FIG. 30A ), 2 μg ACP04 (FIG. 30B ), 10 μg ACP04 (FIG. 30C ), 17.5 μg ACP11 (FIG. 30D ), 175 μg ACP11 (FIG. 30E ), and 525 μg ACP11 (FIG. 30F ). Each line represents a single mouse. -
FIGS. 31A-31C depicts three graphs showing results of analyzing ACP16 and ACP124 in a tumor xenograft model.FIG. 31A shows tumor volume over time in mice treated with 4.4 μg ACP16 (squares), 17 μg ACP16 (triangles), 70 μg ACP16 (downward triangles), 232 μg ACP16 (dark circles), and as acomparator 12 μg wild type IL-2 (dashed line, triangles) and 36 μg wild type IL-2 (dashed line, diamonds). Vehicle alone is indicated by large open circles. The data show tumor volume decreasing over time in a dose-dependent manner in mice treated with ACP16 at higher concentrations.FIG. 31B shows tumor volume over time in mice treated with 17 μg ACP124 (squares), 70 μg ACP124 (triangles), 230 μg ACP124 (downward triangles), and 700 μg ACP124 (diamonds). Vehicle alone is indicated by large open circles.FIG. 31C shows tumor volume over time in mice treated with 17 μg ACP16 (dashed line, triangles), 70 μg ACP16 (circles), 232 μg ACP16 (dark circles), and as a comparator 17 μg ACP124 (downward triangles) 70 μg ACP124 (dashed line, diamonds), 230 μg ACP124 (dashed line, stars). Vehicle alone is indicated by dark downward triangles. The data show tumor volume decreasing over time in a dose-dependent manner in mice treated with ACP16, but not ACP124. -
FIGS. 32A-32U are a series of spaghetti plots showing activity of fusion proteins in an MC38 mouse xenograft model. Each line in the plots is a single mouse. -
FIG. 33 is a graph showing tumor volume over time in a mouse xenograft model showing tumor growth in control mice (open circles) and AP16-treated mice (squares). -
FIGS. 34A-34D are a series of survival plots showing survival of mice over time after treatment with cleavable fusion proteins.FIG. 34A shows data for mice treated with vehicle alone (dotted line), 17 μg ACP16 (dark line), and 17 μg ACP124 (dashed line).FIG. 34B shows data for mice treated with vehicle alone (dotted line), 70 μg ACP16 (dark line), and 70 μg ACP124 (dashed line).FIG. 34C shows data for mice treated with vehicle alone (dotted line), 232 μg ACP16 (dark line), and 230 μg ACP124 (dashed line).FIG. 34D shows data for mice treated with vehicle alone (dotted line), 232 μg ACP16 (dark line), and 700 μg ACP124 (dashed line). -
FIGS. 35A-35M are a series of spaghetti plots showing activity of fusion proteins in an MC38 mouse xenograft model. All mouse groups were given four doses total except for the highest three doses of APC132, wherein fatal toxicity was detected after 1 week/2 doses.FIG. 35A shows tumor volume over time for mice treated with vehicle.FIGS. 35B-3E shows tumor volume over tume in mice treated with ACP16 at 17 μg/animal (FIG. 35B ), 55 μg/animal (FIG. 35C ), 70 μg/animal (FIG. 35C ), and 230 μg/animal 35E.FIGS. 35F-35I shows tumor volume over time for mice treated with ACP132 at 9 μg/animal (FIG. 35F ), 28 μg/animal (FIG. 35G ). 36 μg/animal (FIG. 35H ), and 119 μg/animal (FIG. 35I ).FIGS. 35J-35M shows tumor volume over time for mice treated with ACP21 at 13 μg/animal (FIG. 35J ), 42 μg/animal (FIG. 35K ), 54 μg/animal (FIG. 35L ), and 177 μg/animal (FIG. 35M ). Each line in the plots represents an individual animal. -
FIG. 36 is a schematic illustrating substrate cleavage activity in conditioned complete (+FBS) media by FRET endpoint assay across four cell lines. The ratio of tumor vs control activity was approximated by averaging the three tumor cell lines and comparing to the control myofibroblast cell line where signal was detectable.FIG. 36 discloses SEQ ID NOs: 201, 198, 197, 196 and 195, respectively, in order of appearance. -
FIG. 37 is a schematic illustrating ADAM17_2 substrate kinetics in cell culture conditioned media.FIG. 37 discloses SEQ ID NO: 235. -
FIG. 38 is a schematic illustrating FAPα_1 substrate kinetics in conditioned media.FIG. 38 discloses SEQ ID NO: 197. -
FIG. 39 is a schematic illustrating FAPα_1 substrate kinetics in cell lysates.FIG. 39 discloses SEQ ID NO: 197. -
FIG. 40 is a schematic illustrating MMP9_1 substrate kinetics in cell lysates.FIG. 40 discloses SEQ ID NO: 196. -
FIG. 41 is a schematic illustrating a substrate cleavage activity in cell lysates by FRET endpoint assay.FIG. 41 discloses SEQ ID NOS 198 and 197, respectively, in order of appearance. -
FIG. 42 is a schematic illustrating CTSL1_1 substrate kinetics in cell lysates.FIG. 42 discloses SEQ ID NO: 198. -
FIG. 43 is a schematic illustrating MMP14_1 substrate kinetics in cell lysates.FIG. 43 discloses SEQ ID NO: 195. -
FIG. 44 is a schematic illustrating calculated concentration of enzyme equivalents per cell culture-derived sample.FIG. 44 discloses SEQ ID NOS 201, 198, 197, 197, 196 and 195, respectively, in order of appearance. -
FIG. 45 is a schematic illustrating an enzyme progress curve for CTSL1 cleavage of CSTL1_2 vs CTSL1_1.FIG. 45 discloses SEQ ID NOS 198, 199 and 236, respectively, in order of appearance. -
FIG. 46 is a schematic illustrating 30-mer cleavage of CTSL1_1 (ALFKSSFP, SEQ ID NO: 198) vs CTSL1_2 (ALFFSSPP, SEQ ID NO: 199). -
FIG. 47 is a schematic illustrating susceptibility of the CTSL1 FRET substrates to CTSK cleavage. Rates of product formation were measured as a specific activity in units pmol min−1 μg−1. The threshold value for the reference substrate, Z-LR-AMC is shown as a dashed line.FIG. 47 discloses SEQ ID NOS 198 and 199, respectively, in order of appearance. -
FIG. 48 is a schematic illustrating 30-mer Substrate Degradation by MMP9 Ranking of the substrates by relative rates of degradation are shown with “+”; uncleaved substrates are indicated as “−”.FIG. 48 discloses SEQ ID NOS 204, 205, 214, 216, 202, 217, 203, 211, 219, 207, 215, 212, 213, 206, 208, 209, 210, 218 and 220, respectively, in order of appearance. -
FIG. 49 is a schematic illustrating tandem MMP14_1 Motif degradation by MMP9. Top: substrate degradation traces, modeled with first-order kinetics. Bottom: product formation traces showing complex kinetics.FIG. 49 discloses SEQ ID NOS 202-205, respectively, in order of appearance. -
FIG. 50 is a schematic illustrating 30-mer Substrate Degradation by FAPα Ranking of the substrates by relative rates of degradation are shown with “+”; uncleaved substrates are indicated as “−”.FIG. 50 discloses SEQ ID NOS 205, 204, 206, 217, 203, 218, 219, 213, 216, 207, 214, 210, 202, 211, 208, 209, 212, 215 and 220, respectively, in order of appearance. -
FIG. 51 is a schematic illustrating 30-mer Substrate Degradation by CTSL1 Ranking of the substrates by relative rates of degradation are shown with “+”; uncleaved substrates are indicated as “−”.FIG. 51 discloses SEQ ID NOS 207, 208, 202, 218, 219, 212, 215, 217, 211, 209, 214, 206, 213, 210, 216, 203, 204, 205 and 220, respectively, in order of appearance. -
FIG. 52 is a schematic illustrating 30-mer Substrate Degradation by ADAM17 Ranking of the substrates by relative rates of degradation are shown with “+”; uncleaved substrates are indicated as “−”.FIG. 52 discloses SEQ ID NOS 208, 209, 211, 214, 217, 219, 213, 218, 215, 210, 212, 216, 207, 206, 202, 203, 204, 205 and 220, respectively, in order of appearance. -
FIG. 53 is a schematic illustrating 30-mer Substrate Degradation by Factor Xa Ranking of the substrates by relative rates of degradation are shown with “+”; uncleaved substrates are indicated as “−”.FIG. 53 discloses SEQ ID NOS 220, 206, 202, 214, 208, 209, 215, 210, 218, 217, 207, 213, 216, 211, 212, 219, 203, 204 and 205, respectively, in order of appearance. -
FIG. 54 is a schematic illustrating 30-mer Substrate Degradation by Thrombin Ranking of the substrates by relative rates of degradation are shown with “+”; uncleaved substrates are indicated as “−”.FIG. 54 discloses SEQ ID NOS 220, 204, 202, 207, 205, 211, 212, 215, 209, 218, 219, 217, 210, 213, 216, 214, 208, 206 and 203, respectively, in order of appearance. -
FIG. 55 is a schematic illustrating 30-mer Substrate Degradation by hepsin Ranking of the substrates by relative rates of degradation are shown with “+”; uncleaved substrates are indicated as “−”.FIG. 55 discloses SEQ ID NOS 220, 209, 216, 215, 210, 213, 206, 214, 212, 207, 217, 208, 211, 218, 219, 202, 203, 204 and 205, respectively, in order of appearance. -
FIGS. 56A-56C show western blots probed with an IL-2 antibody demonstrating the stability of ACP16 (FIG. 56A ), ACP153 (FIG. 56B ), and ACP157 (FIG. 56C ) in 90% serum. Serum was pooled from three human donors. Constructs of interest were incubated with PBS, Serum, or MMP9 protease and cleavage was assessed at T=0 hours and at T=24 hours. -
FIG. 57A-57D show western blots probed with an IL-2 antibody demonstrating the stability of ACP153, ACP155, ACP156, ACP16, and ACP372 in 90% serum. Serum was pooled from three human donors. Constructs of interest were incubated with PBS, Serum, or MMP9 protease and cleavage was assessed at T=24 hours and at T=72 hours.FIGS. 57A-57B shows the result using serum from a human donor andFIGS. 57C-57D shows the result using serum from a mouse donor. -
FIGS. 58A-58D show a series of spaghetti plots showing activity of fusion proteins in an MC38 mouse xenograft model. Shown are vehicle alone (FIG. 58A , top), 17, 55, and 230 μg ACP16 (FIG. 58A ), 55 and 230 μg ACP153 (FIG. 58B ), 55 and 230 μg ACP155 (FIG. 58C ), and 55 and 230 μg ACP156 (FIG. 58D ). Each line in the plots represents an individual animal. -
FIG. 59 shows a graph depicting results from a STAT activation reporter assay performed on IL-2 fusion proteins and recombinant human IL2 (Rec hIL-2). Analysis was performed based on quantification of Secreted Alkaline Phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). -
FIG. 60 shows a graph depicting results from a STAT activation reporter assay performed on IL-2 fusion proteins and recombinant human IL2 (Rec hIL-2). Analysis was performed based on quantification of Secreted Alkaline Phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). -
FIG. 61 shows a graph depicting results from a STAT activation reporter assay performed on IL-2 fusion proteins and recombinant human IL2 (Rec hIL-2). Analysis was performed based on quantification of Secreted Alkaline Phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). -
FIG. 62B shows a graph depicting results from a STAT activation reporter assay performed on IL-2 fusion proteins and recombinant human IL2 (Rec hIL-2). Analysis was performed based on quantification of Secreted Alkaline Phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen).FIG. 62A shows an SDS-PAGE gel of construct cleavage by MMP9 protease. -
FIGS. 63A-63B shows tables reporting engineered cleavage substrates described herein and the extent of cleavage observed using relevant proteases Flanking sequences are lower case, the first cleavable sequence is underlined, the second is in bold font, and the third cleavable sequence is in italics. In some cases, there is overlap between cleavable sequences, which are indicated accordingly.FIGS. 63A-63B discloses SEQ ID NOS 202-220, respectively, in order of appearance. -
FIG. 64 is a schematic of an inducible tetravalent antibody format. -
FIGS. 65A-65B show that multivalent 4-1BB antibodies are able to inducibly agonize 4-1BB. - This disclosure relates to novel separation moieties or linkers and to polypeptides, such as fusion proteins, that contain the linkers. The linkers are preferably protease cleavable and link a first amino acid sequence of interest (e.g. a first domain of interest) to a second amino acid sequence of interest (e.g. a second domain of interest).
- The disclosed separation moieties confer site-selectivity with regard to the action of the attached payload or payloads. The payload can be a therapeutic agent, a half-life extender, a blocking agent and the like, or any combination thereof. The separation moieties may be used to attach any payload of interest, including e.g. cytokines, antibodies, cell-based therapies, etc. The separation moieties may be used individually or be used in tandem, triplicate, quadruplicate, and so forth, as long as the separation moiety is smaller than about 100 amino acids. Individual separation moieties may be directly joined to each other, or may be interspersed with non-cleavable linkers, whichever promotes high efficiency and site-specificity.
- The various embodiments of the present disclosure are further described in detail in the paragraphs below.
- Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodologies by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer-defined protocols and conditions unless otherwise noted.
- “Cytokine” is a well-known term of art that refers to any of a class of immunoregulatory proteins (such as interleukin or interferon) that are secreted by cells especially of the immune system and that are modulators of the immune system. Cytokine polypeptides that can be used in the fusion proteins disclosed herein include, but are not limited to transforming growth factors, such as TGF-α and TGF-β (e.g., TGFbeta1, TGFbeta2, TGFbeta3); interferons, such as interferon-α, interferon-β, interferon-γ, interferon-kappa and interferon-omega; interleukins, such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21 and IL-25; tumor necrosis factors, such as tumor necrosis factor alpha and lymphotoxin; transforming growth factor beta (TGFbeta) family proteins, chemokines (e.g., C—X—C motif chemokine 10 (CXCL10), CCL19, CCL20, CCL21), and granulocyte macrophage-colony stimulating factor (GM-CS), as well as fragments of such polypeptides that active the cognate receptors for the cytokine (i.e., functional fragments of the foregoing). “Chemokine” is a term of art that refers to any of a family of small cytokines with the ability to induce directed chemotaxis in nearby responsive cells.
- Cytokines are well-known to have short serum half-lives that frequently are only a few minutes. Even forms of cytokines that have altered amino acid sequences intended to extend the serum half-life yet retain receptor agonist activity typically also have short serum half-lives. As used herein, a “short-half-life cytokine” refers to a cytokine that has a substantially brief half-life circulating in the serum of a subject, such as a serum half-life that is less than 10, less than 15, less than 30, less than 60, less than 90, less than 120, less than 240, or less than 480 minutes. As used herein, a short half-life cytokine includes cytokines which have not been modified in their sequence to achieve a longer than usual half-life in the body of a subject and polypeptides that have altered amino acid sequences intended to extend the serum half-life yet retain receptor agonist activity. This latter case is not meant to include the addition of heterologous protein domains, such as a bona fide half-life extension element, such as serum albumin.
- A “conservative” amino acid substitution, as used herein, generally refers to substitution of one amino acid residue with another amino acid residue from within a recognized group which can change the structure of the peptide but biological activity of the peptide is substantially retained. Conservative substitutions of amino acids are known to those skilled in the art. Conservative substitutions of amino acids can include, but not limited to, substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. For instance, a person of ordinary skill in the art reasonably expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the biological activity of the resulting molecule.
- “Sortases” are transpeptidases that modify proteins by recognizing and cleaving a carboxyl-terminal sorting signal embedded in or terminally attached to a target protein or peptide. Sortase A catalyzes the cleavage of the LPXTG motif (where X is any standard amino acid) (SEQ ID NO: 237) between the Thr and Gly residue on the target protein, with transient attachment of the Thr residue to the active site Cys residue on the enzyme, forming an enzyme-thioacyl intermediate. To complete transpeptidation and create the peptide-monomer conjugate, a biomolecule with an N-terminal nucleophilic group, typically an oligoglycine motif, attacks the intermediate, displacing Sortase A and joining the two molecules.
- As used herein, the term “steric blocker” refers to a polypeptide or polypeptide moiety that can be covalently bonded to a cytokine polypeptide directly or indirectly through other moieties such as linkers, for example in the form of a chimeric polypeptide (fusion protein), but otherwise does not covalently bond to the cytokine polypeptide. A steric blocker can non-covalently bond to the cytokine polypeptide, for example though electrostatic, hydrophobic, ionic or hydrogen bonding. A steric blocker typically inhibits or blocks the activity of the cytokine moiety due to its proximity to the cytokine moiety and comparative size.
- As used and described herein, a “half-life extension element” is a part of the chimeric polypeptide that increases the serum half-life and improve pK, for example, by altering its size (e.g., to be above the kidney filtration cutoff), shape, hydrodynamic radius, charge, or parameters of absorption, biodistribution, metabolism, and elimination.
- The term “separation moiety” or “linker” as used herein refers to an amino acid sequence typically less than about 100 amino acids that connects or links a first amino acid sequence of interest (e.g., an amino acid sequence that folds to form a first protein domain) to a second amino acid sequence of interest (e.g., an amino acid sequence that folds to form a second protein domain) in a contiguous polypeptide chain. The separation moiety or linker typically include one or more protease cleavage sites and thus is protease cleavable. A “tandem linker” refers to a linker that comprises two or more protease cleavages sites which can be cleaved by the same or different proteases, and which can be arranged in any desired orientation, such as one cleavage site adjacent to another cleavage site, one cleavage site overlapping another cleavage site, one cleavage site following by another cleavage site with intervening amino acids between the two cleavage sites.
- As used herein, the terms “activatable,” “activate,” “induce,” and “inducible” refer to the ability of a protein, i.e. a cytokine, that is part of a conjugate, to bind its receptor and effectuate activity upon cleavage of additional elements from the conjugate.
- As used herein, “plasmids” or “viral vectors” are agents that transport the disclosed nucleic acids into the cell without degradation and include a promoter yielding expression of the nucleic acid molecule and/or polypeptide in the cells into which it is delivered.
- As used herein, the terms “peptide”, “polypeptide”, or “protein” are used broadly to mean two or more amino acids linked by a peptide bond. Protein, peptide, and polypeptide are also used herein interchangeably to refer to amino acid sequences. It should be recognized that the term polypeptide is not used herein to suggest a particular size or number of amino acids comprising the molecule and that a peptide of the invention can contain up to several amino acid residues or more.
- As used throughout, “subject” can be a vertebrate, more specifically a mammal (e g a human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig), birds, reptiles, amphibians, fish, and any other animal. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered.
- As used herein, “patient” or “subject” may be used interchangeably and can refer to a subject with a disease or disorder (e.g. cancer). The term patient or subject includes human and veterinary subjects.
- As used herein the terms “treatment”, “treat”, “treating,” or grammatically related terms refer to a method of reducing the effects of a disease or condition or symptom of the disease or condition. Thus, in the disclosed method, treatment can refer to at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or substantially complete reduction in the severity of an established disease or condition or symptom of the disease or condition. For example, a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject as compared to a control. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is well understood in the art that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition. 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.
- As used herein, the terms “prevent”, “preventing”, and “prevention” of a disease or disorder refers to an action, for example, administration of the chimeric polypeptide or nucleic acid sequence encoding the chimeric polypeptide, that occurs before or at about the same time a subject begins to show one or more symptoms of the disease or disorder, which inhibits or delays onset or exacerbation of one or more symptoms of the disease or disorder.
- As used herein, references to “decreasing”, “reducing”, or “inhibiting” include a change of at least about 10%, of at least about 20%, of at least about 30%, of at least about 40%, of at least about 50%, of at least about 60%, of at least about 70%, of at least about 80%, of at least about 90% or greater as compared to a suitable control level. Such terms can include but do not necessarily include complete elimination of a function or property, such as agonist activity.
- An “attenuated cytokine receptor agonist” is a cytokine receptor agonist that has decreased receptor agonist activity as compared to the cytokine receptor's naturally occurring agonist. An attenuated cytokine agonist may have at least about 10×, at least about 50×, at least about 100×, at least about 250×, at least about 500×, at least about 1000× or less agonist activity as compared to the receptor's naturally occurring agonist. When a fusion protein that contains a cytokine polypeptide as described herein is described as “attenuated” or having “attenuated activity”, it is meant that the fusion protein is an attenuated cytokine receptor agonist.
- An “intact fusion protein” is a fusion protein in which no domain has been removed from the fusion protein, for example by protease cleavage. A domain may be removable by protease cleavage or other enzymatic activity, but when the fusion protein is “intact”, this has not occurred.
- As used herein “moiety” refers to a portion of a molecule that has a distinct function within that molecule, and that function may be performed by that moiety in the context of another molecule. A moiety may be a chemical entity with a particular function, or a portion of a biological molecule with a particular function. For example, a “blocking moiety” within a fusion protein is a portion of the fusion protein which is capable of blocking the activity of some or all of the fusion polypeptide. This may be a protein domain, such as serum albumin.
- A. Separation Moiety or Linker
- The disclosure relates to novel protease cleavable separation moieties. As described herein, the protease cleavable separation moieties were designed so that the separation moieties are cleaved with high efficiency by proteases at a desired location (e.g., proteases that are selectively expressed or expressed at high levels in the tumor microenvironment) but are stable and not cleaved or cleaved with low efficiency in other locations (e.g., in the periphery, for example healthy tissue or serum).
- The protease cleavable separation moieties were designed using a process that included prioritizing proteases that would be suitable for cleaving the separation moieties based on expression in target indications, such as, expression in particular types of tumors (e.g., colon, lung, breast, melanoma). Multiple data sources for increased expression or specific expression of proteases in the target indications were used, including mRNA, proteomics and tissue staining data. The proteases were also prioritized based on their specific activity as well as intrinsic specificity, with high specific activity and high intrinsic activity preferred. Stability in the serum is an important design consideration, and to avoid potential off-target cleavage of the separation moieties by serum proteases, proteases that are not dependent on arginine in their substrate were selected, since many off-target enzymes are active towards arginine residues.
- Starting sequences for the design process were selected using a diverse peptide library as substrates for proteases with mass spectrometric detection of protease cleaved products to identify preferred sequence motifs for each candidate protease. For selected initial motifs, a new peptide library that was tailored to the preferred sequence motif for the candidate protease was designed, created and analyzed. The peptide motifs were also counter-screened for cleavage by the serum proteases thrombin and Factor Xa as well as the liver/kidney protease hepsin. This process yielded peptides containing sequence motifs that are cleaved by certain tumor-associated proteases (e.g., Matrix Metaloprotease 9 (MMP9), MMP14 and/or Cathepsin L) with high efficiency, but are stable (not cleaved or cleaved with low efficiency) in the serum or normal healthy tissue (e.g., by thrombin, Factor Xa, hepsin and the like). The separation moieties disclosed herein are efficient cleavage by human tumors and minimal cleavage by normal tissues or serum.
- This disclosure relates to separation moieties or linkers that connect a first amino acid sequence of interest (e.g. a first domain of interest) to a second amino acid sequence of interest (e.g. a second domain of interest). Typically, the first amino acid sequence of interest and the second amino acid sequence of interest are not found together in a naturally occurring protein. For example, the separation moiety can connect or link a first domain of interest to a second domain of interest in a fusion protein. The separation moiety is an amino acid sequence that can be of any suitable length, and preferably can be cleaved by a protease.
- The separation moieties disclosed herein can confer functionality, including flexibility as well as the ability to be cleaved. Flexible linkers are usually applied when joined domains requires a certain degree of movement or interaction. Cleavable linkers are introduced to release free and functional domains in vivo at a target site. The separation moieties disclosed herein serve to connect at least two domains of interest. The separation moieties can maintain cooperative inter-domain interactions or preserving biological activity. The separation moieties can join functional domains (e.g., a payload and half-life extension element) that are released from the separation moiety at a target site (e.g. a tumor microenvironment).
- In a preferred embodiment, the separation moiety is cleavable by a cleaving agent, e.g., an enzyme. Preferably, the separation moiety comprises a protease cleavage site. In some cases, the separation moiety comprises one or more cleavage sites. The separation moiety can comprise a single protease cleavage site. The separation moiety can also comprise 2 or more protease cleavage sites. For example, 2 cleavage sites, 3 cleavage sites, 4, cleavage sites, 5 cleavage sites, or more. In cases the separation moiety comprises 2 or more protease cleavage sites, the cleavage sites can be cleaved by the same protease or different proteases. A separation moiety comprising two or more cleavage sites is referred to as a “tandem linker.” The two or more cleavage sites can be arranged in any desired orientation, including, but not limited tom one cleavage site adjacent to another cleavage site, one cleavage site overlapping another cleavage site, or one cleavage site following by another cleavage site with intervening amino acids between the two cleavage sites.
- Of particular interest in the present invention are disease specific protease-cleavable linkers. Also preferred are protease-cleavable linkers that are preferentially cleaved at a desired location in the body, such as the tumor microenvironment, relative to the peripheral circulation. For example, the rate at which the protease-cleavable linker is cleaved in the tumor microenvironment can be at least about 10 times, at least about 100 times, at least about 1000 times or at least about 10,000 times faster in the desired location in the body, e.g., the tumor microenvironment, in comparison to in the peripheral circulation (e.g., in plasma).
- Proteases known to be associated with diseased cells or tissues include but are not limited to serine proteases, cysteine proteases, aspartate proteases, threonine proteases, glutamic acid proteases, metalloproteases, asparagine peptide lyases, serum proteases, cathepsins, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin G, Cathepsin K, Cathepsin L, kallikreins, hK1, hK10, hK15, plasmin, collagenase, Type IV collagenase, stromelysin, Factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidain, bromelain, calpain, caspases, caspase-3, Mirl-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin, nepenthesin, metalloexopeptidases, metalloendopeptidases, matrix metalloproteases (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, urokinase plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin-1β converting enzyme, thrombin, FAP (FAPcx), dipeptidyl peptidase, meprins, granzymes and dipeptidyl peptidase IV (DPPIV/CD26). Proteases capable of cleaving linker amino acid sequences (which can be encoded by the chimeric nucleic acid sequences provided herein) can, for example, be selected from the group consisting of a prostate specific antigen (PSA), a matrix metalloproteinase (MMP), an A Disintigrin and a Metalloproteinase (ADAM), a plasminogen activator, a cathepsin, a caspase, a tumor cell surface protease, and an elastase. The MMP can, for example, be matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), matrix metalloproteinase 14 (MMP14). In addition, or alternatively, the linker can be cleaved by a cathepsin, such as, Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin G, Cathepsin K and/or Cathepsin L. Preferably, the linker can be cleaved by MMP14 or Cathepsin L.
- Proteases useful for cleavage of linkers and for use in the methods disclosed herein are presented in Table 1, and exemplary proteases and their cleavage site are presented in Table 1a:
-
TABLE 1 Proteases relevant to inflammation and cancer Protease Specificity Other aspects Secreted by killer T cells: Granzyme B (grB) Cleaves after Asp Type of serine protease; strongly residues (asp-ase) implicated in inducing perforin-dependent target cell apoptosis Granzyme A (grA) trypsin-like, cleaves after Type of serine protease; basic residues Granzyme H (grH) Unknown substrate Type of serine protease; specificity Other granzymes are also secreted by killer T cells, but not all are present in humans Caspase-8 Cleaves after Asp Type of cysteine protease; plays essential residues role in TCR-induced cellular expansion- exact molecular role unclear Mucosa-associated Cleaves after arginine Type of cysteine protease; likely acts both lymphoid tissue residues as a scaffold and proteolytically active (MALT1) enzyme in the CBM-dependent signaling pathway Tryptase Targets: angiotensin I, Type of mast cell-specific serine protease; fibrinogen, prourokinase, trypsin-like; resistant to inhibition by TGFβ; preferentially macromolecular protease inhibitors cleaves proteins after expressed in mammals due to their lysine or arginine tetrameric structure, with all sites facing residues narrow central pore; also associated with inflammation Associated with inflammation: Thrombin Targets: FGF-2, Type of serine protease; modulates HB-EGF, Osteo-pontin, activity of vascular growth factors, PDGF, VEGF chemokines and extracellular proteins; strengthens VEGF-induced proliferation; induces cell migration; angiogenic factor; regulates hemostasis Chymase Exhibit chymotrypsin- Type of mast cell-specific serine protease like specificity, cleaving proteins after aromatic amino acid residues Carboxypeptidase A Cleaves amino acid Type of zinc-dependent metalloproteinase (MC-CPA) residues from C-terminal end of peptides and proteins Kallikreins Targets: high molecular Type of serine protease; modulate weight relaxation response; contribute to kininogen, pro-urokinase inflammatory response; fibrin degradation Elastase Targets: E-cadherin, GM- Type of neutrophil serine protease; CSF, IL-1, IL-2, IL-6, degrades ECM components; regulates IL8, p38MAPK, TNFα, VE- inflammatory response; activates pro- cadherin apoptotic signaling Cathepsin G Targets: EGF, ENA-78, Type of serine protease; degrades ECM IL-8, MCP-1, MMP-2, components; chemo-attractant of MT1-MMP, leukocytes; regulates inflammatory PAI-1, RANTES, TGFβ, response; promotes apoptosis TNFα PR-3 Targets: ENA-78, IL-8, Type of serine protease; promotes IL-18, JNK, p38MAPK inflammatory response; activates pro- TNFα apoptotic signaling Granzyme M (grM) Cleaves after Met and Type of serine protease; only expressed in other long, unbranched NK cells hydrophobic residues Calpains Cleave between Arg and Family of cysteine proteases; calcium- Gly dependent; activation is involved in the process of numerous inflammation- associated diseases -
TABLE 1a Exemplary Proteases and Protease Recognition Sequences Protease Cleavage Domain Sequence SEQ ID NO: MMP7 KRALGLPG 3 MMP7 (DE)8RPLALWRS(DR)8 4 MMP9 PR(S/T)(L/I)(S/T) 5 MMP9 LEATA 6 MMP11 GGAANLVRGG 7 MMP14 SGRIGFLRTA 8 MMP PLGLAG 9 MMP PLGLAX 10 MMP PLGC(me) AG 11 MMP ESPAYYTA 12 MMP RLQLKL 13 MMP RLQLKAC 14 MMP2, MMP9, MMP14 EP(Cit)G(Hof) YL 15 Urokinase plasminogen activator (uPA) SGRSA 16 Urokinase plasminogen activator (uPA) DAFK 17 Urokinase plasminogen activator (uPA) GGGRR 18 Lysosomal Enzyme GFLG 19 Lysosomal Enzyme ALAL 20 Lysosomal Enzyme FK 21 Cathepsin B NLL 22 Cathepsin D PIC(Et) FF 23 Cathepsin K GGPRGLPG 24 Prostate Specific Antigen HSSKLQ 25 Prostate Specific Antigen HSSKLQL 26 Prostate Specific Antigen HSSKLQEDA 27 Herpes Simplex Virus Protease LVLASSSFGY 28 HIV Protease GVSQNYPIVG 29 CMV Protease GVVQASCRLA 30 Thrombin F(Pip)RS 31 Thrombin DPRSFL 32 Thrombin PPRSFL 33 Caspase-3 DEVD 34 Caspase-3 DEVDP 35 Caspase-3 KGSGDVEG 36 Interleukin 1ß converting enzyme GWEHDG 37 Enterokinase EDDDDKA 38 FAP KQEQNPGST 39 Kallikrein 2GKAFRR 40 Plasmin DAFK 41 Plasmin DVLK 42 Plasmin DAFK 43 TOP ALLLALL 44 GPLGVRG 221 IPVSLRSG 222 VPLSLYSG 223 SGESPAYYTA 224 - Exemplary protease linkers include, but are not limited to kallikrein cleavable linkers, thrombin cleavable linkers, chymase cleavable linkers, carboxypeptidase A cleavable linkers, cathepsin cleavable linkers, elastase cleavable linkers, FAP cleavable linkers, ADAM cleavable linkers, PR-3 cleavable linkers, granzyme M cleavable linkers, a calpain cleavable linkers, a matrix metalloproteinase (MMP) cleavable linkers, a plasminogen activator cleavable linkers, a caspase cleavable linkers, a tryptase cleavable linkers, or a tumor cell surface protease. Specifically, MMP9 cleavable linkers, ADAM cleavable linkers, CTSL1 cleavable linkers, FAPα cleavable linkers, and cathepsin cleavable linkers. Some preferred protease-cleavable linkers are cleaved by a MMP and/or a cathepsin.
- The separation moieties disclosed herein are typically less than 100 amino acids. Such separation moieties can be of different lengths, such as from 1 amino acid (e.g., Gly) to 30 amino acids, from 1 amino acid to 40 amino acids, from 1 amino acid to 50 amino acids, from 1 amino acid to 60 amino acids, from 1 to 70 amino acids, from 1 to 80 amino acids, from 1 to 90 amino acids, and from 1 to 100 amino acids. In some embodiments, the linker is at least about 1, about 2, about 3, about 4, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 amino acids in length. Preferred linkers are typically from about 5 amino acids to about 30 amino acids.
- Preferably the lengths of linkers vary from 2 to 30 amino acids, optimized for each condition so that the linker does not impose any constraints on the conformation or interactions of the linked domains.
- In some embodiments, the separation moiety comprises the sequence GPAGLYAQ (SEQ ID NO: 195); GPAGMKGL (SEQ ID NO: 196); PGGPAGIG (SEQ ID NO: 197); ALFKSSFP (SEQ ID NO: 198); ALFFSSPP (SEQ ID NO: 199); LAQRLRSS (SEQ ID NO: 200); LAQKLKSS (SEQ ID NO; 201); GALFKSSFPSGGGPAGLYAQGGSGKGGSGK (SEQ ID NO: 202); RGSGGGPAGLYAQGSGGGPAGLYAQGGSGK (SEQ ID NO: 203); KGGGPAGLYAQGPAGLYAQGPAGLYAQGSR (SEQ ID NO: 204); RGGPAGLYAQGGPAGLYAQGGGPAGLYAQK (SEQ ID NO: 205); KGGALFKSSFPGGPAGIGPLAQKLKSSGGS (SEQ ID NO: 206); SGGPGGPAGIGALFKSSFPLAQKLKSSGGG (SEQ ID NO: 207); RGPLAQKLKSSALFKSSFPGGPAGIGGGGK (SEQ ID NO: 208); GGGALFKSSFPLAQKLKSSPGGPAGIGGGR (SEQ ID NO: 209); RGPGGPAGIGPLAQKLKSSALFKSSFPGGG (SEQ ID NO: 210); RGGPLAQKLKSSPGGPAGIGALFKSSFPGK (SEQ ID NO: 211); RSGGPAGLYAQALFKSSFPLAQKLKSSGGG (SEQ ID NO: 212); GGPLAQKLKSSALFKSSFPGPAGLYAQGGR (SEQ ID NO: 213); GGALFKSSFPGPAGLYAQPLAQKLKSSGGK (SEQ ID NO: 214); RGGALFKSSFPLAQKLKSSGPAGLYAQGGK (SEQ ID NO: 215); RGGGPAGLYAQPLAQKLKSSALFKSSFPGG (SEQ ID NO: 216); SGPLAQKLKSSGPAGLYAQALFKSSFPGSK (SEQ ID NO: 217); KGGPGGPAGIGPLAQRLRSSALFKSSFPGR (SEQ ID NO: 218); KSGPGGPAGIGALFFSSPPLAQKLKSSGGR (SEQ ID NO: 219); or SGGFPRSGGSFNPRTFGSKRKRRGSRGGGG (SEQ ID NO: 220)
- Certain preferred separation moieties comprises the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198). The separation moieties disclosed herein can comprise one or more cleavage motif or functional variants that are the same or different. The separation moieties can comprise 1, 2, 3, 4, 5, or more cleavage motifs or functional variants. Separation moieties comprising 30 amino acids can contain 2 cleavage motifs or functional variants, 3 cleavage motifs or functional variants or more. A “functional variant” of a separation moiety retains the ability to be cleaved with high efficiency at a target site (e.g., a tumor microenvironment that expresses high levels of the protease) and are not cleaved or cleaved with low efficiency in the periphery (e.g., serum). For example, the functional variants retain at least about 50%, about 55%, about 60%, about 70%, about 80%, about 85%, about 95% or more of the cleavage efficiency of a separation moiety comprising any one of SEQ ID NOs. 195-220.
- The separation moieties comprising more than one cleavage motif can be selected from SEQ ID NOs: 195-201 and combinations thereof. Preferred separation moieties comprising more than one cleavage motif comprise the amino acids selected from SEQ ID NO: 202-220.
- The separation moiety can comprise both ALFKSSFP (SEQ ID NO: 198) and GPAGLYAQ (SEQ ID NO: 195). The separation moiety can comprise two cleavage motifs that each have the sequence GPAGLYAQ (SEQ ID NO: 195). Alternatively or additionally, the separation moiety can comprise two cleavage motifs that each have the sequence ALFKSSFP (SEQ ID NO: 198). The separation moiety can comprise a third cleavage motif that is the same or different.
- In some embodiments, the separation moiety comprises an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99% identical to SEQ ID NOs: 195 to SEQ ID NO: 220 over the full length of SEQ ID NO: 195-220.
- The disclosure also relates to functional variants of separation moieties comprising SEQ ID NOs. 195-220. The functional variants of separation moieties comprising SEQ ID NOs: 195-220 generally differ from SEQ ID NOs. 195-220 by one or a few amino acids (including substitutions, deletions, insertions, or any combination thereof), and substantially retain their ability to be cleaved by a protease.
- The functional variants can contain at least one or more amino acid substitutions, deletions, or insertions relative to the separation moieties comprising SEQ ID NOs. 195-220. The functional variant can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations comparted to the separation moieties comprising SEQ ID NOs. 195-220. In some preferred embodiments, the functional variant differs from the separation moiety comprising SEQ ID NOs. 195-220 by less than 10, less, than 8, less than 5, less than 4, less than 3, less than 2, or one amino acid alterations, e.g., amino acid substitutions or deletions. In other embodiments, the functional variant may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to SEQ ID NOs. 195-220. The amino acid substitution can be a conservative substitution or a non-conservative substitution, but preferably is a conservative substitution.
- In other embodiments, the functional variants of the separation moieties may comprise 1, 2, 3, 4, or 5 or more non-conservative amino acid substitutions compared the separation moieties comprising SEQ ID NOs: 195-220. Non-conservative amino acid substitutions could be recognized by one of skill in the art. The functional variant of the separation moiety preferably contains no more than 1, 2, 3, 4, or 5 amino acid deletions.
- The amino acid sequences disclosed in the separation moieties can be described by the relative linear position in the separation moiety with respect to the sissile bond. As will be well-understood by persons skilled in the art, separation moieties comprising 8 amino acid protease substrates (e.g., SEQ ID Nos: 195-201) contain amino acid at positions P4, P3, P2, P1, P1′, P2′, P3′, P4′, wherein the sissile bond is between P1 and P1′. For example, amino acid positions for the separation moiety comprising the sequence GPAGLYAQ (SEQ ID NO: 195) can be described as follows:
-
G P A G L Y A Q P4 P3 P2 P1 P1′ P2′ P3′ P4′ - Amino acids positions for the separation moiety comprising the sequence ALFKSSFP (SEQ ID NO: 198) can be described as follows:
-
A L F K S S F P P4 P3 P2 P1 P1′ P2′ P3′ P4′ - Preferably, the amino acids surrounding the cleavage site (e.g., positions P1 and P1′ for SEQ ID NOs: 195-201) are not substituted.
- In embodiments, the separation moiety comprises the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) or a functional variant of SEQ ID NO: 195 or a function variant of SEQ ID NO: 198. As described herein, a functional variant of PAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) can comprise one or more amino acid substitutions, and substantially retain their ability to be cleaved by a protease. Specifically, the functional variants of GPAGLYAQ (SEQ ID NO: 195) is cleaved by MMP14, and the functional variant of ALFKSSFP (SEQ ID NO: 198) is cleaved by Capthepsin L (CTSL1). The functional variants also retain their ability to be cleaved with high efficiency at a target site (e.g., a tumor microenvironment that expresses high levels of the protease). For example, the functional variants of GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) retain at least about 50%, about 55%, about 60%, about 70%, about 80%, about 85%, about 95% or more of the cleavage efficiency of a separation moiety comprising amino acid sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198), respectively.
- Preferably, the functional variant of GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) comprise no more than 1, 2, 3, 4, or 5 conservative amino acid substitutions compared to GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198). Preferably, the amino acids at position P1 and P1′ are not substituted. The amino acids at positions P1 and P1′ in SEQ ID NO: 195 are G and L, and the amino acids at positions P1 and P1′ in SEQ ID NO: 198 are K and S.
- The functional variant of GPAGLYAQ (SEQ ID NO: 195) can preferably comprise one or more of the following: a) an arginine amino acid substitution at position P4, b) a leucine, valine, asparagine, or proline amino acid substitution at position P3, c) a asparagine amino acid substitution at position P2, d) a histidine, asparagine, or glycine amino acid substitution at position P1, e) a asparagine, isoleucine, or leucine amino acid substitution at position P1′, f) a tyrosine or arginine amino acid substitution at position P2′, g) a glycine, arginine, or alanine amino acid substitution at position P3′, h) or a serine, glutamine, or lysine amino acid substitution at position P4′. The following amino acid substitutions are disfavored in functional variants of GPAGLYAQ (SEQ ID NO: 195): a) arginine or isoleucine at position P3, b) alanine at position P2, c) valine at position P1, d) arginine, glycine, asparagine, or threonine at position P1′, e) aspartic acid or glutamic acid at position P2′, f) isoleucine at position P3′, g) valine at position P4′. In some embodiments, the functional variant of GPAGLYAQ (SEQ ID NO: 195) does not comprise an amino acid substitution at position P1 and/or P1′.
- The amino acid substitution of the functional variant of GPAGLYAQ (SEQ ID NO: 195) preferably comprises an amino acid substitution at position P4 and/or P4′. For example, the functional variant of GPAGLYAQ (SEQ ID NO: 195) can comprises a leucine at position P4, or serine, glutamine, lysine, or phenylalanine at position P4. Alternatively or additionally, the functional variant of GPAGLYAQ (SEQ ID NO: 195) can comprises a glycine, phenylalanine, or a proline at position P4′.
- In some embodiments, the amino acid substitutions at position P2 or P2′ of GPAGLYAQ (SEQ ID NO: 195) are not preferred.
- In some embodiments, the functional variant of GPAGLYAQ (SEQ ID NO: 195) comprises the amino acid sequence selected from SEQ ID NOs: 258-331. Specific functional variants of GPAGLYAQ (SEQ ID NO: 195) include GPLGLYAQ (SEQ ID NO: 295), and GPAGLKGA (SEQ ID NO: 285).
- The functional variants of LFKSSFP (SEQ ID NO: 198) preferably comprises hydrophobic amino acid substitutions. The functional variant of LFKSSFP (SEQ ID NO: 198) can preferably comprise one or more of the following: (a) lysine, histidine, serine, glutamine, leucine, proline, or phenylalanine at position P4; (b) lysine, histidine, glycine, proline, asparagine, phenylalanine at position P3; (c) arginine, leucine, alanine, glutamine, or histatine at position P2; (d) phenylalanine, histidine, threonine, alanine, or glutamine at position P1; (e) histidine, leucine, lysine, alanine, isoleucine, arginine, phenylalanine, asparagine, glutamic acid, or glycine at position P1′, (f) phenylalanine, leucine, isoleucine, lysine, alanine, glutamine, or proline at position P2′; (g) phenylalanine, leucine, glycine, serine, valine, histidine, alanine, or asparagine at position P3′; and phenylalanine, histidine, glycine, alanine, serine, valine, glutamine, lysine, or leucine.
- The inclusion of aspartic acid and/or glutamic acid in functional variants of SEQ ID NO:198 are generally disfavored and avoided. The following amino acid substitutions are also disfavored in functional variants of LFKSSFP (SEQ ID NO: 198): (a) alanine, serine, or glutamic acid at position P3; (b) proline, threonine, glycine, or aspartic acid at position P2; (c) proline at position P1; (d) proline at position P1′; (e) glycine at position P2′; (f) lysine or glutamic acid at position P3′; (g) aspartic acid at position P4′.
- The amino acid substitution of the functional variant of LFKSSFP (SEQ ID NO: 198) preferably comprises an amino acid substitution at position P4 and/or P1. In some embodiments, an amino acid substitution of the functional variant of LFKSSFP (SEQ ID NO: 198) at position P4′ is not preferred.
- In some embodiments, the functional variant of LFKSSFP (SEQ ID NO: 198) comprises the amino acid sequence selected from SEQ ID NOs: 332-408. Specific functional variants of LFKSSFP (SEQ ID NO: 198) include ALFFSSPP (SEQ ID NO: 199), ALFKSFPP (SEQ ID NO: 381), ALFKSLPP (SEQ ID NO: 382); ALFKHSPP (SEQ ID NO: 370); ALFKSIPP (SEQ ID NO: 383); ALFKSSLP (SEQ ID NO: 390); or SPFRSSRQ (SEQ ID NO: 333).
- The separation moieties disclosed herein can form a stable complex under physiological conditions with the amino acid sequences (e.g. domains) that they link, while being capable of being cleaved by a protease. For example, the separation moiety is stable (e.g., not cleaved or cleaved with low efficiency) in the circulation and cleaved with higher efficiency at a target site (i.e. a tumor microenvironment). Accordingly, fusion polypeptides that include the linkers disclosed herein can, if desired, have a prolonged circulation half-life and/or lower biological activity in the circulation in comparison to the components of the fusion polypeptide as separate molecular entities. Yet, when in the desired location (e.g., tumor microenvironment) the linkers can be efficiently cleaved to release the components that are joined together by the linker and restoring or nearly restoring the half-life and biological activity of the components as separate molecular entities.
- The separation moiety desirably remains stable in the circulation for at least 2 hours, at least 5, hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 24 hours, at least 30 hours, at least 35 hours, at least 40 hours, at least 45 hours, at least 50 hours, at least 60 hours, at least 65 hours, at least 70 hours, at least 80 hours, at least 90 hours, or longer.
- In some embodiments, the separation moiety is cleaved by less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 20%, 5%, or 1% in the circulation as compared to the target location. The separation moiety is also stable in the absence of an enzyme capable of cleaving the linker. However, upon expose to a suitable enzyme (i.e., a protease), the separation moiety is cleaved resulting in separation of the linked domain.
- B. Polypeptides and Compositions Comprising a Separation Moiety
- The separation moieties disclosed herein can be used in a wide range of applications. Without limitation, they are suitable for us in fusion proteins. As further described herein, the separation moieties are particularly useful for preparing therapeutic fusion proteins in which the therapeutic of biological activity of the fusion protein is attenuated and the attenuation is removed upon cleavage of the separation moiety. The separation moieties can also be used to conjugate a variety of payloads such as therapeutic and/or diagnostic agents to carriers or targeting agents (e.g., antibodies and fragments of antibodies, nanoparticles). Suitable methods for prepare such conjugates are well-known in the art, see for example, Bioconjugate Techniques, Third Ed., G. T. Hermanson (Ed.) Academic Press 2013. Exemplary payloads include, but are not limited to cytokines, antibodies, cell based-therapies, antibiotics, cytotoxic drugs, or other recombinant polypeptide complexes. Of specific interest are separation moieties that are suitable for use in conjugation with payloads that target or are targeted to tumor microenvironments.
- This disclosure relates recombinant polypeptides in which a separation moiety as disclosed herein links a first amino acid sequence of interest (e.g. a first domain of interest) to a second amino acid sequence of interest (e.g. a second domain of interest). Typically, the first amino acid sequence of interest and the second amino acid sequence of interest are not found together in a naturally occurring protein. Preferred linkers are SEQ ID NOS:195-220. In embodiments, at least one of the first amino acid sequence of interest and the second amino acid sequence of interest is the amino acid sequence of a therapeutic polypeptide. In some embodiments in which at least one of the first amino acid sequence of interest and the second amino acid sequence of interest is the amino acid sequence of a therapeutic polypeptide, the other amino acid sequence of interest can be the amino acid sequence of a targeting polypeptide, a half-life extending polypeptide and/or a blocking polypeptide.
- The polypeptides that contain a separation moiety can be represented by Formula I: [D1]-[L1]-[D2], wherein D1 is a first amino acid sequence of interest (e.g., a domain of interest), L1 is a separation moiety that connects or links D1 to D2; and D2 is a second amino acid sequence of interest (e.g., a second domain of interest). Preferably, L1 is a protease-cleavable separation moiety, and more preferably L1 comprises or consists of any of SEQ ID NOS: 195-220.
- The polypeptides can also be represented by Formula II: [D1]-[L1]-[D2]-[L2]-[D3], wherein D1 is a first amino acid of interest (e.g., a domain of interest), L1 and L2 are each, independently, a linker; D2 is a second amino acid sequence of interest (e.g., a domain of interest), and D3 is a third amino acid of interest (e.g., a domain of interest), wherein at least one of L1 and L2 is a protease-cleavable separation moiety, and preferably at least one of L1 and L2 comprises or consists of any of SEQ ID NOS: 195-220.
- The polypeptide can also be represented by Formula III: [D1]-[L1]-[D2]-[L2]-[D3]-[L3]-[D4], wherein D1 is a first amino acid of interest (e.g., a domain of interest), L1, L2 and L3 are each, independently, a linker; D2 is a second amino acid sequence of interest (e.g., a domain of interest), D3 is a third amino acid of interest (e.g., a domain of interest); and wherein D4 is a fourth amino acid of interest (e.g., a domain of interest), wherein at least one of L1, L2 and L3 is a protease-cleavable separation moiety, and preferably at least one of L1, L2 and L3 comprises or consists of any of SEQ ID NOS: 195-220.
- Additional specific applications of the separation moieties are described in more detail herein.
- i. Delivery of Payloads
- The separation moieties as described herein can be used to attach a therapeutic drug-moiety. In this approach, a therapeutic drug moiety is attached to the separation moiety to make a therapeutic drug moiety complex. The separate drug moiety complex can be a prodrug that is inactive until the target protease cleaves the prodrug, releasing the free drug.
- ii. Antibody-Drug Conjugates
- Another example of use of the separation moiety is in the field of antibody-drug conjugates (ADCs) that are mainly directed toward the treatment of cancer. ADCs typically are an antibody that is linked to a cytotoxic moiety, such as a cytotoxic drug. The ADCs discriminate between the healthy and diseased cells and provide targeted delivery of drug (e.g. cytotoxic drug) to diseased cells. ADCs typically comprise an antibody that targets a tumor marker that is specific to tumor cells, whereupon the antibody attaches itself to the tumor cell, causing the ADC to be absorbed into the cell, which enables the cytotoxic component to be released to kill the tumor cell. A key aspect of ADCs is the provision of a stable linker between the antibody component and the cytotoxic agent. In such applications, linkers may be cleavable or non-cleavable. For non-cleavable linkers, the antibody, linker and cytotoxic unit is incorporated into the tumor cell. The nature of the linker typically determines the release profile of the cytotoxic agent. For example, cleavable linkers between the antibody and the cytotoxic agent are typically catalyzed by enzymes in the tumor cell or in the tumor microenvironment, wherein the antibody and the cytotoxic agents are cleaved to release the cytotoxic agent.
- In certain embodiments, the separation moiety disclosed herein links or connects a drug moiety to an antibody moiety.
- iii. Peptide-Drug Conjugates
- The separation moieties disclosed herein are suitable for use in peptide-drug conjugates. These compounds typically comprise a cytotoxic payload and linker; however, instead of antibodies, peptide-drug conjugates are equipped with peptides that have the ability to penetrate tumors, thus allowing the cargo to be delivered inside the tumor. In some embodiments, the separation moiety links or connects a cytotoxic payload with a peptide. The peptide-drug conjugate remains stable and has no biological activity until the target protease cleaves the separation moiety.
- iv. Inducible Adoptive Cell Therapy
- The separation moieties disclosed herein are suitable for use in constructs engineered for use in adoptive cell transfer (ACT) therapies. The field of adoptive cell transfer (ACT) is currently comprised of chimeric antigen receptor (CAR) engineered T cells (and next generation therapies) that target T cells to cell surface expressed targets (e.g., tumor cells expressing surface targets), and T cell receptor (TCR) engineered T cells that can target intracellular antigens.
- In one embodiment, the separation moiety is used to tether a targeting moiety to a CAR construct. A CAR replaces the endogenous TCR complex with a new receptor that uses a fragment of a human or mouse antibody to bind to targets outside of a cancer cell. The antibody fragment is linked to various signaling proteins inside T-cell which mediate receptor activation when the CAR binds to its target. Porter et al., (2011) NEJM, 365:725-733; Grupp et al., (2013) NEJM, 368:1509-1518; U.S. Ser. No. 10/221,245/WO/2014/153270, Treatment of cancer using humanized anti-CD19 chimeric antigen receptor.
- In another embodiment, the separation moiety is used to tether a targeting moiety to a TCR construct. A TCR is based on the gene for the protein receptor that is already naturally present in T-cells. The gene for a desired TCR can be discovered in a single patient, e.g., a patient that is able to mount an effective immune response against a type of cancer. This gene can then be introduced into other patients by incorporating it into TCR T cell constructs or reengineered to improve the binding interaction with its MHC target. Guy et al., (2013) Nat Immunol., 14(3):262-70; Kuhns et al., (2012) Front Immunol., 25; 3:159; Fesnak et al., (2016) Nat Rev Cancer, 16(9): 566-581.
- These types of engineered T cells, whether autologous or allogenic, comprise engineered T cell receptor components that comprise a targeting agent such as an isolated human or humanized antibody. In CAR-T and TCR-T cells, the binding affinity of the targeting moiety may be affected by the steric, chemical, or flexibility properties of the separation moiety tethering the targeting moiety to the rest of the construct and the T cell. The separation moieties disclosed herein are suitable for use with engineered constructs for making CAR T and TCR T cells.
- v. Antigen-Binding Proteins
- The separation moieties disclosed herein are suitable for use in antigen-binding proteins. An “antigen-binding protein” (ABP) is a protein comprising one or more antigen-binding domains that specifically bind to an antigen or epitope. In some embodiments, the antigen-binding domain binds the antigen or epitope with specificity and affinity similar to that of naturally occurring antibodies. Typically, the separation moieties link a polypeptide that blocks the antigen-binding site of the ABP from binding to its cognate antigen. But when the separation moiety is cleaved the blocking polypeptide can diffuse away from the ABP antigen binding site and the ABP can bind to its cognate antigen. Exemplary binding polypeptides that can block the antigen binding site of an ABP include steric blocker such as human serum albumin, and peptides that interact with one or more of the complementarity determining regions (CDRs) in the antigen binding site of the ABP. Such blocking peptides can be obtained by screening libraries or by screening peptide fragments of the cognate antigen of an ABP of interest. Typically, when the ABP contains and an antigen binding site of an antibody, the separation moiety and blocker will be bonded to the amino terminus of the antibody light chain, or the amino terminus of the antibody heavy chain, so that the blocker is tethered close to and will readily block the antigen binding site. Suitable variations of this approach are used when the ABP contains an alternative scaffold for the binding site. Similarly, when single chain antibody binding sites are use, such as scFV of dAb, the blocker-separation moiety is typically bonded to the amino terminus near the antigen binding site. In certain embodiments, the ABP comprises an antibody binding site that comprises a VH and a VL, and the blocker-separation moiety is bonded to the amino terminus of the VL.
- The ABP can be an antibody (e.g., the first and second antigen binding domains are in the form of an antibody). Preferably, at least one antigen binding domain of the ABP is in the form of an antibody. In another preferred embodiment, a first or second antigen binding domain is in the form of an antibody, and a first or second antigen binding domain is in the form of an antigen-binding fragment (e.g., the first antigen binding domain is an antibody and the second antigen binding domain is an antigen-binding fragment. Alternatively, the first antigen binding domain is an antigen binding fragment and the second antigen binding domain is an antibody).
- In some embodiments, the ABP consists of an antibody. In some embodiments, the ABP consists essentially of an antibody. In some embodiments, the ABP comprises an alternative scaffold. In some embodiments, the ABP consists of an alternative scaffold. In some embodiments, the ABP consists essentially of an alternative scaffold. In some embodiments, the ABP comprises an antibody fragment. In some embodiments, the ABP consists of an antibody fragment. In some embodiments, the ABP consists essentially of an antibody fragment.
- In some embodiments, the separation moieties disclosed herein are suitable for use in conjunction with an antibody. The term “antibody” is used herein in its broadest sense and includes certain types of immunoglobulin molecules comprising one or more antigen-binding domains that specifically bind to an antigen or epitope. An antibody specifically includes intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multi-specific antibodies. An antibody is one type of ABP.
- In some embodiments, the separation moieties disclosed herein are suitable for use in conjunction with an antigen binding protein comprising an alternative scaffold. The term “alternative scaffold” refers to a molecule in which one or more regions may be diversified to produce one or more antigen-binding domains that specifically bind to an antigen or epitope.
- In some embodiments, the antigen-binding domain binds the antigen or epitope with specificity and affinity similar to that of an antibody. Exemplary alternative scaffolds include those derived from fibronectin (e.g., Adnectins™), the β-sandwich (e.g., iMab), lipocalin (e.g., Anticalins®), EETI-II/AGRP, BPTI/LACI-D1/ITI-D2 (e.g., Kunitz domains), thioredoxin peptide aptamers, protein A (e.g., Affibody®), ankyrin repeats (e.g., DARPins), gamma-B-crystallin/ubiquitin (e.g., Affilins), CTLD3 (e.g., Tetranectins), Fynomers, and (LDLR-A module) (e.g., Avimers). Additional information on alternative scaffolds is provided in Binz et al., Nat. Biotechnol., 2005 23:1257-1268; Skerra, Current Opin. in Biotech., 2007 18:295-304; and Silacci et al., J. Biol. Chem., 2014, 289:14392-14398; each of which is incorporated by reference in its entirety. An alternative scaffold is one type of ABP.
- In some embodiments, the separation moieties disclosed herein are suitable for use in conjunction with an antibody fragment. An “antibody fragment” comprises a portion of an intact antibody, such as the antigen-binding or variable region of an intact antibody. Antibody fragments include, for example, Fv fragments, Fab fragments, F(ab′)2 fragments, Fab′ fragments, scFv (sFv) fragments, and scFv-Fc fragments.
- In some embodiments, the separation moieties disclosed herein are suitable for use in conjunction with one or more Fv, Fab, or F(ab′)2 fragments. “Fv” fragments comprise a non-covalently-linked dimer of one heavy chain variable domain and one light chain variable domain. “Fab” fragments comprise, in addition to the heavy and light chain variable domains, the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments may be generated, for example, by recombinant methods or by papain digestion of a full-length antibody. “F(ab′)2” fragments contain two Fab′ fragments joined, near the hinge region, by disulfide bonds. F(ab′)2 fragments may be generated, for example, by recombinant methods or by pepsin digestion of an intact antibody. The F(ab′) fragments can be dissociated, for example, by treatment with 1-mercaptoethanol.
- In some embodiments, the separation moieties disclosed herein are suitable for use in conjunction with an scFv or scFv-Fc. “Single-chain Fv” or “sFv” or “scFv” antibody fragments comprise a VH domain and a VL domain in a single polypeptide chain. The VH and VL are generally linked by a peptide linker. See Plückthun A. (1994). Any suitable linker may be used.
- In some embodiments, the linker is a (GGGGS)n (SEQ ID NO: 231). In some embodiments, n=1, 2, 3, 4, 5, or 6. See Antibodies from Escherichia coli. In Rosenberg M. & Moore G. P. (Eds.), The Pharmacology of Monoclonal Antibodies vol. 113 (pp. 269-315). Springer-Verlag, New York, incorporated by reference in its entirety. “scFv-Fc” fragments comprise an scFv attached to an Fc domain. For example, an Fc domain may be attached to the C-terminal of the scFv. The Fc domain may follow the VH or VL, depending on the orientation of the variable domains in the scFv (i.e., VH-VL or VL-VH). Any suitable Fc domain known in the art or described herein may be used. In some cases, the Fc domain comprises an IgG4 Fc domain.
- In some embodiments, the separation moieties disclosed herein are suitable for use in conjunction with a single domain antibody. The term “single domain antibody” refers to a molecule in which one variable domain of an antibody specifically binds to an antigen without the presence of another variable domain. Single domain antibodies, and fragments thereof, are described in Arabi Ghahroudi et al., FEBS Letters, 1998, 414:521-526 and Muyldermans et al., Trends in Biochem. Sci., 2001, 26:230-245, each of which is incorporated by reference in its entirety. Single domain antibodies are also known as sdAbs or nanobodies.
- In some embodiments, the separation moieties disclosed herein are suitable for use in conjunction with a monospecific ABP. A “monospecific ABP” is an ABP that comprises one or more binding sites that specifically bind to the same epitope. An example of a monospecific ABP is a naturally occurring IgG molecule which, while divalent (i.e., having two antigen-binding domains), recognizes the same epitope at each of the two antigen-binding domains. The binding specificity may be present in any suitable valency.
- In some embodiments, the separation moieties disclosed herein are suitable for use in conjunction with a monoclonal antibody. The term “monoclonal antibody” refers to an antibody from a population of substantially homogeneous antibodies. A population of substantially homogeneous antibodies comprises antibodies that are substantially similar and that bind the same epitope(s), except for variants that may normally arise during production of the monoclonal antibody. Such variants are generally present in only minor amounts. A monoclonal antibody is typically obtained by a process that includes the selection of a single antibody from a plurality of antibodies. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, yeast clones, bacterial clones, or other recombinant DNA clones. The selected antibody can be further altered, for example, to improve affinity for the TNFR superfamily member proteins (“affinity maturation”), to humanize the antibody, to improve its production in cell culture, and/or to reduce its immunogenicity in a subject.
- In some embodiments, the separation moieties disclosed herein are suitable for use in conjunction with a chimeric antibody. 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.
- In some embodiments, the separation moieties disclosed herein are suitable for use in conjunction with a humanized antibody. “Humanized” forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. A humanized antibody is generally a human antibody (recipient antibody) in which residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody having a desired specificity, affinity, or biological effect. In some instances, selected framework region residues of the recipient antibody are replaced by the corresponding framework region residues from the donor antibody Humanized antibodies may also comprise residues that are not found in either the recipient antibody or the donor antibody. Such modifications may be made to further refine antibody function. For further details, see Jones et al., Nature, 1986, 321:522-525; Riechmann et al., Nature, 1988, 332:323-329; and Presta, Curr. Op. Struct. Biol., 1992, 2:593-596, each of which is incorporated by reference in its entirety.
- In some embodiments, the separation moieties disclosed herein are suitable for use in conjunction with a human antibody. A “human antibody” is one which possesses an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or derived from a non-human source that utilizes a human antibody repertoire or human antibody-encoding sequences (e.g., obtained from human sources or designed de novo). Human antibodies specifically exclude humanized antibodies.
- In some embodiments, the ABPs provided herein specifically bind to the extracellular domain of a TNFR Superfamily Protein. In some embodiments, the TNFR superfamily protein is CD27, CD137, CD40, GITR, LT-betaR, CD30, HVEM, TNFR1, TNFR2, or OX-40. The TNFR superfamily protein may be expressed on the surface of any suitable target cell. In some embodiments, the target cell is a T cell. In some embodiments, the target cell is an effector T cell. In some embodiments, the target cell is a regulatory T cell. In some embodiments, the target cell is a natural killer (NK) cell. In some embodiments, the target cell is a natural killer T (NKT) cell. In some embodiments, the target cell is a B cell. In some embodiments, the target cell is a myeloid derived cell. In some embodiments, the target cell is a myeloid derived suppressor cell. In some embodiments, the target cell is a dendritic cell.
- In some embodiments, an ABP provided herein is an antibody. In some embodiments, an ABP provided herein is an antibody fragment. In some embodiments, an ABP provided herein is an alternative scaffold.
- In some embodiments, the ABPs provided herein comprise an immunoglobulin molecule. In some embodiments, the ABPs provided herein consist of an immunoglobulin molecule. In some embodiments, the ABPs provided herein consist essentially of an immunoglobulin molecule. In some aspects, the immunoglobulin molecule comprises an antibody. In some aspects, the immunoglobulin molecule consists of an antibody. In some aspects, the immunoglobulin molecule consists essentially of an antibody.
- In some embodiments, the ABPs provided herein comprise a light chain. In some aspects, the light chain is a kappa light chain. In some aspects, the light chain is a lambda light chain.
- In some embodiments, the ABPs provided herein comprise a heavy chain. In some aspects, the heavy chain is an IgA. In some aspects, the heavy chain is an IgD. In some aspects, the heavy chain is an IgE. In some aspects, the heavy chain is an IgG. In some aspects, the heavy chain is an IgM. In some aspects, the heavy chain is an IgG1. In some aspects, the heavy chain is an IgG2. In some aspects, the heavy chain is an IgG3. In some aspects, the heavy chain is an IgG4. In some aspects, the heavy chain is an IgA1. In some aspects, the heavy chain is an IgA2.
- In some embodiments, the separation moieties connect to a further blocking moiety.
- In some embodiments, the separation moieties are positioned such that movement of the antigen-binding protein subunits is restricted.
- a. Multispecific and Monospecific Multivalent Antigen Binding Proteins
- In some embodiments, the separation moieties disclosed herein are suitable for use in conjunction with a multi-specific antigen binding protein (ABP). Multispecific ABP's provided herein bind more than one antigen. For instance, a multispecific antibody can bind 2 antigens, 3 antigens, binds 4 antigens, 5 antigens, or more. Alternatively, the multispecific ABP can bind 2 or more different epitopes. When the ABP binds two or more epitopes, the two or more different epitopes may be epitopes on the same antigen (e.g., a single TNFR superfamily protein molecule expressed by a cell) or on different antigens (e.g., different TNFR superfamily member protein molecules expressed by the same cell, or a TNFR superfamily member protein molecule and a non-TNFR Superfamily member protein molecule). In some aspects, a multi-specific ABP binds two different epitopes (i.e., a “bispecific ABP”). In some aspects, a multi-specific ABP binds three different epitopes (i.e., a “trispecific ABP”). In some aspects, a multi-specific ABP binds four different epitopes (i.e., a “quadspecific ABP”). In some aspects, a multi-specific ABP binds five different epitopes (i.e., a “quintspecific ABP”). In some aspects, a multi-specific ABP binds 6, 7, 8, or more different epitopes. Each binding specificity may be present in any suitable valency.
- In various embodiments, the antigen binding protein comprises a blocking domain. The separation moiety disclosed herein links the blocking domain to a first antigen binding domain and consequently the blocking domain inhibits (a) binding affinity or avidity of the antigen binding protein on the epitope and/or (b) agonist or antagonist activity of the antigen binding protein on the epitope. Preferably the separation moiety comprises a cleavage site. Upon cleavage of the separation moiety by a disease-specific enzyme (i.e., a protease) (a) the binding affinity or avidity of the antigen binding protein on the epitope and/or (b) agonist or antagonist activity of the antigen binding protein on the epitope is increased. In some embodiments, the antigen binding protein comprises an additional separation moiety that links or connects an additional blocking domain to an antigen binding domain. The additional separation moiety comprises a cleavage site.
- In various embodiments, the antigen binding protein comprises an additional blocking domain (e.g., a second, third, or fourth blocking domain) that is/are operably linked to an antigen binding domain (e.g., the second, third, or fourth antigen binding domain) by a cleavable separation moiety as disclosed herein. The blocking domain can be a steric blocker or a specific blocker. In some embodiments, the steric blocker is independently selected from the group consisting of a fragment of the extracellular portion of the antibody binding protein, serum albumin, a fragment of serum albumin, and an antibody or antigen binding fragment thereof that binds serum albumin. In some embodiments, the specific blocker is independently selected from a fragment of an antibody or antigen-binding fragment thereof that binds to the first, second, third or fourth antigen binding domain of the antigen binding protein described herein. Preferably, the blocking domain is serum albumin or a fragment thereof, or an antibody or antigen binding fragment thereof that binds serum albumin.
- The antigen binding domain may further comprise a third antigen binding domain and a fourth antigen binding domain each with binding specificity for a target antigen. TNFR superfamily target antigens are well known in the art and are encompassed by this disclosure. Exemplary TNFR superfamily members include, but are not limited to CD27, CD30, CD137 (4-1BB), TNFR1 (CD120a), TNFR2 (CD120b), CD40, CD95 (Fas/Apo-1), HVEM, LT-betaR, GITR, nerve growth factor receptor or OX-40 (CD34). Preferred TNFR superfamily target antigens are CD27, CD137, and OX40.
- The antigen binding domains with specificity for an antigen can have binding specificity for the same epitope or for a different epitope on the same antigen. For example, the first, second and third antigen binding domains can have binding specificity for the same epitope, for a different epitope, two of the antigen binding domains may binding specificity for the same epitope, or three of the antigen binding domains may have binding specificity for the same epitope. In some embodiments, the antigen binding protein may further comprise a fifth antigen binding domain with specificity for a tumor antigen.
- In some embodiments, the antigen binding polypeptide comprises an Fc region. In one format, two antigen binding domains can be located on each end of the Fc region. Another format comprises two antibody arms attached to the N terminus of the Fc region, each arm comprising 2 antigen binding domains. The antigen binding domain can also comprise a half-life extension domain. The half-life extension domain can be albumin, an antigen binding domain that recruits albumin, or an immunoglobulin Fc, or a fragment thereof. In some embodiments, the half-life extension domain is operably linked to the antigen binding polypeptide by a cleavable linker.
- The disclosure also relates to an antigen binding protein comprising at least a first polypeptide and a second polypeptide. The first polypeptide comprises at least a portion of an antibody heavy chain constant region and an antibody heavy chain variable region (VH). The second polypeptide comprises at least a portion of an antibody light chain constant region and an antibody light chain variable region (VL). At least one of the first and second polypeptide further comprises a blocking domain that is operably linked to the VH or VL through a protease cleavable linker. The first polypeptide associates with the second polypeptide, and VH and VL form an antigen binding site with binding specificity for a target antigen (e.g., 4-1BB or CS27). The blocking domain inhibits binding of the antigen binding site to the target antigen (e.g., 4-1BB or CD27). In some embodiments, the first polypeptide further comprises a second VH, and the first polypeptide associates with two of said second polypeptides to form two VH/VL antigen binding sites that each have specificity for human 4-1BB. In some embodiments, the antigen binding protein is a dimer of the first polypeptide with the associated second polypeptides. The antigen binding protein can further comprise a third, fourth, fifth, or a sixth polypeptide.
- In some embodiments the ABP is monospecific multivalent ABP. Such formats can have a variety of structures and be prepared using suitable antibody engineering techniques. For example, a dual Fab antibody that contains a heavy chain with the structure VH—CH1-noncleavable liner-VH—CH1-CH2-CH3 can be prepare. Such a heavy chain can be expressed and can pair with two light chains. The heavy chain can dimerize through conventional interchain disulfide bonds to form an antibody format that contains 4 Fab antigen binding sites. In such formats, the separation moieties described herein can be bonded to the amino terminus of the light chain polypeptides to link a blocker to each of the Fab antigen binding sites. Other suitable monospecific, multivalent ABP formats can be readily envisioned by those of skill in the art. In one such example a heavy chain is prepared that has the structure VH—CH1-CH2-CH3-VH—CH1. Two such heavy chains and dimerize and associate with four light chains to form a monospecific tetravalent ABP format. In such formats, the separation moieties described herein can be bonded to the amino terminus of the light chain polypeptides to link a blocker to each of the Fab antigen binding sites. The binding activity of such monospecific tetravalent ABPs is masked by the blocking domain, and the mask is removed upon cleavage of the separation domain (e.g., in a tumor microenvironment) and the ABP is able to bind its cognate antigen.
- The monospecific multivalent ABP formats can have binding specificity for any desired antigen. In some embodiments, the monospecific multivalent ABP formats specifically bind to the extracellular domain of a TNFR Superfamily Protein, such as CD27, CD30, CD137 (4-1BB), TNFR1 (CD120a), TNFR2 (CD120b), CD40, CD95 (Fas/Apo-1), HVEM, LT-betaR, GITR, nerve growth factor receptor or OX-40 (CD34).
- In embodiments, the multivalent antigen binding protein can comprise a first antigen binding site with specificity for a target antigen (e.g., CD27 or TNFR1), second antigen binding site with specificity for the target antigen, a blocking polypeptide, at least one protease cleavable linker, and an optional half-life extension element. In such embodiments, a first blocking polypeptide is operably linked to the first antigen binding site by a protease cleavable linker and, optionally, a second blocking polypeptide is operably linked to the second antigen binding site by a protease cleavable linker. Preferably, the blocking polypeptide is operably linked to the second antigen binding site by a protease cleavable linker. The blocking polypeptide inhibits (a) binding affinity or avidity of the antigen binding protein on the target antigen and/or (b) agonist or antagonist activity of the antigen binding protein on the target antigen and upon cleavage of the protease cleavable linker (a) the binding affinity or avidity of the antigen binding protein on the target antigen and/or (b) agonist activity of the antigen binding protein on the target antigen is increased. The optional half-life extension element can be operably linked to the first antigen binding site and/or second antigen binding site through a optionally protease cleavable linker. In such embodiments, the first and second binding sites can have specificity for the same epitope or different epitopes.
- In embodiments, the antigen binding protein further comprises a third antigen binding site with specificity for the same target antigen. The antigen binding protein can further comprise a fourth antigen binding site with specificity for the same target antigen as the first and second and third antigen binding sites. The third and fourth antigen binding sites can each further comprise a blocking polypeptide that is operable linked to the antigen-binding sight through a protease clevable linker.
- This disclosure also relates to a tetravalent antigen binding protein that can comprise a first polypeptide that comprises at least a portion of an antibody heavy chain constant region and an antibody heavy chain variable region (VH) and a second polypeptide that comprises at least a portion of an antibody light chain constant region, an antibody light chain variable region (VL). The first polypeptide associates with the second polypeptide and the VH and VL form an antigen binding site with binding specificity for a target antigen. The tetravalent antigen binding protein further comprises a blocking polypeptide that is operably linked to the VH or VL through a protease cleavable linker. The blocking polypeptide inhibits binding of the antigen binding site to the target antigen. In some embodiments, the first polypeptide can further comprise a second VH. The first polypeptide associates with two of the second polypeptides to form two VH/VL antigen binding site that each have specificity for the target antigen.
- The tetravalent antigen binding protein can further comprise a third polypeptide, a fourth polypeptide, a fifth polypeptide, a sixth polypeptide, a seventh polypeptide, and an eight polypeptide. The third polypeptide can comprise at least a portion of an antibody heavy chain constant region and an antibody heavy chain variable region (VH). The fourth polypeptide can comprise at least a portion of an antibody light chain constant region, an antibody light chain variable region (VL). The firth polypeptide can comprise at least portion of an antibody heavy chain constant region and an antibody heavy chain variable region (VH). The sixth polypeptide can comprise at least a portion of an antibody light chain constant region, an antibody light chain variable region (VL). The seventh polypeptide can comprise at least portion of an antibody heavy chain constant region and an antibody heavy chain variable region (VH). The eighth polypeptide can comprise at least a portion of an antibody light chain constant region, an antibody light chain variable region (VL).
- In some embodiments at least one of the third and fourth polypeptide further comprises a blocking polypeptide that is operably linked to the VH or VL through a protease cleavable linker. The third polypeptide associates with the fourth polypeptide and VH and VL form an antigen binding site with binding specificity for a target antigen, and the blocking domain inhibits binding of the antigen binding site to the target antigen.
- In some embodiments, the fifth and sixth polypeptide further comprises a blocking polypeptide that is operably linked to the VH or VL through a protease cleavable linker. The fifth polypeptide associates with the sixth polypeptide and VH and VL form an antigen binding site with binding specificity for a target antigen, and the blocking domain inhibits binding of the antigen binding site to the target antigen.
- In some embodiments, the seventh and eighth polypeptide further comprises a blocking polypeptide that is operably linked to the VH or VL through a protease cleavable linker. The seventh polypeptide associates with the eighth polypeptide and VH and VL form an antigen binding site with binding specificity for a target antigen, and the blocking domain inhibits binding of the antigen binding site to the target antigen.
- vi. Inducible Cytokines
- Disclosed herein are methods and compositions to engineer and use constructs comprising inducible cytokines. Cytokines are potent immune agonists, which lead to them being considered promising therapeutic agents for oncology. However, cytokines have a very narrow therapeutic window. Cytokines have short serum half-lives and are also considered to be highly potent. Consequently, therapeutic administration of cytokines produces undesirable systemic effects and toxicities. These were exacerbated by the need to administer large quantities of cytokine in order to achieve the desired levels of cytokine at the intended site of cytokine action (e.g., a tumor). Unfortunately, due to the biology of cytokines and inability to effectively target and control their activity, cytokines have not achieved the hoped-for clinical advantages in the treatment of tumors.
- Disclosed herein are fusion proteins that overcome the toxicity and short half-life problems that have severely limited the clinical use of cytokines in oncology. The fusion proteins contain cytokine polypeptides that have receptor agonist activity. But in the context of the fusion protein, the cytokine receptor agonist activity is attenuated, and the circulating half-life is extended. The fusion proteins include protease cleave sites, which are cleaved by proteases that are associated with a desired site of cytokine activity (e.g., a tumor), and are typically enriched or selectively present at the site of desired activity. Thus, the fusion proteins are preferentially (or selectively) and efficiently cleaved at the desired site of activity to limit cytokine activity substantially to the desired site of activity, such as the tumor microenvironment. Protease cleavage at the desired site of activity, such as in a tumor microenvironment, releases a form of the cytokine from the fusion protein that is much more active as a cytokine receptor agonist than the fusion protein (typically at least about 100× more active than the fusion protein). The form of the cytokine that is released upon cleavage of the fusion protein typically has a short half-life, which is often substantially similar to the half-life of the naturally occurring cytokine, further restricting cytokine activity to the tumor microenvironment. Even though the half-life of the fusion protein is extended, toxicity is dramatically reduced or eliminated because the circulating fusion protein is attenuated, and active cytokine is targeted to the tumor microenvironment. The fusion proteins described herein, for the first time, enable the administration of an effective therapeutic dose of a cytokine to treat tumors with the activity of the cytokine substantially limited to the tumor microenvironment, and dramatically reduces or eliminates unwanted systemic effects and toxicity of the cytokine.
- In general, the therapeutic use of cytokines is strongly limited by their systemic toxicity. TNF, for example, was originally discovered for its capacity of inducing the hemorrhagic necrosis of some tumors, and for its in vitro cytotoxic effect on different tumoral lines, but it subsequently proved to have strong pro-inflammatory activity, which can, in case of overproduction conditions, dangerously affect the human body. As the systemic toxicity is a fundamental problem with the use of pharmacologically active amounts of cytokines in humans, novel derivatives and therapeutic strategies are now under evaluation, aimed at reducing the toxic effects of this class of biological effectors while keeping their therapeutic efficacy.
- IL-2 exerts both stimulatory and regulatory functions in the immune system and is, along with other members of the common γ chain (γc) cytokine family, central to immune homeostasis. IL-2 mediates its action by binding to IL-2 receptors (IL-2R), consisting of either trimeric receptors made of IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (γc, CD132) chains or dimeric βγ IL-2Rs (1, 3). Both IL-2R variants are able to transmit signal upon IL-2 binding. However, trimeric αβγ IL-2Rs have a roughly 10-100 times higher affinity for IL-2 than dimeric βγ IL-2Rs (3), implicating that CD25 confers high-affinity binding of IL-2 to its receptor but is not crucial for signal transduction. Trimeric IL-2Rs are found on activated T cells and CD4+ forkhead box P3 (FoxP3)+ T regulatory cells (Treg), which are sensitive to IL-2 in vitro and in vivo. Conversely, antigen-experienced (memory) CD8+, CD44 high memory-phenotype (MP) CD8+, and natural killer (NK) cells are endowed with high levels of dimeric βγ IL-2Rs, and these cells also respond vigorously to IL-2 in vitro and in vivo.
- Expression of the high-affinity IL-2R is critical for endowing T cells to respond to low concentrations of IL-2 that is transiently available in vivo. IL-2Rα expression is absent on naive and memory T cells but is induced after antigen activation. IL-2Rβ is constitutively expressed by NK, NKT, and memory CD8+ T cells but is also induced on naive T cells after antigen activation. γc is much less stringently regulated and is constitutively expressed by all lymphoid cells. Once the high-affinity IL-2R is induced by antigen, IL-2R signaling upregulates the expression of IL-2Rα in part through Stat5-dependent regulation of Il2ra transcription (Kim et al., 2001). This process represents a mechanism to maintain expression of the high-affinity IL-2R and sustain IL-2 signaling while there remains a source of IL-2.
- IL-2 is captured by IL-2Rα through a large hydrophobic binding surface surrounded by a polar periphery that results in a relatively weak interaction (Kd 10-8 M) with rapid on-off binding kinetics. However, the IL-2Rα-IL-2 binary complex leads to a very small conformational change in IL-2 that promotes association with IL-2Rβ through a distinct polar interaction between IL-2 and IL-2Rβ. The pseudo-high affinity of the IL2/α/β trimeric complex (i.e. Kd ˜300 pM) clearly indicates that the trimeric complex is more stable than either IL2 bound to the α chain alone (Kd=10 nM) or to the β chain alone (Kd=450 nM). In any event, the IL2/α/β trimer then recruits the γ chain into the quaternary complex capable of signaling, which is facilitated by the large composite binding site on the IL2-bound β chain for the γ chain.
- In other words, the ternary IL-2Rα-IL-2Rβ-IL-2 complex then recruits γc through a weak interaction with IL-2 and a stronger interaction with IL-2Rβ to produce a stable quaternary high-affinity IL-2R (Kd 10-11 M which is 10 pM). The formation of the high-affinity quaternary IL-2-IL-2R complex leads to signal transduction through the tyrosine kinases Jak1 and Jak3, which are associated with IL-2Rβ and γc, respectively (Nelson and Willerford, 1998). The quaternary IL-2-IL-2R complex is rapidly internalized, where IL-2, IL-2Rβ, and γc are rapidly degraded, but IL-2Rα is recycled to the cell surface (Hémar et al., 1995; Yu and Malek, 2001). Thus, those functional activities that require sustained IL-2R signaling require a continued source of IL-2 to engage IL-2Rα and form additional IL-2-IL-2R signaling complexes.
- Interleukin-15 (IL-15), another member of the 4-alpha-helix bundle family of cytokines, has also emerged as an immunomodulator for the treatment of cancer. IL-15 is initially captured via IL-15Rα, which is expressed on antigen-presenting dendritic cells, monocytes and macrophages. IL-15 exhibits broad activity and induces the differentiation and proliferation of T, B and natural killer (NK) cells via signaling through the IL-15/IL-2-R-β (CD122) and the common γ chain (CD132). It also enhances cytolytic activity of CD8+ T cells and induces long-lasting antigen-experienced CD8+CD44 memory T cells. IL-15 stimulates differentiation and immunoglobulin synthesis by B cells and induces maturation of dendritic cells. It does not stimulate immunosuppressive T regulatory cells (Tregs). Thus, boosting IL-15 activity selectively in the tumor micro-environment could enhance innate and specific immunity and fight tumors (Waldmann et al., 2012). IL-15 was initially identified for its ability to stimulate T cell proliferation in an IL-2-like manner through common receptor components (IL-2R/15Rβ-γc) and signaling through JAK1/JAK3 and STAT3/STAT5. Like IL-2, IL-15 has been shown to stimulate proliferation of activated CD4−CD8−, CD4+CD8+, CD4+ and CD8+ T cells as well as facilitate the induction of cytotoxic T-lymphocytes, and the generation, proliferation and activation of NK cells (Waldmann et al., 1999). However, unlike IL-2 which is required to maintain forkhead box P3 (FOXP3)-expressing CD4+CD25+ Treg cells and for the retention of these cells in the periphery, IL-15 has little effect on Tregs (Berger et al., 2009). This is important as FOXP3-expressing CD4+CD25+ Tregs inhibit effector T cells, thereby inhibiting immune responses including those directed against the tumor. IL-2 also has a crucial role in initiating activation induced cell death (AICD), a process that leads to the elimination of self-reactive T cells, whereas IL-15 is an anti-apoptotic factor for T cells (Marks-Konczalik et al., 2000). IL-15 co-delivered with HIV peptide vaccines has been shown to overcome CD4+ T cell deficiency by promoting longevity of antigen-specific CD8+ T cells and blocking TRAIL-mediated apoptosis (Oh et al., 2008). Furthermore, IL-15 promotes the long-term maintenance of CD8+CD44hi memory T cells (Kanegane et al., 1996).
- The importance of IL-15 and IL-15Rα to T and NK cell development is further highlighted by the phenotype of IL-15Rα−/− and IL-15−/− mice. Knockout mice demonstrate decreased numbers of total CD8+ T cells, and are deficient in memory-phenotype CD8+ T cells, NK cells, NK/T cells and some subsets of intestinal intraepithelial lymphocytes, indicating that IL-15 provides essential positive homeostatic functions for these subsets of cells (Lodolce et al., 1996; Kennedy et al., 1998). The similarities in the phenotypes of these two strains of knockout mice suggest the importance of IL-15Rα in maintaining physiologically relevant IL-15 signals.
- IL-15 is presented in trans by the IL-15 receptor alpha-chain to the IL-15Rβγc complex displayed on the surface of T cells and natural killer (NK) cells (Han et al., 2011). The IL-15Ra-chain plays a role of chaperone protein, stabilizes, and increases IL-15 activity (Desbois et al., 2016). It has been shown that exogenous IL-15 may have a limited impact on patients with cancer due to its dependency on IL-15Ra frequently downregulated in cancer patients. Therefore, the fusion protein RLI, composed of the sushi+ domain of IL15Ra coupled via a linker to IL-15, has been suggested as an alternative approach to IL15 therapy (Bessard et al., 2009). It was found that administration of soluble IL-15/IL-15Rα complexes greatly enhanced IL-15 serum half-life and bioavailability in vivo (Stoklasek et al., 2010).
- In addition to the effects on T and NK cells, IL-15 also has several effects on other components of the immune system. IL-15 protects neutrophils from apoptosis, modulates phagocytosis and stimulates the secretion of IL-8 and IL-1R antagonist. It functions through the activation of JAK2, p38 and ERK1/2 MAPK, Syk kinase and the NF-kB transcriptional factor (Pelletier et al., 2002). In mast cells, IL-15 can act as a growth factor and an inhibitor of apoptosis. In these cells IL-15 activates the JAK2/STAT5 pathway without the requirement of γc binding (Tagaya et al., 1996). IL-15 also induces B lymphocyte proliferation and differentiation, and increases immunoglobulin secretion (Armitage et al., 1995). It also prevents Fas-mediated apoptosis and allows induction of antibody responses partially independent of CD4-help (Demerci et al., 2004; Steel et al., 2010). Monocytes, macrophages and dendritic cells effectively transcribe and translate IL-15. They also respond to IL-15 stimulation. Macrophages respond by increasing phagocytosis, inducing IL-8, IL-12 and MCP-1 expression, and secreting IL-6, IL-8 and TNFα (Budagian et al., 2006). Dendritic cells incubated with IL-15 demonstrate maturation with increased CD83, CD86, CD40, and MHC class II expression, are also resistant to apoptosis, and show enhanced interferon-γ secretion (Anguille et al., 2009).
- IL-15 has also been shown to have effects on non-hematological cells including myocytes, adipocytes, endothelial and neural cells. IL-15 has an anabolic effect on muscle and may support muscle cell differentiation (Quinn et al., 1995). It stimulates myocytes and muscle fibers to accumulate contractile protein and is able to slow muscle wasting in rats with cancer-related cachexia (Figueras et al., 2004). IL-15 has also been shown to stimulate angiogenesis (Angiolillo et al., 1997) and induce microglial growth and survival (Hanisch et al., 1997).
- Interleukin-7 (IL-7), also of the IL-2/IL-15 family, is a well-characterized pleiotropic cytokine, and is expressed by stromal cells, epithelial cells, endothelial cells, fibroblasts, smooth muscle cells and keratinocytes, and following activation, by dendritic cells (Alpdogan et al., 2005). Although it was originally described as a growth and differentiation factor for precursor B lymphocytes, subsequent studies have shown that IL-7 is critically involved in T-lymphocyte development and differentiation. Interleukin-7 signaling is essential for optimal CD8 T-cell function, homeostasis and establishment of memory (Schluns et al., 2000); it is required for the survival of most T-cell subsets, and its expression has been proposed to be important for regulating T-cell numbers.
- IL-7 binds to a dimeric receptor, including IL-7Rα and γc to form a ternary complex that plays fundamental roles in extracellular matrix remodeling, development, and homeostasis of T and B cells (Mazzucchelli and Durum, 2007). IL-7Rα also cross-reacts to form a ternary complex with thymic stromal lymphopoietin (TSLP) and its receptor (TSLPR), and activates the TSLP pathway, resulting in T and dendritic cell proliferation in humans and further B cell development in mice (Leonard, 2002). Tight regulation of the signaling cascades activated by the complexes are therefore crucial to normal cellular function. Under-stimulation of the IL-7 pathway caused by mutations in the IL-7Rα ectodomain inhibits T and B cell development, resulting in patients with a form of severe combined immunodeficiency (SCID) (Giliani et al., 2005; Puel et al., 1998).
- IL-7 has a potential role in enhancing immune reconstitution in cancer patients following cytotoxic chemotherapy. IL-7 therapy enhances immune reconstitution and can augment even limited thymic function by facilitating peripheral expansion of even small numbers of recent thymic emigrants. Therefore, IL-7 therapy could potentially repair the immune system of patients who have been depleted by cytotoxic chemotherapy (Capitini et al., 2010).
- Interleukin-12 (IL-12) is a disulfide-linked heterodimer of two separately encoded subunits (p35 and p40), which are linked covalently to give rise to the so-called bioactive heterodimeric (p70) molecule (Lieschke et al., 1997; Jana et al., 2014). Apart from forming heterodimers (IL-12 and IL-23), the p40 subunit is also secreted as a monomer (p40) and a homodimer (p402). It is known in the art that synthesis of the heterodimer as a single chain with a linker connecting the p35 to the p40 subunit preserves the full biological activity of the heterodimer. IL-12 plays a critical role in the early inflammatory response to infection and in the generation of Th1 cells, which favor cell-mediated immunity. It has been found that overproduction of IL-12 can be dangerous to the host because it is involved in the pathogenesis of a number of autoimmune inflammatory diseases (e.g. MS, arthritis,
type 1 diabetes). - The IL-12 receptor (IL-12R) is a heterodimeric complex consisting of IL-12Rβ1 and IL-12Rβ2 chains expressed on the surface of activated T-cells and natural killer cells (Trinchieri et al., 2003). The IL-12Rβ1 chain binds to the IL-12p40 subunit, whereas IL-12p35 in association with IL-12Rβ2 confers an intracellular signaling ability (Benson et al., 2011). Signal transduction through IL-12R induces phosphorylation of Janus kinase (Jak2) and tyrosine kinase (Tyk2), that phosphorylate and activate signal transducer and activator of transcription (STAT)1, STAT3, STAT4, and STAT5. The specific cellular effects of IL-12 are due mainly to activation of STAT4. IL-12 induces natural killer and T-cells to produce cytokines, in particular interferon (IFN)γ, that mediate many of the proinflammatory activities of IL-12, including CD4+ T-cell differentiation toward the Th1 phenotype (Montepaone et al., 2014).
- Treg cells actively suppress activation of the immune system and prevent pathological self-reactivity and consequent autoimmune disease. Developing drugs and methods to selectively activate regulatory T cells for the treatment of autoimmune disease is the subject of intense research and, until the development of the present invention, which can selectively deliver active interleukins at the site of inflammation, has been largely unsuccessful. Treg are a class of CD4+CD25+ T cells that suppress the activity of other immune cells. Treg are central to immune system homeostasis and play a major role in maintaining tolerance to self-antigens and in modulating the immune response to foreign antigens. Multiple autoimmune and inflammatory diseases, including
Type 1 Diabetes (T1D), Systemic Lupus Erythematosus (SLE), and Graft-versus-Host Disease (GVHD) have been shown to have a deficiency of Treg cell numbers or Treg function. - Consequently, there is great interest in the development of therapies that boost the numbers and/or function of Treg cells. One treatment approach for autoimmune diseases being investigated is the transplantation of autologous, ex vivo-expanded Treg cells (Tang, Q., et al, 2013, Cold Spring Harb. Perspect. Med., 3:1-15). While this approach has shown promise in treating animal models of disease and in several early stage human clinical trials, it requires personalized treatment with the patient's own T cells, is invasive, and is technically complex. Another approach is treatment with low dose Interleukin-2 (IL-2). Treg cells characteristically express high constitutive levels of the high affinity IL-2 receptor, IL2Rαβγ, which is composed of the subunits IL2Rα(CD25), IL2Rβ (CD122), and IL2Rγ (CD132), and Treg cell growth has been shown to be dependent on IL-2 (Malek, T. R., et al., 2010, Immunity, 33:153-65).
- Conversely, immune activation has also been achieved using IL-2, and recombinant IL-2 (Proleukin®) has been approved to treat certain cancers. High-dose IL-2 is used for the treatment of patients with metastatic melanoma and metastatic renal cell carcinoma with a long-term impact on overall survival.
- Clinical trials of low-dose IL-2 treatment of chronic GVHD (Koreth, J., et al., 2011, N Engl J Med., 365:2055-66) and HCV-associated autoimmune vasculitis patients (Saadoun, D., et al., 2011, N Engl J Med., 365:2067-77) have demonstrated increased Treg levels and signs of clinical efficacy. New clinical trials investigating the efficacy of IL-2 in multiple other autoimmune and inflammatory diseases have been initiated. The rationale for using so-called low dose IL-2 was to exploit the high IL-2 affinity of the trimeric IL-2 receptor which is constitutively expressed on Tregs while leaving other T cells which do not express the high affinity receptor in the inactivated state. Aldesleukin (marketed as Proleukin® by Prometheus Laboratories, San Diego, Calif.), the recombinant form of IL-2 used in these trials, is associated with high toxicity. Aldesleukin is approved for the treatment of metastatic melanoma and metastatic renal cancer, but its side effects are so severe that its use is only recommended in a hospital setting with access to intensive care (Web address: www.proleukin com/assets/pdf/proleukin.pdf).
- The clinical trials of IL-2 in autoimmune diseases have employed lower doses of IL-2 in order to target Treg cells, because Treg cells respond to lower concentrations of IL-2 than many other immune cell types due to their expression of IL2R alpha (Klatzmann D, 2015 Nat Rev Immunol. 15:283-94). However, even these lower doses resulted in safety and tolerability issues, and the treatments used have employed daily subcutaneous injections, either chronically or in intermittent 5-day treatment courses. Therefore, there is a need for an autoimmune disease therapy that potentiates Treg cell numbers and function, that targets Treg cells more specifically than IL-2, that is safer and more tolerable, and that is administered less frequently.
- One approach that has been suggested for improving the therapeutic index of IL-2-based therapy is to use variants of IL-2 that are selective for Treg cells relative to other immune cells. IL-2 receptors are expressed on a variety of different immune cell types, including T cells, NK cells, eosinophils, and monocytes, and this broad expression pattern likely contributes to its pleiotropic effect on the immune system and high systemic toxicity. In particular, activated T effector cells express IL2Rαβγ, as do pulmonary epithelial cells. But, activating T effector cells runs directly counter to the goal of down-modulating and controlling an immune response, and activating pulmonary epithelial cells leads to known dose-limiting side effects of IL-2 including pulmonary edema. In fact, the major side effect of high-dose IL-2 immunotherapy is vascular leak syndrome (VLS), which leads to accumulation of intravascular fluid in organs such as lungs and liver with subsequent pulmonary edema and liver cell damage. 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.
- According to the literature, VLS is believed to be caused by the release of proinflammatory cytokines from IL-2-activated NK cells. However, there is strong evidence that pulmonary edema results from direct binding of IL-2 to lung endothelial cells, which expressed low to intermediate levels of functional αβγ IL-2Rs. And, the pulmonary edema associated with interaction of IL-2 with lung endothelial cells was abrogated by blocking binding to CD25 with an anti-CD25 monoclonal antibody (mAb), in CD25-deficient host mice, or by the use of CD122-specific IL-2/anti-IL-2 mAb (IL-2/mAb) complexes, thus preventing VLS.
- Treatment with interleukin cytokines other than IL-2 has been more limited. IL-15 displays immune cell stimulatory activity similar to that of IL-2 but without the same inhibitory effects, thus making it a promising immunotherapeutic candidate. Clinical trials of recombinant human IL-15 for the treatment of metastatic malignant melanoma or renal cell cancer demonstrated appreciable changes in immune cell distribution, proliferation, and activation and suggested potential antitumor activity (Conlon et. al., 2014). IL-15 is currently in clinical trials to treat various forms of cancer. However, IL-15 therapy is known to be associated with undesired and toxic effects, such as exacerbating certain leukemias, graft-versus-host disease, hypotension, thrombocytopenia, and liver injury. (Mishra A., et al., Cancer Cell, 2012, 22(5):645-55; Alpdogan O. et al., Blood, 2005, 105(2):866-73; Conlon K C et al., J Clin Oncol, 2015, 33(1):74-82.)
- IL-7 promotes lymphocyte development in the thymus and maintains survival of naive and memory T cell homeostasis in the periphery. Moreover, it is important for the organogenesis of lymph nodes (LN) and for the maintenance of activated T cells recruited into the secondary lymphoid organs (SLOs) (Gao et. al., 2015). In clinical trials of IL-7, patients receiving IL-7 showed increases in both CD4+ and CD8+ T cells, with no significant increase in regulatory T cell numbers as monitored by FoxP3 expression (Sportes et al., 2008). In clinical trials reported in 2006, 2008 and 2010, patients with different kinds of cancers such as metastatic melanoma or sarcoma were injected subcutaneously with different doses of IL-7. Little toxicity was seen except for transient fevers and mild erythema. Circulating levels of both CD4+ and CD8+ T cells increased significantly and the number of Treg reduced. TCR repertoire diversity increased after IL-7 therapy. However, the anti-tumor activity of IL-7 was not well evaluated (Gao et. al., 2015). Results suggest that IL-7 therapy could enhance and broaden immune responses.
- IL-12 is a pleiotropic cytokine, the actions of which create an interconnection between the innate and adaptive immunity. IL-12 was first described as a factor secreted from PMA-induced EBV-transformed B-cell lines. Based on its actions, IL-12 has been designated as cytotoxic lymphocyte maturation factor and natural killer cell stimulatory factor. Due to bridging the innate and adaptive immunity and potently stimulating the production of IFNγ, a cytokine coordinating natural mechanisms of anticancer defense, IL-12 seemed ideal candidate for tumor immunotherapy in humans. However, severe side effects associated with systemic administration of IL-12 in clinical investigations and the very narrow therapeutic index of this cytokine markedly tempered enthusiasm for the use of this cytokine in cancer patients (Lasek et. al., 2014). Approaches to IL-12 therapy in which delivery of the cytokine is tumor-targeted, which may diminish some of the previous issues with IL-12 therapy, are currently in clinical trials for cancers.
- The direct use of IL-2 as an agonist to bind the IL-2R and modulate immune responses therapeutically has been problematic due its well-documented therapeutic risks, e.g., its short serum half-life and high toxicity. These risks have also limited the therapeutic development and use of other cytokines. New forms of cytokines that reduce these risks are needed. Disclosed herein are compositions and methods comprising IL-2 and IL-15 and other cytokines, functional fragments and muteins of cytokines as well as conditionally active cytokines designed to address these risks and provide needed immunomodulatory therapeutics.
- The present invention is designed to address the shortcomings of direct IL-2 therapy and therapy using other cytokines, for example using cytokine blocking moieties, e.g. steric blocking polypeptides, serum half-life extending polypeptides, targeting polypeptides, linking polypeptides, including protease cleavable linkers, and combinations thereof. Cytokines, including interleukins (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-21 IL-23), interferons (IFNs, including IFNα, IFNβ and IFNγ), tumor necrosis factors (e.g., TNFα, lymphotoxin), transforming growth factors (e.g., TGFβ1, TGFβ2, TGFβ3), chemokines (C—X—C motif chemokine 10 (CXCL10), CCL19, CCL20, CCL21), and granulocyte macrophage-colony stimulating factor (GM-CS) are highly potent when administered to patients. As used herein, “chemokine” means a family of small cytokines with the ability to induce directed chemotaxis in nearby responsive cells Cytokines can provide powerful therapy but are accompanied by undesired effects that are difficult to control clinically and which have limited the clinical use of cytokines. This disclosure relates to new forms of cytokines that can be used in patients with reduced or eliminated undesired effects. In particular, this disclosure relates to pharmaceutical compositions including chimeric polypeptides (fusion proteins), nucleic acids encoding fusion proteins and pharmaceutical formulations of the foregoing that contain cytokines or active fragments or muteins of cytokines that have decreased cytokine receptor activating activity in comparison to the corresponding cytokine. However, under selected conditions or in a selected biological environment the chimeric polypeptides activate their cognate receptors, often with the same or higher potency as the corresponding naturally occurring cytokine. As described herein, this is typically achieved using a cytokine blocking moiety that blocks or inhibits the receptor activating function of the cytokine, active fragment or mutein thereof under general conditions but not under selected conditions, such as those present at the desired site of cytokine activity (e.g., an inflammatory site or a tumor).
- The chimeric polypeptides and nucleic acids encoding the chimeric polypeptides can be made using any suitable method. For example, nucleic acids encoding a chimeric polypeptide can be made using recombinant DNA techniques, synthetic chemistry or combinations of these techniques, and expressed in a suitable expression system, such as in CHO cells. Chimeric polypeptides can similarly be made, for example by expression of a suitable nucleic acid, using synthetic or semi-synthetic chemical techniques, and the like. In some embodiments, the blocking moiety can be attached to the cytokine polypeptide via sortase-mediated conjugation. “Sortases” are transpeptidases that modify proteins by recognizing and cleaving a carboxyl-terminal sorting signal embedded in or terminally attached to a target protein or peptide. Sortase A catalyzes the cleavage of the LPXTG motif (where X is any standard amino acid) (SEQ ID NO: 237) between the Thr and Gly residue on the target protein, with transient attachment of the Thr residue to the active site Cys residue on the enzyme, forming an enzyme-thioacyl intermediate. To complete transpeptidation and create the peptide-monomer conjugate, a biomolecule with an N-terminal nucleophilic group, typically an oligoglycine motif, attacks the intermediate, displacing Sortase A and joining the two molecules.
- To form the cytokine-blocking moiety conjugate, the cytokine polypeptide is first tagged at the N-terminus with a polyglycine sequence, or alternatively, with at the C-terminus with a LPXTG (SEQ ID NO: 237) motif. The blocking moiety or other element has respective peptides attached that serve as acceptor sites for the tagged polypeptides. For conjugation to domains carrying a LPXTG (SEQ ID NO: 237) acceptor peptide attached via its N-terminus, the polypeptide will be tagged with an N-terminal poly-glycine stretch. For conjugation to domain carrying a poly-glycine peptide attached via its C-terminus, the polypeptide will be tagged at its C-terminus with a LPXTG (SEQ ID NO: 237) sortase recognition sequence. Recognizing poly-glycine and LPXTG (SEQ ID NO: 237) sequences, sortase will form a peptide bond between polymer-peptide and tagged polypeptides. The sortase reaction cleaves off glycine residues as intermediates and occurs at room temperature.
- A variety of mechanisms can be exploited to remove or reduce the inhibition caused by the blocking moiety. For example, the pharmaceutical compositions can include a cytokine moiety and a blocking moiety, e.g. a steric blocking moiety, with a protease cleavable linker comprising a protease cleavage site located between the cytokine and cytokine blocking moiety or within the cytokine blocking moiety. When the protease cleavage site is cleaved, the blocking moiety can dissociate from cytokine, and the cytokine can then activate cytokine receptor.
- Any suitable linker can be used. For example, the linker can comprise glycine-glycine, a sortase-recognition motif, or a sortase-recognition motif and a peptide sequence (Gly4Ser)n(SEQ ID NO: 238) or (Gly3Ser)n(SEQ ID NO: 239), wherein n is 1, 2, 3, 4 or 5. Typically, the sortase-recognition motif comprises a peptide sequence LPXTG (SEQ ID NO: 237), where X is any amino acid. In some embodiments, the covalent linkage is between a reactive lysine residue attached to the C-terminal of the cytokine polypeptide and a reactive aspartic acid attached to the N-terminal of the blocker or other domain. In other embodiments, the covalent linkage is between a reactive aspartic acid residue attached to the N-terminal of the cytokine polypeptide and a reactive lysine residue attached to the C-terminal of the blocker or another domain.
- Accordingly, as described in detail herein, the cytokine blocking moieties used can be steric blockers. As used herein, a “steric blocker” refers to a polypeptide or polypeptide moiety that can be covalently bonded to a cytokine polypeptide directly or indirectly through other moieties such as linkers, for example in the form of a chimeric polypeptide (fusion protein), but otherwise does not covalently bond to the cytokine polypeptide. A steric blocker can non-covalently bond to the cytokine polypeptide, for example though electrostatic, hydrophobic, ionic or hydrogen bonding. A steric blocker typically inhibits or blocks the activity of the cytokine moiety due to its proximity to the cytokine moiety and comparative size. The steric inhibition of the cytokine moiety can be removed by spatially separating the cytokine moiety from the steric blocker, such as by enzymatically cleaving a fusion protein that contains a steric blocker and a cytokine polypeptide at a site between the steric blocker and the cytokine polypeptide.
- As described in greater detail herein, the blocking function can be combined with or due to the presence of additional functional components in the pharmaceutical composition, such as a targeting domain, a serum half-life extension element, and protease-cleavable linking polypeptides. For example, a serum half-life extending polypeptide can also be a steric blocker.
- In the interest of presenting a concise disclosure of the full scope of the invention, aspects of the invention are described in detail using the cytokine IL-2 as an exemplary cytokine. However, the invention and this disclosure are not limited to IL-2. It will be clear to a person of skill in the art that this disclosure, including the disclosed methods, polypeptides and nucleic acids, adequately describes and enables the use of other cytokines, fragments and muteins, such as IL-2, IL-7, IL-12, IL-15, IL-18, IL-21 IL-23, IFNα, IFNβ, IFNγ, TNFα, lymphotoxin, TGF-β1, TGFβ2, TGFβ3, GM-CSF, CXCL10, CCL19, CCL20, CCL21 and functional fragments or muteins of any of the foregoing.
- Various elements ensure the delivery and activity of IL-2 preferentially at the site of desired IL-2 activity and to severely limit systemic exposure to the interleukin via a blocking and/or a targeting strategy preferentially linked to a serum half-life extension strategy. In this serum half-life extension strategy, the blocked version of interleukin circulates for extended times (preferentially 1-2 or more weeks) but the activated version has the typical serum half-life of the interleukin.
- By comparison to a serum half-life extended version, the serum half-life of IL-2 administered intravenously is only ˜10 minutes due to distribution into the total body extracellular space, which is large, ˜15 L in an average sized adult. Subsequently, IL-2 is metabolized by the kidneys with a half-life of ˜2.5 hours. (Smith, K. “
Interleukin 2 immunotherapy.” Therapeutic Immunology 240 (2001)). By other measurements, IL-2 has a very short plasma half-life of 85 minutes for intravenous administration and 3.3 hours subcutaneous administration (Kirchner, G. I., et al., 1998, Br J Clin Pharmacol. 46:5-10). In some embodiments of this invention, the half-life extension element is linked to the interleukin via a linker which is cleaved at the site of action (e.g. by inflammation-specific proteases) releasing the interleukin's full activity at the desired site and also separating it from the half-life extension of the uncleaved version. In such embodiments, the fully active and free interleukin would have very different pharmacokinetic (pK) properties—a half-life of hours instead of weeks. In addition, exposure to active cytokine is limited to the site of desired cytokine activity (e.g., an inflammatory site or tumor) and systemic exposure to active cytokine, and associated toxicity and side effects, are reduced. - Other cytokines envisioned in this invention have similar pharmacology (e.g. IL-15 as reported by Blood 2011 117:4787-4795; doi: doi.org/10.1182/blood-2010-10-311456) as IL-2 and accordingly, the designs of this invention address the shortcomings of using these agents directly, and provide chimeric polypeptides that can have extended half-life and/or be targeted to a site of desired activity (e.g., a site of inflammation or a tumor).
- If desired, IL-2 can be engineered to bind the IL-2R complex generally or one of the three IL-2R subunits specifically with an affinity that differs from that of the corresponding wild-type IL-2, for example to selectively activate Tregs or Teff (Effector T Cell). For example, IL-2 polypeptides that are said to have higher affinity for the trimeric form of the IL-2 receptor relative to the dimeric beta/gamma form of the 11-2 receptor in comparison to wild type IL-2 can have an amino acid sequence that includes one of the following sets of mutations with respect to SEQ ID NO: 1 (a mature IL-2 protein comprising amino acids 21-153 of human IL-2 having the UniProt Accession No. P60568-1): (a) K64R, V69A, and Q74P; (b) V69A, Q74P, and T101A; (c) V69A, Q74P, and I128T; (d) N30D, V69A, Q74P, and F103S; (e) K49E, V69A, A73V, and K76E; (f) V69A, Q74P, T101A, and T133N; (g) N30S, V69A, Q74P, and I128A; (h) V69A, Q74P, N88D, and S99P; (i) N30S, V69A, Q74P, and I128T; (j) K9T, Q11R, K35R, V69A, and Q74P; (k) A1T, M46L, K49R, E61D, V69A, and H79R; (1) K48E, E68D, N71T, N90H, F103S, and 1114V; (m) S4P, T10A, Q11R, V69A, Q74P, N88D, and T133A; (n) E15K, N30S Y31H, K35R, K48E, V69A, Q74P, and I92T; (o) N30S, E68D, V69A, N71A, Q74P, 575P, K76R, and N90H; (p) N30S, Y31C, T37A, V69A, A73V, Q74P, H79R, and I128T; (q) N26D, N29S, N30S, K54R, E67G, V69A, Q74P, and I92T; (r) K8R, Q13R, N26D, N30T, K35R, T37R, V69A, Q74P, and I92T; and (s) N29S, Y31H, K35R, T37A, K48E, V69A, N71R, Q74P, N88D, and I89V. This approach can also be applied to prepare muteins of other cytokines including interleukins (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-23), interferons (IFNs, including IFNalpha, IFNbeta and IFNgamma), tumor necrosis factors (e.g., TNFalpha, lymphotoxin), transforming growth factors (e.g., TGFbeta1, TGFbeta2, TGFbeta3) and granulocyte macrophage-colony stimulating factor (GM-CS). For example, muteins can be prepared that have desired binding affinity for a cognate receptor.
- As noted above, any of the mutant IL-2 polypeptides disclosed herein can include the sequences described; they can also be limited to the sequences described and otherwise identical to SEQ ID NO:1. Moreover, any of the mutant IL-2 polypeptides disclosed herein can optionally include a substitution of the cysteine residue at
position 125 with another residue (e.g., serine) and/or can optionally include a deletion of the alanine residue atposition 1 of SEQ ID NO:1. - Another approach to improving the therapeutic index of an IL-2 based therapy is to optimize the pharmacokinetics of the molecule to maximally activate Treg cells. Early studies of IL-2 action demonstrated that IL-2 stimulation of human T cell proliferation in vitro required a minimum of 5-6 hours exposure to effective concentrations of IL-2 (Cantrell, D. A., et. al., 1984, Science, 224: 1312-1316). When administered to human patients, IL-2 has a very short plasma half-life of 85 minutes for intravenous administration and 3.3 hours subcutaneous administration (Kirchner, G. I., et al., 1998, Br J Clin Pharmacol. 46:5-10). Because of its short half-life, maintaining circulating IL-2 at or above the level necessary to stimulate T cell proliferation for the necessary duration necessitates high doses that result in peak IL-2 levels significantly above the EC50 for Treg cells or will require frequent administration. These high IL-2 peak levels can activate IL2Rβγ receptors and have other unintended or adverse effects, for example VLS as noted above. An IL-2 analog, or a multifunctional protein with IL-2 attached to a domain that enables binding to the FcRn receptor, with a longer circulating half-life than IL-2 can achieve a target drug concentration for a specified period of time at a lower dose than IL-2, and with lower peak levels. Such an IL-2 analog will therefore require either lower doses or less frequent administration than IL-2 to effectively stimulate Treg cells. Less frequent subcutaneous administration of an IL-2 drug will also be more tolerable for patients. A therapeutic with these characteristics will translate clinically into improved pharmacological efficacy, reduced toxicity, and improved patient compliance with therapy. Alternatively, IL-2 or muteins of IL-2 (herein, “IL-2*”) can be selectively targeted to the intended site of action (e.g. sites of inflammation). This targeting can be achieved by one of several strategies, including the addition of domains to the administered agent that comprise blockers of the IL-2 (or muteins) that are cleaved away or by targeting domains or a combination of the two.
- In some embodiments, IL-2* partial agonists can be tailored to bind with higher or lower affinity depending on the desired target; for example, an IL-2* can be engineered to bind with enhanced affinity to one of the receptor subunits and not the others. These types of partial agonists, unlike full agonists or complete antagonists, offer the ability to tune the signaling properties to an amplitude that elicits desired functional properties while not meeting thresholds for undesired properties. Given the differential activities of the partial agonists, a repertoire of IL-2 variants could be engineered to exhibit an even finer degree of distinctive signaling activities, ranging from almost full to partial agonism to complete antagonism.
- In some embodiments, the IL-2* has altered affinity for IL-2Rα. In some embodiments, the IL-2* has a higher affinity for IL-2Rα than wild-type IL-2. In other embodiments, the IL-2* has altered affinity for IL-2Rβ. In one embodiment, IL-2* has enhanced binding affinity for IL-2Rβ, e.g., the N-terminus of IL-2Rβ, that eliminates the functional requirement for IL-2Rα. In another embodiment, an IL-2* is generated that has increased binding affinity for IL-2Rβ but that exhibited decreased binding to IL-2Rγ, and thereby is defective IL-2Rβγ heterodimerization and signaling.
- Blocking moieties, described in further detail below, can also be used to favor binding to or activation of one or more receptors. In one embodiment, blocking moieties are added such that IL-2Rβγ binding or activation is blocked but IL-2Rα binding or activation is not changed. In another embodiment, blocking moieties are added such that IL-2Rα binding or activation is diminished. In another embodiment, blocking moieties are added such that binding to and or activation of all three receptors is inhibited. This blocking may be relievable by removal of the blocking moieties in a particular environment, for example by proteolytic cleavage of a linker linking one or more blocking moieties to the cytokine.
- A similar approach can be applied to improve other cytokines, particularly for use as immunostimulatory agents, for example for treating cancer. For example, in this aspect, the pharmacokinetics and/or pharmacodynamics of the cytokine (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-21 IL-23, IFNα, IFNβ and IFNγ, TNFα, lymphotoxin, TGF-β1, TGFβ2, TGFβ3, GM-CSF, CXCL10, CCL19, CCL20, and CCL21 can be tailored to maximally activate effector cells (e.g., effect T cells, NK cells) and/or cytotoxic immune response promoting cells (e.g., induce dendritic cell maturation) at a site of desired activity, such as in a tumor, but preferably not systemically.
- Thus, provided herein are pharmaceutical compositions comprising at least one cytokine polypeptide, such as interleukins (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23), interferons (IFNs, including IFNα, IFNβ and IFNγ), tumor necrosis factors (e.g., TNFα, lymphotoxin), transforming growth factors (e.g., TGF-β1, TGFβ2, TGFβ3), chemokines (e.g. CXCL10, CCL19, CCL20, CCL21) and granulocyte macrophage-colony stimulating factor (GM-CS) or a functional fragment or mutein of any of the foregoing. The polypeptide typically also includes at least one linker amino acid sequence, wherein the amino acid sequence is in certain embodiments capable of being cleaved by an endogenous protease. In one embodiment, the linker comprises an amino acid sequence comprising HSSKLQ (SEQ ID NO: 25), GPLGVRG (SEQ ID NO: 221), IPVSLRSG (SEQ ID NO: 222), VPLSLYSG (SEQ ID NO: 223), or SGESPAYYTA (SEQ ID NO: 224). In other embodiments, the chimeric polypeptide further contains a blocking moiety, e.g. a steric blocking polypeptide moiety, capable of blocking the activity of the interleukin polypeptide. The blocking moiety, for example, can comprise a human serum albumin (HSA) binding domain or an optionally branched or multi-armed polyethylene glycol (PEG). Alternatively, the pharmaceutical composition comprises a first cytokine polypeptide or a fragment thereof, and blocking moiety, e.g. a steric blocking polypeptide moiety, wherein the blocking moiety blocks the activity of the cytokine polypeptide on the cytokine receptor, and wherein the blocking moiety in certain embodiments comprises a protease cleavable domain. In some embodiments, blockade and reduction of cytokine activity is achieved simply by attaching additional domains with very short linkers to the N or C terminus of the interleukin domain. In such embodiments, it is anticipated the blockade is relieved by protease digestion of the blocking moiety or of the short linker that tethers the blocker to the interleukin. Once the domain is clipped or is released, it will no longer be able to achieve blockade of cytokine activity.
- The pharmaceutical composition e.g., chimeric polypeptide can comprise two or more cytokines, which can be the same cytokine polypeptide or different cytokine polypeptides. For example, the two or more different types of cytokines have complementary functions. In some examples, a first cytokine is IL-2 and a second cytokine is IL-12. In some embodiments, each of the two or more different types of cytokine polypeptides have activities that modulate the activity of the other cytokine polypeptides. In some examples of chimeric polypeptides that contain two cytokine polypeptides, a first cytokine polypeptide is T-cell activating, and a second cytokine polypeptide is non-T-cell-activating. In some examples of chimeric polypeptides that contain two cytokine polypeptides, a first cytokine is a chemoattractant, e.g., CXCL10, and a second cytokine is an immune cell activator.
- Preferably, the cytokine polypeptides (including functional fragments) that are included in the fusion proteins disclosed herein are not mutated or engineered to alter the properties of the naturally occurring cytokine, including receptor binding affinity and specificity or serum half-life. However, changes in amino acid sequence from naturally occurring (including wild type) cytokine are acceptable to facilitate cloning and to achieve desired expression levels, for example.
- a. Blocking Moiety
- The blocking moiety can be any moiety that inhibits the ability of the cytokine to bind and/or activate its receptor. The blocking moiety can inhibit the ability of the cytokine to bind and/or activate its receptor sterically blocking and/or by noncovalently binding to the cytokine. Examples of suitable blocking moieties include the full length or a cytokine-binding fragment or mutein of the cognate receptor of the cytokine. Antibodies and fragments thereof including, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody a single chain variable fragment (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain of camelid-type nanobody (VHH), a sdAb and the like that bind the cytokine can also be used. Other suitable antigen-binding domain that bind the cytokine can also be used, include non-immunoglobulin proteins that mimic antibody binding and/or structure such as, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin and CTLA4 scaffolds. Further examples of suitable blocking polypeptides include polypeptides that sterically inhibit or block binding of the cytokine to its cognate receptor. Advantageously, such moieties can also function as half-life extending elements. For example, a peptide that is modified by conjugation to a water-soluble polymer, such as PEG, can sterically inhibit or prevent binding of the cytokine to its receptor. Polypeptides, or fragments thereof, that have long serum half-lives can also be used, such as serum albumin (human serum albumin), immunoglobulin Fc, transferring and the like, as well as fragments and muteins of such polypeptides.
- Antibodies and antigen-binding domains that bind to, for example, a protein with a long serum half-life such as HSA, immunoglobulin or transferrin, or to a receptor that is recycled to the plasma membrane, such as FcRn or transferrin receptor, can also inhibit the cytokine, particularly when bound to their antigen. Examples of such antigen-binding polypeptides include a single chain variable fragment (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain of camelid-type nanobody (VHH), a sdAb and the like. Other suitable antigen-binding domain that bind the cytokine can also be used, include non-immunoglobulin proteins that mimic antibody binding and/or structure such as, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin and CTLA4 scaffolds.
- In illustrative examples, when IL-2 is the cytokine in the chimeric polypeptide, the blocking moiety can be the full length or fragment or mutein of the alpha chain of IL-2 receptor (IL-2Rα) or beta (IL-2Rβ) or gamma chain of IL-2 receptor (IL-2Rγ), an anti-IL-2 single-domain antibody (sdAb) or scFv, an anti-CD25 antibody or fragment thereof, and anti-HSA sdAb or scFv, and the like.
- b. In Vivo Half-Life Extension Elements
- Preferably, the chimeric polypeptides comprise an in vivo half-life extension element. Increasing the in vivo half-life of therapeutic molecules with naturally short half-lives allows for a more acceptable and manageable dosing regimen without sacrificing effectiveness. As used herein, a “half-life extension element” is a part of the chimeric polypeptide that increases the in vivo half-life and improve pK, for example, by altering its size (e.g., to be above the kidney filtration cutoff), shape, hydrodynamic radius, charge, or parameters of absorption, biodistribution, metabolism, and elimination. An exemplary way to improve the pK of a polypeptide is by expression of an element in the polypeptide chain that binds to receptors that are recycled to the plasma membrane of cells rather than degraded in the lysosomes, such as the FcRn receptor on endothelial cells and transferrin receptor. Three types of proteins, e.g., human IgGs, HSA (or fragments), and transferrin, persist for much longer in human serum than would be predicted just by their size, which is a function of their ability to bind to receptors that are recycled rather than degraded in the lysosome. These proteins, or fragments of them that retain the FcRn binding are routinely linked to other polypeptides to extend their serum half-life. In one embodiment, the half-life extension element is a human serum albumin (HSA) binding domain. HSA (SEQ ID NO:2) may also be directly bound to the pharmaceutical compositions or bound via a short linker. Fragments of HSA may also be used. HSA and fragments thereof can function as both a blocking moiety and a half-life extension element. Human IgGs can also carry out a similar function.
- The serum half-life extension element can also be antigen-binding polypeptide that binds to a protein with a long serum half-life such as serum albumin, transferrin and the like. Examples of such polypeptides include antibodies and fragments thereof including, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody a single chain variable fragment (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain of camelid-type nanobody (VHH), a sdAb and the like. Other suitable antigen-binding domain include non-immunoglobulin proteins that mimic antibody binding and/or structure such as, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin and CTLA4 scaffolds. Further examples of antigen-binding polypeptides include a ligand for a desired receptor, a ligand-binding portion of a receptor, a lectin, and peptides that binds to or associates with one or more target antigens.
- Some preferred serum half-life extension elements are polypeptides that comprise complementarity determining regions (CDRs), and optionally non-CDR loops. Advantageously, such serum half-life extension elements can extend the serum half-life of the cytokine, and also function as inhibitors of the cytokine (e.g., via steric blocking, non-covalent interaction or combination thereof) and/or as targeting domains. In some instances, the serum half-life extension elements are domains derived from an immunoglobulin molecule (Ig molecule) or engineered protein scaffolds that mimic antibody structure and/or binding activity. The Ig may be of any class or subclass (IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM etc.). A polypeptide chain of an Ig molecule folds into a series of parallel beta strands linked by loops. In the variable region, three of the loops constitute the “complementarity determining regions” (CDRs) which determine the antigen binding specificity of the molecule. An IgG molecule comprises at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding fragment thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs) with are hypervariable in sequence and/or involved in antigen recognition and/or usually form structurally defined loops, interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments of this disclosure, at least some or all of the amino acid sequences of FR1, FR2, FR3, and FR4 are part of the “non-CDR loop” of the binding moieties described herein. A variable domain of an immunoglobulin molecule has several beta strands that are arranged in two sheets. The variable domains of both light and heavy immunoglobulin chains contain three hypervariable loops, or complementarity-determining regions (CDRs). The three CDRs of a V domain (CDR1, CDR2, CDR3) cluster at one end of the beta barrel. The CDRs are the loops that connect beta strands B-C, C′-C″, and F-G of the immunoglobulin fold, whereas the bottom loops that connect beta strands AB, CC′, C″-D and E-F of the immunoglobulin fold, and the top loop that connects the D-E strands of the immunoglobulin fold are the non-CDR loops. In some embodiments of this disclosure, at least some amino acid residues of a constant domain, CH1, CH2, or CH3, are part of the “non-CDR loop” of the binding moieties described herein. Non-CDR loops comprise, in some embodiments, one or more of AB, CD, EF, and DE loops of a C1-set domain of an Ig or an Ig-like molecule; AB, CC′, EF, FG, BC, and EC′ loops of a C2-set domain of an Ig or an Ig-like molecule; DE, BD, GF, A(A1A2)B, and EF loops of I(Intermediate)-set domain of an Ig or Ig-like molecule.
- Within the variable domain, the CDRs are believed to be responsible for antigen recognition and binding, while the FR residues are considered a scaffold for the CDRs. However, in certain cases, some of the FR residues play an important role in antigen recognition and binding. Framework region residues that affect Ag binding are divided into two categories. The first are FR residues that contact the antigen, thus are part of the binding-site, and some of these residues are close in sequence to the CDRs. Other residues are those that are far from the CDRs in sequence but are in close proximity to it in the 3-D structure of the molecule, e.g., a loop in heavy chain.
- The binding moieties are any kinds of polypeptides. For example, in certain instances the binding moieties are natural peptides, synthetic peptides, or fibronectin scaffolds, or engineered bulk serum proteins. The bulk serum protein comprises, for example, albumin, fibrinogen, or a globulin. In some embodiments, the binding moieties are engineered scaffolds. Engineered scaffolds comprise, for example, a sdAb, a scFv, a Fab, a VHH, a fibronectin type III domain, immunoglobulin-like scaffold (as suggested in Halaby et al., 1999. Prot Eng 12(7):563-571), DARPin, cystine knot peptide, lipocalin, three-helix bundle scaffold, protein G-related albumin-binding module, or a DNA or RNA aptamer scaffold.
- In some cases, the serum half-life extending element comprises a binding site for a bulk serum protein. In some embodiments, the CDRs provide the binding site for the bulk serum protein. The bulk serum protein is, in some examples, a globulin, albumin, transferrin, IgG1, IgG2, IgG4, IgG3, IgA monomer, Factor XIII, Fibrinogen, IgE, or pentameric IgM. In some embodiments, the CDR form a binding site for an immunoglobulin light chain, such as an Igκ free light chain or an Igλ free light chain.
- The serum half-life extension element can be any type of binding domain, including but not limited to, domains from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody. In some embodiments, the binding moiety is a single chain variable fragment (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of camelid derived nanobody. In other embodiments, the binding moieties are non-Ig binding domains, i.e., antibody mimetic, such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, and monobodies.
- In other embodiments, the serum half-life extension element can be a water-soluble polymer or a peptide that is conjugated to a water-soluble polymer, such as PEG. “PEG,” “polyethylene glycol” and “poly(ethylene glycol)” as used herein, are interchangeable and encompass any nonpeptidic water-soluble poly(ethylene oxide). The term “PEG” also means a polymer that contains a majority, that is to say, greater than 50%, of —OCH2CH2— repeating subunits. With respect to specific forms, the PEG can take any number of a variety of molecular weights, as well as structures or geometries such as “branched,” “linear,” “forked,” “multifunctional,” and the like, to be described in greater detail below. The PEG is not limited to a particular structure and can be linear (e.g., an end capped, e.g., alkoxy PEG or a bifunctional PEG), branched or multi-armed (e.g., forked PEG or PEG attached to a polyol core), a dendritic (or star) architecture, each with or without one or more degradable linkages. Moreover, the internal structure of the PEG can be organized in any number of different repeat patterns and can be selected from the group consisting of homopolymer, alternating copolymer, random copolymer, block copolymer, alternating tripolymer, random tripolymer, and block tripolymer. PEGs can be conjugated to polypeptide and peptides through any suitable method. Typically a reactive PEG derivative, such as N-hydroxysuccinamidyl ester PEG, is reacted with a peptide or polypeptide that includes amino acids with a side chain that contains an amine, sulfhydryl, carboxylic acid or hydroxyl functional group, such as cysteine, lysine, asparagine, glutamine, threonine, tyrosine, serine, aspartic acid, and glutamic acid.
- c. Targeting and Retention Domains
- For certain applications, it may be desirable to maximize the amount of time the construct is present in its desired location in the body. This can be achieved by including one further domain in the chimeric polypeptide (fusion protein) to influence its movements within the body. For example, the chimeric nucleic acids can encode a domain that directs the polypeptide to a location in the body, e.g., tumor cells or a site of inflammation; this domain is termed a “targeting domain” and/or encode a domain that retains the polypeptide in a location in the body, e.g., tumor cells or a site of inflammation; this domain is termed a “retention domain” In some embodiments a domain can function as both a targeting and a retention domain. In some embodiments, the targeting domain and/or retention domain are specific to a protease-rich environment. In some embodiments, the encoded targeting domain and/or retention domain are specific for regulatory T cells (Tregs), for example targeting the CCR4 or CD39 receptors. Other suitable targeting and/or retention domains comprise those that have a cognate ligand that is overexpressed in inflamed tissues, e.g., the IL-1 receptor, or the IL-6 receptor. In other embodiments, the suitable targeting and/or retention domains comprise those who have a cognate ligand that is overexpressed in tumor tissue, e.g., Epcam, CEA or mesothelin. In some embodiments, the targeting domain is linked to the interleukin via a linker which is cleaved at the site of action (e.g. by inflammation or cancer specific proteases) releasing the interleukin full activity at the desired site. In some embodiments, the targeting and/or retention domain is linked to the interleukin via a linker which is not cleaved at the site of action (e.g. by inflammation or cancer specific proteases), causing the cytokine to remain at the desired site.
- Antigens of choice, in some cases, are expressed on the surface of a diseased cell or tissue, for example a tumor or a cancer cell. Antigens useful for tumor targeting and retention include but are not limited to EpCAM, EGFR, HER-2, HER-3, c-Met, FoIR, and CEA. Pharmaceutical compositions disclosed herein, also include proteins comprising two targeting and/or retention domains that bind to two different target antigens known to be expressed on a diseased cell or tissue. Exemplary pairs of antigen binding domains include but are not limited to EGFR/CEA, EpCAM/CEA, and HER-2/HER-3.
- Suitable targeting and/or retention domains include antigen-binding domains, such as antibodies and fragments thereof including, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody a single chain variable fragment (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain of camelid-type nanobody (VHH), a sdAb and the like. Other suitable antigen-binding domain include non-immunoglobulin proteins that mimic antibody binding and/or structure such as, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin and CTLA4 scaffolds. Further examples of antigen-binding polypeptides include a ligand for a desired receptor, a ligand-binding portion of a receptor, a lectin, and peptides that binds to or associates with one or more target antigens.
- In some embodiments, the targeting and/or retention domains specifically bind to a cell surface molecule. In some embodiments, the targeting and/or retention domains specifically bind to a tumor antigen. In some embodiments, the targeting polypeptides specifically and independently bind to a tumor antigen selected from at least one of EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FoIR. In some embodiments, the targeting polypeptides specifically and independently bind to two different antigens, wherein at least one of the antigens is a tumor antigen selected from EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FoIR.
- The targeting and/or retention antigen can be a tumor antigen expressed on a tumor cell. Tumor antigens are well known in the art and include, for example, EpCAM, EGFR, HER-2, HER-3, c-Met, FolR, PSMA, CD38, BCMA, and CEA. 5T4, AFP, B7-H3, Cadherin-6, CAIX, CD117, CD123, CD138, CD166, CD19, CD20, CD205, CD22, CD30, CD33, CD352, CD37, CD44, CD52, CD56, CD70, CD71, CD74, CD79b, DLL3, EphA2, FAP, FGFR2, FGFR3, GPC3, gpA33, FLT-3, gpNMB, HPV-16 E6, HPV-16 E7, ITGA2, ITGA3, SLC39A6, MAGE, mesothelin, Mucl, Muc16, NaPi2b, Nectin-4, P-cadherin, NY-ESO-1, PRLR, PSCA, PTK7, ROR1, SLC44A4, SLTRKS, SLTRK6, STEAP1, TIM1, Trop2, WT1.
- The targeting and/or retention antigen can be an immune checkpoint protein. Examples of immune checkpoint proteins include but are not limited to CD27, CD137, 2B4, TIGIT, CD155, ICOS, HVEM, CD40L, LIGHT, TIM-1, OX40, DNAM-1, PD-L1, PD1, PD-L2, CTLA-4, CD8, CD40, CEACAM1, CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA, IDO1, IDO2, TDO, KIR, LAG-3, TIM-3, or VISTA.
- The targeting and/or retention antigen can be a cell surface molecule such as a protein, lipid or polysaccharide. In some embodiments, a targeting and/or retention antigen is a on a tumor cell, virally infected cell, bacterially infected cell, damaged red blood cell, arterial plaque cell, inflamed or fibrotic tissue cell. The targeting and/or retention antigen can comprise an immune response modulator. Examples of immune response modulator include but are not limited to granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 12 (IL-12), interleukin 15 (IL-15), B7-1 (CD80), B7-2 (CD86), GITRL, CD3, or GITR.
- The targeting and/or retention antigen can be a cytokine receptor. Examples, of cytokine receptors include but are not limited to Type I cytokine receptors, such as GM-CSF receptor, G-CSF receptor, Type I IL receptors, Epo receptor, LIF receptor, CNTF receptor, TPO receptor; Type II Cytokine receptors, such as IFN-alpha receptor (IFNAR1, IFNAR2), IFB-beta receptor, IFN-gamma receptor (IFNGR1, IFNGR2), Type II IL receptors; chemokine receptors, such as CC chemokine receptors, CXC chemokine receptors, CX3C chemokine receptors, XC chemokine receptors; tumor necrosis receptor superfamily receptors, such as TNFRSF5/CD40, TNFRSF8/CD30, TNFRSF7/CD27, TNFRSF1A/TNFR1/CD120a, TNFRSF1B/TNFR2/CD120b; TGF-beta receptors, such as TGF-
beta receptor 1, TGF-beta receptor 2; Ig super family receptors, such as IL-1 receptors, CSF-1R, PDGFR (PDGFRA, PDGFRB), SCFR. - d. Linkers
- As stated above, the pharmaceutical compositions comprise one or more linker sequences. A linker sequence serves to provide flexibility between polypeptides, such that, for example, the blocking moiety is capable of inhibiting the activity of the cytokine polypeptide. The linker sequence can be located between any or all of the cytokine polypeptide, the serum half-life extension element, and/or the blocking moiety. As described herein, at least one of the linkers is protease cleavable, and contains a (one or more) cleavage site for a (one or more) desired protease. Preferably, the desired protease is enriched or selectively expressed at the desired site of cytokine activity (e.g., the tumor microenvironment). Thus, the fusion protein is preferentially or selectively cleaved at the site of desired cytokine activity.
- The orientation of the components of the pharmaceutical composition, are largely a matter of design choice and it is recognized that multiple orientations are possible, and all are intended to be encompassed by this disclosure. For example, a blocking moiety can be located C-terminally or N-terminally to a cytokine polypeptide.
- Provided herein are pharmaceutical compositions comprising polypeptide sequences. As with all peptides, polypeptides, and proteins, including fragments thereof, it is understood that additional modifications in the amino acid sequence of the chimeric polypeptides (amino acid sequence variants) can occur that do not alter the nature or function of the peptides, polypeptides, or proteins. Such modifications include conservative amino acid substitutions and are discussed in greater detail below.
- The compositions provided herein have a desired function. The compositions are comprised of at least a cytokine polypeptide, such as IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, or IFNγ, or a chemokine, such as CXCL10, CCL19, CCL20, CCL21, a blocking moiety, e.g. a steric blocking polypeptide, and an optional serum half-life extension element, and an optional targeting polypeptide, with one or more linkers connecting each polypeptide in the composition. The first polypeptide, e.g., an IL-2 mutein, is provided to be an active agent. The blocking moiety is provided to block the activity of the interleukin. The linker polypeptide, e.g., a protease cleavable polypeptide, is provided to be cleaved by a protease that is specifically expressed at the intended target of the active agent. Optionally, the blocking moiety blocks the activity of the first polypeptide by binding the interleukin polypeptide. In some embodiments, the blocking moiety, e.g. a steric blocking peptide, is linked to the interleukin via a protease-cleavable linker which is cleaved at the site of action (e.g. by inflammation specific proteases) releasing the cytokine full activity at the desired site.
- In some embodiments, the linker comprises glycine-glycine, a sortase-recognition motif, or a sortase-recognition motif and a peptide sequence (Gly4Ser)n(SEQ ID NO: 238) or (Gly3Ser)n (SEQ ID NO: 239), wherein n is 1, 2, 3, 4 or 5. In one embodiment, the sortase-recognition motif comprises a peptide sequence LPXTG, where X is any amino acid (SEQ ID NO: 237). In one embodiment, the covalent linkage is between a reactive lysine residue attached to the C-terminal of the cytokine polypeptide and a reactive aspartic acid attached to the N-terminal of the blocking or other moiety. In one embodiment, the covalent linkage is between a reactive aspartic acid residue attached to the N-terminal of the cytokine polypeptide and a reactive lysine residue attached to the C-terminal of the blocking or other moiety.
- e. Cleavage and Inducibility
- As described herein, the activity of the cytokine polypeptide the context of the fusion protein is attenuated, and protease cleavage at the desired site of activity, such as in a tumor microenvironment, releases a form of the cytokine from the fusion protein that is much more active as a cytokine receptor agonist than the fusion protein. For example, the cytokine-receptor activating (agonist) activity of the fusion polypeptide can be at least about 10×, at least about 50×, at least about 100×, at least about 250×, at least about 500×, or at least about 1000× less than the cytokine receptor activating activity of the cytokine polypeptide as a separate molecular entity. The cytokine polypeptide that is part of the fusion protein exists as a separate molecular entity when it contains an amino acid that is substantially identical to the cytokine polypeptide and does not substantially include additional amino acids and is not associated (by covalent or non-covalent bonds) with other molecules. If necessary, a cytokine polypeptide as a separate molecular entity may include some additional amino acid sequences, such as a tag or short sequence to aid in expression and/or purification.
- In other examples, the cytokine-receptor activating (agonist) activity of the fusion polypeptide is at least about 10×, at least about 50×, at least about 100×, at least about 250×, at least about 500×, or about 1000× less than the cytokine receptor activating activity of the polypeptide that contains the cytokine polypeptide that is produced by cleavage of the protease cleavable linker in the fusion protein. In other words, the cytokine receptor activating (agonist) activity of the polypeptide that contains the cytokine polypeptide that is produced by cleavage of the protease cleavable linker in the fusion protein is at least about 10×, at least about 50×, at least about 100×, at least about 250×, at least about 500×, or at least about 1000× greater than the cytokine receptor activating activity of the fusion protein. In other examples, a recombinant polypeptide is in conjugation with a cleavable moiety wherein the cleavable moiety is cleaved with reduced catalytic efficiency by one or more proteases than a reference polypeptide sequence.
- In some embodiments, the cleavable moiety is resistant to proteolytic cleavage by one or more proteases. A cleavable moiety is resistant to a protease if the sequence comprises a binding site that is altered from the canonical cleavable motif sequence for the specific protease. In some embodiments, a binding site is altered compared to a reference sequence by making one or more substitutions in the protease cleavage motif sequence. For example, the protease cathepsin S cleaves the sequence GAVVRGA (SEQ ID NO: 240); a sequence is made to substitution can be made to substitute the arginine (R) with a glutamine (Q), thus changing a charged residue to a shorter, polar residue, reducing the ability of cathepsin S to bind and cleave the sequence. Such semi-conservative amino acid substitutions in protease target sequence motifs can lead to reduced binding ability, and such altered sequence motifs are therefore protease resistant. An uncleavable moiety can be made by inserting a disruptive amino acid into the protease target sequence motif, such as a proline (causes curves in the secondary structure of the peptide) or histidine (causes steric interference with other amino acid side chains). As used herein, a “protease-resistant” peptide linker is one with reduced or undetectable cleavage by one or more specified proteases. Exemplary protease-resistant peptide linkers can be tested, e.g., in vitro by incubation with a specific protease followed by analysis of the digestion products by western blot.
- f. Polypeptide Substitutions
- The polypeptides described herein can include components (e.g., the cytokine, the blocking moiety) that have the same amino acid sequence of the corresponding naturally occurring protein (e.g., IL-2, IL-15, HSA) or can have an amino acid sequence that differs from the naturally occurring protein so long as the desired function is maintained. It is understood that one way to define any known modifications and derivatives or those that might arise, of the disclosed proteins and nucleic acids that encode them is through defining the sequence variants in terms of identity to specific known reference sequences. Specifically disclosed are polypeptides and nucleic acids which have at least, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent identity to the chimeric polypeptides provided herein. For example, provided are polypeptides or nucleic acids that have at least, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent identity to the sequence of any of the nucleic acids or polypeptides described herein. This includes polypeptides or nucleic acids that have at least, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent identity to the sequence of the cleavable linkers provided herein. This also includes variants of the linker or inducible polypeptides that comprise 1, 2, 3, 4, 5, or 6 variants from the cleavage domain sequences. Those of skill in the art readily understand how to determine the identity of two polypeptides or two nucleic acids. For example, the identity can be calculated after aligning the two sequences so that the identity is at its highest level.
- Another way of calculating identity can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman Adv. Appl. Math. 2:482 (1981), by the identity alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection.
- The same types of identity can be obtained for nucleic acids by, for example, the algorithms disclosed in Zuker, Science 244:48-52 (1989); Jaeger et al., Proc. Natl. Acad. Sci. USA 86:7706-7710 (1989); Jaeger et al., Methods Enzymol. 183:281-306 (1989), which are herein incorporated by reference for at least material related to nucleic acid alignment. It is understood that any of the methods typically can be used and that in certain instances the results of these various methods may differ, but the skilled artisan understands if identity is found with at least one of these methods, the sequences would be said to have the stated identity, and be disclosed herein.
- Protein modifications include amino acid sequence modifications. Modifications in amino acid sequence may arise naturally as allelic variations (e.g., due to genetic polymorphism), may arise due to environmental influence (e.g., by exposure to ultraviolet light), or may be produced by human intervention (e.g., by mutagenesis of cloned DNA sequences), such as induced point, deletion, insertion and substitution mutants. These modifications can result in changes in the amino acid sequence, provide silent mutations, modify a restriction site, or provide other specific mutations. Amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional or deletional modifications. Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about from 2 to 6 residues are deleted at any one site within the protein molecule Amino acid substitutions are typically of single residues but can occur at a number of different locations at once; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct. The mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. Substitutional modifications are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Table 2 and are referred to as conservative substitutions.
-
TABLE 2 Exemplary amino acid substitutions Amino Acid Exemplary Substitutions Ala Ser, Gly, Cys Arg Lys, Gln, Met, Ile Asn Gln, His, Glu, Asp Asp Glu, Asn, Gln Cys Ser, Met, Thr Gln Asn, Lys, Glu, Asp Glu Asp, Asn, Gln Gly Pro, Ala His Asn, Gln Ile Leu, Val, Met Leu Ile, Val, Met Lys Arg, Gln, Met, Ile Met Leu, Ile, Val Phe Met, Leu, Tyr, Trp, His Ser Thr, Met, Cys Thr Ser, Met, Val Trp Tyr, Phe Tyr Trp, Phe, His Val Ile, Leu, Met - Modifications, including the specific amino acid substitutions, are made by known methods. For example, modifications are made by site specific mutagenesis of nucleotides in the DNA encoding the polypeptide, thereby producing DNA encoding the modification, and thereafter expressing the DNA in recombinant cell culture. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example M13 primer mutagenesis and PCR mutagenesis.
- Modifications can be selected to optimize binding. For example, affinity maturation techniques can be used to alter binding of the scFv by introducing random mutations inside the complementarity determining regions (CDRs). Such random mutations can be introduced using a variety of techniques, including radiation, chemical mutagens or error-prone PCR. Multiple rounds of mutation and selection can be performed using, for example, phage display.
- The disclosure also relates to nucleic acids that encode the chimeric polypeptides described herein, and to the use of such nucleic acids to produce the chimeric polypeptides and for therapeutic purposes. For example, the invention includes DNA and RNA molecules (e.g., mRNA, self-replicating RNA) that encode a chimeric polypeptide and to the therapeutic use of such DNA and RNA molecules.
- g. Exemplary Compositions
- Exemplary fusion proteins of the invention combine the above described elements in a variety of orientations. The orientations described in this section are meant as examples and are not to be considered limiting.
- In some embodiments, the fusion protein comprises a cytokine, a blocking moiety and a half-life extension element. In some embodiments, the cytokine is positioned between the half-life extension element and the blocking moiety. In some embodiments, the cytokine is N-terminal to the blocking moiety and the half-life extension element. In some such embodiments, the cytokine is proximal to the blocking moiety; in some such embodiments, the cytokine is proximal to the half-life extension element. At least one protease-cleavable linker must be included in all embodiments, such that the cytokine may be active upon cleavage. In some embodiments, the cytokine is C-terminal to the blocking moiety and the half-life extension element. Additional elements may be attached to one another by a cleavable linker, a non-cleavable linker, or by direct fusion.
- In some embodiments, the blocking domains used are capable of extending half-life, and the cytokine is positioned between two such blocking domains. In some embodiments, the cytokine is positioned between two blocking domains, one of which is capable of extending half-life.
- In some embodiments, two cytokines are included in the same construct. In some embodiments, the cytokines are connected to two blocking domains each (three in total in one molecule), with a blocking domain between the two cytokine domains. In some embodiments, one or more additional half-life extension domains may be included to optimize pharmacokinetic properties.
- In some embodiments, three cytokines are included in the same construct. In some embodiments, the third cytokine may function to block the other two in place of a blocking domain between the two cytokines.
- Preferred half-life extension elements for use in the fusion proteins are human serum albumin (HSA), an antibody or antibody fragment (e.g., scFV, dAb) which binds serum albumin, a human or humanized IgG, or a fragment of any of the foregoing. In some preferred embodiments, the blocking moiety is human serum albumin (HSA), or an antibody or antibody fragment which binds serum albumin, an antibody which binds the cytokine and prevents activation of binding or activation of the cytokine receptor, another cytokine, or a fragment of any of the foregoing. In preferred embodiments comprising an additional targeting domain, the targeting domain is an antibody which binds a cell surface protein which is enriched on the surface of cancer cells, such as EpCAM, FOLR1, and Fibronectin.
- vii. Other Uses
- The separation moieties disclosed herein can be used for antibody-antibiotic conjugates. The separation moiety disclosed herein connects or links an antimicrobial antibiotic to an antibody specific for a bacterial strain (e.g., Staphylococcus aureus Ab). The antibody-antibiotic conjugate does not display antibacterial activity when the antibody is linked to the antibiotic. However, upon internalization into host cells, the separation moiety is cleaved by proteases releasing the free antibiotic. The free antibiotic kills the intracellular bacteria.
- The separation moieties disclosed herein can be used for applications in chemical probes use for detection and isolation of proteins. Chemical probes are designed based on small molecule interaction with proteins. The probes typically comprise a covalent binding motif in order for the probe to interact with the target protein, a detection/purification tag for visualization/purification of the protein target and a linker group. The separation moieties described herein can be incorporated to enable the target protein to be detected and isolated.
- C. Methods of Treatment and Pharmaceutical Compositions
- Further provided are methods of treating a subject with or at risk of developing an of a disease or disorder, such as proliferative disease, a tumorous disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, or graft-versus-host disease. The methods administering to a subject in need thereof an effective amount of a fusion protein as disclosed herein that is typically administered as a pharmaceutical composition. In some embodiments, the method further comprises selecting a subject with or at risk of developing such a disease or disorder. The pharmaceutical composition preferably comprises a blocked cytokine, fragment or mutein thereof that is activated at a site of inflammation. In one embodiment, the chimeric polypeptide comprises a cytokine polypeptide, fragment or mutein thereof and a serum half-life extension element. In another embodiment, the chimeric polypeptide comprises a cytokine polypeptide, fragment or mutein thereof and a blocking moiety, e.g. a steric blocking polypeptide, wherein the steric blocking polypeptide is capable of sterically blocking the activity of the cytokine polypeptide, fragment or mutein thereof. In another embodiment, the chimeric polypeptide comprises a cytokine polypeptide, fragment or mutein thereof, a blocking moiety, and a serum half-life extension element.
- Inflammation is part of the complex biological response of body tissues to harmful stimuli, such as pathogens, damaged cells, or irritants, and is a protective response involving immune cells, blood vessels, and molecular mediators. The function of inflammation is to eliminate the initial cause of cell injury, clear out necrotic cells and tissues damaged from the original insult and the inflammatory process, and to initiate tissue repair. Inflammation can occur from infection, as a symptom or a disease, e.g., cancer, atherosclerosis, allergies, myopathies, HIV, obesity, or an autoimmune disease. An autoimmune disease is a chronic condition arising from an abnormal immune response to a self-antigen. Autoimmune diseases that may be treated with the polypeptides disclosed herein include but are not limited to lupus, celiac disease,
diabetes mellitus type 1, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus. - The pharmaceutical composition can comprise one or more protease-cleavable linker sequences. The linker sequence serves to provide flexibility between polypeptides, such that each polypeptide is capable of inhibiting the activity of the first polypeptide. The linker sequence can be located between any or all of the cytokine polypeptide, fragment or mutein thereof, the blocking moiety, and serum half-life extension element. Optionally, the composition comprises, two, three, four, or five linker sequences. The linker sequence, two, three, or four linker sequences can be the same or different linker sequences. In one embodiment, the linker sequence comprises GGGGS (SEQ ID NO: 232), GSGSGS (SEQ ID NO: 233), or G(SGGG)2SGGT (SEQ ID NO: 234). In another embodiment, the linker comprises a protease-cleavable sequence selected from group consisting of HSSKLQ (SEQ ID NO: 25), GPLGVRG (SEQ ID NO:221), IPVSLRSG (SEQ ID NO: 222), VPLSLYSG (SEQ ID NO: 223), or SGESPAYYTA (SEQ ID NO: 224). In some embodiments, the linker is cleaved by a protease selected from the group consisting of a kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin G, an elastase, PR-3, granzyme M, a calpain, a matrix metalloproteinase (MMP), a plasminogen activator, a cathepsin, a caspase, a tryptase, or a tumor cell surface protease.
- Further provided are methods of treating a subject with or at risk of developing cancer. The methods comprise administering to the subject in need thereof an effective amount of a chimeric polypeptide (a fusion protein) as disclosed herein that is typically administered as a pharmaceutical composition. In some embodiments, the method further comprises selecting a subject with or at risk of developing cancer. The pharmaceutical composition preferably comprises a blocked cytokine, fragment or mutein thereof that is activated at a tumor site. Preferably, the tumor is a solid tumor. The cancer may be a colon cancer, a lung cancer, a melanoma, a sarcoma, a renal cell carcinoma, a breast cancer,
- The method can further involve the administration of one or more additional agents to treat cancer, such as chemotherapeutic agents (e.g., Adriamycin, Cerubidine, Bleomycin, Alkeran, Velban, Oncovin, Fluorouracil, Thiotepa, Methotrexate, Bisantrene, Noantrone, Thiguanine, Cytaribine, Procarabizine), immuno-oncology agents (e.g., anti-PD-L1, anti-CTLA4, anti-PD-1, anti-CD47, anti-GD2), cellular therapies (e.g, CAR-T, T-cell therapy), oncolytic viruses and the like.
- Provided herein are pharmaceutical formulations or compositions containing the chimeric polypeptides and a pharmaceutically acceptable carrier. The herein provided compositions are suitable for administration in vitro or in vivo. By pharmaceutically acceptable carrier is meant a material that is not biologically or otherwise undesirable, i.e., the material is administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical formulation or composition in which it is contained. The carrier is selected to minimize degradation of the active ingredient and to minimize adverse side effects in the subject.
- Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 21st Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005). Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic, although the formulate can be hypertonic or hypotonic if desired. Examples of the pharmaceutically acceptable carriers include, but are not limited to, sterile water, saline, buffered solutions like Ringer's solution, and dextrose solution. The pH of the solution is generally about 5 to about 8 or from about 7 to 7.5. Other carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the immunogenic polypeptides. Matrices are in the form of shaped articles, e.g., films, liposomes, or microparticles. Certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. Carriers are those suitable for administration of the chimeric polypeptides or nucleic acid sequences encoding the chimeric polypeptides to humans or other subjects.
- The pharmaceutical formulations or compositions are administered in a number of ways depending on whether local or systemic treatment is desired and, on the area to be treated. The compositions are administered via any of several routes of administration, including topically, orally, parenterally, intravenously, intra-articularly, intraperitoneally, intramuscularly, subcutaneously, intracavity, transdermally, intrahepatically, intracranially, nebulization/inhalation, or by installation via bronchoscopy. In some embodiments, the compositions are administered locally (non-systemically), including intratumorally, intra-articularly, intrathecally, etc.
- Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives are optionally present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
- Formulations for topical administration include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners and the like are optionally necessary or desirable.
- Compositions for oral administration include powders or granules, suspension or solutions in water or non-aqueous media, capsules, sachets, or tables. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders are optionally desirable.
- Optionally, the chimeric polypeptides or nucleic acid sequences encoding the chimeric polypeptides are administered by a vector. There are a number of compositions and methods which can be used to deliver the nucleic acid molecules and/or polypeptides to cells, either in vitro or in vivo via, for example, expression vectors. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non-viral based delivery systems. Such methods are well known in the art and readily adaptable for use with the compositions and methods described herein. Such compositions and methods can be used to transfect or transduce cells in vitro or in vivo, for example, to produce cell lines that express and preferably secrete the encoded chimeric polypeptide or to therapeutically deliver nucleic acids to a subject. The components of the chimeric nucleic acids disclosed herein typically are operably linked in frame to encode a fusion protein.
- As used herein, plasmid or viral vectors are agents that transport the disclosed nucleic acids into the cell without degradation and include a promoter yielding expression of the nucleic acid molecule and/or polypeptide in the cells into which it is delivered. Viral vectors are, for example, Adenovirus, Adeno-associated virus, herpes virus, Vaccinia virus, Polio virus, Sindbis, and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviral vectors, in general are described by Coffin et al., Retroviruses, Cold Spring Harbor Laboratory Press (1997), which is incorporated by reference herein for the vectors and methods of making them. The construction of replication-defective adenoviruses has been described (Berkner et al., J. Virol. 61:1213-20 (1987); Massie et al., Mol. Cell. Biol. 6:2872-83 (1986); Haj-Ahmad et al., J. Virol. 57:267-74 (1986); Davidson et al., J. Virol. 61:1226-39 (1987); Zhang et al., BioTechniques 15:868-72 (1993)). The benefit and the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell types, since they can replicate within an initial infected cell, but are unable to form new infectious viral particles. Recombinant adenoviruses have been shown to achieve high efficiency after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma, and a number of other tissue sites. Other useful systems include, for example, replicating and host-restricted non-replicating vaccinia virus vectors.
- The provided polypeptides and/or nucleic acid molecules can be delivered via virus like particles. Virus like particles (VLPs) consist of viral protein(s) derived from the structural proteins of a virus. Methods for making and using virus like particles are described in, for example, Garcea and Gissmann, Current Opinion in Biotechnology 15:513-7 (2004).
- The provided polypeptides can be delivered by subviral dense bodies (DBs). DBs transport proteins into target cells by membrane fusion. Methods for making and using DBs are described in, for example, Pepperl-Klindworth et al., Gene Therapy 10:278-84 (2003).
- The provided polypeptides can be delivered by tegument aggregates. Methods for making and using tegument aggregates are described in International Publication No. WO 2006/110728.
- Non-viral based delivery methods can include expression vectors comprising nucleic acid molecules and nucleic acid sequences encoding polypeptides, wherein the nucleic acids are operably linked to an expression control sequence. Suitable vector backbones include, for example, those routinely used in the art such as plasmids, artificial chromosomes, BACs, YACs, or PACs. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, Wis.), Clonetech (Pal Alto, Calif.), Stratagene (La Jolla, Calif.), and Invitrogen/Life Technologies (Carlsbad, Calif.). Vectors typically contain one or more regulatory regions. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5′ and 3′ untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, and introns. Such vectors can also be used to make the chimeric polypeptides by expression is a suitable host cell, such as CHO cells.
- Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis B virus, and most preferably cytomegalovirus (CMV), or from heterologous mammalian promoters, e.g. β-actin promoter or EF1α promoter, or from hybrid or chimeric promoters (e.g., CMV promoter fused to the β-actin promoter). Of course, promoters from the host cell or related species are also useful herein.
- Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5′ or 3′ to the transcription unit. Furthermore, enhancers can be within an intron as well as within the coding sequence itself. They are usually between 10 and 300 base pairs (bp) in length, and they function in cis. Enhancers usually function to increase transcription from nearby promoters. Enhancers can also contain response elements that mediate the regulation of transcription. While many enhancer sequences are known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
- The promoter and/or the enhancer can be inducible (e.g. chemically or physically regulated). A chemically regulated promoter and/or enhancer can, for example, be regulated by the presence of alcohol, tetracycline, a steroid, or a metal. A physically regulated promoter and/or enhancer can, for example, be regulated by environmental factors, such as temperature and light. Optionally, the promoter and/or enhancer region can act as a constitutive promoter and/or enhancer to maximize the expression of the region of the transcription unit to be transcribed. In certain vectors, the promoter and/or enhancer region can be active in a cell type specific manner. Optionally, in certain vectors, the promoter and/or enhancer region can be active in all eukaryotic cells, independent of cell type. Preferred promoters of this type are the CMV promoter, the SV40 promoter, the β-actin promoter, the EF1α promoter, and the retroviral long terminal repeat (LTR).
- The vectors also can include, for example, origins of replication and/or markers. A marker gene can confer a selectable phenotype, e.g., antibiotic resistance, on a cell. The marker product is used to determine if the vector has been delivered to the cell and once delivered is being expressed. Examples of selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, puromycin, and blasticidin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. Examples of other markers include, for example, the E. coli lacZ gene, green fluorescent protein (GFP), and luciferase. In addition, an expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as GFP, glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or FLAG™ tag (Kodak; New Haven, Conn.) sequences typically are expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino terminus.
- As used herein, the terms peptide, polypeptide, or protein are used broadly to mean two or more amino acids linked by a peptide bond. Protein, peptide, and polypeptide are also used herein interchangeably to refer to amino acid sequences. It should be recognized that the term polypeptide is not used herein to suggest a particular size or number of amino acids comprising the molecule and that a peptide of the invention can contain up to several amino acid residues or more. As used throughout, subject can be a vertebrate, more specifically a mammal (e g a human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig), birds, reptiles, amphibians, fish, and any other animal. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. As used herein, patient or subject may be used interchangeably and can refer to a subject with a disease or disorder (e.g. cancer). The term patient or subject includes human and veterinary subjects.
- A subject at risk of developing a disease or disorder can be genetically predisposed to the disease or disorder, e.g., have a family history or have a mutation in a gene that causes the disease or disorder, or show early signs or symptoms of the disease or disorder. A subject currently with a disease or disorder has one or more than one symptom of the disease or disorder and may have been diagnosed with the disease or disorder.
- The methods and agents as described herein are useful for both prophylactic and therapeutic treatment. For prophylactic use, a therapeutically effective amount of the chimeric polypeptides or chimeric nucleic acid sequences encoding the chimeric polypeptides described herein are administered to a subject prior to onset (e.g., before obvious signs of cancer or inflammation) or during early onset (e.g., upon initial signs and symptoms of cancer or inflammation). Prophylactic administration can occur for several days to years prior to the manifestation of symptoms of cancer or inflammation. Prophylactic administration can be used, for example, in the preventative treatment of subjects diagnosed with a genetic predisposition to cancer. Therapeutic treatment involves administering to a subject a therapeutically effective amount of the chimeric polypeptides or nucleic acid sequences encoding the chimeric polypeptides described herein after diagnosis or development of cancer or inflammation (e.g., an autoimmune disease). Prophylactic use may also apply when a patient is undergoing a treatment, e.g., a chemotherapy, in which inflammation is expected.
- According to the methods taught herein, the subject is administered an effective amount of the agent (e.g., a chimeric polypeptide). The terms effective amount and effective dosage are used interchangeably. The term effective amount is defined as any amount necessary to produce a desired physiologic response. Effective amounts and schedules for administering the agent may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for administration are those large enough to produce the desired effect in which one or more symptoms of the disease or disorder are affected (e.g., reduced or delayed). The dosage should not be so large as to cause substantial adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex, type of disease, the extent of the disease or disorder, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosages can vary and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.
- As used herein the terms treatment, treat, or treating refers to a method of reducing the effects of a disease or condition or symptom of the disease or condition. Thus, in the disclosed method, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or condition or symptom of the disease or condition. For example, a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject as compared to a control. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.
- As used herein, the terms prevent, preventing, and prevention of a disease or disorder refers to an action, for example, administration of the chimeric polypeptide or nucleic acid sequence encoding the chimeric polypeptide, that occurs before or at about the same time a subject begins to show one or more symptoms of the disease or disorder, which inhibits or delays onset or exacerbation of one or more symptoms of the disease or disorder. As used herein, references to decreasing, reducing, or inhibiting include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater as compared to a control level. Such terms can include but do not necessarily include complete elimination.
- IL-2 variants have been developed that are selective for IL2Rαβγ relative to IL2Rβγ (Shanafelt, A. B., et al., 2000, Nat Biotechnol. 18:1197-202; Cassell, D. J., et. al., 2002, Curr Pharm Des., 8:2171-83). These variants have amino acid substitutions which reduce their affinity for IL2RB. Because IL-2 has undetectable affinity for IL2RG, these variants consequently have reduced affinity for the IL2Rβγ receptor complex and reduced ability to activate IL2Rβγ-expressing cells but retain the ability to bind IL2RA and the ability to bind and activate the IL2Rαβγ receptor complex.
- One of these variants, IL2/N88R (Bay 50-4798), was clinically tested as a low-toxicity version of IL-2 as an immune system stimulator, based on the hypothesis that IL2Rβγ-expressing NK cells are a major contributor to toxicity. Bay 50-4798 was shown to selectively stimulate the proliferation of activated T cells relative to NK cells, and was evaluated in phase I/II clinical trials in cancer patients (Margolin, K., et. al., 2007, Clin Cancer Res., 13:3312-9) and HIV patients (Davey, R. T., et. al., 2008, J Interferon Cytokine Res., 28:89-100). These clinical trials showed that Bay 50-4798 was considerably safer and more tolerable than aldesleukin, and also showed that it increased the levels of CD4+CD25+ T cells, a cell population enriched in Treg cells. Subsequent to these trials, research in the field more fully established the identity of Treg cells and demonstrated that Treg cells selectively express IL2Rαβγ (reviewed in Malek, T. R., et al., 2010, Immunity, 33:153-65). Based on this new research, it can now be understood that IL2Rαβγ selective agonists should be selective for Treg cells.
- In addition, mutants can be made that selectively alter the affinity for the CD25 chain relative to native 11-2.
- IL-2 can be engineered to produce mutants that bind the IL-2R complex generally or the IL-2Rα subunit specifically with an affinity that differs from that of the corresponding wild-type IL-2 or of a presently available mutant (referred to as C125 S, as the cysteine residue at
position 125 is replaced with a serine residue). - Accordingly, the present invention features mutant interleukin-2 (IL-2*) polypeptides that include an amino acid sequence that is at least 80% identical to wild-type IL-2 (e.g., 85, 87, 90, 95, 97, 98, or 99% identical) and that bind, as compared to WT IL-2, with higher to the IL-2 trimeric receptor relative to the dimeric IL-2 receptor. Typically, the muteins will also bind an IL-2 receptor a subunit (IL-2Rα) with an affinity that is greater than the affinity with which wild type IL-2 binds the IL-2Rα. The amino acid sequence within mutant IL-2 polypeptides can vary from SEQ ID NO:1 (UniProtKB accession number P60568) by virtue of containing (or only containing) one or more amino acid substitutions, which may be considered conservative or non-conservative substitutions. Non-naturally occurring amino acids can also be incorporated. Alternatively, or in addition, the amino acid sequence can vary from SEQ ID NO:1 (which may be considered the “reference” sequence) by virtue of containing and addition and/or deletion of one or more amino acid residues. More specifically, the amino acid sequence can differ from that of SEQ ID NO:1 by virtue of a mutation at least one of the following positions of SEQ ID NO:1: 1, 4, 8, 9, 10, 11, 13, 15, 26, 29, 30, 31, 35, 37, 46, 48, 49, 54, 61, 64, 67, 68, 69, 71, 73, 74, 75, 76, 79, 88, 89, 90, 92, 99, 101, 103, 114, 125, 128, or 133 (or combinations thereof). As noted, as few as one of these positions may be altered, as may two, three, four, five, six, seven, eight, nine, ten, or 11 or more (including up to all) of the positions. For example, the amino acid sequence can differ from SEQ ID NO:1 at positions 69 and 74 and further at one or more of
positions 30, 35, and 128. The amino acid sequence can also differ from SEQ ID NO:2 (as disclosed in U.S. Pat. No. 7,569,215, incorporated herein by reference) at one of the following sets of positions: (a) positions 64, 69, and 74; (b) positions 69, 74, and 101; (c) positions 69, 74, and 128; (d) positions 30, 69, 74, and 103; (e) positions 49, 69, 73, and 76; (f) positions 69, 74, 101, and 133; (g) positions 30, 69, 74, and 128; (h) positions 69, 74, 88, and 99; (i) positions 30, 69, 74, and 128; (j) positions 9, 11, 35, 69, and 74; (k) positions 1, 46, 49, 61, 69, and 79; (1) positions 48, 68, 71, 90, 103, and 114; (m) positions 4, 10, 11, 69, 74, 88, and 133; (n) positions 15, 30 31, 35, 48, 69, 74, and 92; (0) positions 30, 68, 69, 71, 74, 75, 76, and 90; (p) positions 30, 31, 37, 69, 73, 74, 79, and 128; (q) positions 26, 29, 30, 54, 67, 69, 74, and 92; (r) positions 8, 13, 26, 30, 35, 37, 69, 74, and 92; and (s) positions 29, 31, 35, 37, 48, 69, 71, 74, 88, and 89. Aside from mutations at these positions, the amino acid sequence of the mutant IL-2 polypeptide can otherwise be identical to SEQ ID NO:1. With respect to specific substitutions, the amino acid sequence can differ from SEQ ID NO:1 by virtue of having one or more of the following mutations: A1T, S4P, K8R, K9T, T10A, Q11R, Q13R, E15K, N26D, N29S, N30S, N30D, N30T, Y31H, Y31C, K35R, T37A, T37R, M46L, K48E, K49R, K49E, K54R, E61D, K64R, E67G, E68D, V69A, N71T, N71A, N71R, A73V, Q74P, 575P, K76E, K76R, H79R, N88D, I89V, N90H, I92T, S99P, T101A, F103S, I114V, I128T, I128A, T133A, or T133N. Our nomenclature is consistent with that of the scientific literature, where the single letter code of the amino acid in the wild-type or reference sequence is followed by its position within the sequence and then by the single letter code of the amino acid with which it is replaced. Thus, A1T designates a substitution of the alanine residue aposition 1 with threonine. Other mutant polypeptides within the scope of the invention include those that include a mutant of SEQ ID NO:2 having substitutions at V69 (e.g. A) and Q74 (e.g., P). For example, the amino acid sequence can include one of the following sets of mutations with respect to SEQ ID NO:2: (a) K64R, V69A, and Q74P; (b) V69A, Q74P, and T101A; (c) V69A, Q74P, and I128T; (d) N30D, V69A, Q74P, and F103S; (e) K49E, V69A, A73V, and K76E; (f) V69A, Q74P, T101A, and T133N; (g) N30S, V69A, Q74P, and I128A; (h) V69A, Q74P, N88D, and S99P; (i) N30S, V69A, Q74P, and I128T; (j) K9T, Q11R, K35R, V69A, and Q74P; (k) A1T, M46L, K49R, E61D, V69A, and H79R; (1) K48E, E68D, N71T, N90H, F103S, and I114V; (m) S4P, T10A, Q11R, V69A, Q74P, N88D, and T133A; (n) E15K, N30S Y31H, K35R, K48E, V69A, Q74P, and I92T; (o) N30S, E68D, V69A, N71A, Q74P, S75P, K76R, and N90H; (p) N30S, Y31C, T37A, V69A, A73V, Q74P, H79R, and I128T; (q) N26D, N29S, N30S, K54R, E67G, V69A, Q74P, and I92T; (r) K8R, Q13R, N26D, N30T, K35R, T37R, V69A, Q74P, and I92T; and (s) N29S, Y31H, K35R, T37A, K48E, V69A, N71R, Q74P, N88D, and I89V. SEQ ID NO:2 is disclosed in U.S. Pat. No. 7,569,215, which is incorporated herein by reference as an exemplary IL-2 polypeptide sequence that can be used in the invention. - As noted above, any of the mutant IL-2 polypeptides disclosed herein can include the sequences described; they can also be limited to the sequences described and otherwise identical to SEQ ID NO:1. Moreover, any of the mutant IL-2 polypeptides described herein can optionally include a substitution of the cysteine residue at
position 125 with another residue (e.g., serine) and/or can optionally include a deletion of the alanine residue atposition 1 of SEQ ID NO:1. - The mutant IL-2 polypeptides disclosed herein can bind to the IL-2Rα subunit with a Kd of less than about 28 nM (e.g., less than about 25 nM; less than about 5 nM; about 1 nM; less than about 500 pM; or less than about 100 pM). More specifically, a mutant IL-2 polypeptide can have an affinity equilibrium constant less than 1.0 nM (e.g., about 0.8, 0.6, 0.4, or 0.2 nM). Affinity can also be expressed as a relative rate of dissociation from an IL-2Rα subunit or from an IL-2 receptor complex (e.g., a complex expressed on the surface of a cell or otherwise membrane bound). For example, the mutant IL-2 polypeptides can dissociate from, e.g., IL-2Rα, at a decreased rate relative to a wild-type polypeptide or to an IL-2 based therapeutic, e.g., IL-2*. Alternatively, affinity can be characterized as the time, or average time, an IL-2* polypeptide persists on, for example, the surface of a cell expressing an IL-2R. For example, an IL-2*polypeptide can persist on the receptor for at least about 2, 5, 10, 50, 100, or 250 times (or more).
- Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a method is disclosed and discussed and a number of modifications that can be made to a number of molecules including the method are discussed, each and every combination and permutation of the method, and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.
- Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties.
- All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. However, the citation of a reference herein should not be construed as an acknowledgement that such reference is prior art to the present invention. To the extent that any of the definitions or terms provided in the references incorporated by reference differ from the terms and discussion provided herein, the present terms and definitions control.
- 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 herein.
- IL-2 mutein is detected with a commercially available antibody, e.g., the anti-IL-2 monoclonal (JES6-1A12) (BD Pharmingen; San Jose, Calif.). A positive control is used to show whether the monoclonal antibody recognizes the cytokine or mutein. Antibodies against IL-2Rα and IL-2Ry chain are also used. Wells of a 96-well plate are coated with an antibody (2.5 μg/ml) in PBS. Wells are blocked with 5% non-fat milk in PBS with 0.2% Tween®20 (PBS-M-Tw) and fusion proteins are added for 1-2 hours at 37° C. After washing, an anti-IL-2 biotin-labeled antibody, e.g., JES5H4 (BD Pharmingen) is added and binding is detected using Streptavidin HRP (Southern Biotechnology Associates; Birmingham, Ala.). The ELISA plate is developed by adding 50 μl O-phenylenediamine (OPD) (Sigma-Aldrich) in 0.1M Citrate pH 4.5 and 0.04% H2O2, stopped by adding 50 μl/well 2N H2SO4 and the absorbance was read at 490 nm.
- One of skill in the art would be familiar with methods of setting up protein cleavage assay. 100 μg of protein in 1×PBS pH 7.4 were cleaved with 1 μg active MMP9 (Sigma catalog #SAE0078-50 or Enzo catalog BML-SE360) and incubated at room temperature for up to 16 hours. Digested protein is subsequently used in functional assays or stored at −80° C. prior to testing. Extent of cleavage was monitored by SDS PAGE using methods well known in the art. As shown in
FIGS. 10, 13, 18A, 18 b, and 27A full cleavage of the fusion proteins by MMP9 protease is seen. - CTLL2 cells (ATCC) were plated in suspension at a concentration of 500,000 cells/well in culture media with or without 40 mg/ml human serum albumin (HSA) and stimulated with a dilution series of recombinant hIL2 or activatable hIL2 for 72 hours at 37° C. and 5% CO2. Activity of uncleaved and cleaved activatable hIL2 was tested. Cleaved activatable hIL2 was generated by incubation with active MMP9. Cell activity was assessed using a CellTiter-Glo® (Promega) luminescence-based cell viability assay. Results are shown in
FIGS. 8A-8F ,FIGS. 9A-9Z ,FIG. 25C . - The IL-2 mutein fusion proteins are biochemically characterized before and after cleavage with a protease, e.g., PSA Immunoblot analyses will show that the fusion proteins can be cleaved by PSA and that there is an increase in intensity of the predicted low molecular weight cleavage product of approximately 20 kDa reactive with an anti-IL-2 antibody after treatment of the samples with PSA. The degree of cleavage is dependent upon the amount of PSA as well as the time of incubation. Interestingly, when the fusion protein is analyzed before and after PSA treatment by ELISA, it was found that the apparent amount of IL-2 is increased after PSA cleavage. In this experiment, there is an approximately 2 or 4-fold increase in the apparent amount of IL-2 detected using this sandwich ELISA depending on the construct, suggesting that the antibody binding is partially hindered in the intact fusion protein. Aliquots of the same samples are also analyzed after PSA treatment using the CTLL-2 cell line that requires IL-2 for growth and survival and the viability of cells can be ascertained using the colorimetric MTT assay. In this assay, the more a supernatant can be diluted, the more biologically active IL-2 it contains, and there is an increase in the amount of biologically active IL-2 after PSA cleavage. The amount of IL-2 mutein increase will suggest that after PSA cleavage there is an increase in the predicted low molecular weight cleavage fragment of approximately 20 kDa reactive with an anti-IL-2 antibody, an increase in antibody accessibility, and most importantly, an increase in the amount of biologically active IL-2 mutein.
- The chimeric polypeptide is examined to determine if it could have biological effects in vivo. For these experiments a system is used in which tumor cells injected intraperitoneally rapidly and preferentially attach and grow initially on the milky spots, a series of organized immune aggregates found on the omentum (Gerber et al., Am. J. Pathol. 169:1739-52 (2006)). This system offers a convenient way to examine the effects of fusion protein treatment on tumor growth since fusion proteins can be delivered intraperitoneally multiple times and tumor growth can be analyzed by examining the dissociated omental cells. For these experiments, the Colon 38 cell line, a rapidly growing tumor cell line that expresses both MMP2 and MMP9 in vitro, may be used. The omental tissue normally expresses a relatively small amount of MMP2 and MMP9, but, when Colon 38 tumor is present on the omentum, MMP levels increase. Using this tumor model, the ability of IL-2 mutein fusion proteins to affect tumor growth is examined Colon 38 cells are injected intraperitoneally, allowed to attach and grow for 1 day, and then treated daily with fusion protein intraperitoneally. At
day 7, the animals are sacrificed and the omenta examined for tumor growth using flow cytometry and by a colony-forming assay. - Activatable interleukin proteins are tested for their binding affinities to human CD20+ cells and cynomolgus CD20+ cells.
- CD20+ cells are incubated with 100 μL of serial dilutions of the activatable interleukin proteins and at least one protease. After washing three times with FACS buffer the cells are incubated with 0.1 mL of 10 μg/mL mouse monoclonal anti-idiotype antibody in the same buffer for 45 min on ice. After a second washing cycle, the cells are incubated with 0.1 mL of 15 μg/mL FITC-conjugated goat anti-mouse IgG antibodies under the same conditions as before. As a control, cells are incubated with the anti-His IgG followed by the FITC-conjugated goat anti-mouse IgG antibodies without the activatable IL2 proteins. The cells were then washed again and resuspended in 0.2 mL of FACS buffer containing 2 μg/mL propidium iodide (PI) in order to exclude dead cells. The fluorescence of 1×104 living cells is measured using a Beckman-Coulter FC500 MPL flow cytometer using the MXP software (Beckman-Coulter, Krefeld, Germany) or a Millipore Guava EasyCyte flow cytometer using the Incyte software (Merck Millipore, Schwalbach, Germany). Mean fluorescence intensities of the cell samples are calculated using CXP software (Beckman-Coulter, Krefeld, Germany) or Incyte software (Merck Millipore, Schwalbach, Germany). After subtracting the fluorescence intensity values of the cells stained with the secondary and tertiary reagents alone the values are then used for calculation of the KD values with the equation for one-site binding (hyperbola) of the GraphPad Prism (version 6.00 for Windows, GraphPad Software, La Jolla Calif. USA).
- CD20 binding and crossreactivity are assessed on the human CD20+ tumor cell lines. The KD ratio of crossreactivity is calculated using the KD values determined on the CHO cell lines expressing either recombinant human or recombinant cynomolgus antigens.
- Activatable interleukin protein is evaluated in vitro on its mediation of immune response to CD20+ target cells.
- Fluorescence labeled CD20+ REC-1 cells (a Mantle cell lymphoma cell line, ATCC CRL-3004) are incubated with isolated PBMC of random donors or CB15 T-cells (standardized T-cell line) as effector cells in the presence of the activatable IL2 protein and at least one protease. After incubation for 4 h at 37° C. in a humidified incubator, the release of the fluorescent dye from the target cells into the supernatant is determined in a spectro-fluorimeter. Target cells incubated without the activatable IL2 protein and target cells totally lysed by the addition of saponin at the end of the incubation serve as negative and positive controls, respectively.
- Based on the measured remaining living target cells, the percentage of specific cell lysis is calculated according to the following formula: [1−(number of living targets(sample)/number of living targets(spontaneous)]×100%. Sigmoidal dose response curves and EC50 values are calculated by non-linear regression/4-parameter logistic fit using the GraphPad Software. The lysis values obtained for a given antibody concentration are used to calculate sigmoidal dose-response curves by 4 parameter logistic fit analysis using the Prism® GraphPad® software.
- Activatable interleukin protein is evaluated for half-time elimination in animal studies.
- The activatable IL2 protein is administered to cynomolgus monkeys as a 0.5 mg/kg bolus injection into the saphenous vein. Another cynomolgus monkey group receives a comparable IL2 construct in size but lacking a serum half-life extension element. A third and fourth group receive an IL2 construct with serum half-life extension element and a cytokine with CD20 and serum half-life extension elements respectively, and both comparable in size to the activatable interleukin protein. Each test group consists of 5 monkeys. Serum samples are taken at indicated time points, serially diluted, and the concentration of the proteins is determined using a binding ELISA to CD20.
- Pharmacokinetic analysis is performed using the test article plasma concentrations. Group mean plasma data for each test article conforms to a multi-exponential profile when plotted against the time post-dosing. The data are fit by a standard two-compartment model with bolus input and first-order rate constants for distribution and elimination phases. The general equation for the best fit of the data for i.v. administration is: c(t)=Ae−αt+Be−βt, where c(t) is the plasma concentration at time t, A and B are intercepts on the Y-axis, and α and β are the apparent first-order rate constants for the distribution and elimination phases, respectively. The α-phase is the initial phase of the clearance and reflects distribution of the protein into all extracellular fluid of the animal, whereas the second or β-phase portion of the decay curve represents true plasma clearance. Methods for fitting such equations are well known in the art. For example, A=D/V(α−k21)/(α−β), B=D/V(β−k21)/(α−β), and α and β (for α>β) are roots of the quadratic equation: r2+(k12+k21+k10)r+k21k10=0 using estimated parameters of V=volume of distribution, k10=elimination rate, k12=transfer rate from
compartment 1 tocompartment 2 and k21=transfer rate fromcompartment 2 tocompartment 1, and D=the administered dose. - Data analysis: Graphs of concentration versus time profiles are made using KaleidaGraph (KaleidaGraph™ V. 3.09 Copyright 1986-1997. Synergy Software. Reading, Pa.). Values reported as less than reportable (LTR) are not included in the PK analysis and are not represented graphically. Pharmacokinetic parameters are determined by compartmental analysis using WinNonlin software (WinNonlin® Professional V. 3.1 WinNonlin™ Copyright 1998-1999. Pharsight Corporation. Mountain View, Calif.). Pharmacokinetic parameters are computed as described in Ritschel W A and Kearns G L, 1999, IN: Handbook of Basic Pharmacokinetics Including Clinical Applications, 5th edition, American Pharmaceutical Assoc., Washington, D.C.
- It is expected that the activatable interleukin protein has improved pharmacokinetic parameters such as an increase in elimination half-time as compared to proteins lacking a serum half-life extension element.
- Activatable IL2 protein is evaluated in a xenograft model.
- Female immune-deficient NOD/scid mice are sub-lethally irradiated (2 Gy) and subcutaneously inoculated with 4×106 Ramos RA1 cells into the right dorsal flank. When tumors reach 100 to 200 mm3, animals are allocated into 3 treatment groups.
Groups 2 and 3 (8 animals each) are intraperitoneally injected with 1.5×107 activated human T-cells. Three days later, animals fromGroup 3 are subsequently treated with a total of 9 intravenous doses of 50 μg activatable interleukin protein.Groups - It is expected that animals treated with the activatable interleukin protein have a statistically significant delay in tumor growth in comparison to the respective vehicle-treated control group.
- WEHI279 cells (ATCC) were plated in suspension at a concentration of 25,000 cells/well in culture media with or without 1.5% human serum albumin (HSA) and stimulated with a dilution series of recombinant mIFNγ or inducible mIFNγ for 72 hours at 37° C. and 5% CO2. Activity of uncleaved and cleaved inducible mIFNγ was tested. Cleaved inducible mIFNγ was generated by incubation with active MMP9. Cell survival was assessed using a CellTiter-Glo (Promega) luminescence-based cell viability assay. The EC50 values for cleaved inducible mIFNγ molecules were at least 100× more potent than un-cleaved inducible mIFNγ molecules. As shown in
FIGS. 16A-16 , greater inducibility was seen in assays wherein the culture medium contained human serum albumin. - B16-Blue IFNγ cells (InvivoGen) were plated at a concentration of 75,000 cells/well in culture media with or without 1.5% human serum albumin (HSA) and stimulated with a dilution series of recombinant mIFNγ or inducible mIFNγ for 24 hours at 37° C. and 5% CO2. Activity of uncleaved and cleaved inducible mIFNγ was tested. Cleaved inducible mIFNγ was generated by incubation with active MMP9. Supernatants were harvested, and SEAP activation was assessed by adding QUANTI-Blue Reagent (InvivoGen), incubating at 37° C. for 2 hours, and measuring absorbance at 620 nm. The EC50 values for cleaved inducible mIFNγ molecules were at least 100× more potent than un-cleaved inducible mIFNγ molecules. Results are shown in e.g.,
FIG. 19A-19B ,FIGS. 22A-22B ,FIGS. 23A-23B . This experiment was repeated with for IFNα conjugates using B16-Blue IFNα/β cells. The EC50 values for cleaved inducible mIFNα molecules were at least 100× more potent than un-cleaved inducible mIFNα molecules. SeeFIGS. 20A-20B . - The chimeric polypeptide is examined to determine if it could have biological effects in vivo. For these experiments a system is used in which tumor cells injected intraperitoneally rapidly and preferentially attach and grow initially on the milky spots, a series of organized immune aggregates found on the omentum (Gerber et al., Am. J. Pathol. 169:1739-52 (2006)). This system offers a convenient way to examine the effects of fusion protein treatment on tumor growth since fusion proteins can be delivered intraperitoneally multiple times and tumor growth can be analyzed by examining the dissociated omental cells. For these experiments, the Colon 38 cell line, a rapidly growing tumor cell line that expresses both MMP2 and MMP9 in vitro, may be used. The omental tissue normally expresses a relatively small amount of MMP2 and MMP9, but, when Colon 38 tumor is present on the omentum, MMP levels increase. Using this tumor model, the ability of IFN fusion proteins to affect tumor growth is examined Colon 38 cells are injected intraperitoneally, allowed to attach and grow for 1 day, and then treated daily with fusion protein intraperitoneally. At
day 7, the animals are sacrificed and the omenta examined for tumor growth using flow cytometry and by a colony-forming assay. - The MC38 cell line, a rapidly growing colon adenocarcinoma cell line that expresses MMP9 in vitro, was used. Using this tumor model, the ability of IFNγ fusion proteins to affect tumor growth was examined MC38 cells were injected subcutaneously, allowed to grow for 10-14 days, and then treated with fusion protein twice weekly intraperitoneally for a total of four doses. As a comparator, wild type mIFNγ was administered at the dose levels indicated, twice daily for 2 weeks on a 5 day on/2 day off schedule (10 total doses). Tumor growth and body weight were monitored approximately twice per week for two weeks.
- An IFNγ polypeptide capable of binding to CD20 polypeptide present in a tumor or on a tumor cell is produced as follows. A nucleic acid is produced that contains nucleic acid sequences: (1) encoding an IFNγ polypeptide sequence and (2) one or more polypeptide linkers. Activatable IFNγ plasmid constructs can have optional Flag, His or other affinity tags, and are electroporated into HEK293 or other suitable human or mammalian cell lines and purified. Validation assays include T cell activation assays using T cells responsive to IFNγ stimulation in the presence of a protease.
- CD20 is one of the cell surface proteins present on B-lymphocytes. CD20 antigen is found in normal and malignant pre-B and mature B lymphocytes, including those in over 90% of B-cell non-Hodgkin's lymphomas (NHL). The antigen is absent in hematopoietic stem cells, activated B lymphocytes (plasma cells) and normal tissue. As such, several antibodies mostly of murine origin have been described: 1F5, 2B8/C2B8, 2H7, and 1H4.
- Human or humanized anti-CD20 antibodies are therefore used to generate scFv sequences for CD20 binding domains of an activatable IFNγ protein. DNA sequences coding for human or humanized VL and VH domains are obtained, and the codons for the constructs are, optionally, optimized for expression in cells from Homo sapiens. The order in which the VL and VH domains appear in the scFv is varied (i.e., VL-VH, or VH-VL orientation), and three copies of the “G4S” (SEQ ID NO: 241) or “G4S” (SEQ ID NO: 241) subunit (G4S)3 (SEQ ID NO: 242) connect the variable domains to create the scFv domain. Anti-CD20 scFv plasmid constructs can have optional Flag, His or other affinity tags, and are electroporated into HEK293 or other suitable human or mammalian cell lines and purified. Validation assays include binding analysis by FACS, kinetic analysis using Proteon, and staining of CD20-expressing cells.
- The activatable IFNγ construct with protease cleavage site domains is used to construct an activatable IFNγ protein in combination with an anti-CD20 scFv domain and a serum half-life extension element (e.g., a HSA binding peptide or VH domain). For expression of an activatable IFNγ protein in CHO cells, coding sequences of all protein domains are cloned into a mammalian expression vector system. In brief, gene sequences encoding the activatable IFNγ domain, serum half-life extension element, and CD20 binding domain along with peptide linkers L1 and L2 are separately synthesized and subcloned. The resulting constructs are then ligated together in the order of CD20 binding domain-L1-IFNγ subunit 1-L2-protease cleavage domain-L3-IFNγ subunit2-L4-anti-CD20 scFv-L5-serum half-life extension element to yield a final construct. All expression constructs are designed to contain coding sequences for an N-terminal signal peptide and a C-terminal hexahistidine (6×His)-tag (SEQ ID NO: 243) to facilitate protein secretion and purification, respectively.
- A CHO cell expression system (Flp-In®, Life Technologies), a derivative of CHO—K1 Chinese Hamster ovary cells (ATCC, CCL-61) (Kao and Puck, Proc. Natl. Acad. Sci. USA 1968; 60(4):1275-81), is used. Adherent cells are subcultured according to standard cell culture protocols provided by Life Technologies.
- For adaption to growth in suspension, cells are detached from tissue culture flasks and placed in serum-free medium. Suspension-adapted cells are cryopreserved in medium with 10% DMSO.
- Recombinant CHO cell lines stably expressing secreted activatable IFNγ proteins are generated by transfection of suspension-adapted cells. During selection with the antibiotic Hygromycin B viable cell densities are measured twice a week, and cells are centrifuged and resuspended in fresh selection medium at a maximal density of 0.1×106 viable cells/mL. Cell pools stably expressing activatable IFNγ proteins are recovered after 2-3 weeks of selection at which point cells are transferred to standard culture medium in shake flasks. Expression of recombinant secreted proteins is confirmed by performing protein gel electrophoresis or flow cytometry. Stable cell pools are cryopreserved in DMSO containing medium.
- Activatable IFNγ proteins are produced in 10-day fed-batch cultures of stably transfected CHO cell lines by secretion into the cell culture supernatant. Cell culture supernatants are harvested after 10 days at culture viabilities of typically >75%. Samples are collected from the production cultures every other day and cell density and viability are assessed. On day of harvest, cell culture supernatants are cleared by centrifugation and vacuum filtration before further use.
- Protein expression titers and product integrity in cell culture supernatants are analyzed by SDS-PAGE.
- Activatable IFNγ proteins are purified from CHO cell culture supernatants in a two-step procedure. The constructs are subjected to affinity chromatography in a first step followed by preparative size exclusion chromatography (SEC) on
Superdex 200 in a second step. Samples are buffer-exchanged and concentrated by ultrafiltration to a typical concentration of >1 mg/mL. Purity and homogeneity (typically >90%) of final samples are assessed by SDS PAGE under reducing and non-reducing conditions, followed by immunoblotting using an anti-HSA or anti idiotype antibody as well as by analytical SEC, respectively. Purified proteins are stored at aliquots at −80° C. until use. - The activatable IFNγ proteins are tested for their binding affinities to human CD20+ cells and cynomolgus CD20+ cells.
- CD20+ cells are incubated with 100 μL of serial dilutions of the activatable IFNγ proteins and at least one protease. After washing three times with FACS buffer the cells are incubated with 0.1 mL of 10 μg/mL mouse monoclonal anti-idiotype antibody in the same buffer for 45 min on ice. After a second washing cycle, the cells are incubated with 0.1 mL of 15 μg/mL FITC-conjugated goat anti-mouse IgG antibodies under the same conditions as before. As a control, cells are incubated with the anti-His IgG followed by the FITC-conjugated goat anti-mouse IgG antibodies without the activatable IFNγ proteins. The cells were then washed again and resuspended in 0.2 mL of FACS buffer containing 2 μg/mL propidium iodide (PI) in order to exclude dead cells. The fluorescence of 1×104 living cells is measured using a Beckman-Coulter FC500 MPL flow cytometer using the MXP software (Beckman-Coulter, Krefeld, Germany) or a Millipore Guava EasyCyte flow cytometer using the Incyte software (Merck Millipore, Schwalbach, Germany). Mean fluorescence intensities of the cell samples are calculated using CXP software (Beckman-Coulter, Krefeld, Germany) or Incyte software (Merck Millipore, Schwalbach, Germany). After subtracting the fluorescence intensity values of the cells stained with the secondary and tertiary reagents alone the values are then used for calculation of the KD values with the equation for one-site binding (hyperbola) of the GraphPad Prism (version 6.00 for Windows, GraphPad Software, La Jolla Calif. USA).
- CD20 binding and cross-reactivity are assessed on the human CD20+ tumor cell lines. The KD ratio of cross-reactivity is calculated using the KD values determined on the CHO cell lines expressing either recombinant human or recombinant cynomolgus antigens.
- The activatable IFNγ protein is evaluated in vitro on its mediation of immune response to CD20+ target cells.
- Fluorescence labeled CD20+ REC-1 cells (a Mantle cell lymphoma cell line, ATCC CRL-3004) are incubated with isolated PBMC of random donors or CB15 T-cells (standardized T-cell line) as effector cells in the presence of the activatable IFNγ protein and at least one protease. After incubation for 4 h at 37° C. in a humidified incubator, the release of the fluorescent dye from the target cells into the supernatant is determined in a spectrofluorometer. Target cells incubated without the activatable IFNγ protein and target cells totally lysed by the addition of saponin at the end of the incubation serve as negative and positive controls, respectively.
- Based on the measured remaining living target cells, the percentage of specific cell lysis is calculated according to the following formula: [1−(number of living targets(sample)/number of living targets(spontaneous)]×100%. Sigmoidal dose response curves and EC50 values are calculated by non-linear regression/4-parameter logistic fit using the GraphPad Software. The lysis values obtained for a given antibody concentration are used to calculate sigmoidal dose-response curves by 4 parameter logistic fit analysis using the Prism software.
- The activatable IFNγ protein is evaluated for half-time elimination in animal studies.
- The activatable IFNγ protein is administered to cynomolgus monkeys as a 0.5 mg/kg bolus injection into the saphenous vein. Another cynomolgus monkey group receives a comparable cytokine in size but lacking a serum half-life extension element. A third and fourth group receive a cytokine with serum half-life extension elements and a cytokine with CD20 and serum half-life extension elements respectively, and both comparable in size to the activatable IFNγ protein. Each test group consists of 5 monkeys. Serum samples are taken at indicated time points, serially diluted, and the concentration of the proteins is determined using a binding ELISA to CD20.
- Pharmacokinetic analysis is performed using the test article plasma concentrations. Group mean plasma data for each test article conforms to a multi-exponential profile when plotted against the time post-dosing. The data are fit by a standard two-compartment model with bolus input and first-order rate constants for distribution and elimination phases. The general equation for the best fit of the data for i.v. administration is: c(t)=Ae−αt+Be−βt, where c(t) is the plasma concentration at time t, A and B are intercepts on the Y-axis, and α and β are the apparent first-order rate constants for the distribution and elimination phases, respectively. The α-phase is the initial phase of the clearance and reflects distribution of the protein into all extracellular fluid of the animal, whereas the second or β-phase portion of the decay curve represents true plasma clearance. Methods for fitting such equations are well known in the art. For example, A=D/V(α−k21)/(α−β), B=D/V(β−k21)/(α−β), and α and β (for α>β) are roots of the quadratic equation: r2+(k12+k21+k10)r+k21k10=0 using estimated parameters of V=volume of distribution, k10=elimination rate, k12=transfer rate from
compartment 1 tocompartment 2 and k21=transfer rate fromcompartment 2 tocompartment 1, and D=the administered dose. - Data analysis: Graphs of concentration versus time profiles are made using KaleidaGraph (KaleidaGraph™ V. 3.09 Copyright 1986-1997. Synergy Software. Reading, Pa.). Values reported as less than reportable (LTR) are not included in the PK analysis and are not represented graphically. Pharmacokinetic parameters are determined by compartmental analysis using WinNonlin software (WinNonlin® Professional V. 3.1 WinNonlin™ Copyright 1998-1999. Pharsight Corporation. Mountain View, Calif.). Pharmacokinetic parameters are computed as described in Ritschel W A and Kearns G L, 1999, IN: Handbook of Basic Pharmacokinetics Including Clinical Applications, 5th edition, American Pharmaceutical Assoc., Washington, D.C.
- It is expected that the activatable IFNγ protein has improved pharmacokinetic parameters such as an increase in elimination half-time as compared to proteins lacking a serum half-life extension element.
- The activatable IFNγ protein is evaluated in a xenograft model.
- Female immune-deficient NOD/scid mice are sub-lethally irradiated (2 Gy) and subcutaneously inoculated with 4×10δ Ramos RA1 cells into the right dorsal flank. When tumors reach 100 to 200 mm3, animals are allocated into 3 treatment groups.
Groups 2 and 3 (8 animals each) are intraperitoneally injected with 1.5×107 activated human T-cells. Three days later, animals fromGroup 3 are subsequently treated with a total of 9 intravenous doses of 50 μg activatable IFNγ protein.Groups - It is expected that animals treated with the activatable IFNγ protein have a statistically significant delay in tumor growth in comparison to the respective vehicle-treated control group.
- While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
- HEK-Blue IL12 cells (InvivoGen) were plated in suspension at a concentration of 250,000 cells/well in culture media with or without 40 mg/ml human serum albumin (HSA) and stimulated with a dilution series of recombinant hIL12, chimeric IL12 (mouse p35/human p40) or activatable hIL12 for 24 hours at 37° C. and 5% CO2. Activity of uncleaved and cleaved activatable hIL12 was tested. Cleaved inducible hIL12 was generated by incubation with active MMP9. IL12 activity was assessed by quantification of Secreted Alkaline Phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen), a colorimetric based assay.
- HEK-Blue IL2 cells (InvivoGen) were plated in suspension at a concentration of 50,000 cells/well in culture media with or without 15-40 mg/ml human serum albumin (HSA) and stimulated with a dilution series of recombinant hIL2 or activatable hIL2 for 24 hours at 37° C. and 5% CO2. Activity of uncleaved and cleaved activatable hIL2 was tested. Cleaved inducible hIL2 was generated by incubation with active MMP9 or another protease. IL2 activity was assessed by quantification of Secreted Alkaline Phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen), a colorimetric based assay. Results are shown in
FIGS. 59-62 . - T-Blasts were induced from murine splenocytes with a 6-day incubation with PHA and a 24 hr incubation with recombinant hIL12. T-blasts were then plated in suspension at a concentration of 200,000 cells/well in culture media with or without 40 mg/ml human serum albumin (HSA) and stimulated with a dilution series of recombinant hIL12 or chimeric IL12 (mouse p35/human p40) or mouse IL12 for 72 hours at 37° C. and 5% CO2. Activity of uncleaved and cleaved IL12 was tested. Cleaved inducible hIL12 was generated by incubation with active MMP9. IL12 activity was assessed by downstream quantification of IFNγ production using a mIFNγ alphaLISA.
- The chimeric polypeptide is examined to determine if it could have biological effects in vivo. For these experiments a system is used in which tumor cells injected intraperitoneally rapidly and preferentially attach and grow initially on the milky spots, a series of organized immune aggregates found on the omentum (Gerber et al., Am. J. Pathol. 169:1739-52 (2006)). This system offers a convenient way to examine the effects of fusion protein treatment on tumor growth since fusion proteins can be delivered intraperitoneally multiple times and tumor growth can be analyzed by examining the dissociated omental cells. For these experiments, the Colon 38 cell line, a rapidly growing tumor cell line that expresses both MMP2 and MMP9 in vitro, may be used. The omental tissue normally expresses a relatively small amount of MMP2 and MMP9, but, when Colon 38 tumor is present on the omentum, MMP levels increase. Using this tumor model, the ability of IL-2 mutein fusion proteins to affect tumor growth is examined Colon 38 cells are injected intraperitoneally, allowed to attach and grow for 1 day, and then treated daily with fusion protein intraperitoneally. At
day 7, the animals are sacrificed and the omenta examined for tumor growth using flow cytometry and by a colony-forming assay. - The human IL-12p35 chain canonical sequence is UniProt Accession No. P29459. The human IL-12p40 chain canonical sequence is UniProt Accession No. P29460. IL-12p35 and IL-12p40 are cloned into an expression construct. A protease cleavage site is included between the IL-12p35 and IL-12p40 domains. An IL-12 polypeptide capable of binding to CD20 polypeptide present in a tumor or on a tumor cell is produced as follows. A nucleic acid is produced that contains nucleic acid sequences: (1) encoding an IFNγ polypeptide sequence and (2) one or more polypeptide linkers. Activatable interleukin plasmid constructs can have optional Flag, His or other affinity tags, and are electroporated into HEK293 or other suitable human or mammalian cell lines and purified. Validation assays include T cell activation assays using T cells responsive to IL-12 stimulation in the presence of a protease.
- CD20 is one of the cell surface proteins present on B-lymphocytes. CD20 antigen is found in normal and malignant pre-B and mature B lymphocytes, including those in over 90% of B-cell non-Hodgkin's lymphomas (NHL). The antigen is absent in hematopoietic stem cells, activated B lymphocytes (plasma cells) and normal tissue. As such, several antibodies mostly of murine origin have been described: 1F5, 2B8/C2B8, 2H7, and 1H4.
- Human or humanized anti-CD20 antibodies are therefore used to generate scFv sequences for CD20 binding domains of an activatable interleukin protein. DNA sequences coding for human or humanized VL and VH domains are obtained, and the codons for the constructs are, optionally, optimized for expression in cells from Homo sapiens. The order in which the VL and VH domains appear in the scFv is varied (i.e., VL-VH, or VH-VL orientation), and three copies of the “G4S” (SEQ ID NO: 241) or “G4S” (SEQ ID NO: 241) subunit (G4S)3 (SEQ ID NO: 242) connect the variable domains to create the scFv domain. Anti-CD20 scFv plasmid constructs can have optional Flag, His or other affinity tags, and are electroporated into HEK293 or other suitable human or mammalian cell lines and purified. Validation assays include binding analysis by FACS, kinetic analysis using Proteon, and staining of CD20-expressing cells.
- The activatable interleukin construct with protease cleavage site domains are used to construct an activatable interleukin protein in combination with an anti-CD20 scFv domain and a serum half-life extension element (e.g., a HSA binding peptide or VH domain). For expression of an activatable interleukin protein in CHO cells, coding sequences of all protein domains are cloned into a mammalian expression vector system. In brief, gene sequences encoding the activatable interleukin domain, serum half-life extension element, and CD20 binding domain along with peptide linkers L1 and L2 are separately synthesized and subcloned. The resulting constructs are then ligated together in the order of CD20 binding domain-L1-IL-12p35-L2-protease cleavage domain-L3-IL-12p40-L4-anti-CD20 scFv-L5-serum half-life extension element to yield a final construct. All expression constructs are designed to contain coding sequences for an N-terminal signal peptide and a C-terminal hexahistidine (6×His)-tag (SEQ ID NO: 243) to facilitate protein secretion and purification, respectively.
- A CHO cell expression system (Flp-In®, Life Technologies), a derivative of CHO—K1 Chinese Hamster ovary cells (ATCC, CCL-61) (Kao and Puck, Proc. Natl. Acad Sci USA 1968; 60(4):1275-81), is used. Adherent cells are subcultured according to standard cell culture protocols provided by Life Technologies.
- For adaption to growth in suspension, cells are detached from tissue culture flasks and placed in serum-free medium. Suspension-adapted cells are cryopreserved in medium with 10% DMSO.
- Recombinant CHO cell lines stably expressing secreted activatable interleukin proteins are generated by transfection of suspension-adapted cells. During selection with the antibiotic Hygromycin B viable cell densities are measured twice a week, and cells are centrifuged and resuspended in fresh selection medium at a maximal density of 0.1×106 viable cells/mL. Cell pools stably expressing activatable interleukin proteins are recovered after 2-3 weeks of selection at which point cells are transferred to standard culture medium in shake flasks. Expression of recombinant secreted proteins is confirmed by performing protein gel electrophoresis or flow cytometry. Stable cell pools are cryopreserved in DMSO containing medium.
- Activatable interleukin proteins are produced in 10-day fed-batch cultures of stably transfected CHO cell lines by secretion into the cell culture supernatant. Cell culture supernatants are harvested after 10 days at culture viabilities of typically >75%. Samples are collected from the production cultures every other day and cell density and viability are assessed. On day of harvest, cell culture supernatants are cleared by centrifugation and vacuum filtration before further use.
- Protein expression titers and product integrity in cell culture supernatants are analyzed by SDS-PAGE.
- Activatable interleukin proteins are purified from CHO cell culture supernatants in a two-step procedure. The constructs are subjected to affinity chromatography in a first step followed by preparative size exclusion chromatography (SEC) on
Superdex 200 in a second step. Samples are buffer-exchanged and concentrated by ultrafiltration to a typical concentration of >1 mg/mL. Purity and homogeneity (typically >90%) of final samples are assessed by SDS PAGE under reducing and non-reducing conditions, followed by immunoblotting using an anti-HSA or anti idiotype antibody as well as by analytical SEC, respectively. Purified proteins are stored at aliquots at −80° C. until use. - Activatable interleukin proteins are tested for their binding affinities to human CD20+ cells and cynomolgus CD20+ cells.
- CD20+ cells are incubated with 100 μL of serial dilutions of the activatable interleukin proteins and at least one protease. After washing three times with FACS buffer the cells are incubated with 0.1 mL of 10 μg/mL mouse monoclonal anti-idiotype antibody in the same buffer for 45 min on ice. After a second washing cycle, the cells are incubated with 0.1 mL of 15 μg/mL FITC-conjugated goat anti-mouse IgG antibodies under the same conditions as before. As a control, cells are incubated with the anti-His IgG followed by the FITC-conjugated goat anti-mouse IgG antibodies without the activatable interleukin proteins. The cells were then washed again and resuspended in 0.2 mL of FACS buffer containing 2 μg/mL propidium iodide (PI) in order to exclude dead cells. The fluorescence of 1×104 living cells is measured using a Beckman-Coulter FC500 MPL flow cytometer using the MXP software (Beckman-Coulter, Krefeld, Germany) or a Millipore Guava EasyCyte flow cytometer using the Incyte software (Merck Millipore, Schwalbach, Germany). Mean fluorescence intensities of the cell samples are calculated using CXP software (Beckman-Coulter, Krefeld, Germany) or Incyte software (Merck Millipore, Schwalbach, Germany). After subtracting the fluorescence intensity values of the cells stained with the secondary and tertiary reagents alone the values are then used for calculation of the KD values with the equation for one-site binding (hyperbola) of the GraphPad Prism (version 6.00 for Windows, GraphPad Software, La Jolla Calif. USA).
- CD20 binding and cross-reactivity are assessed on the human CD20+ tumor cell lines. The KD ratio of cross-reactivity is calculated using the KD values determined on the CHO cell lines expressing either recombinant human or recombinant cynomolgus antigens.
- The activatable interleukin protein is evaluated in vitro on its mediation of immune response to CD20+ target cells.
- Fluorescence labeled CD20+ REC-1 cells (a Mantle cell lymphoma cell line, ATCC CRL-3004) are incubated with isolated PBMC of random donors or CB15 T-cells (standardized T-cell line) as effector cells in the presence of the activatable interleukin protein and at least one protease. After incubation for 4 h at 37° C. in a humidified incubator, the release of the fluorescent dye from the target cells into the supernatant is determined in a spectrofluorometer. Target cells incubated without the activatable interleukin protein and target cells totally lysed by the addition of saponin at the end of the incubation serve as negative and positive controls, respectively.
- Based on the measured remaining living target cells, the percentage of specific cell lysis is calculated according to the following formula: [1−(number of living targets(sample)/number of living targets(spontaneous)]×100%. Sigmoidal dose response curves and EC50 values are calculated by non-linear regression/4-parameter logistic fit using the GraphPad Software. The lysis values obtained for a given antibody concentration are used to calculate sigmoidal dose-response curves by 4 parameter logistic fit analysis using the Prism software.
- The activatable interleukin protein is evaluated for half-time elimination in animal studies.
- The activatable interleukin protein is administered to cynomolgus monkeys as a 0.5 mg/kg bolus injection into the saphenous vein. Another cynomolgus monkey group receives a comparable cytokine in size but lacking a serum half-life extension element. A third and fourth group receive a cytokine with serum half-life extension elements and a cytokine with CD20 and serum half-life extension elements respectively, and both comparable in size to the activatable interleukin protein. Each test group consists of 5 monkeys. Serum samples are taken at indicated time points, serially diluted, and the concentration of the proteins is determined using a binding ELISA to CD20.
- Pharmacokinetic analysis is performed using the test article plasma concentrations. Group mean plasma data for each test article conforms to a multi-exponential profile when plotted against the time post-dosing. The data are fit by a standard two-compartment model with bolus input and first-order rate constants for distribution and elimination phases. The general equation for the best fit of the data for i.v. administration is: c(t)=Ae−αt+Be−βt, where c(t) is the plasma concentration at time t, A and B are intercepts on the Y-axis, and α and β are the apparent first-order rate constants for the distribution and elimination phases, respectively. The α-phase is the initial phase of the clearance and reflects distribution of the protein into all extracellular fluid of the animal, whereas the second or β-phase portion of the decay curve represents true plasma clearance. Methods for fitting such equations are well known in the art. For example, A=D/V(α−k21)/(α−β), B=D/V(β−k21)/(α−β), and α and β (for α>β) are roots of the quadratic equation: r2+(k12+k21+k10)r+k21k10=0 using estimated parameters of V=volume of distribution, k10=elimination rate, k12=transfer rate from
compartment 1 tocompartment 2 and k21=transfer rate fromcompartment 2 tocompartment 1, and D=the administered dose. - Data analysis: Graphs of concentration versus time profiles are made using KaleidaGraph (KaleidaGraph™ V. 3.09 Copyright 1986-1997. Synergy Software. Reading, Pa.). Values reported as less than reportable (LTR) are not included in the PK analysis and are not represented graphically. Pharmacokinetic parameters are determined by compartmental analysis using WinNonlin software (WinNonlin® Professional V. 3.1 WinNonlin™ Copyright 1998-1999. Pharsight Corporation. Mountain View, Calif.). Pharmacokinetic parameters are computed as described in Ritschel W A and Kearns G L, 1999, IN: Handbook of Basic Pharmacokinetics Including Clinical Applications, 5th edition, American Pharmaceutical Assoc., Washington, D.C.
- It is expected that the activatable interleukin protein has improved pharmacokinetic parameters such as an increase in elimination half-time as compared to proteins lacking a serum half-life extension element.
- Activatable interleukin protein is evaluated in a xenograft model.
- Female immune-deficient NOD/scid mice are sub-lethally irradiated (2 Gy) and subcutaneously inoculated with 4×106 Ramos RA1 cells into the right dorsal flank. When tumors reach 100 to 200 mm3, animals are allocated into 3 treatment groups.
Groups 2 and 3 (8 animals each) are intraperitoneally injected with 1.5×107 activated human T-cells. Three days later, animals fromGroup 3 are subsequently treated with a total of 9 intravenous doses of 50 μg activatable interleukin protein.Groups -
TABLE 3 Summary of the treatment modes Formulation Gr. N Agent dose Route Schedule 1# 10 Vehicle — ip biwk × 3 2 7 ACP16 700 μg/animal ip biwk × 3 3 7 ACP16 230 μg/animal ip biwk × 3 4 7 ACP16 70 μg/animal ip biwk × 3 5 7 ACP16 55 μg/animal ip biwk × 3 6 7 ACP16 17 μg/animal ip biwk × 3 7 7 ACP132 361 μg/animal ip biwk × 3 8 7 ACP132 119 μg/animal ip biwk × 3 9 7 ACP132 36 μg/animal ip biwk × 3 10 7 ACP132 28 μg/animal ip biwk × 3 11 7 ACP132 9 μg/animal ip biwk × 3 12 7 ACP21 540 μg/animal ip biwk × 3 13 7 ACP21 177 μg/animal ip biwk × 3 14 7 ACP21 54 μg/animal ip biwk × 3 15 7 ACP21 42 μg/animal ip biwk × 3 16 7 ACP21 13 μg/animal ip biwk × 3 17 7 ACP133 210 μg/animal ip bid × 5 then 2-day pause then bid × 5 then 2-day pause 18 7 ACP133 105 μg/animal ip bid × 5 then 2-day pause then bid × 5 then 2-day pause 19 7 ACP133 40 μg/animal ip bid × 5 then 2-day pause then bid × 5 then 2-day pause 20 7 ACP133 3 μg/animal ip bid × 5 then 2-day pause then bid × 5 then 2-day pause #Control Group - It is expected that animals treated with the activatable interleukin protein have a statistically significant delay in tumor growth in comparison to the respective vehicle-treated control group.
- While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
- The MC38 cell line, a rapidly growing colon adenocarcinoma cell line that expresses MMP9 in vitro, was used. Using this tumor model, the ability of fusion proteins to affect tumor growth was examined.
- Additional studies were carried out in non-tumor bearing animals as described below.
-
TABLE 4 Summarizes the treatment regime. Gr. N Agent Formulation dose Route Schedule 1 8 Vehicle — ip biwk × 4 2 8 ACP16 700 μg/animal ip biwk × 4 3 8 ACP16 230 μg/animal ip biwk × 4 4 8 ACP16 70 μg/animal ip biwk × 4 8 8 ACP153 700 μg/animal ip biwk × 4 9 8 ACP153 230 μg/animal ip biwk × 4 10 8 ACP153 70 μg/animal ip biwk × 4 11 8 ACP154 700 μg/animal ip biwk × 4 12 8 ACP154 230 μg/animal ip biwk × 4 13 8 ACP154 70 μg/animal ip biwk × 4 14 8 ACP155 700 μg/animal ip biwk × 4 15 8 ACP155 230 μg/animal ip biwk × 4 16 8 ACP155 70 μg/animal ip biwk × 4 17 8 ACP156 700 μg/animal ip biwk × 4 18 8 ACP156 230 μg/animal ip biwk × 4 19 8 ACP156 70 μg/animal ip biwk × 4 20 8 ACP157 700 μg/animal ip biwk × 4 21 8 ACP157 230 μg/animal ip biwk × 4 22 8 ACP157 70 μg/animal ip biwk × 4 -
TABLE 5 Describes the constructs used in the MC38 IL-2POC animal study. Construct Name Description MW ACP16 IL2-X-HSA-LX-blocker Fusion protein- 58256 6 × His ACP133 IL-2 with C term 6 × His16462 ACP132 IL2-L-HSA 29996 ACP21 IL2-XL-blocker Fusion protein-6 × His 44843 -
-
TABLE 6 Summarizes the treatment regime. Gr. N Agent Formulation dose Route Schedule 1# 12 Vehicle — ip biwk × 2 2 8 ACP16 4.4 μg/animal ip biwk × 2 3 8 ACP16 17 μg/animal ip biwk × 2 4 8 ACP16 70 μg/animal ip biwk × 2 5 8 ACP16 232 μg/animal ip biwk × 2 6 8 ACP130 19 μg/animal ip biwk × 2 7 8 ACP130 45 μg/animal ip biwk × 2 8 8 ACP130 180 μg/animal ip biwk × 2 9 8 ACP130 600 μg/animal ip biwk × 1 12 8 ACP124 17 μg/animal ip biwk × 2 13 8 ACP124 70 μg/animal ip biwk × 2 14 8 ACP124 230 μg/animal ip biwk × 2 15 8 ACP124 700 μg/animal ip biwk × 2 16 8 IL-2- 12 μg/animal ip bid × 5 then 2-day pause WTI then bid × 5 then 2-day pause 17 8 IL-2- 36 μg/animal ip bid × 5 then 2-day pause WTI then bid × 5 then 2-day pause #Control Group - Mice were anaesthetized with isoflurane for implant of cells to reduce the ulcerations. 308 CR female C57BL/6 mice were set up with 5×105 MC38 tumor cells in 0% Matrigel sc in flank. Cell Injection Volume was 0.1 mL/mouse. Mouse age at start date was 8 to 12 weeks. Pair matches were performed when tumors reach an average size of 100-150 mm3 and begin treatment. Body weights were taken at initiation and then biweekly to the end. Caliper measurements were taken biweekly to the end. Any adverse reactions were to be reported immediately. Any individual animal with a single observation of >than 30% body weight loss or three consecutive measurements of >25% body weight loss was euthanized Any group with a mean body weight loss of >20% or >10% mortality stopped dosing; the group was not euthanized and recovery is allowed. Within a group with >20% weight loss, individuals hitting the individual body weight loss endpoint were euthanized. If the group treatment related body weight loss is recovered to within 10% of the original weights, dosing resumed at a lower dose or less frequent dosing schedule. Exceptions to non-treatment body weight % recovery were allowed on a case-by-case basis. Endpoint was tumor growth delay (TGD). Animals were monitored individually. The endpoint of the experiment was a tumor volume of 1500 mm3 or 45 days, whichever comes first. Responders were followed longer. When the endpoint was reached, the animals are to be euthanized.
- No compounds in salt form were used. The amount needed per week was calculated, aliquoted accordingly, and stored at −20 C. For each week of dosing, one aliquot was thawed, stored at 4 C, and diluted in the required amount with PBS right before each injection. IL-2-WTI required protection from light; pre-formulation stored at −4° C., post-formulation—stored at −20° C. Lyophilized material was reconstituted as directed by instructions, similar to above. ACP16, ACP130, ACP124, and IL-2 WTI were prepared for dosing in PBS. IL-2-WTI indicates Proleukin (aldesleukin) in PBS; the vehicle was PBS.
- Dosing volume was 0.2 mL/mouse for IL-2-WTI; 0.3 mL for ACP16, ACP130; 0.5 mL for ACP124. Do not adjust for body weight.
- ACP16: current amount of required compound—13.45 mg
- ACP130: current amount of required compound −25.83 mg
- ACP124: current amount of required compound −42.31 mg
- IL-2-WTI: current amount of required compound −9.98 mg
- Necropsy was to be performed in case of unexpected toxicity
-
-
TABLE 7 Summarizes the treatment regime. Gr. N Agent Formulation dose Route Schedule 1# 12 Vehicle ip biwk × 3 2 8 ACP11 17.5 μg/animal ip biwk × 3 3 8 ACP11 175 μg/animal ip biwk × 3 4 8 ACP11 525 μg/animal ip biwk × 3 5 8 ACP31 33 μg/animal ip biwk × 3 6 8 ACP31 110 μg/animal ip biwk × 3 7 8 ACP31 330 μg/animal ip biwk × 3 8 8 ACP131 1 μg/animal ip bid × 5 then 2-day pause then bid × 5 then 2-day pause 9 8 ACP131 10 μg/animal ip bid × 5 then 2-day pause then bid × 5 then 2-day pause 10 8 ACP131 30 μg/animal ip bid × 5 then 2-day pause then bid × 5 then 2-day pause 11 8 mIFNal- 1 μg/animal ip bid × 5 then 2-day pause then bid × WTI 5 then 2-day pause 12 8 mIFNal- 10 μg/animal ip bid × 5 then 2-day pause then bid × WTI 5 then 2-day pause 13 8 IL-12- 2 μg/animal ip bid × 5 then 2-day pause then bid × HM- 5 then 2-day pause WTI 14 8 IL-12- 10 μg/animal ip bid × 5 then 2-day pause then bid × HM- 5 then 2-day pause WTI 15 8 ACP131 5 μg/animal itu bid × 5 then 2-day pause then bid × 5 then 2-day pause #Control Group - Mice were anaesthetized with isoflurane for implant of cells to reduce the ulcerations. 308 CR female C57BL/6 mice were set up with 5×105 MC38 tumor cells in 0% Matrigel sc in flank. Cell Injection Volume was 0.1 mL/mouse. Mouse age at start date was 8 to 12 weeks. Pair matches were performed when tumors reach an average size of 100-150 mm3 and begin treatment. Body weights were taken at initiation and then biweekly to the end. Caliper measurements were taken biweekly to the end. Any adverse reactions were to be reported immediately. Any individual animal with a single observation of >than 30% body weight loss or three consecutive measurements of >25% body weight loss was euthanized Any group with a mean body weight loss of >20% or >10% mortality stopped dosing; the group was not euthanized and recovery is allowed. Within a group with >20% weight loss, individuals hitting the individual body weight loss endpoint were euthanized. If the group treatment related body weight loss is recovered to within 10% of the original weights, dosing resumed at a lower dose or less frequent dosing schedule. Exceptions to non-treatment body weight % recovery were allowed on a case-by-case basis. Endpoint was tumor growth delay (TGD). Animals were monitored individually. The endpoint of the experiment was a tumor volume of 1500 mm3 or 45 days, whichever comes first. Responders were followed longer. When the endpoint was reached, the animals are to be euthanized.
- No compounds in salt form were used. Prepared dosing solutions were as follows: IL-12-HM-WTI was stored to provide protection from light; pre-formulation at −4° C., post-formulation at −20° C. mIFNa1-WTI was stored at −20° C., protected from light; pre-formulation stored at −20° C., post-formulation stored at −4° C. Lyophilized material was reconstituted as directed in instructions. The amount needed per week was calculated, aliquoted accordingly, and stored at −20 C. For each week of dosing, one aliquot was thawed and stored at 4 C, the required amount was diluted with PBS right before each injection.
- For ACP11, ACP31, ACP131, the amount needed per week was calculated, aliquoted accordingly, and stored at −20 C. For each week of dosing, one aliquot was thawed and stored at 4 C, the required amount was diluted with PBS right before each injection.
- PBS was used as the vehicle for all tests.
- Intraperitoneal (ip) dosing volume ACP131, mIFNa1-WTI, IL-12-HM-WTI=0.2 mL/mouse. Dosing volume for ACP11=0.4 mL (before Day 18, Feb. 26, 2019) and 0.55 mL (starting on Day 18, Feb. 26, 2019).
- Dosing volume for ACP31=0.3 mL. Dosage was not adjusted for body weight. Intratumoral (itu) dosing volume for Gr.16 mIFNa1-WTI and Gr.17 ACP131=0.05 mL/mouse. Dosage was not adjusted for body weight.
-
-
TABLE 8 Summarizes the treatment regime. Gr. N Agent Formulation dose Route Schedule 1# 33 No — — — Treatment 2 7 ACP16 70 μg/animal ip MC38- e415 Gr 4 An 1-4, 6-8 (ACP16 biwk × 2) 3 8 ACP16 232 μg/animal ip MC38- e415 Gr 5 An 1-8 (ACP16 biwk × 2) 5 5 IL-2- WTI 12 μg/animal ip MC38- e415 Gr 16 An 1,3-6 (IL-2-WTI bid × 5 then2-day pause then bid × 5 then 2-day pause) 6 7 IL-2-WTI 36 μg/animal ip MC38-e415 Gr 17 An 1-7 (IL-2-WTI bid × 5 then 2- day pause then bid × 5 then 2-day pause) #Control Group - Mice were anaesthetized with isoflurane for implant of cells to reduce the ulcerations. This portion of the study began on the day of implant (Day 1).
Group 1 consisted of 33 CR female C57BL/6 mice set up with 5×105 MC38 tumor cells in 0% Matrigel subcutaneously in the flank. Groups 2-6 consisted of 33 CR female C57BL/6 mice set up with 5×105 MC38 tumor cells in 0% Matrigel sc in the left flank. The tumors from the previous MC38 experiment (example 25×) were implanted in the right flank of each animal Cell Injection Volume was 0.1 mL/mouse. Age of control mice at initiation was 14 to 17 weeks. These mice were age matched to mice from the previous MC38 experiment (example 25×). No dosing of active agent occurred during rechallenge. Body Weights were taken biweekly until end, as were caliper measurements. Any adverse reactions or death were reported immediately. Any individual animal with a single observation of >than 30% body weight loss or three consecutive measurements of >25% body weight loss was euthanized Endpoint was tumor growth delay (TGD). Animals were monitored individually. The endpoint of the experiment was a tumor volume of 1000 mm3 or 45 days, whichever comes first. Responders were followed longer when possible. When the endpoint is reached, the animals were euthanized. -
-
TABLE 9 Summarizes the treatment regime. Gr. N Agent Formulation dose Route Schedule 1# 12 Vehicle — ip biwk × 2 2 8 ACP16 17 μg/animal ip biwk × 2 3 8 ACP16 55 μg/animal ip biwk × 2 4 8 ACP16 230 μg/animal ip biwk × 2 5 8 ACP155 55 μg/animal ip biwk × 2 6 8 ACP155 230 μg/animal ip biwk × 2 7 8 ACP153 55 μg/animal ip biwk × 2 8 8 ACP153 230 μg/animal ip biwk × 2 9 8 ACP156 55 μg/animal ip biwk × 2 10 8 ACP156 230 μg/animal ip biwk × 2 - Mice were anaesthetized with isoflurane for implant of cells to reduce the ulcerations. CR female C57BL/6 mice were set up with 5×105 MC38 tumor cells in 0% Matrigel sc in flank. Cell Injection Volume was 0.1 mL/mouse. Mouse age at start date was 8 to 12 weeks. Pair matches were performed when tumors reach an average size of 100-150 mm3 and begin treatment. ACP16 was dosed at 17, 55 or 230 μg/animal; ACP153, ACP155 and ACP156 were dosed at 55 or 230 μg/animal Body weights were taken at initiation and then biweekly to the end. Caliper measurements were taken biweekly to the end. Any adverse reactions were to be reported immediately. Any individual animal with a single observation of >than 30% body weight loss or three consecutive measurements of >25% body weight loss was euthanized Any group with a mean body weight loss of >20% or >10% mortality stopped dosing; the group was not euthanized and recovery is allowed. Within a group with >20% weight loss, individuals hitting the individual body weight loss endpoint were euthanized. If the group treatment related body weight loss is recovered to within 10% of the original weights, dosing resumed at a lower dose or less frequent dosing schedule. Exceptions to non-treatment body weight % recovery were allowed on a case-by-case basis. Endpoint was tumor growth delay (TGD). Animals were monitored individually. The endpoint of the experiment was a tumor volume of 1500 mm3 or 45 days, whichever comes first. Responders were followed longer. When the endpoint was reached, the animals are to be euthanized Results are shown in
FIG. 58A-58D . - Proteolytic activity was screened in the conditioned media samples by fluorescent resonance energy transfer (FRET) assays with the substrates listed in Table 10.
-
TABLE 10 Substrate motif sequences. Experimentally Tested Substrate P4-P4′ SEQ ID in vitro Conditioned 30-mer Name Sequence NO: kinetics media MSP-MS MMP14_1 GPAGLYAQ 195 ✓ ✓ ✓ MMP9_1 GPAGMKGL 196 ✓ ✓ ✓ FAPα_1 PGGPAGIG 197 ✓ ✓ ✓ CTSL1_1 ALFKSSFP 198 ✓ ✓ ✓ CTSL1_2 ALFFSSPP 199 ✓ ✓ ✓ ADAM17_1 LAQRLRSS 200 ✓ ✓ ✓ ADAM17_2 LAQKLKSS 201 ✓ ✓ ✓ - Protease specificity screening was performed using Multiplexed Substrate Profiling by Mass Spectrometry (MSP-MS), the method described in, e.g. O'Donoghue A. J. et al., Nat Methods., 2012; 9(11): 1095-1100. This method employs a physico-chemically diverse peptide library as substrates for proteases, and reactions are monitored over time with mass spectrometric detection of cleaved products. The resulting cleavages are assessed for specific cleavage in the enzyme-treated sample by comparison with results from a no-enzyme control incubation. Sequence logos for motif analysis are generated with iceLogo software (v.1.2) (iomics.ugent.be/icelogoserver/).
- Recombinant human enzymes were sourced from R&D Systems (Bio-techne): CTSL1 (#952-CY), ADAM17 (a.k.a. TACE, TNF-alpha Converting Enzyme) (#930-ADB), FAP-alpha (#930-ADB-010), MMP-14 (#918-MP-010), furin (#1503-SE-010), MMP-9 (#911-MP-010), thrombin (#2196-SE-200), thermolysin (#3097-ZN), Factor Xa (#1063-SE-010), hepsin (#4776-SE) and ADAM-TS1 (#2197-AD). Enzymes were activated and assayed following the manufacturer's recommendations.
- For CTSL1, enzyme was activated by pre-incubation in assay buffer (50 mM MES, 5 mM DTT, 1 mM EDTA, pH=6) for 15 min. MSP-MS reactions were then started with the mixing of enzyme and substrate, at final CTSL1 concentration of 0.04 ng/μl (0.77 nM), and peptide substrate concentration at 500 nM.
- ADAM17 activity was monitored following manufacturer's recommended conditions using the FRET substrate Mca-PLAQAV-Dpa-RSSSR—NH2 (SEQ ID NO: 244), in the recommended assay buffer: 25 mM TRIS, 2.5 μM ZnCl2 at the optimal pH 9.0, and also at physiological pH 7.4. ADAM17 enzyme specificity was profiled in the MSP-MS experiment at pH 9.0 using 10 nM enzyme, and at pH 7.4 using 50 nM enzyme.
- MMP-14 was activated following manufacturer's recommended conditions by pre-incubation with the enzyme furin, at a molar ratio of 1:100 furin to MMP-14, at 37° C. and pH 9.0 for 1.5 hours. FRET assays were performed using 20 nM MMP-14 at 37° C. in activation buffer: 50 mM TRIS HCl, 3 mM CaCl2, 1 μM ZnCl2 at pH 8.5.
- MMP9 was activated by incubation with 1 mM p-amino phenyl mercuric acetate (prepared from a stock at 100 mM in DMSO) for 24h at 37° C. in activation buffer: 50 mM TRIS HCl (pH 7.5), 10 mM CaCl2, 150 mM NaCl, 0.05% Brij35. The final enzyme concentration used for fluorescence assays was 2.5 nM.
- FAPα activity was tested with fluorescence detection using a generic Z-GP-AMC substrate. For FRET assays, the final enzyme concentration was 5 nM and the buffer was 50 mM TRIS HCl, 1 M NaCl, 1 mg/mL BSA, pH 7.5.
- Thrombin was activated following the manufacturer's recommended conditions by pre-incubation with the enzyme with thermolysin for 15 min, and then the thermolysin was quenched with 1,10 phenanthroline treatment. Activated thrombin was assayed at 1.2 nM in activation buffer: 50 mM TRIS HCl, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5.
- Factor Xa was assayed at 4.7 nM enzyme, at 37° C. in the manufacturer recommended buffer: 50 mM TRIS, 10 mM CaCl2, 150 mM NaCl, pH 7.5.
- Hepsin was pre-activated overnight at 37° C. in the manufacturer recommended buffer: 100 mM TRIS, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35, pH 8.0. Activity was measured with 2.4 or 24 nM enzyme in the buffer: 50 mM TRIS HCl at pH 7.4.
- ADAM-TS1 was assayed at 5 μM enzyme concentration in the manufacturer recommended buffer: 50 mM TRIS, 10 mM CaCl2, 150 mM NaCl at pH 7.5.
- Conditioned media and cell lysate samples were received from Charles River Laboratories for the murine colon cancer cell lines: MC38 (epithelial), CT26 (fibroblast), and CT26 transduced with a lentivirus for doxycyclin-inducible expression of MMP9 (CT26 pLVX MMP9-5T4 +dox). An immortalized mouse intestinal myofibroblast cell line (ABM Good #T0565) was used as a control stromal cell line, grown under manufacturer recommended conditions. Conditioned media and cell lysate samples were also received for screening purposes from ABM Good for the immortalized mouse colonic epithelial cell line YAMC (ABM Good #T0567).
- Conditioned media samples were prepared according to the standard protocol using logarithmic phase cells. Briefly, cells were grown for 16h in either serum-free media or in complete media containing 10% fetal bovine serum (FBS). After conditioned media collection, the adherent cells were washed then lysed on plate with non-denaturing lysis buffer to produce a lysed “cell pellet” to capture any activity that remained associated with the cells. All cell culture-derived conditioned media samples, cell lysates from Charles River Laboratories, and cell lysates derived internally were processed with identical methods, to allow comparison between samples.
- The resulting conditioned media was buffer exchanged with PBS and concentrated to 10× the original titer as a stock solution.
- Method development experiments with these samples showed that conditioned media containing fetal bovine serum (FBS) were amenable to FRET screening, although a small amount of background fluorescence for each substrate was obtained in the presence of FBS. FBS background controls were therefore used for baseline subtraction at each concentration of substrate throughout the FRET experiments.
- In the end-point screening assays, a fixed titer of 10× concentrated sample and a substrate concentration of 10 μM was used. Values reported in the end-point experiments are initial velocity measurements, in relative fluorescence units (RFU) per second. End-point measurements are a starting point for a screen, but the rate of the reaction is non-linearly related to substrate concentration. Therefore, a more accurate representation of activity would be given by steady-state kinetic measurements covering a range of substrate concentrations. To compare activity across cell lines, the data were treated with a Michaelis-Menten model, and the concentration of equivalent enzyme units [E0] was solved.
- Accordingly, initial velocity was measured as a function of substrate concentration, using a 2-fold dilution series from 250 to 1 μM. Data were fitted using non-linear least squares fitting with GraphPad Prism software v 8.0 to the Michaelis-Menten equation:
-
- where kcat is the rate of product formation under steady-state conditions in units (s−1), and KM is the Michaelis constant that gives the substrate concentration in molar units (M) at which half maximum velocity of the reaction is produced for a given enzyme, [E0] is the enzyme concentration, and [S] the substrate concentration in (M).
- In the assays where purified recombinant enzyme is used, the Michaelis-Menten parameters are as classically defined above. For conditioned media samples, the effective titer of conditioned media per assay was adjusted to approximately a 2× concentrate of the original unprocessed media samples to assure that the detection range matched the signal produced. The enzyme component in the calculation is redefined as a mixture of possible proteases that contribute to the cumulative observed activity, each with intrinsic reactivity toward a substrate. In this case, it is more appropriate to consider kcat and KM as macroscopic constants that represent the overall efficiency of catalysis, and E0 is an enzyme equivalent, expressed in units of concentration. To solve the data fit, kcat/KM can be fixed at that of the recombinant enzyme and enzyme equivalents per cell culture volume can be output. The effective titer of enzyme equivalents in this case can be used to compare the activity produced by the different cell lines.
- The ratios of enzyme equivalents between tumor and control cell lines may be a contributing factor to therapeutic index in a pro-drug employing protease-cleavable linkers.
- For all six motif substrates, greater activity was detected in the conditioned media from the tumor cell lines than from the control myofibroblast cell line (
FIG. 36 ). - The ADAM17 substrate with the sequence LAQKLKSS (ADAM17_2) (SEQ ID NO: 201), based on the results in the end-point screen experiments, had approximately 3-fold greater activity in the conditioned media produced from the three tumor cell lines, compared to the myofibroblast cell line (
FIG. 36 , black bars). For reference, the activity of recombinant ADAM17 at 12 nM is shown in the same assay format. The steady state kinetic curves for ADAM17 substrate are shown inFIG. 37 . There was essentially undetectable ADAM17_2 background activity in the myofibroblast sample. - The CTSL1 substrate, ALFKSSFP (SEQ ID NO: 198) (CTSL1_1), based on the results in the end-point screen experiments, had undetectable activity in the control myofibroblast cell line, as well had low activity in the MC38 and CT26 parental cell lines, and had undetectable activity in the CT26-MMP9+ cell line (
FIG. 36 ). This assay was benchmarked with CTSL1 at 1.5 nM, showing that the extracellular concentration of secreted CTSL1 activity was below this effective concentration across the four cell lines. Re-analysis of conditioned media with the CTSL1_1 substrate using the steady-state kinetics analysis yielded no measurable activity above background. - The secreted activity for FAPα, based on the results in the end-point screen experiments, was very low, but was still detected in all three tumor cell lines (
FIG. 36 , blue bars). The FAPα_1 activity was benchmarked with 5 nM FAPα enzyme. This enzyme is also found in a plasma membrane-associated form, therefore FAPα activity was also tested in the cell pellet fraction that was collected after the media conditioning procedure. These results will be discussed below. - As observed in the end-point screen, re-analysis of the conditioned media for activity toward the FAPα_1 substrate showed lower activity than that associated with the cell pellet, measured in cell lysates (
FIG. 38 andFIG. 39 ). Myofibroblasts had no measurable secreted activity but did have cell-associated activity (FIG. 38 andFIG. 39 ). - The MMP substrates MMP9_1 and MMP14_1 all had high activity in the end point screen, and the activity was significantly higher in the tumor cell lines than in the myofibroblast control cells. MMP9_1 (second column from right,
FIG. 38 ) had approximately 4-fold greater activity in the tumor lines than the myofibroblast control; and MMP14_1 had approximately 57-fold greater activity in the tumor lines. The MMP14_1 substrate had the lowest activity in the myofibroblast cells, thus contributing to the higher ratio of tumor vs control in this assay. Secreted activity was measured for the MMP9_1 substrate in the conditioned media for each of the tumor cell lines using the steady-state kinetics analysis (FIG. 36 ). The myofibroblast cell line had an artefact in analysis of MMP9_1 secreted activity (FIG. 36 ); this will be addressed with a repeat analysis at higher enzyme titer. As with the MMP9 substrates, the MMP14_1 substrate had significant measurable activity in the conditioned media from the tumor cells but not in the myofibroblast cell line. - Taken together, the results of the FRET screens of conditioned media demonstrated tumor-specific activity for the enzymes ADAM17, CTSL1, MMP9 and MMP14. Soluble FAP-alpha activity was low.
- To test for cell pellet-associated FAPα activity, the cell lysates were clarified by ultracentrifugation and then assayed neat in the FRET assay. As shown in
FIG. 41 , FAPα_1 cleavage activity was detected in all four cell lines (blue bars, right hand bar of each pair). A modest 2-fold greater activity was detected in the three tumor cell lines over the myofibroblast cell line. For comparison, CTSL1 activity was also tested in the cell lysates. CTSL1 is a lysosomal enzyme; therefore, activity toward CTSL1_1 was expected to be similarly abundant in all four cell lines. The ratio of CTSL1_1 cleavage in tumor vs control cell lines in this screening format was ˜1× (grey bars, left-hand bar of each pair,FIG. 41 ). FAPα (fibroblast activating protein-alpha) is a marker for fibroblast (CT26) and myofibroblast cell lines. Therefore, an additional non-fibroblast, epithelial cell line was obtained as an alternate negative control cell line, comparable to the MC38 epithelial cell line. - Cell-associated activity toward CTSL1 substrate was detected in all of the cell lysates, indicating cell-associated activity for this enzyme. The activity was significantly greater in MC38 cells over the myofibroblast, CT26 parental, and CT26-MMP9+ cells (
FIG. 42 ). - Low background-corrected fluorescence was observed in all the myofibroblast reactions. Thus, it can be concluded that the immortalized myofibroblast cell line had low conditioned media activity.
- An Immortalized Mouse Colonic Epithelial Cell Line (YAMC) was used to test activity in the presence of additional control “normal” epithelial cells. YAMC's are given the calculated enzyme equivalents (nM) resulting from all of these steady state experiments, to allow for comparison of the amount of equivalent activity per sample. For example, the YAMC control epithelial cell line produced activity with the ADAM17_2 substrate equivalent to that of 0.52±0.03 nM of ADAM17 recombinant enzyme. This cell line compares directly to MC38, also of epithelial origin, which had activity equivalent to that of 0.32±0.03 nM of ADAM17 recombinant enzyme. The results of the screening show that the YAMC cell line generally had more background activity than did the myofibroblast cell line with all of the substrates. This cell line required non-standard cell culture conditions, including growth with murine IFNγ, and at low temperature to maintain, and may not be fully representative of epithelial cell lines.
- Additional in vitro kinetic experiments were performed to characterize a new candidate motif for CTSL1 enzyme; the CTSL1_2 motif has the sequence ALFFSSPP (SEQ ID NO: 199). CTSL1 cleaves the FRET substrate bearing the CTSL1_1 motif highly efficiently (
FIG. 46 ). Yet, initial experiments with the CTSL1_2 FRET substrate were inconclusive, possibly due to artifacts in the FRET assay; therefore, a new FRET peptide construct was designed with the 9-mer sequence ALFFSSPPS (SEQ ID NO: 236). This 9-mer FRET substrate bearing the CTSL1_2 motif produced comparable catalytic efficiency to the CTSL1_1 motif. - All of the FRET substrates used in these studies have a methoxycoumarin (Mca) fluorophore and dinitrophenyl-lysyl (Dnp) quencher pair at the N- and C-termini of the sequences, respectively. The motif ALFFSSPP (SEQ ID NO: 199) was efficiently cleaved in previous experiments at ALFF|SSPP (SEQ ID NO: 199) (where indicates the scissile bond) within 14-mer peptide constructs that were used in the MSP-MS tailored library assay. It was cleaved also at the ALF|FSSPP (SEQ ID NO: 199) and ALFFS|SPP (SEQ ID NO: 199) sites within 14-mer peptides, indicating that the enzyme recognition in the CTSL1_2 motif may be shifted up- or down-sequence by +/−1 site in the 14-mer peptide experiments. This also suggests that the 8-mer design using Mca-ALFFSSPPK-Dnp (SEQ ID NO: 245) may have unfavorable interactions (steric or otherwise) with the fluor/quencher pair in the shifted P4 or P4′ positions that were alleviated by the redesign as a 9-mer substrate using Mca-ALFFSSPPSK-Dnp (SEQ ID NO: 246). This FRET experiment indicated that cleavage of this sequence most likely requires binding recognition at the +/−1 positionally-shifted site and that the fluor or quencher may interfere with this binding.
- This hypothesis was tested in the context of the hybrid linker design experiment. Shown in
FIG. 46 are the results of cleavage of two matched 30-mer peptides with the following sequences: -
(CTSL1_1) (SEQ ID NO: 207) SGGPGGPAGIGALFKSSFPLAQKLKSSGGG (CTSL1_2) (SEQ ID NO: 219) KSGPGGPAGIGALFFSSPPLAQKLKSSGGR - Both peptides showed rapid disappearance of substrate (squares) and product formation from cleavage at the target scissile bond (
site 0, blue triangles,FIG. 46 ). Where the peptides differed is in the permissive cleavage at the −1 and +1 sites. CTSL1_1 had a −1 site product at ALF|KSSFP (SEQ ID NO: 198) (downward triangles) but CTSL1_2 did not, and the +1 site product was more rapidly formed from the CTSL1_2 substrate (ALFFS|SPP (SEQ ID NO: 199)) than with the CTSL1_1 substrate (ALFKS|SFP) (SEQ ID NO: 198). - Counter-screening of CTSL1_1 and CTSL1_2 was performed with the enzyme Cathepsin K (CTSK). The substrate CTSL1_2, although lacking a basic P1 residue (ALFFSSPP (SEQ ID NO: 199)) was measurably cleaved by CTSK but at a 15× lower level than the CTSL1_1 substrate (ALFKSSFP (SEQ ID NO: 198))
FIG. 47 . For comparison, the specific activity for reference substrates such as Z-LR-AMC and Z-FR-AMC is 15,000-25,000 pmol/min/μg, with the average value indicated by a dashed line inFIG. 47 . - Thus, these results show that the new CTSL1_2 motif was not cleaved in the context of an 8-mer FRET peptide, but it was cleaved as a 9-mer FRET peptide and it was rapidly cleaved in the context of an extended tandem linker.
- To analyze the catalytic efficiency of candidate tandem linker designs, a tailored library approach using MSP-MS was applied. For the tailored library, a set of nineteen synthetic peptides, 30 amino acids in length (30-mers), were designed to test whether sequence context affects the efficiency of cleavage. Peptides were assayed in a multiplex format together as a substrate library with individual recombinant enzymes, using MSP-MS. For quantitative comparisons, kinetic analysis was performed over a time course, and results are reported either as a catalytic efficiency kcat/KM for well-behaved first-order kinetics, or as observed rates as kobs for more complex kinetic behavior.
- The library of tandem linker sequences was designed to incorporate three individual protease motifs within the context of a longer, 30-mer peptide sequence. Table 11 lists the sequences and motif arrangement of the 30-mer peptides.
-
TABLE 11 30-mer peptide sequences. Substrate SEQ ID Name Sequence NO: ALU30-1 GALFKSSFPSGGGPAGLYAQGGSGKGGSGK 202 ALU30-2 RGSGGGPAGLYAQGSGGGPAGLYAQGGSGK 203 ALU30-3 KGGGPAGLYAQGPAGLYAQGPAGLYAQGSR 204 ALU30-4 RGGPAGLYAQGGPAGLYAQGGGPAGLYAQK 205 ALU30-5 KGGALFKSSFPGGPAGIGPLAQKLKSSGGS 206 ALU30-6 SGGPGGPAGIGALFKSSFPLAQKLKSSGGG 207 ALU30-7 RGPLAQKLKSSALFKSSFPGGPAGIGGGGK 208 ALU30-8 GGGALFKSSFPLAQKLKSSPGGPAGIGGGR 209 ALU30-9 RGPGGPAGIGPLAQKLKSSALFKSSFPGGG 210 ALU30-10 RGGPLAQKLKSSPGGPAGIGALFKSSFPGK 211 ALU30-11 RSGGPAGLYAQALFKSSFPLAQKLKSSGGG 212 ALU30-12 GGPLAQKLKSSALFKSSFPGPAGLYAQGGR 213 ALU30-13 GGALFKSSFPGPAGLYAQPLAQKLKSSGGK 214 ALU30-14 RGGALFKSSFPLAQKLKSSGPAGLYAQGGK 215 ALU30-15 RGGGPAGLYAQPLAQKLKSSALFKSSFPGG 216 ALU30-16 SGPLAQKLKSSGPAGLYAQALFKSSFPGSK 217 ALU30-17 KGGPGGPAGIGPLAQRLRSSALFKSSFPGR 218 ALU30-18 KSGPGGPAGIGALFFSSPPLAQKLKSSGGR 219 ALU30-19 SGGFPRSGGSFNPRTFGSKRKRRGSRGGGG 220 - To test for additive effects of having one, two, or three repeat MMP14_1 motifs with small differences in spacing between the motifs, a set of four tandem linkers were designed (ALU30-1 to -4).
- To test whether neighboring effects from adjacent sites could alter the catalytic efficiency of a given protease toward its target motif, the three motifs in a set were arranged in all permutations. For example, the combination of motifs A, B, and C could be arranged as: A-B-C, C-A-B, B-C-A, B-A-C, A-C-B, and C-B-A. These permutations were tested for the tandem combinations of MMP14_1, ADAM17_2, and CTSL1_1 as a broad set of motifs susceptible to matrix metalloprotease (MMP) activity (ALU30-11 to -16), and for FAPa_1 with ADAM17_2, and CTSL1_1 as a set with lower MMP sensitivity (ALU30-5 to -10).
- Also tested within the library were two alternate substrates for ADAM17 and CTSL1. Two additional peptides were designed, swapping the ADAM17_2 motif with ADAM17_1 (in ALU30-17), or CTSL1_1 with CTSL1_2 (in ALU30-18), demonstrating a strategy for testing further tandem linker designs with new individual protease motifs.
- Counter-screening was performed against the enzymes Thrombin, Factor Xa and Hepsin using this library as well, and for calibration of activity, a positive-control peptide bearing favorable motifs for these enzymes was also designed (ALU30-19).
- Outside of these motifs, minor variations were made to the bracketing sequences at the N- or C-terminus of each tandem linker peptide to generate unique sequences that allow for differentiation of peptides using mass spectrometric detection.
- Catalytic efficiency (kcat/KM) estimations were used to rank the top cleaved substrates in the MSP-MS reaction. Peptides were quantified by mass spectrometric label-free quantitation from the MS1 precursor ion peak areas for each peptide. Enzyme progress curves were modeled from six-time point measurements in GraphPad Prism. Data were fitted using non-linear least squares fitting to the first order kinetics equation:
-
- where Y=percent product formation or substrate consumption, and t=time. The observed rate is a function of the enzyme concentration [E0], and an observed catalytic efficiency (kcat/KM) in units M−1 s−1
- Analyses with the 30-mer tandem linker library showed specific cleavages with each of the recombinant enzymes. In this experiment, the rates of substrate degradation and of product formation were both useful comparisons for understanding the efficiency of linker cleavage.
- For example, the substrate degradation traces for the 30-mer library peptides are shown from MMP9-treatment in
FIG. 55 . The most efficient MMP9 substrates were those that bore three-repeats of the MMP14_1 motif. The next most efficient substrates contained the pair of motifs where MMP14_1 was followed directly by ADAM17_2. The single or double MMP14_1 motif-bearing peptides were next in the order. An unanticipated result was that the FAPa_1 motif was also cleaved within the peptides bearing FAPa_1 directly followed by the CTSL1_1 motif at PGGPAG|IGALF (SEQ ID NO: 247); these were lower efficiency cleavages. The FAPa_1 motif was not cleaved by MMP9, however, when followed either by the ADAM17_2 motif or the spacing residues GG. The slowest peptides bear the MMP14_1 motif near the C-terminus of the 30-mer peptide. From this experiment, a trend emerged that MMP9 has additional sequence preferences in the downstream prime-side positions, and that the combination of MMP14_1 upstream of ADAM17_2 is most efficient. - To better understand the intrinsic rates of product formation with MMP9 treatment, it is also possible to monitor the cleavage products at a specific peptide bond in these experiments. A comparison of peptides bearing one, two or three MMP14_1 tandem motifs is shown in
FIG. 50 . The most rapidly degraded peptides were those bearing three repeat motifs of MMP14_1 (top panel). Overall, the peptides bearing one or two MMP14_1 motifs were degraded at approximately the same rate (top panel). However, considering intrinsic rates of bond cleavage at individual sites, the cleavage product of Alu30-2 atbond 9 appeared more rapidly than the cleavage product of Alu30-1 atbond 16 or of Alu30-2 at bond 21 (bottom panel). Thus, the specific cleavage of the Alu30-2 peptide was more efficient than the Alu30-1 peptide. These individual bond cleavage events are monitored by tracking unique peptide fragments, and although too complex for data fitting, the ranking of products is possible. - FAPα was able to cleave both FAPα_1 and MMP14_1 motifs, at PGGP|AGIG (SEQ ID NO: 197) and GP|AGLYAQ (SEQ ID NO: 195) respectively. Degradation of the 30-mer peptides by FAPα showed highest cleavage activity toward the peptides bearing tandem MMP14_1 motifs (
FIG. 50 , Alu30-4 or Alu30-3). The peptide bearing two MMP14_1 motifs (Alu30-2) was also efficiently cleaved. FAPα_1 motifs were cleaved more efficiently when followed by the ADAM17_1 or ADAM17_2 motifs than when followed by CTSL1_1 or CTSL1_2. Among these peptides, the Lys-bearing ADAM17_2 or CTSL1_1 motifs were lower efficiency than the new motifs tested in this experiment, ADAM17_1 and CTSL1_2. - Another trend was a preference for cleavage of the FAPα_1 motif in the first or second position relative to the N-terminus of these peptides. When the FAPα_1 motif was in the third position closest to the C-terminus, these peptides were not cleaved (Alu30-7 or Alu30-8). The MMP14_1 motif was readily cleaved in the third position closest to the C-terminus, but this was most evident in the tandem MMP14_1 motif-bearing peptides, where N-terminal cleavages assist in shortening the 30-mer. Thus, it may be that FAPα could serve as a secondary-cleaving enzyme after activation with a first enzyme elsewhere in a hybrid linker design.
- CTSL1 treatment of the 30-mer peptide library produced cleavages in all peptides bearing a CTSL1_1 or CTSL1_2 motif (
FIG. 51 ). The most efficiently cleaved peptides were those containing the FAPa_1, CTSL1_1 and ADAM17_2 motifs, in multiple orders. Peptides bearing the MMP14_1 motif with CTSL1_1 and ADAM17_2 were slightly lower efficiency. CTSL1 motifs in the middle of the 30-mer peptides were also favorable, thus CTSL1 may be able to serve as a first cleavage in the hybrid motifs. - ADAM17 produced cleavages in all peptides bearing an ADAM17_1 or ADAM17_2 motif as well (
FIG. 52 ). The most efficient peptide cleavages occurred in the peptides containing ADAM17_2, CTSL1_1 and FAPa_1 motifs. Slightly lower efficiency cleavages were obtained in peptides containing the MMP14_1 with ADAM17_2 and CTSL1_1. Peptides bearing only the MMP14_1 motif were not cleaved. Peptides Alu30-9 and Alu30-17, containing the ADAM17_2 and ADAM17_1 motifs respectively, had similar cleavage efficiencies. - Counter-screening with Thrombin, Factor Xa and Hepsin was also performed following the same analysis. The library peptide Alu30-19 was designed to include authentic sites for each enzyme, for comparison of overall cleavage efficiency as well as cleavage site specificity within each motif. The substrate designed for Factor Xa has the motif FNPR|TFGS (SEQ ID NO: 248), derived from the Factor Xa cleavage site in Thrombin. The substrate motif for Thrombin was FPR|, a common tool substrate motif. The substrate motif for hepsin was RKRR|GSRG (SEQ ID NO: 249) from filaggrin.
- Shown in
FIG. 53 are the results of Factor Xa cleavage. The authentic cleavage site motif was Peptide Alu30-19 with this motif was completely degraded before the 5 min time point. Products of the Alu30-19 cleavage were formed from all three sites in this peptide. In comparison, the only library peptide with significant cleavage was Alu30-5, cleaved at the CTSL1_1 motif ALFKS|SFP (SEQ ID NO: 198), upstream of a FAPa_1 motif. - Otherwise, all other peptides bearing a CTSL1_1 motif were cleaved by Factor Xa at either ALF|KSSFP (SEQ ID NO: 198) or ALFKS|SFP (SEQ ID NO: 198). The ADAM17 motifs were also cleaved at KLK|SSGP (SEQ ID NO: 250), KLKSS|ALF (SEQ ID NO: 251), or RLR|SSALF SEQ ID NO: 252), but these cleavages were all lower efficiency. The peptide with the motif CTSL1_2 was cleaved at the lowest efficiency.
- Thrombin showed higher activity than Factor Xa, and it cleaved each of the 30-mer peptides. The MMP14_1 motifs were cleaved at GPAG|LYAQ (SEQ ID NO: 195), the CTSL1_1 motifs at ALFK|SSFP (SEQ ID NO: 198), and the ADAM17_2 motifs at LAQK|LKSS (SEQ ID NO: 201) or LAQKLK|SS (SEQ ID NO: 201) (or LAQR|LRSS (SEQ ID NO: 200) and LAQRL|SS (SEQ ID NO: 253) in ADAM17_1). The two alternate ADAM17 motifs were cleaved with similar efficiency (peptides Alu30-17 vs Alu30-9). However, the CTSL1_2 motif had lower cleavage activity than did the CTSL1_1 motif (peptides Alu30-18 vs Alu30-6). The peptides Alu30-1 and Alu30-3 were the most rapidly cleaved in the library, and Alu30-4 was also an efficient substrate, showing the susceptibility of MMP14_1 motifs in this experiment. The peptide Alu30-2, bearing two MMP14_1 motifs, was less susceptible to thrombin, however, potentially due to altered spacing with additional glycine residues between motifs. Also, the peptides Alu30-12, Alu30-13 and Alu30-15, with MMP141 motifs in combination with ADAM17_2 and CTSL1_1, were cleaved at lower efficiency by thrombin. Thus, increased spacing between the motifs or specific arrangements may rescue a thrombin-susceptible linker design (
FIG. 54 ). - The favored arrangements for reducing thrombin susceptibility include using ADAM17_2 upstream of CTSL1_1, and CTSL1_1 upstream of FAPa_1.
- Finally, hepsin treatment of the 30-mer library produced lower efficiency cleavages overall (
FIG. 55 ). The kinetics of these peptide degradation reactions were complex due to the formation of multiple cleavage produces; for example, Alu30-19 was cleaved at all three motifs within the first 15 min of the reaction at RKRR| (SEQ ID NO: 254), FPR| and FNPR| (SEQ ID NO: 255). The literature value for the catalytic efficiency of a FRET peptide bearing this motif was 3.5×105 M−1s−1. The authentic peptide Alu30-19, as well as the top cleaved peptides Alu30-8,-9, -12, and -14 all had apparent catalytic efficiencies on the same order of 105 The features that made peptides more susceptible to hepsin appear to be placement of an ADAM17_2 motif in the second motif position of the peptide, or in the combination of ADAM17_2 followed by CTSL1_1. In general, the library peptides were all cleaved at P1 Lys sites, QKLK| (SEQ ID NO: 256) or ALFK| (SEQ ID NO: 257). The ADAM17_1 motif was less efficiently cleaved than ADAM172, and the alternate CTSL1_2 motif was also cleaved at a much slower rate than CTSL1_1. - To conclude, the tandem linker analysis with mass spectrometric detection revealed motif order preferences and unexpected side-reactions.
- To generate the most efficient tandem linker, the cleavage efficiencies for each of the targeted enzymes toward the 30-mer library peptides can be compared (
FIG. 64 ). - The tandem linker with the arrangement of ADAM17_2-MMP14_1-CTSL1_1 in the peptide Alu30-16 had generally high activity toward the full set of five targeted enzymes, as well as the lowest susceptibility to thrombin, Factor Xa, and hepsin. The next best configuration was MMP14_1-ADAM17_2-CTSL1_1 in Alu30-15 which, although it had slightly higher hepsin susceptibility and lower CTSL1 activity may be rescued by the replacement with the CTSL1_2 motif. Replacement of the ADAM17_2 motif with ADAM17_1 may also enhance activity toward FAP-alpha and CTSL1, even if ADAM17 activity enhancement is minor, and hepsin susceptibility would be predicted to be reduced.
- The stability of constructs of interest was measured in human or mouse serum. Each construct (approximately 30 mg/mL) was combined with serum (1:9 ratio of construct to serum), MMP9, or PBS. The mixture was incubated at 37° C. for 24 or 72 hours. Samples were taken at T=0 hours for comparison. After incubation, samples were diluted 1:500 (in human serum,
FIG. 56 ) or 1:200 (in mouse serum,FIG. 57 ) in PBS and run on an SDS PAGE gel for western blot analysis. A polyclonal anti-IL-2 antibody (R&D Systems) was used to probe the blots. Results are shown inFIG. 56 andFIG. 57 . - The stability of IL2 fusion proteins in human serum (normal and cancer patient) was measured using capillary electrophoresis-based immunoassays (Jess instrument, Protein Simple). Fusion proteins were concentrated to 10 mg/ml and incubated with serum (90%). A time zero sample was immediately stored on ice while 24 hour and 72 hour samples were placed at 37° C. Post incubation, samples were diluted 1:1000 with 0.1× sample buffer (Protein Simple) and loaded on the Jess cartridge per the manufacturer's protocol. The primary antibody was a monoclonal human IL2 antibody (R&D Systems, cat #AF-202-NA, stock concentration 0.2 mg/ml, working concentration 1:100), and the secondary antibody was a peroxidase-conjugated AffiiniPure Bovine Anti Goat IgG (H+L) (Jackson Immuno Research, cat #805-035-18, reconstituted at 0.8 mg/ml, working concentration 1:5000 dilution). All antibodies were diluted in milk free diluent (Protein Simple). Quantitation of IL2 containing species was quantitated using Compass software (Protein Simple) to determine the extent of cleavage (i.e. amount of IL2 containing species smaller than the input fusion protein). Results are shown in
FIGS. 24A-24B . - Primary human lung epithelial cells and renal epithelial cells were obtained from ATCC. Primary hepatocytes were obtained from Lonza and Sigma. Cells were thawed, counted, and plated at 1e4 cells per well in a 96 well round bottom plate in their respective growth medias. Polypeptides containing recombinant human IL-2 and the sequence for Linker-2 and Linker-3 were incubated with cells or media alone for 24 and 72 hours at a
concentration 5 μg/mL. Cell culture supernatants were collected, and cells were discarded. Protein cleavage was measured by western blot for IL-2 using the protein simple JESS system and Compass software. Results are shown inFIG. 5 . - Primary human lung fibroblasts were obtained from ATCC. Prior to the processing assay, human PBMCs were stimulated with 5 ug/mL PHA for 72 hours to generate T blasts, which were then frozen and used to measure polypeptide processing. Primary human fibroblasts were thawed, counted, and plated at 1e5 cells per well in a 96 well round bottom plate in
X-Vivo 15 media. Polypeptides containing recombinant human IL-2 and the sequence for Linker-1 (GPAGMKGL, SEQ ID NO: 196), Linker-2 (GPAGLYAQ, SEQ ID NO: 195), or Linker-3 (ALFKSSFP, SEQ ID NO: 198), or a non-cleavable sequence were incubated with or without fibroblasts cells for 48 hours at a concentration of either A) 3.3 nM or B) 0.33 nM. As a positive control, some wells were also incubated with a polypeptide that was pre-cut in vitro overnight with 1 μg pre-activated MMP9 enzyme at 37° C. After 48 hours, cell culture supernatants were collected and healthy fibroblasts were discarded. Polypeptide processing was measured either by A) western blot for IL-2 using the protein simple JESS system, or by B) measuring the capacity of the cell culture supernatants to stimulate T blast proliferation. Briefly, T blasts were incubated for 72 hours with cell culture supernatants containing polypeptides previously exposed to healthy human fibroblasts, before T blast proliferation was measured by Cell Titer Glow analysis. Results are shown inFIG. 6A-6B . - Cleavage efficiency of Linker-2 (GPAGLYAQ, SEQ ID NO: 195) was evaluated in tumor tissue derived from human samples. A total of 66 samples were analyzed from seven solid tumor types. The tumor types are shown in Table 5 below.
- Briefly, frozen dissociated tumor cells (DTC) were thawed, counted and plated in X-Vivo media. Cells or “No Cell Control” wells were incubated with an inducible IL-2 cytokine containing Linker 1 (a cleavable linker that was not designed using the processes described herein) or Linker-2 (GPAGLYAQ, SEQ ID NO: 195). These inducible IL-2 proteins have no or minimal IL-2 biological activity when the linker is intact, and IL-2 biological activity is induced when the linker is cleaved. Cell culture supernatants were collected and frozen before analysis for IL-2 biological activity using T-Blast proliferation and Jess protein assays.
- The minimum fold change in IL-2 activity is 1.00, and the maximum is 2.50. A 1.3 fold change is indicative of a significant increase in biological activity relative to the uncleaved control. Table 5 shows the average fold change for Linker-2 (GPAGLYAQ, SEQ ID NO: 195) in comparison to an uncleaved control, a cleaved control, and Linker-1. In Table 12 the symbol “−” indicate essentially no increase in activity; “+/−” indicates some increase in activity but not significant; “+,” “++,” and “+++” indicate the relative significant increase in activity compared to the uncleavable control. Linker-2 is sufficiently cleaved in human tumor types to induce IL-2 biological activity.
-
TABLE 12 Cleavage efficiency of Linker-2 in human tumor types Average Fold Change Over Non-Cleavable Uncleav- Diagnosis able Linker 1 Linker 2Pre-cut Melanoma (n = 8) − − +/− +++ Kidney Cancer, Renal Cell − − + +++ Carcinoma (n = 11) Head and Neck Cancer, − +/− +++ +++ Squamous Cell (n = 6) Colorectal Cancer, − +/− + +++ Adenocarcinoma (n = 10) Lung Cancer, Squamous − − ++ +++ Cell Carcinoma (n = 13) Lung Cancer, − − + +++ Adenocarcinoma (n = 10) Breast Cancer (n = 5) − − +/− +++ - Cleavage efficiency of Linker-3 (ALFKSSFP, SEQ ID NO: 198) was evaluated in tumor tissue derived from human samples. A total of 66 samples were analyzed from seven solid tumor types. The tumor types are shown in Table 6 below.
- Briefly, frozen DTC cells were thawed, counted and plated in X-Vivo media. Cells or “No Cell Control” wells were incubated with an inducible IL-2 cytokine containing Linker 1 (a cleavable linker that was not designed using the processes described herein) or Linker-2 (GPAGLYAQ, SEQ ID NO: 195). These inducible IL-2 proteins have no or minimal IL-2 biological activity when the linker is intact, and IL-2 biological activity is induced when the linker is cleaved. Cell culture supernatants were collected and frozen before analysis for IL-2 biological activity using T-Blast proliferation and Jess protein assays.
- The minimum fold change in IL-2 activity is 1.00, and the maximum is 2.50. A 1.3 fold change is indicative of a significant increase in biological activity relative to the uncleaved control. Table 6 shows the average fold change for Linker-3 (ALFKSSFP, SEQ ID NO: 198) in comparison to an uncleaved control, a cleaved control, and
Linker 1. In Table 13 the symbol “−” indicate essentially no increase in activity; “+/−” indicates some increase in activity but not significant; “+,” “++,” and “+++” indicate the relative significant increase in activity compared to the uncleavable control. Linker-3 (ALFKSSFP, SEQ ID NO: 198) is sufficiently cleaved in human tumor types to induce IL-2 biological activity. -
TABLE 13 Cleavage efficiency of Linker-3 in human tumor types Average Fold Change Over Non-Cleavable Uncleav- Diagnosis able Linker 1 Linker 3Pre-cut Melanoma (n = 8) − − + +++ Kidney Cancer, Renal Cell − − + +++ Carcinoma (n = 11) Head and Neck Cancer, − +/− +++ +++ Squamous Cell (n = 6) Colorectal Cancer, − +/− +++ +++ Adenocarcinoma (n = 10) Lung Cancer, Squamous − − +++ +++ Cell Carcinoma (n = 13) Lung Cancer, − − +++ +++ Adenocarcinoma (n = 10) Breast Cancer (n = 5) − − + +++ - A stable HT-1080 cell line expressing human 4-1BB was established. Agonism of human-4-1BB in these cells resulted in increased secretion of IL-8. Tetravalent monospecific antibodies, inducible format tetravalent monospecific antibodies (protease cleaved or uncleaved), or trimeric ligands were tested for ability to agonize 4-1BB.
- Before addition to the cultured cells, some samples of the antibody to be tested were incubated with an appropriate protease under suitable conditions for proteolysis. Some samples were maintained in an uncleaved state. The extent of inducibility was determined by comparison of uncleaved (uninduced) inducible format tetravalent monospecific antibody with the corresponding protease cleaved inducible format tetravalent monospecific antibody.
- After incubation at 37° C. and 5% CO2 for 6 hours, the agonistic activities of the cleaved and uncleaved antibodies were evaluated by the quantification of IL-8 production using an IL-8 AlphaLISA or ELISA. The EC50s and maximum IL-8 levels were compared. Results are shown in
FIG. 65A-65B . - One of skill in the art would be familiar with methods of setting up protein cleavage assay. 100 μg of protein in 1×PBS pH 7.4 were cleaved with 1 jig active MMP9 (Sigma catalog #SAE0078-50 or Enzo catalog BML-SE360) and incubated at room temperature for up to 16 hours. Digested protein is subsequently used in functional assays or stored at −80° C. prior to testing. The extent of cleavage was monitored by SDS PAGE using methods well known in the art.
- One of skill in an be replaced with Linker-3. the art would be familiar with methods of setting up protein cleavage assay. 100 μg of protein in 1×PBS pH 7.4 are cleaved with 1 μg active MMP14 (R&D Systems Catalog #9518-MP-010) or CTSL1 (R&D Systems Catalog #952-CY) and incubated at room temperature for up to 16 hours. Digested protein are subsequently used in functional assays or stored at −80° C. prior to testing. The extent of cleavage is monitored by SDS PAGE using methods well known in the art.
- The disclosure set forth above may encompass multiple distinct inventions with independent utility. Although each of these inventions has been disclosed in its preferred form(s), the specific embodiments thereof as disclosed and illustrated herein are not to be considered in a limiting sense, because numerous variations are possible. The subject matter of the inventions includes all novel and nonobvious combinations and subcombinations of the various elements, features, functions, and/or properties disclosed herein. The following claims particularly point out certain combinations and subcombinations regarded as novel and nonobvious. Inventions embodied in other combinations and subcombinations of features, functions, elements, and/or properties may be claimed in this application, in applications claiming priority from this application, or in related applications. Such claims, whether directed to a different invention or to the same invention, and whether broader, narrower, equal, or different in scope in comparison to the original claims, also are regarded as included within the subject matter of the inventions of the present disclosure.
- Exemplary polypeptide constructs are detailed herein in Appendix A. While the exemplary polypeptides that contain Linker-2 (GPAGLYAQ, SEQ ID NO: 195), or Linker-3 (ALFKSSFP, SEQ ID NO: 198) or other cleavable linkers are disclosed in Appendix A, for each construct, the disclosed linker can be replaced with either Linker-2 (GPAGLYAQ, SEQ ID NO: 195), or Linker-3 (ALFKSSFP, SEQ ID NO: 198) or other cleavable linkers disclosed herein. For example construct
- Blocker_(Blocker=VHVL.F2.high.A02_Vh\Vl_A46S;X=MMP14-1) can contain Linker-2, however it can be replaced with Linker-3. Alternatively, construct ACPT464 (IgG4_Fc(S228P)—X-IL2-LX-blocker_(Blocker=VHVL.F2.high.A02_Vh/Vl_VH105-VL43_disulfidel;X=CTSL1-1) can contain Linker-3, however, it can be replaced with Linker-2.
- The elements of the polypeptide constructs provided in Appendix A contain the abbreviations as follows: “L,” “X,” “LX,” and “XL” each refer to a linker. “X” refers to a cleavable linker. “L” refers a linker that is optionally cleavable. When L is the only linker in a polypeptide, L is cleavable. “LX” or “XL” each refer to a cleavable linker with an extended non-cleavable sequence adjacent to it. Cleav. Lin. Also refers to a cleavable linker. Other abbreviations used include: “mIFNg” for mouse interferon gamma (IFNg); (5) “hAlbumin” for human serum albumin (HSA); and (6) “mAlbumin” indicates mouse serum albumin.
Claims (14)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US18/315,927 US20230272028A1 (en) | 2019-05-14 | 2023-05-11 | Separation moieties and methods of use thereof |
Applications Claiming Priority (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201962847914P | 2019-05-14 | 2019-05-14 | |
US201962938786P | 2019-11-21 | 2019-11-21 | |
PCT/US2020/032988 WO2020232305A1 (en) | 2019-05-14 | 2020-05-14 | Separation moieties and methods and use thereof |
US17/082,955 US20210115102A1 (en) | 2019-05-14 | 2020-10-28 | Separation moieties and methods of use thereof |
US17/677,473 US11739132B2 (en) | 2019-05-14 | 2022-02-22 | Separation moieties and methods of use thereof |
US18/315,927 US20230272028A1 (en) | 2019-05-14 | 2023-05-11 | Separation moieties and methods of use thereof |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/677,473 Division US11739132B2 (en) | 2019-05-14 | 2022-02-22 | Separation moieties and methods of use thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
US20230272028A1 true US20230272028A1 (en) | 2023-08-31 |
Family
ID=73289342
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/082,955 Pending US20210115102A1 (en) | 2019-05-14 | 2020-10-28 | Separation moieties and methods of use thereof |
US17/677,473 Active US11739132B2 (en) | 2019-05-14 | 2022-02-22 | Separation moieties and methods of use thereof |
US18/315,927 Pending US20230272028A1 (en) | 2019-05-14 | 2023-05-11 | Separation moieties and methods of use thereof |
Family Applications Before (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/082,955 Pending US20210115102A1 (en) | 2019-05-14 | 2020-10-28 | Separation moieties and methods of use thereof |
US17/677,473 Active US11739132B2 (en) | 2019-05-14 | 2022-02-22 | Separation moieties and methods of use thereof |
Country Status (12)
Country | Link |
---|---|
US (3) | US20210115102A1 (en) |
EP (1) | EP3969035A4 (en) |
JP (1) | JP2022532217A (en) |
KR (1) | KR20220023988A (en) |
CN (1) | CN114450022A (en) |
AU (1) | AU2020275877A1 (en) |
BR (1) | BR112021022666A2 (en) |
CA (1) | CA3137512A1 (en) |
IL (1) | IL287977A (en) |
MX (1) | MX2021013766A (en) |
SG (1) | SG11202112541RA (en) |
WO (1) | WO2020232305A1 (en) |
Families Citing this family (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2022502088A (en) | 2018-09-27 | 2022-01-11 | エクシリオ デベロップメント, インコーポレイテッド | Masked cytokine polypeptide |
JP2023503258A (en) * | 2019-11-14 | 2023-01-27 | ウェアウルフ セラピューティクス, インコーポレイテッド | Activatable cytokine polypeptides and methods of use thereof |
CN110862459A (en) * | 2019-11-18 | 2020-03-06 | 温州医科大学 | HPV16E7 affibody-loaded granzyme B affoxin targeting molecule and application thereof |
MX2022012592A (en) | 2020-04-10 | 2022-12-07 | Cytomx Therapeutics Inc | Activatable cytokine constructs and related compositions and methods. |
JP2023550880A (en) | 2020-10-26 | 2023-12-06 | サイチューン ファーマ | IL-2/IL-15Rβγ agonist for treating non-melanoma skin cancer |
AU2021367887A1 (en) | 2020-10-26 | 2023-06-01 | Cytune Pharma | IL-2/IL-15Rβү AGONIST FOR TREATING SQUAMOUS CELL CARCINOMA |
WO2022140388A1 (en) | 2020-12-21 | 2022-06-30 | Allogene Therapeutics, Inc. | Protease-activating cd45-gate car |
IL303853A (en) | 2021-01-29 | 2023-08-01 | Allogene Therapeutics Inc | KNOCKDOWN OR KNOCKOUT OF ONE OR MORE OF TAP2, NLRC5, β2m, TRAC, RFX5, RFXAP AND RFXANK TO MITIGATE T CELL RECOGNITION OF ALLOGENEIC CELL PRODUCTS |
CA3211924A1 (en) | 2021-03-16 | 2022-09-22 | Sayantan Mitra | Masked activatable cytokine constructs and related compositions and methods |
WO2023150735A2 (en) * | 2022-02-04 | 2023-08-10 | The Board Of Trustees Of The Leland Stanford Junior University | Surrogate cytokine polypeptides |
WO2023161853A1 (en) | 2022-02-23 | 2023-08-31 | Bright Peak Therapeutics Ag | Activatable il-18 polypeptides |
WO2024026445A1 (en) | 2022-07-29 | 2024-02-01 | Allogene Therapeutics Inc. | Engineered cells with reduced gene expression to mitigate immune cell recognition |
Family Cites Families (180)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4419446A (en) | 1980-12-31 | 1983-12-06 | The United States Of America As Represented By The Department Of Health And Human Services | Recombinant DNA process utilizing a papilloma virus DNA as a vector |
US4601978A (en) | 1982-11-24 | 1986-07-22 | The Regents Of The University Of California | Mammalian metallothionein promoter system |
US4560655A (en) | 1982-12-16 | 1985-12-24 | Immunex Corporation | Serum-free cell culture medium and process for making same |
US4657866A (en) | 1982-12-21 | 1987-04-14 | Sudhir Kumar | Serum-free, synthetic, completely chemically defined tissue culture media |
US4767704A (en) | 1983-10-07 | 1988-08-30 | Columbia University In The City Of New York | Protein-free culture medium |
US4965199A (en) | 1984-04-20 | 1990-10-23 | Genentech, Inc. | Preparation of functional human factor VIII in mammalian cells using methotrexate based selection |
GB8516415D0 (en) | 1985-06-28 | 1985-07-31 | Celltech Ltd | Culture of animal cells |
US4676980A (en) | 1985-09-23 | 1987-06-30 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Target specific cross-linked heteroantibodies |
GB8601597D0 (en) | 1986-01-23 | 1986-02-26 | Wilson R H | Nucleotide sequences |
US4927762A (en) | 1986-04-01 | 1990-05-22 | Cell Enterprises, Inc. | Cell culture medium with antioxidant |
EP0307434B2 (en) | 1987-03-18 | 1998-07-29 | Scotgen Biopharmaceuticals, Inc. | Altered antibodies |
US5770701A (en) | 1987-10-30 | 1998-06-23 | American Cyanamid Company | Process for preparing targeted forms of methyltrithio antitumor agents |
US5606040A (en) | 1987-10-30 | 1997-02-25 | American Cyanamid Company | Antitumor and antibacterial substituted disulfide derivatives prepared from compounds possessing a methyl-trithio group |
DE68925971T2 (en) | 1988-09-23 | 1996-09-05 | Cetus Oncology Corp | CELL GROWING MEDIUM FOR INCREASED CELL GROWTH, FOR INCREASING LONGEVITY AND EXPRESSION OF PRODUCTS |
KR0184860B1 (en) | 1988-11-11 | 1999-04-01 | 메디칼 리써어치 카운실 | Single domain ligands receptors comprising said ligands methods for their production and use of said ligands |
US5089261A (en) | 1989-01-23 | 1992-02-18 | Cetus Corporation | Preparation of a polymer/interleukin-2 conjugate |
SE509359C2 (en) | 1989-08-01 | 1999-01-18 | Cemu Bioteknik Ab | Use of stabilized protein or peptide conjugates for the preparation of a drug |
US5208020A (en) | 1989-10-25 | 1993-05-04 | Immunogen Inc. | Cytotoxic agents comprising maytansinoids and their therapeutic use |
US6673986B1 (en) | 1990-01-12 | 2004-01-06 | Abgenix, Inc. | Generation of xenogeneic antibodies |
US6075181A (en) | 1990-01-12 | 2000-06-13 | Abgenix, Inc. | Human antibodies derived from immunized xenomice |
DE69120146T2 (en) | 1990-01-12 | 1996-12-12 | Cell Genesys Inc | GENERATION OF XENOGENIC ANTIBODIES |
US6150584A (en) | 1990-01-12 | 2000-11-21 | Abgenix, Inc. | Human antibodies derived from immunized xenomice |
US5891693A (en) | 1990-01-25 | 1999-04-06 | Alusuisse Holdings A.G. | Recombinant DNA methods vectors and host cells |
EP0547163B1 (en) | 1990-08-29 | 2002-02-06 | Centre Hospitalier Regional De Nantes | Protein polyligands joined to a stable protein core |
US5122469A (en) | 1990-10-03 | 1992-06-16 | Genentech, Inc. | Method for culturing Chinese hamster ovary cells to improve production of recombinant proteins |
US5508192A (en) | 1990-11-09 | 1996-04-16 | Board Of Regents, The University Of Texas System | Bacterial host strains for producing proteolytically sensitive polypeptides |
US5264365A (en) | 1990-11-09 | 1993-11-23 | Board Of Regents, The University Of Texas System | Protease-deficient bacterial strains for production of proteolytically sensitive polypeptides |
US5650150A (en) | 1990-11-09 | 1997-07-22 | Gillies; Stephen D. | Recombinant antibody cytokine fusion proteins |
US5571894A (en) | 1991-02-05 | 1996-11-05 | Ciba-Geigy Corporation | Recombinant antibodies specific for a growth factor receptor |
US7018809B1 (en) | 1991-09-19 | 2006-03-28 | Genentech, Inc. | Expression of functional antibody fragments |
US5587458A (en) | 1991-10-07 | 1996-12-24 | Aronex Pharmaceuticals, Inc. | Anti-erbB-2 antibodies, combinations thereof, and therapeutic and diagnostic uses thereof |
WO1993008829A1 (en) | 1991-11-04 | 1993-05-13 | The Regents Of The University Of California | Compositions that mediate killing of hiv-infected cells |
US5288931A (en) * | 1991-12-06 | 1994-02-22 | Genentech, Inc. | Method for refolding insoluble, misfolded insulin-like growth factor-I into an active conformation |
AU675929B2 (en) | 1992-02-06 | 1997-02-27 | Curis, Inc. | Biosynthetic binding protein for cancer marker |
WO1993022332A2 (en) | 1992-04-24 | 1993-11-11 | Board Of Regents, The University Of Texas System | Recombinant production of immunoglobulin-like domains in prokaryotic cells |
CA2149329C (en) | 1992-11-13 | 2008-07-15 | Darrell R. Anderson | Therapeutic application of chimeric and radiolabeled antibodies to human b lymphocyte restricted differentiation antigen for treatment of b cell lymphoma |
US5635483A (en) | 1992-12-03 | 1997-06-03 | Arizona Board Of Regents Acting On Behalf Of Arizona State University | Tumor inhibiting tetrapeptide bearing modified phenethyl amides |
US5780588A (en) | 1993-01-26 | 1998-07-14 | Arizona Board Of Regents | Elucidation and synthesis of selected pentapeptides |
JPH08511420A (en) | 1993-06-16 | 1996-12-03 | セルテック・セラピューテイクス・リミテッド | Body |
US5773001A (en) | 1994-06-03 | 1998-06-30 | American Cyanamid Company | Conjugates of methyltrithio antitumor agents and intermediates for their synthesis |
US5639635A (en) | 1994-11-03 | 1997-06-17 | Genentech, Inc. | Process for bacterial production of polypeptides |
US5731168A (en) | 1995-03-01 | 1998-03-24 | Genentech, Inc. | Method for making heteromultimeric polypeptides |
US5641870A (en) | 1995-04-20 | 1997-06-24 | Genentech, Inc. | Low pH hydrophobic interaction chromatography for antibody purification |
US5869046A (en) | 1995-04-14 | 1999-02-09 | Genentech, Inc. | Altered polypeptides with increased half-life |
US5739277A (en) | 1995-04-14 | 1998-04-14 | Genentech Inc. | Altered polypeptides with increased half-life |
US5714586A (en) | 1995-06-07 | 1998-02-03 | American Cyanamid Company | Methods for the preparation of monomeric calicheamicin derivative/carrier conjugates |
US5712374A (en) | 1995-06-07 | 1998-01-27 | American Cyanamid Company | Method for the preparation of substantiallly monomeric calicheamicin derivative/carrier conjugates |
GB9603256D0 (en) | 1996-02-16 | 1996-04-17 | Wellcome Found | Antibodies |
AU2660397A (en) | 1996-04-05 | 1997-10-29 | Board Of Regents, The University Of Texas System | Methods for producing soluble, biologically-active disulfide bond-containing eukaryotic proteins in bacterial cells |
US5834597A (en) | 1996-05-20 | 1998-11-10 | Protein Design Labs, Inc. | Mutated nonactivating IgG2 domains and anti CD3 antibodies incorporating the same |
DE19701141C1 (en) | 1997-01-16 | 1998-04-09 | Hoechst Ag | Gene construct for treatment of disease involving protease, particularly tumours and inflammation |
US6277375B1 (en) | 1997-03-03 | 2001-08-21 | Board Of Regents, The University Of Texas System | Immunoglobulin-like domains with increased half-lives |
US6083715A (en) | 1997-06-09 | 2000-07-04 | Board Of Regents, The University Of Texas System | Methods for producing heterologous disulfide bond-containing polypeptides in bacterial cells |
DE69830315T2 (en) | 1997-06-24 | 2006-02-02 | Genentech Inc., San Francisco | GALACTOSYLATED GLYCOPROTEIN CONTAINING COMPOSITIONS AND METHOD FOR THE PRODUCTION THEREOF |
ATE419009T1 (en) | 1997-10-31 | 2009-01-15 | Genentech Inc | METHODS AND COMPOSITIONS CONSISTING OF GLYCOPROTEIN GLYCOFORMS |
ATE375365T1 (en) | 1998-04-02 | 2007-10-15 | Genentech Inc | ANTIBODIES VARIANTS AND FRAGMENTS THEREOF |
AU3657899A (en) | 1998-04-20 | 1999-11-08 | James E. Bailey | Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity |
EP2275540B1 (en) | 1999-04-09 | 2016-03-23 | Kyowa Hakko Kirin Co., Ltd. | Method for controlling the activity of immunologically functional molecule |
GB9912350D0 (en) * | 1999-05-26 | 1999-07-28 | European Molecular Biology Lab Embl | Modified cytokine |
IT1306736B1 (en) | 1999-10-29 | 2001-10-02 | Benito Salmi | CONTAINMENT AND DISTRIBUTION DEVICE FOR BALLS FOR ALVOLO SHOOTS. |
AU767394C (en) | 1999-12-29 | 2005-04-21 | Immunogen, Inc. | Cytotoxic agents comprising modified doxorubicins and daunorubicins and their therapeutic use |
EP1276849A4 (en) | 2000-04-12 | 2004-06-09 | Human Genome Sciences Inc | Albumin fusion proteins |
US20040014652A1 (en) | 2000-06-01 | 2004-01-22 | Andre Trouet | Tumor activated prodrug compounds and methods of making and using the same |
DE10045592A1 (en) | 2000-09-15 | 2002-03-28 | Klaus Pfizenmaier | An antibody-TNF-TNF inhibitor fusion protein (TNF selectokine) as a target-specific procytokine for tumor therapy |
US7064191B2 (en) | 2000-10-06 | 2006-06-20 | Kyowa Hakko Kogyo Co., Ltd. | Process for purifying antibody |
JP3523245B1 (en) | 2000-11-30 | 2004-04-26 | メダレックス,インコーポレーテッド | Transgenic chromosome-introduced rodents for the production of human antibodies |
EP2341060B1 (en) | 2000-12-12 | 2019-02-20 | MedImmune, LLC | Molecules with extended half-lives, compositions and uses thereof |
US6942853B2 (en) | 2001-01-09 | 2005-09-13 | Queen Mary And Westfield College | Latent fusion protein |
CA2440582A1 (en) | 2001-03-09 | 2002-10-03 | Dyax Corp. | Serum albumin binding moieties |
FI117667B (en) * | 2001-07-05 | 2007-01-15 | Univ Zuerich | A pharmaceutical composition suitable for use in orthopedics and dentistry |
NZ592087A (en) | 2001-08-03 | 2012-11-30 | Roche Glycart Ag | Antibody glycosylation variants having increased antibody-dependent cellular cytotoxicity |
AU2002355955A1 (en) | 2001-08-13 | 2003-03-03 | University Of Southern California | Interleukin-2 mutants with reduced toxicity |
ES2326964T3 (en) | 2001-10-25 | 2009-10-22 | Genentech, Inc. | GLICOPROTEIN COMPOSITIONS. |
EP2277889B1 (en) | 2001-12-21 | 2014-07-09 | Human Genome Sciences, Inc. | Fusion proteins of albumin and interferon beta |
EP1463752A4 (en) | 2001-12-21 | 2005-07-13 | Human Genome Sciences Inc | Albumin fusion proteins |
US20040093621A1 (en) | 2001-12-25 | 2004-05-13 | Kyowa Hakko Kogyo Co., Ltd | Antibody composition which specifically binds to CD20 |
US7691568B2 (en) | 2002-04-09 | 2010-04-06 | Kyowa Hakko Kirin Co., Ltd | Antibody composition-containing medicament |
AU2003236018A1 (en) | 2002-04-09 | 2003-10-20 | Kyowa Hakko Kirin Co., Ltd. | METHOD OF ENHANCING ACTIVITY OF ANTIBODY COMPOSITION OF BINDING TO FcGamma RECEPTOR IIIa |
AU2003236019A1 (en) | 2002-04-09 | 2003-10-20 | Kyowa Hakko Kirin Co., Ltd. | Drug containing antibody composition appropriate for patient suffering from Fc Gamma RIIIa polymorphism |
ATE503829T1 (en) | 2002-04-09 | 2011-04-15 | Kyowa Hakko Kirin Co Ltd | CELL WITH REDUCED OR DELETED ACTIVITY OF A PROTEIN INVOLVED IN GDP-FUCOSE TRANSPORT |
CA2481837A1 (en) | 2002-04-09 | 2003-10-16 | Kyowa Hakko Kogyo Co., Ltd. | Production process for antibody composition |
ES2425013T3 (en) * | 2002-06-12 | 2013-10-10 | Symphony Evolution, Inc. | Human ADAM-10 inhibitors |
AU2003265866A1 (en) | 2002-09-03 | 2004-03-29 | Vit Lauermann | Targeted release |
CN1703423A (en) | 2002-10-14 | 2005-11-30 | 豪夫迈-罗氏公司 | Antagonists il-15 |
US7361740B2 (en) | 2002-10-15 | 2008-04-22 | Pdl Biopharma, Inc. | Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis |
JP2006524036A (en) | 2002-11-08 | 2006-10-26 | アブリンクス エン.ヴェー. | Single domain antibodies targeting tumor necrosis factor alpha and uses thereof |
DE60332957D1 (en) | 2002-12-16 | 2010-07-22 | Genentech Inc | IMMUNOGLOBULIN VARIANTS AND ITS USES |
TWI364295B (en) | 2002-12-26 | 2012-05-21 | Mountain View Pharmaceuticals | Polymer conjugates of cytokines, chemokines, growth factors, polypeptide hormones and antagonists thereof with preserved receptor-binding activity |
WO2004090135A2 (en) | 2003-04-09 | 2004-10-21 | Asterion Limited | Cytokine polypeptides and antibodies containing a signal sequence for the attachement of glycosylphosphatidylinositol |
US20080241884A1 (en) | 2003-10-08 | 2008-10-02 | Kenya Shitara | Fused Protein Composition |
AU2004280065A1 (en) | 2003-10-09 | 2005-04-21 | Kyowa Hakko Kirin Co., Ltd. | Process for producing antibody composition by using RNA inhibiting the function of alpha1,6-fucosyltransferase |
KR101520209B1 (en) | 2003-11-06 | 2015-05-13 | 시애틀 지네틱스, 인크. | Monomethylvaline compounds capable of conjugation to ligands |
JPWO2005053742A1 (en) | 2003-12-04 | 2007-06-28 | 協和醗酵工業株式会社 | Medicament containing antibody composition |
BRPI0507026A (en) | 2004-02-09 | 2007-04-17 | Human Genome Sciences Inc | albumin fusion proteins |
AU2004101073B4 (en) | 2004-12-21 | 2005-08-18 | Kerry William O'Hanlon | A Beverage Container Holder |
US10011858B2 (en) | 2005-03-31 | 2018-07-03 | Chugai Seiyaku Kabushiki Kaisha | Methods for producing polypeptides by regulating polypeptide association |
WO2006110728A2 (en) | 2005-04-12 | 2006-10-19 | The Uab Research Foundation | Immunogenic cmv tegument aggregates |
US20070269422A1 (en) | 2006-05-17 | 2007-11-22 | Ablynx N.V. | Serum albumin binding proteins with long half-lives |
AU2007258609B2 (en) | 2006-06-07 | 2013-01-24 | Human Genome Sciences, Inc. | Albumin fusion proteins |
US7939632B2 (en) * | 2006-06-14 | 2011-05-10 | Csl Behring Gmbh | Proteolytically cleavable fusion proteins with high molar specific activity |
EP1867660A1 (en) | 2006-06-14 | 2007-12-19 | CSL Behring GmbH | Proteolytically cleavable fusion protein comprising a blood coagulation factor |
US20110252501A1 (en) * | 2006-08-17 | 2011-10-13 | Monsanto Technology Llc | Transgenic plants with enhanced agronomic traits |
EP2068905A4 (en) | 2006-09-14 | 2009-12-30 | Human Genome Sciences Inc | Albumin fusion proteins |
ATE554859T1 (en) | 2007-05-24 | 2012-05-15 | Univ California | INTEGRATED FLUIDIC DEVICES WITH MAGNETIC SORTING |
US20090304719A1 (en) | 2007-08-22 | 2009-12-10 | Patrick Daugherty | Activatable binding polypeptides and methods of identification and use thereof |
JP2011512134A (en) | 2008-02-19 | 2011-04-21 | アステリオン・リミテッド | Modified linker |
US8575102B2 (en) | 2008-08-01 | 2013-11-05 | Nektar Therapeutics | Conjugates having a releasable linkage |
GB0815216D0 (en) | 2008-08-21 | 2008-09-24 | Asterion Ltd | Interleukin |
RU2636046C2 (en) | 2009-01-12 | 2017-11-17 | Сайтомкс Терапьютикс, Инк | Modified antibodies composition, methods of production and application |
BRPI1011384A2 (en) | 2009-02-23 | 2016-03-15 | Cytomx Therapeutics Inc | proproteins and their methods of use |
EP2456787A4 (en) | 2009-07-24 | 2013-01-30 | Univ Leland Stanford Junior | Cytokine compositions and methods of use thereof |
US20130064788A1 (en) | 2009-10-10 | 2013-03-14 | Eleven Biotherapeutics, Inc. | Il-17 family cytokine compositions and uses |
US8734774B2 (en) * | 2010-04-02 | 2014-05-27 | University Of Rochester | Protease activated cytokines |
CN106977608A (en) | 2010-04-09 | 2017-07-25 | 阿尔布麦狄克斯公司 | Albumin derivant and variant |
WO2012054929A2 (en) | 2010-10-22 | 2012-04-26 | Protox Therapeutics Corp. | Use of human serum albumin to decrease antigenicity of therapeutic proteins |
WO2012059486A1 (en) | 2010-11-01 | 2012-05-10 | Novozymes Biopharma Dk A/S | Albumin variants |
LT2637694T (en) | 2010-11-12 | 2021-05-25 | Nektar Therapeutics | Conjugates of an il-2 moiety and a polymer |
CU23923B1 (en) | 2010-11-12 | 2013-07-31 | Ct De Inmunología Molecular | POLYPEPTIDES DERIVED FROM IL-2 WITH AGONIST ACTIVITY |
WO2012106615A1 (en) * | 2011-02-03 | 2012-08-09 | Xoma Technology Ltd. | Methods and materials for enhancing functional protein expression in bacteria |
WO2013163631A2 (en) | 2012-04-27 | 2013-10-31 | Cytomx Therapeutics, Inc. | Activatable antibodies that bind epidermal growth factor receptor and methods of use thereof |
AU2013278075B2 (en) | 2012-06-22 | 2018-05-17 | Cytomx Therapeutics, Inc. | Anti-jagged 1/Jagged 2 cross-reactive antibodies, activatable anti-Jagged antibodies and methods of use thereof |
WO2013192546A1 (en) | 2012-06-22 | 2013-12-27 | Cytomx Therapeutics, Inc. | Activatable antibodies having non-binding steric moieties and mehtods of using the same |
IN2015KN00329A (en) | 2012-08-09 | 2015-07-10 | Univ Leland Stanford Junior | |
EP2882844B1 (en) | 2012-08-10 | 2018-10-03 | Cytomx Therapeutics Inc. | Protease-resistant systems for polypeptide display and methods of making and using thereof |
WO2014052462A2 (en) | 2012-09-25 | 2014-04-03 | Cytomx Therapeutics, Inc. | Activatable antibodies that bind interleukin-6 receptor and methods of use thereof |
WO2014100014A1 (en) | 2012-12-17 | 2014-06-26 | The Board Of Trustees Of The Leland Stanford Junior University | Super-2 complexes with antibody to augment il-2 therapy |
JP6605957B2 (en) | 2013-01-04 | 2019-11-13 | シトムクス セラピューティクス,インコーポレイティド | Compositions and methods for detecting protease activity in biological systems |
WO2014120555A1 (en) | 2013-01-31 | 2014-08-07 | The Regents Of The University Of California | Antibodies specific for urokinase-type plasminogen activator and methods of use thereof |
WO2014165093A2 (en) | 2013-03-13 | 2014-10-09 | Bristol-Myers Squibb Company | Fibronectin based scaffold domains linked to serum albumin or a moiety binding thereto |
US9517276B2 (en) | 2013-06-04 | 2016-12-13 | Cytomx Therapeutics, Inc. | Compositions and methods for conjugating activatable antibodies |
HRP20220553T1 (en) | 2013-07-25 | 2022-06-10 | Cytomx Therapeutics Inc. | Multispecific antibodies, multispecific activatable antibodies and methods of using the same |
CA3214529A1 (en) | 2013-09-25 | 2015-04-02 | Cytomx Therapeutics, Inc. | Matrix metalloproteinase substrates and other cleavable moieties and methods of use thereof |
US9540440B2 (en) | 2013-10-30 | 2017-01-10 | Cytomx Therapeutics, Inc. | Activatable antibodies that bind epidermal growth factor receptor and methods of use thereof |
WO2015089283A1 (en) | 2013-12-11 | 2015-06-18 | Cytomx Therapeutics, Inc. | Antibodies that bind activatable antibodies and methods of use thereof |
AU2015210862B2 (en) | 2014-01-31 | 2020-04-23 | Cytomx Therapeutics, Inc | Matriptase and u-plasminogen activator substrates and other cleavable moieties and methods of use thereof |
IL247320B (en) | 2014-02-21 | 2022-09-01 | Nektar Therapeutics | Use of il -2r beta – selective agonists in combination with anti ctla – 4 or pd - 1 antibodies for treating cancer |
US10150802B2 (en) | 2014-04-24 | 2018-12-11 | The Board Of Trustees Of The Leland Stanford Junior University | Superagonists, partial agonists and antagonists of interleukin-2 |
WO2016014974A2 (en) | 2014-07-25 | 2016-01-28 | Cytomx Therapeutics, Inc. | Anti-cd3 antibodies, activatable anti-cd3 antibodies, multispecific anti-cd3 antibodies, multispecific activatable anti-cd3 antibodies, and methods of using the same |
MA41374A (en) | 2015-01-20 | 2017-11-28 | Cytomx Therapeutics Inc | MATRIX METALLOPROTEASE CLIVABLE AND SERINE PROTEASE CLIVABLE SUBSTRATES AND METHODS OF USE THEREOF |
SG11201707383PA (en) | 2015-03-13 | 2017-10-30 | Cytomx Therapeutics Inc | Anti-pdl1 antibodies, activatable anti-pdl1 antibodies, and methods of use thereof |
EP3292149B1 (en) | 2015-05-04 | 2021-12-01 | CytomX Therapeutics, Inc. | Activatable anti-cd71 antibodies, and methods of use thereof |
AU2016258988A1 (en) | 2015-05-04 | 2017-12-07 | Cytomx Therapeutics, Inc | Anti-ITGa3 antibodies, activatable anti-ITGa3 antibodies, and methods of use thereof |
CA2984948A1 (en) | 2015-05-04 | 2016-11-10 | Cytomx Therapeutics, Inc. | Anti-cd166 antibodies, activatable anti-cd166 antibodies, and methods of use thereof |
IL293719B2 (en) | 2015-05-21 | 2023-07-01 | Harpoon Therapeutics Inc | Trispecific binding proteins and methods of use |
WO2016200645A1 (en) | 2015-06-12 | 2016-12-15 | Tianxin Wang | Methods for protein modification in pharmaceutical applications |
KR20180040138A (en) | 2015-07-13 | 2018-04-19 | 싸이톰스 테라퓨틱스, 인크. | Anti-PD-1 antibodies, activatable anti-PD-1 antibodies, and methods of using them |
MA43816A (en) | 2016-03-08 | 2018-11-28 | Maverick Therapeutics Inc | INDUCTIBLE BINDING PROTEINS AND METHODS OF USE |
US10100106B2 (en) | 2016-05-20 | 2018-10-16 | Harpoon Therapeutics, Inc. | Single domain serum albumin binding protein |
US20190225702A1 (en) | 2016-10-14 | 2019-07-25 | Harpoon Therapeutics, Inc. | Innate immune cell trispecific binding proteins and methods of use |
AU2017342559B2 (en) | 2016-10-14 | 2022-03-24 | Xencor, Inc. | Bispecific heterodimeric fusion proteins containing IL-15/IL-15Ralpha Fc-fusion proteins and PD-1 antibody fragments |
PE20191131A1 (en) | 2016-11-03 | 2019-09-02 | Bristol Myers Squibb Co | ACTIVABLE CYTOTOXIC T LYMPHOCYTE 4 ANTI ANTIGEN (CTLA-4) ANTIBODIES AND THEIR USES |
WO2018136725A1 (en) | 2017-01-19 | 2018-07-26 | Harpoon Therapeutics, Inc. | Innate immune cell inducible binding proteins and methods of use |
EP3589662A4 (en) | 2017-02-28 | 2020-12-30 | Harpoon Therapeutics, Inc. | Inducible monovalent antigen binding protein |
CN110366421A (en) | 2017-03-01 | 2019-10-22 | 尼克塔治疗公司 | Using Interleukin 2 Receptor α, the combined tumor immunotherapy treatment method of beta selective agonists and adoptive cell transfer therapy |
AU2018231127A1 (en) | 2017-03-09 | 2019-09-19 | Cytomx Therapeutics, Inc. | CD147 antibodies, activatable CD147 antibodies, and methods of making and use thereof |
KR20190141760A (en) | 2017-05-02 | 2019-12-24 | 넥타르 테라퓨틱스 | Immunotherapy Tumor Treatment Methods |
EP3619234A4 (en) * | 2017-05-03 | 2021-05-26 | Harpoon Therapeutics, Inc. | Compositions and methods for adoptive cell therapies |
MX2019013621A (en) | 2017-05-15 | 2020-01-13 | Nektar Therapeutics | Long-acting interleukin-15 receptor agonists and related immunotherapeutic compositions and methods. |
BR112019025188A2 (en) | 2017-06-01 | 2020-06-23 | Cytomx Therapeutics, Inc. | ACTIVABLE ANTI-PDL1 ANTIBODIES AND METHODS OF USE OF THE SAME |
EP3641829A4 (en) | 2017-06-20 | 2021-04-21 | The Board of Regents of The University of Texas System | Interferon prodrug for the treatment of cancer |
EA202090247A1 (en) | 2017-07-14 | 2020-05-12 | Цитомкс Терапьютикс, Инк. | ANTIBODIES AGAINST CD166 AND THEIR APPLICATION |
JP2020530554A (en) | 2017-07-20 | 2020-10-22 | シートムエックス セラピューティクス,インコーポレイテッド | Methods and Uses for Qualitative and / or Quantitative Analysis of Activating Antibody Properties |
EP3668548A2 (en) | 2017-08-17 | 2020-06-24 | Nektar Therapeutics | Immunotherapeutic tumor treatment method |
MX2020002198A (en) | 2017-08-30 | 2020-07-20 | Cytomx Therapeutics Inc | Activatable anti-cd166 antibodies and methods of use thereof. |
PE20212205A1 (en) * | 2017-09-08 | 2021-11-18 | Maverick Therapeutics Inc | RESTRICTED CONDITIONALLY ACTIVATED BINDING PROTEINS |
CN111315773A (en) * | 2017-09-08 | 2020-06-19 | 马弗里克治疗公司 | Conditionally active binding moieties comprising an Fc region |
JP7438106B2 (en) | 2017-10-14 | 2024-02-26 | シートムエックス セラピューティクス,インコーポレイテッド | Antibodies, activatable antibodies, bispecific antibodies, and bispecific activatable antibodies and methods of use thereof |
EP3706770A4 (en) | 2017-11-07 | 2021-10-27 | Nektar Therapeutics | Immunotherapeutic combination for treating cancer |
JP2021515599A (en) | 2018-03-09 | 2021-06-24 | アスクジーン・ファーマ・インコーポレイテッドAskGene Pharma, Inc. | New cytokine prodrug |
CU24554B1 (en) | 2018-05-07 | 2021-11-04 | Ct Inmunologia Molecular | FUSION PROTEINS COMPOSED OF INTERLEUKIN 2 MUTEIN AND TYPE 1 INTERFERON |
ES2955511T3 (en) * | 2018-05-14 | 2023-12-04 | Werewolf Therapeutics Inc | Activatable interleukin 2 polypeptides and methods of use thereof |
JP2021524756A (en) | 2018-05-14 | 2021-09-16 | ウェアウルフ セラピューティクス, インコーポレイテッド | Activateable cytokine polypeptides and how to use them |
KR20210038548A (en) | 2018-06-22 | 2021-04-07 | 큐진 인크. | Cytokine-based bioactive drugs and methods of use thereof |
JP2022502088A (en) | 2018-09-27 | 2022-01-11 | エクシリオ デベロップメント, インコーポレイテッド | Masked cytokine polypeptide |
KR20220020879A (en) | 2019-06-12 | 2022-02-21 | 에스크진 파마, 아이엔씨. | New IL-15 prodrugs and how to use them |
EP4004026A4 (en) | 2019-07-25 | 2023-11-15 | Trutino Biosciences Inc. | Il-2 cytokine prodrugs comprising a cleavable linker |
US20220306714A1 (en) | 2019-08-12 | 2022-09-29 | AskGene Pharma, Inc. | Il-2 fusion proteins that preferentially bind il-2ralpha |
JP2023520517A (en) | 2020-04-01 | 2023-05-17 | エクシリオ デベロップメント, インコーポレイテッド | Masked IL-2 cytokines and their cleavage products |
TW202204386A (en) | 2020-04-01 | 2022-02-01 | 美商艾希利歐發展股份有限公司 | Masked il-12 cytokines and their cleavage products |
BR112022019789A2 (en) | 2020-04-01 | 2022-11-16 | Xilio Dev Inc | IL-15 MASKED CYTOKINES, CLEAVAGE PRODUCTS, NUCLEIC ACID, VECTOR, HOST CELL, COMPOSITION, PHARMACEUTICAL COMPOSITION, KIT AND METHODS OF PRODUCING A MASKED IL-15 CYTOKINE AND TREATMENT OR PREVENTION OF CANCER |
-
2020
- 2020-05-14 JP JP2021568018A patent/JP2022532217A/en active Pending
- 2020-05-14 SG SG11202112541RA patent/SG11202112541RA/en unknown
- 2020-05-14 AU AU2020275877A patent/AU2020275877A1/en active Pending
- 2020-05-14 CA CA3137512A patent/CA3137512A1/en active Pending
- 2020-05-14 MX MX2021013766A patent/MX2021013766A/en unknown
- 2020-05-14 CN CN202080050736.3A patent/CN114450022A/en active Pending
- 2020-05-14 BR BR112021022666A patent/BR112021022666A2/en unknown
- 2020-05-14 WO PCT/US2020/032988 patent/WO2020232305A1/en unknown
- 2020-05-14 EP EP20804817.3A patent/EP3969035A4/en active Pending
- 2020-05-14 KR KR1020217040418A patent/KR20220023988A/en unknown
- 2020-10-28 US US17/082,955 patent/US20210115102A1/en active Pending
-
2021
- 2021-11-10 IL IL287977A patent/IL287977A/en unknown
-
2022
- 2022-02-22 US US17/677,473 patent/US11739132B2/en active Active
-
2023
- 2023-05-11 US US18/315,927 patent/US20230272028A1/en active Pending
Also Published As
Publication number | Publication date |
---|---|
KR20220023988A (en) | 2022-03-03 |
US20210115102A1 (en) | 2021-04-22 |
JP2022532217A (en) | 2022-07-13 |
EP3969035A1 (en) | 2022-03-23 |
MX2021013766A (en) | 2022-02-21 |
SG11202112541RA (en) | 2021-12-30 |
BR112021022666A2 (en) | 2022-03-29 |
AU2020275877A1 (en) | 2021-12-23 |
WO2020232305A1 (en) | 2020-11-19 |
US20220220181A1 (en) | 2022-07-14 |
CN114450022A (en) | 2022-05-06 |
EP3969035A4 (en) | 2023-06-21 |
US11739132B2 (en) | 2023-08-29 |
IL287977A (en) | 2022-01-01 |
CA3137512A1 (en) | 2020-11-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11739132B2 (en) | Separation moieties and methods of use thereof | |
US11535658B2 (en) | Activatable interleukin-2 polypeptides and methods of use thereof | |
US20220324930A1 (en) | Activatable interleukin 12 polypeptides and methods of use thereof | |
US20240033326A1 (en) | Activatable Cytokine Polypeptides and Methods of Use Thereof | |
US20210130430A1 (en) | Activatable cytokine polypeptides and methods of use thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: WEREWOLF THERAPEUTICS, INC., MASSACHUSETTS Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:ALAUNUS BIOSCIENCES, INC.;REEL/FRAME:063647/0942 Effective date: 20201028 Owner name: MPM ASSET MANAGEMENT LLC, MASSACHUSETTS Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:EVNIN, LUKE;BHASKAR, VINAY;REEL/FRAME:063647/0881 Effective date: 20200520 Owner name: ALAUNUS BIOSCIENCES, INC., CALIFORNIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:KNUDSEN, GISELLE;REEL/FRAME:063649/0295 Effective date: 20200608 Owner name: WEREWOLF THERAPEUTICS, INC., MASSACHUSETTS Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:MPM ASSET MANAGEMENT LLC;REEL/FRAME:063647/0906 Effective date: 20200520 Owner name: WEREWOLF THERAPEUTICS, INC., MASSACHUSETTS Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:WINSTON, WILLIAM;HICKLIN, DANIEL;SEIDEL-DUGAN, CYNTHIA;AND OTHERS;SIGNING DATES FROM 20200520 TO 20200521;REEL/FRAME:063649/0253 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |