US20240026032A1 - Antibody Immune Cell Inhibitor Fusion Proteins - Google Patents
Antibody Immune Cell Inhibitor Fusion Proteins Download PDFInfo
- Publication number
- US20240026032A1 US20240026032A1 US18/187,928 US202318187928A US2024026032A1 US 20240026032 A1 US20240026032 A1 US 20240026032A1 US 202318187928 A US202318187928 A US 202318187928A US 2024026032 A1 US2024026032 A1 US 2024026032A1
- Authority
- US
- United States
- Prior art keywords
- seq
- amino acid
- acid sequences
- domain
- immune cell
- 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
- 210000002865 immune cell Anatomy 0.000 title claims abstract description 236
- 239000003112 inhibitor Substances 0.000 title claims abstract description 210
- 108020001507 fusion proteins Proteins 0.000 title claims abstract description 163
- 102000037865 fusion proteins Human genes 0.000 title claims abstract description 162
- 239000000427 antigen Substances 0.000 claims abstract description 162
- 108091007433 antigens Proteins 0.000 claims abstract description 158
- 102000036639 antigens Human genes 0.000 claims abstract description 158
- 230000027455 binding Effects 0.000 claims abstract description 138
- 210000004027 cell Anatomy 0.000 claims abstract description 99
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 95
- 229920001184 polypeptide Polymers 0.000 claims abstract description 92
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 92
- 230000004913 activation Effects 0.000 claims abstract description 66
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 52
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 51
- 102000005962 receptors Human genes 0.000 claims abstract description 41
- 108020003175 receptors Proteins 0.000 claims abstract description 41
- 238000000034 method Methods 0.000 claims abstract description 35
- 239000012642 immune effector Substances 0.000 claims abstract description 33
- 229940121354 immunomodulator Drugs 0.000 claims abstract description 33
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 21
- 102100024216 Programmed cell death 1 ligand 1 Human genes 0.000 claims description 148
- 108010074708 B7-H1 Antigen Proteins 0.000 claims description 122
- 108060003951 Immunoglobulin Proteins 0.000 claims description 109
- 102000018358 immunoglobulin Human genes 0.000 claims description 109
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 68
- 102000013463 Immunoglobulin Light Chains Human genes 0.000 claims description 45
- 108010065825 Immunoglobulin Light Chains Proteins 0.000 claims description 45
- 239000000203 mixture Substances 0.000 claims description 43
- 102000006496 Immunoglobulin Heavy Chains Human genes 0.000 claims description 34
- 108010019476 Immunoglobulin Heavy Chains Proteins 0.000 claims description 34
- 102100024213 Programmed cell death 1 ligand 2 Human genes 0.000 claims description 18
- 102100038929 V-set domain-containing T-cell activation inhibitor 1 Human genes 0.000 claims description 18
- 108010061593 Member 14 Tumor Necrosis Factor Receptors Proteins 0.000 claims description 17
- 102000009093 Dihydrolipoyllysine-residue acetyltransferase Human genes 0.000 claims description 16
- 108010073112 Dihydrolipoyllysine-residue acetyltransferase Proteins 0.000 claims description 16
- 108700030875 Programmed Cell Death 1 Ligand 2 Proteins 0.000 claims description 16
- 102100029527 Natural cytotoxicity triggering receptor 3 ligand 1 Human genes 0.000 claims description 14
- 102100038078 CD276 antigen Human genes 0.000 claims description 13
- 108010079206 V-Set Domain-Containing T-Cell Activation Inhibitor 1 Proteins 0.000 claims description 13
- 230000028993 immune response Effects 0.000 claims description 12
- 208000008439 Biliary Liver Cirrhosis Diseases 0.000 claims description 11
- 101001098352 Homo sapiens OX-2 membrane glycoprotein Proteins 0.000 claims description 11
- 102100037589 OX-2 membrane glycoprotein Human genes 0.000 claims description 11
- 208000012654 Primary biliary cholangitis Diseases 0.000 claims description 11
- 101710201161 Natural cytotoxicity triggering receptor 3 ligand 1 Proteins 0.000 claims description 10
- 101710185679 CD276 antigen Proteins 0.000 claims description 9
- 239000012636 effector Substances 0.000 claims description 8
- 239000003937 drug carrier Substances 0.000 claims description 4
- 238000007911 parenteral administration Methods 0.000 claims description 4
- 231100000433 cytotoxic Toxicity 0.000 claims description 3
- 230000001472 cytotoxic effect Effects 0.000 claims description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims 78
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims 3
- 102000012220 Member 14 Tumor Necrosis Factor Receptors Human genes 0.000 claims 2
- 238000011282 treatment Methods 0.000 abstract description 12
- 239000011324 bead Substances 0.000 description 107
- 230000004927 fusion Effects 0.000 description 81
- 230000006044 T cell activation Effects 0.000 description 79
- 230000005764 inhibitory process Effects 0.000 description 76
- 241000699666 Mus <mouse, genus> Species 0.000 description 57
- 101100519207 Mus musculus Pdcd1 gene Proteins 0.000 description 53
- 235000018102 proteins Nutrition 0.000 description 48
- 101001117317 Homo sapiens Programmed cell death 1 ligand 1 Proteins 0.000 description 39
- 241001529936 Murinae Species 0.000 description 38
- 238000007619 statistical method Methods 0.000 description 36
- 238000003556 assay Methods 0.000 description 32
- 239000013642 negative control Substances 0.000 description 25
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 24
- 229960003767 alanine Drugs 0.000 description 24
- 235000004279 alanine Nutrition 0.000 description 24
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 21
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Natural products NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 20
- 102100028785 Tumor necrosis factor receptor superfamily member 14 Human genes 0.000 description 20
- 102000048776 human CD274 Human genes 0.000 description 18
- 230000007896 negative regulation of T cell activation Effects 0.000 description 18
- 238000006467 substitution reaction Methods 0.000 description 18
- 239000013598 vector Substances 0.000 description 16
- 239000003446 ligand Substances 0.000 description 15
- 230000001225 therapeutic effect Effects 0.000 description 15
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 13
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 13
- 125000000539 amino acid group Chemical group 0.000 description 13
- 229960001230 asparagine Drugs 0.000 description 13
- 235000009582 asparagine Nutrition 0.000 description 13
- 238000009472 formulation Methods 0.000 description 13
- 229960001153 serine Drugs 0.000 description 13
- 235000004400 serine Nutrition 0.000 description 13
- 201000010099 disease Diseases 0.000 description 12
- 229940079593 drug Drugs 0.000 description 12
- 239000003814 drug Substances 0.000 description 12
- 229960002885 histidine Drugs 0.000 description 12
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 12
- 235000014304 histidine Nutrition 0.000 description 12
- 210000001519 tissue Anatomy 0.000 description 12
- 239000004471 Glycine Substances 0.000 description 11
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 11
- 230000000694 effects Effects 0.000 description 11
- 229960002449 glycine Drugs 0.000 description 11
- 102100039498 Cytotoxic T-lymphocyte protein 4 Human genes 0.000 description 10
- 101000889276 Homo sapiens Cytotoxic T-lymphocyte protein 4 Proteins 0.000 description 10
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 10
- 239000004472 Lysine Substances 0.000 description 10
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 10
- 238000010494 dissociation reaction Methods 0.000 description 10
- 230000005593 dissociations Effects 0.000 description 10
- 238000005516 engineering process Methods 0.000 description 10
- 230000006870 function Effects 0.000 description 10
- 230000014509 gene expression Effects 0.000 description 10
- 229960003646 lysine Drugs 0.000 description 10
- 238000011201 multiple comparisons test Methods 0.000 description 10
- 229960004295 valine Drugs 0.000 description 10
- 239000004474 valine Substances 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 9
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 9
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 9
- 239000004473 Threonine Substances 0.000 description 9
- 235000001014 amino acid Nutrition 0.000 description 9
- 150000001413 amino acids Chemical class 0.000 description 9
- 229960005261 aspartic acid Drugs 0.000 description 9
- 235000003704 aspartic acid Nutrition 0.000 description 9
- 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 9
- 238000012575 bio-layer interferometry Methods 0.000 description 9
- 230000004069 differentiation Effects 0.000 description 9
- 238000009792 diffusion process Methods 0.000 description 9
- 208000035475 disorder Diseases 0.000 description 9
- 229960002989 glutamic acid Drugs 0.000 description 9
- 229960000310 isoleucine Drugs 0.000 description 9
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 9
- 230000003389 potentiating effect Effects 0.000 description 9
- 229960002429 proline Drugs 0.000 description 9
- 235000013930 proline Nutrition 0.000 description 9
- 241000894007 species Species 0.000 description 9
- 229960002898 threonine Drugs 0.000 description 9
- 238000012384 transportation and delivery Methods 0.000 description 9
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 8
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 8
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 description 8
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 8
- 102100026878 Interleukin-2 receptor subunit alpha Human genes 0.000 description 8
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 8
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 8
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 8
- 239000000872 buffer Substances 0.000 description 8
- 230000001268 conjugating effect Effects 0.000 description 8
- 235000013922 glutamic acid Nutrition 0.000 description 8
- 239000004220 glutamic acid Substances 0.000 description 8
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 8
- 229960002743 glutamine Drugs 0.000 description 8
- 235000004554 glutamine Nutrition 0.000 description 8
- 229960003136 leucine Drugs 0.000 description 8
- 229930182817 methionine Natural products 0.000 description 8
- 229960004452 methionine Drugs 0.000 description 8
- 229960005190 phenylalanine Drugs 0.000 description 8
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 8
- 235000008729 phenylalanine Nutrition 0.000 description 8
- 230000035755 proliferation Effects 0.000 description 8
- 210000000130 stem cell Anatomy 0.000 description 8
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 7
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 7
- 101710094000 Programmed cell death 1 ligand 1 Proteins 0.000 description 7
- 102100033279 Prostaglandin-H2 D-isomerase Human genes 0.000 description 7
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 description 7
- 239000012634 fragment Substances 0.000 description 7
- 238000002347 injection Methods 0.000 description 7
- 239000007924 injection Substances 0.000 description 7
- 230000003993 interaction Effects 0.000 description 7
- 208000023275 Autoimmune disease Diseases 0.000 description 6
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 6
- 108010007707 Hepatitis A Virus Cellular Receptor 2 Proteins 0.000 description 6
- 102000007346 Hepatitis A Virus Cellular Receptor 2 Human genes 0.000 description 6
- 101001137987 Homo sapiens Lymphocyte activation gene 3 protein Proteins 0.000 description 6
- 101000955999 Homo sapiens V-set domain-containing T-cell activation inhibitor 1 Proteins 0.000 description 6
- 102100026120 IgG receptor FcRn large subunit p51 Human genes 0.000 description 6
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 6
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 6
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 6
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 6
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 6
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 6
- 102100038282 V-type immunoglobulin domain-containing suppressor of T-cell activation Human genes 0.000 description 6
- 230000004071 biological effect Effects 0.000 description 6
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 239000013604 expression vector Substances 0.000 description 6
- 238000000684 flow cytometry Methods 0.000 description 6
- 210000000987 immune system Anatomy 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 230000003472 neutralizing effect Effects 0.000 description 6
- 210000003289 regulatory T cell Anatomy 0.000 description 6
- 230000008685 targeting Effects 0.000 description 6
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 6
- 229960004441 tyrosine Drugs 0.000 description 6
- 239000003981 vehicle Substances 0.000 description 6
- 239000004475 Arginine Substances 0.000 description 5
- 101000884279 Homo sapiens CD276 antigen Proteins 0.000 description 5
- 101001122360 Homo sapiens Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial Proteins 0.000 description 5
- 101710177940 IgG receptor FcRn large subunit p51 Proteins 0.000 description 5
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 5
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 5
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 5
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 5
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 5
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 5
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 5
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 5
- 101710187780 Tumor necrosis factor receptor superfamily member 14 Proteins 0.000 description 5
- 210000000612 antigen-presenting cell Anatomy 0.000 description 5
- 229960003121 arginine Drugs 0.000 description 5
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 5
- 235000009697 arginine Nutrition 0.000 description 5
- 102000050261 human DLAT Human genes 0.000 description 5
- 238000002513 implantation Methods 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 230000035772 mutation Effects 0.000 description 5
- 230000036961 partial effect Effects 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 5
- 238000004448 titration Methods 0.000 description 5
- 229960004799 tryptophan Drugs 0.000 description 5
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 4
- 108050005493 CD3 protein, epsilon/gamma/delta subunit Proteins 0.000 description 4
- 108091026890 Coding region Proteins 0.000 description 4
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 4
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 101000611936 Homo sapiens Programmed cell death protein 1 Proteins 0.000 description 4
- 101000666896 Homo sapiens V-type immunoglobulin domain-containing suppressor of T-cell activation Proteins 0.000 description 4
- 102000002698 KIR Receptors Human genes 0.000 description 4
- 108010043610 KIR Receptors Proteins 0.000 description 4
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 4
- 102100020862 Lymphocyte activation gene 3 protein Human genes 0.000 description 4
- 101100407308 Mus musculus Pdcd1lg2 gene Proteins 0.000 description 4
- 101001117316 Mus musculus Programmed cell death 1 ligand 1 Proteins 0.000 description 4
- -1 PDCD1L1 Proteins 0.000 description 4
- 229940024606 amino acid Drugs 0.000 description 4
- 230000001363 autoimmune Effects 0.000 description 4
- 210000003719 b-lymphocyte Anatomy 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 231100000673 dose–response relationship Toxicity 0.000 description 4
- 229910052739 hydrogen Inorganic materials 0.000 description 4
- 230000006698 induction Effects 0.000 description 4
- 239000012678 infectious agent Substances 0.000 description 4
- 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 4
- 210000001616 monocyte Anatomy 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000000600 sorbitol Substances 0.000 description 4
- 210000000952 spleen Anatomy 0.000 description 4
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 4
- 238000013268 sustained release Methods 0.000 description 4
- 239000012730 sustained-release form Substances 0.000 description 4
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 description 4
- 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 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 3
- 206010016654 Fibrosis Diseases 0.000 description 3
- 101000633520 Homo sapiens Natural cytotoxicity triggering receptor 3 ligand 1 Proteins 0.000 description 3
- 241000578472 Human endogenous retrovirus H Species 0.000 description 3
- 229930195725 Mannitol Natural products 0.000 description 3
- 102100027159 Membrane primary amine oxidase Human genes 0.000 description 3
- 101710132836 Membrane primary amine oxidase Proteins 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 102000015728 Mucins Human genes 0.000 description 3
- 108010063954 Mucins Proteins 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 108020004511 Recombinant DNA Proteins 0.000 description 3
- 102100031988 Tumor necrosis factor ligand superfamily member 6 Human genes 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 230000001594 aberrant effect Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 229960002224 eculizumab Drugs 0.000 description 3
- 210000003162 effector t lymphocyte Anatomy 0.000 description 3
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 108091006086 inhibitor proteins Proteins 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 239000002502 liposome Substances 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 239000000594 mannitol Substances 0.000 description 3
- 235000010355 mannitol Nutrition 0.000 description 3
- 210000004379 membrane Anatomy 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 239000000546 pharmaceutical excipient Substances 0.000 description 3
- 108091033319 polynucleotide Proteins 0.000 description 3
- 102000040430 polynucleotide Human genes 0.000 description 3
- 239000002157 polynucleotide Substances 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
- 238000000746 purification Methods 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- UNFWWIHTNXNPBV-WXKVUWSESA-N spectinomycin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- RUDATBOHQWOJDD-UHFFFAOYSA-N (3beta,5beta,7alpha)-3,7-Dihydroxycholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)CC2 RUDATBOHQWOJDD-UHFFFAOYSA-N 0.000 description 2
- WRMNZCZEMHIOCP-UHFFFAOYSA-N 2-phenylethanol Chemical compound OCCC1=CC=CC=C1 WRMNZCZEMHIOCP-UHFFFAOYSA-N 0.000 description 2
- 102100028187 ATP-binding cassette sub-family C member 6 Human genes 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 102000005427 Asialoglycoprotein Receptor Human genes 0.000 description 2
- 102100026293 Asialoglycoprotein receptor 2 Human genes 0.000 description 2
- 101710200901 Asialoglycoprotein receptor 2 Proteins 0.000 description 2
- 108091008875 B cell receptors Proteins 0.000 description 2
- 102000008096 B7-H1 Antigen Human genes 0.000 description 2
- 101100463133 Caenorhabditis elegans pdl-1 gene Proteins 0.000 description 2
- 241000701022 Cytomegalovirus Species 0.000 description 2
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 102000018651 Epithelial Cell Adhesion Molecule Human genes 0.000 description 2
- 108010066687 Epithelial Cell Adhesion Molecule Proteins 0.000 description 2
- 239000004593 Epoxy Substances 0.000 description 2
- 208000007465 Giant cell arteritis Diseases 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 102100035943 HERV-H LTR-associating protein 2 Human genes 0.000 description 2
- 101000955037 Homo sapiens Homeobox protein MOX-2 Proteins 0.000 description 2
- 101100460850 Homo sapiens NCR3LG1 gene Proteins 0.000 description 2
- 101001117312 Homo sapiens Programmed cell death 1 ligand 2 Proteins 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 208000028622 Immune thrombocytopenia Diseases 0.000 description 2
- 102000017578 LAG3 Human genes 0.000 description 2
- 241000829100 Macaca mulatta polyomavirus 1 Species 0.000 description 2
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 2
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 2
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 2
- 208000003250 Mixed connective tissue disease Diseases 0.000 description 2
- 108010008707 Mucin-1 Proteins 0.000 description 2
- 102000007298 Mucin-1 Human genes 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 description 2
- 108050003738 Neural cell adhesion molecule 1 Proteins 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 102000002508 Peptide Elongation Factors Human genes 0.000 description 2
- 108010068204 Peptide Elongation Factors Proteins 0.000 description 2
- 102100040678 Programmed cell death protein 1 Human genes 0.000 description 2
- 102000012751 Pyruvate Dehydrogenase Complex Human genes 0.000 description 2
- 108010090051 Pyruvate Dehydrogenase Complex Proteins 0.000 description 2
- 108010071390 Serum Albumin Proteins 0.000 description 2
- 102000007562 Serum Albumin Human genes 0.000 description 2
- 108010063829 Solute Carrier Organic Anion Transporter Family Member 1B3 Proteins 0.000 description 2
- 102100027233 Solute carrier organic anion transporter family member 1B1 Human genes 0.000 description 2
- 101710091462 Solute carrier organic anion transporter family member 1B1 Proteins 0.000 description 2
- 102100027239 Solute carrier organic anion transporter family member 1B3 Human genes 0.000 description 2
- 206010072148 Stiff-Person syndrome Diseases 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 108091008874 T cell receptors Proteins 0.000 description 2
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 2
- 208000031981 Thrombocytopenic Idiopathic Purpura Diseases 0.000 description 2
- 206010043561 Thrombocytopenic purpura Diseases 0.000 description 2
- 206010051526 Tolosa-Hunt syndrome Diseases 0.000 description 2
- 102100026143 Transferrin receptor protein 2 Human genes 0.000 description 2
- 101710145355 Transferrin receptor protein 2 Proteins 0.000 description 2
- 108050002568 Tumor necrosis factor ligand superfamily member 6 Proteins 0.000 description 2
- 102000006668 UniProt protein families Human genes 0.000 description 2
- 108020004729 UniProt protein families Proteins 0.000 description 2
- 101710113286 V-type immunoglobulin domain-containing suppressor of T-cell activation Proteins 0.000 description 2
- 239000012491 analyte Substances 0.000 description 2
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 108010006523 asialoglycoprotein receptor Proteins 0.000 description 2
- 210000002469 basement membrane Anatomy 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- RYYVLZVUVIJVGH-UHFFFAOYSA-N caffeine Chemical compound CN1C(=O)N(C)C(=O)C2=C1N=CN2C RYYVLZVUVIJVGH-UHFFFAOYSA-N 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 238000002737 cell proliferation kit Methods 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 230000007882 cirrhosis Effects 0.000 description 2
- 208000019425 cirrhosis of liver Diseases 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000021615 conjugation Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- IJJVMEJXYNJXOJ-UHFFFAOYSA-N fluquinconazole Chemical compound C=1C=C(Cl)C=C(Cl)C=1N1C(=O)C2=CC(F)=CC=C2N=C1N1C=NC=N1 IJJVMEJXYNJXOJ-UHFFFAOYSA-N 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 description 2
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 2
- 102000048362 human PDCD1 Human genes 0.000 description 2
- 230000036737 immune function Effects 0.000 description 2
- 229940072221 immunoglobulins Drugs 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 238000001802 infusion Methods 0.000 description 2
- 238000010212 intracellular staining Methods 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- LXCFILQKKLGQFO-UHFFFAOYSA-N methylparaben Chemical compound COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 2
- 206010063344 microscopic polyangiitis Diseases 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 210000000822 natural killer cell Anatomy 0.000 description 2
- 108010068617 neonatal Fc receptor Proteins 0.000 description 2
- 229940021182 non-steroidal anti-inflammatory drug Drugs 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 229920000747 poly(lactic acid) Polymers 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 2
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 2
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 2
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 2
- 230000012743 protein tagging Effects 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000012064 sodium phosphate buffer Substances 0.000 description 2
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- 210000004988 splenocyte Anatomy 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 206010043207 temporal arteritis Diseases 0.000 description 2
- AYEKOFBPNLCAJY-UHFFFAOYSA-O thiamine pyrophosphate Chemical compound CC1=C(CCOP(O)(=O)OP(O)(O)=O)SC=[N+]1CC1=CN=C(C)N=C1N AYEKOFBPNLCAJY-UHFFFAOYSA-O 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- RUDATBOHQWOJDD-UZVSRGJWSA-N ursodeoxycholic acid Chemical compound C([C@H]1C[C@@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 RUDATBOHQWOJDD-UZVSRGJWSA-N 0.000 description 2
- 229960001661 ursodiol Drugs 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- JNYAEWCLZODPBN-JGWLITMVSA-N (2r,3r,4s)-2-[(1r)-1,2-dihydroxyethyl]oxolane-3,4-diol Chemical class OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O JNYAEWCLZODPBN-JGWLITMVSA-N 0.000 description 1
- XMQUEQJCYRFIQS-YFKPBYRVSA-N (2s)-2-amino-5-ethoxy-5-oxopentanoic acid Chemical compound CCOC(=O)CC[C@H](N)C(O)=O XMQUEQJCYRFIQS-YFKPBYRVSA-N 0.000 description 1
- HSINOMROUCMIEA-FGVHQWLLSA-N (2s,4r)-4-[(3r,5s,6r,7r,8s,9s,10s,13r,14s,17r)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2-methylpentanoic acid Chemical compound C([C@@]12C)C[C@@H](O)C[C@H]1[C@@H](CC)[C@@H](O)[C@@H]1[C@@H]2CC[C@]2(C)[C@@H]([C@H](C)C[C@H](C)C(O)=O)CC[C@H]21 HSINOMROUCMIEA-FGVHQWLLSA-N 0.000 description 1
- WHBMMWSBFZVSSR-GSVOUGTGSA-N (R)-3-hydroxybutyric acid Chemical compound C[C@@H](O)CC(O)=O WHBMMWSBFZVSSR-GSVOUGTGSA-N 0.000 description 1
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 101710150021 ATP-binding cassette sub-family C member 6 Proteins 0.000 description 1
- QCHQVPPBLFSODM-MZWCVTMLSA-N Ac-Asp-Lys-Ala-Thr-Ile-Gly-Phe-Glu-Val-Gln-Glu-Glu Chemical compound OC(=O)C[C@H](NC(C)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O)CC1=CC=CC=C1 QCHQVPPBLFSODM-MZWCVTMLSA-N 0.000 description 1
- 206010002556 Ankylosing Spondylitis Diseases 0.000 description 1
- 208000003343 Antiphospholipid Syndrome Diseases 0.000 description 1
- 206010003827 Autoimmune hepatitis Diseases 0.000 description 1
- 206010069002 Autoimmune pancreatitis Diseases 0.000 description 1
- 208000031212 Autoimmune polyendocrinopathy Diseases 0.000 description 1
- 208000022106 Autoimmune polyendocrinopathy type 2 Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000023328 Basedow disease Diseases 0.000 description 1
- 208000009137 Behcet syndrome Diseases 0.000 description 1
- 239000005711 Benzoic acid Substances 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 108700031361 Brachyury Proteins 0.000 description 1
- 108091058539 C10orf54 Proteins 0.000 description 1
- 108010084313 CD58 Antigens Proteins 0.000 description 1
- 229940045513 CTLA4 antagonist Drugs 0.000 description 1
- 102000000905 Cadherin Human genes 0.000 description 1
- 108050007957 Cadherin Proteins 0.000 description 1
- GHXZTYHSJHQHIJ-UHFFFAOYSA-N Chlorhexidine Chemical compound C=1C=C(Cl)C=CC=1NC(N)=NC(N)=NCCCCCCN=C(N)N=C(N)NC1=CC=C(Cl)C=C1 GHXZTYHSJHQHIJ-UHFFFAOYSA-N 0.000 description 1
- 206010008635 Cholestasis Diseases 0.000 description 1
- 102000012422 Collagen Type I Human genes 0.000 description 1
- 108010022452 Collagen Type I Proteins 0.000 description 1
- 102000000503 Collagen Type II Human genes 0.000 description 1
- 108010041390 Collagen Type II Proteins 0.000 description 1
- 102000001187 Collagen Type III Human genes 0.000 description 1
- 108010069502 Collagen Type III Proteins 0.000 description 1
- 102000004266 Collagen Type IV Human genes 0.000 description 1
- 108010042086 Collagen Type IV Proteins 0.000 description 1
- 108010078018 Complement C3d Proteins 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- 208000011231 Crohn disease Diseases 0.000 description 1
- 229920000858 Cyclodextrin Polymers 0.000 description 1
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 1
- 102000012192 Cystatin C Human genes 0.000 description 1
- 108010061642 Cystatin C Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 102000010170 Death domains Human genes 0.000 description 1
- 108050001718 Death domains Proteins 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 101710205862 Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial Proteins 0.000 description 1
- 102100027152 Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial Human genes 0.000 description 1
- 208000021866 Dressler syndrome Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 239000001116 FEMA 4028 Substances 0.000 description 1
- 108010087819 Fc receptors Proteins 0.000 description 1
- 102000009109 Fc receptors Human genes 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 206010018364 Glomerulonephritis Diseases 0.000 description 1
- 102000008214 Glutamate decarboxylase Human genes 0.000 description 1
- 108091022930 Glutamate decarboxylase Proteins 0.000 description 1
- 208000024869 Goodpasture syndrome Diseases 0.000 description 1
- 208000015023 Graves' disease Diseases 0.000 description 1
- 208000035895 Guillain-Barré syndrome Diseases 0.000 description 1
- 101710203114 HERV-H LTR-associating protein 2 Proteins 0.000 description 1
- 206010019663 Hepatic failure Diseases 0.000 description 1
- 101000986621 Homo sapiens ATP-binding cassette sub-family C member 6 Proteins 0.000 description 1
- 101001021491 Homo sapiens HERV-H LTR-associating protein 2 Proteins 0.000 description 1
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 1
- 101000611183 Homo sapiens Tumor necrosis factor Proteins 0.000 description 1
- 101000638161 Homo sapiens Tumor necrosis factor ligand superfamily member 6 Proteins 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 201000009794 Idiopathic Pulmonary Fibrosis Diseases 0.000 description 1
- 208000010159 IgA glomerulonephritis Diseases 0.000 description 1
- 206010021263 IgA nephropathy Diseases 0.000 description 1
- 208000021330 IgG4-related disease Diseases 0.000 description 1
- 229940076838 Immune checkpoint inhibitor Drugs 0.000 description 1
- 208000031781 Immunoglobulin G4 related sclerosing disease Diseases 0.000 description 1
- 208000004187 Immunoglobulin G4-Related Disease Diseases 0.000 description 1
- 102000037984 Inhibitory immune checkpoint proteins Human genes 0.000 description 1
- 108091008026 Inhibitory immune checkpoint proteins Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- LPHGQDQBBGAPDZ-UHFFFAOYSA-N Isocaffeine Natural products CN1C(=O)N(C)C(=O)C2=C1N(C)C=N2 LPHGQDQBBGAPDZ-UHFFFAOYSA-N 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- 241000283953 Lagomorpha Species 0.000 description 1
- 102100030984 Lymphocyte function-associated antigen 3 Human genes 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 206010049567 Miller Fisher syndrome Diseases 0.000 description 1
- 101001043827 Mus musculus Interleukin-2 Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 229920000954 Polyglycolide Polymers 0.000 description 1
- 206010036105 Polyneuropathy Diseases 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 108010071690 Prealbumin Proteins 0.000 description 1
- 102000007584 Prealbumin Human genes 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 101100327358 Rattus norvegicus Cdk12 gene Proteins 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 108091006207 SLC-Transporter Proteins 0.000 description 1
- 102000037054 SLC-Transporter Human genes 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 206010039705 Scleritis Diseases 0.000 description 1
- 206010039710 Scleroderma Diseases 0.000 description 1
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 1
- 208000021386 Sjogren Syndrome Diseases 0.000 description 1
- DWAQJAXMDSEUJJ-UHFFFAOYSA-M Sodium bisulfite Chemical compound [Na+].OS([O-])=O DWAQJAXMDSEUJJ-UHFFFAOYSA-M 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 210000000662 T-lymphocyte subset Anatomy 0.000 description 1
- 208000001106 Takayasu Arteritis Diseases 0.000 description 1
- 206010071574 Testicular autoimmunity Diseases 0.000 description 1
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 1
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 1
- 229940127174 UCHT1 Drugs 0.000 description 1
- 206010046865 Vaccinia virus infection Diseases 0.000 description 1
- 206010047115 Vasculitis Diseases 0.000 description 1
- 229960003697 abatacept Drugs 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 229910052783 alkali metal Inorganic materials 0.000 description 1
- 150000001340 alkali metals Chemical class 0.000 description 1
- 206010002022 amyloidosis Diseases 0.000 description 1
- 230000002529 anti-mitochondrial effect Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000013011 aqueous formulation Substances 0.000 description 1
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 230000006470 autoimmune attack Effects 0.000 description 1
- 208000006424 autoimmune oophoritis Diseases 0.000 description 1
- 201000009780 autoimmune polyendocrine syndrome type 2 Diseases 0.000 description 1
- 206010071578 autoimmune retinopathy Diseases 0.000 description 1
- LMEKQMALGUDUQG-UHFFFAOYSA-N azathioprine Chemical compound CN1C=NC([N+]([O-])=O)=C1SC1=NC=NC2=C1NC=N2 LMEKQMALGUDUQG-UHFFFAOYSA-N 0.000 description 1
- 229960002170 azathioprine Drugs 0.000 description 1
- 229960000686 benzalkonium chloride Drugs 0.000 description 1
- 235000010233 benzoic acid Nutrition 0.000 description 1
- 229960004365 benzoic acid Drugs 0.000 description 1
- CADWTSSKOVRVJC-UHFFFAOYSA-N benzyl(dimethyl)azanium;chloride Chemical compound [Cl-].C[NH+](C)CC1=CC=CC=C1 CADWTSSKOVRVJC-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 description 1
- 235000011175 beta-cyclodextrine Nutrition 0.000 description 1
- 229960004853 betadex Drugs 0.000 description 1
- 239000003613 bile acid Substances 0.000 description 1
- 210000000013 bile duct Anatomy 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 239000004067 bulking agent Substances 0.000 description 1
- DQXBYHZEEUGOBF-UHFFFAOYSA-N but-3-enoic acid;ethene Chemical compound C=C.OC(=O)CC=C DQXBYHZEEUGOBF-UHFFFAOYSA-N 0.000 description 1
- 229960001948 caffeine Drugs 0.000 description 1
- VJEONQKOZGKCAK-UHFFFAOYSA-N caffeine Natural products CN1C(=O)N(C)C(=O)C2=C1C=CN2C VJEONQKOZGKCAK-UHFFFAOYSA-N 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 229960003260 chlorhexidine Drugs 0.000 description 1
- 230000007870 cholestasis Effects 0.000 description 1
- 231100000359 cholestasis Toxicity 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 150000001860 citric acid derivatives Chemical class 0.000 description 1
- 229940096422 collagen type i Drugs 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 238000012875 competitive assay Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000008139 complexing agent Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000013270 controlled release Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 239000003246 corticosteroid Substances 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 238000013211 curve analysis Methods 0.000 description 1
- 229960004397 cyclophosphamide Drugs 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 239000003405 delayed action preparation Substances 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- VLYUGYAKYZETRF-UHFFFAOYSA-N dihydrolipoamide Chemical compound NC(=O)CCCCC(S)CCS VLYUGYAKYZETRF-UHFFFAOYSA-N 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940000406 drug candidate Drugs 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 208000030172 endocrine system disease Diseases 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 210000003979 eosinophil Anatomy 0.000 description 1
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 1
- 239000005038 ethylene vinyl acetate Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 229940093915 gynecological organic acid Drugs 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 102000048770 human CD276 Human genes 0.000 description 1
- 102000048679 human NCR3LG1 Human genes 0.000 description 1
- 102000057041 human TNF Human genes 0.000 description 1
- 102000057058 human VSIR Human genes 0.000 description 1
- 102000055298 human VTCN1 Human genes 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 239000000017 hydrogel Substances 0.000 description 1
- 229960002163 hydrogen peroxide Drugs 0.000 description 1
- 229920001477 hydrophilic polymer Polymers 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 230000005931 immune cell recruitment Effects 0.000 description 1
- 230000006058 immune tolerance Effects 0.000 description 1
- 239000012274 immune-checkpoint protein inhibitor Substances 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 229940088592 immunologic factor Drugs 0.000 description 1
- 239000000367 immunologic factor Substances 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 230000001861 immunosuppressant effect Effects 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 108091008042 inhibitory receptors Proteins 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 229940047124 interferons Drugs 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 238000000185 intracerebroventricular administration Methods 0.000 description 1
- 210000003228 intrahepatic bile duct Anatomy 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 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 1
- 239000000644 isotonic solution Substances 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 229940067606 lecithin Drugs 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
- 230000000670 limiting effect Effects 0.000 description 1
- 150000002632 lipids Chemical group 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 231100000835 liver failure Toxicity 0.000 description 1
- 208000007903 liver failure Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 206010025135 lupus erythematosus Diseases 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 description 1
- 239000004292 methyl p-hydroxybenzoate Substances 0.000 description 1
- 229960002216 methylparaben Drugs 0.000 description 1
- 239000003094 microcapsule Substances 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 201000008383 nephritis Diseases 0.000 description 1
- 208000008795 neuromyelitis optica Diseases 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000002616 plasmapheresis Methods 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229920001983 poloxamer Polymers 0.000 description 1
- 229920001200 poly(ethylene-vinyl acetate) Polymers 0.000 description 1
- 201000006292 polyarteritis nodosa Diseases 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 239000004633 polyglycolic acid Substances 0.000 description 1
- 229920002338 polyhydroxyethylmethacrylate Polymers 0.000 description 1
- 239000004626 polylactic acid Substances 0.000 description 1
- 230000007824 polyneuropathy Effects 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000244 polyoxyethylene sorbitan monooleate Substances 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 210000004896 polypeptide structure Anatomy 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229940068977 polysorbate 20 Drugs 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 229940068968 polysorbate 80 Drugs 0.000 description 1
- 229940068965 polysorbates Drugs 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 235000010232 propyl p-hydroxybenzoate Nutrition 0.000 description 1
- 239000004405 propyl p-hydroxybenzoate Substances 0.000 description 1
- 229960003415 propylparaben Drugs 0.000 description 1
- 239000001990 protein-drug conjugate Substances 0.000 description 1
- 208000005069 pulmonary fibrosis Diseases 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 229960004889 salicylic acid Drugs 0.000 description 1
- 238000013391 scatchard analysis Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229940079827 sodium hydrogen sulfite Drugs 0.000 description 1
- 235000010267 sodium hydrogen sulphite Nutrition 0.000 description 1
- 229940001482 sodium sulfite Drugs 0.000 description 1
- 235000010265 sodium sulphite Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000003153 stable transfection Methods 0.000 description 1
- 238000011146 sterile filtration Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 150000005846 sugar alcohols Chemical class 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000375 suspending agent 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
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 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 1
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 1
- 229940033663 thimerosal Drugs 0.000 description 1
- 238000001196 time-of-flight mass spectrum Methods 0.000 description 1
- 239000012443 tonicity enhancing agent Substances 0.000 description 1
- 231100000048 toxicity data Toxicity 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 238000003146 transient transfection Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- ODLHGICHYURWBS-LKONHMLTSA-N trappsol cyclo Chemical compound CC(O)COC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](COCC(C)O)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)COCC(O)C)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1COCC(C)O ODLHGICHYURWBS-LKONHMLTSA-N 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 229960000281 trometamol Drugs 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 208000007089 vaccinia Diseases 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
-
- 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
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70532—B7 molecules, e.g. CD80, CD86
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
-
- 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
- C07K2317/71—Decreased effector function due to an Fc-modification
-
- 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
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
-
- 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
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
Definitions
- the disclosure relates to antibody immune cell inhibitor fusion proteins comprising four polypeptide chains that form two antigen binding sites and at least two immune cell receptor binding sites that inhibit or diminish activation of an immune effector cell when bound to a target antigen.
- the disclosure also relates to antibody immune cell inhibitor fusion proteins comprising two polypeptide chains that form one antigen binding site and at least one immune cell receptor binding site that inhibit or diminish activation of an immune effector cell when bound to a target antigen.
- the disclosure further relates to pharmaceutical compositions and kits that comprise such antibody immune cell inhibitor fusion proteins, and methods of treatment using such proteins.
- Autoimmune disorders are diseases caused by a dysfunction of the immune system, wherein the body produces an inappropriate immune response against its own tissues.
- the immune system creates B and T lymphocytes, autoantibodies, monocytes, NK cells, antigen presenting cells, and other immune factors that attack or facilitate immune responses to an individual's own cells, tissues, and/or organs.
- autoimmune diseases are among the most prevalent diseases in the United States.
- Some of the current treatments for autoimmune diseases include administration of corticosteroid drugs, non-steroidal anti-inflammatory drugs (NSAIDs), or more powerful immunosuppressant drugs such as cyclophosphamide, methotrexate, and azathioprine that suppress the immune response and stop the progression of the disease.
- NSAIDs non-steroidal anti-inflammatory drugs
- azathioprine that suppress the immune response and stop the progression of the disease.
- Radiation of the lymph nodes and plasmapheresis are other ways of treating an autoimmune disease. However, these treatments often have devastating long-term side effects.
- PBC Primary Biliary Cholangitis
- Ursodeoxycholic acid is the only FDA approved treatment for PBC, which ameliorates symptoms of the disease by primarily sequestering bile acid. Once symptoms manifest in an individual, life expectancy is less than 10 years, unless a liver transplant becomes available.
- AMAs anti-mitochondrial antibodies
- Most AMAs are reported to react with dihydrolipoamide 5-acetyltransferase (DLAT), the E2 subunit of the mitochondrial pyruvate dehydrogenase complex.
- DLAT dihydrolipoamide 5-acetyltransferase
- Aberrant expression of DLAT or mimic antigens on biliary epithelial cells provide the target for an autoimmune attack in PBC.
- the disclosure provides an antibody immune cell inhibitor fusion protein comprising four polypeptide chains that form two antigen binding sites and at least two immune cell receptor binding sites; wherein two polypeptide chains have a structure represented by the formula II 1 -V L -C L -II 2 , and two polypeptide chains have a structure represented by the formula II 3 -V H -C H1 -Fc-II 4 ; wherein:
- the disclosure also provides an antibody immune cell inhibitor fusion protein comprising four polypeptide chains that form two antigen binding sites and at least two immune cell receptor binding sites; wherein two polypeptide chains have a structure represented by the formula II 1 -L 1 -V L -C L -L 2 -II 2 , and two polypeptide chains have a structure represented by the formula II 3 -L 3 -V H -C H1 -Fc-L 4 -II 4 ; wherein:
- the disclosure further provides an antibody immune cell inhibitor fusion protein comprising two polypeptide chains that form one antigen binding site and at least one immune cell receptor binding site; wherein one polypeptide chain has a structure represented by the formula II 1 -V L -C L -II 2 , and one polypeptide chain has a structure represented by the formula II 3 -V H -C H1 -II 4 ; wherein:
- the disclosure further provides an antibody immune cell inhibitor fusion protein comprising two polypeptide chains that form one antigen binding site and at least one immune cell receptor binding site; wherein one polypeptide chain has a structure represented by the formula II 1 -L 1 -V L -C L -L 2 -II 2 , and one polypeptide chain has a structure represented by the formula II 3 -L 3 -V H -C H1 -L 4 -II 4 ; wherein:
- FIGS. 1 A- 1 G show bio-layer interferometry results of PD-1 binding to PD-L1 fusion proteins.
- FIG. 1 A illustrates that TPP-993 (PD2 Fab having no PD-L1 domain and no linker) did not bind to PD-1 Fc because TPP-993 lacks a PD-L1 domain.
- FIGS. 1 B and 1D illustrate that TPP-994 (PD2 with PD-L1 fused to the C-terminus of the heavy chain) and TPP-999 (PD2 with PD-L1 fused to the C-terminus of the light chain) exhibited very little binding to PD-1 Fc.
- FIGS. 1 A illustrates that TPP-993 (PD2 Fab having no PD-L1 domain and no linker) did not bind to PD-1 Fc because TPP-993 lacks a PD-L1 domain.
- FIGS. 1 B and 1D illustrate that TPP-994 (PD2 with PD-L1 fused to the C
- FIG. 1 C and 1 E illustrate that TPP-995 (PD2 with PD-L1 fused to the N-terminus of the heavy chain) and TPP-1001 (PD2 with PD-L1 fused to the N-terminus of the light chain) both exhibited binding to PD-1 Fc.
- FIG. 1 F illustrates that PD-L1 Fc (a dimeric protein having two PD-L1 proteins fused to a human Fc) bound PD-1 very strongly.
- FIG. 1 G illustrates that PD-1 Fc did not bind to itself, demonstrating that the Fc portion of PD-L1 Fc is not interacting with the tips.
- FIGS. 2 A- 2 C show bio-layer interferometry results of mouse IgG1 chimera anti-DLAT antibodies to DLAT antigen.
- FIG. 2 A illustrates slow on-rates and fast off-rates for the TPP-1003 PD2 antibody.
- FIGS. 2 B and 2 C illustrate slow off-rates for the TPP-1004 and TPP-1005 anti-DLAT antibodies.
- FIGS. 3 A- 3 E show bio-layer interferometry results of simultaneous binding of human IgG2/4 anti-DLAT antibody PD-L1 V-like domain fusions to DLAT antigen and PD-1 Fc.
- FIG. 3 A illustrates that TPP-985 (human IgG2/4 PD5 antibody) bound to the DLAT antigen, but did not bind to PD-1 Fc.
- FIGS. 3 B and 3 D illustrate that TPP-986 and TPP-992 (fusions of the PD-L1 V-like domain to the N-terminus of anti-DLAT antibodies) both exhibited the ability to simultaneously bind the DLAT antigen and PD-1 Fc.
- FIG. 3 A illustrates that TPP-985 (human IgG2/4 PD5 antibody) bound to the DLAT antigen, but did not bind to PD-1 Fc.
- FIGS. 3 B and 3 D illustrate that TPP-986 and TPP-992 (fusions of the PD-L1 V-like domain
- FIG. 3 C illustrates that TPP-990 (PD-L1 V-like domain fused to the C-terminus of an anti-DLAT antibody) bound the DLAT antigen, but did not bind PD-1 Fc.
- FIG. 3 E illustrates that the PD-L1 Fc control bound to PD-1 Fc, but did not bind to the DLAT antigen.
- FIGS. 4 A- 4 F show human T-cell activation for targeted PD-L1 fusion inhibition. Representative histograms of one of three replicates are shown for each sample at 260 nM and 65 nM concentrations of antibody fusions or non-targeted PD-L1 Fc.
- FIG. 4 A illustrates that NC beads exhibited no activation of T-cells, whereas the DLAT-aCD3 beads demonstrated clear activation of the pan T-cells.
- FIG. 4 B illustrates that TPP-985 (an anti-DLAT IgG2-4 antibody with no PD-L1 variable-like domain) exhibited no significant inhibition of T-cell activation.
- FIG. 4 C illustrates that TPP-986 (an anti-DLAT antibody with PD-L1 fused at the N-terminus of the heavy chain) exhibited significant inhibition of T-cell activation at both the 65 nM and 260 nM concentrations.
- FIG. 4 D illustrates that TPP-992 (an anti-DLAT antibody with PD-L1 fused at the N-terminus of the light chain) exhibited the most significant inhibition of T-cell activation, with complete inhibition at the 260 nM concentration.
- FIG. 4 E illustrates that the non-targeting PD-L1 Fc chimera exhibited no significant inhibition of T-cell activation.
- FIG. 4 F illustrates that the titration of TPP-992 demonstrated a dose-dependent inhibition of T-cell activation with an EC50 of 17 nM ⁇ 4.5nM.
- FIGS. 5 A- 5 C show that targeting by the PD-L1 fusion is necessary for inhibition of T-cell activation.
- FIG. 5 B illustrates that there was no significant inhibition for any of the PD-L1 anti-DLAT fusions using TA2 beads that lack the DLAT antigen.
- FIG. 5 C illustrates that TA2 bead activation showed no significant difference between samples.
- FIGS. 6 A- 6 D show the statistical analysis of the human T-cell activation results.
- FIG. 6 B illustrates the statistical analysis summary of all samples in the T-cell activation assay.
- FIG. 6 C illustrates that at the 65 nM concentration, a lower level of T-cell activation inhibition was observed for all samples.
- TPP-992 demonstrated the most significant inhibition of T-cell activation.
- FIG. 6 D illustrates the statistical analysis summary of T-cell activation inhibition for anti-DLAT antibody PD-L1 fusion proteins at the 65 nM concentration.
- FIGS. 7 A- 7 B illustrate a schematic for T-cell activation and inhibition assays using anti-DLAT antibody PD-L1 fusion proteins.
- FIG. 7 A is a table listing the samples used in the T-cell activation and inhibition assays.
- FIG. 7 B illustrates exemplary antibody immune cell inhibitor fusion proteins or fragments thereof.
- FIG. 8 is a schematic illustrating DLAT-aCD3 beads, T-cell activation beads (TA2 beads), and negative control (NC) beads used in human T-cell activation assays.
- DLAT-aCD3 beads were prepared by conjugating anti-human CD3 antibody and human DLAT recombinant protein
- TA2 beads were prepared by conjugating anti-human CD3 antibody-coated beads with human IgG Fc
- NC beads were prepared by conjugating human IgG Fc with uncoated beads to prepare a no-activation control reagent.
- FIG. 9 depicts the binding affinity of TPP-992 (PD5 with PD-L1 V-like domain fused to the N-terminus of the light chain).
- TPP-992 had an affinity of 184 pM as determined using a single curve analysis by BIAcore.
- FIG. 10 depicts an electrospray ionization—time of flight mass spectra for TPP-985 and TPP-992.
- FIGS. 11 A- 11 D illustrate the BIAcore kinetics results for TPP-1003, TPP-1004, and TPP-1005.
- FIG. 11 A illustrates that TPP-1003 and TPP-1004 had pico-molar binding to the DLAT antigen and TPP-1005 had a single digit nano-molar affinity to the DLAT antigen.
- FIGS. 11 B, 11 C and 11 D illustrate the BIAcore kinetics for TPP-1003, TPP-1004, and TPP 1005, respectively.
- FIGS. 12 A- 12 F show mouse T-cell activation for targeted PD-L1 fusion inhibition. Samples were gated to exclude the low Forward Scatter (FSC) and the FL-4+ (SYTOXTM Red) dead cells. The same gate was utilized to analyze all samples. The histograms shown are gated on the live mouse T-cells. The decrease in signal of the FL-1 mean fluorescence measurement represents the activated T-cells that have expanded new generations of T-cells, with decreasing dye concentration in each respective generation.
- FIG. 12 A illustrates complete inhibition of mouse T-cell activation for TPP-1986 at 500 nM with an incomplete inhibition observed for 100 nM.
- FIG. 12 A illustrates complete inhibition of mouse T-cell activation for TPP-1986 at 500 nM with an incomplete inhibition observed for 100 nM.
- FIG. 12 B illustrates that TPP-1694 had a significant difference compared to the NC beads, but showed no significant difference compared to TPP-1986 at 500 nM. A significant difference was observed at 100 nM for human TPP-1986 and TPP-992 versus mouse PD-L1-TPP-1694.
- FIG. 12 C illustrates that TPP-1695 demonstrated a lower potency compared to the PDS human PD-L1 fusion variants at 500 nM and 100 nM concentrations with similar results observed for TPP-1694.
- FIG. 12 D illustrates complete inhibition of mouse T-cell activation for TPP-992 at 500 nM with an incomplete inhibition for 100 nM.
- TPP-1697 non-targeted isotype control with human PD-L1
- TPP-1004 murine IgG1 PDS with no PD-L1 variable-like domain present
- FIGS. 13 A and 13 B show the statistical analysis of the mouse T-cell activation results at 500 nM.
- FIG. 13 A illustrates complete inhibition of TPP-1986 and TPP-992 fusions containing human PD-L1 demonstrated by a not significant difference compared to the NC beads.
- An expected decrease in the geometric mean fluorescence intensity (Geo MFI) of FL-1 representing the CFSE dye diffusion into progeny cells for the 1X beads, TPP-1697, and TPP-1004 was observed, illustrating activation of the murine T-cells with no significant differences between samples.
- FIG. 13 B is a table of statistical analysis between samples at 500 nM concentrations using Tukey's multiple comparisons test.
- FIGS. 14 A and 14 B show the statistical analysis of the mouse T-cell activation results at 500 nM.
- FIG. 14 A illustrates complete inhibition of TPP-1986 and TPP-992 fusions containing human PD-L1 demonstrated by a not significant difference compared to the NC beads and incomplete inhibition of TPP-1504 containing hB7-H4.
- An expected decrease in the geometric mean fluorescence intensity (Geo MFI) of FL-1 representing the CFSE dye diffusion into progeny cells for the 1X beads, TPP-1697 and TPP-1505 was observed, illustrating activation of the murine T-cells.
- FIG. 14 B is a table of statistical analysis between samples at 500 nM concentrations using Tukey's multiple comparisons test.
- FIGS. 15 A and 15 B show the statistical analysis of the mouse T-cell activation results at 100 nM.
- FIG. 15 A illustrates incomplete inhibition using fusions containing human PD-L1 compared to the NC beads. A significant difference of human versus murine PD-L1 containing fusions was observed, suggesting human PD-L1 results in greater inhibition of mouse T-cells than mouse PD-L1 V-like domain. An expected decrease in the geometric mean fluorescence intensity (Geo MFI) of FL-1 representing the CFSE dye diffusion into progeny cells for the 1X beads, TPP-1697, and TPP-1004 was observed, illustrating activation of the murine T-cells with no significant differences between samples.
- FIG. 15 B is a table of statistical analysis between samples at 100 nM concentrations using Tukey's multiple comparisons test.
- FIGS. 16 A and 16 B show the statistical analysis of the mouse T-cell activation results at 100 nM.
- FIG. 16 A illustrates incomplete inhibition using fusions containing human PD-L1 and hB7-H4 as compared to the NC beads.
- An expected decrease in the geometric mean fluorescence intensity (Geo MFI) of FL-1 representing the CFSE dye diffusion into progeny cells for the 1X beads, TPP-1697 and TPP 1505 was observed illustrating activation of the murine T-cells.
- FIG. 16 B is a table of statistical analysis between samples at 100 nM concentrations using Tukey's multiple comparisons test.
- FIG. 17 illustrates that mouse PD-L1(TPP-1964) binds to human PD-1 with a faster on-rate than human PD-L1.
- PD-1 Fc R&D Systems
- msPDL1 and TPP-1986 huPDL1 bound PD-1 Fc.
- B1 sensor The increased slope observed for TPP-1964 (B1 sensor) suggests the mouse PD-L1 has a faster on-rate than the human PD-L1 on the TPP-1986 fusion (G1 Fusion). This binding steps followed by the dissociation in PBS buffer.
- FIGS. 18 A- 18 D show the statistical analysis of the human T-cell activation results.
- FIG. 18 A illustrates that at the 500 nM concentration of the anti-DLAT PD-L1 (TPP-1986), B7H4 (TPP-1504), HVEM (TPP-1506), B7H6 (TPP-1894) and CTLA4 (TPP-1898) fusions tested at day 7 inhibited T-cell activation.
- FIG. 18 B illustrates the statistical analysis summary of all samples in the T-cell activation assay.
- FIG. 18 A illustrates that at the 500 nM concentration of the anti-DLAT PD-L1 (TPP-1986), B7H4 (TPP-1504), HVEM (TPP-1506), B7H6 (TPP-1894) and CTLA4 (TPP-1898) fusions tested at day 7 inhibited T-cell activation.
- FIG. 18 B illustrates the statistical analysis summary of all samples in the T-cell activation assay.
- FIG. 18 C illustrates that at the 500 nM concentration of the anti-DLAT fusions only PDL-2 (TPP-2215), CD200 (TPP-2216), TIM-3 (TPP-2220) and PD-L1 (TTP-992) showed inhibition of T cell activation at day 4.
- FIG. 18 D illustrates the statistical analysis summary of T-cell activation inhibition for anti-DLAT antibody fusion proteins.
- FIGS. 19 A and 19 B illustrates human T-cell activation at 4 days for 500 nM concentration of PD-L1 Fab fusions.
- FIG. 19 B illustrates the statistical analysis summary of T-cell activation inhibition for PD-L1 fab fusions.
- V V-like domain of PDL1;
- V ⁇ C V- +C-like domains of PDL1;
- HC PDL1 fused to N-terminus of Heavy Chain and
- LC PDL1 fused to N-terminus of Light Chain.
- FIGS. 20 A- 20 D show the statistical analysis of the mouse CD4 + T-cell activation results.
- FIG. 20 A illustrates that at 500 nM concentration of the anti-DLAT PD-L1 (TPP-1984), PD-5 (TPP-1694), and CTLA4 (TPP-1898) fusions tested at day 7 inhibited T-cell activation.
- FIG. 20 B illustrates the statistical analysis summary of all samples in the T-cell activation assay.
- FIG. 20 C illustrates that at the 125 nM a lower level of T-cell activation inhibition was observed with TPP-1898 showing the highest level of inhibition.
- FIG. 20 D illustrates the statistical analysis summary of T-cell activation inhibition for anti-DLAT antibody fusion proteins.
- FIGS. 21 A and 21 B demonstrate the induction of regulatory T-cells (Tregs—CD4+, CD25+, FoxP3+) with the PD-L1 fusions anti-DLAT_PDL1 (labeled DLAT DRUG) and anti-VAP-1_PDL1 (labeled VAP-1 DRUG) combined with the respective activation beads.
- FIG. 21 A illustrate flow cytometry results demonstrating low levels of CD25 for the no activation control (labeled NO BEADS) with 1.71% in quadrant 2 (Q2) representing the CD4+, CD25+ population.
- Q2 quadrant 2
- the histogram shown below the dot plot represents the intracellular staining with FoxP3 of the Q2 gated events.
- FIG. 21 B illustrates inhibition of the Teff population shown as 7% in Q2 with the addition of TPP-992 (labeled DLAT DRUG) to the DLAT activation beads, compared to 32% for DLAT beads only.
- TPP-992 labeled DLAT DRUG
- the FoxP3 positive events went from 7% to 16% with the drug.
- Anti-VAP-1_PDL1 previously showed weaker inhibition in the T-cell activation assay using proliferation dye compared to the anti-DLAT_PDL1 (data not shown). This intermediate effect of anti-VAP-1 fusion was confirmed by the results of this assay; a higher percentage of Q2 events were observed (18%), with a lower induction of Tregs 12% compared to anti-DLAT_PDL1.
- FIGS. 22 A- 22 C illustrate histograms showing itration from 250 nM to 50 nM of anti-CD80_mPDL1 fusion incubated with CD80 activation beads for 4 days with human T-cells. The results demonstrate complete inhibition at 250 nM, partial inhibition at 100 nM and no significant inhibition at 50 nM.
- FIG. 22 D shows the dilution of proliferation dye (CFSE) into the progeny T-cells observed by the multiple peaks on the histograms which indicate that the controls including no activation (labeled No Beads) had no activation and the activation control with the CD80 beads had significant activation.
- FIG. 22 E is the graphical overview of the described results from above, the geometric mean of the CFSE is plotted vs the samples incubated.
- FIG. 22 F is the statistical analysis of the results using Dunnett's multiple comparisons test.
- FIGS. 23 A- 23 C demonstrate the titration of anti-CD20_mPDL1 fusion with CD20 activation beads from 250 nM to 50 nM incubated for 4 days with human T-cells. The results demonstrate complete inhibition at 250 nM, partial inhibition at 100 nM, and less inhibition at 50 nM.
- FIG. 23 D illustrates the dilution of proliferation dye (CFSE) into the progeny T-cells observed by the multiple peaks indicating that activation of the T-cells by the CD20 beads occurred.
- FIG. 23 E represents the graphical overview of the geometric mean of the proliferation dye CFSE.
- FIG. 23 F is the statistical analysis of the described data demonstrating that all of the concentrations of the anti-CD20 mPDL1 were statistically significantly different from the CD20 beads showing inhitibion of T-cell activation.
- the disclosure provides antibody immune cell inhibitor fusion proteins comprising four polypeptide chains that form two antigen binding sites and at least two immune cell receptor binding sites and that inhibit or diminish activation of an immune effector cell when bound to a target antigen.
- the disclosure also provides antibody immune cell inhibitor fusion proteins comprising two polypeptide chains that form one antigen binding site and at least one immune cell receptor binding site that inhibit or diminish activation of an immune effector cell when bound to a target antigen.
- the disclosure further provides pharmaceutical compositions and kits that comprise such antibody immune cell inhibitor fusion proteins, and methods of treatment using such proteins.
- Standard recombinant DNA methodologies are used to construct the polynucleotides that encode the polypeptides that form the antibody immune cell inhibitor fusion proteins of the disclosure, incorporate these polynucleotides into recombinant expression vectors, and introduce such vectors into host cells. See e.g., Green and Sambrook, 2012, M OLECULAR C LONING : A L ABORATORY M ANUAL (Cold Spring Harbor Laboratory Press, 4th ed.). Enzymatic reactions and purification techniques may be performed according to manufacturer's protocols, as commonly accomplished in the art, or as described herein.
- antibody immune cell inhibitor fusion protein refers to a non-naturally occurring (or recombinant) molecule which comprises four polypeptide chains that form two antigen binding sites and at least two immune cell receptor binding sites; wherein two polypeptide chains have a structure represented by the formula II 1 -V L -C L -II 2 , and two polypeptide chains have a structure represented by the formula II 3 -V H -C H1 -Fc-II 4 , wherein:
- antibody immune cell inhibitor fusion protein as used herein also refers to a non-naturally occurring (or recombinant) molecule which comprises four polypeptide chains that form two antigen binding sites and at least two immune cell receptor binding sites; wherein two polypeptide chains have a structure represented by the formula II 1 -L 1 -V L -C L -L 2 -II 2 , and two polypeptide chains have a structure represented by the formula II 3 -L 3 -V H -C H1 -Fc-L 4 -II 4 ; wherein:
- antibody immune cell inhibitor fusion protein as used herein also refers to a non-naturally occurring (or recombinant) molecule which comprises two polypeptide chains that form one antigen binding site and at least one immune cell receptor binding site; wherein one polypeptide chain has a structure represented by the formula II 1 -V L -C L -II 2 , and one polypeptide chain has a structure represented by the formula II 3 -V H -C H1 -II 4 , wherein:
- antibody immune cell inhibitor fusion protein as used herein also refers to a non-naturally occurring (or recombinant) molecule which comprises two polypeptide chains that form one antigen binding site and at least one immune cell receptor binding site, wherein one polypeptide chain has a structure represented by the formula II 1 -L 1 -V L -C L -L 2 -II 2 , and one polypeptide chain has a structure represented by the formula II 3 -L 3 -V H -C H1 -L 4 -II 4 , wherein:
- antibody refers to a protein that is capable of recognizing and specifically binding to an antigen.
- Ordinary or conventional mammalian antibodies comprise a tetramer, which is typically composed of two identical pairs of polypeptide chains, each pair consisting of one “light” chain (typically having a molecular weight of about 25 kDa) and one “heavy” chain (typically having a molecular weight of about 50-70 kDa).
- each light and heavy chain typically includes a variable domain of about 100 to 110 or more amino acids that typically is responsible for antigen recognition.
- the variable domain may be subjected to further protein engineering to humanize the framework regions if the antibody was derived from a non-human source.
- the carboxyl-terminal portion of each chain typically defines a constant domain responsible for effector function.
- a full-length heavy chain immunoglobulin polypeptide includes a variable domain (V H ) and three constant domains (C H1 , C H2 , and C H3 ) and a hinge region between C H1 and C H2 , wherein the V H domain is at the amino-terminus of the polypeptide and the C H3 domain is at the carboxyl-terminus, and a full-length light chain immunoglobulin polypeptide includes a variable domain (V L ) and a constant domain (C L ), wherein the V L domain is at the amino-terminus of the polypeptide and the C L domain is at the carboxyl-terminus.
- variable and constant domains typically are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids.
- the variable regions of each light/heavy chain pair typically form an antigen binding site.
- the variable domains of naturally occurring antibodies typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs.
- the CDRs from the two chains of each pair typically are aligned by the framework regions, which may enable binding to a specific epitope.
- both light and heavy chain variable domains typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
- IgG2/4 refers to the non-naturally occurring, protein-engineered, heavy chain developed for use in eculizumab (C H1 -Hinge-C H2 -C H3 ) and designed to reduce immune effector function and immune cell activation, minimize immunogenicity, and contribute to a longer half-life.
- This non-IgG 1, 2, 3 or 4 heavy chain is a chimera containing sequence elements of IgG4 and IgG2 including two disulfide bonds in the hinge and changes in the constant regions C H1 and C H2 .
- human antibody as used herein includes antibodies having variable and constant regions substantially corresponding to circulating human antibodies and human germline immunoglobulin sequences.
- human antibodies are produced in non-human transgenic mammals, including, but not limited to, rodents, such as mice and rats, and lagomorphs, such as rabbits.
- rodents such as mice and rats
- lagomorphs such as rabbits.
- human antibodies are produced in hybridoma cells.
- human antibodies are produced recombinantly.
- antibody fragment refers to a portion of an intact or full-length chain or an antibody, generally the target binding or variable region.
- antibody fragments include, but are not limited to, F ab , F ab′ , F (ab′)2 and F v fragments.
- functional fragment is generally synonymous with “antibody fragment,” and with respect to antibodies, can refer to antibody fragments such as F v , F ab , F (ab′)2 .
- autoantibody refers to a naturally obtained antibody produced by an individual, wherein the antibody recognizes and binds to an antigen derived from or which mimics an antigen derived from a human tissue.
- An autoantibody can facilitate an immune response against a self-tissue or self-antigen (i.e., antigens that are native to the individual, e.g., an antigen on a cell or tissue, or an endogenous peptide or protein).
- Autoantibodies frequently arise or are triggered by an infection with an infectious agent such as a virus, bacteria or parasite, where the infectious agent carried a structure that induces antibodies to the structure.
- the induced antibodies can become harmful in the infection aftermath where that inducing biologic structure is also found on naturally occurring human tissues, thus creating an aberrant and continuing autoantibody response in the aftermath of the infection.
- the autoantibody can be redesigned and repurposed, as described herein, from the natural autoantibody to modify the original structure found in a diseased individual.
- the autoantibody may be re-humanized and the heavy chain may be modified to alter the isotype or to otherwise modulate harmful secondary immune functions of the autoantibody.
- the modified autoantibody can be used to produce a therapeutic fusion protein to treat the disorder created by an aberrant autoantibody response.
- the autoantibody may also be re-engineered and/or re-derived from or into a single chain Camelid VHH antibody format.
- antigen or “target antigen” as used herein refers to a molecule or a portion of a molecule that is capable of being recognized by and bound by an antibody or the antigen binding portion of the antibody immune cell inhibitor fusion proteins of the disclosure.
- the target antigen is capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen.
- a target antigen may have one or more epitopes.
- the antibody or the antigen binding portion of the fusion protein is capable of competing with an intact antibody that recognizes the target antigen.
- epitope refers to a region or structural element of an antigen that is recognized and bound by an antibody or the antigen binding portion of the antibody immune cell inhibitor fusion protein of the disclosure. More precisely, the epitope is the specific structure that is bound by the CDRs of the antibody. Epitopes can comprise protein structural elements, carbohydrates or even portions of lipid structures found in membranes. An antibody or the antigen binding portion of the antibody immune cell inhibitor fusion protein is said to specifically bind an antigen when it preferentially recognizes its antigen target in a complex mixture of proteins and/or macromolecules.
- the term “specifically binds,” as used herein, refers to the ability of an antibody or antigen binding portion of the antibody immune cell inhibitor fusion protein to bind to an antigen containing an epitope with an Kd of at least about 1 ⁇ 10 ⁇ 6 M, 1 ⁇ 10 ⁇ 7 M, 1 ⁇ 10 ⁇ 8 M, 1 ⁇ 10 ⁇ 9 M, 1 ⁇ 10 ⁇ 10 M, 1 ⁇ 10 ⁇ 11 M, 1 ⁇ 10 ⁇ 12 M, or more, and/or to bind to an epitope with an affinity that is at least two-fold greater than its affinity for a nonspecific antigen.
- antigen binding site refers to a site created on the surface of an antibody or the antigen binding portion of the antibody immune cell inhibitor fusion protein of the disclosure where an antigen or an epitope on an antigen is bound.
- the antigen binding site of an antibody or the antigen binding portion or surface of the antibody immune cell inhibitor fusion protein is typically described by reference to the loop structures created by complementarity determining regions (CDRs) of the antibody or antibody immune cell inhibitor fusion protein.
- ligand refers to a chemical molecule or biological molecule that can bind readily to a receptor with a specific binding affinity constant.
- the ligand may be natural or synthetic.
- receptor refers to a protein capable of interacting (binding) with a ligand. In some embodiments, such a protein is capable of transmitting information resulting from interaction with a ligand, into a cell.
- the terms ligand and receptor may be interchangeable because both the ligand and receptor may be surface bound proteins on different cells that interact with each other.
- a receptor on one cell is a ligand of another receptor on a different cell however if the cell which is the focal point is reversed, so is the receptor ligand relationship.
- both interacting molecules are surface bound and the receptor-ligand relationship is not strict, but merely designates two different molecules interacting with another and causing cell signaling consequences.
- native Fc refers to a molecule comprising the sequence of a non-antigen binding fragment resulting from digestion of an antibody or produced by other means, whether in monomeric or multimeric form, and can contain the hinge region.
- the original immunoglobulin source of the native Fc is preferably of human origin and can be any of the immunoglobulins.
- Native Fc molecules are made up of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association.
- the number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2).
- class e.g., IgG, IgA, and IgE
- subclass e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2
- One example of a native Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG.
- native Fc as used herein is generic to the monomeric, dimeric, and multimeric forms.
- Fc variant refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn (neonatal Fc receptor). Exemplary Fc variants, and their interaction with the salvage receptor, are known in the art. Thus, the term “Fc variant” can comprise a molecule or sequence that is humanized from a non-human native Fc. Furthermore, a native Fc comprises regions that can be removed or mutated to produce an Fc variant to alter certain residues that provide structural features or biological activity that are not required for the antibody immune cell inhibitor fusion proteins of the disclosure.
- Fc variant comprises a molecule or sequence that lacks one or more native Fc sites or residues, or in which one or more Fc sites or residues has been modified, that affect or are involved in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).
- ADCC antibody-dependent cellular cytotoxicity
- Fc domain encompasses native Fc and Fc variants and sequences as defined above. As with Fc variants and native Fc molecules, the term “Fc domain” includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by other means.
- an antibody or antibody immune cell inhibitor fusion protein of the disclosure described herein can, in some embodiments, comprise a variant human Fc constant region that binds to human neonatal Fc receptor (FcRn) with greater affinity than that of the native human Fc constant region from which the variant human Fc constant region was derived.
- the Fc constant region can comprise one or more (e.g., two, three, four, five, six, seven, or eight or more) amino acid substitutions relative to the native human Fc constant region from which the variant human Fc constant region was derived.
- the substitutions can increase the binding affinity of an IgG antibody or antibody immune cell inhibitor fusion protein containing the variant Fc constant region to FcRn at pH 6.0, while maintaining the pH dependence of the interaction.
- substitutions that enhance the binding affinity of an antibody Fc constant region for FcRn include, for example: (1) the M252Y/S254T/T256E triple substitution described by Dall'Acqua et al., 2006 , J. Biol. Chem. 281(33): 23514-24; (2) the M428L or T250Q/M428L substitutions described in Hinton et al., 2004 , J. Biol. Chem. 279(8): 6213-16, and Hinton et al., 2006 , J. Immunol.
- the variant constant region has a substitution at EU amino acid residue 255 for valine. In some embodiments, the variant constant region has a substitution at EU amino acid residue 309 for asparagine. In some embodiments, the variant constant region has a substitution at EU amino acid residue 312 for isoleucine. In some embodiments, the variant constant region has a substitution at EU amino acid residue 386 for leucine.
- the variant Fc constant region comprises no more than (e.g., no more than 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2) amino acid substitutions, insertions, or deletions relative to the native constant region from which it was derived.
- the variant Fc constant region comprises one or more amino acid substitutions selected from the group consisting of M252Y, S254T, T256E, N434S, M428L, V259I, T250I, and V308F.
- the variant human Fc constant region comprises a methionine at position 428 and an asparagine at position 434, each in EU numbering.
- the variant Fc constant region comprises a 428L/4345 double substitution as described in, for example, U.S. Pat. No. 9,079,949.
- the 428L/434S mutations are shifted to 429L/435S as a result of the IgG2/4 chimerization engineering.
- the precise mutations in the eculizumab heavy chain are Met-429-Leu and Asn-435-Ser. See U.S. Pat. No. 9,079,949, the disclosure of which is hereby incorporated by reference in its entirety.
- the variant constant region comprises a substitution at amino acid position 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434, or 436 (EU numbering) relative to the native human Fc constant region.
- the substitution is selected from the group consisting of methionine for glycine at position 237; alanine for proline at position 238; lysine for serine at position 239; isoleucine for lysine at position 248; alanine, phenylalanine, isoleucine, methionine, glutamine, serine, valine, tryptophan, or tyrosine for threonine at position 250; phenylalanine, tryptophan, or tyrosine for methionine at position 252; threonine for serine at position 254; glutamic acid for arginine at position 255; aspartic acid, glutamic acid, or glutamine for threonine at position 256; alanine, glycine, isoleucine, leucine, methionine, asparagine, serine, threonine, or valine for proline at position 257; histidine for glut
- an “immune cell inhibitor domain” as used herein refers to an immunoglobulin domain of an immunoglobulin superfamily member that can cause suppression of immune responses upon binding to the specific receptor on an immune system cell, including inhibition of T-cells, B-cells, monocytes, and antigen presenting cells, inhibition of a particular immune cell function, including cytotoxicity, or any combination of the above responses.
- the immune cell inhibitor domains of the antibody immune cell inhibitor fusion proteins of the disclosure are referred to herein as follows: II 1 , which, when present, is fused (with or without an intervening linker) to the N-terminal end of the light chain; II 2 , which, when present, is fused (with or without an intervening linker) to the C-terminal end of the light chain; II 3 , which, when present, is fused (with or without an intervening linker) to the N-terminal end of the heavy chain; and II 4 , which, when present, (with or without an intervening linker) is fused to the C-terminal end of the heavy chain.
- Linkers may or may not be needed depending on the where the stop and start residues of protein fusions are chosen because often natural linkers are found between immunoglobulin domains.
- immunoglobulin superfamily member refers to a class of proteins that are associated with the adhesion, binding, and recognition processes of cells.
- exemplary members of the immunoglobulin superfamily include, but are not limited to, Programmed Death Ligand 1 (PD-L1), also known as B7 Homolog 1 (B7-H1), or CD274; Programmed Death Ligand 2 (PD-L2), also known as B7-DC; B7 Homolog 3 (B7-H3), as known as CD276; B7 Homolog 4 (B7-H4), also known as V-set Domain Containing T-cell Activation Inhibitor 1 (VTCN1), B7 Superfamily, Member 1 (B7S1), or B7x; Herpesvirus Entry Mediator (HVEM), also known as Herpesvirus Entry Mediator A (HVEA), Tumor Necrosis Factor Receptor Superfamily, Member 14 (TNFRSF14), or CD270; V-type Immuno
- immune effector cell refers to the cells of the immune system that mount immune responses to an antigen. Suitable effector cells include but are not limited to populations of antigen presenting cells, cytotoxic T-cells, and T helper cells that mediate cellular immunity. In addition to antigen-specific effector T-cells, the effector cell populations may include, but are not limited to, other cytotoxic immune cells against a selected antigen such as natural killer cells, lymphocytes, monocytes, macrophages, neutrophils, and eosinophils.
- linker refers to one or more amino acid residues inserted between immunoglobulin domains and/or immune cell inhibitor domains of the antibody immune cell inhibitor fusion proteins of the disclosure.
- a linker may be inserted between an immunoglobulin domain and an immune cell inhibitor domain, at the sequence level.
- the precise location of a domain transition can be determined by locating peptide stretches that do not form secondary structural elements such as beta-sheets or alpha-helices as demonstrated by experimental data or as can be assumed by techniques of modeling or secondary structure prediction.
- Linkers may or may not be needed depending on the where the stop and start residues of protein fusions are chosen because often natural linkers are found between immunoglobulin domains.
- the linkers of the antibody immune cell inhibitor fusion proteins of the disclosure are referred to herein as follows: L 1 , which, when present, is located on the light chain between the Ili and V L domains; L 2 , which, when present, is located on the light chain between the C L and II 2 domains; L 3 , which, when present, is located on the heavy chain between the II 3 and L 3 domains; and L 4 , which, when present, is located on the heavy chain between the Fc and II 4 domains.
- the linkers L 1 , L 2 , L 3 , and L 4 are independent, but in some embodiments of the antibody immune cell inhibitor fusion proteins of the disclosure may have the same sequence and/or length.
- non-naturally occurring refers to the fact that the object can be found in nature and has not been manipulated by man.
- a polynucleotide or polypeptide that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man is naturally occurring.
- non-naturally occurring refers to a protein molecule that is not found in nature or that has been structurally modified through protein engineering and synthesized, manufactured, or produced by man using recombinant DNA technologies in appropriate cells such as, for example, CHO cells.
- a “recombinant” molecule is one that has been prepared, expressed, created, or isolated by recombinant DNA technology means.
- fusion protein refers to protein constructs comprising an immunoglobulin domain and an immune cell inhibitor protein.
- the immune cell inhibitor protein in some fusion proteins of the disclosure may not constitute the entire natural protein but may be limited to an active domain of the entire protein responsible for binding to a corresponding receptor on the surface of an immune function cell.
- an immunoglobulin domain in some fusion proteins of the disclosure may not constitute the entire natural immunoglobulin domain but may be limited to a portion of the natural immunoglobulin domain responsible for specifically binding a target antigen or epitope or conferring other properties of the natural immunoglobulin domain.
- the fragment of the immune cell inhibitor protein or immunoglobulin domain in some fusion proteins of the disclosure would not be naturally occurring as the fragment, but may retain the same protein sequence for the fragment and incorporated into a therapeutic fusion protein.
- inhibitor or “diminish” as used herein refer to a complete or partial arrest of immune effector cell activation.
- substantially pure or substantially purified refer to a compound or species that is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition).
- a substantially purified fraction is a composition wherein the species comprises at least about 50% (on a molar basis) of all macromolecular species present.
- a substantially pure composition will comprise more than about 80%, 85%, 90%, 95%, or 99% of all macromolar species present in the composition.
- the species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.
- biological property in reference to an antibody or an antibody immune cell inhibitor fusion protein of the disclosure are used interchangeably herein and include, but are not limited to, epitope affinity and specificity, ability to antagonize the activity of the antigen target (or targeted polypeptide), the in vivo stability of the antibody or antibody immune cell inhibitor fusion protein, and the immunogenic properties of the antibody or antibody immune cell inhibitor fusion protein.
- Other identifiable biological properties or characteristics of an antibody or an antibody immune cell inhibitor fusion protein include, for example, cross-reactivity, (i.e., with non-human homologs of the antigen target, or with other antigen targets or tissues, generally), and ability to preserve high expression levels of protein in mammalian cells. The aforementioned properties or characteristics can be observed or measured using art-recognized techniques.
- a “neutralizing effect” of an antibody immune cell inhibitor fusion protein as used herein refers to a fusion protein of an antibody and an immune cell inhibitor ligand that is able to first, bind an antigen for which the antigen binding portion of the antibody immune cell inhibitor fusion protein specifically recognizes, then, through simultaneous binding of the fused immune cell inhibitor domain to a specific receptor, to block or substantially reduce an unwanted deleterious or autoimmune effector function carried out by the cell expressing the immune cell inhibitor receptor.
- substantially reduce means at least about 60%, preferably at least about 70%, more preferably at least about 75%, even more preferably at least about 80%, still more preferably at least about 85%, most preferably at least about 90% reduction of the unwanted or autoimmune effector function of the cell carrying the ligand's receptor.
- neutralizing antibody refers to an antibody that can neutralize the function of the protein or infectious agent the antibody specifically recognizes and binds.
- neutralizing antibodies refer to antibodies specific for viral, bacterial, or other infectious agents.
- therapeutic antibodies are used as drug candidates to treat human disease
- neutralizing antibodies can arise as anti-drug antibodies (ADA) but have the undesirable function of neutralizing the therapeutic benefit of the therapeutic antibody.
- Neutralizing antibodies to the antigen binding portions of the fusion proteins of the present disclosure would represent an undesirable development.
- An antibody or antibody immune cell inhibitor fusion protein of the disclosure typically has a dissociation constant (K D ) of 10 ⁇ 5 to 10 ⁇ 12 moles/liter or less, or 10 ⁇ 7 to 10 ⁇ 12 moles/liter or less, or 10 ⁇ 3 to 10 ⁇ 12 moles/liter, and/or with a binding affinity of at least 10 7 M ⁇ 1 , or at least 10 8 M ⁇ 1 , or at least 10 9 M ⁇ 1 , or at least 10 12 M ⁇ 1 .
- K D dissociation constant
- Any K D value greater than 10 ⁇ 4 moles/liter is generally considered to indicate non-specific binding. Therefore, the lower the K D value, the greater the affinity.
- a monovalent antibody or antibody immune cell inhibitor fusion protein of the disclosure will bind to a desired antigen with an affinity less than 500 nM, or less than 200 nM, or less than 10 nM, or less than 500 pM.
- High affinity or very strong binding is often associated with greater efficacy, but it is not always the case that the greater the affinity the greater the efficacy.
- the dissociation constant (K D ) can be determined, for example, by surface plasmon resonance (SPR).
- SPR surface plasmon resonance
- surface plasmon resonance analysis measures real-time binding interactions (both on rate and off rate) between a ligand (a target antigen on a biosensor matrix) and an analyte by surface plasmon resonance using, for example, the BIAcore system (Pharmacia Biosensor; Piscataway, NJ).
- Surface plasmon analysis can also be performed by immobilizing the analyte and presenting the ligand.
- Specific binding of an antibody or an antibody immune cell inhibitor fusion protein of the disclosure to an antigen or antigenic determinant can also be determined in any suitable manner known in the art, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme linked immunosorbent assays (ELISA), enzyme immunoassays (EIA), and sandwich competition assays.
- Scatchard analysis and/or competitive binding assays such as radioimmunoassays (RIA), enzyme linked immunosorbent assays (ELISA), enzyme immunoassays (EIA), and sandwich competition assays.
- vector refers to any molecule (e.g., nucleic acid, plasmid, or virus) that is used to transfer coding information to a host cell.
- plasmid refers to a circular double-stranded DNA molecule into which additional DNA segments may be inserted.
- viral vector Another type of vector is a viral vector, wherein additional DNA segments may be inserted into the viral genome.
- Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
- vectors e.g., non-episomal mammalian vectors
- vectors can be integrated into the genome of a host cell upon introduction into the host cell and thereby are replicated along with the host genome.
- certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
- flanking sequence operably linked is used herein to refer to an arrangement of flanking sequences wherein the flanking sequences are configured or assembled so as to perform their usual function.
- a flanking sequence operably linked to a coding sequence may be capable of effecting the replication, transcription, and/or translation of the coding sequence.
- a coding sequence is operably linked to a promoter when the promoter is capable of directing transcription of that coding sequence.
- host cell refers to a cell into which an expression vector has been introduced.
- a host cell is intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but such cells are still included within the scope of the term “host cell” as used herein.
- a wide variety of host cell expression systems can be used to express the antibody immune cell inhibitor fusion proteins of the disclosure, including bacterial, yeast, baculoviral, and mammalian expression systems (as well as phage display expression systems).
- cultured mammalian cell lines include Chinese Hamster ovary (CHO) simian cells such as COS, murine cell lines such as NSO, and human cell lines such as HEK and HeLa, which may be used to produce the antibody immune cell inhibitor fusion proteins of the disclosure.
- Vectors are transfected into the cells and the DNA may be integrated into the genome by homologous recombination in the case of stable transfection, or the cells may be transiently transfected.
- mammalian expression vectors include adenoviral vectors, the pSV and the pCMV series of plasmid vectors, vaccinia and retroviral vectors, as well as baculovirus.
- CMV cytomegalovirus
- SV40 simian virus 40
- Non-viral promoters such as the elongation factor (EF)-1 promoter, may also be used.
- nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form an antibody immune cell inhibitor fusion protein of the disclosure.
- Another embodiment of the disclosure provides expression vectors comprising nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form the antibody immune cell inhibitor fusion proteins of the disclosure.
- host cells that express such antibody immune cell inhibitor fusion proteins (i.e., comprising nucleic acid molecules or vectors encoding polypeptide chains that form such antibody immune cell inhibitor fusion proteins).
- the disclosure provides methods for preparing an antibody immune cell inhibitor fusion protein of the disclosure, wherein such methods comprise cultivating or maintaining a host cell under conditions such that the host cell produces or expresses such antibody immune cell inhibitor fusion proteins, and optionally further comprises isolating the antibody immune cell inhibitor fusion protein so produced.
- a skilled artisan will be able to determine suitable variants of the polypeptide chains of the antibody immune cell inhibitor fusion proteins of the disclosure using well-known techniques. For example, one skilled in the art may identify suitable areas of a polypeptide chain that may be changed without destroying activity by targeting regions not believed to be important for activity. Alternatively, one skilled in the art can identify residues and portions of the molecules that are conserved among similar polypeptides. In addition, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.
- patient includes human and animal subjects.
- a “disorder” is any condition that would benefit from treatment using the antibody immune cell inhibitor fusion proteins of the disclosure. “Disorder” and “condition” are used interchangeably herein and include chronic and acute disorders or diseases, including those pathological conditions that predispose a patient to the disorder in question.
- a “T-cell mediated-condition” is a condition directly or indirectly effected by the T-cells of the immune system.
- treatment refers to both therapeutic treatment and prophylactic or preventative measures.
- Those in need of treatment include those having a disorder as well as those prone to have the disorder or those in which the disorder is to be prevented.
- composition or “therapeutic composition” as used herein refer to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.
- a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of at least one antibody immune cell inhibitor fusion protein of the disclosure.
- pharmaceutically acceptable carrier or “physiologically acceptable carrier” as used herein refers to one or more formulation materials suitable for accomplishing or enhancing the delivery of one or more antibody immune cell inhibitor fusion proteins of the disclosure.
- a therapeutically effective amount when used in reference to a pharmaceutical composition comprising one or more antibody immune cell inhibitor fusion proteins of the disclosure refer to an amount or dosage sufficient to produce a desired therapeutic result. More specifically, a therapeutically effective amount is an amount of an antibody immune cell inhibitor fusion protein sufficient to inhibit, for some period of time, one or more of the clinically defined pathological processes associated with the condition being treated. The effective amount may vary depending on the specific antibody immune cell inhibitor fusion protein that is being used, and may also depend on a variety of factors and conditions related to the patient being treated and the severity of the disorder.
- the antibody immune cell inhibitor fusion protein is to be administered in vivo, factors such as the age, weight, and health of the patient as well as dose response curves and toxicity data obtained in preclinical animal work would be among those factors considered.
- factors such as the age, weight, and health of the patient as well as dose response curves and toxicity data obtained in preclinical animal work would be among those factors considered.
- the determination of an effective amount or therapeutically effective amount of a given pharmaceutical composition is within the ability of those skilled in the art.
- the antibody immune cell inhibitor fusion protein comprises four polypeptide chains that form two antigen binding sites at least two immune cell receptor binding sites, wherein two polypeptide chains have a structure represented by the formula II 1 -V L -C L -II 2 , and two polypeptide chains have a structure represented by the formula II 3 -V H -C H1 -Fc-II 4 ; wherein:
- the antibody immune cell inhibitor fusion protein comprises four polypeptide chains that form two antigen binding sites and at least two immune cell receptor binding sites, wherein two polypeptide chains have a structure represented by the formula II 1 -L 1 -V L -C L -L 2 -II 2 , and two polypeptide chains have a structure represented by the formula II 3 -L 3 -V H -C H1 -Fc-L 4 -II 4 ; wherein:
- the antibody immune cell inhibitor fusion protein comprises two polypeptide chains that form one antigen binding site and at least one immune cell receptor binding site; wherein one polypeptide chain has a structure represented by the formula II 1 -V L -C L -II 2 , and one polypeptide chain has a structure represented by the formula II 3 -V H -C H1 -II 4 ; wherein:
- the antibody immune cell inhibitor fusion protein comprises two polypeptide chains that form one antigen binding site and at least one immune cell receptor binding site; wherein one polypeptide chain has a structure represented by the formula II 1 -L 1 -V L -C L -L 2 -II 2 , and one polypeptide chain has a structure represented by the formula II 3 -L 3 -V H -C H1 -L 4 -II 4 ; wherein:
- the antibody immune cell inhibitor fusion proteins of the disclosure may be prepared using domains or sequences obtained or derived from any human or non-human antibody, including, for example, human, murine, or humanized antibodies.
- the V L , V H , C L , C H1 , and/or Fc domains of the antibody immune cell inhibitor fusion protein may not constitute the entire natural immunoglobulin domain, provided, however, that the portion of the V L , V H , C L , C H1 , and/or Fc domain used in the antibody immune cell inhibitor fusion protein is capable of functioning in the same manner as the full-length natural immunoglobulin domain.
- the antibody immune cell inhibitor fusion proteins of the disclosure may further comprise additional V L , V H , C L , C H1 , and/or Fc domains.
- one or more of II 1 , II 2 , II 3 , and II 4 is an immune cell inhibitor domain.
- at least one of the immune cell inhibitor domains can inhibit or diminish activation of an immune effector cell involved in the immune response to self-tissue.
- at least one of the immune cell inhibitor domains can inhibit or diminish activation of an immune effector cell involved in the immune response to self-tissue when an autoantibody is bound to the antigen at the site of an ongoing disease process.
- at least one of II 1 , II 2 , II 3 , and II 4 is an immune cell inhibitor domain of an immunoglobulin domain superfamily member.
- at least one of II 1 , II 2 , II 3 and II 4 is absent.
- the immune cell inhibitor domain is obtained or derived from a member of the immunoglobulin superfamily.
- the immune cell inhibitor domain comprises a Programmed Death Ligand 1 (PD-L1), B7 Homolog 1 (B7-H1), or CD274 domain; Programmed Death Ligand 2 (PD-L2) or B7-DC domain; B7 Homolog 3 (B7-H3) or CD276 domain; B7 Homolog 4 (B7-H4), V-set Domain-Containing T-cell Activation Inhibitor 1 (VTCN1), B7 Superfamily, Member 1 (B7S1), or B7x domain; Herpesvirus Entry Mediator (HVEM), Herpesvirus Entry Mediator A (HVEA), Tumor Necrosis Factor Receptor Superfamily, Member 14 (TNFRSF14), or CD270 domain (also known as Tumor necrosis factor receptor superfamily member 14); V-type Immunoglobulin Domain-Containing Suppressor
- the immune cell inhibitor domain comprises a PD-L1 extracellular domain (wherein PD-L1 is also known as CD274, B7-H, B7H1, PDCD1L 1 , or PDCD1LG1) or a PD-L2 extracellular domain (wherein PD-L2 is also known as CD273).
- PD-L1 when bound to Programmed Death 1 (PD-1) inhibitory receptor (also known as CD279) acts as an immune checkpoint inhibitor of B-cells, T-cells, monocytes, and antigen presenting cells.
- PD-1 inhibitory receptor also known as CD279
- an activated T-cell expresses PD-1 on its surface upon antigen recognition and produces interferons, which induce expression of PD-L1 in multiple tissues.
- Binding of PD-1 to its ligand limits T-cell activity. Under normal conditions, the PD-1/PD-L1 pathway prevents excessive stimulation and maintains the immune tolerance to self-antigens by negatively regulating the immune response (Riella et al., 2012, Am. J. Transplant. 12(10): 2575-87).
- the antibody immune cell inhibitor fusion protein comprises four polypeptide chains that form two antigen binding sites and at least two PD-1 binding sites; wherein two polypeptide chains have a structure represented by the formula II 1 -V L -C L -II 2 , and two polypeptide chains have a structure represented by the formula II 3 -V H -C H1 -Fc-II 4 , wherein:
- the antibody immune cell inhibitor fusion protein comprises four polypeptide chains that form two antigen binding sites and at least two PD-1 binding sites; wherein two polypeptide chains have a structure represented by the formula II 1 -L 1 -V L -C L -L 2 -II 2 , and two polypeptide chains have a structure represented by the formula II 3 -L 3 -V H -C H1 -Fc-L 4 -II 4 ; wherein:
- the antibody immune cell inhibitor fusion protein comprises two polypeptide chains that form one antigen binding site and at least one PD-1 binding site; wherein one polypeptide chain has a structure represented by the formula II 1 -V L -C L -II 2 , and one polypeptide chain has a structure represented by the formula II 3 -V H -C H1 -II 4 ; wherein:
- the antibody immune cell inhibitor fusion protein comprises two polypeptide chains that form one antigen binding site and at least one PD-1 binding site; wherein one polypeptide chain has a structure represented by the formula II 1 -L 1 -V L -C L -L 2 -II 2 , and one polypeptide chain has a structure represented by the formula II 3 -L 3 -V H -C H1 -L 4 -II 4 , wherein:
- the V L , V H , C L , and C H1 domains of the antibody immune cell inhibitor fusion proteins of the disclosure form an autoantibody that recognizes and binds an antigen derived from or mimicking an antigen derived from a human tissue.
- the autoantibody facilitates an immune response to a self-tissue.
- the antibody immune cell inhibitor fusion proteins of the disclosure are capable of specifically binding one or more antigen targets.
- the antibody immune cell inhibitor fusion protein is capable of specifically binding at least one antigen target that is Human serum albumin, Bone mineral (hydroxyapatite), Complement component 3d (C3d), Glutamic acid decarboxylase 65 (GAD65), Asialoglycoprotein receptor (ASGPR), Asialoglycoprotein receptor 2 (ASGPR2) Transferrin receptor protein 2 (TFR2), Solute carrier family 2, facilitated glucose (SLC2A2), Solute carrier organic anion transporter family member 1B1 (SLCO1B1), Solute carrier organic anion transporter family member 1B3 (SLCO1B3), Multidrug resistance-associated protein 6 (ABCC6), Major histocompatibility complex I (MHC I), Major histocompatibility complex II (MHC II), T-cell receptor (TCR), B-Cell receptor (BCR), Transforming growth factor beta, Cluster differentiation 4 (
- the antibody immune cell inhibitor fusion proteins of the disclosure comprise a linker joining at least one immune cell inhibitor domain to at least one immunoglobulin light chain or heavy chain or domain.
- L 1 when present, may be 5 to 30 amino acid residues in length
- L 2 when present, may be 5 to 30 amino acid residues in length
- L 3 when present, may be 5 to 30 amino acid residues in length
- L 4 when present, may be 5 to 30 amino acid residues in length.
- At least one of L 1 , L 2 , L 3 and L 4 is absent.
- the identity and sequence of amino acid residues in the linker may vary depending on the type of secondary structural element necessary to be achieved. For example, glycine, serine, and alanine are best for linkers having maximum flexibility. Some combination of glycine, proline, threonine, and serine are useful if a more rigid and extended linker is necessary. Any amino acid residue may be considered as a linker in combination with one or more other amino acid residues, which may be the same as or different as the first amino acid residue, to construct larger peptide linkers as necessary depending on the desired properties.
- Therapeutic or pharmaceutical compositions comprising one or more antibody immune cell inhibitor fusion proteins of the disclosure are within the scope of the disclosure.
- Such therapeutic or pharmaceutical compositions can comprise a therapeutically effective amount of an antibody immune cell inhibitor fusion protein, or antibody immune cell inhibitor fusion protein-drug conjugate, in admixture with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration.
- Acceptable formulation materials are preferably nontoxic to recipients at the dosages and concentrations employed.
- the pharmaceutical composition can contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition.
- Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite, or sodium hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as serum albumin, gelatin, or immunoglobulins), coloring, flavoring and diluting agents, emuls
- compositions will be determined by a skilled artisan depending upon, for example, the intended route of administration, delivery format, and desired dosage. Such compositions can influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the antibody immune cell inhibitor fusion proteins of the disclosure.
- the primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature.
- a suitable vehicle or carrier for injection can be water, physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration.
- Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.
- Other exemplary pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can further include sorbitol or a suitable substitute.
- antibody immune cell inhibitor fusion protein compositions can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents in the form of a lyophilized cake or an aqueous solution. Further, the antibody immune cell inhibitor fusion protein can be formulated as a lyophilizate using appropriate excipients such as sucrose.
- compositions of the disclosure can be selected for parenteral delivery.
- the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally.
- the preparation of such pharmaceutically acceptable compositions is within one of skill in the art.
- the formulation components are present in concentrations that are acceptable to the site of administration.
- buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.
- the therapeutic compositions for use in this disclosure can be in the form of a pyrogen-free, parenterally acceptable, aqueous solution comprising the desired antibody immune cell inhibitor fusion protein in a pharmaceutically acceptable vehicle.
- a suitable vehicle for parenteral injection is sterile distilled water in which an antibody immune cell inhibitor fusion protein of the disclosure is formulated as a sterile, isotonic solution, properly preserved.
- Yet another preparation can involve the formulation of the antibody immune cell inhibitor fusion protein with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, that provides for the controlled or sustained release of the product which can then be delivered via a depot injection.
- Hyaluronic acid can also be used, and this can have the effect of promoting sustained duration in the circulation.
- Other suitable means for the introduction of the antibody immune cell inhibitor fusion protein include implantable drug delivery devices.
- compositions of the disclosure will be evident to those skilled in the art, including formulations involving antibody immune cell inhibitor fusion proteins in sustained- or controlled-delivery formulations.
- Techniques for formulating a variety of other sustained- or controlled-delivery means such as liposome carriers, bio-erodible microparticles, or porous beads and depot injections, are also known to those skilled in the art.
- Additional examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules.
- Sustained release matrices can include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid.
- Sustained-release compositions can also include liposomes, which can be prepared by any of several methods known in the art.
- compositions of the disclosure to be used for in vivo administration typically must be sterile. This can be accomplished by filtration through sterile filtration membranes. Where the composition is lyophilized, sterilization using this method can be conducted either prior to, or following, lyophilization and reconstitution.
- the composition for parenteral administration can be stored in lyophilized form or in a solution.
- parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
- the pharmaceutical composition can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder.
- Such formulations can be stored either in a ready-to-use form or in a form (e.g., lyophilized) requiring reconstitution prior to administration.
- kits for producing a single-dose administration unit can each contain both a first container having a dried protein and a second container having an aqueous formulation. Also included within the scope of this disclosure are kits containing single and multi-chambered pre-filled syringes (e.g., liquid syringes and lyosyringes).
- the effective amount of pharmaceutical composition containing an antibody immune cell inhibitor fusion protein to be employed therapeutically will depend, for example, upon the therapeutic context and objectives.
- One skilled in the art will appreciate that the appropriate dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication for which the antibody immune cell inhibitor fusion protein is being used, the route of administration, and the size (body weight, body surface, or organ size) and condition (the age and general health) of the patient. Accordingly, the clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.
- a typical dosage can range from about 0.1 mg/kg to up to about 100 mg/kg or more, depending on the factors mentioned above.
- the dosage can range from 0.1 mg/kg up to about 100 mg/kg; or 1 mg/kg up to about 100 mg/kg; or 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, up to about 100 mg/kg.
- Dosing frequency will depend upon the pharmacokinetic parameters of the antibody immune cell inhibitor fusion protein in the formulation being used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect.
- the composition can therefore be administered as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages can be ascertained through use of appropriate dose-response data.
- the route of administration of the pharmaceutical composition is in accord with known methods, e.g., orally; through injection by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; by sustained release systems; or by implantation devices.
- the compositions can be administered by bolus injection or continuously by infusion, or by implantation device.
- composition can also be administered locally via implantation of a membrane, sponge, or other appropriate material onto which the desired molecule has been absorbed or encapsulated.
- a membrane, sponge, or other appropriate material onto which the desired molecule has been absorbed or encapsulated.
- the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration.
- the antibody immune cell inhibitor fusion proteins of the disclosure are used to treat a patient having Primary Biliary Cholangitis (PBC), Type 1 Diabetes, Goodpasture's syndrome, Amyloidosis, Ankylosing spondylitis, Anti-glomerular basement membrane/anti-tubular basement membrane nephritis, Antiphospholipid syndrome, Autoimmune hepatitis, Autoimmune oophoritis Autoimmune pancreatitis, Autoimmune retinopathy, Behcet's disease, Crohn's disease, Devic's disease, Lupus, Dressler's syndrome, Fibrosing alveolitis, Glomerulonephritis, Graves' disease, Guillain-Barre syndrome, IgA Nephropathy, IgG4-related sclerosing disease, Immune thrombocytopenic purpura (ITP), Microscopic polyangiitis (MPA), Mixed connective tissue disease (MCTD), Multiple sclerosis, Polyn
- PBC Primary Bili
- An antibody immune inhibitor fusion protein was designed comprising a human autoimmune antibody to a self-antigen identified as driving the disease process known as Primary Biliary Cholangitis (PBC), previously considered cirrhosis.
- PBC Primary Biliary Cholangitis
- the self-antigen recognized by the autoimmune antibody was shown to be Pyruvate Dehydrogenase Complex subunit E2 (PDC-E2, also known as Dihydrolipoamide Acetyltransferase or DLAT).
- the original fusion protein fused the extracellular domain of Programmed Death Ligand 1 (PD-L1, also known as CD274, or B7 Homolog 1 (B7-H1)), to each of the N- and C-termini of an engineered autoantibody containing the V regions of the autoantibody and an engineered Fc variant known as IgG2/4 to minimize effector function.
- P-L1 Programmed Death Ligand 1
- B7-H1 B7 Homolog 1
- the PD-L1 domain was fused to the antibody to interact with Programmed Death 1 (PD-1, CD279) receptor on T-cells believed to be driving the disease process, thereby inhibiting activation of the T-cells and subsequent activation of other immune system actors such as antigen presenting cells, B-cells, and any associated excreted cytokines to thus ameliorate the immune response causing the disease.
- PD-1 Programmed Death 1
- variable-like domain of the extracellular domain (ECD) of PD-L1 linked to either the C-terminus or the N-terminus of the heavy and/or light chains.
- ECD extracellular domain
- Sequences for the constructs are shown in Table 3. The sequences are shown as monomeric sequences, but all full-length antibodies expressed and purified were, of course, dimeric.
- the heavy chain of the antibody is shown in regular text, the light chain of the antibody is shown in underlined text, the linker is shown in italicized text, and the immune cell inhibitor domain of an immunoglobulin superfamily member are shown in bold text.
- PD2, PD5, and DWZ Three of the anti-DLAT antibodies referenced in Thomson et al., 1998, J. Hepatol. 28(4): 582-94, designated PD2, PD5, and DWZ, were selected for cloning as: (i) human full-length antibodies were constructed with the autoantibody V H -V C1 and V L -C L regions fused to a Fc variant region (IgG2/4); (ii) murine IgG1 Fc; (iii) PDS as a murine IgG1 Fc variant with mutations to remove effector functions.
- TPP-1986 was ordered from Absolute Antibody as a custom production consisting of their murine IgG1 Fc Silent isotype with PDS variable region and human PD-L1 variable-like domain.
- All murine light chain sequences are kappa light chains.
- the reference sequence sources for these antibodies are shown in Table 4, the sequences comprising the heavy and light chains are shown in Table 3 above, and the construct descriptions are shown in Table 5.
- PD-1 Fc chimera (R&D Systems, Catalog # 1086-PD-050) was captured on the anti-human Fc tips (ForteBio, Catalog # 18-5064) at 10 ⁇ g/mL in PBS, followed by binding to PD2 Fab fusion variants or Programmed Death Ligand 1 (PD-L1) Fc chimera (R&D Systems, Catalog # 156-B7-100) at 10 ⁇ g/mL or ⁇ g/mL in PBS, respectively.
- Final dissociation in PBS was performed in the last step on the Bio-Layer Interferometry (BLI) data.
- TPP-993 did not bind to PD-1 Fc because TPP-993 lacks a PD-L1 domain ( FIG. 1 A ).
- TPP-994 and TPP-999 exhibited very little binding to PD-1 Fc ( FIGS. 1 B and 1 D ) while TPP-995 and TPP-1001 both exhibited binding to PD-1 Fc (FIGS. 1 C and E).
- PD-L1 Fc a dimeric protein having two PD-L1 proteins fused to a human Fc, bound PD-1 very strongly to PD-1 Fc ( FIG. 1 F ), but did not bind to itself, demonstrating that the Fc portion of PD-L1 Fc did not non-specifically interact with the tips ( FIG. 1 G ).
- the three anti-DLAT murine IgG1 antibodies described in Example 2 were diluted to 40 ⁇ g/mL in PBS and loaded onto anti-mouse IgG1 Fc tips (ForteBio, Catalog # 18-5090).
- Human DLAT recombinant protein ProSpec, Catalog # ENZ-082
- Binding was determined by the association of the DLAT antigen with the respective antibodies using Bio-Layer Interferometry (BLI) deflections the Octet Red (ForteBio).
- BKI Bio-Layer Interferometry
- a dissociation step in PBS was performed to determine dissociation from the bound antibodies.
- the on- and off-rates of the anti-DLAT murine IgG1 antibodies to DLAT antigen are shown in FIG. 2 .
- Slow on-rates and fast off-rates observed for TPP-1003 suggested a weak affinity
- a slow off-rate observed for TPP-1004 ( FIG. 2 B ) and TPP-1005 ( FIG. 2 C ) suggested a high affinity to the DLAT antigen.
- Octet Red (ForteBio) was utilized for Bio-Layer Interferometry (BLI) to determine binding of the anti-DLAT human IgG2/4 antibodies to antibody PD-L1 fusions to associate with the DLAT antigen followed by binding to PD-1 Fc.
- This assay was utilized to determine the ability of the targeted PD-L1 fusions to simultaneously bind the DLAT antigen by the antibody and PD-1 Fc by the PD-L1 variable-like domain.
- Antibodies and antibody fusions were diluted to 16 ⁇ g/mL in PBS and loaded onto anti-human IgG Fc tips (ForteBio, Catalog # 18-5064).
- Human DLAT recombinant protein ProSpec, Catalog # ENZ-082
- PD-L1 Fc chimera R&D Systems, Catalog # 156-B7-100
- PD-1 Fc chimera R&D Systems, Catalog # 1086-PD-050
- Results are summarized in Table 6 below and shown in FIGS. 3 A-E .
- the fusion proteins lacking PD-L1, or having PD-L1 fused to the antibody C-terminus ( FIG. 3 C ) of the light chain did not bind well to PD-1 Fc for this particular combination of antibody and immune inhibitor.
- Dihydrolipoamide Acetyltransferase (DLAT) human T-cell activation beads were prepared by conjugating 120 ⁇ g anti-human CD3, clone UCHT1 (Stemcell Technologies, Catalog # 60011) to 3 mL of Dynabeads M-450 Epoxy (ThermoFisher, Catalog # 14011) for 18 hours rotating at room temperature in 0.1M sodium phosphate buffer, pH 8.0. See FIG. 8 for visual representations of the various bead constructs.
- T-cell activation beads (“TA2 beads”) were prepared by conjugating anti-CD3-coated beads (as prepared in making the human DLAT-aCD3 beads) with negative control irrelevant human IgG Fc (Bethyl Labs, Catalog # P80-104).
- Negative control (“NC”) beads were prepared by conjugating human IgG Fc with uncoated beads for a no-activation control reagent. Bead conjugation was verified by flow cytometry prior to performing the T-cell activation assay.
- Human peripheral primary human pan-T-cells including CD4+and CD8+T-cells as well as some gamma/delta T-cell subsets (Stemcell Technologies, Catalog # 70024) were stained with the CFSE Cell Proliferation Kit (ThermoFisher, Catalog # C34554) and then activated using DLAT-aCD3 beads, TA2 beads or NC beads, in the presence or absence of the anti-DLAT antibody or antibody PD-L1 fusions, TPP-985, TPP-986, TPP-992, or the PD-L1 Fc chimera as a non-targeted control (R&D Systems, Catalog #156-B7-100) at 260 nM or 65 nM in replicates of three.
- TPP-992 was titrated starting from 260 nM by two-fold dilutions down to 2 nM and combined with the DLAT-aCD3 beads as described. This assay was performed in a 96-well U-bottom tissue culture plate (Corning, Catalog # 3799) with 1.5 ⁇ 10 5 cells/well with a 1:1.5 bead to cell ratio for 4 days at 37° C. with 5% CO 2 .
- TPP-985 human IgG2/4 PDS antibody bound to the DLAT antigen, but did not bind to PD-1 Fc ( FIG. 3 A ).
- TPP-986 and TPP-992 fusions of the PD-L1 V-like domain to the N-terminus of anti-DLAT antibodies) both exhibited the ability to simultaneously bind the DLAT antigen and PD-1 Fc ( FIGS. 3 B and 3 D ).
- TPP-990 (PD-L1 V-like domain fused to the C-terminus of an anti-DLAT antibody) bound the DLAT antigen, but did not bind PD-1 Fc ( FIG. 3 C ).
- the PD-L1 Fc control bound to PD-1 Fc, but did not bind to the DLAT antigen ( FIG. 3 E ).
- the decreased signal in the FL-1 mean fluorescence measurement represents the activated T-cells that have expanded into new generations of T-cells, as demonstrated by the decreasing dye concentration in each respective generation ( FIG. 4 A ).
- Samples were gated to exclude the low Forward Scatter (FSC) and the FL-4+(SYTOXTM Red) dead cells. The same gate was utilized to analyze all samples.
- the histograms shown in FIG. 4 were gated on the live T-cells.
- FIG. 4 B illustrates that TPP-985, an anti-DLAT IgG2-4 with no PD-L1 variable-like domain present, showed no significant inhibition of T-cell activation.
- FIGS. 4 A- 4 F show human T-cell activation for targeted PD-L1 fusion inhibition. Representative histograms of one of three replicates are shown for each sample at 260 nM and 65 nM concentrations of antibody fusions or non-targeted PD-L1 Fc.
- FIG. 4 A illustrates that NC beads exhibited no activation of T-cells, whereas the DLAT-aCD3 beads demonstrated clear activation of the pan T-cells.
- FIG. 4 B illustrates that TPP-985 (an anti-DLAT IgG2/4 antibody with no PD-L1 variable-like domain) exhibited no significant inhibition of T-cell activation.
- FIG. 4 C illustrates that TPP-986 (an anti-DLAT antibody with PD-L1 fused at the N-terminus of the heavy chain) exhibited significant inhibition of T-cell activation at both the 65 nM and 260 nM concentrations.
- FIG. 4 D illustrates that TPP-992 (an anti-DLAT antibody with PD-L1 fused at the N-terminus of the light chain) exhibited the most significant inhibition of T-cell activation, with complete inhibition at the 260 nM concentration.
- FIG. 4 E illustrates that the non-targeting PD-L1 Fc chimera exhibited no significant inhibition of T-cell activation.
- FIG. 4 F illustrates that the titration of TPP-992 demonstrated a dose-dependent inhibition of T-cell activation with an EC50 of 17 nM ⁇ 4.5 nM.
- N-terminal fusion of PD-L1 is the most effective design.
- TPP-986 which is an anti-DLAT IgG2/4 fused with PD-L1 on the N-terminus of the heavy chain
- TPP-992 ( FIG. 4 D ) which is an anti-DLAT IgG2/4 fused with PD-L1 on the N-terminus of the light chain, showed the most significant inhibition of human T-cell activation of all of the samples tested, with nearly complete inhibition at the 260 nM concentration.
- FIGS. 22 A- 22 C illustrate histograms showing titration from 250 nM to 50 nM of anti-CD80_mPDL1 fusion incubated with CD80 activation beads for 4 days with human T-cells. The results demonstrate complete inhibition at 250 nM, partial inhibition at 100 nM and no significant inhibition at 50 nM.
- FIG. 22 D shows the dilution of proliferation dye (CFSE) into the progeny T-cells observed by the multiple peaks on the histograms which indicate that the controls including no activation (labeled No Beads) had no activation and the activation control with the CD80 beads had significant activation.
- FIG. 22 E is the graphical overview of the described results from above, the geometric mean of the CFSE is plotted vs the samples incubated.
- FIG. 22 F is the statistical analysis of the results using Dunnett's multiple comparisons test.
- FIGS. 23 A- 23 C demonstrate the titration of anti-CD20 mPDL1 fusion with CD20 activation beads from 250 nM to 50 nM incubated for 4 days with human T-cells. The results demonstrate complete inhibition at 250 nM, partial inhibition at 100 nM, and less inhibition at 50 nM.
- FIG. 23 D illustrates the dilution of proliferation dye (CFSE) into the progeny T-cells observed by the multiple peaks indicating that activation of the T-cells by the CD20 beads occurred.
- FIG. 23 E represents the graphical overview of the geometric mean of the proliferation dye CFSE.
- FIG. 23 F is the statistical analysis of the described data demonstrating that all of the concentrations of the anti-CD20 mPDL1 were statistically significantly different from the CD20 beads showing inhitibion of T-cell activation.
- the T-cell activation assay described above was performed on a sample combining TPP-985 (an anti-DLAT construct having no PD-L1 variable-like domain) with TPP-992 (an anti-DLAT IgG2/4 fused with PD-L1 on the N-terminus of the light chain) at 260 nM and 31 nM, respectively, in three replicates.
- TPP-985 an anti-DLAT construct having no PD-L1 variable-like domain
- TPP-992 an anti-DLAT IgG2/4 fused with PD-L1 on the N-terminus of the light chain
- FIG. 6 A shows that at the 260 nM concentration of the anti-DLAT PD-L1 fusions tested, TPP-992 (the anti-DLAT IgG2/4 construct with PD-L1 at the N-terminus of the light chain) demonstrated the greatest level of T-cell activation inhibition, with significantly greater activity inhibition than TPP-986 (the anti-DLAT IgG2/4 construct with PD-L1 at the N-terminus of the heavy chain). Both TPP-986 and TPP-992 showed significant inhibition of T-cell activation as compared to the human DLAT-aCD3 beads (referred to in the figure as DLAT Beads). TPP-992 was not significantly different than the no-activation NC beads, whereas TPP-986 was significantly different than the no-activation NC beads.
- FIG. 6 B illustrates the statistical analysis summary of all samples in the T-cell activation assay.
- FIG. 6 C illustrates the statistical analysis summary of T-cell activation inhibition for anti-DLAT antibody PD-L1 fusion proteins at the 65 nM concentration.
- FIG. 18 A illustrates that at 500 nM concentration of the anti-DLAT PD-L1 (TPP-1986), B7H4 (TPP-1504), HVEM (TPP-1506), B7H6 (TPP-1894) and CTLA4 (TPP-1898) fusions tested at day 7, inhibited T-cell activation.
- FIG. 18 B illustrates the statistical analysis summary of all samples in the T-cell activation assay.
- FIG. 18 C illustrates that at 500 nM concentration of the anti-DLAT PDL-2 (TPP-2215), CD200 (TPP-2216), TIM-3 (TPP-2220) and PD-5 (TTP-992) fusions showed inhibition of T cell activation at day 4.
- FIG. 18 D illustrates the statistical analysis summary of T-cell activation inhibition for anti-DLAT antibody fusion proteins.
- Dihydrolipoamide Acetyltransferase mouse T-cell activation beads (“mouse DLAT-aCD3 beads”) were prepared by conjugating 10 ⁇ g anti-mouse CD3, clone 145-2C11 (Stemcell Technologies, Catalog # 60015) to 250 ⁇ L of Dynabeads M-450 Epoxy (ThermoFisher, Catalog # 14011) for 24 hours rotating at room temperature in 0.1M sodium phosphate buffer, pH 8.0.
- mouse DLAT-aCD3 beads were washed and an excess of 50 ⁇ g of human DLAT recombinant protein (ProteinTech, Catalog # Ag22831) antigen was added to the mouse DLAT-aCD3 beads and incubated for an additional 48 hours at room temperature.
- the mouse DLAT-aCD3 beads were then washed and suspended to a concentration of 4 ⁇ 10 7 beads/mL in PBS with EasySep Buffer (Stemcell Technologies, Catalog # 20144) and 250 ⁇ L aliquots were prepared and stored at ⁇ 20° C.
- Negative control (“NC”) beads were previously prepared by conjugating human IgG Fc (Bethyl Labs, Catalog # P80-104) with uncoated beads for a no-activation control reagent. Bead conjugation was verified by flow cytometry prior to performing the mouse T-cell activation assay.
- Splenocytes were adjusted to 1e8 cells/mL using EasySep buffer per Mouse T-cell Isolation Kit (Stemcell Technologies, Catalog # 19851) protocol.
- Mouse T-cells were isolated following the protocol provided with the isolation kit using the Big Easy Magnet (Stemcell Technologies, Cat # 18001).
- Mouse T-cells isolated as described above were stained with 5 ⁇ M CFSE Cell Proliferation Kit (ThermoFisher, Catalog # C34554) dye in 10 mL EasySep buffer, incubated for 20 minutes in a 37° C. water bath, with sporadic mixing. 100 mL of TexMacs Media (Miltenyi Biosciences, Catalog # 130-097-196) was added to stained cells and incubated for 5 minutes at 37° C. water bath to quench dye.
- 5 CFSE Cell Proliferation Kit ThermoFisher, Catalog # C34554
- TexMacs Media (Miltenyi Biosciences, Catalog # 130-097-196) was added to stained cells and incubated for 5 minutes at 37° C. water bath to quench dye.
- mice were centrifuged for 10 minutes at 400 ⁇ g, supernatant discarded, and cell pellet was resuspended to 1.28e6 cells/mL with TexMacs Media supplemented with recombinant mouse IL-2 (Stemcell Technologies, Cat # 78081).
- 3 ⁇ L of mouse DLAT-aCD3 beads were added to a 96-well U-bottom tissue culture plate (Corning, Catalog #3799) followed by monoclonal antibody PD-L1 fusions (mAb-PDL1) to a final concentration of 500 nM and 100 nM in replicates of three.
- the samples of antibody immune cell inhibitor fusion proteins evaluated in the murine T-cell activation assay include molecules are listed in Table 7 below.
- This assay was performed in a 96-well U-bottom tissue culture plate (Corning, Catalog # 3799) with 1.3 ⁇ 10 5 cells/well with a 1:1 bead to cell ratio for 4 days at 37° C. with 5% CO 2 .
- flow cytometry was performed after staining with SYTOXTM Red Dead Cell stain (ThermoFisher, Catalog # S34859) following the manufacturer's protocol using the C6 Accuri Flow Cytometer with C6 Sampler (BD Biosciences) detectors FL-1 (CFSE) and FL-4 (SYTOXTM Red).
- Fab fusion was not as potent as mab, full length PD-L1 ECD not as potent as V-like domain only when tested as Fabs.
- HVEM Inhibited human T-cells as potently as PD-L1 fusion, (CD270) TPP-1506 vs. DLAT beads (P Value 0.0053).
- FIG. 12 A TPP-1986 provided complete inhibition of mouse T-cell activation at 500 nM with incomplete inhibition observed for 100 nM.
- FIG. 12 B illustrates that TPP-1694 showed a significant difference as compared to the NC beads, but showed no significant difference as compared to TPP-1986 at 500 nM.
- FIG. 12 C illustrates that TPP-1695 demonstrated a lower potency as compared to the PDS human PD-L1 fusion variants at 500 nM and 100 nM concentrations with similar results observed for TPP-1694.
- FIG. 12 D illustrates complete inhibition of mouse T-cell activation for TPP-992 at 500 nM with an incomplete inhibition for 100 nM.
- TPP-1697 non-targeted isotype control with human PD-L1
- TPP-1004 murine IgG1 PDS with no PD-L1 variable-like domain present
- FIGS. 13 A and 13 B show the statistical analysis of the mouse T-cell activation results at 500 nM.
- FIG. 13 A illustrates complete inhibition of mouse T-cells with TPP-1986 and TPP-992 fusions containing human PD-L1 as demonstrated by no significant difference as compared to the NC beads.
- FIG. 13 B is a table of statistical analysis between samples at 500 nM concentrations using Tukey's multiple comparisons test.
- FIGS. 14 A and 14 B show the statistical analysis of the mouse T-cell activation results at 500 nM.
- FIG. 14 A illustrates complete inhibition of mouse T-cells with TPP-TPP-992 with no statistically significant difference to TPP-1504 (hB7-H4) fusion and no significant difference as compared to the NC beads.
- TPP-1505 (hB7-H5) fusion shows no significant difference to the 1X beads suggesting it did not inhibit T-cell activation.
- FIG. 14 B is a table of statistical analysis between samples at 500 nM concentrations using Tukey's multiple comparisons test.
- FIGS. 15 and 16 show the statistical analysis of the mouse T-cell activation results at 100 nM.
- FIGS. 15 A and 16 A illustrate incomplete inhibition using fusions containing human PD-L1 and hB7-H4as compared to the NC beads. A significant difference of human versus murine PD-L1 containing fusions was observed, suggesting human PD-L1 results in greater inhibition of mouse T-cells than mouse PD-L1 V-like domain.
- An expected decrease in the geometric mean fluorescence intensity (Geo MFI) of FL-1 representing the CFSE dye diffusion into progeny cells for the 1X beads, TPP-1697, TPP-1004, and TPP-1505 was observed illustrating activation of the murine T-cells with no significant differences between samples.
- FIGS. 15 B and 16 B are tables of statistical analysis between samples at 100 nM concentrations using Tukey's multiple comparisons test.
- FIGS. 20 A- 20 C illustrate the results of mouse T-cell activation at 7 days gated on the CD4+cells.
- FIG. 20 A illustrates that at the 500 nM concentration of the anti-DLAT PD-L1 (TPP-1984) and CTLA4 (TPP-1898) fusions tested at day 7, inhibited mouse T-cell activation completely.
- FIG. 20 B illustrates that at the 125 nM concentration a lower level of T-cell activation inhibition was observed with TPP-1984 but not TPP-1898 that maintains no significant difference to the NC beads.
- FIG. 20 C illustrates the anti-DLAT CTLA4 (TPP-1898) completely inhibits mouse T-cell activation at a 32nM concentration in the 7 day assay.
- FIG. 20 D illustrates the statistical analysis summary of T-cell activation inhibition for anti-DLAT antibody fusion proteins at the three concentrations tested. The statistical analysis demonstrates the anti-DLAT CTLA4 is significantly more potent than the untargeted CTLA4 (Abatacept)
- FIGS. 21 A and 21 B demonstrate the induction of regulatory T-cells (Tregs—CD4+, CD25+, FoxP3+) with the PD-L1 fusions anti-DLAT_PDL1 (labeled DLAT DRUG) and anti-VAP-1_PDL1 (labeled VAP-1 DRUG) combined with the respective activation beads.
- FIG. 21 A illustrate flow cytometry results demonstrating low levels of CD25 for the no activation control (labeled NO BEADS) with 1.71% in quadrant 2 (Q2) representing the CD4+, CD25+ population.
- Q2 quadrant 2
- the histogram shown below the dot plot represents the intracellular staining with FoxP3 of the Q2 gated events.
- FIG. 21 B illustrates inhibition of the Teff population shown as 7% in Q2 with the addition of TPP-992 (labeled DLAT DRUG) to the DLAT activation beads, compared to 32% for DLAT beads only.
- TPP-992 labeled DLAT DRUG
- the FoxP3 positive events went from 7% to 16% with the drug.
- Anti-VAP-1_PDL1 previously showed weaker inhibition in the T-cell activation assay using proliferation dye compared to the anti-DLAT_PDL1 (data not shown). This intermediate effect of anti-VAP-1 fusion was confirmed by the results of this assay; a higher percentage of Q2 events were observed (18%), with a lower induction of Tregs 12% compared to anti-DLAT_PDL1.
Abstract
The disclosure provides antibody immune cell inhibitor fusion proteins comprising four polypeptide chains that form two antigen binding sites and at least two immune cell receptor binding sites that inhibit or diminish activation of an immune effector cell when bound to a target antigen. The disclosure also provides antibody immune cell inhibitor fusion proteins comprising two polypeptide chains that form one antigen binding site and at least one immune cell receptor binding site that inhibit or diminish activation of an immune effector cell when bound to a target antigen. The disclosure further provides pharmaceutical compositions and kits that comprise such antibody immune cell inhibitor fusion proteins, and methods of treatment using such proteins.
Description
- This application is a continuation of U.S. patent application Ser. No. 16/607,183, filed Oct. 22, 2019, which is a national stage application under 35 U.S.C. § 371 of International Application No. PCT/US2018/028963, filed Apr. 23, 2018, which claims priority to U.S. Provisional Patent Application Ser. No. 62/489,027, filed Apr. 24, 2017, the disclosures of which are incorporated by referenced herein in their entirety.
- A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on Mar. 20, 2023 having the file name “16-1519-WO-US.xml” and is 78,154 bytes in size.
- The disclosure relates to antibody immune cell inhibitor fusion proteins comprising four polypeptide chains that form two antigen binding sites and at least two immune cell receptor binding sites that inhibit or diminish activation of an immune effector cell when bound to a target antigen. The disclosure also relates to antibody immune cell inhibitor fusion proteins comprising two polypeptide chains that form one antigen binding site and at least one immune cell receptor binding site that inhibit or diminish activation of an immune effector cell when bound to a target antigen. The disclosure further relates to pharmaceutical compositions and kits that comprise such antibody immune cell inhibitor fusion proteins, and methods of treatment using such proteins.
- Autoimmune disorders are diseases caused by a dysfunction of the immune system, wherein the body produces an inappropriate immune response against its own tissues. As a result, the immune system creates B and T lymphocytes, autoantibodies, monocytes, NK cells, antigen presenting cells, and other immune factors that attack or facilitate immune responses to an individual's own cells, tissues, and/or organs.
- Autoimmune diseases are among the most prevalent diseases in the United States. The National Institutes of Health (NIH) estimates that up to 23.5 million Americans suffer from an autoimmune disease, and that the prevalence of such diseases is rising. Some of the current treatments for autoimmune diseases include administration of corticosteroid drugs, non-steroidal anti-inflammatory drugs (NSAIDs), or more powerful immunosuppressant drugs such as cyclophosphamide, methotrexate, and azathioprine that suppress the immune response and stop the progression of the disease. Radiation of the lymph nodes and plasmapheresis (a procedure that removes the diseased cells and harmful molecules from a patient's blood circulation) are other ways of treating an autoimmune disease. However, these treatments often have devastating long-term side effects.
- Primary Biliary Cholangitis (PBC) is an autoimmune disease of the liver characterized by T-lymphocyte mediated destruction of the intrahepatic bile ducts.
- The continuous attack on the bile duct epithelial cells leads to cholestasis, fibrosis, cirrhosis, liver failure, and death. Symptomatic patients generally become very ill in 3 to 5 years, and require a liver transplant soon thereafter. Ursodeoxycholic acid (UDCA) is the only FDA approved treatment for PBC, which ameliorates symptoms of the disease by primarily sequestering bile acid. Once symptoms manifest in an individual, life expectancy is less than 10 years, unless a liver transplant becomes available.
- The hallmark diagnosis for PBC is through the detection of serum autoantibodies known as anti-mitochondrial antibodies (AMAs) found in >95% of PBC patients. Most AMAs are reported to react with dihydrolipoamide 5-acetyltransferase (DLAT), the E2 subunit of the mitochondrial pyruvate dehydrogenase complex. Aberrant expression of DLAT or mimic antigens on biliary epithelial cells provide the target for an autoimmune attack in PBC.
- The disclosure provides an antibody immune cell inhibitor fusion protein comprising four polypeptide chains that form two antigen binding sites and at least two immune cell receptor binding sites; wherein two polypeptide chains have a structure represented by the formula II1-VL-CL-II2, and two polypeptide chains have a structure represented by the formula II3-VH-CH1-Fc-II4; wherein:
-
- VL is an immunoglobulin light chain variable domain;
- VL is an immunoglobulin heavy chain variable domain;
- CL is an immunoglobulin light chain constant domain;
- CH1 is the immunoglobulin CH1 heavy chain constant domain;
- Fc is the immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and
- II1, II2, II3, and II4 are each independently an immune cell inhibitor domain of an immunoglobulin superfamily member or are absent;
wherein at least one of II1, II2, II3, and II4 is an immune cell inhibitor domain of an immunoglobulin superfamily member; and wherein the antibody immune cell inhibitor fusion protein inhibits or diminishes activation of an immune effector cell only when bound to a target antigen at one or both of the antigen binding sites.
- The disclosure also provides an antibody immune cell inhibitor fusion protein comprising four polypeptide chains that form two antigen binding sites and at least two immune cell receptor binding sites; wherein two polypeptide chains have a structure represented by the formula II1-L1-VL-CL-L2-II2, and two polypeptide chains have a structure represented by the formula II3-L3-VH-CH1-Fc-L4-II4; wherein:
-
- VL is an immunoglobulin light chain variable domain;
- VH is an immunoglobulin heavy chain variable domain;
- CL is an immunoglobulin light chain constant domain;
- CH1 is the immunoglobulin CH1 heavy chain constant domain;
- Fc is the immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains;
- L1, L2, L3 and L4 are each independently a linker domain or are absent; and
- II1, II2, II3, and II4 are each independently an immune cell inhibitor domain of an immunoglobulin superfamily member or are absent;
wherein at least one of L1, L2, L3 and L4 is a linker domain and at least one of II1, II2, II3, and II4 is an immune cell inhibitor domain of an immunoglobulin superfamily member; and wherein the antibody immune cell inhibitor fusion protein inhibits or diminishes activation of an immune effector cell only when bound to a target antigen at one or both of the antigen binding sites.
- The disclosure further provides an antibody immune cell inhibitor fusion protein comprising two polypeptide chains that form one antigen binding site and at least one immune cell receptor binding site; wherein one polypeptide chain has a structure represented by the formula II1-VL-CL-II2, and one polypeptide chain has a structure represented by the formula II3-VH-CH1-II4; wherein:
-
- VL is an immunoglobulin light chain variable domain;
- VH is an immunoglobulin heavy chain variable domain;
- CL is an immunoglobulin light chain constant domain;
- CH1 is the immunoglobulin CH1 heavy chain constant domain; and
- II1, II2, II3, and II4 are each independently an immune cell inhibitor domain of an immunoglobulin superfamily member or are absent;
wherein at least one of II1, II2, II3, and II4 is an immune cell inhibitor domain of an immunoglobulin superfamily member; and wherein the antibody immune cell inhibitor fusion protein inhibits or diminishes activation of an immune effector cell only when bound to a target antigen at the antigen binding site.
- The disclosure further provides an antibody immune cell inhibitor fusion protein comprising two polypeptide chains that form one antigen binding site and at least one immune cell receptor binding site; wherein one polypeptide chain has a structure represented by the formula II1-L1-VL-CL-L2-II2, and one polypeptide chain has a structure represented by the formula II3-L3-VH-CH1-L4-II4; wherein:
-
- VL is an immunoglobulin light chain variable domain;
- VH is an immunoglobulin heavy chain variable domain;
- CL is an immunoglobulin light chain constant domain;
- CH1 is the immunoglobulin CH1 heavy chain constant domain;
- L1, L2, L3 and L4 are each independently a linker domain or are absent; and
- II1, II2, II3, and II4 are each independently an immune cell inhibitor domain of an immunoglobulin superfamily member or are absent;
wherein at least one of L1, L2, L3 and L4 is a linker domain and at least one of II1, II2, II3, and II4 is an immune cell inhibitor domain of an immunoglobulin superfamily member; and wherein the antibody immune cell inhibitor fusion protein inhibits or diminishes activation of an immune effector cell only when bound to a target antigen at the antigen binding site.
- Specific embodiments of the disclosure will become evident from the following more detailed description of certain embodiments and the claims.
-
FIGS. 1A-1G show bio-layer interferometry results of PD-1 binding to PD-L1 fusion proteins.FIG. 1A illustrates that TPP-993 (PD2 Fab having no PD-L1 domain and no linker) did not bind to PD-1 Fc because TPP-993 lacks a PD-L1 domain.FIGS. 1B and 1D illustrate that TPP-994 (PD2 with PD-L1 fused to the C-terminus of the heavy chain) and TPP-999 (PD2 with PD-L1 fused to the C-terminus of the light chain) exhibited very little binding to PD-1 Fc.FIGS. 1C and 1E illustrate that TPP-995 (PD2 with PD-L1 fused to the N-terminus of the heavy chain) and TPP-1001 (PD2 with PD-L1 fused to the N-terminus of the light chain) both exhibited binding to PD-1 Fc.FIG. 1F illustrates that PD-L1 Fc (a dimeric protein having two PD-L1 proteins fused to a human Fc) bound PD-1 very strongly.FIG. 1G illustrates that PD-1 Fc did not bind to itself, demonstrating that the Fc portion of PD-L1 Fc is not interacting with the tips. -
FIGS. 2A-2C show bio-layer interferometry results of mouse IgG1 chimera anti-DLAT antibodies to DLAT antigen.FIG. 2A illustrates slow on-rates and fast off-rates for the TPP-1003 PD2 antibody.FIGS. 2B and 2C illustrate slow off-rates for the TPP-1004 and TPP-1005 anti-DLAT antibodies. -
FIGS. 3A-3E show bio-layer interferometry results of simultaneous binding of human IgG2/4 anti-DLAT antibody PD-L1 V-like domain fusions to DLAT antigen and PD-1 Fc.FIG. 3A illustrates that TPP-985 (human IgG2/4 PD5 antibody) bound to the DLAT antigen, but did not bind to PD-1 Fc.FIGS. 3B and 3D illustrate that TPP-986 and TPP-992 (fusions of the PD-L1 V-like domain to the N-terminus of anti-DLAT antibodies) both exhibited the ability to simultaneously bind the DLAT antigen and PD-1 Fc.FIG. 3C illustrates that TPP-990 (PD-L1 V-like domain fused to the C-terminus of an anti-DLAT antibody) bound the DLAT antigen, but did not bind PD-1 Fc.FIG. 3E illustrates that the PD-L1 Fc control bound to PD-1 Fc, but did not bind to the DLAT antigen. -
FIGS. 4A-4F show human T-cell activation for targeted PD-L1 fusion inhibition. Representative histograms of one of three replicates are shown for each sample at 260 nM and 65 nM concentrations of antibody fusions or non-targeted PD-L1 Fc.FIG. 4A illustrates that NC beads exhibited no activation of T-cells, whereas the DLAT-aCD3 beads demonstrated clear activation of the pan T-cells.FIG. 4B illustrates that TPP-985 (an anti-DLAT IgG2-4 antibody with no PD-L1 variable-like domain) exhibited no significant inhibition of T-cell activation.FIG. 4C illustrates that TPP-986 (an anti-DLAT antibody with PD-L1 fused at the N-terminus of the heavy chain) exhibited significant inhibition of T-cell activation at both the 65 nM and 260 nM concentrations.FIG. 4D illustrates that TPP-992 (an anti-DLAT antibody with PD-L1 fused at the N-terminus of the light chain) exhibited the most significant inhibition of T-cell activation, with complete inhibition at the 260 nM concentration.FIG. 4E illustrates that the non-targeting PD-L1 Fc chimera exhibited no significant inhibition of T-cell activation.FIG. 4F illustrates that the titration of TPP-992 demonstrated a dose-dependent inhibition of T-cell activation with an EC50 of 17 nM±4.5nM. -
FIGS. 5A-5C show that targeting by the PD-L1 fusion is necessary for inhibition of T-cell activation.FIG. 5A illustrates a significant difference (** P value=0.0084) between the TPP-985 and TPP-992-competed sample versus the TPP-992 sample alone.FIG. 5B illustrates that there was no significant inhibition for any of the PD-L1 anti-DLAT fusions using TA2 beads that lack the DLAT antigen.FIG. 5C illustrates that TA2 bead activation showed no significant difference between samples. -
FIGS. 6A-6D show the statistical analysis of the human T-cell activation results.FIG. 6A illustrates that at the 260 nM concentration of the anti-DLAT PD-L1 fusions tested, TPP-992 (anti-DLAT IgG2-4 with PD-L1 fused at the N-terminus of the light chain) demonstrated the greatest level of T-cell activation inhibition with no significant difference to the NC beads (P value=0.464).FIG. 6B illustrates the statistical analysis summary of all samples in the T-cell activation assay.FIG. 6C illustrates that at the 65 nM concentration, a lower level of T-cell activation inhibition was observed for all samples. TPP-992 demonstrated the most significant inhibition of T-cell activation.FIG. 6D illustrates the statistical analysis summary of T-cell activation inhibition for anti-DLAT antibody PD-L1 fusion proteins at the 65 nM concentration. -
FIGS. 7A-7B illustrate a schematic for T-cell activation and inhibition assays using anti-DLAT antibody PD-L1 fusion proteins.FIG. 7A is a table listing the samples used in the T-cell activation and inhibition assays.FIG. 7B illustrates exemplary antibody immune cell inhibitor fusion proteins or fragments thereof. -
FIG. 8 is a schematic illustrating DLAT-aCD3 beads, T-cell activation beads (TA2 beads), and negative control (NC) beads used in human T-cell activation assays. DLAT-aCD3 beads were prepared by conjugating anti-human CD3 antibody and human DLAT recombinant protein, TA2 beads were prepared by conjugating anti-human CD3 antibody-coated beads with human IgG Fc, and NC beads were prepared by conjugating human IgG Fc with uncoated beads to prepare a no-activation control reagent. -
FIG. 9 depicts the binding affinity of TPP-992 (PD5 with PD-L1 V-like domain fused to the N-terminus of the light chain). TPP-992 had an affinity of 184 pM as determined using a single curve analysis by BIAcore. -
FIG. 10 depicts an electrospray ionization—time of flight mass spectra for TPP-985 and TPP-992. -
FIGS. 11A-11D illustrate the BIAcore kinetics results for TPP-1003, TPP-1004, and TPP-1005.FIG. 11A illustrates that TPP-1003 and TPP-1004 had pico-molar binding to the DLAT antigen and TPP-1005 had a single digit nano-molar affinity to the DLAT antigen.FIGS. 11B, 11C and 11D illustrate the BIAcore kinetics for TPP-1003, TPP-1004, andTPP 1005, respectively. -
FIGS. 12A-12F show mouse T-cell activation for targeted PD-L1 fusion inhibition. Samples were gated to exclude the low Forward Scatter (FSC) and the FL-4+ (SYTOX™ Red) dead cells. The same gate was utilized to analyze all samples. The histograms shown are gated on the live mouse T-cells. The decrease in signal of the FL-1 mean fluorescence measurement represents the activated T-cells that have expanded new generations of T-cells, with decreasing dye concentration in each respective generation.FIG. 12A illustrates complete inhibition of mouse T-cell activation for TPP-1986 at 500 nM with an incomplete inhibition observed for 100 nM.FIG. 12B illustrates that TPP-1694 had a significant difference compared to the NC beads, but showed no significant difference compared to TPP-1986 at 500 nM. A significant difference was observed at 100 nM for human TPP-1986 and TPP-992 versus mouse PD-L1-TPP-1694.FIG. 12C illustrates that TPP-1695 demonstrated a lower potency compared to the PDS human PD-L1 fusion variants at 500 nM and 100 nM concentrations with similar results observed for TPP-1694.FIG. 12D illustrates complete inhibition of mouse T-cell activation for TPP-992 at 500 nM with an incomplete inhibition for 100 nM.FIG. 12E and 12F illustrate that TPP-1697 (non-targeted isotype control with human PD-L1) and TPP-1004 (murine IgG1 PDS with no PD-L1 variable-like domain present), showed no significant inhibition of mouse T-cell activation. -
FIGS. 13A and 13B show the statistical analysis of the mouse T-cell activation results at 500 nM.FIG. 13A illustrates complete inhibition of TPP-1986 and TPP-992 fusions containing human PD-L1 demonstrated by a not significant difference compared to the NC beads. An expected decrease in the geometric mean fluorescence intensity (Geo MFI) of FL-1 representing the CFSE dye diffusion into progeny cells for the 1X beads, TPP-1697, and TPP-1004 was observed, illustrating activation of the murine T-cells with no significant differences between samples.FIG. 13B is a table of statistical analysis between samples at 500 nM concentrations using Tukey's multiple comparisons test. -
FIGS. 14A and 14B show the statistical analysis of the mouse T-cell activation results at 500 nM.FIG. 14A illustrates complete inhibition of TPP-1986 and TPP-992 fusions containing human PD-L1 demonstrated by a not significant difference compared to the NC beads and incomplete inhibition of TPP-1504 containing hB7-H4. An expected decrease in the geometric mean fluorescence intensity (Geo MFI) of FL-1 representing the CFSE dye diffusion into progeny cells for the 1X beads, TPP-1697 and TPP-1505 was observed, illustrating activation of the murine T-cells.FIG. 14B is a table of statistical analysis between samples at 500 nM concentrations using Tukey's multiple comparisons test. -
FIGS. 15A and 15B show the statistical analysis of the mouse T-cell activation results at 100 nM.FIG. 15A illustrates incomplete inhibition using fusions containing human PD-L1 compared to the NC beads. A significant difference of human versus murine PD-L1 containing fusions was observed, suggesting human PD-L1 results in greater inhibition of mouse T-cells than mouse PD-L1 V-like domain. An expected decrease in the geometric mean fluorescence intensity (Geo MFI) of FL-1 representing the CFSE dye diffusion into progeny cells for the 1X beads, TPP-1697, and TPP-1004 was observed, illustrating activation of the murine T-cells with no significant differences between samples.FIG. 15B is a table of statistical analysis between samples at 100 nM concentrations using Tukey's multiple comparisons test. -
FIGS. 16A and 16B show the statistical analysis of the mouse T-cell activation results at 100 nM.FIG. 16A illustrates incomplete inhibition using fusions containing human PD-L1 and hB7-H4 as compared to the NC beads. An expected decrease in the geometric mean fluorescence intensity (Geo MFI) of FL-1 representing the CFSE dye diffusion into progeny cells for the 1X beads, TPP-1697 andTPP 1505 was observed illustrating activation of the murine T-cells.FIG. 16B is a table of statistical analysis between samples at 100 nM concentrations using Tukey's multiple comparisons test. -
FIG. 17 illustrates that mouse PD-L1(TPP-1964) binds to human PD-1 with a faster on-rate than human PD-L1. PD-1 Fc (R&D Systems) was loaded onto anti-human IgG tips illustrated by the first observed deflection. The two fusions TPP-1964 (msPDL1) and TPP-1986 (huPDL1) bound PD-1 Fc. The increased slope observed for TPP-1964 (B1 sensor) suggests the mouse PD-L1 has a faster on-rate than the human PD-L1 on the TPP-1986 fusion (G1 Fusion). This binding steps followed by the dissociation in PBS buffer. -
FIGS. 18A-18D show the statistical analysis of the human T-cell activation results.FIG. 18A illustrates that at the 500 nM concentration of the anti-DLAT PD-L1 (TPP-1986), B7H4 (TPP-1504), HVEM (TPP-1506), B7H6 (TPP-1894) and CTLA4 (TPP-1898) fusions tested atday 7 inhibited T-cell activation.FIG. 18B illustrates the statistical analysis summary of all samples in the T-cell activation assay.FIG. 18C illustrates that at the 500 nM concentration of the anti-DLAT fusions only PDL-2 (TPP-2215), CD200 (TPP-2216), TIM-3 (TPP-2220) and PD-L1 (TTP-992) showed inhibition of T cell activation atday 4.FIG. 18D illustrates the statistical analysis summary of T-cell activation inhibition for anti-DLAT antibody fusion proteins. -
FIGS. 19A and 19B illustrates human T-cell activation at 4 days for 500 nM concentration of PD-L1 Fab fusions.FIG. 19B illustrates the statistical analysis summary of T-cell activation inhibition for PD-L1 fab fusions. V=V-like domain of PDL1; V−C=V- +C-like domains of PDL1; HC=PDL1 fused to N-terminus of Heavy Chain and LC=PDL1 fused to N-terminus of Light Chain. -
FIGS. 20A-20D show the statistical analysis of the mouse CD4+T-cell activation results.FIG. 20A illustrates that at 500 nM concentration of the anti-DLAT PD-L1 (TPP-1984), PD-5 (TPP-1694), and CTLA4 (TPP-1898) fusions tested atday 7 inhibited T-cell activation.FIG. 20B illustrates the statistical analysis summary of all samples in the T-cell activation assay.FIG. 20C illustrates that at the 125 nM a lower level of T-cell activation inhibition was observed with TPP-1898 showing the highest level of inhibition.FIG. 20D illustrates the statistical analysis summary of T-cell activation inhibition for anti-DLAT antibody fusion proteins. -
FIGS. 21A and 21B demonstrate the induction of regulatory T-cells (Tregs—CD4+, CD25+, FoxP3+) with the PD-L1 fusions anti-DLAT_PDL1 (labeled DLAT DRUG) and anti-VAP-1_PDL1 (labeled VAP-1 DRUG) combined with the respective activation beads.FIG. 21A illustrate flow cytometry results demonstrating low levels of CD25 for the no activation control (labeled NO BEADS) with 1.71% in quadrant 2 (Q2) representing the CD4+, CD25+ population. The histogram shown below the dot plot represents the intracellular staining with FoxP3 of the Q2 gated events. The activated T-cells (labeled DLAT) showed a significant increase in effector T-cells (Teff) in Q2, however a small percentage of these CD4+, CD25+ cells were Tregs (FoxP3+).FIG. 21B illustrates inhibition of the Teff population shown as 7% in Q2 with the addition of TPP-992 (labeled DLAT DRUG) to the DLAT activation beads, compared to 32% for DLAT beads only. In addition, from the Q2 population, the FoxP3 positive events went from 7% to 16% with the drug. Anti-VAP-1_PDL1 previously showed weaker inhibition in the T-cell activation assay using proliferation dye compared to the anti-DLAT_PDL1 (data not shown). This intermediate effect of anti-VAP-1 fusion was confirmed by the results of this assay; a higher percentage of Q2 events were observed (18%), with a lower induction ofTregs 12% compared to anti-DLAT_PDL1. -
FIGS. 22A-22C illustrate histograms showing itration from 250 nM to 50 nM of anti-CD80_mPDL1 fusion incubated with CD80 activation beads for 4 days with human T-cells. The results demonstrate complete inhibition at 250 nM, partial inhibition at 100 nM and no significant inhibition at 50 nM.FIG. 22D shows the dilution of proliferation dye (CFSE) into the progeny T-cells observed by the multiple peaks on the histograms which indicate that the controls including no activation (labeled No Beads) had no activation and the activation control with the CD80 beads had significant activation.FIG. 22E is the graphical overview of the described results from above, the geometric mean of the CFSE is plotted vs the samples incubated.FIG. 22F is the statistical analysis of the results using Dunnett's multiple comparisons test. -
FIGS. 23A-23C demonstrate the titration of anti-CD20_mPDL1 fusion with CD20 activation beads from 250 nM to 50 nM incubated for 4 days with human T-cells. The results demonstrate complete inhibition at 250 nM, partial inhibition at 100 nM, and less inhibition at 50 nM.FIG. 23D illustrates the dilution of proliferation dye (CFSE) into the progeny T-cells observed by the multiple peaks indicating that activation of the T-cells by the CD20 beads occurred.FIG. 23E represents the graphical overview of the geometric mean of the proliferation dye CFSE.FIG. 23F is the statistical analysis of the described data demonstrating that all of the concentrations of the anti-CD20 mPDL1 were statistically significantly different from the CD20 beads showing inhitibion of T-cell activation. - The disclosure provides antibody immune cell inhibitor fusion proteins comprising four polypeptide chains that form two antigen binding sites and at least two immune cell receptor binding sites and that inhibit or diminish activation of an immune effector cell when bound to a target antigen. The disclosure also provides antibody immune cell inhibitor fusion proteins comprising two polypeptide chains that form one antigen binding site and at least one immune cell receptor binding site that inhibit or diminish activation of an immune effector cell when bound to a target antigen. The disclosure further provides pharmaceutical compositions and kits that comprise such antibody immune cell inhibitor fusion proteins, and methods of treatment using such proteins.
- Standard recombinant DNA methodologies are used to construct the polynucleotides that encode the polypeptides that form the antibody immune cell inhibitor fusion proteins of the disclosure, incorporate these polynucleotides into recombinant expression vectors, and introduce such vectors into host cells. See e.g., Green and Sambrook, 2012, M
OLECULAR CLONING : A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, 4th ed.). Enzymatic reactions and purification techniques may be performed according to manufacturer's protocols, as commonly accomplished in the art, or as described herein. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Similarly, conventional techniques may be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, delivery, and treatment of patients. - As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
- The term “antibody immune cell inhibitor fusion protein” as used herein refers to a non-naturally occurring (or recombinant) molecule which comprises four polypeptide chains that form two antigen binding sites and at least two immune cell receptor binding sites; wherein two polypeptide chains have a structure represented by the formula II1-VL-CL-II2, and two polypeptide chains have a structure represented by the formula II3-VH-CH1-Fc-II4, wherein:
-
- VL is an immunoglobulin light chain variable domain;
- VH is an immunoglobulin heavy chain variable domain;
- CL is an immunoglobulin light chain constant domain;
- CH1 is the immunoglobulin CH1 heavy chain constant domain;
- Fc is the immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and
- II1, II2, II3, and II4 are each independently an immune cell inhibitor domain of an immunoglobulin superfamily member or are absent;
wherein at least one of II1, II2, II3, and II4 is an immune cell inhibitor domain of an immunoglobulin superfamily member; and wherein the antibody immune cell inhibitor fusion protein inhibits or diminishes activation of an immune effector cell only when bound to a target antigen at one or both of the antigen binding sites.
- The term “antibody immune cell inhibitor fusion protein” as used herein also refers to a non-naturally occurring (or recombinant) molecule which comprises four polypeptide chains that form two antigen binding sites and at least two immune cell receptor binding sites; wherein two polypeptide chains have a structure represented by the formula II1-L1-VL-CL-L2-II2, and two polypeptide chains have a structure represented by the formula II3-L3-VH-CH1-Fc-L4-II4; wherein:
-
- VL is an immunoglobulin light chain variable domain;
- VH is an immunoglobulin heavy chain variable domain;
- CL is an immunoglobulin light chain constant domain;
- CH1 is the immunoglobulin CH1 heavy chain constant domain;
- Fc is the immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains;
- L1, L2, L3 and L4 are each independently a linker domain or are absent; and
- II2, II3, and II4 are each independently an immune cell inhibitor domain of an immunoglobulin superfamily member or are absent;
wherein at least one of L1, L2, L3 and L4 is a linker domain and at least one of II1, II2, II3, and II4 is an immune cell inhibitor domain of an immunoglobulin superfamily member; and wherein the antibody immune cell inhibitor fusion protein inhibits or diminishes activation of an immune effector cell only when bound to a target antigen at one or both of the antigen binding sites.
- The term “antibody immune cell inhibitor fusion protein” as used herein also refers to a non-naturally occurring (or recombinant) molecule which comprises two polypeptide chains that form one antigen binding site and at least one immune cell receptor binding site; wherein one polypeptide chain has a structure represented by the formula II1-VL-CL-II2, and one polypeptide chain has a structure represented by the formula II3-VH-CH1-II4, wherein:
-
- VL is an immunoglobulin light chain variable domain;
- VH is an immunoglobulin heavy chain variable domain;
- CL is an immunoglobulin light chain constant domain;
- CH1 is the immunoglobulin CH1 heavy chain constant domain;
- II1, II2, II3, and II4 are each independently an immune cell inhibitor domain of an immunoglobulin superfamily member or are absent;
wherein at least one of II2, II3, and II4 is an immune cell inhibitor domain of an immunoglobulin superfamily member; and wherein the antibody immune cell inhibitor fusion protein inhibits or diminishes activation of an immune effector cell only when bound to a target antigen at the antigen binding site.
- The term “antibody immune cell inhibitor fusion protein” as used herein also refers to a non-naturally occurring (or recombinant) molecule which comprises two polypeptide chains that form one antigen binding site and at least one immune cell receptor binding site, wherein one polypeptide chain has a structure represented by the formula II1-L1-VL-CL-L2-II2, and one polypeptide chain has a structure represented by the formula II3-L3-VH-CH1-L4-II4, wherein:
-
- VL is an immunoglobulin light chain variable domain;
- VH is an immunoglobulin heavy chain variable domain;
- CL is an immunoglobulin light chain constant domain;
- CH1 is the immunoglobulin CH1 heavy chain constant domain;
- L1, L2, L3 and L4 are each independently a linker domain or are absent; and
- II2, II3, and II4 are each independently an immune cell inhibitor domain of an immunoglobulin superfamily member or are absent;
wherein at least one of L1, L2, L3 and L4 is a linker domain and at least one of II1, II2, II3, and II4 is an immune cell inhibitor domain of an immunoglobulin superfamily member; and wherein the antibody immune cell inhibitor fusion protein inhibits or diminishes activation of an immune effector cell only when bound to a target antigen at the antigen binding site.
- The term “antibody” as used herein refers to a protein that is capable of recognizing and specifically binding to an antigen. Ordinary or conventional mammalian antibodies comprise a tetramer, which is typically composed of two identical pairs of polypeptide chains, each pair consisting of one “light” chain (typically having a molecular weight of about 25 kDa) and one “heavy” chain (typically having a molecular weight of about 50-70 kDa). The terms “heavy chain” and “light chain,” as used herein, refer to any immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen. The amino-terminal portion of each light and heavy chain typically includes a variable domain of about 100 to 110 or more amino acids that typically is responsible for antigen recognition. The variable domain may be subjected to further protein engineering to humanize the framework regions if the antibody was derived from a non-human source. The carboxyl-terminal portion of each chain typically defines a constant domain responsible for effector function. Thus, in a naturally occurring antibody, a full-length heavy chain immunoglobulin polypeptide includes a variable domain (VH) and three constant domains (CH1, CH2, and CH3) and a hinge region between CH1 and CH2, wherein the VH domain is at the amino-terminus of the polypeptide and the CH3 domain is at the carboxyl-terminus, and a full-length light chain immunoglobulin polypeptide includes a variable domain (VL) and a constant domain (CL), wherein the VL domain is at the amino-terminus of the polypeptide and the CL domain is at the carboxyl-terminus.
- Within full-length light and heavy chains, the variable and constant domains typically are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. The variable regions of each light/heavy chain pair typically form an antigen binding site. The variable domains of naturally occurring antibodies typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair typically are aligned by the framework regions, which may enable binding to a specific epitope. From the amino-terminus to the carboxyl-terminus, both light and heavy chain variable domains typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
- The term “IgG2/4” as used herein refers to the non-naturally occurring, protein-engineered, heavy chain developed for use in eculizumab (CH1-Hinge-CH2-CH3) and designed to reduce immune effector function and immune cell activation, minimize immunogenicity, and contribute to a longer half-life. This non-IgG 1, 2, 3 or 4 heavy chain is a chimera containing sequence elements of IgG4 and IgG2 including two disulfide bonds in the hinge and changes in the constant regions CH1 and CH2.
- The term “human antibody” as used herein includes antibodies having variable and constant regions substantially corresponding to circulating human antibodies and human germline immunoglobulin sequences. In some embodiments, human antibodies are produced in non-human transgenic mammals, including, but not limited to, rodents, such as mice and rats, and lagomorphs, such as rabbits. In other embodiments, human antibodies are produced in hybridoma cells. In still other embodiments, human antibodies are produced recombinantly.
- The term “antibody fragment” refers to a portion of an intact or full-length chain or an antibody, generally the target binding or variable region. Examples of antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)2 and Fv fragments. As used herein, the term “functional fragment” is generally synonymous with “antibody fragment,” and with respect to antibodies, can refer to antibody fragments such as Fv, Fab, F(ab′)2.
- The term “autoantibody” as used herein refers to a naturally obtained antibody produced by an individual, wherein the antibody recognizes and binds to an antigen derived from or which mimics an antigen derived from a human tissue. An autoantibody can facilitate an immune response against a self-tissue or self-antigen (i.e., antigens that are native to the individual, e.g., an antigen on a cell or tissue, or an endogenous peptide or protein). Autoantibodies frequently arise or are triggered by an infection with an infectious agent such as a virus, bacteria or parasite, where the infectious agent carried a structure that induces antibodies to the structure. The induced antibodies can become harmful in the infection aftermath where that inducing biologic structure is also found on naturally occurring human tissues, thus creating an aberrant and continuing autoantibody response in the aftermath of the infection. The autoantibody can be redesigned and repurposed, as described herein, from the natural autoantibody to modify the original structure found in a diseased individual. The autoantibody may be re-humanized and the heavy chain may be modified to alter the isotype or to otherwise modulate harmful secondary immune functions of the autoantibody. The modified autoantibody can be used to produce a therapeutic fusion protein to treat the disorder created by an aberrant autoantibody response. The autoantibody may also be re-engineered and/or re-derived from or into a single chain Camelid VHH antibody format.
- The term “antigen” or “target antigen” as used herein refers to a molecule or a portion of a molecule that is capable of being recognized by and bound by an antibody or the antigen binding portion of the antibody immune cell inhibitor fusion proteins of the disclosure. The target antigen is capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen. A target antigen may have one or more epitopes. With respect to each target antigen recognized by an antibody or the antigen binding portion of the antibody immune cell inhibitor fusion protein, the antibody or the antigen binding portion of the fusion protein is capable of competing with an intact antibody that recognizes the target antigen.
- The term “epitope” as used herein refers to a region or structural element of an antigen that is recognized and bound by an antibody or the antigen binding portion of the antibody immune cell inhibitor fusion protein of the disclosure. More precisely, the epitope is the specific structure that is bound by the CDRs of the antibody. Epitopes can comprise protein structural elements, carbohydrates or even portions of lipid structures found in membranes. An antibody or the antigen binding portion of the antibody immune cell inhibitor fusion protein is said to specifically bind an antigen when it preferentially recognizes its antigen target in a complex mixture of proteins and/or macromolecules. The term “specifically binds,” as used herein, refers to the ability of an antibody or antigen binding portion of the antibody immune cell inhibitor fusion protein to bind to an antigen containing an epitope with an Kd of at least about 1×10−6 M, 1×10−7 M, 1×10−8 M, 1×10−9 M, 1×10−10 M, 1×10−11 M, 1×10−12 M, or more, and/or to bind to an epitope with an affinity that is at least two-fold greater than its affinity for a nonspecific antigen.
- The term “antigen binding site” as used herein refers to a site created on the surface of an antibody or the antigen binding portion of the antibody immune cell inhibitor fusion protein of the disclosure where an antigen or an epitope on an antigen is bound. The antigen binding site of an antibody or the antigen binding portion or surface of the antibody immune cell inhibitor fusion protein is typically described by reference to the loop structures created by complementarity determining regions (CDRs) of the antibody or antibody immune cell inhibitor fusion protein.
- The term “ligand” as used herein refers to a chemical molecule or biological molecule that can bind readily to a receptor with a specific binding affinity constant. The ligand may be natural or synthetic.
- The term “receptor” as used herein refers to a protein capable of interacting (binding) with a ligand. In some embodiments, such a protein is capable of transmitting information resulting from interaction with a ligand, into a cell.
- In some circumstances, the terms ligand and receptor may be interchangeable because both the ligand and receptor may be surface bound proteins on different cells that interact with each other. Depending on which cell is the focal point, a receptor on one cell is a ligand of another receptor on a different cell however if the cell which is the focal point is reversed, so is the receptor ligand relationship. In some circumstances both interacting molecules are surface bound and the receptor-ligand relationship is not strict, but merely designates two different molecules interacting with another and causing cell signaling consequences.
- The term “native Fc” as used herein refers to a molecule comprising the sequence of a non-antigen binding fragment resulting from digestion of an antibody or produced by other means, whether in monomeric or multimeric form, and can contain the hinge region. The original immunoglobulin source of the native Fc is preferably of human origin and can be any of the immunoglobulins. Native Fc molecules are made up of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). One example of a native Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG. The term “native Fc” as used herein is generic to the monomeric, dimeric, and multimeric forms.
- The term “Fc variant” as used herein refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn (neonatal Fc receptor). Exemplary Fc variants, and their interaction with the salvage receptor, are known in the art. Thus, the term “Fc variant” can comprise a molecule or sequence that is humanized from a non-human native Fc. Furthermore, a native Fc comprises regions that can be removed or mutated to produce an Fc variant to alter certain residues that provide structural features or biological activity that are not required for the antibody immune cell inhibitor fusion proteins of the disclosure. Thus, the term “Fc variant” comprises a molecule or sequence that lacks one or more native Fc sites or residues, or in which one or more Fc sites or residues has been modified, that affect or are involved in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).
- The term “Fc domain” as used herein encompasses native Fc and Fc variants and sequences as defined above. As with Fc variants and native Fc molecules, the term “Fc domain” includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by other means.
- An antibody or antibody immune cell inhibitor fusion protein of the disclosure described herein can, in some embodiments, comprise a variant human Fc constant region that binds to human neonatal Fc receptor (FcRn) with greater affinity than that of the native human Fc constant region from which the variant human Fc constant region was derived. For example, the Fc constant region can comprise one or more (e.g., two, three, four, five, six, seven, or eight or more) amino acid substitutions relative to the native human Fc constant region from which the variant human Fc constant region was derived. The substitutions can increase the binding affinity of an IgG antibody or antibody immune cell inhibitor fusion protein containing the variant Fc constant region to FcRn at pH 6.0, while maintaining the pH dependence of the interaction. See, e.g., Hinton et al., 2004, J. Biol. Chem. 279(8): 6213-16; and Datta-Mannan et al., 2007, Drug Metab. Dispos. 35(1): 86-94. Methods for testing whether one or more substitutions in the Fc constant region of an antibody increases the affinity of the Fc constant region for FcRn at pH 6.0 (while maintaining pH dependence of the interaction) are known in the art. See, e.g., Datta-Mannan et al., 2007, J. Biol. Chem. 282(3): 1709-17; International Publication Nos. WO 98/23289 and WO 97/34631; and U.S. Pat. No. 6,277,375, the disclosures of each of which are incorporated herein by reference in their entirety.
- Substitutions that enhance the binding affinity of an antibody Fc constant region for FcRn are known in the art and include, for example: (1) the M252Y/S254T/T256E triple substitution described by Dall'Acqua et al., 2006, J. Biol. Chem. 281(33): 23514-24; (2) the M428L or T250Q/M428L substitutions described in Hinton et al., 2004, J. Biol. Chem. 279(8): 6213-16, and Hinton et al., 2006, J. Immunol. 176(1): 346-56; and (3) the N434A or T307/E380A/N434A substitutions described in Petkova et al., 2006, Int. Immunol. 18(12): 1759-69, the disclosures of which are incorporated herein by reference in their entirety. The additional substitution pairings P257I/Q311I, P257I/N434H, and D376V/N434H are described in, for example, Datta-Mannan et al., 2007, J. Biol. Chem. 282(3): 1709-17, the disclosure of which is incorporated herein by reference in its entirety.
- Many mutations to modify Fc biological properties have been identified and may be useful depending on the biology of the disease being treated. In some embodiments, the variant constant region has a substitution at EU amino acid residue 255 for valine. In some embodiments, the variant constant region has a substitution at EU amino acid residue 309 for asparagine. In some embodiments, the variant constant region has a substitution at EU amino acid residue 312 for isoleucine. In some embodiments, the variant constant region has a substitution at EU amino acid residue 386 for leucine.
- In some embodiments, the variant Fc constant region comprises no more than (e.g., no more than 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2) amino acid substitutions, insertions, or deletions relative to the native constant region from which it was derived. In some embodiments, the variant Fc constant region comprises one or more amino acid substitutions selected from the group consisting of M252Y, S254T, T256E, N434S, M428L, V259I, T250I, and V308F. In some embodiments, the variant human Fc constant region comprises a methionine at position 428 and an asparagine at position 434, each in EU numbering. In some embodiments, the variant Fc constant region comprises a 428L/4345 double substitution as described in, for example, U.S. Pat. No. 9,079,949.
- In some embodiments, when the eculizumab heavy chain is used as the starting heavy chain, the 428L/434S mutations are shifted to 429L/435S as a result of the IgG2/4 chimerization engineering. Furthermore, the precise mutations in the eculizumab heavy chain are Met-429-Leu and Asn-435-Ser. See U.S. Pat. No. 9,079,949, the disclosure of which is hereby incorporated by reference in its entirety.
- In some embodiments, the variant constant region comprises a substitution at amino acid position 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434, or 436 (EU numbering) relative to the native human Fc constant region. In some embodiments, the substitution is selected from the group consisting of methionine for glycine at position 237; alanine for proline at position 238; lysine for serine at position 239; isoleucine for lysine at position 248; alanine, phenylalanine, isoleucine, methionine, glutamine, serine, valine, tryptophan, or tyrosine for threonine at position 250; phenylalanine, tryptophan, or tyrosine for methionine at position 252; threonine for serine at position 254; glutamic acid for arginine at position 255; aspartic acid, glutamic acid, or glutamine for threonine at position 256; alanine, glycine, isoleucine, leucine, methionine, asparagine, serine, threonine, or valine for proline at position 257; histidine for glutamic acid at position 258; alanine for aspartic acid at position 265; phenylalanine for aspartic acid at position 270; alanine, or glutamic acid for asparagine at position 286; histidine for threonine at position 289; alanine for asparagine at position 297; glycine for serine at position 298; alanine for valine at position 303; alanine for valine at position 305; alanine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, valine, tryptophan, or tyrosine for threonine at position 307; alanine, phenylalanine, isoleucine, leucine, methionine, proline, glutamine, or threonine for valine at position 308; alanine, aspartic acid, glutamic acid, proline, or arginine for leucine or valine at position 309; alanine, histidine, or isoleucine for glutamine at position 311; alanine or histidine for aspartic acid at position 312; lysine or arginine for leucine at position 314; alanine or histidine for asparagine at position 315; alanine for lysine at position 317; glycine for asparagine at position 325; valine for isoleucine at position 332; leucine for lysine at position 334; histidine for lysine at position 360; alanine for aspartic acid at position 376; alanine for glutamic acid at position 380; alanine for glutamic acid at position 382; alanine for asparagine or serine at position 384; aspartic acid or histidine for glycine at position 385; proline for glutamine at position 386; glutamic acid for proline at position 387; alanine or serine for asparagine at position 389; alanine for serine at position 424;alanine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan, or tyrosine for methionine at position 428; lysine for histidine at position 433; alanine, phenylalanine, histidine, serine, tryptophan, or tyrosine for asparagine at position 434; and histidine for tyrosine or phenylalanine at position 436 (all in EU numbering).
- An “immune cell inhibitor domain” as used herein refers to an immunoglobulin domain of an immunoglobulin superfamily member that can cause suppression of immune responses upon binding to the specific receptor on an immune system cell, including inhibition of T-cells, B-cells, monocytes, and antigen presenting cells, inhibition of a particular immune cell function, including cytotoxicity, or any combination of the above responses. The immune cell inhibitor domains of the antibody immune cell inhibitor fusion proteins of the disclosure are referred to herein as follows: II1, which, when present, is fused (with or without an intervening linker) to the N-terminal end of the light chain; II2, which, when present, is fused (with or without an intervening linker) to the C-terminal end of the light chain; II3, which, when present, is fused (with or without an intervening linker) to the N-terminal end of the heavy chain; and II4, which, when present, (with or without an intervening linker) is fused to the C-terminal end of the heavy chain. Linkers may or may not be needed depending on the where the stop and start residues of protein fusions are chosen because often natural linkers are found between immunoglobulin domains.
- The term “immunoglobulin superfamily member” as used herein refers to a class of proteins that are associated with the adhesion, binding, and recognition processes of cells. Exemplary members of the immunoglobulin superfamily include, but are not limited to, Programmed Death Ligand 1 (PD-L1), also known as B7 Homolog 1 (B7-H1), or CD274; Programmed Death Ligand 2 (PD-L2), also known as B7-DC; B7 Homolog 3 (B7-H3), as known as CD276; B7 Homolog 4 (B7-H4), also known as V-set Domain Containing T-cell Activation Inhibitor 1 (VTCN1), B7 Superfamily, Member 1 (B7S1), or B7x; Herpesvirus Entry Mediator (HVEM), also known as Herpesvirus Entry Mediator A (HVEA), Tumor Necrosis Factor Receptor Superfamily, Member 14 (TNFRSF14), or CD270; V-type Immunoglobulin Domain-Containing Suppressor of T-cell Activation; Chromosome 10 Open reading Frame 54 (C10orf54), also known as Death Domain 1-Alpha (DD1-Alpha); B7 Homolog 6 (B7-H6), also known as Natural Cytotoxicity Triggering Receptor 3 ligand 1; Human Endogenous Retrovirus-H Long Terminal Repeat-Associating 2 (HHLA2), also known as B7 Homolog 7 (B7-H7), B7 Homolog 5 (B7-H5), or HERV-H LTR-Associating 2; Cytotoxic T Lymphocyte-Associated 4 (CTLA-4), also known as CD152; CD200, also known as Membrane Glycoprotein MRC OX-2 or MOX2; Killer-cell Immunoglobulin-Like Receptor (KIR); T-cell Immunoglobulin and Mucin Domains-Containing Protein 3 (TIM-3), also known as Hepatitis A Virus Cellular Receptor 2 (HAVCR2); and Lymphocyte Activation Gene 3 (LAG3), also known as CD223.
- The term “immune effector cell” as used herein refers to the cells of the immune system that mount immune responses to an antigen. Suitable effector cells include but are not limited to populations of antigen presenting cells, cytotoxic T-cells, and T helper cells that mediate cellular immunity. In addition to antigen-specific effector T-cells, the effector cell populations may include, but are not limited to, other cytotoxic immune cells against a selected antigen such as natural killer cells, lymphocytes, monocytes, macrophages, neutrophils, and eosinophils.
- The term “linker” as used herein refers to one or more amino acid residues inserted between immunoglobulin domains and/or immune cell inhibitor domains of the antibody immune cell inhibitor fusion proteins of the disclosure. For example, a linker may be inserted between an immunoglobulin domain and an immune cell inhibitor domain, at the sequence level. The precise location of a domain transition can be determined by locating peptide stretches that do not form secondary structural elements such as beta-sheets or alpha-helices as demonstrated by experimental data or as can be assumed by techniques of modeling or secondary structure prediction. Linkers may or may not be needed depending on the where the stop and start residues of protein fusions are chosen because often natural linkers are found between immunoglobulin domains. The linkers of the antibody immune cell inhibitor fusion proteins of the disclosure are referred to herein as follows: L1, which, when present, is located on the light chain between the Ili and VL domains; L2, which, when present, is located on the light chain between the CL and II2 domains; L3, which, when present, is located on the heavy chain between the II3 and L3 domains; and L4, which, when present, is located on the heavy chain between the Fc and II4 domains. The linkers L1, L2, L3, and L4 are independent, but in some embodiments of the antibody immune cell inhibitor fusion proteins of the disclosure may have the same sequence and/or length.
- The term “naturally occurring” as used herein and applied to an object refers to the fact that the object can be found in nature and has not been manipulated by man. For example, a polynucleotide or polypeptide that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man is naturally occurring. Similarly, “non-naturally occurring” as used herein refers to a protein molecule that is not found in nature or that has been structurally modified through protein engineering and synthesized, manufactured, or produced by man using recombinant DNA technologies in appropriate cells such as, for example, CHO cells.
- A “recombinant” molecule is one that has been prepared, expressed, created, or isolated by recombinant DNA technology means.
- The term “fusion protein” as used herein refers to protein constructs comprising an immunoglobulin domain and an immune cell inhibitor protein. The immune cell inhibitor protein in some fusion proteins of the disclosure may not constitute the entire natural protein but may be limited to an active domain of the entire protein responsible for binding to a corresponding receptor on the surface of an immune function cell. In addition, an immunoglobulin domain in some fusion proteins of the disclosure may not constitute the entire natural immunoglobulin domain but may be limited to a portion of the natural immunoglobulin domain responsible for specifically binding a target antigen or epitope or conferring other properties of the natural immunoglobulin domain. Importantly, the fragment of the immune cell inhibitor protein or immunoglobulin domain in some fusion proteins of the disclosure would not be naturally occurring as the fragment, but may retain the same protein sequence for the fragment and incorporated into a therapeutic fusion protein.
- The terms “inhibit” or “diminish” as used herein refer to a complete or partial arrest of immune effector cell activation.
- The terms “substantially pure” or “substantially purified” as used herein refer to a compound or species that is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition). In some embodiments, a substantially purified fraction is a composition wherein the species comprises at least about 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition will comprise more than about 80%, 85%, 90%, 95%, or 99% of all macromolar species present in the composition. In still other embodiments, the species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.
- The phrases “biological property,” “biological characteristic,” and the term “activity” in reference to an antibody or an antibody immune cell inhibitor fusion protein of the disclosure are used interchangeably herein and include, but are not limited to, epitope affinity and specificity, ability to antagonize the activity of the antigen target (or targeted polypeptide), the in vivo stability of the antibody or antibody immune cell inhibitor fusion protein, and the immunogenic properties of the antibody or antibody immune cell inhibitor fusion protein. Other identifiable biological properties or characteristics of an antibody or an antibody immune cell inhibitor fusion protein include, for example, cross-reactivity, (i.e., with non-human homologs of the antigen target, or with other antigen targets or tissues, generally), and ability to preserve high expression levels of protein in mammalian cells. The aforementioned properties or characteristics can be observed or measured using art-recognized techniques.
- A “neutralizing effect” of an antibody immune cell inhibitor fusion protein as used herein refers to a fusion protein of an antibody and an immune cell inhibitor ligand that is able to first, bind an antigen for which the antigen binding portion of the antibody immune cell inhibitor fusion protein specifically recognizes, then, through simultaneous binding of the fused immune cell inhibitor domain to a specific receptor, to block or substantially reduce an unwanted deleterious or autoimmune effector function carried out by the cell expressing the immune cell inhibitor receptor. As used herein, “substantially reduce” means at least about 60%, preferably at least about 70%, more preferably at least about 75%, even more preferably at least about 80%, still more preferably at least about 85%, most preferably at least about 90% reduction of the unwanted or autoimmune effector function of the cell carrying the ligand's receptor.
- The term “neutralizing antibody” refers to an antibody that can neutralize the function of the protein or infectious agent the antibody specifically recognizes and binds. Typically, neutralizing antibodies refer to antibodies specific for viral, bacterial, or other infectious agents. Where therapeutic antibodies are used as drug candidates to treat human disease, neutralizing antibodies can arise as anti-drug antibodies (ADA) but have the undesirable function of neutralizing the therapeutic benefit of the therapeutic antibody. Neutralizing antibodies to the antigen binding portions of the fusion proteins of the present disclosure would represent an undesirable development.
- The term “KD,” as used herein, refers to the dissociation constant (KD=[A]×[B]/[AB]) of the interaction between an antibody or an antibody immune cell inhibitor fusion protein of the disclosure and an antigen target and has the units of moles/liter.
- An antibody or antibody immune cell inhibitor fusion protein of the disclosure typically has a dissociation constant (KD) of 10−5 to 10−12 moles/liter or less, or 10−7 to 10−12 moles/liter or less, or 10−3 to 10−12 moles/liter, and/or with a binding affinity of at least 107 M−1, or at least 108 M−1, or at least 109 M−1, or at least 1012 M−1. Any KD value greater than 10−4 moles/liter is generally considered to indicate non-specific binding. Therefore, the lower the KD value, the greater the affinity. In some embodiments, a monovalent antibody or antibody immune cell inhibitor fusion protein of the disclosure will bind to a desired antigen with an affinity less than 500 nM, or less than 200 nM, or less than 10 nM, or less than 500 pM. High affinity or very strong binding is often associated with greater efficacy, but it is not always the case that the greater the affinity the greater the efficacy.
- The dissociation constant (KD) can be determined, for example, by surface plasmon resonance (SPR). Generally, surface plasmon resonance analysis measures real-time binding interactions (both on rate and off rate) between a ligand (a target antigen on a biosensor matrix) and an analyte by surface plasmon resonance using, for example, the BIAcore system (Pharmacia Biosensor; Piscataway, NJ). Surface plasmon analysis can also be performed by immobilizing the analyte and presenting the ligand. Specific binding of an antibody or an antibody immune cell inhibitor fusion protein of the disclosure to an antigen or antigenic determinant can also be determined in any suitable manner known in the art, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme linked immunosorbent assays (ELISA), enzyme immunoassays (EIA), and sandwich competition assays.
- The term “vector,” as used herein, refers to any molecule (e.g., nucleic acid, plasmid, or virus) that is used to transfer coding information to a host cell. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA molecule into which additional DNA segments may be inserted. Another type of vector is a viral vector, wherein additional DNA segments may be inserted into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
- The term “operably linked” is used herein to refer to an arrangement of flanking sequences wherein the flanking sequences are configured or assembled so as to perform their usual function. Thus, a flanking sequence operably linked to a coding sequence may be capable of effecting the replication, transcription, and/or translation of the coding sequence. For example, a coding sequence is operably linked to a promoter when the promoter is capable of directing transcription of that coding sequence.
- The term “host cell,” as used herein, refers to a cell into which an expression vector has been introduced. A host cell is intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but such cells are still included within the scope of the term “host cell” as used herein. A wide variety of host cell expression systems can be used to express the antibody immune cell inhibitor fusion proteins of the disclosure, including bacterial, yeast, baculoviral, and mammalian expression systems (as well as phage display expression systems).
- Examples of cultured mammalian cell lines include Chinese Hamster ovary (CHO) simian cells such as COS, murine cell lines such as NSO, and human cell lines such as HEK and HeLa, which may be used to produce the antibody immune cell inhibitor fusion proteins of the disclosure. Vectors are transfected into the cells and the DNA may be integrated into the genome by homologous recombination in the case of stable transfection, or the cells may be transiently transfected. Examples of mammalian expression vectors include adenoviral vectors, the pSV and the pCMV series of plasmid vectors, vaccinia and retroviral vectors, as well as baculovirus. The promoters for cytomegalovirus (CMV) and simian virus 40 (SV40) are commonly used in mammalian expression vectors to drive gene expression. Non-viral promoters, such as the elongation factor (EF)-1 promoter, may also be used.
- One embodiment of the disclosure provides nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form an antibody immune cell inhibitor fusion protein of the disclosure. Another embodiment of the disclosure provides expression vectors comprising nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form the antibody immune cell inhibitor fusion proteins of the disclosure. Yet another embodiment of the disclosure provides host cells that express such antibody immune cell inhibitor fusion proteins (i.e., comprising nucleic acid molecules or vectors encoding polypeptide chains that form such antibody immune cell inhibitor fusion proteins).
- In some embodiments, the disclosure provides methods for preparing an antibody immune cell inhibitor fusion protein of the disclosure, wherein such methods comprise cultivating or maintaining a host cell under conditions such that the host cell produces or expresses such antibody immune cell inhibitor fusion proteins, and optionally further comprises isolating the antibody immune cell inhibitor fusion protein so produced.
- A skilled artisan will be able to determine suitable variants of the polypeptide chains of the antibody immune cell inhibitor fusion proteins of the disclosure using well-known techniques. For example, one skilled in the art may identify suitable areas of a polypeptide chain that may be changed without destroying activity by targeting regions not believed to be important for activity. Alternatively, one skilled in the art can identify residues and portions of the molecules that are conserved among similar polypeptides. In addition, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.
- The term “patient” as used herein includes human and animal subjects.
- A “disorder” is any condition that would benefit from treatment using the antibody immune cell inhibitor fusion proteins of the disclosure. “Disorder” and “condition” are used interchangeably herein and include chronic and acute disorders or diseases, including those pathological conditions that predispose a patient to the disorder in question. A “T-cell mediated-condition” is a condition directly or indirectly effected by the T-cells of the immune system.
- The terms “treatment” or “treat” as used herein refer to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those having a disorder as well as those prone to have the disorder or those in which the disorder is to be prevented.
- The terms “pharmaceutical composition” or “therapeutic composition” as used herein refer to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient. One embodiment of the disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of at least one antibody immune cell inhibitor fusion protein of the disclosure.
- The term “pharmaceutically acceptable carrier” or “physiologically acceptable carrier” as used herein refers to one or more formulation materials suitable for accomplishing or enhancing the delivery of one or more antibody immune cell inhibitor fusion proteins of the disclosure.
- The terms “effective amount” and “therapeutically effective amount” when used in reference to a pharmaceutical composition comprising one or more antibody immune cell inhibitor fusion proteins of the disclosure refer to an amount or dosage sufficient to produce a desired therapeutic result. More specifically, a therapeutically effective amount is an amount of an antibody immune cell inhibitor fusion protein sufficient to inhibit, for some period of time, one or more of the clinically defined pathological processes associated with the condition being treated. The effective amount may vary depending on the specific antibody immune cell inhibitor fusion protein that is being used, and may also depend on a variety of factors and conditions related to the patient being treated and the severity of the disorder. For example, if the antibody immune cell inhibitor fusion protein is to be administered in vivo, factors such as the age, weight, and health of the patient as well as dose response curves and toxicity data obtained in preclinical animal work would be among those factors considered. The determination of an effective amount or therapeutically effective amount of a given pharmaceutical composition is within the ability of those skilled in the art.
- In one embodiment of the disclosure, the antibody immune cell inhibitor fusion protein comprises four polypeptide chains that form two antigen binding sites at least two immune cell receptor binding sites, wherein two polypeptide chains have a structure represented by the formula II1-VL-CL-II2, and two polypeptide chains have a structure represented by the formula II3-VH-CH1-Fc-II4; wherein:
-
- VL is an immunoglobulin light chain variable domain;
- VH is an immunoglobulin heavy chain variable domain;
- CL is an immunoglobulin light chain constant domain;
- CH1 is the immunoglobulin CH1 heavy chain constant domain;
- Fc is the immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and
- II1, II2, II3, and II4 are each independently an immune cell inhibitor domain of an immunoglobulin superfamily member or are absent;
wherein at least one of II1, II2, II3, and II4 is an immune cell inhibitor domain of an immunoglobulin superfamily member; and wherein the antibody immune cell inhibitor fusion protein inhibits or diminishes activation of an immune effector cell only when bound to a target antigen at one or both of the antigen binding sites.
- In another embodiment of the disclosure, the antibody immune cell inhibitor fusion protein comprises four polypeptide chains that form two antigen binding sites and at least two immune cell receptor binding sites, wherein two polypeptide chains have a structure represented by the formula II1-L1-VL-CL-L2-II2, and two polypeptide chains have a structure represented by the formula II3-L3-VH-CH1-Fc-L4-II4; wherein:
-
- VL is an immunoglobulin light chain variable domain;
- VH is an immunoglobulin heavy chain variable domain;
- CL is an immunoglobulin light chain constant domain;
- CH1 is the immunoglobulin CH1 heavy chain constant domain;
- Fc is the immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains;
- L1, L2, L3 and L4 are each independently a linker domain or are absent; and
- II1, II2, II3, and II4 are each independently an immune cell inhibitor domain of an immunoglobulin superfamily member or are absent;
wherein at least one of L1, L2, L3 and L4 is a linker domain and at least one of II1, II2, II3, and II4 is an immune cell inhibitor domain of an immunoglobulin superfamily member; and wherein the antibody immune cell inhibitor fusion protein inhibits or diminishes activation of an immune effector cell only when bound to a target antigen at one or both of the antigen binding sites.
- In another embodiment of the disclosure, the antibody immune cell inhibitor fusion protein comprises two polypeptide chains that form one antigen binding site and at least one immune cell receptor binding site; wherein one polypeptide chain has a structure represented by the formula II1-VL-CL-II2, and one polypeptide chain has a structure represented by the formula II3-VH-CH1-II4; wherein:
-
- VL is an immunoglobulin light chain variable domain; VH is an immunoglobulin heavy chain variable domain; CL is an immunoglobulin light chain constant domain;
- CH1 is the immunoglobulin CH1 heavy chain constant domain; and
- II1, II2, II3, and II4 are each independently an immune cell inhibitor domain of an immunoglobulin superfamily member or are absent;
wherein at least one of II1, II2, II3, and II4 is an immune cell inhibitor domain of an immunoglobulin superfamily member; and wherein the antibody immune cell inhibitor fusion protein inhibits or diminishes activation of an immune effector cell only when bound to a target antigen at the antigen binding site.
- In another embodiment of the disclosure, the antibody immune cell inhibitor fusion protein comprises two polypeptide chains that form one antigen binding site and at least one immune cell receptor binding site; wherein one polypeptide chain has a structure represented by the formula II1-L1-VL-CL-L2-II2, and one polypeptide chain has a structure represented by the formula II3-L3-VH-CH1-L4-II4; wherein:
-
- VL is an immunoglobulin light chain variable domain;
- VH is an immunoglobulin heavy chain variable domain;
- CL is an immunoglobulin light chain constant domain;
- CH1 is the immunoglobulin CH1 heavy chain constant domain;
- L1, L2, L3 and L4 are each independently a linker domain or are absent; and
- II1, II2, II3, and II4 are each independently an immune cell inhibitor domain of an immunoglobulin superfamily member or are absent;
wherein at least one of L1, L2, L3 and L4 is a linker domain, and at least one of II1, II2, II3, and II4 is an immune cell inhibitor domain of an immunoglobulin superfamily member; and wherein the antibody immune cell inhibitor fusion protein inhibits or diminishes activation of an immune effector cell only when bound to a target antigen at the antigen binding site.
- The antibody immune cell inhibitor fusion proteins of the disclosure may be prepared using domains or sequences obtained or derived from any human or non-human antibody, including, for example, human, murine, or humanized antibodies. In some antibody immune cell inhibitor fusion proteins of the disclosure, the VL, VH, CL, CH1, and/or Fc domains of the antibody immune cell inhibitor fusion protein may not constitute the entire natural immunoglobulin domain, provided, however, that the portion of the VL, VH, CL, CH1, and/or Fc domain used in the antibody immune cell inhibitor fusion protein is capable of functioning in the same manner as the full-length natural immunoglobulin domain. In other embodiments, the antibody immune cell inhibitor fusion proteins of the disclosure may further comprise additional VL, VH, CL, CH1, and/or Fc domains.
- In some antibody immune cell inhibitor fusion proteins of the disclosure, one or more of II1, II2, II3, and II4 is an immune cell inhibitor domain. In some embodiments, at least one of the immune cell inhibitor domains can inhibit or diminish activation of an immune effector cell involved in the immune response to self-tissue. In other embodiments, at least one of the immune cell inhibitor domains can inhibit or diminish activation of an immune effector cell involved in the immune response to self-tissue when an autoantibody is bound to the antigen at the site of an ongoing disease process. In other embodiments, at least one of II1, II2, II3, and II4 is an immune cell inhibitor domain of an immunoglobulin domain superfamily member. In other embodiments, at least one of II1, II2, II3 and II4 is absent.
- In some embodiments, the immune cell inhibitor domain is obtained or derived from a member of the immunoglobulin superfamily. In certain embodiments, the immune cell inhibitor domain comprises a Programmed Death Ligand 1 (PD-L1), B7 Homolog 1 (B7-H1), or CD274 domain; Programmed Death Ligand 2 (PD-L2) or B7-DC domain; B7 Homolog 3 (B7-H3) or CD276 domain; B7 Homolog 4 (B7-H4), V-set Domain-Containing T-cell Activation Inhibitor 1 (VTCN1), B7 Superfamily, Member 1 (B7S1), or B7x domain; Herpesvirus Entry Mediator (HVEM), Herpesvirus Entry Mediator A (HVEA), Tumor Necrosis Factor Receptor Superfamily, Member 14 (TNFRSF14), or CD270 domain (also known as Tumor necrosis factor receptor superfamily member 14); V-type Immunoglobulin Domain-Containing Suppressor of T-cell Activation domain; B7 Homolog 6 (B7-H6) or Natural cytotoxicity triggering receptor 3 ligand 1 domain; Human Endogenous Retrovirus-H Long Terminal Repeat-Associating 2 (HHLA2), B7 Homolog 7 (B7-H7), B7 Homolog 5 (B7-H5), or HERV-H LTR-associating protein 2 domain; Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA-4) or CD152 domain; CD200, Membrane Glycoprotein MRC OX-2, or MOX2 domain; Killer-cell Immunoglobulin-like Receptor (KIR) domain; T-cell Immunoglobulin and Mucin Domains-Containing Protein 3 (TIM-3) or Hepatitis A Virus Cellular Receptor 2 (HAVCR2) domain; or Lymphocyte Activation Gene-3 (LAG3) or CD223 domain.
- In some embodiments, the immune cell inhibitor domain comprises a PD-L1 extracellular domain (wherein PD-L1 is also known as CD274, B7-H, B7H1, PDCD1L1, or PDCD1LG1) or a PD-L2 extracellular domain (wherein PD-L2 is also known as CD273). PD-L1 when bound to Programmed Death 1 (PD-1) inhibitory receptor (also known as CD279) acts as an immune checkpoint inhibitor of B-cells, T-cells, monocytes, and antigen presenting cells. For example, an activated T-cell expresses PD-1 on its surface upon antigen recognition and produces interferons, which induce expression of PD-L1 in multiple tissues. Binding of PD-1 to its ligand limits T-cell activity. Under normal conditions, the PD-1/PD-L1 pathway prevents excessive stimulation and maintains the immune tolerance to self-antigens by negatively regulating the immune response (Riella et al., 2012, Am. J. Transplant. 12(10): 2575-87).
- In one embodiment of the disclosure, the antibody immune cell inhibitor fusion protein comprises four polypeptide chains that form two antigen binding sites and at least two PD-1 binding sites; wherein two polypeptide chains have a structure represented by the formula II1-VL-CL-II2, and two polypeptide chains have a structure represented by the formula II3-VH-CH1-Fc-II4, wherein:
-
- VL is an immunoglobulin light chain variable domain;
- VH is an immunoglobulin heavy chain variable domain;
- CL is an immunoglobulin light chain constant domain;
- CH1 is the immunoglobulin CH1 heavy chain constant domain;
- Fc is the immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and
- II1, II2, II3, and II4 are each independently an immune cell inhibitor domain of an immunoglobulin superfamily member capable of binding PD-1 or are absent;
wherein at least one of II1, II2, II3, and II4 is an immune cell inhibitor domain of an immunoglobulin superfamily member capable of binding PD-1; and wherein the antibody immune cell inhibitor fusion protein inhibits or diminishes activation of an immune effector cell only when bound to a target antigen at one or both of the antigen binding sites.
- In another embodiment of the disclosure, the antibody immune cell inhibitor fusion protein comprises four polypeptide chains that form two antigen binding sites and at least two PD-1 binding sites; wherein two polypeptide chains have a structure represented by the formula II1-L1-VL-CL-L2-II2, and two polypeptide chains have a structure represented by the formula II3-L3-VH-CH1-Fc-L4-II4; wherein:
-
- VL is an immunoglobulin light chain variable domain;
- VH is an immunoglobulin heavy chain variable domain;
- CL is an immunoglobulin light chain constant domain;
- CH1 is the immunoglobulin CH1 heavy chain constant domain;
- Fc is the immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains;
- L1, L2, L3 and L4 are each independently a linker domain or are absent; and
- II2, II3, and II4 are each independently an immune cell inhibitor domain of an immunoglobulin superfamily member capable of binding PD-1 or are absent;
wherein at least one of L1, L2, L3 and L4 is a linker domain and at least one of II1, II2, II3, and II4 is an immune cell inhibitor domain of an immunoglobulin superfamily member capable of binding PD-1; and wherein the antibody immune cell inhibitor fusion protein inhibits or diminishes activation of an immune effector cell only when bound to a target antigen at one or both of the antigen binding sites.
- In another embodiment of the disclosure, the antibody immune cell inhibitor fusion protein comprises two polypeptide chains that form one antigen binding site and at least one PD-1 binding site; wherein one polypeptide chain has a structure represented by the formula II1-VL-CL-II2, and one polypeptide chain has a structure represented by the formula II3-VH-CH1-II4; wherein:
-
- VL is an immunoglobulin light chain variable domain;
- VH is an immunoglobulin heavy chain variable domain;
- CL is an immunoglobulin light chain constant domain;
- CH1 is the immunoglobulin CH1 heavy chain constant domain;
- II1, II2, II3, and II4 are each independently an immune cell inhibitor domain of an immunoglobulin superfamily member capable of binding PD-1 or are absent;
wherein at least one of II1, II2, II3, and II4 is an immune cell inhibitor domain of an immunoglobulin superfamily member capable of binding PD-1; and wherein the antibody immune cell inhibitor fusion protein inhibits or diminishes activation of an immune effector cell only when bound to a target antigen at the antigen binding site.
- In another embodiment of the disclosure, the antibody immune cell inhibitor fusion protein comprises two polypeptide chains that form one antigen binding site and at least one PD-1 binding site; wherein one polypeptide chain has a structure represented by the formula II1-L1-VL-CL-L2-II2, and one polypeptide chain has a structure represented by the formula II3-L3-VH-CH1-L4-II4, wherein:
-
- VL is an immunoglobulin light chain variable domain;
- VH is an immunoglobulin heavy chain variable domain;
- CL is an immunoglobulin light chain constant domain;
- CH1 is the immunoglobulin CH1 heavy chain constant domain;
- L1, L2, L3 and L4 are each independently a linker domain or are absent; and
- II1, II2, II3, and II4 are each independently an immune cell inhibitor domain of an immunoglobulin superfamily member capable of binding PD-1 or are absent;
wherein at least one of L1, L2, L3 and L4 is a linker domain and at least one of II1, II2, II3, and II4 is an immune cell inhibitor domain of an immunoglobulin superfamily member capable of binding PD-1; and wherein the antibody immune cell inhibitor fusion protein inhibits or diminishes activation of an immune effector cell only when bound to a target antigen at the antigen binding site.
- In some embodiments, the VL, VH, CL, and CH1 domains of the antibody immune cell inhibitor fusion proteins of the disclosure form an autoantibody that recognizes and binds an antigen derived from or mimicking an antigen derived from a human tissue. In some embodiments, the autoantibody facilitates an immune response to a self-tissue.
- In some embodiments of the disclosure, the antibody immune cell inhibitor fusion proteins of the disclosure are capable of specifically binding one or more antigen targets. In certain embodiments of the disclosure, the antibody immune cell inhibitor fusion protein is capable of specifically binding at least one antigen target that is Human serum albumin, Bone mineral (hydroxyapatite), Complement component 3d (C3d), Glutamic acid decarboxylase 65 (GAD65), Asialoglycoprotein receptor (ASGPR), Asialoglycoprotein receptor 2 (ASGPR2) Transferrin receptor protein 2 (TFR2), Solute carrier family 2, facilitated glucose (SLC2A2), Solute carrier organic anion transporter family member 1B1 (SLCO1B1), Solute carrier organic anion transporter family member 1B3 (SLCO1B3), Multidrug resistance-associated protein 6 (ABCC6), Major histocompatibility complex I (MHC I), Major histocompatibility complex II (MHC II), T-cell receptor (TCR), B-Cell receptor (BCR), Transforming growth factor beta, Cluster differentiation 4 (CD4), Cluster differentiation 8 (CD8), Cluster differentiation 11c (CD11c), cluster differentiation 14 (CD14), Fas ligand/Tumor necorsis factor super family 6 (Fas Ligand/TNFSF6), Fibrinogen (1F3), collagen type I, collagen type II, collagen type III, collagen type IV, Cystatin C, Cluster differentiation 133 (CD133), Cluster differentiation 10 (CD10), Cluster differentiation 13 (CD13), Cluster differentiation 20 (CD20), Cluster differentiation 80 (CD80), Neural cell adhesion molecule 1 (NCAM1), E-cadherin, Epithelial cell adhesion molecule (EPCAM), Epithelial Membrane Antigen (EMA), Transthyretin, Vascular adhesion protein 1, Lymphocyte function-associated antigen 3, or Dihydrolipoamide acetyltransferase. In some embodiments of the disclosure, the antibody immune cell inhibitor fusion protein is capable of inhibiting the function of one or more of the antigen targets.
- In some embodiments, the antibody immune cell inhibitor fusion proteins of the disclosure comprise a linker joining at least one immune cell inhibitor domain to at least one immunoglobulin light chain or heavy chain or domain. In some antibody immune cell inhibitor fusion proteins, L1, when present, may be 5 to 30 amino acid residues in length; L2, when present, may be 5 to 30 amino acid residues in length; L3, when present, may be 5 to 30 amino acid residues in length; and L4, when present, may be 5 to 30 amino acid residues in length.
- In some antibody immune cell inhibitor fusion proteins of the disclosure, at least one of L1, L2, L3 and L4 is absent.
- The identity and sequence of amino acid residues in the linker may vary depending on the type of secondary structural element necessary to be achieved. For example, glycine, serine, and alanine are best for linkers having maximum flexibility. Some combination of glycine, proline, threonine, and serine are useful if a more rigid and extended linker is necessary. Any amino acid residue may be considered as a linker in combination with one or more other amino acid residues, which may be the same as or different as the first amino acid residue, to construct larger peptide linkers as necessary depending on the desired properties.
- Therapeutic or pharmaceutical compositions comprising one or more antibody immune cell inhibitor fusion proteins of the disclosure are within the scope of the disclosure. Such therapeutic or pharmaceutical compositions can comprise a therapeutically effective amount of an antibody immune cell inhibitor fusion protein, or antibody immune cell inhibitor fusion protein-drug conjugate, in admixture with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration.
- Acceptable formulation materials are preferably nontoxic to recipients at the dosages and concentrations employed.
- The pharmaceutical composition can contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite, or sodium hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as serum albumin, gelatin, or immunoglobulins), coloring, flavoring and diluting agents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorbitan esters; polysorbates such as polysorbate 20 or polysorbate 80; triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancing agents (such as sucrose or sorbitol), tonicity enhancing agents (such as alkali metal halides—preferably sodium or potassium chloride—or mannitol sorbitol), delivery vehicles, diluents, excipients and/or pharmaceutical adjuvants (see, e.g., R
EMINGTON'S PHARMACEUTICAL SCIENCES (18th Ed., A. R. Gennaro, ed., Mack Publishing Company 1990), and subsequent editions of the same, incorporated herein by reference in their entirety for any purpose). - The optimal pharmaceutical composition will be determined by a skilled artisan depending upon, for example, the intended route of administration, delivery format, and desired dosage. Such compositions can influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the antibody immune cell inhibitor fusion proteins of the disclosure.
- The primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier for injection can be water, physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can further include sorbitol or a suitable substitute. In one embodiment of the disclosure, antibody immune cell inhibitor fusion protein compositions can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents in the form of a lyophilized cake or an aqueous solution. Further, the antibody immune cell inhibitor fusion protein can be formulated as a lyophilizate using appropriate excipients such as sucrose.
- The pharmaceutical compositions of the disclosure can be selected for parenteral delivery. Alternatively, the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within one of skill in the art.
- The formulation components are present in concentrations that are acceptable to the site of administration. For example, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.
- When parenteral administration is contemplated, the therapeutic compositions for use in this disclosure can be in the form of a pyrogen-free, parenterally acceptable, aqueous solution comprising the desired antibody immune cell inhibitor fusion protein in a pharmaceutically acceptable vehicle. One suitable vehicle for parenteral injection is sterile distilled water in which an antibody immune cell inhibitor fusion protein of the disclosure is formulated as a sterile, isotonic solution, properly preserved. Yet another preparation can involve the formulation of the antibody immune cell inhibitor fusion protein with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, that provides for the controlled or sustained release of the product which can then be delivered via a depot injection. Hyaluronic acid can also be used, and this can have the effect of promoting sustained duration in the circulation. Other suitable means for the introduction of the antibody immune cell inhibitor fusion protein include implantable drug delivery devices.
- Additional pharmaceutical compositions of the disclosure will be evident to those skilled in the art, including formulations involving antibody immune cell inhibitor fusion proteins in sustained- or controlled-delivery formulations. Techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles, or porous beads and depot injections, are also known to those skilled in the art. Additional examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Sustained-release compositions can also include liposomes, which can be prepared by any of several methods known in the art.
- Pharmaceutical compositions of the disclosure to be used for in vivo administration typically must be sterile. This can be accomplished by filtration through sterile filtration membranes. Where the composition is lyophilized, sterilization using this method can be conducted either prior to, or following, lyophilization and reconstitution. The composition for parenteral administration can be stored in lyophilized form or in a solution. In addition, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
- Once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations can be stored either in a ready-to-use form or in a form (e.g., lyophilized) requiring reconstitution prior to administration.
- Also encompassed by the disclosure are kits for producing a single-dose administration unit. The kits can each contain both a first container having a dried protein and a second container having an aqueous formulation. Also included within the scope of this disclosure are kits containing single and multi-chambered pre-filled syringes (e.g., liquid syringes and lyosyringes).
- The effective amount of pharmaceutical composition containing an antibody immune cell inhibitor fusion protein to be employed therapeutically will depend, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication for which the antibody immune cell inhibitor fusion protein is being used, the route of administration, and the size (body weight, body surface, or organ size) and condition (the age and general health) of the patient. Accordingly, the clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect. A typical dosage can range from about 0.1 mg/kg to up to about 100 mg/kg or more, depending on the factors mentioned above. In other embodiments, the dosage can range from 0.1 mg/kg up to about 100 mg/kg; or 1 mg/kg up to about 100 mg/kg; or 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, up to about 100 mg/kg.
- Dosing frequency will depend upon the pharmacokinetic parameters of the antibody immune cell inhibitor fusion protein in the formulation being used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect. The composition can therefore be administered as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages can be ascertained through use of appropriate dose-response data.
- The route of administration of the pharmaceutical composition is in accord with known methods, e.g., orally; through injection by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; by sustained release systems; or by implantation devices. Where desired, the compositions can be administered by bolus injection or continuously by infusion, or by implantation device.
- The composition can also be administered locally via implantation of a membrane, sponge, or other appropriate material onto which the desired molecule has been absorbed or encapsulated. Where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration.
- In some embodiments of the disclosure, the antibody immune cell inhibitor fusion proteins of the disclosure are used to treat a patient having Primary Biliary Cholangitis (PBC),
Type 1 Diabetes, Goodpasture's syndrome, Amyloidosis, Ankylosing spondylitis, Anti-glomerular basement membrane/anti-tubular basement membrane nephritis, Antiphospholipid syndrome, Autoimmune hepatitis, Autoimmune oophoritis Autoimmune pancreatitis, Autoimmune retinopathy, Behcet's disease, Crohn's disease, Devic's disease, Lupus, Dressler's syndrome, Fibrosing alveolitis, Glomerulonephritis, Graves' disease, Guillain-Barre syndrome, IgA Nephropathy, IgG4-related sclerosing disease, Immune thrombocytopenic purpura (ITP), Microscopic polyangiitis (MPA), Mixed connective tissue disease (MCTD), Multiple sclerosis, Polyneuropathy, Organomegaly, Endocrinopathy, Monoclonal syndrome (POEMS), Polyarteritis nodosa, Rheumatoid arthritis, Schmidt syndrome, Scleritis, Scleroderma, Sjögren's syndrome Sperm & testicular autoimmunity, Stiff person syndrome (SPS), Takayasu's arteritis, Temporal arteritis, Giant cell arteritis, Thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), Vasculitis or a T-cell mediated condition. - The Examples that follow are illustrative of specific embodiments of the disclosure, and various uses thereof. They are set forth for explanatory purposes only, and should not be construed as limiting the scope of the disclosure in any way.
- An antibody immune inhibitor fusion protein was designed comprising a human autoimmune antibody to a self-antigen identified as driving the disease process known as Primary Biliary Cholangitis (PBC), previously considered cirrhosis. The self-antigen recognized by the autoimmune antibody was shown to be Pyruvate Dehydrogenase Complex subunit E2 (PDC-E2, also known as Dihydrolipoamide Acetyltransferase or DLAT). The original fusion protein fused the extracellular domain of Programmed Death Ligand 1 (PD-L1, also known as CD274, or B7 Homolog 1 (B7-H1)), to each of the N- and C-termini of an engineered autoantibody containing the V regions of the autoantibody and an engineered Fc variant known as IgG2/4 to minimize effector function. See Pascual et al., 1994, J. Immunol. 152(5): 2577-85; and Chen et al., 1998, J. Autoimmun. 11(2): 151-61. The PD-L1 domain was fused to the antibody to interact with Programmed Death 1 (PD-1, CD279) receptor on T-cells believed to be driving the disease process, thereby inhibiting activation of the T-cells and subsequent activation of other immune system actors such as antigen presenting cells, B-cells, and any associated excreted cytokines to thus ameliorate the immune response causing the disease.
- Initially, multiple anti-DLAT antibodies were identified in the literature (Thomson et al., 1998, J. Hepatol. 28(4): 582-94). Two of them (designated PD2 and PD5) were initially selected for cloning as: (i) human full-length antibodies and were constructed with the autoantibody VH and VL regions fused with a non-naturally occurring engineered heavy chain including CH1-hinge-CH1-CH1 (SEQ ID NO: 35)(IgGp2/4); and (ii) human CH1 Fabs. Each of these antibodies or antibody fragments were constructed with or without the variable-like domain of the extracellular domain (ECD) of PD-L1 linked to either the C-terminus or the N-terminus of the heavy and/or light chains. Upon review of the crystal structure of the PD-1/PD-L1 complex, it was determined that the variable-like domain of PD-L1 was the binding portion, and thus the variable-like domain of PD-L1 was fused to each of the N- and C-termini of the anti-DLAT antibody to determine which orientation was most effective for this particular antibody-immune inhibitor pair. See Zak et al., 2015, Structure 23(12): 2341-48.
- Using Geneious 8.1.5 software, reference sequences (see Table 1) were imported and modified for construct design. Constructs were synthesized and cloned into suitable vectors and were sequence-verified prior to transient transfections. Protein expression was performed by transfection of constructs shown in Table 2 using the Invitrogen EXPI293F system. Protein A columns were used for purification of antibodies and antibody fusions or Nickel NTA was used for purification of the 6X His-tagged Fab proteins using commercial columns.
- Sequences for the constructs are shown in Table 3. The sequences are shown as monomeric sequences, but all full-length antibodies expressed and purified were, of course, dimeric. The heavy chain of the antibody is shown in regular text, the light chain of the antibody is shown in underlined text, the linker is shown in italicized text, and the immune cell inhibitor domain of an immunoglobulin superfamily member are shown in bold text.
-
TABLE 1 Reference Sequences Reference Sequences Accession # Source Human PD-L1 Reference Protein Sequence Q9NZQ7 UniProt Human PD-L1 Reference Protein Sequence NP_054862.1 NCBI Murine PD-L1 Reference Protein Sequence NP_068693.1 NCBI Murine PD-L1 Reference Protein Sequence Q9EP73 UniProt Crystal Structure of human PD-1 4ZQK Protein and its ligand PD-L1 Data Bank PD-1 binding domain from human PD-L1 5C3T Protein Data Bank DWZ Heavy Chain Reference Protein Sequence AJ001167.1 GenBank DWZ Light Chain Reference Protein Sequence AJ001168.1 GenBank PD2 Heavy Chain Reference Protein Sequence AJ001171.1 GenBank PD2 Light Chain Reference Protein Sequence AJ001172.1 GenBank Human B7 Dendritic Cell Q9BQ51 UniProt Molecule (B7-DC), Programmed cell death 1 ligand 2 (PD-L2) Human B7-Homolog 3 (B7-H3), (CD276) Q5ZPR3 UniProt Human B7-H4, V-set Q7Z7D3 UniProt domain-containing T-cell activation inhibitor 1 (VTCN1) Human B7-H5, V-type Q9H7M9 UniProt immunoglobulin domain-containing suppressor of T-cell activation (VISTA) Human B7-H6 (NCR3LG1), Q68D85 UniProt Natural cytotoxicity triggering receptor 3 ligand 1 Human B7-H7, HERV-H LTR-associating Q9UM44 UniProt protein 2 (HHLA2) Human Cytotoxic T-lymphocyte P16410 UniProt protein 4 (CTLA-4) Human Tumor necrosis factor receptor Q92956 UniProt superfamily member 14 (TNFRSF14), Herpesvirus Entry Mediator (HVEM) Human T-Cell Immunoglobulin Q8TDQ0 UniProt Mucin Family Member 3 (TIM-3), Hepatitis A virus cellular receptor 2 (HAVCR2) -
TABLE 2 Construct Descriptions Mole- Pro- cular tein Transfection Weight ID Isotype Constructs (kDa) Description TPP- Human IgG2/4 pME074 + 144 PD5_IgG2-4 985 pME090 TPP- Human IgG2/4 pME074 + 172.8 N-Terminus_PDL1_ 986 pME095 HC_PD5 TPP- Human IgG2/4 pME076 + 172.8 N-terminus_PDL1_ 992 pME090 LC_PD5 TPP- Human IgG2/4 pME075 + 172.8 PD5_LC_ 990 pME090 C-terminus_PDL1 TPP- Human IgG2/4 pME080 + 174.4 N-Terminus_PDL1_ 996 pME092 HC_PD2 TPP- Human IgG2/4 pME082 + 175 N-terminus_PDL1_ 1002 pME089 LC_PD2 TPP- Human IgG2/4 pME091 + 144 DWZ_IgG2-4 1007 pME102 TPP- Human IgG2/4 pME089 + 145.7 PD2_IgG2-4 1006 pME080 TPP- Human Fab pME071 + 46.7 PD5_Human_Fab 955 pME074 TPP- Human Fab pME072 + 61.1 PD5_LC_C-terminus 983 pME074 linker PDL1 TPP- Human Fab pME073 + 61.1 N-terminus_PDL1_ 984 pME074 LC_PD5 TPP- Human Fab pME076 + 61.1 N-terminus_PDL1_ 991 pME071 HC_PD5 TPP- Human Fab pME075 + 61.1 PD5_HC_C-terminus_ 989 pME071 PDL1 TPP- Human Fab pME077 + 48.4 PD2 Fab 993 pME080 TPP- Human Fab pME078 + 62.8 PD2_HC_C-terminus_ 994 pME080 PDL1 TPP- Human Fab pME079 + 62.8 N-terminus_PDL1_ 995 pME080 HC_PD2 TPP- Human Fab pME081 + 62.8 PD2_LC_ 999 pME077 C-terminus_PDL1 TPP- Human Fab pME077 + 62.8 N-terminus_PDL1_ 1001 pME082 LC_PD2 TPP- Murine IgG1 pME083 + 147.4 mlgG1 PD2 Ab 1003 pME084 TPP- Murine IgG1 pME085 + 145.4 mIgG1 PD5 Ab 1004 pME086 TPP- Murine IgG1 pME087 + 144.9 mlgG1 DWZ Ab 1005 pME088 TPP- N/A pME098 15.2 Human_PDL1_V-domain_ 1008 N-terminus TPP- N/A pME099 26 Human_PDL1_ 1009 V_C_Domain NP_054862.1 TPP- N/A pME100 13.9 Human_PDL1_V-domain 1010 NP_054862.1 TPP- N/A pME101 14.4 Human_PDL1_V-domain_ 1011 C-Terminus TPP- Murine IgG1 N/A 173 N-terminus_PDL1_ 1986 Fc Silent LC_PD5 TPP- Murine IgG1 pME085 + 173.8 N-terminus_ 1694 pADL0128 mPDL1_LC_PD5 TPP- Murine IgG1 pME087 + 173.3 N-terminus_ 1695 pADL0129 mPDL1_LC_DWZ TPP- Human IgG2/4 pADL0131 + 172.8 N-terminus_PDL1_ 1697 PADL0132 LC_12B4 TPP- Human IgG2/4 PADL0123 + 199.7 N-terminus_B7- 1504 pME090 H4_LC_PD5 TPP- Human IgG2/4 PADL0124 + 184.3 N-terminus_B7- 1505 pME090 H5_LC_PD5 TPP- Human IgG2/4 PADL0125 + 182.8 N-terminus_ 1506 pME090 HVEM_LC_PD5 TPP- Human IgG2/4 PADL0141 + 201.4 N-terminus_B7- 1894 pME090 H6_LC_PD5 TPP- Human IgG2/4 PADL0145 + 175.1 N-terminus_CTLA- 1898 pME090 4_LC_PD5 TPP- Human IgG2/4 PADL0126 + 170.8 N-terminus_PDL2_V- 1507 pME090 like_domain_LC_PD5 TPP- Human IgG2/4 PADL0176 + 193.2 N- 2215 pME090 terminus_PDL2_LC_PD5 TPP- Human IgG2/4 PADL0177 + 193 N-terminus_ 2216 pME090 CD200_LC_PD5 TPP- Human IgG2/4 PADL0179 + 242.2 N-terminus_B7- 2218 pME090 H3_LC_PD5 TPP- Human IgG2/4 pADL0181 + 188.1 N-terminus_TIM- 2220 pME090 3_LC_PD5 -
TABLE 3 Construct Sequences SEQ ID Sequence Construct NO: Description Name Sequence 1 PD5 HC Fab pME071 QLQLQESGPGLVKPSETLSLTCIVSGGSISRNSYYWG WIRQPPGKGLEWIGSIVYSGSTYHQPSLKSRVTIFLD TSKNQFFLKLTSVTAADTAVYYCARGTRATTWPPPIG YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVE PKSC 2 PD5 HC PDL1 pME072 QLQLQESGPGLVKPSETLSLTCIVSGGSISRNSYYWG Fab WIRQPPGKGLEWIGSIVYSGSTYHQPSLKSRVTIFLD TSKNQFFLKLTSVTAADTAVYYCARGTRATTWPPPIG YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVE PKSCGGSSRSSSSGGGGSGGGG FTVTVPKDLYVVEYG SNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHG EEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQ DAGVYRCMISYGGADYKRITVKVNA 3 PDL1 HC PD5 pME073 FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIV Fab YWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQL SLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVK VNA GGSSRSSSSGGGGSGGGGQLQLQESGPGLVKPSE TLSLTCIVSGGSISRNSYYWGWIRQPPGKGLEWIGSI VYSGSTYHQPSLKSRVTIFLDTSKNQFFLKLTSVTAA DTAVYYCARGTRATTWPPPIGYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKRVEPKSC 4 PD5 LC Fab pME074 QSVLTQPPSVSAAPGQKVTVSCFGSSSNIGNYFASWY Human Lamda LC QQLPGAAPRLLIYGNNERPSGIPDRESGSKSGTSATL VITGLQTGDEAAYYCATWDSSLSAVVEGGGTKLTVLG QPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAV TVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLT PEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 5 PD5 LC PDLI pME075 QSVLTQPPSVSAAPGQKVTVSCFGSSSNIGNYFASWY QQLPGAAPRLLIYGNNERPSGIPDRESGSKSGTSATL VITGLQTGDEAAYYCATWDSSLSAVVFGGGTKLTVLG QPKANPTVTLEPPSSEELQANKATLVCLISDFYPGAV TVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLT PEQWKSHRSYSCQVTHEGSTVEKTVAPTECS GGSSRS SSSGGGGSGGGG FTVTVPKDLYVVEYGSNMTIECKFP VEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSS YRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMIS YGGADYKRITVKVNA 6 PDL1 LC PD5 pME076 FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIV YWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQL SLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVK VNA GGSSRSSSSGGGGSGGGG QSVLTQPPSVSAAPGQ KVTVSCFGSSSNIGNYFASWYQQLPGAAPRLLIYGNN ERPSGIPDRESGSKSGTSATLVITGLQTGDEAAYYCA TWDSSLSAVVEGGGTKLTVLGQPKANPTVTLEPPSSE ELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVE TTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTH EGSTVEKTVAPTECS 7 PD2 HC Fab pME077 EVQLVESGGGLVKPGGSLRLSCAASGFTFGSYSMTWV RQAPGKGLEWVSFISTVSTYIYYPDSVKGRFTISRDN AKNSLYLQMNSLRAEDTAVYYCARVSGRGSAARDGEK GTYYYYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTEP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKRVEPKSC 8 PD2 HC PDL 1 Fab pME078 EVQLVESGGGLVKPGGSLRLSCAASGFTFGSYSMTWV RQAPGKGLEWVSFISTVSTYIYYPDSVKGRFTISRDN AKNSLYLQMNSLRAEDTAVYYCARVSGRGSAARDGEK GTYYYYMDVWGKGTTVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTEP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKRVEPKSCGGSSRSSSSGGGGSGGGG FTVTVPK DLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDK NIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAAL QITDVKLQDAGVYRCMISYGGADYKRITVKVNA 9 PD2 HC PDL 1 Fab pME079 FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIV YWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQL SLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVK VNA GGSSRSSSSGGGGSGGGGEVQLVESGGGLVKPGG SLRLSCAASGFTFGSYSMTWVRQAPGKGLEWVSFIST VSTYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARVSGRGSAARDGEKGTYYYYMDVWGKGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC 10 PD2 Human Lambda pME080 SSELTQDPAVSVALGQTVTITCQGDSLRSYYASWYQQ LC KPGQAPVLVIFGKNNRPSGIPDRESGSRSGNTASLTI TGAQAEDEADYFCDSRDSSANHWVEGGGTKLTVLQPK AAPSVTLEPPSSEELQANKATLVCLISDFYPGAVTVA WKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKSHKSYSCQVTHEGSTVEKTVAPTECS 11 PD2 LC PDL1 Fab pME081 SSELTQDPAVSVALGQTVTITCQGDSLRSYYASWYQQ KPGQAPVLVIFGKNNRPSGIPDRESGSRSGNTASLTI TGAQAEDEADYFCDSRDSSANHWVEGGGTKLTVLQPK AAPSVTLEPPSSEELQANKATLVCLISDFYPGAVTVA WKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKSHKSYSCQVTHEGSTVEKTVAPTECS GGSSRSSSS GGGGSGGGG FTVTVPKDLYVVEYGSNMTIECKFPVEK QLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQ RARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGG ADYKRITVKVNA 12 PDL1 LC PD2 Fab pME082 FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIV YWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQL SLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVK VNAGGSSRSSSSGGGGSGGGG SSELTQDPAVSVALGQ TVTITCQGDSLRSYYASWYQQKPGQAPVLVIFGKNNR PSGIPDRESGSRSGNTASLTITGAQAEDEADYFCDSR DSSANHWVFGGGTKLTVLQPKAAPSVTLEPPSSEELQ ANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTT PSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGS TVEKTVAPTECS 13 PD2 HC Hu IgG2/4 pME089 EVQLVESGGGLVKPGGSLRLSCAASGFTFGSYSMTWV RQAPGKGLEWVSFISTVSTYIYYPDSVKGRFTISRDN AKNSLYLQMNSLRAEDTAVYYCARVSGRGSAARDGEK GTYYYYMDVWGKGTTVTVSSASTKGPSVFPLAPCSRS TSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSN TKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLSLGK 14 PD5 HC Hu IgG2/4 pME090 QLQLQESGPGLVKPSETLSLTCIVSGGSISRNSYYWG WIRQPPGKGLEWIGSIVYSGSTYHQPSLKSRVTIFLD TSKNQFFLKLTSVTAADTAVYYCARGTRATTWPPPIG YWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVE RKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS LSLGK 15 DWZ HC Hu IgG2/4 pME091 EVQLLESGGDVVQPGGSLRLSCVASGFTLTNYVTAWV RRRPGKGLEWISGMTHGAVATYYSDSVRGRESSSRDT SRSTLYLQMNDLTVEDTAIYYCAKSLHRSGTSLSYWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKC CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL GK 16 PDL1 HC PD2 pME092 FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIV YWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQL SLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVK VNA GGSSRSSSSGGGGSGGGGEVQLVESGGGLVKPGG SLRLSCAASGFTFGSYSMTWVRQAPGKGLEWVSFIST VSTYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARVSGRGSAARDGEKGTYYYYMDVWGKGTTV TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECP PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 17 PDL1 HC PD5 pME095 FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIV YWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQL SLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVK VNA GGSSRSSSSGGGGSGGGGQLQLQESGPGLVKPSE TLSLTCIVSGGSISRNSYYWGWIRQPPGKGLEWIGSI VYSGSTYHQPSLKSRVTIFLDTSKNQFFLKLTSVTAA DTAVYYCARGTRATTWPPPIGYWGQGTLVTVSSASTK GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT QTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVA GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSLGK 18 DWZLC pME102 QSVLTQPPSASGTPGQNINISCSGTTSNIGGSNVDWY QHVPGTAPKLFIHSNNQRPSGVPARESASKSGTSASL AISGLQSEDEADYYCATWDVRLLAYVFGSATEVTVLR HQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGA VTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSL TPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 19 PD2 mIgG1 HC pME083 EVQLVESGGGLVKPGGSLRLSCAASGFTFGSYSMTWV RQAPGKGLEWVSFISTVSTYIYYPDSVKGRFTISRDN AKNSLYLQMNSLRAEDTAVYYCARVSGRGSAARDGEK GTYYYYMDVWGKGTTVTVSSAKTTPPSVYPLAPGSAA QTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTEP AVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASST KVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVL TITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQ TKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNS AAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDK VSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMD TDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHH TEKSLSHSPGK 20 PD2 mu kappa LC pME084 SSELTQDPAVSVALGQTVTITCQGDSLRSYYASWYQQ KPGQAPVLVIFGKNNRPSGIPDRESGSRSGNTASLTI TGAQAEDEADYFCDSRDSSANHWVEGGGTKLTVLQPK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDI NVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTL TKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 21 PD5 mlgG1 HC pME085 QLQLQESGPGLVKPSETLSLTCIVSGGSISRNSYYWG WIRQPPGKGLEWIGSIVYSGSTYHQPSLKSRVTIFLD TSKNQFFLKLTSVTAADTAVYYCARGTRATTWPPPIG YWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTL GCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLY TLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVP RDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKV TCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQI NSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIE KTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMIT NFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVY SKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHS PGK 22 PD5 mu kappa LC pME086 QSVLTQPPSVSAAPGQKVTVSCFGSSSNIGNYEASWY QQLPGAAPRLLIYGNNERPSGIPDRESGSKSGTSATL VITGLQTGDEAAYYCATWDSSLSAVVFGGGTKLTVLG QPRADAAPTVSIFPPSSEQLTSGGASVVCELNNFYPK DINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTL TLTKDEYERHNSYTCEATHKTSTSPIVKSENRNEC 23 DWZ mIgG1 HC pME087 EVQLLESGGDVVQPGGSLRLSCVASGFTLTNYVTAWV RRRPGKGLEWISGMTHGAVATYYSDSVRGRESSSRDT SRSTLYLQMNDLTVEDTAIYYCAKSLHRSGTSLSYWG QGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCL VKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLS SSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDC GCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCV VVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQINST FRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTI SKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFF PEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKL NVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK 24 DWZ mu kappa LC pME088 QSVLTQPPSASGTPGQNINISCSGTTSNIGGSNVDWY QHVPGTAPKLFIHSNNQRPSGVPARESASKSGTSASL AISGLQSEDEADYYCATWDVRLLAYVFGSATEVTVLR HRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKD INVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLT LTKDEYERHNSYTCEATHKTSTSPIVKSENRNEC 25 PD5 mu Lambda LC N/A FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIV YWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQL SLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVK VNAGGSSRSSSSGGGGSGGGG QSVLTQPPSVSAAPGQ KVTVSCFGSSSNIGNYFASWYQQLPGAAPRLLIYGNN ERPSGIPDRESGSKSGTSATLVITGLQTGDEAAYYCA TWDSSLSAVVEGGGTKLTVLGQPKSSPSVTLEPPSSE ELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGME TTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTH EGHTVEKSLSRADCS 26 PD5 mu IgG1 Fc N/A QLQLQESGPGLVKPSETLSLTCIVSGGSISRNSYYWG Silent ™ WIRQPPGKGLEWIGSIVYSGSTYHQPSLKSRVTIFLD TSKNQFFLKLTSVTAADTAVYYCARGTRATTWPPPIG YWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTL GCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLY TLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVP RDCGCKPCICTVPEQSSVFIFPPKPKDVLMISLTPKV TCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQI NSTFRSVSELPILHQDWLNGKEFKCRVNSAAFPAPIE KTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMIT NFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVY SKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHS PGK 27 12B4_HC PADL0131 LEVQLQQSGAELVRPGSSVKISCKASGYVFSNYWMNW VKQRPGQGLEWIGQIYPGDGDTNYNGKFKGKATLTAD KSSSTAYMQLSSLTSEDSAVYFCARREIYFDYWGQGT TVTVASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECP PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 28 PDL1_LC_12B4 pADL0132 FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIV YWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQL SLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVK VNA GGSSRSSSSGGGGSGGGG DILLTQSPAILSVSPG ERVSESCRASQSIGTSIHWYQQRTNGSPRLLIEYASE SISGIPSRESGSGSGTDETLSINSVESEDEAYYYCQQ SNGWPITFGGGTKLEIKRTVAAPSVEIFPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSENRGEC 29 Linker_PDL1_V- pME098 GGSSRSSSSGGGGSGGGG FTVTVPKDLYVVEYGSNMT like_domain IECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDL KVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGV YRCMISYGGADYKRITVKVNA 30 PDL1_ECD pME099 FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIV YWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQL SLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVK VNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVI WTSSDHQVLSGKTTTTNSKREEKLENVTSTLRINTTT NEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER 31 PDL1_V- pME100 FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIV like_domain YWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQL SLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVK VNA 32 PDL1_V- pME101 FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIV like_domain_ YWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQL Linker_Hu_IgG2/4 SLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVK VNA GGSSRSSSSGGGGSGGGGASTKGPSVFPLAPCSR STSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPS NTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK 33 mPDL1_LC_PD5 pADL0128 FTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVV YWEKEDEQVIQFVAGEEDLKPQHSNFRGRASLPKDQL LKGNAALQITDVKLQDAGVYCCIISYGGADYKRITLK VNA GGSSRSSSSGGGGSGGGG QSVLTQPPSVSAAPGQ KVTVSCFGSSSNIGNYFASWYQQLPGAAPRLLIYGNN ERPSGIPDRESGSKSGTSATLVITGLQTGDEAAYYCA TWDSSLSAVVFGGGTKLTVLGQPRADAAPTVSIFPPS SEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNG VLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCE ATHKTSTSPIVKSENRNEC 34 mPDL1_LC_DWZ pADL0129 FTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVV YWEKEDEQVIQFVAGEEDLKPQHSNFRGRASLPKDQL LKGNAALQITDVKLQDAGVYCCIISYGGADYKRITLK VNA GGSSRSSSSGGGGSGGGG QSVLTQPPSASGTPGQ NINISCSGTTSNIGGSNVDWYQHVPGTAPKLFIHSNN QRPSGVPARESASKSGTSASLAISGLQSEDEADYYCA TWDVRLLAYVFGSATEVTVLRHRADAAPTVSIFPPSS EQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGV LNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEA THKTSTSPIVKSENRNEC 35 N-terminus_CTLA- pADL0145 KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVT 4_LC_PD5 (TPP- VLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSG 1898) NQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNG TQIYVIDPEPCPDSD GGSSRSSSSGGGGSGGGG QSVL TQPPSVSAAPGQKVTVSCEGSSSNIGNYEASWYQQLP GAAPRLLIYGNNERPSGIPDRESGSKSGTSATLVITG LQTGDEAAYYCATWDSSLSAVVFGGGTKLTVLGQPRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 36 N-terminus PDL2_ pADL0126 FTVTVPKELYIIEHGSNVTLECNEDTGSHVNLGAITA V-like domain_LC_ SLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQV PD5 RDEGQYQCIIIYGVAWDYKYLTLKVKA GGSSRSSSSG GGGSGGGG QSVLTQPPSVSAAPGQKVTVSCFGSSSNI GNYFASWYQQLPGAAPRLLIYGNNERPSGIPDRESGS KSGTSATLVITGLQTGDEAAYYCATWDSSLSAVVEGG GTKLTVLGQPRTVAAPSVFIFPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS ENRGEC 37 N- PADL0176 FTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITA terminus_PDL2_ SLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQV LC_PD5 RDEGQYQCIIIYGVAWDYKYLTLKVKASYRKINTHIL KVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSR TPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLA SIDLQSQMEPRTHPT GGSSRSSSSGGGGSGGGG QSVL TQPPSVSAAPGQKVTVSCFGSSSNIGNYFASWYQQLP GAAPRLLIYGNNERPSGIPDRESGSKSGTSATLVITG LQTGDEAAYYCATWDSSLSAVVFGGGTKLTVLGQPRT VAAPSVEIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSENRGEC 38 N- pADL0177 QVQVVTQDEREQLYTPASLKCSLQNAQEALIVTWQKK terminus_CD200_ KAVSPENMVTFSENHGVVIQPAYKDKINITQLGLQNS LC_PD5 TITFWNITLEDEGCYMCLENTFGFGKISGTACLTVYV QPIVSLHYKFSEDHLNITCSATARPAPMVFWKVPRSG IENSTVTLSHPNGTTSVTSILHIKDPKNQVGKEVICQ VLHLGTVTDFKQTVNK GGGSSRSSSSGGGGSGGGG QS VLTQPPSVSAAPGQKVTVSCEGSSSNIGNYFASWYQQ LPGAAPRLLIYGNNERPSGIPDRESGSKSGTSATLVI TGLQTGDEAAYYCATWDSSLSAVVFGGGTKLTVLGQP RTVAAPSVEIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSENRGEC 39 N-terminus B7- pADL0179 LEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNL H3_LC_PD5 IWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQ GNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVA APYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEV FWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLG ANGTYSCLVRNPVLQQDAHSSVTITPQRSPTGAVEVQ VPEDPVVALVGTDATLRCSFSPEPGFSLAQLNLIWQL TDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNAS LRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYS KPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQD GQGVPLTGNVTTSQMANEQGLEDVHSVLRVVLGANGT YSCLVRNPVLQQDAHGSVTITGQPMTFPPEA GGSSRS SSSGGGGSGGGG QSVLTQPPSVSAAPGQKVTVSCFGS SSNIGNYFASWYQQLPGAAPRLLIYGNNERPSGIPDR FSGSKSGTSATLVITGLQTGDEAAYYCATWDSSLSAV VFGGGTKLTVLGQPRTVAAPSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSENRGEC 40 N-terminus_TIM- pADL0181 SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGAC 3_LC_PD5 PVFECGNVVLRTDERDVNYWTSRYWLNGDERKGDVSL TIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPA KVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLP DINLTQISTLANELRDSRLANDLRDSGATIRI GGGSS RSSSSGGGGSGGGG QSVLTQPPSVSAAPGQKVTVSCE GSSSNIGNYFASWYQQLPGAAPRLLIYGNNERPSGIP DRESGSKSGTSATLVITGLQTGDEAAYYCATWDSSLS AVVFGGGTKLTVLGQPRTVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSENRGEC 41 N-terminus_B7- PADL0141 DLKVEMMAGGTQITPLNDNVTIFCNIFYSQPLNITSM H6_LC_PD5 GITWFWKSLTFDKEVKVFEFFGDHQEAFRPGAIVSPW RLKSGDASLRLPGIQLEEAGEYRCEVVVTPLKAQGTV QLEVVASPASRLLLDQVGMKENEDKYMCESSGFYPEA INITWEKQTQKFPHPIEISEDVITGPTIKNMDGTENV TSCLKLNSSQEDPGTVYQCVVRHASLHTPLRSNFTLT AARHSLSETEKTDNF SGGSSRSSSSGGGGSGGGG QSV LTQPPSVSAAPGQKVTVSCFGSSSNIGNYEASWYQQL PGAAPRLLIYGNNERPSGIPDRFSGSKSGTSATLVIT GLQTGDEAAYYCATWDSSLSAVVFGGGTKLTVLGQPR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSENRGEC 42 N-terminus_B7- pADL0123 LIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPD H4_LC_PD5 IKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMER GRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITS KGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPR WFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMK VVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIK RRSHLQLLNSKA SGGSSRSSSSGGGGSGGGG QSVLTQ PPSVSAAPGQKVTVSCFGSSSNIGNYEASWYQQLPGA APRLLIYGNNERPSGIPDRESGSKSGTSATLVITGLQ TGDEAAYYCATWDSSLSAVVEGGGTKLTVLGQPRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSENRGEC 43 N-terminus_B7- PADL0124 FKVATPYSLYVCPEGQNVTLTCRLLGPVDKGHDVTFY H5_LC_PD5 KTWYRSSRGEVQTCSERRPIRNLTFQDLHLHHGGHQA ANTSHDLAQRHGLESASDHHGNFSITMRNLTLLDSGL YCCLVVEIRHHHSEHRVHGAMELQVQTGKDAPSNCVV YPSSSQDSENITAA GGSSRSSSSGGGGSGGGG QSVLT QPPSVSAAPGQKVTVSCFGSSSNIGNYFASWYQQLPG AAPRLLIYGNNERPSGIPDRESGSKSGTSATLVITGL QTGDEAAYYCATWDSSLSAVVFGGGTKLTVLGQPRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSENRGEC 44 N- pADL0125 LPSCKEDEYPVGSECCPKCSPGYRVKEACGELTGTVC terminus_HVEM_ EPCPPGTYIAHLNGLSKCLQCQMCDPAMGLRASRNCS LC_PD5 RTENAVCGCSPGHFCIVQDGDHCAACRAYATSSPGQR VQKGGTESQDTLCQNCPPGTFSPNGTLEECQHQTKCS WLVTKAGAGTSSSHWV GGSSRSSSSGGGGSGGGG QSV LTQPPSVSAAPGQKVTVSCFGSSSNIGNYFASWYQQL PGAAPRLLIYGNNERPSGIPDRESGSKSGTSATLVIT GLQTGDEAAYYCATWDSSLSAVVFGGGTKLTVLGQPR TVAAPSVEIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSENRGEC 45 Anti-CD20 HC Human QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWV IgG1 Fc KQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADK SSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVW GAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPEQSGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKENWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK 46 mPDL1_LC_Anti- Human FTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVV CD20 kappa light YWEKEDEQVIQFVAGEEDLKPQHSNFRGRASLPKDQL chain LKGNAALQITDVKLQDAGVYCCIISYGGADYKRITLK VNA GGSSRSSSSGGGGAGGGG QIVLSQSPAILSASPG EKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNL ASGVPVRESGSGSGTSYSLTISRVEAEDAATYYCQQW TSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSENRGEC 47 Anti-CD80 HC Human QVQLQESGPGLVKPSETLSLTCAVSGGSISGGYGWGW IgG1 Fc IRQPPGKGLEWIGSFYSSSGNTYYNPSLKSQVTISTD TSKNQFSLKLNSMTAADTAVYYCVRDRLFSVVGMVYN NWFDVWGPGVLVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPEQSGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK 48 mPDL1_LC_Anti- Human FTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVV CD80 lambda YWEKEDEQVIQFVAGEEDLKPQHSNFRGRASLPKDQL light chain LKGNAALQITDVKLQDAGVYCCIISYGGADYKRITLK VNA GGSSRSSSSGGGGAGGGG ESVLTQPPSVSGAPGQ KVTISCTGSTSNIGGYDLHWYQQLPGTAPKLLIYDIN KRPSGISDRESGSKSGTAASLAITGLQTEDEADYYCQ SYDSSLNAQVFGGGTRLTVLGQPKAAPSVTLFPPSSE ELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVE TTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTH EGSTVEKTVAPTECS - Three of the anti-DLAT antibodies referenced in Thomson et al., 1998, J. Hepatol. 28(4): 582-94, designated PD2, PD5, and DWZ, were selected for cloning as: (i) human full-length antibodies were constructed with the autoantibody VH-VC1 and VL-CL regions fused to a Fc variant region (IgG2/4); (ii) murine IgG1 Fc; (iii) PDS as a murine IgG1 Fc variant with mutations to remove effector functions. Each of these antibodies was constructed with or without the human or mouse variable-like domain of the extracellular domain (ECD) of Programmed Death-Ligand 1 (PD-L1) linked to the N-terminus of the light chains. TPP-1986 was ordered from Absolute Antibody as a custom production consisting of their murine IgG1 Fc Silent isotype with PDS variable region and human PD-L1 variable-like domain.
- All murine light chain sequences are kappa light chains. The reference sequence sources for these antibodies are shown in Table 4, the sequences comprising the heavy and light chains are shown in Table 3 above, and the construct descriptions are shown in Table 5.
-
TABLE 4 Reference Sequences Reference Sequences Accession # Source DWZ Heavy Chain Reference Protein Sequence AJ001167.1 GenBank DWZ Light Chain Reference Protein Sequence AJ001168.1 GenBank PD2 Heavy Chain Reference Protein Sequence AJ001171.1 GenBank PD2 Light Chain Reference Protein Sequence AJ001172.1 GenBank PD5 Heavy Chain Reference Protein Sequence AJ001175.1 GenBank PD5 Light Chain Reference Protein Sequence AJ001176.1 GenBank Murine PD-L1 Reference Protein Sequence NP_068693.1 NCBI Murine PD-L1 Reference Protein Sequence Q9EP73 Uniprot -
TABLE 5 Construct Descriptions Molecular Protein Transfection Weight ID Isotype Constructs (kDa) Description TPP-1003 Murine pME083 + pME084 147.4 mIgG1 PD2 Ab IgG1 TPP-1004 Murine pME085 + pME086 145.4 mIgG1 PD5 Ab IgG1 TPP-1005 Murine pME087 + pME088 144.9 mlgG1 DWZ Ab IgG1 TPP-1986 Murine N/A 173 N-terminus_ IgG1 PDL1_LC_PD5 Fc Silent TPP-1694 Murine pME085 + 173.8 N-terminus_ IgG1 pADL0128 mPDLI_LC_PD5 TPP-1695 Murine pME087 + 173.8 N-terminus_ IgG1 pADL0129 mPDL1_LC_ DWZ - Programmed Death 1 (PD-1) Fc chimera (R&D Systems, Catalog # 1086-PD-050) was captured on the anti-human Fc tips (ForteBio, Catalog # 18-5064) at 10 μg/mL in PBS, followed by binding to PD2 Fab fusion variants or Programmed Death Ligand 1 (PD-L1) Fc chimera (R&D Systems, Catalog # 156-B7-100) at 10 μg/mL or μg/mL in PBS, respectively. Final dissociation in PBS was performed in the last step on the Bio-Layer Interferometry (BLI) data.
- TPP-993 did not bind to PD-1 Fc because TPP-993 lacks a PD-L1 domain (
FIG. 1A ). TPP-994 and TPP-999 exhibited very little binding to PD-1 Fc (FIGS. 1B and 1D ) while TPP-995 and TPP-1001 both exhibited binding to PD-1 Fc (FIGS. 1C and E). PD-L1 Fc, a dimeric protein having two PD-L1 proteins fused to a human Fc, bound PD-1 very strongly to PD-1 Fc (FIG. 1F ), but did not bind to itself, demonstrating that the Fc portion of PD-L1 Fc did not non-specifically interact with the tips (FIG. 1G ). - The three anti-DLAT murine IgG1 antibodies described in Example 2 were diluted to 40 μg/mL in PBS and loaded onto anti-mouse IgG1 Fc tips (ForteBio, Catalog # 18-5090). Human DLAT recombinant protein (ProSpec, Catalog # ENZ-082) was diluted into PBS starting at 200 nM with three-fold dilutions down to 2.5 nM. Binding was determined by the association of the DLAT antigen with the respective antibodies using Bio-Layer Interferometry (BLI) deflections the Octet Red (ForteBio). Finally, a dissociation step in PBS was performed to determine dissociation from the bound antibodies.
- The on- and off-rates of the anti-DLAT murine IgG1 antibodies to DLAT antigen are shown in
FIG. 2 . Slow on-rates and fast off-rates observed for TPP-1003 suggested a weak affinity, whereas a slow off-rate observed for TPP-1004 (FIG. 2B ) and TPP-1005 (FIG. 2C ) suggested a high affinity to the DLAT antigen. - As shown in Table 6 below and
FIG. 3 , Octet Red (ForteBio) was utilized for Bio-Layer Interferometry (BLI) to determine binding of the anti-DLAT human IgG2/4 antibodies to antibody PD-L1 fusions to associate with the DLAT antigen followed by binding to PD-1 Fc. This assay was utilized to determine the ability of the targeted PD-L1 fusions to simultaneously bind the DLAT antigen by the antibody and PD-1 Fc by the PD-L1 variable-like domain. - Antibodies and antibody fusions were diluted to 16 μg/mL in PBS and loaded onto anti-human IgG Fc tips (ForteBio, Catalog # 18-5064). Human DLAT recombinant protein (ProSpec, Catalog # ENZ-082) was diluted into PBS to 10 μg/mL. PD-L1 Fc chimera (R&D Systems, Catalog # 156-B7-100) and PD-1 Fc chimera (R&D Systems, Catalog # 1086-PD-050) were diluted to 12.5 μg/mL in PBS. Finally, a dissociation step in PBS was performed to determine dissociation from the bound antibodies.
- Results are summarized in Table 6 below and shown in
FIGS. 3A-E . The two fusions of the PD-L1 variable-like domain to the N-terminus of the anti-DLAT antibodies, TPP-986 (FIG. 3B ) and TPP-992 (FIG. 3D ), were able to simultaneously bind the DLAT antigen and the PD-1 Fc. The fusion proteins lacking PD-L1, or having PD-L1 fused to the antibody C-terminus (FIG. 3C ) of the light chain did not bind well to PD-1 Fc for this particular combination of antibody and immune inhibitor. -
TABLE 6 Binding to DLAT Antigen and/or PD-1 Fc Bound to DLAT Bound to Construct Antigen? PD-1 Fc? TPP-985, human IgG2/4 PD5 antibody Yes No TPP-986, human IgG2/4 PD5 antibody, Yes Yes N-terminus PD-L1 HC TPP-990, human IgG2/4 PD5 antibody, Yes No C-terminus PD-L1 LC TPP-992, human IgG2/4 PD5 antibody, Yes Yes N-terminus PD-L1 LC PD-L1 Fc control No Yes - Dihydrolipoamide Acetyltransferase (DLAT) human T-cell activation beads were prepared by conjugating 120 μg anti-human CD3, clone UCHT1 (Stemcell Technologies, Catalog # 60011) to 3 mL of Dynabeads M-450 Epoxy (ThermoFisher, Catalog # 14011) for 18 hours rotating at room temperature in 0.1M sodium phosphate buffer, pH 8.0. See
FIG. 8 for visual representations of the various bead constructs. The next day, the beads were washed and an excess of 546 μg of human DLAT recombinant protein antigen (ProSpec, Catalog # ENZ-082) was added to the anti-CD3-coated beads and incubated for an additional 24 hours at room temperature. The DLAT and anti-CD3-coated beads (“human DLAT-aCD3 Beads” or “DLAT beads”) (FIG. 8 ) were then washed and suspended to a concentration of 4×108 beads/mL in PBS with 0.1% w/v Bovine Serum Albumin. 100 μL aliquots were prepared and stored at −20° C. These human DLAT-aCD3 Beads were constructed to facilitate T-cell activation. - As shown in
FIG. 8 , T-cell activation beads (“TA2 beads”) were prepared by conjugating anti-CD3-coated beads (as prepared in making the human DLAT-aCD3 beads) with negative control irrelevant human IgG Fc (Bethyl Labs, Catalog # P80-104). - Negative control (“NC”) beads were prepared by conjugating human IgG Fc with uncoated beads for a no-activation control reagent. Bead conjugation was verified by flow cytometry prior to performing the T-cell activation assay.
- Human peripheral primary human pan-T-cells including CD4+and CD8+T-cells as well as some gamma/delta T-cell subsets (Stemcell Technologies, Catalog # 70024) were stained with the CFSE Cell Proliferation Kit (ThermoFisher, Catalog # C34554) and then activated using DLAT-aCD3 beads, TA2 beads or NC beads, in the presence or absence of the anti-DLAT antibody or antibody PD-L1 fusions, TPP-985, TPP-986, TPP-992, or the PD-L1 Fc chimera as a non-targeted control (R&D Systems, Catalog #156-B7-100) at 260 nM or 65 nM in replicates of three. In addition, TPP-992 was titrated starting from 260 nM by two-fold dilutions down to 2 nM and combined with the DLAT-aCD3 beads as described. This assay was performed in a 96-well U-bottom tissue culture plate (Corning, Catalog # 3799) with 1.5×105 cells/well with a 1:1.5 bead to cell ratio for 4 days at 37° C. with 5% CO2. On the fourth day, flow cytometry was performed after staining with SYTOX™ Red Dead Cell stain (ThermoFisher, Catalog # S34859) following the manufacturer's protocol using the C6 Accuri Flow Cytometer with C6 Sampler (BD Biosciences) detectors FL-1 (CFSE) and FL-4 (SYTOX™ Red).
- TPP-985 (human IgG2/4 PDS antibody) bound to the DLAT antigen, but did not bind to PD-1 Fc (
FIG. 3A ). TPP-986 and TPP-992 (fusions of the PD-L1 V-like domain to the N-terminus of anti-DLAT antibodies) both exhibited the ability to simultaneously bind the DLAT antigen and PD-1 Fc (FIGS. 3B and 3D ). TPP-990 (PD-L1 V-like domain fused to the C-terminus of an anti-DLAT antibody) bound the DLAT antigen, but did not bind PD-1 Fc (FIG. 3C ). The PD-L1 Fc control bound to PD-1 Fc, but did not bind to the DLAT antigen (FIG. 3E ). - As shown in
FIG. 4 , the decreased signal in the FL-1 mean fluorescence measurement represents the activated T-cells that have expanded into new generations of T-cells, as demonstrated by the decreasing dye concentration in each respective generation (FIG. 4A ). Samples were gated to exclude the low Forward Scatter (FSC) and the FL-4+(SYTOX™ Red) dead cells. The same gate was utilized to analyze all samples. The histograms shown inFIG. 4 were gated on the live T-cells.FIG. 4B illustrates that TPP-985, an anti-DLAT IgG2-4 with no PD-L1 variable-like domain present, showed no significant inhibition of T-cell activation. - In summary,
FIGS. 4A-4F show human T-cell activation for targeted PD-L1 fusion inhibition. Representative histograms of one of three replicates are shown for each sample at 260 nM and 65 nM concentrations of antibody fusions or non-targeted PD-L1 Fc.FIG. 4A illustrates that NC beads exhibited no activation of T-cells, whereas the DLAT-aCD3 beads demonstrated clear activation of the pan T-cells.FIG. 4B illustrates that TPP-985 (an anti-DLAT IgG2/4 antibody with no PD-L1 variable-like domain) exhibited no significant inhibition of T-cell activation.FIG. 4C illustrates that TPP-986 (an anti-DLAT antibody with PD-L1 fused at the N-terminus of the heavy chain) exhibited significant inhibition of T-cell activation at both the 65 nM and 260 nM concentrations.FIG. 4D illustrates that TPP-992 (an anti-DLAT antibody with PD-L1 fused at the N-terminus of the light chain) exhibited the most significant inhibition of T-cell activation, with complete inhibition at the 260 nM concentration.FIG. 4E illustrates that the non-targeting PD-L1 Fc chimera exhibited no significant inhibition of T-cell activation.FIG. 4F illustrates that the titration of TPP-992 demonstrated a dose-dependent inhibition of T-cell activation with an EC50 of 17 nM±4.5 nM. - For this particular antibody immune inhibitor fusion protein, N-terminal fusion of PD-L1 is the most effective design. To reiterate, the results shown in
FIG. 4C show that TPP-986, which is an anti-DLAT IgG2/4 fused with PD-L1 on the N-terminus of the heavy chain, showed significant inhibition of human T-cell activation at both the 65 nM and 260 nM concentrations. TPP-992, (FIG. 4D ) which is an anti-DLAT IgG2/4 fused with PD-L1 on the N-terminus of the light chain, showed the most significant inhibition of human T-cell activation of all of the samples tested, with nearly complete inhibition at the 260 nM concentration. -
FIGS. 22A-22C illustrate histograms showing titration from 250 nM to 50 nM of anti-CD80_mPDL1 fusion incubated with CD80 activation beads for 4 days with human T-cells. The results demonstrate complete inhibition at 250 nM, partial inhibition at 100 nM and no significant inhibition at 50 nM.FIG. 22D shows the dilution of proliferation dye (CFSE) into the progeny T-cells observed by the multiple peaks on the histograms which indicate that the controls including no activation (labeled No Beads) had no activation and the activation control with the CD80 beads had significant activation.FIG. 22E is the graphical overview of the described results from above, the geometric mean of the CFSE is plotted vs the samples incubated.FIG. 22F is the statistical analysis of the results using Dunnett's multiple comparisons test. -
FIGS. 23A-23C demonstrate the titration of anti-CD20 mPDL1 fusion with CD20 activation beads from 250 nM to 50 nM incubated for 4 days with human T-cells. The results demonstrate complete inhibition at 250 nM, partial inhibition at 100 nM, and less inhibition at 50 nM.FIG. 23D illustrates the dilution of proliferation dye (CFSE) into the progeny T-cells observed by the multiple peaks indicating that activation of the T-cells by the CD20 beads occurred.FIG. 23E represents the graphical overview of the geometric mean of the proliferation dye CFSE.FIG. 23F is the statistical analysis of the described data demonstrating that all of the concentrations of the anti-CD20 mPDL1 were statistically significantly different from the CD20 beads showing inhitibion of T-cell activation. - To determine whether the PD-L1 fusion needed to bind to surface bound DLAT in order to inhibit T-cell activation, the T-cell activation assay described above was performed on a sample combining TPP-985 (an anti-DLAT construct having no PD-L1 variable-like domain) with TPP-992 (an anti-DLAT IgG2/4 fused with PD-L1 on the N-terminus of the light chain) at 260 nM and 31 nM, respectively, in three replicates. The T-cell activation beads (TA2) were also tested with all of the fusion variants to determine if binding to the DLAT antigen was necessary for inhibiting T-cell activation.
- The results are shown in
FIG. 5 . These results show that the anti-DLAT PD-L1 fusion must bind to the DLAT on the surface of the beads in order to effectively inhibit T-cell activation. - Statistical analysis was performed using the average geometric mean fluorescence of FL-1 (CFSE) intensity using FlowJo X software with gating as described, using the
GraphPad Prism 7 software to calculate P values by One-way ANOVA multiple comparisons test. This method compares all samples to each other, providing the P values between all samples in the assay.GraphPad Prism 7 software was also utilized for all graphs displayed in figures, and all P value calculations shown. Results are shown inFIG. 6 . -
FIG. 6A shows that at the 260 nM concentration of the anti-DLAT PD-L1 fusions tested, TPP-992 (the anti-DLAT IgG2/4 construct with PD-L1 at the N-terminus of the light chain) demonstrated the greatest level of T-cell activation inhibition, with significantly greater activity inhibition than TPP-986 (the anti-DLAT IgG2/4 construct with PD-L1 at the N-terminus of the heavy chain). Both TPP-986 and TPP-992 showed significant inhibition of T-cell activation as compared to the human DLAT-aCD3 beads (referred to in the figure as DLAT Beads). TPP-992 was not significantly different than the no-activation NC beads, whereas TPP-986 was significantly different than the no-activation NC beads.FIG. 6B illustrates the statistical analysis summary of all samples in the T-cell activation assay. - No significant difference was observed when comparing the results with the DLAT-aCD3 beads in the presence or absence of the non-targeting PD-L1 Fc or TPP-985 (anti-DLAT IgG2/4 antibody with no PD-L1 fusion). At the 65 nM concentration, a lower level of T-cell activation inhibition was observed for all samples (see
FIG. 6C ). TPP-992 again demonstrated the most significant inhibition of T-cell activation, and showed significantly greater T-cell activation inhibition than the other fusion samples. In addition, the anti-DLAT PD-L1 fusion T-cell activation inhibition results demonstrated that TPP-986 was no longer significantly different from the PD-L1 Fc chimera at the 65 nM concentration.FIG. 6D illustrates the statistical analysis summary of T-cell activation inhibition for anti-DLAT antibody PD-L1 fusion proteins at the 65 nM concentration. -
FIG. 18A illustrates that at 500 nM concentration of the anti-DLAT PD-L1 (TPP-1986), B7H4 (TPP-1504), HVEM (TPP-1506), B7H6 (TPP-1894) and CTLA4 (TPP-1898) fusions tested atday 7, inhibited T-cell activation.FIG. 18B illustrates the statistical analysis summary of all samples in the T-cell activation assay.FIG. 18C illustrates that at 500 nM concentration of the anti-DLAT PDL-2 (TPP-2215), CD200 (TPP-2216), TIM-3 (TPP-2220) and PD-5 (TTP-992) fusions showed inhibition of T cell activation atday 4.FIG. 18D illustrates the statistical analysis summary of T-cell activation inhibition for anti-DLAT antibody fusion proteins. - Dihydrolipoamide Acetyltransferase mouse T-cell activation beads (“mouse DLAT-aCD3 beads”) were prepared by conjugating 10 μg anti-mouse CD3, clone 145-2C11 (Stemcell Technologies, Catalog # 60015) to 250 μL of Dynabeads M-450 Epoxy (ThermoFisher, Catalog # 14011) for 24 hours rotating at room temperature in 0.1M sodium phosphate buffer, pH 8.0. The next day the mouse DLAT-aCD3 beads were washed and an excess of 50 μg of human DLAT recombinant protein (ProteinTech, Catalog # Ag22831) antigen was added to the mouse DLAT-aCD3 beads and incubated for an additional 48 hours at room temperature. The mouse DLAT-aCD3 beads were then washed and suspended to a concentration of 4×107 beads/mL in PBS with EasySep Buffer (Stemcell Technologies, Catalog # 20144) and 250 μL aliquots were prepared and stored at −20° C.
- Negative control (“NC”) beads were previously prepared by conjugating human IgG Fc (Bethyl Labs, Catalog # P80-104) with uncoated beads for a no-activation control reagent. Bead conjugation was verified by flow cytometry prior to performing the mouse T-cell activation assay.
- Mouse T-cell Isolation from Spleens
- Two six-week old female C57 mice were sacrificed and the spleens harvested into EasySep buffer (Stemcell Technologies, Catalog # 20144). Splenocytes were harvested by dissociation of spleens by grinding between two frosted slides (ThermoFisher Catalog # 6776214) into 5 mL EasySep buffer in a petri dish, filtered through a 40 μM Cell Strainer (Fisher Catalog # 352340) into a 50 mL conical tube. The process was repeated for a second spleen and the combined cells were centrifuged at 300×g for 10 minutes. Splenocytes were adjusted to 1e8 cells/mL using EasySep buffer per Mouse T-cell Isolation Kit (Stemcell Technologies, Catalog # 19851) protocol. Mouse T-cells were isolated following the protocol provided with the isolation kit using the Big Easy Magnet (Stemcell Technologies, Cat # 18001).
- Mouse T-cells isolated as described above were stained with 5 μM CFSE Cell Proliferation Kit (ThermoFisher, Catalog # C34554) dye in 10 mL EasySep buffer, incubated for 20 minutes in a 37° C. water bath, with sporadic mixing. 100 mL of TexMacs Media (Miltenyi Biosciences, Catalog # 130-097-196) was added to stained cells and incubated for 5 minutes at 37° C. water bath to quench dye. Cells were centrifuged for 10 minutes at 400×g, supernatant discarded, and cell pellet was resuspended to 1.28e6 cells/mL with TexMacs Media supplemented with recombinant mouse IL-2 (Stemcell Technologies, Cat # 78081). 3 μL of mouse DLAT-aCD3 beads were added to a 96-well U-bottom tissue culture plate (Corning, Catalog #3799) followed by monoclonal antibody PD-L1 fusions (mAb-PDL1) to a final concentration of 500 nM and 100 nM in replicates of three. The samples of antibody immune cell inhibitor fusion proteins evaluated in the murine T-cell activation assay include molecules are listed in Table 7 below. This assay was performed in a 96-well U-bottom tissue culture plate (Corning, Catalog # 3799) with 1.3×105 cells/well with a 1:1 bead to cell ratio for 4 days at 37° C. with 5% CO2. On the fourth day, flow cytometry was performed after staining with SYTOX™ Red Dead Cell stain (ThermoFisher, Catalog # S34859) following the manufacturer's protocol using the C6 Accuri Flow Cytometer with C6 Sampler (BD Biosciences) detectors FL-1 (CFSE) and FL-4 (SYTOX™ Red).
-
TABLE 7 Antibody Immune Cell Inhibitor Fusion Proteins Evaluated in Mouse T cell Activation Assay Protein ID Isotype Description TPP-1986 mIgG1 Human PDL1_Mouse PD5 TPP-992 Human IgG2/4 Human PDL1_Human PD5 TPP-1694 Mouse IgG1 Mouse PDL1_Mouse PD5 TPP-1695 Mouse IgG1 Mouse PDL1_Mouse DWZ TPP-1697 Human IgG2/4 Human PDL1_Human 12B4 Isotype control TPP-1504 Human IgG2/4 Human B7-H4_Human PD5 TPP-1505 Human IgG2/4 Human B7-H5_Human PD5 TPP-1004 Mouse IgG1 Mouse PD5 TTP-1984 Mouse IgG1 Mouse PDL1_Mouse PD5 TTP-1985 Mouse IgG1 Mouse PDL1_Mouse 12b4 TTP-1898 Human IgG2/4 CTLA4_PD5 TTP-2246 Mouse IgG1 Mouse_PD5 -
TABLE 8 Summary of Antibody Immune Cell Inhibitor Fusion Proteins Evaluated in Mouse or Human T cell Activation Assays Fusion to anti- DLAT (PD5) Result of T-cell Activation Inhibition CTLA-4 Inhibited human T-cells as potently as PD-L1 fusion in-vitro, TPP-1898 vs. DLAT beads (P Value = 0.0001). Complete inhibition observed for human & mouse T-cells. (B7-H1) Inhibited human & mouse T-cells, TPP-992 vs. DLAT PD-L1 beads (P Value = <0.0001). Fab fusion was not as potent as mab, full length PD-L1 ECD not as potent as V-like domain only when tested as Fabs. Complete inhibition observed for human & mouse T-cells. HVEM Inhibited human T-cells as potently as PD-L1 fusion, (CD270) TPP-1506 vs. DLAT beads (P Value = 0.0053). Complete inhibition observed for human & mouse T-cells. B7-H4 Inhibited mouse & human T-cells. Not as (VTCN1, potent as PD-L1 fusion, but close to complete B7x) inhibition observed in 7 day human T-cell assay. TPP-1504 vs. DLAT beads (P value = .3416). Complete inhibition observed in mouse T-cell assay. B7-H6 Not as potent as PD-L1 fusion, but close to complete (NCR3LG1) inhibition observed in 7 day human T-cell assay. Inhibited human T-cells ( Day 7 was notsignificant w 95% CI), TPP-1894 vs. DLAT beads (P Value = 0.202). Similar potency to HVEM fusion. TIM-3 Full length TIM-3 fusion expressed well. Significantly inhibited human T-cells, not as potent as PD-L1 fusion, IC50 ~500 nM in 4 day assay. TPP-2220 vs. DLAT beads (P Value = <0.0001). (B7-DC) Full length PD-L2 ECD fusion expressed well, V-like PD-L2 domain only did not express. Significantly inhibited human T-cells; TPP-2215 vs. DLAT beads (P Value = <0.0001). Not as potent as PD-L1 fusion. Low levels of inhibition observed, only slightly more potent than PDL2 Fc. CD200 Full length ECD of CD200 as a fusion expressed well, v-like domain did not express as fusion. Statistically significant TPP-2216 vs. DLAT beads (P value = 0.0338) inhibition of human T-cells as targeted & non-targeted fusion (CD200_Fc). Not as potent as PD-L1 or other fusions. B7-H3 Full length B7-H3 fusion expressed well. No (CD276) significant inhibition observed. B7-H5 Expressed well as a fusion. No significant inhibition (VISTA) observed in repeated assays. B7-H7 Did not express as individual V-like domains (HHLA2) fused to PD5 or as full length ECD fusion.
As shown inFIG. 12A , TPP-1986 provided complete inhibition of mouse T-cell activation at 500 nM with incomplete inhibition observed for 100 nM.FIG. 12B illustrates that TPP-1694 showed a significant difference as compared to the NC beads, but showed no significant difference as compared to TPP-1986 at 500 nM. A significant difference was observed at 100 nM for human TPP-1986 and TPP-992 versus mouse PD-L1-TPP-1694.FIG. 12C illustrates that TPP-1695 demonstrated a lower potency as compared to the PDS human PD-L1 fusion variants at 500 nM and 100 nM concentrations with similar results observed for TPP-1694.FIG. 12D illustrates complete inhibition of mouse T-cell activation for TPP-992 at 500 nM with an incomplete inhibition for 100 nM.FIGS. 12E and 12F illustrate that TPP-1697 (non-targeted isotype control with human PD-L1) and TPP-1004 (murine IgG1 PDS with no PD-L1 variable-like domain present), showed no significant inhibition of T-cell activation. -
FIGS. 13A and 13B show the statistical analysis of the mouse T-cell activation results at 500 nM.FIG. 13A illustrates complete inhibition of mouse T-cells with TPP-1986 and TPP-992 fusions containing human PD-L1 as demonstrated by no significant difference as compared to the NC beads. An expected decrease in the geometric mean fluorescence intensity (Geo MFI) of FL-1 representing the CFSE dye diffusion into progeny cells for the 1X beads, TPP-1697, TPP-1004, was observed, illustrating activation of the murine T-cells with no significant differences observed between samples.FIG. 13B is a table of statistical analysis between samples at 500 nM concentrations using Tukey's multiple comparisons test. -
FIGS. 14A and 14B show the statistical analysis of the mouse T-cell activation results at 500 nM.FIG. 14A illustrates complete inhibition of mouse T-cells with TPP-TPP-992 with no statistically significant difference to TPP-1504 (hB7-H4) fusion and no significant difference as compared to the NC beads. An expected decrease in the geometric mean fluorescence intensity (Geo MFI) of FL-1 representing the CFSE dye diffusion into progeny cells for the 1X beads, TPP-1697, or NC beads, was observed, illustrating activation of the murine T-cells with no significant differences observed between samples. TPP-1505 (hB7-H5) fusion shows no significant difference to the 1X beads suggesting it did not inhibit T-cell activation.FIG. 14B is a table of statistical analysis between samples at 500 nM concentrations using Tukey's multiple comparisons test. -
FIGS. 15 and 16 show the statistical analysis of the mouse T-cell activation results at 100 nM.FIGS. 15A and 16A illustrate incomplete inhibition using fusions containing human PD-L1 and hB7-H4as compared to the NC beads. A significant difference of human versus murine PD-L1 containing fusions was observed, suggesting human PD-L1 results in greater inhibition of mouse T-cells than mouse PD-L1 V-like domain. An expected decrease in the geometric mean fluorescence intensity (Geo MFI) of FL-1 representing the CFSE dye diffusion into progeny cells for the 1X beads, TPP-1697, TPP-1004, and TPP-1505 was observed illustrating activation of the murine T-cells with no significant differences between samples.FIGS. 15B and 16B are tables of statistical analysis between samples at 100 nM concentrations using Tukey's multiple comparisons test. -
FIGS. 20A-20C illustrate the results of mouse T-cell activation at 7 days gated on the CD4+cells.FIG. 20A illustrates that at the 500 nM concentration of the anti-DLAT PD-L1 (TPP-1984) and CTLA4 (TPP-1898) fusions tested atday 7, inhibited mouse T-cell activation completely.FIG. 20B illustrates that at the 125 nM concentration a lower level of T-cell activation inhibition was observed with TPP-1984 but not TPP-1898 that maintains no significant difference to the NC beads.FIG. 20C illustrates the anti-DLAT CTLA4 (TPP-1898) completely inhibits mouse T-cell activation at a 32nM concentration in the 7 day assay. 1X beads, TPP-1985, and TPP-2246 all show the expected decrease of signal representing the expected decrease in the geometric mean fluorescence intensity (Geo MFI) of FL-1 representing the CFSE dye diffusion into progeny cells was observed illustrating activation of the murine T-cells with no significant differences between samples.FIG. 20D illustrates the statistical analysis summary of T-cell activation inhibition for anti-DLAT antibody fusion proteins at the three concentrations tested. The statistical analysis demonstrates the anti-DLAT CTLA4 is significantly more potent than the untargeted CTLA4 (Abatacept) -
FIGS. 21A and 21B demonstrate the induction of regulatory T-cells (Tregs—CD4+, CD25+, FoxP3+) with the PD-L1 fusions anti-DLAT_PDL1 (labeled DLAT DRUG) and anti-VAP-1_PDL1 (labeled VAP-1 DRUG) combined with the respective activation beads.FIG. 21A illustrate flow cytometry results demonstrating low levels of CD25 for the no activation control (labeled NO BEADS) with 1.71% in quadrant 2 (Q2) representing the CD4+, CD25+ population. The histogram shown below the dot plot represents the intracellular staining with FoxP3 of the Q2 gated events. The activated T-cells (labeled DLAT) showed a significant increase in effector T-cells (Teff) in Q2, however a small percentage of these CD4+, CD25+ cells were Tregs (FoxP3+).FIG. 21B illustrates inhibition of the Teff population shown as 7% in Q2 with the addition of TPP-992 (labeled DLAT DRUG) to the DLAT activation beads, compared to 32% for DLAT beads only. In addition, from the Q2 population, the FoxP3 positive events went from 7% to 16% with the drug. Anti-VAP-1_PDL1 previously showed weaker inhibition in the T-cell activation assay using proliferation dye compared to the anti-DLAT_PDL1 (data not shown). This intermediate effect of anti-VAP-1 fusion was confirmed by the results of this assay; a higher percentage of Q2 events were observed (18%), with a lower induction ofTregs 12% compared to anti-DLAT_PDL1.
Claims (20)
1. An antibody immune cell inhibitor fusion protein comprising two polypeptide chains that form one antigen binding site and at least one immune cell receptor binding site:
wherein one polypeptide chain has a structure represented by the formula. II1-VL-CL-II2, and one polypeptide chain has a structure represented by the formula II3-VH-CH1-II4;
wherein:
VL is an immunoglobulin light chain variable domain;
VH is an immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CH1 is the immunoglobulin CH1 heavy chain constant domain;
II1, II2, II3, and II4 are each independently an immune cell inhibitor domain of an immunoglobulin superfamily member or are absent;
wherein at least one of II1, II2, II3, and II4 is an immune cell inhibitor domain of an immunoglobulin superfamily member; and
wherein the antibody immune cell inhibitor fusion protein inhibits or diminishes activation of an immune cell effector cell only when bound to the target antigen at the antigen binding site.
2. The antibody immune cell inhibitor fusion protein of claim 1 , wherein the polypeptide chains comprise:
(a) the amino acid sequences of SEQ ID NO: 4 and SEQ ID NO: 17;
(b) the amino acid sequences of SEQ ID NO: 6 and SEQ ID NO: 14;
(c) the amino acid sequences of SEQ ID NO: 5 and SEQ ID NO: 14;
(d) the amino acid sequences of SEQ ID NO: 10 and SEQ ID NO: 16;
(e) the amino acid sequences of SEQ ID NO: 12 and SEQ ID NO: 13;
(f) the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 4;
(g) the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 4;
(h) the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 6;
(i) the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 5;
(j) the amino acid sequences of SEQ ID NO: 8 and SEQ ID NO: 10;
(k) the amino acid sequences of SEQ ID NO: 9 and SEQ ID NO: 10;
(l) the amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 11;
(m) the amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 12;
(n) the amino acid sequences of SEQ ID NO: 27 and SEQ ID NO: 28;
(o) the amino acid sequences of SEQ ID NO: 21 and SEQ ID NO: 33;
(p) the amino acid sequences of SEQ ID NO: 23 and SEQ ID NO: 34;
(q) the amino acid sequences of SEQ ID NO: 35 and SEQ ID NO: 14;
(r) the amino acid sequences of SEQ ID NO: 36 and SEQ ID NO: 14;
(s) the amino acid sequences of SEQ ID NO: 37 and SEQ ID NO: 14;
(t) the amino acid sequences of SEQ ID NO: 38 and SEQ ID NO: 14;
(u) the amino acid sequences of SEQ ID NO: 39 and SEQ ID NO: 14;
(v) the amino acid sequences of SEQ ID NO: 40 and SEQ ID NO: 14;
(w) the amino acid sequences of SEQ ID NO: 41 and SEQ ID NO: 14;
(x) the amino acid sequences of SEQ ID NO: 42 and SEQ ID NO: 14;
(y) the amino acid sequences of SEQ ID NO: 43 and SEQ ID NO: 14; or
(z) the amino acid sequences of SEQ ID NO: 44 and SEQ ID NO: 14.
3. A pharmaceutical composition comprising a therapeutically effective amount of the antibody immune cell inhibitor fusion protein of claim 2 and a pharmaceutically acceptable carrier.
4. A kit comprising the antibody immune cell inhibitor fusion protein of claim 2 .
5. An antibody immune cell inhibitor fusion protein comprising two polypeptide chains that form one antigen binding site and at least one immune cell receptor binding site;
wherein one polypeptide chain has a structure represented by the formula II1-L1-VL-CL-L2-II2, and one polypeptide chain has a structure represented by the formula II3-L3-VH-CH1-L4-II4;
wherein:
VL is an immunoglobulin light chain variable domain;
VH is an immunoglobulin heavy chain variable domain;
CL is an immunoglobulin light chain constant domain;
CH1 is the immunoglobulin CH1 heavy chain constant domain; L1, L2, L3, and L4 are each independently a linker domain or are absent; and
II1, II2, II3, and II4 are each independently an immune cell inhibitor domain of an immunoglobulin superfamily member or are absent;
wherein at least one of L1, L2, L3, and L4 is a linker domain and at least one of II1, II2, II3, and II4 a is an immune cell inhibitor domain of an immunoglobulin superfamily member; and
wherein the antibody immune cell inhibiter fusion protein inhibits or diminishes activation of an immune effector cell only when bound to a target antigen at the antigen binding site.
6. The antibody immune cell inhibitor fusion protein of claim 1 , wherein the polypeptide chains comprise:
(a) the amino acid sequences of SEQ ID NO: 4 and SEQ ID NO: 17;
(b) the amino acid sequences of SEQ ID NO: 6 and SEQ ID NO: 14;
(c) the amino acid sequences of SEQ ID NO: 5 and SEQ ID NO: 14;
(d) the amino acid sequences of SEQ ID NO: 10 and SEQ ID NO: 16;
(e) the amino acid sequences of SEQ ID NO: 12 and SEQ ID NO: 13;
(f) the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 4;
(g) the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 4;
(h) the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 6;
(i) the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 5;
(j) the amino acid sequences of SEQ ID NO: 8 and SEQ ID NO: 10;
(k) the amino acid sequences of SEQ ID NO: 9 and SEQ ID NO: 10;
(l) the amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 11;
(m) the amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 12;
(n) the amino acid sequences of SEQ ID NO: 27 and SEQ ID NO: 28;
(o) the amino acid sequences of SEQ ID NO: 21 and SEQ ID NO: 33;
(p) the amino acid sequences of SEQ ID NO: 23 and SEQ ID NO: 34;
(q) the amino acid sequences of SEQ ID NO: 35 and SEQ ID NO: 14;
(r) the amino acid sequences of SEQ ID NO: 36 and SEQ ID NO: 14;
(s) the amino acid sequences of SEQ ID NO: 37 and SEQ ID NO: 14;
(t) the amino acid sequences of SEQ ID NO: 38 and SEQ ID NO: 14;
(u) the amino acid sequences of SEQ ID NO: 39 and SEQ ID NO: 14;
(v) the amino acid sequences of SEQ ID NO: 40 and SEQ ID NO: 14;
(w) the amino acid sequences of SEQ ID NO: 41 and SEQ ID NO: 14;
(x) the amino acid sequences of SEQ ID NO: 42 and SEQ ID NO: 14;
(y) the amino acid sequences of SEQ ID NO: 43 and SEQ ID NO: 14; or
(z) the amino acid sequences of SEQ ID NO: 44 and SEQ ID NO: 14.
7. A pharmaceutical composition comprising a therapeutically effective amount of the antibody immune cell inhibitor fusion protein of claim 6 and a pharmaceutically acceptable carrier.
8. A kit comprising the antibody immune cell inhibitor fusion protein of claim 6 .
9. A method of treating a patient having primary biliary cholangitis, comprising:
administering an antibody immune cell inhibitor fusion protein to the patient, wherein the antibody immune cell inhibitor fusion protein comprises:
(A) four polypeptide chains that form two antigen binding sites and at least two immune cell receptor binding sites; wherein two polypeptide chains have a structure represented by the formula II1-VL-CL, and two polypeptide chains have a structure represented by the formula II3-VH-CH1-Fc; wherein: VL is an immunoglobulin light chain variable domain that specifically binds to dihydrolipoamide acetyltransferase; VH is an immunoglobulin heavy chain variable domain that specifically binds to dihydrolipoamide acetyltransferase; CL is an immunoglobulin light chain constant domain; CH1 is the immunoglobulin CH1 heavy chain constant domain; Fc is the immunoglobulin hinge region and CH2 and CH3immunoglobulin heavy chain constant domains; and II1 and II3 are each independently an immune cell inhibitor domain of an immunoglobulin superfamily member or are absent; wherein at least one of II1 and II3 is an immune cell inhibitor domain of an immunoglobulin superfamily member; wherein the antibody immune cell inhibitor fusion protein specifically binds to dihydrolipoamide acetyltransferase and inhibits or diminishes activation of an immune effector cell only when bound to a target antigen at one or both of the antigen binding sites;
(B) four polypeptide chains that form two antigen binding sites and at least two immune cell receptor binding sites; wherein two polypeptide chains have a structure represented by the formula II1-L1-VL-CL and two polypeptide chains have a structure represented by the formula II3-L3-VH-CH1-Fc; wherein: VL is an immunoglobulin light chain variable domain that specifically binds to dihydrolipoamide acetyltransferase; VH is an immunoglobulin heavy chain variable domain that specifically binds to dihydrolipoamide acetyltransferase; CL is an immunoglobulin light chain constant domain; CH1 is the immunoglobulin CH1 heavy chain constant domain; Fc is the immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; L1 and L3 are each independently a linker domain or are absent; and II1 and II3 are each independently an immune cell inhibitor domain of an immunoglobulin superfamily member or are absent; wherein at least one of L1 and L3 is a linker domain and at least one of II1 and II3 is an immune cell inhibitor domain of an immunoglobulin superfamily member; wherein the antibody immune cell inhibitor fusion protein specifically binds to dihydrolipoamide acetyltransferase and inhibits or diminishes activation of an immune effector cell only when bound to a target antigen at one or both of the antigen binding sites;
(C) four polypeptide chains that form two antigen binding sites and at least two immune cell receptor binding sites; wherein two polypeptide chains have a structure represented by the formula II1-VL-CL, and two polypeptide chains have a structure represented by the formula II3-VH-CH1-Fc; wherein: VL is an immunoglobulin light chain variable domain that specifically binds to dihydrolipoamide acetyltransferase; VH is an immunoglobulin heavy chain variable domain that specifically hinds to dihydrolipoamide acetyltransferase; CL is an immunoglobulin light chain constant domain; CH1 is the immunoglobulin CH1 heavy chain constant domain; Fc is the immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; and II1 and II3 are each independently an immune cell inhibitor domain of an immunoglobulin superfamily member or are absent; wherein at least one of II1 and II3 is an immune cell inhibitor domain of an immunoglobulin superfamily member; wherein the antibody immune cell inhibitor fusion protein specifically binds to dihydrolipoamide acetyltransferase and inhibits or diminishes activation of an immune effector cell only when bound to a target antigen at one or both of the antigen binding sites; or
(D) four polypeptide chains that form two antigen binding sites and at least two immune cell receptor binding sites; wherein two polypeptide chains have a structure represented by the formula II1-L1-VL-CL and two polypeptide chains have a structure represented by the formula II3-L3-VH-CH1-fc; wherein: VL is an immunoglobulin light chain variable domain that specifically binds to dihydrolipoamide acetyltransferase; is an immunoglobulin heavy chain variable domain that specifically binds to dihydrolipoamide acetyltransferase; CL is an immunoglobulin light chain constant domain; CH1 is the immunoglobulin CH1 heavy chain constant domain; Fc is the immunoglobulin hinge region and CH2 and CH3 immunoglobulin heavy chain constant domains; L1 and L3 are each independently a linker domain or are absent; and II1 and II3 are each independently an immune cell inhibitor domain of an immunoglobulin superfamily member or are absent; wherein at least one of L1 and L3 is a linker domain and at least one of II1 and II3 is an immune cell inhibitor domain of an immunoglobulin superfamily member; wherein the antibody immune cell inhibitor fusion protein specifically binds to dihydrolipoamide acetyltransferase and inhibits or diminishes activation of an immune effector cell only when bound to a target antigen at one or both of the antigen binding sites; and
wherein, for (A), (B), (C), and (D), the polypeptide chains comprise:
(a) the amino acid sequences of SEQ ID NO: 4 and SEQ ID NO: 17;
(b) the amino acid sequences of SEQ ID NO: 6 and SEQ ID NO: 14;
(c) the amino acid sequences of SEQ ID NO: 5 and SEQ ID NO: 14;
(d) the amino acid sequences of SEQ ID NO: 10 and SEQ ID NO: 16;
(e) the amino acid sequences of SEQ ID NO: 12 and SEQ ID NO: 13;
(f) the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 4;
(g) the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 4;
(h) the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 6;
(i) the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 5;
(j) the amino acid sequences of SEQ ID NO: 8 and SEQ ID NO: 10;
(k) the amino acid sequences of SEQ ID NO: 9 and SEQ ID NO: 10;
(l) the amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 11;
(m) the amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 12;
(n) the amino acid sequences of SEQ ID NO: 27 and SEQ ID NO: 28;
(o) the amino acid sequences of SEQ ID NO: 21 and SEQ ID NO: 33;
(p) the amino acid sequences of SEQ ID NO: 23 and SEQ ID NO: 34;
(q) the amino acid sequences of SEQ ID NO: 35 and SEQ ID NO: 14;
(r) the amino acid sequences of SEQ ID NO: 36 and SEQ ID NO: 14;
(s) the amino acid sequences of SEQ ID NO: 37 and SEQ ID NO: 14;
(t) the amino acid sequences of SEQ ID NO: 38 and SEQ ID NO: 14;
(u) the amino acid sequences of SEQ ID NO: 39 and SEQ ID NO: 14;
(v) the amino acid sequences of SEQ ID NO: 40 and SEQ ID NO: 14;
(w) the amino acid sequences of SEQ ID NO: 41 and SEQ ID NO: 14;
(x) the amino acid sequences of SEQ ID NO: 42 and SEQ ID NO: 14;
(y) the amino acid sequences of SEQ ID NO: 43 and SEQ ID NO: 14; or
(z) the amino acid sequences of SEQ ID NO: 44 and SEQ ID NO: 14.
10. The method of claim 9 , wherein the administering of the antibody immune cell inhibitor fusion protein to the patient comprises administering a composition comprising the antibody immune cell inhibitor fusion protein.
11. The method of claim 9 , wherein the administering of the antibody immune cell inhibitor fusion protein comprises the administration of (C), and wherein each of II1 and II3 is an immune cell inhibitor domain.
12. The method of claim 9 , wherein the administering of the antibody immune cell inhibitor fusion protein comprises the administration of (C), and wherein at least one of II1 and II3 is an immune cell inhibitor domain and inhibits or diminishes activation of an immune effector cell involved in the immune response to self-tissue.
13. The method of claim 9 , wherein the administering of the antibody immune cell inhibitor fusion protein comprises the administration of (C), and wherein at least one of II1 and II3 is absent.
14. The method of claim 9 , wherein the administering of the antibody immune cell inhibitor fusion protein comprises the administration of (C), wherein the immune cell inhibitor domain comprises a Programmed Death Ligand 1 (PD-L1) domain, Programmed Death Ligand 2 (PD-L2) domain, B7 Homolog 3 (B7-H3) domain, B7 Homolog 4 (B7-H4) domain, Herpesvirus Entry Mediator (HVEM) domain, B7 Homolog 5 (B7-H5) domain, B7 Homolog 6 (B7-H6) domain, B7 Homolog 7 (B7-H7) domain, Cytotoxic T Lymphocyte-Associated 4 (CTLA-4) domain, CD200 domain, T-cell immunoglobulin or Mucin Domains-Containing Protein 3 (TIM-3) domain.
15. The method of claim 9 , wherein the administering of the antibody immune cell inhibitor fusion protein comprises the administration of (D), wherein at least one of L1 and L3 is absent.
16. The method of claim 9 , where the administering comprises parenteral administration.
17. The method of claim 9 , where the administering comprises the administration of a dosage from about 0.1 mg/kg to up to about 100 mg/kg of a composition comprising the antibody immune cell inhibitor fusion protein of (A), (B), (C), or (D).
18. The method of claim 17 , where the composition has a pH range from about 5 to about 8.
19. The method of claim 17 , where the administering comprises the administration of (A) or (B).
20. The method of claim 17 , where the administering comprises the administration of (C) or (D).
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US18/187,928 US20240026032A1 (en) | 2017-04-24 | 2023-03-22 | Antibody Immune Cell Inhibitor Fusion Proteins |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201762489027P | 2017-04-24 | 2017-04-24 | |
PCT/US2018/028963 WO2018200422A1 (en) | 2017-04-24 | 2018-04-23 | Antibody immune cell inhibitor fusion proteins |
US201916607183A | 2019-10-22 | 2019-10-22 | |
US18/187,928 US20240026032A1 (en) | 2017-04-24 | 2023-03-22 | Antibody Immune Cell Inhibitor Fusion Proteins |
Related Parent Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2018/028963 Continuation WO2018200422A1 (en) | 2017-04-24 | 2018-04-23 | Antibody immune cell inhibitor fusion proteins |
US16/607,183 Continuation US11643473B2 (en) | 2017-04-24 | 2018-04-23 | Antibody immune cell inhibitor fusion proteins |
Publications (1)
Publication Number | Publication Date |
---|---|
US20240026032A1 true US20240026032A1 (en) | 2024-01-25 |
Family
ID=62196700
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/607,183 Active 2038-08-22 US11643473B2 (en) | 2017-04-24 | 2018-04-23 | Antibody immune cell inhibitor fusion proteins |
US18/187,928 Pending US20240026032A1 (en) | 2017-04-24 | 2023-03-22 | Antibody Immune Cell Inhibitor Fusion Proteins |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/607,183 Active 2038-08-22 US11643473B2 (en) | 2017-04-24 | 2018-04-23 | Antibody immune cell inhibitor fusion proteins |
Country Status (3)
Country | Link |
---|---|
US (2) | US11643473B2 (en) |
EP (1) | EP3615564A1 (en) |
WO (1) | WO2018200422A1 (en) |
Family Cites Families (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4931385A (en) * | 1985-06-24 | 1990-06-05 | Hygeia Sciences, Incorporated | Enzyme immunoassays and immunologic reagents |
DE69731289D1 (en) | 1996-03-18 | 2004-11-25 | Univ Texas | IMMUNGLOBULIN-LIKE DOMAIN WITH INCREASED HALF-VALUE TIMES |
WO1998023289A1 (en) | 1996-11-27 | 1998-06-04 | The General Hospital Corporation | MODULATION OF IgG BINDING TO FcRn |
US6277375B1 (en) | 1997-03-03 | 2001-08-21 | Board Of Regents, The University Of Texas System | Immunoglobulin-like domains with increased half-lives |
WO2002076406A2 (en) * | 2001-03-27 | 2002-10-03 | Gershwin M Eric | Antibodies against autoantigens of primary biliary cirrhosis and methods of making and using them |
ES2638521T5 (en) * | 2010-03-05 | 2021-03-02 | Univ Johns Hopkins | Compositions and Methods for Targeted Immunomodulatory Antibodies and Fusion Proteins |
NZ711451A (en) | 2014-03-07 | 2016-05-27 | Alexion Pharma Inc | Anti-c5 antibodies having improved pharmacokinetics |
HUE049982T2 (en) | 2014-11-14 | 2020-11-30 | Hoffmann La Roche | Antigen binding molecules comprising a tnf family ligand trimer |
-
2018
- 2018-04-23 US US16/607,183 patent/US11643473B2/en active Active
- 2018-04-23 EP EP18725702.7A patent/EP3615564A1/en active Pending
- 2018-04-23 WO PCT/US2018/028963 patent/WO2018200422A1/en unknown
-
2023
- 2023-03-22 US US18/187,928 patent/US20240026032A1/en active Pending
Also Published As
Publication number | Publication date |
---|---|
WO2018200422A1 (en) | 2018-11-01 |
US20210261684A1 (en) | 2021-08-26 |
EP3615564A1 (en) | 2020-03-04 |
US11643473B2 (en) | 2023-05-09 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP7387780B2 (en) | Anti-CD38 antibody and method of use | |
US20210301028A1 (en) | Composition and methods for anti-tnfr2 antibodies | |
JP6682426B2 (en) | Anti-B7-H5 antibody and use thereof | |
US11713356B2 (en) | Modified bifunctional anti-human signal regulatory protein alpha (SIRPa) antibody and method of use thereof for treating cancer | |
CN113557245B (en) | Humanized anti-human PD-1 antibodies | |
CN110913904A (en) | Pharmaceutical compositions comprising bispecific antibody constructs for improved storage and administration | |
EA031849B1 (en) | Anti-ox40 antibodies and methods of using the same | |
US20220056135A1 (en) | Bifunctional anti-pd-1/sirpa molecule | |
AU2020406083A1 (en) | Bifunctional molecules comprising an IL-7 variant | |
RU2694412C2 (en) | Monoclonal antibodies and methods of using them | |
RU2725807C2 (en) | Soluble universal reinforcing adcc synthetic fused gene, peptide technology and use thereof | |
JP2022514187A (en) | Multimer hybrid Fc protein for the replacement of IVIG | |
WO2023143547A1 (en) | Anti-cd28 antibody and use thereof | |
WO2020048454A1 (en) | Fusion protein and its application in preparing medicine for treating tumor and/or viral infection | |
US20220348689A1 (en) | ADOPTIVE T-CELL THERAPY USING EMPD-SPECIFIC CHIMERIC ANTIGEN RECEPTORS FOR TREATING lgE-MEDIATED ALLERGIC DISEASES | |
US20240026032A1 (en) | Antibody Immune Cell Inhibitor Fusion Proteins | |
CN116323671A (en) | Multi-targeting bispecific antigen binding molecules with increased selectivity | |
EP4077364A1 (en) | Bifunctional molecules comprising an il-7 variant | |
TW201702263A (en) | Anti-CD20-/anti-BAFF bispecific antibodies |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: ALEXION PHARMACEUTICALS, INC., CONNECTICUT Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:CHANDLER, JULIAN;REEL/FRAME:063418/0175 Effective date: 20180424 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |