human antibodies to human tnf (tl1a) ligand 1a

专利摘要:
HUMAN ANTIBODIES TO HUMAN TNF (TL1A) BINDER 1A. The present invention relates to a fully human antibody or antigen-binding fragment of a human antibody that specifically binds and inhibits the human TNF ligand 1A (hTL1A) is provided. Human anti-hTL1A antibodies are useful in the treatment of diseases or disorders associated with TL1A, such as inflammatory diseases and disorders, such as inflammatory bowel diseases, including ulcerative colitis and Crohn's disease, rheumatoid arthritis and the like, auto diseases or disorders -immune, such as multiple sclerosis, diabetes and the like and allergic reactions, such as asthma and allergic lung inflammation.
公开号:BR112013011245A2
申请号:R112013011245-0
申请日:2011-11-08
公开日:2020-08-04
发明作者:Brendan J. CLASSON；Dimitris SKOKOS
申请人:Regeneron Pharmaceuticals, Inc.；
IPC主号:

专利说明:

Invention Patent Descriptive Report for "HUMAN ANTICORPOS TO HUMAN TNF (TL1A) TYPE BINDER 1A".
FIELD OF THE INVENTION The present invention relates to human antibodies and antigen-binding fragments of human antibodies that specifically bind to the human TNF 1A-type ligand (hTL1A) and therapeutic methods of using these antibodies.
STATEMENT OF RELATED TECHNIQUES TL1A is a type II cell membrane protein of the tumor necrosis factor (Tumor Necrosis Factor Superfamily - TNFSF) superfamily and also designated as TNFSF15. It is expressed on the surface of endothelial cells and activated cells of the hematopoietic lineage, including monocytes, macrophages, lymphocytes, lamina propria mononuclear cells, dendritic cells and plasma cells (Tan, KB et al., 1997, Gene 204 : 35-46; Prehn, JL et al., 2007, J Immunol 178: 4033-4038). It is also expressed in the kidney, lung, prostate and thymus (Tan et al., 1997, supra). In endothelial cells, expression of TL1A is up-regulated by IL-1α and TNFγ (Migone, T.S. et al., 2002, Immunity 16: 479-492). In monocytes of fresh human blood and monocyte-derived dendritic cells, expression of TL1A is over-regulated by Fc receptor-mediated signaling (R or Toll-like (TLR)) (Prehn et al., 2007, supra ; Meylan, F. et al., 2008, Immunity 29: 79-89). TL1A can be cleaved from the cell membrane through a mechanism analogous to TNF (and a form of TL1A with soluble ectodomain has been reported ( Migone et al., 2002, supra; Kim, S. et al., 2005, J Immunol Methods 298: 1-8; Yang, CR et al., 2004, Cancer Res 64: 1122-1129). that this form of TL1A is released after cleavage of the membrane-anchored precursor between residues Ala-71 and Leu-72 (Migone et al., 2002, supra). Two variant cDNAs that potentially encode N-terminally truncated versions of TL1A have been identified : VEGI-174 (or TL1) (Zhai, Y. et al., 1999, FASEB J. 13: 181-189) and VEGI-192 (Chew, LJ et al., 2002, FASEB J. 16: 742-744 ). Data published in the literature are suggest that biologically active products
of the TL1A gene are the full-length type II transmembrane protein (residues 1-251) and its proteolytically cleaved ectodomain (residues 72-251) (Migone et al., 2002, supra; Jin et al., 2007, Biochem Bio-phys Res Commun 364: 1-6). A variant of hTL1A, designated as 5 "Fhm", which contains a single amino acid substitution of Gln-167 for Arg, is described in US 6,521,422. TL1A mediates signals via its cognate receptor Death Receptor 3 (Death Receptor 3 - DR3; also known as TNFRSF25; the nucleic acid and amino acid sequences of SEQ ID NOs: 251 and 252, respectively), resulting in the promotion of cell survival and secretion of pro-inflammatory cytokines or that promote apoptosis in a context-dependent manner.
TL1A is one of three known ligands (in addition to FasL and LIGHT) that are linked by the endogenous soluble decoy receptor DcR3 (also known as TR6, NTR3 or TNFRSF2; the nucleic acid and amino acid sequences of SEQ ID NOs: 253 and 254, respectively) (Migone et al., 2002, supra; Yang, CR et al., 2004, Cancer Res. 64: 1122-1129). DR3 is a receptor related to the death domain related to the TNF receptor expressed in most activated T lymphocytes and NK cells (Migone et al., 2002, supra; Screaton, GR et al., 1997, Proc Natl Acad Sci ( USA) 94: 4615-4619). TL1A fits DR3 on T cells, increasing its responsiveness to IL-2 (Migone et al., 2002, supra), potentiating the proliferation of T cells and the release of IFN-γ and GM-CSF under co-conditions. sub-optimal stimulation (Migone et al., 2002, supra; Me-ylan et al., 2008, supra). It has also been shown that TL1A synergizes with sub-optimal levels of IL-12 / IL-18 to induce IFN production (by CD4 + T cells (Papadakis, K.A. et al., 2004, J.
Immunol 172: 7002-7007; Prehn, J.L. et al., 2004, Clin Immunol 112: 66-77; Papadakis, K.A. et al., 2005, J Immunol 174: 4985-4990 ;. Cassatella, M.A. et al., 2007, J Immunol 178: 7325-7333). TL1A has been shown to be involved in several inflammatory diseases and / or autoimmune diseases, including inflammatory bowel diseases [eg, ulcerative colitis (Ulcerative Colitis - UC) and
Crohn's (Crohn's Disease - DC)], rheumatoid arthritis, multiple sclerosis (Multi- ple Sclerosis - MS), atherosclerosis and the like (see Bayry, J., 2010, Nature Reviews / Rheumatology 6: 67-68; Takedatsu, H. et al., 2008, Gastroenterological 135: 552-567; Prehn et al., 2004, supra; Bamias, G. et al., 2008, Clin 5 Immunol 129: 249-255; Bull, MJ et al., 2008 , J Exp Med 205: 2457-2464; Pappu, BP et al., 2008, J Exp Med 205: 1049-1062; Bamias, G. et al., 2003, J Immunol 171: 4868-4874; Kang, Y. et al., 2005, Cytokine 29: 229-235). Although most of the published data are consistent with a central role for TL1A in enabling differentiation of TH1 and TH17 effector cells, a recent study has proposed a role for the TL1A / DR3 interaction in the development of TH2 T cell responses in asthma models ( Fang, L. et al., 2008, J Exp. Med. 205: 1037-1048). Thus, the use of TL1A inhibitors, such as fully human antibodies against TL1A with high affinities and neutralizing activity, alone or in combination with currently available anti-inflammatory agents, immunosuppressants (eg TNF- (, cortisone antagonists) or steroids and the like) and / or antiallergic agents, constitutes an effective treatment for these diseases and disorders.The nucleic acid and amino acid sequences of human TL1A are shown in SEQ ID NOS: 243 and 244, respectively and those of Fhm are shown in SEQ ID NOS: 245 and 246, respectively, Antibodies to TL1A are described, for example, in US 7,597,886, US
7,820,798 and US 2009/0280116.
BRIEF SUMMARY OF THE INVENTION In a first aspect, the invention provides fully human monoclonal antibodies (mAbs) and antigen-binding fragments thereof that specifically bind and neutralize the activity of human TL1A (hTL1A). Antibodies can be full-length (for example, an IgG1 or IgG4 antibody) or can comprise only an antigen-binding portion (for example, a Fab, F (ab ') 2 or scFv fragment) and can be modified to affect functionality, for example, to eliminate
to demonstrate residual effector functions (Reddy et al., 2000, J.
Immunol. 164: 1925-1933). In one embodiment, the invention is characterized by an antibody or antigen-binding fragment of an antibody comprising a heavy chain variable region (HCVR) selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 118, 134, 138, 154, 158, 174, 178, 194, 198, 214, 218 and 234 or a sequence substantially similar to them that is at least 90%, at least at least 95%, at least 98% or at least 99% sequence identity.
In another embodiment, the antibody or antigen-binding fragment thereof comprises an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 134, 174 and 234 In yet another embodiment, the antibody or fragment thereof comprises an HCVR comprising SEQ ID NOs: 2, 18, 174 or 234. In one embodiment, the antibody or fragment thereof also comprises a variable region of light chain (Light Chain Variable Region - LCVR) selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226 and 236 or a sequence substantially similar to those that have at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
In another embodiment, the antibody or antigen-binding portion of an antibody comprises an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 136, 176 and 236 In yet another embodiment, the antibody or fragment thereof comprises an LCVR comprising SEQ ID NOs: 10, 26, 176 or 236. In other embodiments, the antibody or fragment thereof comprises a pair of HCVR and LCVR sequences ( HCVR / LCVR) selected from the group consisting of SEQ ID NOs: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/116, 118 / 126, 134/136, 138/146, 154/156, 158/166, 174/176, 178/186, 194/196, 198/206, 214/216, 218/226 and 234/236.
In one embodiment, the antibody or fragment thereof comprises an HCVR and LCVR selected from pairs of amino acid sequences of SEQ ID NOs: 2/10, 18/26, 34/42, 50/58, 66/74, 134/136 , 174/176 and 234/236. In another embodiment, the antibody or fragment thereof comprises an HCVR / LCVR pair comprising SEQ ID NOs: 2/10, 18/26, 174/176 or 234/236. In a second aspect, the invention is characterized by an antibody or antigen-binding fragment of an antibody that comprises an amino acid sequence from the Heavy Chain Complementarity Determining Region - HCDR3) selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 124, 144, 164, 184, 204 and 224 or a sequence substantially similar to those that have at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a sequence of light chain CDR3 amino acids (Light Chain Complementary Determining Region - LCDR3) selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 132, 152, 172 , 192, 212 and 232 or sequences substantially similar to those having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
In one embodiment, the antibody or fragment thereof comprises a pair of HCDR3 / LCDR3 amino acid sequences comprising SEQ ID NOs: 8/16, 24/32, 40/48, 56/64, 72/80, 88/96, 104/112 124/132, 144/152, 164/172, 184/192, 204/212 or 224/232. In another embodiment, the antibody or fragment thereof comprises a pair of HCDR3 / LCDR3 amino acid sequences comprising SEQ ID NOs: 8/16, 24/32, 40/48, 56/64, 72/80, 124/132, 164/172 or 224/232. In yet another embodiment, the antibody or fragment thereof comprises a pair of HCDR3 / LCDR3 amino acid sequences comprising SEQ ID NOs: 8/16, 24/32, 164/172 or 224/232. In another embodiment, the invention is characterized by an antibody or fragment thereof, further comprising a sequence of heavy chain CDR1 amino acids (HCDR1) selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52 , 68, 84, 100, 120, 140, 160, 180, 200 and 220 or a sequence substantially similar to the same ones that is at least 90%, at least 95%, at least 98% or at least 99% identical of sequence, an amino acid sequence of heavy chain CDR2 (HCDR2) selected from the group consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 122, 142, 162, 182, 202 and 222 or a sequence substantially similar to those that have at least 90%, at least 95%, at least 98% or at least 99% sequence identity and / or a CDR1 light chain amino acid sequence (LCDR1) selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 128, 148, 168, 188, 208 and 228 or a sequence substantially similar to those that have at least in 90%, at least 95%, at least 98% or at least 99% sequence identity and / or a light chain CDR2 (LCDR2) amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30 , 46, 62, 78, 94, 110, 130, 150, 170, 190, 210 and 230 or a sequence substantially similar to them that has at least 90%, at least 95%, at least 98% or at least 99% string identity.
In one embodiment, the antibody or fragment thereof comprises a combination of HCDR1 / HCDR2 / HCDR3 selected from the group consisting of SEQ ID NOs: 4/6/8, 20/22/24, 36/38/40, 52 / 54/56, 68/70/72, 84/86/88, 100/102/104, 120/122/124, 140/142/144, 160/162/164, 180/182/184, 200/202 / 204 and 220/222/224 and / or a combination of LCDR1 / LCDR2 / LC-DR3 selected from the group consisting of SEQ ID NOs: 12/14/16, 28/30/32, 44/46/48, 60 / 62/64, 76/78/80, 92/94/96, 108/110/112, 128/130/132, 148/150/152, 168/170/172, 188/190/192, 208/210 / 212 and 228/230/232. In another mode, the heavy and light chain CDR amino acid sequences comprise a combination of CDR sequences selected from the group consisting of SEQ ID NOs: 4/6/8/12/14/16, 20 / 22/24/28/30/32, 36/38/40 / 44/46/48, 52/54/56/60/62/64, 68/70/72/76/78/80, 84/86 / 88/92/94/96, 100/102 / 104/108/110/112, 120/122/124/128/130/132, 140/142/144/148/150/152, 160/162/164 / 168/170/172, 180/182/184/188/190/192, 200/202/204/208/210/212 and 220/222/224/228/230/232. In another embodiment, the antibody or antigen-binding fragment thereof comprises the heavy and light chain CDR sequences of SEQ ID NOs: 4/6/8/12/14/16, 20/22/24/28/30 / 32, 36/38/40/44/46/48, 52/54/56/60/62/64, 68/70/72/76/78/80, 120/122/124/128 / 130/132 , 160/162/164/168/170/172 or 220/222/224/228/230/232. In yet another embodiment, the heavy and light chain CDR amino acid sequences comprise a combination of CDR sequences selected from SEQ ID NO: 4/6/8/12/14/16, 20/22/24/28/30 / 32, 160/162/164/168/170/172 or 220/222/224/228/230/232. In a related embodiment, the invention comprises an antibody or antigen-binding fragment of an antibody that specifically binds to hTL1A, wherein the antibody or fragment thereof comprises the CDR domains contained within pairs of heavy chain sequences and light selected from the group consisting of SEQ ID NOs: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/116, 118/126, 134 / 136, 138/146, 154/156, 158/166, 174/176, 178/186, 194/196, 198/206, 214/216, 218/226 and 234/236. Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are known in the art and can be applied to identify CDRs within the specified HCVR and / or LCVR amino acid sequences described here.
Conventional definitions that can be applied to identify CDR limits include the definition of Kabat, the definition of Chothia and the definition of AbM.
In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the structural regions of the loop, and the AbM definition is a compromise between the Kabat and Chothia approaches.
See, for example, Kabat, "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991), Al-Lazikani et al., J.
Mol.
Biol. 273: 927-948 (1997) and Martin et al., Proc.
Natl.
Acad.
Sci.
USA 86: 9268-9272 (1989). Public databases are also available for the identification of CDR sequences within an antibody.
In one embodiment, the antibody or fragment thereof comprises CDR sequences contained within an HCVR and LCVR pair selected from the group consisting of the amino acid sequence pairs of SEQ ID
NOs: 2/10, 18/26, 34/42, 50/58, 66/74, 134/136, 174/176 and 234/236. In another embodiment, the antibody or fragment thereof comprises CDR sequences contained within the HCVR and LCVR sequence pair of SEQ ID NOs: 2/10, 18/26, 174/176 or 234/236. In another related embodiment, the invention provides an antibody or antigen-binding fragment thereof that competes for specific binding to hTL1A with an antibody or antigen-binding fragment comprising heavy and light chain CDR sequences of SEQ ID NOs : 4/6/8/12/14/16, 20/22/24/28/30/32, 36/38/40/44/46/48, 52/54 / 56/60/62/64, 68 / 70/72/76/78/80, 120/122/124/128/130/132, 160/162/164/168 / 170/172 or 220/222/224/228/230/232. In one embodiment, the antibody or antigen binding fragment thereof competes for specific binding to hTL1A with an antibody or antigen binding fragment comprising heavy and light chain CDR sequences of SEQ ID NOs: 4/6/8 / 12/14/16, 20/22/24/28/30/32, 160/162/164/168/170/172 or 220/222 / 224/228/230/232. In another embodiment, the antibody of the invention or antigen-binding fragment competes for specific binding to hTL1A with an antibody or antigen-binding fragment comprising a pair of HCVR / LCVR sequences of SEQ ID NOs: 2/10, 18 / 26, 34/42, 50/58, 66/74, 134/136, 174/176 or 234/236. In yet another embodiment, the antibody or antigen-binding fragment thereof competes for specific binding to hTL1A with an antibody or antigen-binding fragment comprising a pair of HCVR / LCVR sequences of SEQ ID NOs: 2 / 10, 18/26, 174/176 or 234/236. In another related embodiment, the invention provides an antibody or antigen-binding fragment thereof that binds to the same epitope on hTL1A that is recognized by an antibody or fragment thereof that comprises the light and heavy chain CDR sequences of SEQ ID NOs: 4/6/8/12/14/16, 20/22/24/28/30/32, 36/38/40/44 / 46/48, 52/54/56/60/62 / 64, 68/70/72/76/78/80, 120/122/124/128/130/132, 160 / 162/164/168/170/172 or 220/222/224/228/230/232. In one embodiment, the antibody or antigen-binding fragment of the same binds to the same epitope on hTL1A that is recognized by an antibody or antigen-binding fragment that comprises the heavy and light chain CDR sequences of SEQ ID NOs : 4/6/8/12/14/16, 20/22/24/28/30/32, 160/162 / 164/168/170/172 or 220/222/224/228/230/232. In another embodiment, the antibody of the invention or antigen binding fragment recognizes the same epitope on hTL1A that is recognized by an antibody or antigen binding fragment comprising a pair of HCVR / LCVR sequences of SEQ ID NO: SEQ ID NOs: 2/10, 18/26, 34/42, 50/58, 66/74, 134/136, 174/176 or 234/236. In yet another embodiment, the antibody or antigen-binding fragment thereof recognizes the same epitope on hTL1A that is recognized by an antibody or antigen-binding fragment comprising a HCVR / LCVR sequence pair of SEQ ID NOs: 2 / 10, 18/26, 174/176 or 234/236. In a third aspect, the invention provides nucleic acid molecules that encode anti-TL1A antibodies or fragments thereof as described above.
Recombinant expression vectors that carry the nucleic acids of the invention and isolated host cells, for example, bacterial cells, such as E. coli cells or mammalian cells, such as CHO cells, into which such vectors have been introduced, are also covered by invention, as well as methods of producing antibodies by culturing host cells under conditions that allow the production of antibodies and recovery of the antibodies produced.
In one embodiment, the invention provides an antibody or fragment thereof comprising an HCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 17, 33, 49, 65, 81, 97, 113, 117, 133, 137, 153, 157, 173, 177, 193, 197, 213, 217 and 233 or a substantially identical sequence that is at least 90%, at least 95%, at least 98% or at least 99 % homology with them.
In another embodiment, the antibody or fragment thereof comprises an HCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 17, 33, 49, 65, 133, 173 and 233. In yet another embodiment , the antibody or fragment thereof comprises an HCVR encoded by the nucleic acid sequence of SEQ ID NOs: 1, 17, 173 or 233. In one embodiment, the antibody or fragment thereof further comprises an LCVR encoded by a sequence of 5 nucleic acid selected from the group consisting of SEQ ID NOs: 9, 25, 41, 57, 73, 89, 105, 115, 125, 135, 145, 155, 165, 175, 185, 195, 205, 215, 225 and 235 or a substantially identical sequence which has at least 90%, at least 95%, at least 98% or at least 99% homology thereto.
In another embodiment, the antibody or fragment thereof comprises an LCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 9, 25, 41, 57, 73, 135, 175 and 235. In yet another embodiment, the antibody or fragment thereof comprises an LCVR encoded by the nucleic acid sequence of SEQ ID NOs: 9, 25, 175 or 235. In other embodiments, the antibody or fragment thereof comprises a pair of HCVR sequences and LCVR (HCVR / LCVR) encoded by a pair of nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1/9, 17/25, 33/41, 49/57, 65/73, 81 / 89, 97/105, 113/115, 117/125, 133/135, 137/145, 153/155, 157/165, 173/175, 177/185, 193/195, 197/205, 213/215, 217/225 and 233/235. In one embodiment, the antibody or fragment thereof comprises a pair of HC-VR / LCVR sequences encoded by a pair of nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1/9, 17 / 25, 33/41, 49/57, 65/73, 133/135, 173/175 and 233/235. In yet another embodiment, the antibody or fragment thereof comprises a pair of HCVR / LCVR sequences encoded by a pair of nucleic acid sequences of SEQ ID NOs: 1/9, 17/25, 173/175 or 233/235. In one embodiment, the invention is characterized by an antibody or antigen-binding fragment of an antibody comprising an HCDR3 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7, 23, 39, 55, 71, 87, 103, 123, 143, 163, 183, 203 and 223 or a substantially identical sequence that has at least 90%, at least 95%, at least 98% or at least 99% homology with the same and an LCDR3 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 15, 31, 47, 63, 79, 95, 111, 131, 151, 171, 191, 211 and 231 or a sequence substantially identical which has at least 90%, at least 95%, at least 98% or at least 99% homology to them.
In another embodiment, the antibody or fragment thereof comprises a pair of LCDR3 and HCDR3 sequences encoded by the nucleic acid sequence pair of SEQ ID NOs: 7/15, 23/31, 39/47, 55/63, 71 / 79, 87/95, 103/111, 123/131, 143/151, 163/171, 183/191, 203/211 or 223/231. In another embodiment, the antibody or fragment thereof comprises a pair of LCDR3 and HCDR3 sequences encoded by the pair of nucleic acid sequences of SEQ ID NOs: 7/15, 23/31, 39/47, 55/63, 71/79, 123/131, 163/171 or 223/231. In yet another embodiment, the HC-DR3 / LCDR3 sequence pair is encoded by the nucleic acid sequence pair of SEQ ID NOs: 7/15, 23/31, 163/171 or 223/231. In another embodiment, the antibody or fragment thereof also comprises an HCDR1 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 19, 35, 51, 67, 83, 99 , 119, 139, 159, 179, 199 and 219 or a substantially identical sequence that has at least 90%, at least 95%, at least 98% or at least 99% homology with them; an HCDR2 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 5, 21, 37, 53, 69, 85, 101, 121, 141, 161, 181, 201 and 221 or a substantially identical sequence which has at least 90%, at least 95%, at least 98% or at least 99% homology with them; an LCDR1 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 11, 27, 43, 59, 75, 91, 107, 127, 147, 167, 187, 207 and 227 or a subscript substantially identical which has at least 90%, at least 95%, at least 98% or at least 99% homology with them; and an LCDR2 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13, 29, 45, 61, 77, 93, 109, 129, 149, 169, 189, 209 and 229 or a substantially identical that has at least 90%, at least 95%, at least 98% or at least 99% homology with them.
In one embodiment, the antibody or fragment 5 thereof comprises a combination of HCDR1 / HCDR2 / HCDR3 encoded by SEQ ID NOs: 3/5/7, 19/21/23, 35/37/39, 51/53 / 55, 67/69/71, 83/85/87, 99/101/103, 119/121/123, 139/141/143, 159/161/163, 179/181/183, 199/201/203 or 219/221/223, and a combination of LCDR1 / LCDR2 / LCDR3 encoded by SEQ ID NOs: 11/13/15, 27/29/31, 43/45/47, 59/61/63, 75/77/79 , 91/93/95, 107/109/111, 127/129/131, 147/149/151, 167/169/171, 187/189/191, 207/209/211 or 227/229/231. In one embodiment, the antibody or fragment thereof comprises heavy and light chain CDR sequences encoded by a combination of nucleic acid sequences selected from the group consisting of SEQ ID NOs: 3/5/7/11/13 / 15, 19/21 / 23/27/29/31, 35/37/39/43/45/47, 51/53/55/59/61/63, 67/69/71/75/77/79, 83/85 / 87/91/93/95 99/101/103/107/109/111, 119/121/123/127/129/131, 139/141 / 143/147/149/151, 159/161 / 163/167/169/171, 179/181/183/187/189/191, 199 / 201/203/207/209/211 and 219/221/223/227/229/231. In another embodiment, the antibody or antigen binding portion thereof comprises heavy and light chain CDR sequences encoded by a combination of nucleic acid sequences of SEQ ID NOs: 3/5/7/11/13/15 , 19/21/23 / 27/29/31, 35/37/39/43/45/47, 51/53/55/59/61/63, 67/69/71/75/77/79, 119 / 121 / 123/127/129/131 or 159/161/163/167/169/171 219/221/223/227/229/231. In yet another embodiment, the antibody or antigen binding portion thereof comprises heavy and light chain CDR sequences encoded by a combination of nucleic acid sequences of SEQ ID NOs: 3/5/7/11/13 / 15, 19/21/23/27/29/31, 159/161/163/167/169/171 or 219/221 / 223/227/229/231. In a fourth aspect, the invention is characterized by an isolated antibody or antigen-binding fragment of an antibody that specifically binds to hTL1A comprising an HCDR3 and LCDR3, wherein HCDR3 comprises an amino acid sequence of the formula X1 -
X2 - X3 - X4 - X5 - X6 - X7 - X8 - X9 - X10 - X11 - X12 - X13 - X14 - X15 - X16 (SEQ ID NO: 239), where X1 is Thr or Ala, X2 is Lys, Arg or is absent, X3 is Glu, Gly or is absent, X4 is Asp, Pro or is absent, X5 is Leu or is absent, X6 is Arg, Tyr, Glu or is absent, X7 is Gly, Asp, Ala or is au - 5 sit, X8 is Asp, Ser or Tyr, X9 is Tyr or Trp, X10 is Tyr or Asp, X11 is Tyr, Lys or Ile, X12 is Gly, Tyr, Asn, or Ser, X13 is Val, Gly or Ser , X14 is Phe or Met, X15 is Asp and X16 is Tyr or Val; and LCDR3 comprises an amino acid sequence of the formula X1 - X2 - X3 - X4 - X5 - X6 - X7 - X8 - X9 (SEQ ID NO: 242), where X1 is Gln, X2 is Gln, X3 is Tyr, Leu or Phe, X4 is His, Tyr or Asn, X5 is Arg or Ser, X6 is Ser, Thr or Tyr, X7 is Trp or Pro, X8 is Phe, Leu or is absent and X9 is Thr.
In another embodiment, the antibody or fragment thereof further comprises an HCDR1 sequence comprising an amino acid sequence of the formula X1 - X2 - X3 - X4 - X5 - X6 - X7 - X8 (SEQ ID NO: 237), where X1 is Gly, X2 is Phe, X3 is Thr, X4 is Phe, X5 is Ser, X6 is Thr, Ser or Asn, X7 is Tyr, and X8 is Gly, Trp, Val or Ala; an HC-DR2 sequence comprising an amino acid sequence of the formula X1 - X2 - X3 - X4 - X5 - X6 - X7 - X8 (SEQ ID NO: 238), where X1 is Ile or Val, X2 is Ser or Lys, X3 is Gly or Glu, X4 is Thr, Asp, Ser or Arg, X5 is Gly, X6 is Arg, Ser or Gly, X7 is Thr, Glu or Ser, and X8 is Thr or Lys; an LCDR1 sequence comprising an amino acid sequence of the formula X1 - X2 - X3 - X4 - X5 - X6 - X7 - X8 - X9 - X10 - X11 - X12 (SEQ ID NO: 240), where X1 is Gln, X2 is Thr, Ser, Ala or Gly, X3 is Ile, X4 is Ser or Leu, X5 is Tyr or is absent, X6 is Ser or is absent, X7 is Ser or is absent, X8 is Asn or is absent, X9 is Asn or is absent, X10 is Lys or is absent, X11 is Ser, Asn or Thr, and X12 is Trp or Tyr; and an LDR2 sequence comprising an amino acid sequence of the formula X1 - X2 - X3 (SEQ ID NO: 241) where X1 is Ala, Trp or Ser, X2 is Ala or Thr, and X3 is Ser.
In a fifth aspect, the invention is characterized by a human anti-TL1A antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) encoded by segments of nucleotide sequences derived from lipid sequences.
germline VH, DH and JH and a light chain variable region (LCVR) encoded by segments of nucleotide sequences derived from VK and JK germline sequences. In certain modalities, the antibody or antigen-binding fragment of the same comprises 5 HCVR and LCVR encoded by segments of nucleotide sequences derived from a combination of genes from the germline selected from the group consisting of: (i) VH3-23, DH2-21, JH4, VK1-5 and JK1; (ii) VH3-7, DH1-7, JH6, VK4-1 and JK3; (iii) VH3-23, DH2-2, JH6, VK1-9 and JK2; (iv) VH3-23, DH6-6, JH4, VK1-9 and JK4; (v) VH1-2, DH2-15, JH3, VK1-12 and JK4; (vi) VH4-34, DH3-9, JH4, VK3-20 and JK4; (vii) VH4-34, DH1-1, JH4, VK3-20 and JK4; and (viii) VH4-34, DH3-3, JH4, VK2-24 and JK4. In a sixth aspect, the invention is characterized by an antibody or antigen-binding fragment that specifically binds to hTL1A or Fhm with an equilibrium dissociation constant (KD) of about 1 nM or less, as measured by the surface plasmon resonance assay (eg, BIACORETM). In certain embodiments, the antibody of the invention exhibits a KD of about 800 pM or less, about 700 pM or less, about 600 pM or less, about 500 pM or less, about 400 pM or less , about 300 pM or less, about 200 pM or less, about 150 pM or less, about 100 pM or less, about 90 pM or less, about 80 pM or less, about 50 pM or less or 30 pM or less. In a seventh aspect, the present invention provides an anti-hTL1A antibody or antigen-binding fragment thereof that binds to the hTL1A protein of SEQ ID NO: 244, but does not cross-react with a variant thereof, such as as Fhm of SEQ ID NO: 246, as determined, for example, by means of ELISA, surface plasmon resonance assay or Luminex® Technology or xMAP, as described here. Fhm contains a single amino acid substitution at position 167, which corresponds to Gln in hTL1A, with Arg (see U.S. Patent No.
6,521,422). In related embodiments, the invention also provides an anti-hTL1A antibody or antigen-binding fragment thereof that binds to an hTL1A protein and cross-reacts with an Fhm.
In another related embodiment, the invention provides an anti-hTL1A antibody or antigen-binding fragment of the same that does not cross-react with mouse TL1A (mTL1A: SEQ ID NO: 250, encoded by the nucleotide sequence of SEQ ID NO: 249), but cross-react with TL1A from monkey Cynomolgus (Macaca fascicularis or MfTL1A: SEQ ID NO: 248, encoded by the nucleotide sequence of SEQ ID NO: 247) or from rhesus monkey (Macaca mulatta: the same amino acid sequence according to MfTL1A). In other related embodiments, the invention provides an anti-hTL1A antibody or antigen-binding fragment thereof that cross-reacts with mTL1A and Mf-TL1A.
The invention encompasses anti-hTL1A antibodies that have an altered glycosylation pattern.
In some applications, modification to remove unwanted glycosylation sites or, for example, removal of a fucus portion and to increase the function of antibody-dependent cell cytotoxicity (Antibody Dependent Cellular Cytotoxicity - ADCC) see Shield and such. (2002) JBC 277: 26733). In other applications, removal of the N-glycosylation site may reduce undesirable immune reactions against therapeutic antibodies or increase antibody affinities.
In yet other applications, modification of galactosylation can be done in order to modify Complement Dependent Cytotoxicity (CDC). In an eighth aspect, the invention is characterized by a pharmaceutical composition comprising a recombinant human antibody or fragment thereof that specifically binds to hTL1A and a pharmaceutically acceptable carrier.
In one embodiment, the invention is characterized by a composition that is a combination of an antibody or antigen-binding fragment thereof and a second therapeutic agent.
The second therapeutic agent can be one or more of any agent, such as immunosuppressive agents, anti-inflammatory agents, analgesic agents, antiallergic agents and the like, many of which can have therapeutic effects overlapping each other.
Immunosuppressants suitable for use in combination with the anti-hTL1A antibodies of the invention include, but are not limited to, glucocorticoids, cyclosporine, methotrexate, interferon β (IFN-β), tacrolimus, sirolimus, azathioprine, 5 mercaptopurine, opioids, mycophenolate, TNF-binding proteins, such as eternacept, infliximab, adalimumab and the like, cytotoxic antibiotics, such as dactinomycin, anthracyclines, mitomycin C, bleomycin, mitramycin and the like, antibodies that target immune cells, such as anti-CD20 antibodies, antibodies anti-CD3 and the like.
Anti-inflammatory and / or analgesic agents suitable for combined therapies with anti-hTL1A antibodies include corticosteroids, non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, ibu-propene, naproxen, COX inhibitors -2 and the like, TNF-α antagonists, IL-1 antagonists, IL-6 antagonists, acetaminophen, morphomimetics and the like.
Suitable antiallergic agents include antihistamines, glucocorticoids, epinephrine (adrenaline), theophylline, sodium chromoline and anti-leukotrienes, as well as anticholinergics, decongestants, mast cell stabilizers and the like.
In a ninth aspect, the invention features methods for inhibiting hTL1A activity using the anti-hTL1A antibody or antigen-binding portion of the antibody of the invention, wherein the therapeutic methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an antibody or antigen-binding fragment of an antibody of the invention and optionally one or more additional therapeutic agents described above.
The disease or disorder treated is any disease or condition which is relieved, better inhibited or prevented or its rate of occurrence is reduced compared to that without treatment with anti-hTL1A antibody by eliminating, reducing or inhibiting the activity of TL1A.
Examples of diseases or disorders treatable by the methods of the invention include, but are not limited to, inflammatory diseases and / or autoimmune diseases, such as inflammatory bowel diseases (Inflammatory Bowel Disease - IBD), including UC and CD, RA,
MS, type 1 and type 2 diabetes, psoriasis, psoriatic arthritis, ankylosing spondylitis, atopic dermatitis and the like; allergic reactions and conditions, including asthma, allergic lung inflammation and the like; atherosclerosis in cancers, infections, neurodegenerative diseases, graft rejection, graft 5 versus host diseases (GVHD), cardiovascular disorders / diseases and the like. Other modalities will be evident from a review of the detailed description below.
DETAILED DESCRIPTION Before the present invention is described in detail, it should be understood that the present invention is not limited to the particular methods and experimental conditions described, since the methods and conditions may vary. It should also be understood that the terminology used here is for the purpose of describing particular modalities only and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. Unless otherwise stated, all technical and scientific terms used here have the same meaning as commonly understood by those versed in the technique to which the present invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are now described. All publications mentioned are incorporated herein by reference in their entirety. Definitions The term "human TNF ligand 1A" or "hTL1A", as used here, refers to hTL1A which has the nucleic acid sequence shown in SEQ ID NO: 243 and the amino acid sequence of SEQ ID NO: 244 or a biologically active fragment thereof, as well as the hTL1A variants, including Fhm, which have the nucleic acid sequence shown in SEQ ID NO: 245 and the amino acid sequence of SEQ ID NO: 246 or a biologically active fragment thereof, unless specifically indicated otherwise. The term "antibody", as used herein, is intended to refer to immunoglobulin molecules comprised of four polypeptide chains, two heavy chains (H) and two light chains (L) interconnected by disulfide bridges.
Each heavy chain is comprised of a heavy chain variable region (HCVR) and a heavy chain constant region (CH; comprised of CH1, CH2 and CH3 domains). Each light chain is comprised of a light chain variable region (LCVR) and a light chain constant region (CL). The HCRV and LCVR can also be subdivided into regions of hypervariability called Complementary Determining Regions (CDR) interspersed with regions that are more conserved called framework regions (FR). Each HCVR and LCVR is composed of three CDRs and four FRs available, from the amino terminus to the carboxy terminus, in the following order: FR1, C-DR1, FR2, CDR2, FR3, CDR3 and FR4. Replacement of one or more CDR residues or omission of one or more CDRs is also possible.
Antibodies have been described in the scientific literature in which one or two CDRs can be dispensed for binding.
Padlan et al. (1995 FASEB J. 9: 133-139) analyzed the contact regions between antibodies and their antigens based on published crystal structures and concluded that only about one fifth to one third of CDR residues actually contact the antigen.
Padlan also discovered many antibodies in which one or two CDRs had no amino acids in contact with an antigen (see also, Vajdos et al., 2002 J Mol Biol 320: 415-428). CDR residues that do not contact the antigen can be identified based on previous studies (for example, H60- H65 residues in CDRH2 are often not needed) from Kabat CDR regions that are outside Chothia CDRs through of molecular and / or empirical modeling.
If a CDR or residue (s) thereof is omitted, it is usually replaced by an amino acid that occupies the corresponding position in another human antibody sequence or a consensus on such sequences.
Substitution positions within CDRs and amino acids to be replaced can also be selected empirically.
Empirical substitutions can be conservative or non-conservative substitutions.
The term "human antibody", as used herein, is intended to include antibodies with variable and constant regions derived from human germline immunoglobulin sequences.
The human mAbs of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (for example, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo ), for example, in CDRs and, in particular, in CDR3. However, the term "human antibody", as used here, is not intended to include mAbs in which CDR sequences derived from the germline of another species of mammal (eg, mouse) have been grafted onto human RF sequences.
The fully human anti-TL1A antibodies described here may comprise one or more substitutions, insertions and / or deletions of amino acids in the framework and / or CDR regions of the variable domains of heavy and light chains when compared to corresponding germline sequences.
Such mutations can be easily determined by comparing the amino acid sequences described here with germline sequences available, for example, from public antibody sequence databases.
The present invention includes antibodies and antigen-binding fragments thereof which are derived from any of the amino acid sequences described here, in which one or more amino acids within one or more framework and / or CDR regions have been mutated to (a) corresponding residue (s) from the germline sequence from which the antibody was derived or to the corresponding residue (s) from another human germline sequence or a substitution conservative amino acid content of the corresponding germline residue (s) (such sequence changes are collectively referred to here as "germline mutations"). Those skilled in the art, starting from the light and heavy chain variable region sequences described here, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline line mutations or combinations of the same.
In certain modalities, all framework and / or CDR residues within the VH and / or VL domains have undergone a reverse mutation to the residues found in the original germline sequence from which the antibody was derived.
In other modalities, only certain residues undergo a reverse mutation to the original germline sequence, for example, only the residues with mutation found within the first eight amino acids of FR1 or within the last 8 amino acids of FR4 or only the residues mutations found within CDR1, CDR2 or CDR3. In other modalities, one or more of the framework and / or CDR residues are mutated to the corresponding residue (s) of a different germline sequence (that is, a lineage sequence germline that is different from the germline sequence from which the antibody was originally derived). In addition, the antibodies of the present invention can contain any combination of two or more germline mutations in the framework and / or CDR regions, for example, in which certain residues mutate individually to the corresponding residues of a germline sequence in particular, while some other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue from a different germline sequence.
Once obtained, antibodies and antigen-binding fragments that contain one or more germline mutations can be easily tested for one or more desired properties, such as enhanced binding specificity, increased binding affinity, antagonistic biological properties or enhanced or increased agonists (as appropriate), reduced immunogenicity, etc.
Antibodies and antigen binding fragments obtained in this general manner are covered by the present invention.
The present invention also includes anti-TL1A antibodies comprising variants of any of the HCVR, LCVR and / or CDR amino acid sequences described herein that have one or more conservative substitutions.
For example, the present invention includes anti-TL1A antibodies having HCVR, LCVR and / or CDR amino acid sequences with, for example, 10 or less, 8 or less, 6 or less, 4 or less, 2 or 1 conservative amino acid substitution (s) in relation to any of the HCVR, LCVR and / or CDR amino acid sequences described here.
Unless specifically indicated otherwise, the term "antibody" (Ab), as used herein, is to be understood as encompassing antibody molecules that comprise two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., molecules "complete" antibodies), as well as antigen-binding fragments from them.
The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic or genetically engineered polypeptide or glycoprotein that binds specifically to an antigen to form a complex.
Antigen-binding fragments of an antibody can be derived, for example, from complete antibody molecules using any suitable conventional techniques, such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding variable and (optionally) constant domains of antibody.
Such DNA is known and / or is readily available, for example, from commercial sources, DNA libraries (including, for example, phage display antibody libraries) or can be synthesized.
DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to arrange one or more variable and / or constant domains in an appropriate configuration or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.
Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F (ab ') 2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv molecules (scFv); (vi) dAb fragments; and (vii) minimal recognition units that consist of amino acid residues that mimic the hypervariable region of an antibody (for example, an isolated Complementary Determining Region - CDR), such as a peptide of CDR3) or a FR3-CDR3-FR4 restricted peptide. Other engineered molecules, such as domain-specific antibodies, antibodies with a single domain, antibodies with deleted domain, chimeric antibodies, antibodies grafted with CDR, diabodies, tribodies, tetribodies, minibodies, nanobodies (for example, monovalent nanobodies, bivalent nanobodies , etc.), small modular immuno-pharmaceutical products (Small Modular Immuno-Pharmaceuticals - SMIPs) and shark variable IgNAR domains are also included within the term "antigen binding fragment" as used here.
An antigen-binding fragment of an antibody will typically comprise at least one variable domain.
The variable domain can be of any size or composition of amino acids and will generally comprise at least one CDR which is adjacent to or in-frame with one or more framework sequences.
In antigen-binding fragments that have a VH domain associated with a VL domain, the VH and VL domains can be located relative to each other in any suitable arrangement.
For example, the variable region can be dimeric and contain VH-VH, VH-VL or VL-VL diameters.
Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.
In certain embodiments, an antigen-binding fragment of an antibody can contain at least one variable domain covalently linked to at least one constant domain.
Exemplary non-limiting configurations of variable and constant domains that can be found within an antigen-binding fragment of an antibody of the present invention include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix)
VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL.
In any configuration of constant and variable domains, including any of the exemplary configurations listed above, the variable and constant domains can be directly linked to each other or can be linked by a total link or hinge region or partial.
A hinge region can consist of at least 2, for example, 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible link between variable domains and / or adjacent constants in a single molecule polypeptide.
In addition, an antigen-binding fragment of an antibody of the present invention may comprise a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with each other and / or with one or more monomeric VH or VL domains (for example, by disulfide bond (s)). According to complete antibody molecules, antigen-binding fragments can be monospecific or multispecific (for example, bispecific). A multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, where each variable domain is capable of binding specifically to a different antigen or to a different epitope on the same antigen.
Any multispecific antibody format, including the exemplary bispecific antibody formats described herein, can be adapted for use in the context of an antigen binding fragment of an antibody of the present invention using routine methods available in the art.
In certain embodiments, the antibody or antibody fragments of the invention can be conjugated to a therapeutic moiety ("immunoconjugate"), such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant or a radioisotope.
The term "specifically bind" or similar means that an antibody or antigen-binding fragment likewise forms a complex with an antigen that is relatively stable under physiological conditions.
Specific binding can be characterized by an equilibrium dissociation constant (KD) of about 3000 nM or less (ie, a smaller KD denotes a firmer connection), about 2000 nM or less, about 1000 nM or less, about 500 nM or less, about 300 nm or less, about 5 200 nm or less, about 100 nm or less, about 50 nm or less, about 1 nm or less or about 0, 5 nm or less.
Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance and the like.
An isolated antibody that specifically binds to hTL1A may, however, exhibit cross-reactivity with other antigens, such as molecules of TL1A from other species, for example, monkey TL1A Cynomolgus (SEQ ID NO: 248) and / or Mouse TL1A (SEQ ID NO: 250) and / or a TL1A variant, such as Fhm (SEQ ID NO: 246). In addition, multi-specific (e.g., bispecific) antibodies that bind to hTL1A and one or more additional antigens are, however, considered to be antibodies that "specifically bind" to hTL1A, as used here.
The term "high affinity" antibody refers to those antibodies that have a binding affinity for hTL1A, expressed as KD, of about 1 x 10-9 M or less, about 0.5 x 10-9 M or less, about 0.25 x 10-9 M or less, about 1 x 10-10 M or less or about 0.5 x 10 -10 M or less, as measured by surface plasmon resonance, for example example, BIACORE®or solution-affinity ELISA.
The term "KD", as used here, is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.
By the term "dissociation rate", "Koff" or "kd" is meant an antibody that dissociates from hTL1A with a rate constant of 1 x 10-3 s-1 or less, preferably 1 x 10-4 s -1 or less, as determined by surface plasmon resonance, for example, BIACORE®. The term "intrinsic affinity constant" or "ka" means an antibody that associates with hTL1A at a constant rate of about 1 x 103 M-1 s-1 or greater, as determined by means of surface plasmon resonance, for example, BIACORE. An "isolated antibody", as used here, is intended to refer to an antibody that is substantially free of other mAbs that have different antigen specificities (for example, an isolated antibody 5 that specifically binds to hTL1A is substantially free Abs that specifically bind to antigens other than hTL1A). An isolated antibody that specifically binds to hTL1A may, however, have a cross-reactivity with other antigens, such as TL1A molecules from other species, such as monkey Cynomolgus and mouse and / or hTL1A variants, such as Fhm.
A "neutralizing antibody", as used here (or an "antibody that neutralizes TL1A activity"), is intended to refer to an antibody whose binding to TL1A results in inhibition of at least one biological activity of TL1A .
This inhibition of biological activity of TL1A can be assessed by measuring one or more indicators of biological activity of TL1A by means of one or more of the various in vitro or in vivo standard assays known in the art (see also examples below). The term "surface plasmon resonance", as used here, refers to an optical phenomenon that allows the analysis of biospecific interactions in real time by detecting changes in protein concentrations within a matrix of biosensor, for example, using the BIACORE system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). The term "epitope" is a region of an antigen that is linked by an antibody.
Epitopes can be defined as structural or functional.
Functional epitopes are, in general, a subset of structural epitopes and have those residues that directly contribute to the affinity of the interaction.
Epitopes can also be conformational, that is, composed of non-linear amino acids.
In certain embodiments, epitopes may include determinants that are groupings with a chemically active surface of molecules, such as amino acids, sugar side chains, phosphoryl groups or sulfonyl groups and, in certain embodiments, may have specific three-dimensional structural characteristics and / or characteristics specific load
The term "substantial identity" or "substantially identical", when referring to a nucleic acid or fragment thereof, indicates 5 that, when perfectly aligned with suitable nucleotide insertions or deletions with another nucleic acid (or its complementary strand ), there is nucleotide sequence identity in at least about 90% and, more preferably, at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any algorithm - known sequence identity hand, such as FASTA, BLAST or GAP, as discussed below.
When applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two sequences of peptides, when perfectly aligned, such as by GAP or BESTFIT programs using default weights by gap, share at least 90% sequence identity, even more preferably at least 95%, 98% or 99% sequence identity.
Preferably, positions of residues which are not identical differ by conservative amino acid substitutions.
A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue with a side chain (group R) with similar chemical properties (for example, charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially alter the functional properties of a protein.
In cases where two or more amino acid sequences differ from one another by conservative substitutions, the percentage or degree of similarity can be adjusted upwards to correct the conservative nature of the substitution.
Means for making this adjustment are well known to those skilled in the art.
See, for example, Pearson (1994) Methods Mol.
Biol. 24: 307-331, which is incorporated by reference.
Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic side chains -
hydroxyl: serine and threonine; 3) side chains containing amide: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) basic side chains: lysine, arginine and histidine; 6) acidic side chains: aspartate and glutamate; and 7) sulfur-containing side chains: cysteine 5 and methionine.
Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate and asparagine-glutamine.
Alternatively, a conservative substitution is any change having a positive value in the PAM250 log-probability matrix described in Gonnet et al. (1992) Science 256: 45-1443, incorporated herein by reference.
A "moderately conservative" substitution is any change having a non-negative value in the PAM250 log-probability matrix. Sequence similarity for polypeptides is typically measured using sequence analysis software.
Protein analysis software combines similar sequences using similarity measures attributed to various substitutions, deletions and other modifications, including conservative amino acid substitutions.
For example, the GCG software contains programs, such as GAP and BESTFIT, that can be used with the default parameters to determine the identity or sequence homology between closely related polypeptide sequences, such as polypeptides from homologues of different species organisms or between a wild-type protein and a mutein in it.
See, for example, GCG Version 6.1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters, a program in GCG Version 6.1. FASTA (for example, FASTA2 and FASTA3) provides alignments and percent sequence identity of regions of the best overlaps between the query and search strings (Pearson, 2000, supra). Another preferred algorithm when comparing a sequence of the invention with a database containing a large number of sequences from different organisms is the BLAST computer program, especially BLASTP and TBLASTN, using default parameters.
See, for example, Altschul et al., 1990, J.
Mol.
Biol.
215: 403-410, 1997; Nucleic Acids Res. 25: 3389-402, each of which is incorporated herein by reference.
By the phrase "therapeutically effective amount" is meant an amount that produces the desired effect for which it is administered.
The exact amount will depend on the purpose of the treatment, the age and size of an individual being treated, the route of administration and the like and will be determinable by those skilled in the art using known methods (see, for example, Lloyd (1999), The Art, Science and Technology of Pharmaceutical Compounding). Preparation of Human Antibodies Methods for generating human antibodies in transgenic mice are known in the art.
Any of the known methods can be used in the context of the present invention to produce human antibodies that specifically bind to TL1A.
Using VELOCIMMUNE® technology or any other known method for generating monoclonal antibodies, chimeric antibodies of high affinity for TL1A are initially isolated having a human variable region and a constant mouse region.
As in the experimental section below, antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope and the like.
In general, the antibodies of the present invention have high affinities, typically having a KD of about 10-12 M to about 10-9 M, when measured by antigen binding, whether immobilized on solid phase or in solution phase. .
The mouse constant regions are replaced with desired human constant regions, for example, wild type IgG1 (SEQ ID NO: 255) or IgG4 (SEQ ID NO: 256) or modified IgG1 or IgG4 (for example, IgG4 with Ser-108 replaced by Pro, as shown in SEQ ID NO: 257), to generate the fully human antibodies of the invention.
Although the selected constant region may vary according to the specific use, the characteristics of antigen binding and specificity for the target of antibodies reside in the variable region.
Epitope Mapping and Related Technologies For screening antibodies that bind to a particular epitope, a routine cross-block assay, such as that described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., 5 New York) can be accomplished.
Other methods include alanine scan mutants, peptide blots (Reineke, 2004, Methodos Mol Biol 248: 443-63) (here specifically incorporated by reference in full) or analysis of peptide cleavage.
In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer, 2000, Protein Science 9: 487-496) (here specifically incorporated by reference in full). The term "epitope" refers to a site on an antigen to which B cells and / or T cells respond.
B cell epitopes can be formed from contiguous amino acids or juxtaposed non-contiguous amino acids by tertiary folding of a protein.
Epitopes formed from contiguous amino acids are typically retained when exposed to denaturing solvents, while epitopes formed by tertiary folding are typically lost when treated with denaturing solvents.
An epitope typically includes at least 3 and, more usually, at least 5 or 8-10 amino acids in a single spatial conformation.
Profile Modification-Assisted Profiling (MAP), also known as Antibody Profile Characterization based on Antigen Structure (Antigen Sctructure-based Antibody Profiling - ASAP), is a method that classifies large numbers of targeted mAbs against the same antigen according to the similarities in the characterization of the binding profile of each antibody against chemically or enzymatically modified surfaces (document US 2004/0101920, here specifically incorporated by reference in its entirety). Each category can reflect a unique epitope, either distinctly different or partially overlapping with an epitope represented by another category.
This technology allows rapid filtration of genetically identical mAbs, so that
characterization can be focused on genetically distinct mAbs.
When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce mAbs that have the desired characteristics.
MAP can be used to classify the anti-TL1A mAbs of the invention into groups of mAbs that bind to different epitopes.
The present invention includes antibodies to hTL1A that bind to the same epitope as any of the specific exemplary antibodies described herein.
Likewise, the present invention also includes anti-hTL1A antibodies that compete for binding to hTL1A or a fragment of hTL1A with any of the specific exemplary antibodies described herein.
One can easily determine whether an antibody binds to the same epitope or competes for binding with a reference anti-hTL1A antibody using routine methods known in the art.
For example, to determine whether a test antibody binds to the same epitope as a reference anti-hTL1A antibody of the invention, the reference antibody is allowed to bind to an hTL1A protein or peptide under saturation conditions. ration.
Then, the ability of a test antibody to bind to the hTL1A molecule is assessed.
If the test antibody is able to bind to hTL1A after saturation binding with the reference anti-hTL1A antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-hTL1A antibody.
On the other hand, if the test antibody is unable to bind to the hTL1A molecule after binding by saturation with the reference anti-hTL1A antibody, then the test antibody can bind to the same epitope as the bound epitope by the reference anti-hTL1A antibody of the invention.
To determine whether an antibody competes for binding with a reference anti-hTL1A antibody, the binding methodology described above is carried out in two orientations: in a first orientation, the reference antibody is allowed to bind to an hTL1A molecule under conditions of saturation, followed by evaluation of the binding of the test antibody to the hTL1A molecule.
In a second orientation, the test antibody is allowed to bind to an hTL1A molecule under saturation conditions, followed by evaluation of binding of the reference antibody to the TL1A molecule.
If, in both orientations, only the first antibody (saturation) is able to bind to the TL1A molecule, then it was concluded that the test antibody and the reference antibody compete for binding to hTL1A.
As will be appreciated by those skilled in the art, an antibody that competes for binding with a reference antibody may not necessarily bind to the identical epitope as the reference antibody, but may sterically block the binding of the reference antibody when binding to an overlapping or adjacent epitope.
Two antibodies bind to the same epitope or overlap the epitope if each competitively inhibits (blocks) the other's binding to the antigen.
That is, an excess of 1, 5, 10, 20 or 100 times of one antibody inhibits the binding of the other by at least 50%, more preferably 75%, 90% or even 99%, measured in a competitive binding assay ( see, for example, Junghans et al., Cancer Res. 1990, 50: 1495-1502). Alternatively, two antibodies have the same epitope if essentially all of the amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other.
Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other.
Additional routine experimentation (eg, peptide mutation and binding analysis) can then be performed to confirm that the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody or whether spinal block (or another phenomenon) is responsible for the absence of observed connection.
Such experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry or any other qualitative or quantitative antibody binding assay available in the art.
Immunoconjugates
The invention encompasses a human anti-TL1A monoclonal antibody conjugated to a therapeutic moiety ("immunoconjugate"), such as a cyto-toxin, a chemotherapeutic drug, an immunosuppressant or a radioisotope.
Cytotoxigenic agents include any agent that is harmful to 5 cells.
Examples of cytotoxigenic agents and chemotherapeutic agents suitable for forming immunoconjugates are known in the art; see, for example, WO 05/103081, here specifically incorporated by reference). Bispecific The antibodies of the present invention can be monospecific, bispecific or multispecific. Multispecific mAbs can be specific for different epitopes of a target polypeptide or can contain antigen-binding domains specific for more than one target polypeptide.
See, for example, Tutt et al., 1991, J.
Immunol. 147: 60-69. Human anti-hTL1A mAbs can be linked to or co-expressed with another functional molecule, for example, another peptide or protein.
For example, an antibody or fragment thereof can be functionally linked (for example, by chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody with a second binding specificity.
An exemplary bispecific antibody format that can be used in the context of the present invention involves the use of a first immunoglobulin (Ig) CH3 domain and a second Ig CH3 domain, where the first and second Ig CH3 domains differ from each other by at least one amino acid and in which at least one amino acid difference reduces the binding of the bispecific antibody to Protein A when compared to a bi-specific antibody that lacks the amino acid difference.
In one embodiment, the first Ig CH3 domain binds to Protein A and the second Ig CH3 domain contains a mutation that reduces or suppresses Protein A binding, such as an H95R modification (by exon numbering
IMGT; H435R by EU numbering). The second CH3 domain may further comprise a modification Y96F (by IMGT; Y436F by the EU). Other modifications that can be found within the second CH3 domain include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, 5 L358M, N384S, K392N, V397M and V422I by the EU) in the case of IgG1; N44S, K52N and V82I (IMGT; N384S, K392N and V422I by the EU) for IgG2 and Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409, E394 V422I by the EU) in the case of IgG4 antibodies. Variations in the format of bispecific antibodies described above are considered within the scope of the present invention.
Bioequivalents The anti-hTL1A antibodies and antibody fragments of the present invention encompass proteins with amino acid sequences that vary from those of the described mAbs, but which retain the ability to bind to human TL1A.
Such variant mAb and antibody fragments comprise one or more amino acid additions, deletions or substitutions when compared to the parental sequence, but exhibit biological activity that is essentially equivalent to that of MAbs.
Likewise, DNA sequences encoding mAb to hTL1A of the present invention contain sequences that comprise one or more nucleotide additions, deletions or substitutions when compared to the described sequence, but which encode an anti-hTL1A antibody or fragment of antibody that is essentially bioequivalent to an anti-hTL1A antibody or antibody fragment of the invention.
Examples of such DNA sequences and variant amino acids are discussed above.
Two antigen-binding proteins or antibodies are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered in the same moiety under similar experimental conditions , either in a single or multiple doses.
Some antibodies will be considered equivalent or pharmaceutical alternatives if they are equivalent in extent to their absorption,
but not its absorption rate and can still be considered bioequivalent because such differences in the absorption rate are intentional and reflected in the marking are not essential to obtain effective concentrations of the drug in the body, for example, when in use chronic, and 5 are considered clinically insignificant for the specific drug product studied.
In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically significant differences in their safety, purity and potency.
In one embodiment, two antigen-binding proteins are bioequivalent if, in a patient, there may be an exchange one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically altered one. significant in immunogenicity or reduced efficacy when compared to continuous therapy without such an exchange.
In one embodiment, two antigen-binding proteins are bioequivalent if both act through a common mechanism or mechanisms of action under the condition or conditions of use, as long as such mechanisms are known.
Bioequivalence can be demonstrated using in vivo and in vitro methods.
Bioequivalence measures include, for example, (a) an in vivo test in humans or other mammals in which the concentration of the antibody or its metabolites is measured in blood, plasma, serum or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably prognostic for human bioavailability data in vivo; (c) an in vivo test on humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) a well-controlled clinical experiment that establishes the safety, efficacy or bioavailability or bioequivalence of an antibody.
Bioequivalent variants of anti-hTL1A antibodies of the invention can be constructed, for example, by making several substitutions of residues or sequences or deleting terminal or internal residues or sequences.
not necessary for biological activity.
For example, cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to avoid the formation of unnecessary or incorrect intramolecular disulfide bridges after renaturation. Therapeutic Formulations and Administration The invention provides therapeutic compositions comprising the anti-hTL1A antibodies or antigen-binding fragments thereof of the present invention and therapeutic methods using the same.
The administration of therapeutic compositions according to the invention will be carried out with vehicles, excipients and other suitable agents that are incorporated into formulations to provide improved transfer, distribution, tolerance and the like.
Multiple appropriate formulations can be found in the form known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.
These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vehicles containing lipids (cationic or anionic) (such as LIPOFECTIN®), DNA conjugates, anhydrous absorption pastes, water-emulsions in-oil and water-in-oil, emulsions in Carbowax (polyethylene glycols of various molecular weights), semi-solid gels and semi-solid mixtures containing Carbowax.
See also Powell et al. "Compendium of Excipients for Parenteral Formulations" PDA, 1998, J Pharm Sci Tecno 52: 238-311. The dose may vary depending on the age and size of an individual to be administered, the target disease, the purpose of treatment, conditions, route of administration and the like.
When the antibody of the present invention is used to treat various conditions and diseases associated directly or indirectly with TL1A, including inflammatory diseases / disorders, autoimmune diseases / disorders, allergic reactions and the like in an adult patient, it is It is advantageous to administer the antibody of the present invention intravenously or subcutaneously in a single dose of about 0.01 to about 20 mg / kg of body weight, more preferably about 0.02 to about 7, about 0.03 at about 5 or about 0.05 to about 3 mg / kg of body weight.
Depending on the severity of the condition, the frequency and duration of treatment can be adjusted.
In certain modalities, the antibody or antigen-binding fragment of the same invention can be administered as an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 500 mg , about 5 to about 300 mg, or about 10 to about 200 mg, about 100 mg, or about 50 mg.
In certain embodiments, the starting dose may be followed by the administration of a second or multiple subsequent doses of the antibody or antigen-binding fragment thereof in an amount that may be approximately the same or less than the starting dose, at which the doses subsequent periods are separated by at least 1 day to 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 12 weeks or at least 14 weeks.
Various delivery systems are known and can be used to administer the pharmaceutical composition of the invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see , for example, Wu et al., 1987, J.
Biol.
Chem. 262: 4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes.
The composition can be administered via any convenient route, for example, through infusion or bolus injection, through absorption through mucocutaneous or epithelial linings (for example, oral, rectal and intestinal mucosa, etc.) and it can be administered together with other biologically active agents.
Administration can be systemic or local.
The pharmaceutical composition can also be distributed in a vesicle, in particular a liposome (see Langer, 1990, Science 249: 1527-1533; Treat et al., 1989, in Liposomes in The Therapy of Infectious
Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss, New York, pages 353-365; Lopez-Berestein, ibid., Pages 317-327; see in general ibid.). In certain situations, the pharmaceutical composition can be distributed in a controlled release system.
In a modality, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit Ref Biomed Eng. 14: 201). In another modality, polymeric materials can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres, Boca Raton, Florida (1974). In yet another modality, a controlled-release system can be placed close to the target of the composition, thus requiring only a fraction of the systemic dose (see, for example, Goodson, in Medical Applications of Control-Led Release, supra, vol. 2, pages 115-138, 1984). Injectable preparations may include dosage forms for administration by intravenous, subcutaneous, intradermal and intramuscular injection, drip infusions, etc.
These injectable preparations can be made using publicly known methods.
For example, injectable preparations can be made, for example, by dissolving, suspending or emulsifying the antibody or its salt previously described in a sterile aqueous medium or an oily medium used conventionally for injectable solutions.
As an aqueous medium for injections there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc. which can be used in combination with an appropriate solubilizing agent, such as an alcohol (eg, ethanol), a polyalcohol (eg, propylene glycol, polyethylene glycol), a nonionic surfactant [eg, polysorbate 80, HCO- 50 (polyoxyethylene adduct (50 moles) of hydrogenated castor oil)], etc.
As the oil medium are used, for example, sesame oil, soy oil, etc., which can be used in combination with a solubilizing agent, such as benzyl benzoate, benzyl alcohol, etc.
The injection thus prepared is preferably filled in a suitable ampoule.
A pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe.
In addition, with regard to subcutaneous delivery, a pen delivery device readily has applications in the delivery of a pharmaceutical composition of the present invention.
Such a pen dispensing device can be reusable or disposable.
A reusable pen dispenser usually uses a replaceable cartridge that contains a pharmaceutical composition.
Once the entire pharmaceutical composition inside the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition.
The pen delivery device can then be reused.
In a disposable pen administration device, there is no replaceable cartridge.
On the contrary, the disposable pen dispensing device is pre-filled with the pharmaceutical composition contained in a reservoir within the device.
Once the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.
Numerous autoinjector and reusable pen dispensing devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention.
Examples include, but certainly are not limited to, AUTOPEN® (Owen Mumford, Inc., Woodstock, United Kingdom), DISETRONIC® pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75 / 25® pen, HUMALOG® pen, HUMALIN 70 / 30® pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN® I, II and III (New Normandy, Copenhagen, Denmark), NOVOPEN JUNIOR® (Novo Nordisk, Copenhagen, Denmark), BD® pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN®, OPTIPEN PRO®, OPTIPEN STARLET® and OPTICLIK® (Sanofi- Aventis, Frankfurt, Germany), to name just a few.
Examples of disposable pen dispensing devices having applications in subcutaneous dispensing of a pharmaceutical composition of the present invention include, but certainly without limitations, the SoloSTAR® (Sanofi- Aventis), FLEXPEN® (Novo Nordisk) and KWIKPEN® (Eli Lilly). Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared in dosage forms in a unit dose suitable to adjust the dose of the active ingredients.
Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.
The amount of the aforementioned antibody contained is, in general, from about 0.1 to 5 to about 800 mg per dosage form in a unit dose; especially in the form of an injection, the antibody referred to above is contained in about 1 to about 500 mg, about 5 to 300 mg, about 8 to 200 mg and about 10 to about 100 mg for the other dosage forms.
Combined Therapies The invention also provides therapeutic methods for treating diseases or disorders that are directly or indirectly associated with hTL1A by administering the mAb to hTL1A or a fragment thereof in combination with one or more additional therapeutic agents .
The additional therapeutic agent can be one or more of any agent that is advantageously combined with the antibody or fragment of the invention, including immunosuppressants, anti-inflammatory agents, analgesic agents, antiallergic agents and the like.
Suitable immunosuppressants include, but are not limited to, glucocorticoids, cyclosporine, methotrexate, interferon β (IFN-β), tacrolimus, sirolimus, azathioprine, mercaptopurine, opioids, mycophenolate, TNF-binding proteins, such as infliximab, eternacept adalimumab and the like, cytotoxic antibiotics, such as dactinomycin, anthracyclines, mitomycin C, bleomycin, mitramycin and the like, antibodies that target immune cells, such as anti-CD20 antibodies, anti-CD3 antibodies and the like.
Anti-inflammatory and / or analgesic agents suitable for therapies combined with anti-hTL1A antibodies include corticosteroids, non-sterodal anti-inflammatory drugs (NSAIDs), such as aspirin, ibuprofen, naproxen, COX-2 inhibitors and the like, TNF-α antagonists (for example, infliximab or REMICADE® by Centocor; Golimumab by Centocor; etanercept or ENBREL® by Amgen / Wyeth; adalimumab or HUMIRA® by Abbott Laboratories and the like), IL-1 antagonists (for example , IL-1-binding fusion proteins, for example, ARCALYST® by Regeneron Pharmaceuticals, Inc .; see US Patent No.
6,927,044; KINERET® by Amgen and the like), IL-6 antagonists (for example, anti-IL6 receptor antibodies, as described in US Patent No.
7,582,298 and ACTEMRA® by Roche), acetaminophen, morphomimetics and the like. Suitable antiallergic agents which can block the action of allergic mediators or prevent cell activation and de-granulation processes include antihistamines, glucocorticoids, epinephrine (adrenaline), theophylline, sodium cromoglycate and antileukotrienes, such as montelu - caste (SINGULAIR® from Merck) or zafirlukast (ACCOLATE® from AstraZenica), as well as anticholinergics, decongestants, mast cell stabilizers and other compounds that can impair eosinophil chemotaxis. The mAb to hTL1A or a fragment thereof and the additional therapeutic agent (s) can be co-administered together or separately. When different dosage formulations are used, the antibody or fragment thereof and the additional agents can be administered simultaneously, separately or at alternate times, that is, sequentially, in appropriate orders.
EXAMPLES The following examples are provided in order to provide those skilled in the art with a complete description and disclosure of how to make and use the methods and compositions of the invention and are not intended to limit the scope of what the inventors regard as their invention. - convention. Efforts have been made to ensure accuracy with respect to the numbers used, but some errors and experimental deviations must be accounted for. Unless otherwise stated, the molecular weight is the average molecular weight, the temperature is in degrees centigrade and the pressure is atmospheric or close. Example 1: Generation of human antibodies to Human TL1A VELOCIMMUNE® mice were immunized with human TL1A and the antibody immune response monitored by antigen-specific immunoassay using serum obtained from these mice. B cells expressing anti-hTL1A antibody collected from the spleens of immunized mice have been shown to have high titers of anti-hTL1A antibodies and have been fused with mouse myeloma cells to form hybrids.
Hybridomas were screened and selected to identify cell lines that express specific antibodies to hTL1A 5 using assays as described below.
The assays identified several cell lines that produced chimeric anti-hTL1A antibodies named H2M1681N, H2M1704N, H2M1804N, H2M1805N, H2M1817N and H2M1818N.
These antibodies were then converted to the hIgG4 isotype by replacing the respective mouse constant regions with the IgG4 amino acid sequence of SEQ ID NO: 257, which contains a S108P mutation in the hinge region and designated as H4H1681N, H4H1704N, H4H1804N, H4H1805N, H4H1817N and H4H1818N, respectively.
Antibodies specific for human TL1A have been isolated directly from B cells immunized with antigen without fusion to myeloma cells, as described in US Patent No. 7.58 2,298, which is incorporated herein by reference in its entirety.
Variable regions of the heavy and light chain have been cloned to generate fully human anti-hTL1A antibodies designated as H4H1719P, H4H1725P, H4H1738P, H4H1742P, H4H1745P, H4H1750P and H4H1752P.
CHO cell lines that express stable recombinant antibodies have been established.
Example 2. Analysis of Variable Gene Use To analyze the structure of the antibodies produced, the nucleic acids encoding variable regions of antibodies were cloned and sequenced.
From the nucleic acid sequence and the predicted amino acid sequence of the antibodies, the use of the gene was identified for each Heavy Chain Variable Region (HCVR) and Light Chain Variable Region (LCVR). Table 1 shows the use of genes for antibodies selected according to the invention.
Table 1
HCVR LCVR Antibody
VH DH JH VK JK H2M1704 3-7 1-7 6 4-1 3 H2M1681 3-23 2-21 4 1-5 1 H2M1817 4-34 3-9 4 3-20 4 H2M1804 4-34 1-1 4 3 -20 4 H2M1818 3-11 4-17 6 4-1 1 H2M1805 4-34 3-3 4 2-24 4 H4H1719 3-9 3-3 6 2-28 2 H4H1725 1-2 2-15 3 1-12 4 H4H1738 3-15 4-4 6 2-28 2 H4H1742 3-23 2-2 6 1-9 2 H4H1745 3-23 6-6 4 1-9 4 H4H1750 3-30 4-17 6 1-17 1 H4H1752 3-23 1-7 4 1-5 1 Table 2 shows the amino acid sequence pairs of the selected region of heavy and light chains of selected anti-hTL1A antibodies and their corresponding antibody identifiers. The designations 5 N and P refer to antibodies that have light and heavy chains with identical CDR sequences, but with sequence variations in the regions that are outside the CDR sequences (that is, in the framework regions). Thus, N and P variants of a particular antibody have identical CDR sequences within their variable regions of heavy and light chains, but contain modifications within the framework regions. Table 2 HCVR / LCVR mAb name HCVR / LCVR mAb name (H2M- or H4H-) SEQ ID NOS (H2M- or H4H-) SEQ ID NOS 1704N 2/10 1738N 138/146 1681N 18/26 1738P 154/156 1804N 34/42 1745N 158/166 1805N 50/58 1745P 174/176 1817N 66/74 1750N 178/186 1818N 82/90 1750P 194/196 1719N 98/106 1752N 198/206 1719P 114/116 1752P 214/216 1725N 118 / 126 1742N 218/226 1725P 134/136 1742P 234/236 Example 3. TL1A Binding Affinity Determination Binding affinities and kinetic constants were determined
by surface plasmon resonance at 25 ° C and 37 ° C, as shown in Tables 3-5 for monoclonal anti-human TL1A antibodies that bind to the following variants of TL1A species: human (h) (expressed as CHO, residues 72-251 of SEQ ID NO: 244, with His6-tag 5 N-terminal), monkey Cynomolgus (Mf) (expressed in E. coli, residues 72-251 of SEQ ID NO: 248, with or without Met N-terminal), monkey Cynomolgus (expressed in CHO, residues 72-251 of SEQ ID NO: 248, with His6-tag N-terminal), mouse (m) (expressed in E. coli, residues 76-252 of SEQ ID NO: 250, with or without N-terminal Met), mouse (expressed in CHO, residues 76-252 of SEQ ID NO: 250, with His6-tag N-terminal) and rat (expressed in CHO; residues 76-252 of SEQ ID NO: 258, with N-terminal His6-tag). Binding constants were also determined for the hTL1A, Fhm variant (expressed in E. coli, residues 72-251 of SEQ ID NO: 246, containing the Q167R substitution, with or without N-terminal Met). Measurements were performed on a BIACORE® T100 instrument. The antibodies, expressed with mouse Fc (designated with the prefix "H2M") or human IgG4 Fc (S108P) (designated with the prefix "H4H"), were captured on an anti-Fc sensing surface and at least three different concentrations of soluble TL1A proteins, ranging from 1.25 nM to 100 nM, were injected onto the sensor surface.
Kinetic association (ka) and dissociation (kd) constants were determined by fitting the data to a 1: 1 binding model using the BIAevaluation 4.1 curve fitting software (BIAcore Life Sciences). Molar concentrations of TL1A / Fhm used in data adaptation admit a monomeric state for TL1A in solution.
The equilibrium bond dissociation constants (KD) and dissociation half-life (t1 / 2) were calculated from the kinetic rate constants as: KD (M) = kd / ka and t1 / 2 (min) = [ ln2 / (60 * kd)]. NB: No connection under the conditions tested; NT: Not tested in this experiment; *: kd values below 1x10-6 (1 / s) are slower than the detection limit under these experimental conditions and therefore the kd values were fixed at 1x10-6 (1 / s ) for the purpose of approximating KD and t1 / 2; **: equilibrium dissociation constants for antibodies were determined under steady state conditions.
As shown in Tables 3 and 4, antibodies bound with high affinity to CHO expressed forms of human and monkey TL1A protein at 25 ° C (13 and 12 antibodies with KD <1 nM, respectively) and at 5 37 ° C (13 and 12 antibodies with KD <1 nM, respectively). H4H1750P bound significantly weaker to the monkey TL1A protein than to the human TL1A protein.
H4H1704N bound to mTL1A expressed in CHO with KD <2nM at 25 ° C and 37 ° C.
H4H1818N bound to mTL1A expressed in CHO at 25 ° C (K D ~ 7Nm), but not at 37 ° C.
Five antibodies, H4H1681 N, H4H1738P, H4H1750P, H4H1752P and H4H1805N, did not bind to rat TL1A expressed in CHO cells at 25 ° C or 37 ° C; the other eight antibodies will bind to rat TL1A at both temperatures with KD ranging from ~ 0.6 pM to ~ 16 nM. As shown in Table 5, three antibodies (H2M1681N, H4H1752P and H2M1805N) showed no link to the Fhm variant expressed in E. coli [hTL1A (Q167R)] under the conditions tested.
Three antibodies (H2M1704N, H4H1725P and H2M1818N) demonstrated poor binding (KD ranging from ~ 60 nM to ~ 170 nM) to mouse TL1A expressed in E. coli, as assessed under steady state conditions, while all other antibodies tested do not bind to mouse TL1A protein under the conditions tested.
Table 3 TL1A mAb expressed in CHO at 25 C hTL1A hTL1A MfTL1A MfTL1A mTL1A mTL1A rTL1A rTL1A KD (pM) t1 / 2 (min) KD (pM) t1 / 2 (min) KD (pM) t1 / 2 (min) K1 (pM) t1 / 2 (min) H4H1681N 263 36 404 25 NB NB NB NB H4H1704N 39.2 453 59.9 276 194 46 404 25 H4H1719P 481 44 417 45 NB NB 364 38 H4H1725P 63.6 185 346 64 NB NB 317 26 H4H1738P 608 64 361 93 NB NB NB NB H4H1742P 60.4 755 115 577 NB NB 78 144 H4H1745P 164 172 115 231 NB NB 2.7 (nM) 4 H4H1750P 15.8 * 11550 * 8.6 (nM) 21 NB NB NB NB H4H1752P 156 197 213 139 NB NB NB NB H4H1804N 291 51 264 49 NB NB 321 43 H4H1805N 365 73 342 74 NB NB NB NB H4H1817N 321 103 356 92 NB NB 2.5 (nM) 25 H4H1818N 124 120 119 122 7 7 7 , 1 (nM) 12 88 92
Table 4 TL1A mAb expressed in CHO at 37 C hTL1A hTL1A MfTL1A MfTL1A mTL1A mTL1A rTL1A rTL1A KD (pM) t1 / 2 (min) KD (pM) t1 / 2 (min) KD (pM) t1 / 2 (min) K1 (pM) t1 / 2 (min) H4H1681N 254 31 226 32 NB NB NB NB H4H1704N 1.00 * 11550 * 0.93 * 11550 * 1.3 (nM) 27 46 133 H4H1719P 7.61 1912 2.01 5784 NB NB 78 86 H4H1725P 23.9 758 17.7 888 NB NB 411 15 H4H1738P 571 51 465 60 NB NB NB NB H4H1742P 4.37 * 11550 * 4.45 * 11550 * NB NB 653 27 H4H1745P 177 129 173 124 NB NB 16 , 5 (nM) 11 H4H1750P 13.8 * 11550 * 17.1 (nM) 11 NB NB NB NB H4H1752P 225 96 223 91 NB NB NB NB H4H1804N 45.8 286 78 167 NB NB 127 88 H4H1805N 299 80 352 68 NB NB NB NB H4H1817N 27.8 1098 25.6 1150 NB NB 1.6 (nM) 26 H4H1818N 0.925 * 11550 * 0.804 * 11550 * NB NB 0.61 * 11550 *
Table 5 Fhm / TL1A expressed in E. coli ooooo 25 C 37 C 25 C 37 C 25 C mAb Fhm Fhm Fhm Fhm MfTL1A MfTL1A MfTL1A MfTL1A mTL1A KD (pM) t1 / 2 (min) KD (pM) t1 / 2 (min ) KD (pM) t1 / 2 (min) KD (pM) t1 / 2 (min) KD (pM) ** H2M1681N NB NB NT NT 546 54 1.5 (nM) 14 NB H2M1704N 282 52 NT NT 285 57 751 16 127 (nM) H4H1719P 109 96 174 42 242 79 289 40 NB H4H1725P 28.9 447 43.6 194 63.7 292 62.2 174 62 (nM) H4H1738P 1020 19 3100 4 1360 18 4.2 (nM) 4 NB H4H1742P 437 129 696 45 593 97 945 35 NB H4H1745P 115 226 207 71 141 221 281 74 NB H4H1750P 204 623 244 335 52.7 (nM) 4 456 (nM) 1 NB H4H1752P NB NB NB NB 274 122 1320 29 NB H2M1804 192 108 NT NT 176 172 218 118 NB H2M1805N NB NB NT NT 345 67 439 36 NB H2M1817N 451 57 NT NT 1250 37 1.0 (nM) 30 NB H2M1818N 1080 17 NT NT 1840 10 4.1 (nM) 3 171 ( nM)
Example 4: Inhibition of TL1A by Anti-hTL1A Antibodies HEK293 cell lines (CRK01573, ATCC) were generated 5 to stably express human DR3 (full length; SEQ ID NO: 252) or mouse DR3 (full length; SEQ ID NO: 259), together with a luciferase reporter [NFκB (5x) -luciferase-IRES-GFP response element]. Activation of NFκB by TL1A has been shown previously (Migone et al., 2002, Immunity 16: 479-492). To test the membrane-bound form of TL1A and variants of TL1A, HEK293 cell lines that stably express full-length human TL1A (SEQ ID NO: 244), full-length human TL1A with Gln-167 replaced by Arg [Fhm ; TL1A (Q167R); SEQ ID NO: 246], Cynomolgus monkey full-length TL1A, Macaca fascicularis (MfTL1A; SEQ ID NO: 248), full-length mouse TL1A (SEQ ID NO: 250) and full-length mouse TL1A ( SEQ ID NO: 258). Stable cell lines were isolated and maintained
in 10% fetal bovine serum (FBS; Irvine Scientific), Dulbecco's Modified Eagle's Medium (DMEM; Irvine Scientific), non-essential amino acids (NE-AA, Irvine Scientific), penicillin / streptomycin (Invitrogen) and G418 (Invitrogen). For the bioassay, human or mouse DR3 reporter cells were cultured in 96 well assay plates at 1 x 104 cells / well in medium with low serum content, ie 0.1% FBS and OPTIMEM® (Invitrogen) and incubated at 37 ° C and 5% CO 2 overnight.
The next day, soluble TL1A or Fhm (sTL1A or sFhm) was serially diluted 1: 3 and added to cells in concentrations ranging from 0.002 nM to 100 nM (in addition to a control buffer that did not contain TL1A). For inhibition, antibodies were serially diluted 1: 3 and added to cells in concentrations ranging from 0.002 nM to 100 nM (in addition to a control buffer that does not contain the antibody) in the presence of constant concentrations of TL1A or Fhm : 800 pM for hTL1A (expressed in CHO cells; residues 72-251 of SEQ ID NO: 244, with the N-terminal Hi6-tag), 100 pM for hTL1A (expressed in E. coli; residues 72-251 of SEQ ID NO: 244, with or without N-terminal Met), 500 pM for Fhm (expressed in CHO cells; residues 72-251 of SEQ ID NO: 246), 400 pM for MfTL1A (expressed in CHO cells; residues 72-251 of SEQ ID NO: 248, with N-terminal Hi6-tag), 400 pM for MfTL1A (expressed in E. coli; residues 72-251 of SEQ ID NO: 248, with or without N-terminal Met), 50 pM for TL1A mouse (expressed in CHO cells; residues 76-252 of SEQ ID NO: 250, with N-terminal Hi6-tag), 20 pM for mouse TL1A (expressed in E. coli; residues 76-252 of SEQ ID NO: 250, with or without N-terminal Met) and 50 pM for TL1A mouse (expressed in CHO cells; residues 76-252 of SEQ ID NO: 258, with Hi6-tag N-terminal). Luciferase activity was detected after 5.5 hours of incubation at 37 ° C and 5% CO 2. The results are shown in Table 6. Control mAb1: positive control (an anti-hTL1A antibody with the variable chain domains heavy and light having amino acid sequences corresponding to SEQ ID NOS: 21 and 27 of US 2009/0280116); control mAb2: Negative control (irrelevant antibody); NB: No connection under the conditions tested; NT: Not tested in this test; *: Inhibition is not the baseline for the highest antibody concentration of 100 nM.
Table 6 hTL1A hTL1A Fhm MfTL1A MfTL1A mTL1A mTL1A rTL1A sTL1A or sFhm (CHO) (E.
Coli) (CHO) (CHO) (E.
Coli) (CHO) (E.
Coli) (CHO) EC50 (nM) 0.63 0.12 0.32 0.86 2.02 0.08 0.01 0.06 TL1A or Fhm constant (pM) 800 100 500 400 400 50 20 50 H4H1681N 0 , 17 0.02 NB 0.03 0.02 NB NB NB H4H1704N 0.13 0.06 0.17 0.01 0.03 NB NB 0.40 H4H1804N 0.07 0.03 0.10 0.03 0 , 02 NB NB 3.64 H4H1805N 0.10 0.04 185.50 * 0.03 0.02 NB NB NB H4H1817N 0.11 0.04 0.12 0.04 0.02 NB NB 23.87 H4H1818N 0 , 37 0.29 0.62 0.13 0.06 NB NB 1.10 H4H1719P 0.06 0.02 0.08 0.01 0.02 NT NB NT H4H1725P 0.05 0.02 0.07 0, 01 0.02 NB NB 63.43 IC50 [nM] H4H1738P 0.39 0.16 0.33 0.39 0.07 NT NB NT
47/55 H4H1742P 0.31 0.19 0.53 0.26 0.07 NB NB 6.12 H4H1745P 0.09 0.06 0.15 0.05 0.03 NB NB NB H4H1750P 0.90 2.17 3.10 154.70 32.47 * NT NB NT H4H1752P 0.36 0.21 NB 0.12 0.05 NT NB NT mAb1 control NB 0.74 NB NB 3.25 NT NB NT mAb2 control NB NB NB NB NB NB NT NB
As shown in Table 6, thirteen anti-TL1A antibodies have been shown to inhibit the stimulation of human DR3 receptor expressed on HEK293 cells induced by human soluble TL1A (expressed in CHO and E. coli), as determined using the luciferase reporter for activation of 5 NFκB.
A positive control antibody (control mAb1) inhibited hTL1A expressed in E. coli, but not expressed in CHO.
Ten antibodies also inhibited the stimulation of cells that express hDR3 by Fhm (hTL1A with Q167R). H4H1681N, H4H1805N and H4H1752P did not completely inhibit Fhm at the highest antibody concentration of 100 nM.
All thirteen antibodies also blocked MfTL1A (Table 6). Mouse TL1A (produced from CHO and E. coli) stimulated NFκB activation in hDR3 reporter cells, however, none of the 13 anti-human TL1A antibodies inhibited mouse TL1A expressed in E. coli in this assay (Table 6). Nine selected antibodies were tested and showed no blockage of mouse TL1A expressed in CHO in this assay (Table 6). To test the ability of TL1A expressed on cells to stimulate signaling in the hDR3 reporter system, bioassays were performed as described above for soluble TL1A with the following modifications: adherent HEK293 / TL1A cells were dissociated using Enzyme-Free Dissociation Solution (Chemicon) and added to the corresponding hDR3 reporter cells after serial dilution of the cells with 1: 2 TL1A starting from 2 x 105 cells to 195 cells (plus a cellless control). To inhibit antibody formation, 1 x 104 cells were added together with antibodies serially diluted from 100 nM to 0.002 nM (in addition to a control not containing the antibody). The results are shown in Table 7. Control mAb1 and mAb2: Same as above assays.
NB: No connection under the conditions tested; NT: Not tested in this test; *: Inhibition is not the baseline for the highest antibody concentration of 100 nM.
Table 7 HEK293 / HEK293 / HEK293 / HEK293 / HEK293 / TL1A or Fhm linked to the hTL1A cell Fhm MfTL1A mTL1A rTL1A EC50 (cells) 23474 47921 8465 12366 9773 Constant TL1A / Fhm 10,000 N6.67H (10,000 cells) NT NT H4H1704N 1.11 1.58 3.76 NB NT H4H1804N 0.56 1.23 2.23 NB 3.99 H4H1805N 0.82 54.10 * 1.93 NB NB H4H1817N 1.11 0.62 5, 04 NB 94.96 * H4H1818N 1.54 1.42 4.29 NT NT H4H1719P 0.82 0.84 3.00 NT NT IC50 [nM] H4H1725P 0.66 0.94 2.82 NB 190.30 * H4H1738P 7.47 7.75 20.25 NT NT H4H1742P 6.55 8.39 18.45 NB NT H4H1745P 0.76 2.12 4.28 NT NT H4H1750P 12.82 29.52 * 107.40 * NT NT H4H1752P 1.99 138.10 * 11.67 NT NT mAb1 control NB NB NB NB NT mAb2 control NB NB NB NB NB
As shown in Table 7, all thirteen antibodies blocked the stimulation of cells that express hDR3 by hTL1A expressed on cells.
With cell-bound Fhm, all antibodies significantly inhibited 5, except H4H1681N, H4H1805N, H4H1750P and H4H1752P, which did not fully inhibit at the highest tested antibody concentration of 100 nM.
With MfTL1A bound to the cell, all antibodies inhibited, except H4H1750, which did not completely inhibit at the highest tested antibody concentration of 100 nM.
Six of the antibodies, H4H1704N, H4H1804N, H4H1805N, H4H1817N, H4H1725P and H4H1742P, were tested for blocking stimulation of mDR3 cells by mTL1A on the cell surface and showed no inhibition.
Reporter cells that express mouse DR3 could also be stimulated by 293 cells that express rTL1A, with an observed EC50 of 9773 cells (Table 7). Four antibodies were tested in the rTL1A / mDR3 assay: three antibodies, H4H1804N, H4H1817N and H4H1725P, blocked, while H4H1805N did not block stimulation of mDR3 cells by rTL1A on the cell surface (Table 7). Control mAb1 blocked stimulation of hDR3 cells by soluble hTL1A and MfTL1A expressed in E. coli, but failed to block CHO-expressed forms of hTL1A and MfTL1A under all conditions tested (Table 6). Control mAb1 also did not inhibit the stimulation of cells that express hDR3 by any of TL1A and Fhm ex-
haste on the cell surface under all conditions tested (Table 7). Example 5: Blocking TL1A to hDR3 and DcR3 by Anti-TL1A Antibodies The ability of antibodies to block the binding of human TL1A to its cognate receptors, the DR3 and DcR3 receptors, was measured using a competitive sandwich ELISA.
In addition, blockade of binding of a human TL1A protein variant, Fhm (human TL1A Q167R) and monkey Cynomolgus (Macaca fascicularis) (MfTL1A) to human DR3 or DcR3 receptors was measured in the same way.
Constant amounts of biotinylated human TL1A or Fhm (both expressed with a 6-His tag in CHO cells) or biotinylated MfTL1A (expressed in CHO cells) were titrated separately with varying amounts of antibodies.
The antibody-protein complexes were incubated in solution (1h, 25 ° C) and then transferred to microtiter plates coated with human DR3 (hDR3) or human DcR3 (hDcR3) expressed as human Fc / IgG1 fusion proteins.
After one hour at 25 ° C, the wells were washed and TL1A from monkey or human bound was detected with streptavidine conjugated to horseradish peroxidase (HRP). The wells were washed with a TMB solution to produce a colorimetric reaction and dissipated with aqueous sulfuric acid, before reading the absorbance at 450 nm on a Perkin-Elmer Plate Reader Victor X5.
A sigmoidal dose-response curve was adapted to the data using the Prism® data analysis package. The calculated IC50 value, defined as the antibody concentration required to block 50% of TL1A binding to hDR3 or hDcR3, was used as an indicator of blocking power.
Both fully human anti-hTL1A mAbs and comparison antibodies, that is, the control mAb1 (an anti-hTL1A antibody with the heavy and light chain variable domains having amino acid sequences corresponding to SEQ ID NOS: 21 and 27 , respectively, US 2009/0280116) and control mAb3 (an anti-hTL1A antibody with variable domains of the heavy and light chain having amino acid sequences corresponding to SEQ ID NOS: 57 and 48, respectively, US 2009/0280116), were included in the study.
The results are shown in Table 8. NB: No connection under the conditions tested; NT: not tested.
Concentrations of soluble biotinylated binders: (1) 150 pM; (2) 500 pM; (3) 10 pM; and (4) 50 pM.
As shown in Table 8, most fully human mAbs show effective blocking of 5 TL1A / hDR3 and TL1A / hDcR3 linkage interaction, with several showing IC 50 values below 50 pM.
Two of the antibodies, H4H1752P and H4H1805N, strongly blocked the binding of human and monkey TL1A to hDR3 and hDcR3, but failed to block Fhm binding to either hDR3 or hDcR3, suggesting that the binding epitope for these two antibodies can involve the region near the Fhm mutation site (hTL1A with Q167R). The hTL1A crystal structure shows that the Q167 residue occurs inside an exposed handle on the surface (Zhan et al., 2009, Biochemistry 48: 7636-7645). Table 8 hDR3 hDR3 hDR3 DcR3 DcR3 DcR3 1 1 2 3 3 4 mAb ID hTL1A (CHO) Fhm (CHO) MfTL1A (CHO) hTL1A (CHO) Fhm (CHO) MfTL1A (CHO) IC50 (pM) IC50 (pM) IC50 (pM) IC50 (pM) IC50 pM) IC50 (pM) IC50 (pM) IC50 (pM) H4H1681N 60> 10000 141 17 90 93 H2M1681N 37> 10000 61 13 234 149 H4H1704N 30 44 42 77 170 110 H2M1704N NT NT NT NT NT NT NT H4H1719P 22 23 46 44 45 61 H4H1725P 15 18 16 68 85 145 H4H1738P 69 152 117 122 150 68 H4H1742P 64 214 240 181 231 127 H4H1745P 18 44 50 58 85 118 H4H1750P 341 589 5209 656 626 NB H4H1752P 104 NB 110 31 NB 56 H4H1804N 40 69 71 175> 34 H2M1804N 46 102 81 120> 10000 9 H4H1805N 14 NB 26 33 436 313 H2M1805N 6 NB 13 12 2241 1138 H4H1817N 114 235 101 270 NT 322 H2M1817N 154 249 137 890 666 776 H4H1818N 119 202 123 232 NT 1102 H2M1818N 154 317 239 55 mAb1 of> 10000 NB 5300> 1000 NT 21000 control mAb3 of> 10000 NB 17000 8600 NT NT control
Example 6: Competition for Cell Surface Binding of Antibodies to TL1A with Soluble hTL1A Stable transfected human embryonic kidney 293 cells to overexpress hTL1A on the cell surface were first scored in a flow cytometry experiment with eight anti-hTL1A antibodies in four concentrations (1, 0.1, 0.01 and 0.003 µg / ml). Human antibodies bound
results were detected using a goat F (ab ') 2 labeled with human allophakocyanin specific for human FCγ [or anti-hFcγ-APC F (ab') 2, Jackson ImmunoRese-arch, # 109-136-170]. The lowest concentration of antibody that provided significant levels of staining was then used in a competitive binding assay. A negative isotype control antibody (human IgG4) was used at 1 µg / ml to define the background signal. For the competition experiment, eight antibody samples, at the minimum concentrations identified above, were first treated with soluble hTL1A expressed from CHO cells in concentrations ranging from 0.03 µg / ml to 10 µg / ml. After pre-incubation for 30 min on ice, the antibody / hTL1A mixture was added to 293 / HEK-hTL1A cells that had been isolated by centrifugation in a 96-well conical plate. After incubating for another 10 minutes on ice, the cells were washed. The secondary reagent, F (ab ') 2 anti-hFcγ-APC, was added to all wells in a 200-fold dilution to detect bound antibodies. The samples were incubated for 15 minutes on ice, protected from light and then washed. The cells were processed on an LSR II BD Flow Cytometer (BD Biosciences) to detect anti-hTL1A antibodies bound to the cell surface and the data were analyzed using FlowJo software (Version 8.8.6; Tree Star Inc.). The results are shown in Table 9. Maximum signal: anti-hTL1A antibody binding in the absence of soluble hTL1A; Minimum Signal: signal recorded when 1 µg / ml isotype control antibody was added instead of anti-hTL1A antibody. NT: Not tested. Table 9 Mean fluorescence intensity for anti-hTL1A (H4H) antibodies that bind to hTL1A on the cell surface hTL1A 1704N 1725P 1742P 1804N 1805N 1817N 1681N 1745P soluble 0.1 g / ml 0.1 g / ml 1 g / ml 0, 1 g / ml 0.1 g / ml 1 g / ml 1 g / ml 1 g / ml (g / ml) 10 NT NT NT NT NT NT 16.9 15 3 22.6 34.9 19.9 28, 9 23.6 25.2 28.5 19.5 1 26 29.2 32.7 33.7 25.1 29.1 80.9 26.9
0.3 31.5 40.7 44.7 33.5 36.8 23.6 236 115
0.1 132 84.5 79.4 51.1 60.2 97.2 327 93.1
0.03 163 207 126 126 156 85.3 318 80 Signal 116 211 126 127 158 110 320 87.2 maximum Signal 27.5 27.5 27.5 27.5 27.5 27.5 17.9 17.9 minimum
As shown in Table 9, signals from the eight antibodies tested competed to lower baseline levels by adding excess soluble hTL1A, demonstrating the specificity of binding antibodies to hTL1A on the cell surface. 5 Example 7: Blocking Stimulation of CD4 + hTL1A-Dependent T-cells By Anti-TL1A Antibodies To determine the ability of anti-hTL1A antibodies to block stimulation of hTL1A-dependent human CD4 + T cells, an in vitro assay was developed in which release of IFN-gamma (IFN-γ) stimulated by hTL1A / anti-CD3 / anti-CD28 was measured in the presence or absence of antibodies.
Human CD4 + T cells were isolated from the fresh buffy coat prepared from human blood samples obtained from the New York Blood Center.
Cells from a single donor were separated from cells from another donor for each assay.
CD4 + T cells were added to the wells of a 96-well plate at 3.5 x 105 cells per well.
To each well, soluble hTL1A was then added (residues 72-251 of NP_005109.2 with an N-terminal hexa-histidine tag, expressed from CHO cells) at a final concentration of 1 µg / ml (16 nM, assuming trimers of formation of hTL1A in solution) in RPMI + 10% FBS, L-glutamine and penicillin / streptomycin.
Anti-hTL1A antibodies or an isotype control antibody in final concentrations of 1.0 µg / ml or 3.0 µg / ml (6.7 nM or 20 nM, respectively) were also added to each well. The samples were incubated for 15 minutes at 4 ° C in the dark, followed by the addition of anti-hCD3 (BS Pharmingen, cat # 555336) and anti-hCD28 (BD Pharmingen, cat # 555725) to each well in final concentrations of 1.0 µg / ml.
The samples were incubated for 24 hours at 37 ° C, the supernatants collected and the levels of IFN-γ (determined by ELISA.
The blocking effect (average of two wells for each condition) of each antibody in each donor sample of human CD4 + T cells is represented as the reduction in the maximum signal divided by the maximum response window; ie Block% = [(Max - Inib) / (Max - Min)] x 100, where "Max", "Inib" and "Min" are concentrations of IFN-γ measured for T cells
Human CD4 + treated as follows: "Max" - treated with [hTL1A + anti - hCD3 + anti-hCD28 + isotype control mAb], "Min:" - treated with [anti-hCD3 + anti-hCD28 + mAb isotype control] and "Inib" - treated with [hTL1A + anti-hCD3 + anti-hCD28 + test anti-hTL1A mAb]. Antibodies 5 for which IFN-γ blocking has exceeded the "Min" baseline level are represented as 100% blocking. Proportion (Max / Min) is the proportion between the concentration of IFN-γ produced from human CD4 + T cells treated under Max and Min conditions, as defined above.
As shown in Table 10, the H4H1725P, H4H1805N, H4H1817N and H4H1804N antibodies significantly blocked hTL1A-stimulated IFN-γ release at both concentrations of 1 µg / ml and 3 µg / ml, with almost complete blockage (> 80 %) observed for most donors in the highest concentration of antibodies.
The results for blocking IFN-γ secretion by thirteen different anti-hTL1A antibodies against T cells from 10 different human CD4 + donors are further summarized in Table 11. SD: standard deviation.
Table 10% IFN-γγ production block in human T cells from 10 Donor donors # D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 Ratio 5 4 10 10 4 3 4 3 8 2 mAb ID (Max / Min) H4H1725P 90 100 70 85 45 80 60 85 50 95 mAb H4H1805N 100 100 90 100 100 100 100 100 90 100 1 g / ml H4H1817N 100 100 90 90 100 45 55 100 55 80 (6.7 nM) H4H1804N 95 100 80 90 100 50 10 100 50 0 H4H1725P 95 100 95 100 90 80 90 100 90 100 mAb H4H1805N 100 100 95 100 80 100 100 100 70 100 3 g / ml H4H1817N 100 100 100 100 100 100 100 100 95 85 (20 nM) H4H1804N 100 100 95 100 100 100 100 100 100 80
Table 11 ID mAb Block% medium Block% medium Block% medium (SD) (SD) 0.1 g / ml mAb (SD) 1 g / ml mAb 3 g / ml mAb H4H1681N 20% (24) 45% (30) 95% (7) H4H1704N 22% (29) 53% (27) 98% (5) H4H1719P 10% (22) 37% (23) 78% (30) H4H1725P 13% (14) 80% (20) 94% (7 ) H4H1738P 25% (30) 41% (35) 81% (18) H4H1742P 10% (22) 58% (33) 83% (16) H4H1745P 22% (27) 36% (36) 91% (7) H4H1750P 11% (14) 42% (35) 89% (15) H4H1752P 18% (25) 52% (35) 71% (30) H4H1804N 26% (30) 68% (38) 98% (6) H4H1805N 21% (28) 98% (4) 94% (10) H4H1817N 25% (33) 81% (22) 98% (5) H4H1818N 25% (33) 42% (33) 71% (35) Isotype control 16% (21) 26% (33) 28% (27)
IFN-γ levels were also measured at six different antibody concentrations (ranging from 0.03 µg / ml to 10 µg / ml) for each of six different antibodies added to CD4 + T cells from twelve human donors.
Adaptation of the curve to the data allowed us to estimate the concentration of antibody in which half of the maximum inhibition was obtained for each antibody for each cell sample of the donors.
The mean concentrations (± SD) to obtain half of the maximum inhibition are given in Table 12. Table 12 mAb IC50 (nM) Donor # H4H1725P H4H1742P H4H1805N H4H1817N H4H1804N H4H1704N _ D1 3.4 24 2.4 6.6 6 6 , 8 _ D2 3.2 4.6 5.5 3.1 8.6 _ D3 5.4 10 3.4 4.7 8.6 _ D4 4.7 13 2.5 2.4 7.7 D5 7.0 12 2.9 8.5 6.7 17 D6 5.4 10 3.4 4.7 8.6 11 D7 13 31 8.0 7.0 12 12 D8 12 27 5.1 7.0 11 19 D9 6.4 56 5.9 9.5 8.1 7.5 D10 4.1 12 2.8 7.3 12 10 D11 6.1 27 3.0 7.3 7.6 11 D12 6.4 14 3.1 9.4 7.5 8.0 average 6.4 20 4.0 6.5 8.8 12 (± SD) (3.1) (14) (1.7) (2.3) (1.9) (4.1)
Four of the antibodies, H4H1725P, H4H1804N, H4H1805N, H4H1817N, exhibited average concentrations for half of the maximum inhibition below 10 nM (ranging from approximately 4-9 nM).

权利要求:
Claims (20)
[1]
1. Isolated human antibody or an antigen-binding fragment thereof that specifically binds and neutralizes the activity of human TNF ligand 1A (hTL1A). 5
[2]
An antigen-binding antibody or fragment according to claim 1, wherein the antibody or antigen-binding fragment thereof binds to hTL1A which has the amino acid sequence of SEQ ID NO: 244 with a dissociation constant in equilibrium (KD) of about 700 pM or less, about 300 pM or less, about 80 pM or less or 50 pM or less.
[3]
3. Isolated human antibody or antigen-binding fragment thereof that specifically binds to human TNF ligand 1A (hTL1A) comprising a heavy chain variable region (HCVR) comprising an amino acid sequence selected from SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 118, 134, 138, 154, 158, 174, 178, 194, 198, 214, 218 and 234.
[4]
4. Isolated human antibody or antigen-binding fragment thereof that specifically binds to human TNF ligand 1A (hTL1A) comprising a light chain variable region (LCVR) comprising a selected amino acid sequence of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226 and 236.
[5]
An antigen-binding antibody or fragment according to claim 3 or 4, comprising an HCVR / LCVR sequence pair selected from SEQ ID NO: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/116, 118/126, 134/136, 138/146, 154/156, 158/166, 174/176, 178/186, 194/196, 198 / 206, 214/216, 218/226 and 234/236.
[6]
An antigen-binding antibody or fragment according to any one of claims 3 to 5, comprising a pair of HCVR / LCVR sequences of SEQ ID NOs: 2/10, 18/26, 174/176 or 234 / 236.
[7]
7. Isolated human antibody or an antigen-binding fragment that specifically binds to hTL1A, comprising
[8]
complementarity determining region (CDR) sequences of heavy and light chains contained in the HCVR / LCVR sequence pair of the antibody or antigen binding fragment according to claim 5 or 6. 5 8. Antibody or antigen binding fragment thereof which competes for binding to hTL1A with the antibody or antigen binding fragment according to claim 6.
[9]
An antibody or antigen-binding fragment thereof that binds to the same epitope on hTL1A that is recognized by the antibody or antigen-binding fragment according to claim 6.
[10]
An antigen-binding antibody or fragment according to any one of claims 3 to 9, wherein the antibody or antigen-binding fragment is cross-reactive with Cynomolgus monkey TL1A (MfTL1A).
[11]
An antigen-binding antibody or fragment according to any one of claims 3 to 10, wherein the antibody or antigen-binding fragment is cross-reactive with Fhm which has the amino acid sequence of SEQ ID NO: 246.
[12]
12. Antigen-binding antibody or fragment according to any one of claims 3 to 10, wherein the antibody or antigen-binding fragment does not cross-react with Fhm which has the amino acid sequence of SEQ ID NO: 246.
[13]
13. Isolated nucleic acid molecule encoding the antibody or antigen binding fragment as defined in any of claims 1 to 12.
[14]
An expression vector comprising the nucleic acid molecule as defined in claim 13.
[15]
15. Method of producing an anti-hTL1A antibody or antigen-binding fragment thereof, comprising culturing a host cell comprising the expression vector as defined in claim 14 under conditions that permit the production of the antibody or fragment thereof and recovering the antibody or fragment thereof produced.
[16]
16. Pharmaceutical composition comprising the antibody or antigen binding fragment as defined in any one of claims 1 to 12 and a pharmaceutically acceptable carrier. 5
[17]
A pharmaceutical composition according to claim 16, further comprising one or more additional therapeutic agents selected from an immunosuppressant, an anti-inflammatory agent, an analgesic and an antiallergic agent.
[18]
18. Use of the antibody or antigen-binding fragment thereof as defined in any one of claims 1 to 12 in the manufacture of a medicament for the treatment of a disease or disorder which is prevented, relieved, improved or inhibited by removal , reduction or inhibition of TL1A activity.
[19]
19. Method for the treatment of a disease or disorder which is prevented, relieved, improved or inhibited by removing, reducing or inhibiting TL1A activity comprising administering, to an individual who needs it, a therapeutically effective amount of the drug. pharmaceutical position as defined in claim 16.
[20]
20. Use according to claim 18 or method as defined in claim 19, wherein said disease or disorder is selected from ulcerative colitis, Crohn's disease, rheumatoid arthritis, multiple sclerosis, diabetes, asthma and lung inflammation allergic.

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法律状态:
2020-08-18| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2020-11-17| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2020-12-29| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2021-03-16| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-06-29| B11B| Dismissal acc. art. 36, par 1 of ipl - no reply within 90 days to fullfil the necessary requirements|

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2021-11-03| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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