BTLA ANTIBODIES
20230235058 · 2023-07-27
Inventors
- Simon John DAVIS (Oxford,, GB)
- Richard John CORNALL (Oxford,, GB)
- Chirstopher Douglas PALUCH (Oxford,, GB)
Cpc classification
A61K39/395
HUMAN NECESSITIES
C07K2317/72
CHEMISTRY; METALLURGY
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
C07K2317/94
CHEMISTRY; METALLURGY
C07K2317/24
CHEMISTRY; METALLURGY
C07K2317/73
CHEMISTRY; METALLURGY
A61P1/00
HUMAN NECESSITIES
C07K2317/34
CHEMISTRY; METALLURGY
C07K16/2896
CHEMISTRY; METALLURGY
A61P37/06
HUMAN NECESSITIES
C07K2317/92
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
C07K16/28
CHEMISTRY; METALLURGY
A61P1/00
HUMAN NECESSITIES
Abstract
This invention relates generally to antibodies that bind to human B and T lymphocyte attenuator (BTLA) and uses thereof. More specifically, the invention relates to agonistic antibodies that bind human BTLA and modulate its activity, and their use in treating inflammatory, autoimmune and proliferative diseases and disorders. Suitably, the antibodies also possess an Fc modification that enhances signalling through FcγR2B.
Claims
1. An isolated antibody that specifically binds to human B and T lymphocyte attenuator (BTLA), wherein said antibody comprises a heavy chain and a light chain, wherein said heavy chain comprises an Fc region that comprises a substitution that results in increased binding to FcγR2B compared to a parent molecule that lacks the substitution.
2. The antibody according to claim 1, wherein said heavy chain comprises an Fc region that comprises one or more of the following amino acids: alanine (A) at position 234, alanine (A) at position 235, aspartic acid (D) at position 236, aspartic acid (D) at position 237, aspartic acid (D) at position 238, alanine (A) at position 265, glutamic acid (E) at position 267, glycine (G) at position 271, arginine (R) at position 330, alanine (A) at position 332, or alanine (A) at position 297 (numbering according to EU Index).
3. The antibody according to claim 1, wherein said heavy chain comprises an Fc region that comprises an aspartic acid at position 238 (EU Index).
4. The antibody according to claim 3, wherein (i) said Fc region binds to FcγR2B with a higher affinity relative to a comparable control antibody that comprises an Fc region with proline at position 238 (EU Index); or (ii) said antibody binds to FcγR2B with an affinity of from about 5 μM to 0.1 μM, as determined by surface plasmon resonance (SPR); or (iii) said Fc region binds to FcγR2A (131R allotype) with a lower or equal affinity relative to a comparable control antibody that comprises an Fc region that comprises a proline at position 238 (EU Index); or (iv) said antibody binds to FcγR2A (131R allotype) with a K.sub.D of at least 20 μM, as determined by surface plasmon resonance (SPR); or (v) said antibody binds to FcγR2A (131H allotype) with a lower or equal affinity relative to a comparable control antibody that comprises an Fc region that comprises a proline at position 238 (EU Index); or (vi) said antibody binds to FcγR2A (131H allotype) with a K.sub.D of at least 50 μM, as determined by surface plasmon resonance (SPR); or (vii) wherein said antibody exhibits an in vivo half-life of at least 10 days.
5. The antibody of any one of claims 1 to 4, wherein said antibody binds to an epitope of human BTLA selected from the group consisting of: (i) D52, P53, E55, E57, E83, Q86, E103, L106 and E92; or (ii) Y39, K41, R42, Q43, E45 and S47; or (iii) D35, T78, K81, S121 and L123; or (iv) H68; or (v) N65 and A64; wherein each position is in relation to the amino acid sequence disclosed in SEQ ID NO:225.
6. The antibody of any one of claims 1 to 5, wherein said antibody exhibits increased agonism of human BTLA expressed on the surface of a human immune cell, such as measured by a BTLA agonist assay selected from a T cell activation assay or a B cell activation assay.
7. The antibody of any one of claims 1 to 6, wherein the heavy chain comprises a heavy chain variable region comprising three complementarity determining regions (CDRs): CDRH1, CDRH2 and CDRH3 and the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, wherein (i) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 1, 17, and 3, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 4, 12, and 6, respectively, with from 0 to 3 amino acid modifications; or (ii) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 20, 21, and 22, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 23, 24, and 25, respectively, with from 0 to 3 amino acid modifications; or (iii) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 30, 31, and 32, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 33, 34, and 35, respectively, with from 0 to 3 amino acid modifications; or (iv) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 45, 46, and 47, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 33, 34, and 35, respectively with from 0 to 3 amino acid modifications; or (v) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 53, 54, and 55, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 56, 57, and 58, respectively, with from 0 to 3 amino acid modifications; or (vi) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 61, 62, and 63, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 64, 65, and 66, respectively, with from 0 to 3 amino acid modifications; or (vii) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 61, 69, and 70, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 71, 72, and 73, respectively, with from 0 to 3 amino acid modifications; or (viii) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 76, 77, and 78, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 79, 80, and 81, respectively, with from 0 to 3 amino acid modifications; or (ix) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 45, 46, and 84, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 33, 34, and 85, respectively, with from 0 to 3 amino acid modifications; or (x) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 88, 89, and 90, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 91, 65, and 92, respectively, with from 0 to 3 amino acid modifications; or (xi) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 95, 96, and 97, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 98, 99, and 100, respectively, with from 0 to 3 amino acid modifications; or (xii) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 103, 104, and 105, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 106, 107, and 108, respectively, with from 0 to 3 amino acid modifications; or (xiii) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 76, 111, and 112, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 113, 114, and 115, respectively, with from 0 to 3 amino acid modifications; or (xiv) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 118, 119, and 120, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 121, 122, and 123, respectively, with from 0 to 3 amino acid modifications; or (xv) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 126, 127, and 128, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 79, 129, and 130, respectively, with from 0 to 3 amino acid modifications; or (xvi) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 133, 134, and 135, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 106, 107, and 136, respectively, with from 0 to 3 amino acid modifications; or (xvii) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 103, 134, and 139, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 106, 107, and 136, respectively, with from 0 to 3 amino acid modifications; or (xviii) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 143, 144, and 145, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 146, 147, and 148, respectively, with from 0 to 3 amino acid modifications; or (xix) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 151, 152, and 153, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 154, 155, and 156, respectively, with from 0 to 3 amino acid modifications; or (xx) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 159, 160, and 161, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 4, 12, and 164, respectively, with from 0 to 3 amino acid modifications; or (xx1) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 167, 168, and 169, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 170, 171, and 172, respectively, with from 0 to 3 amino acid modifications; or (xxii) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 45, 46, and 47, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 170, 171, and 172, respectively, with from 0 to 3 amino acid modifications; or (xxiii) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 45, 46, and 177, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 154, 155, and 178, respectively, with from 0 to 3 amino acid modifications; or (xxiv) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 181, 182, and 183, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 184, 185, and 186, respectively, with from 0 to 3 amino acid modifications; or (xxv) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 76, 77, and 78, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 79, 80, and 189, respectively, with from 0 to 3 amino acid modifications; or (xxvi) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 45, 191, and 192, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 154, 155, and 193, respectively, with from 0 to 3 amino acid modifications; or (xxvii) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 196, 197, and 198, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 199, 200, and 201, respectively, with from 0 to 3 amino acid modifications; or (xxviii) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 204, 205, and 206, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 207, 208, and 209, respectively, with from 0 to 3 amino acid modifications; or (xxix) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 212, 213, and 214, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 215, 34, and 216, respectively, with from 0 to 3 amino acid modifications; or (xxx) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 1, 2, and 3, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 4, 5, and 6, respectively, with from 0 to 3 amino acid modifications; or (xxxi) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 20, 163, and 22, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 23, 176, and 25, respectively, with from 0 to 3 amino acid modifications; or (xxxii) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 30, 48, and 32, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 33, 34, and 35, respectively, with from 0 to 3 amino acid modifications; or (xxxiii) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 1, 11, and 3, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 4, 12, and 6, respectively, with from 0 to 3 amino acid modifications; or (xxxiv) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 1, 11, and 3, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 4, 5, and 6, respectively, with from 0 to 3 amino acid modifications; or (xxxv) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 1, 17, and 3, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 4, 12, and 6, respectively, with from 0 to 3 amino acid modifications; or (xxxvi) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 20, 21, and 22, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 23, 24, and 25, respectively, with from 0 to 3 amino acid modifications; or (xxxvii) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 30, 31, and 32, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 33, 34, and 35, respectively, with from 0 to 3 amino acid modifications; or (xxxviii) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 30, 40, and 32, respectively, with from 0 to 3 amino acid modifications, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 33, 34, and 35, respectively, with from 0 to 3 amino acid modifications; optionally wherein the Fc region comprises an aspartic acid at position 238 (EU Index).
8. An isolated antibody that specifically binds BTLA, comprising a heavy chain and a light chain, wherein (1) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 18, or a sequence with at least 90% identity thereto and an Fc region comprising an aspartic acid at position 238 (EU Index) and the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 14, or a sequence with at least 90% identity thereto; or (2) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 26, or a sequence with at least 90% identity thereto and an Fc region comprising an aspartic acid at position 238 (EU Index) and the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 27, or a sequence with at least 90% identity thereto; or (3) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 36, or a sequence with at least 90% identity thereto and an Fc region comprising an aspartic acid at position 238 (EU Index) and the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 43, or a sequence with at least 90% identity thereto.
9. An isolated antibody that specifically binds BTLA, comprising a heavy chain and a light chain, wherein (1) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 19, or a sequence with at least 90% sequence identity thereto and light chain comprises an amino acid sequence as set forth in SEQ ID NO: 16, or a sequence with at least 90% identity thereto; (2) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 28, or a sequence with at least 90% sequence identity thereto and light chain comprises an amino acid sequence as set forth in SEQ ID NO: 29, or a sequence with at least 90% identity thereto; or (3) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 38, or a sequence with at least 90% sequence identity thereto and light chain comprises an amino acid sequence as set forth in SEQ ID NO: 44, or a sequence with at least 90% identity thereto; wherein the heavy chain comprises an Fc region comprising an aspartic acid at position 238 (EU Index).
10. The antibody of any one of the preceding claims, which is an IgG1, IgG2 or IgG4 antibody.
11. The antibody of any one of the preceding claims, which is selected from the group consisting of: a human antibody, a humanised antibody, a chimeric antibody and a multispecific antibody (such as a bispecific antibody).
12. The antibody of any one of the preceding claims, which is monoclonal.
13. The antibody of any one of the preceding claims, wherein said antibody agonizes human BTLA expressed on the surface of an immune cell, optionally wherein said immune cell is a T cell.
14. The antibody of any one of the preceding claims, wherein binding of said antibody to human BTLA expressed on the surface of an immune cell decreases proliferation of said cell relative to a comparable immune cell not bound by said antibody, optionally wherein said cell is a T cell, optionally wherein said decrease in cell proliferation is at least about 10%, 15%, 20%, 25%, 30%, 40%, or 50%.
15. The antibody of any one of the preceding claims, wherein (i) said antibody specifically binds human B and T Lymphocyte Attenuator (BTLA) with a K.sub.D of less than 10 nM; and/or (ii) wherein said antibody binds cynomolgus BTLA with a K.sub.D of less than 20 nM; and/or (iii) said antibody does not inhibit binding of BTLA to herpes virus entry mediator (HVEM); and/or (iv) said antibody inhibits proliferation of T cells in vitro, as determined by a mixed lymphocyte reaction assay.
16. The antibody of claim 15, wherein said antibody binds human B and T Lymphocyte Attenuator (BTLA) with an on rate of at least 5.0×10.sup.5 (l/Ms) at 37° C. and/or with an off rate of less than 3.0×10.sup.4 (l/s) at 37° C. and/or with a K.sub.D of less than 10 nM, as determined by surface plasmon resonance (SPR) at 37° C.
17. An isolated human antibody that specifically binds B and T lymphocyte attenuator (BTLA), comprising a heavy chain and a light chain, wherein (a) the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2 and CDRH3, wherein CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 17, and SEQ ID NO: 3, respectively, and wherein the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2 and CDRL3, wherein CDRL1 has an amino acid sequence as set forth in SEQ ID NO: 4, CDRL2 has an amino acid sequence as set forth in SEQ ID NO: 12, and CDRL3 has an amino acid sequence as set forth in SEQ ID NO: 6; and/or (b) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 18; or a sequence with at least 90% identity thereto; and/or (c) the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 14, or a sequence with at least 90% identity thereto; wherein the heavy chain comprises an Fc region that comprises an aspartic act at position 238 (EU Index); optionally wherein the antibody is an IgG1, IgG2 or IgG4 antibody.
18. A nucleic acid which comprises one or more nucleotide sequences encoding polypeptides capable of forming an antibody in any of claims 1 to 17.
19. An expression vector comprising the nucleic acid molecule of claim 18.
20. A host cell comprising the nucleic acid sequence of claim 18 or 19.
21. A method of producing an antibody that binds to BTLA, comprising the step of culturing the host cell of claim 20 under conditions for production of said antibody, optionally further comprising isolating and/or purifying said antibody.
22. A method for preparing an antibody that specifically binds BTLA, the method comprising the steps of: (i) providing a host cell comprising one or more nucleic acid molecules encoding the amino acid sequence of a heavy chain and a light chain which when expressed are capable of combining to create an antibody molecule of any one of claims 1 to 17; (ii) culturing the host cell expressing the encoded amino acid sequence; and (iii) isolating the antibody.
23. A pharmaceutical composition comprising a therapeutically effective amount of the antibody of any one of claims 1 to 17 and at least one pharmaceutically acceptable excipient.
24. An antibody in accordance with any one of claims 1 to 17, or the pharmaceutical composition in accordance with claim 23, for use in therapy.
25. An antibody in accordance with any one of claims 1 to 17, or the pharmaceutical composition in accordance with claim 23, for use in the treatment or prevention of inflammatory or autoimmune diseases, and disorders of excessive immune cell proliferation.
26. The antibody for use according to claim 25, wherein the inflammatory or autoimmune disease is selected from Addison's disease, allergy, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, anti-phospholipid syndrome, asthma (including allergic asthma), autoimmune haemolytic anaemia, autoimmune hepatitis, autoimmune pancreatitis, autoimmune polyendocrine syndrome, Behcet's disease, bullous pemphigoid, cerebral malaria, chronic inflammatory demyelinating polyneuropathy, coeliac disease, Crohn's disease, Cushing's Syndrome, dermatomyositis, diabetes mellitus type 1, eosinophilic granulomatosis with polyangiitis, graft versus host disease, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, Hidradenitis Suppurativa, inflammatory fibrosis (e.g., scleroderma, lung fibrosis, and cirrhosis), juvenile arthritis, Kawasaki disease, leukemia, lymphoma, lymphoproliferative disorders, multiple sclerosis, myasthenia gravis, myeloma, neuromyelitis optica, pemphigus, polymyositis, primary biliary cholangitis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, rheumatoid arthritis, sarcoidosis, Sjögren's syndrome, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis, transplant rejection, transverse myelitis, ulcerative colitis, uveitis, vasculitis, vitiligo and Vogt-Koyanagi-Harada Disease.
27. The antibody for use according to claim 25, wherein the disorder of excessive immune cell proliferation is selected from lymphoma, leukemia, systemic mastocytosis, myeloma, or a lymphoproliferative disorder.
28. A method of treating a BTLA-related disease in a patient, comprising administering to the patient a therapeutically effective amount of the antibody of any one of claims 1-17 or the pharmaceutical composition of claim 23.
29. The method of claim 28, wherein the BTLA-related disease is an inflammatory or autoimmune disease, or an immunoproliferative disease or disorder.
30. The method of claim 29, wherein the inflammatory or autoimmune disease is selected from Addison's disease, allergy, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, anti-phospholipid syndrome, asthma (including allergic asthma), autoimmune haemolytic anaemia, autoimmune hepatitis, autoimmune pancreatitis, autoimmune polyendocrine syndrome, Behcet's disease, bullous pemphigoid, cerebral malaria, chronic inflammatory demyelinating polyneuropathy, coeliac disease, Crohn's disease, Cushing's Syndrome, dermatomyositis, diabetes mellitus type 1, eosinophilic granulomatosis with polyangiitis, graft versus host disease, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, Hidradenitis Suppurativa, inflammatory fibrosis (e.g., scleroderma, lung fibrosis, and cirrhosis), juvenile arthritis, Kawasaki disease, leukemia, lymphoma, lymphoproliferative disorders, multiple sclerosis, myasthenia gravis, myeloma, neuromyelitis optica, pemphigus, polymyositis, primary biliary cholangitis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, rheumatoid arthritis, sarcoidosis, Sjögren's syndrome, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis, transplant rejection, transverse myelitis, ulcerative colitis, uveitis, vasculitis, vitiligo and Vogt-Koyanagi-Harada Disease.
31. The method of claim 29, wherein the immunoproliferative disease or disorder is selected from lymphoma, leukemia, systemic mastocytosis, myeloma, or a lymphoproliferative disorder.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0406]
Binding of antibodies to human and cynomolgus BTLA in soluble and cell expressed forms. (a) Surface plasmon resonance (SPR) binding curves for soluble monomeric human BTLA extracellular domain injected at increasing concentrations over immobilized anti-BTLA antibody; graphs show SPR signal after reference and blank subtraction. (b) Association and dissociation rates for binding to human or cynomolgus BTLA as calculated by curve fitting using BiaEvaluation software. (c) Binding of antibody 2.8.6, compared to isotype control antibody, to a human BTLA or cynomolgus BTLA expressing Jurkat cell line (data points represent mean+/−SD of triplicate wells at each antibody concentration). (d) EC50s for antibody binding to transfected cell lines, as calculated by non-linear curve fitting using GraphPad Prism software
[0407]
(a) Blockade of ligand binding by anti-BTLA antibodies was assessed by SPR. Human BTLA extracellular domain was immobilized on the sensor chip. Human HVEM was injected to confirm binding, then allowed to fully dissociate. A saturating concentration of anti-BTLA antibody was then injected, followed immediately by a second injection of HVEM. (b) Equilibrium binding of HVEM after injection of antibody was expressed as a percent of HVEM binding prior to antibody injection. Saturation of BTLA with clone 11.5.1, but not with clone 2.8.6, blocked subsequent binding of ligand.
[0408]
Epitope mapping of anti-BTLA antibodies. (a) HEK293T cells transfected with BTLA constructs in a bicistronic vector also expressing GFP were stained with Pacific Blue conjugated anti-BTLA antibody. Clone 11.5.1 binds to cells transfected with wild-type receptor (left) but not to cells transfected with BTLA having a Y39R mutation (right). (b) Binding to each BTLA mutant construct was expressed as a percentage of binding to wild-type BTLA for clones 2.8.6 and 11.5.1. (c) Mutations Y39R and K41E which selectively eliminate binding of clone 11.5.1 were mapped onto the crystal structure of human BTLA (black residues). Residues critical for binding of the ligand HVEM are highlighted in grey.
[0409]
(a) The crystal structure of human BTLA extracellular domain in complex with the Fab′ fragment of clone 2.8.6. Residues on BTLA which are buried at the interface are highlighted in black. (b) The epitope of antibody 2.8.6 is shown (black residues) in relation to the HVEM binding site (grey residues).
[0410]
(a) Strategy for creation of a chimeric BTLA gene in humanised-BTLA mice. A section of human genomic DNA from the beginning of exon 2 to the end of exon 3 was inserted into the mouse locus replacing the mouse sequence from the beginning of exon 2 to the end of exon 4. The sequences at the exon-intron junction at the beginning of mouse exon 2 and end of mouse exon 4 were left intact to ensure proper splicing.
[0411]
(a) Protocol for T cell transfer assay to assess anti-BTLA antibodies in vivo. A mixture of humanised and wild-type OVA specific CD4 T cells was injected into recipient mice. The next day mice were immunised with ovalbumin in Alum to activate the transferred cells and 24 hours later were dosed with anti-human-BTLA antibody or isotype control. Eight days after initial cell transfer the ratio of humanised to wild-type cells in the transferred population in the spleen was assessed by flow cytometry. (b) Clone 11.5.1 and to a lesser extent 2.8.6 both reduced expansion of the humanised cells relative to the wild-type. Graph shows pooled data from two (for 11.5.1) or three (for 2.8.6) repeat experiments. Each data point represents an individual recipient mouse.
[0412]
Effect of anti-BTLA clone 2.8.6 on CD4 T-cell proliferation in a mixed lymphocyte reaction in vitro. T cells from humanised C57BL/6 mice were stained with CellTraceViolet and added to Mitomycin C treated Balb/c stimulator cells in the presence of anti-BTLA antibody or isotype control. After 96 hours, proliferation of humanised CD4 cells was assessed and normalised to proliferation in the absence of antibody. Clone 2.8.6 inhibited proliferation of humanised cells with an IC50 of 0.029 nM and had a maximal effect of 42% inhibition of proliferation. Data points represent mean+/−SD of triplicate wells at each antibody concentration and are representative of 5 independent experiments.
[0413]
(a) Effect of clone 2.8.6 in a T cell colitis model. RAG knockout recipient mice were injected with CD45RBhiCD25-CD4+ T cells from humanised BTLA mice and treated with 200 μg 2.8.6 or isotype control antibody on days 7, 21 and 35. Isotype control treated mice progressively lost weight from 3 weeks onwards, whilst 2.8.6 treated mice were spared. (b) 8 weeks after cell transfer colons were processed to extract lamina propria lymphocytes and the total number of inflammatory cells extracted per colon was calculated. Isotype control treated mice had significantly more infiltrating immune cells than 2.8.6 treated mice. (c) Colon weight to length ratios were calculated as a marker of inflammation and thickening. 2.8.6 treatment prevented the increase in weight to length ratio seen in isotype control treated mice.
[0414]
(a) Effect of BTLA antibodies in a parent-to-F1 model of GVHD. C57BL/6 splenocytes and bone marrow cells from humanised-BTLA mice were injected into CB6F1 recipient mice, which were then treated with anti-BTLA antibody or isotype control. Untreated mice developed clinical GVHD with progressive weight loss, dermatitis and diarrhea and were culled when they reached pre-specified humane endpoints. 2.8.6 and 11.5.1 antibody treated mice were relatively spared, with survival comparable to control mice reconstituted with syngeneic cells. (b) 5 weeks after cell transfer mice were culled and colon weight to length ratio was calculated as a marker of gut inflammation. 2.8.6 and 11.5.1 treatment prevented the colon thickening seen in untreated mice.
[0415]
(a) Effect of D265A mutated clone 11.5.1 in a T cell transfer assay in vivo. This mutated antibody, which does not bind Fc receptors, no longer inhibited proliferation of humanised BTLA cells, instead lead to enhanced proliferation due to receptor blockade. (b) The D265A mutated 11.5.1 antibody no longer inhibited T cell proliferation in a mixed lymphocyte reaction.
[0416]
Anti-BTLA antibodies do not fix complement. Splenocytes from humanised BTLA mice were incubated with 10% rabbit complement for 1 hour at 37° C. in the presence of 20 μg/ml BTLA antibody, isotype control or positive control (a depleting CD20 antibody). Anti-CD20 antibody depleted the majority of B cells confirming the activity of the rabbit complement, but BTLA antibodies did not deplete either B or T cells, even though both these populations stain positive for BTLA.
[0417]
Anti-BTLA antibodies do not cause antibody-dependent-cell-mediated cytotoxicity. Splenocytes from humanised BTLA mice were incubated for 24 hours at 37° C. in the presence of 20 μg/ml BTLA antibody, isotype control or positive control (a depleting CD20 antibody). Anti-CD20 antibody depleted the majority of B cells by inducing ADCC by effector cells in the mixture, but BTLA antibodies did not deplete either B or T cells, even though both these populations stain positive for BTLA.
[0418]
Anti-BTLA antibodies do not deplete B or T cells in vivo. Humanised BTLA mice were injected with 200 μg of 2.8.6 antibody. At 24 hours spleens and bone marrow were collected and cell populations assessed by flow cytometry. 2.8.6 did not deplete B or T cells in the spleen or affect the frequency of different B cell precursor populations in the bone marrow (n=3 mice per group).
[0419]
BTLA expression levels on B cells or CD4.sup.+ T cells from humanised mice following 6 days of in vivo incubation with antibodies 2.8.6 or 11.5.1, compared to BTLA expression on cells from mice injected with isotype control antibody (n=5 mice per group).
[0420]
Agonist effect of BTLA antibodies in a reporter assay is dependent on Fc receptor binding and isotypes with greater FcγR2B binding are more effective agonists. A Jurkat T cell line expressing GFP under the control of NFkB-responsive transcriptional elements was transfected with human BTLA and stimulated by co-culture with a BW5147 cell line expressing an anti-CD3 ScFv construct on its surface. NFkB signaling was detected by measuring the GFP geomean by flow cytometry after 24 hours of culture. The inhibitory effect of adding BTLA agonist antibodies of different isotypes to the culture was assessed in the condition where the BW5147 cell line was also transfected to express hFcγR2B (a) or in the condition where no Fc receptors were present (b). Data points are the mean+/−SD of triplicate wells at each antibody concentration, and are representative of 3 independent experiments.
[0421]
Humanised anti-BTLA agonist antibodies 2.8.6, 6.2_varC and 3E8 expressed on a P238D isotype have greater efficacy and potency in a reporter assay, compared to an Fc fusion protein of BTLA's ligand HVEM or the prior art BTLA agonist 22B3. A Jurkat T cell line expressing GFP under the control of NFkB-responsive transcriptional elements was transfected with human BTLA and stimulated by co-culture with a BW5147 cell line expressing an anti-CD3 ScFv construct and hFcγR2B on its surface. NFkB signaling was detected by measuring the GFP geomean by flow cytometry after 24 hours of culture. The inhibitory effect of BTLA agonist antibodies added to the co-culture was assessed. Data points are the mean+/−SD of triplicate wells at each antibody concentration and are representative of 3 independent experiments.
[0422]
Humanised anti-BTLA 2.8.6 inhibits CD4 T cell proliferation in a mixed leukocyte reaction. Purified primary human T cells from a blood bank donor were stained with a cell proliferation tracking dye and co-cultured for 5 days with allogeneic monocyte derived dendritic cells from a different donor in a 4:1 ratio, in the presence of BTLA agonist antibodies or hIgG1 P238D isotype control. Cell populations were identified by flow cytometry and proliferation was assessed by dilution of the tracking dye. CD4 proliferation in the presence of BTLA antibody was normalized to the proliferation in the presence of the equivalent concentration of isotype control. Data was collated from 6 independent experiments with different donor pairs. 2.8.6 significantly inhibited CD4 T cell proliferation as a P238D isotype but not in other isotype formats. The prior art molecule 22B3 had no significant effect on CD4 proliferation.
[0423]
Humanised anti-BTLA agonist antibodies 2.8.6, 6.2 varC and 3E8 expressed on a P238D isotype inhibit primary B cell activation in response to the TLR9 agonist ODN2006. Primary human B cells were isolated from healthy donor PBMCs and stimulated with 0.01 μM ODN2006 in the presence or absence of different doses of P238D isotype control antibody or selected BTLA agonist antibodies. After 5 days, IL-10 concentration in the supernatant was assessed by ELISA. Bars represent the mean+/−SD of triplicate wells at each antibody concentration, and are representative of 3 independent experiments.
[0424]
Humanised anti-BTLA agonist antibodies 2.8.6, 6.2 varC and 3E8 expressed on a P238D isotype significantly reduce weight loss in a xenogeneic graft vs host disease model. Irradiated NSG mice were reconstituted IV with 10 million human PBMCs on day 0 and then treated IP on day 1 with 10 mg/kg BTLA antibody or P238D isotype control. Mice were weighed regularly and weight is plotted relative to starting weight (n=9 mice per group, data points represent mean+/−SD).
EXAMPLES
[0425] In the examples that follow it is shown that antibodies such as 11.5.1 and 2.8.6 bind to human BTLA with high affinity. Using transgenic mice expressing the human receptor it is shown that, following binding to BTLA, these antibodies inhibit T cell responses in vitro and in vivo and are able to ameliorate disease in murine models of inflammatory bowel disease and graft-versus-host disease. Whilst these agonist effects are dependent on Fc-receptor binding, the antibodies do not cause depletion of BTLA expressing cells via cytotoxicity and do not induce receptor down-modulation. Introduction of the P238D modification in the heavy chain greatly enhances the agonist signaling of FcγR2B and increases the ratio of signaling of FcγR2B over FcγR2A. Such dual BTLA and FcγR2B agonist antibodies are expected to be of therapeutic utility, particularly in autoimmune and inflammatory disease settings.
Example 1. Generation and Sequencing of Anti-BTLA Antibodies
[0426] Antibodies recognizing the human immune cell receptor BTLA were generated by BioGenes GmbH via immunizing mice with the extracellular region of human BTLA (BTLA.sup.K31-R151). Splenocytes from immunized mice were fused with Sp2/0-Ag14 myeloma cells and resulting hybridomas selected for reactivity with human BTLA by ELISA of supernatants, in conjunction with dilution cloning. Antibodies were isotyped from hybridoma supernatant using a Rapid Mouse Isotyping Kit (RayBiotech). The antibodies produced by clones 2.8.6 and 11.5.1 were both found to be IgG1k.
[0427] To sequence the immunoglobulin variable domains, RNA was extracted from hybridomas using TRIzol Reagent (ThermoFisher) as per the manufacturer's instructions. RNA was reverse transcribed to produce cDNA using primers specific for the first constant domain of the heavy chain or for the constant domain of the light chain, and Super Script II Reverse Transcriptase (Invitrogen) as per manufacturer's instructions.
[0428] PCR was then performed using primers targeting conserved regions of the immunoglobulin locus as previously described (Tiller et al., J Immunol Methods. 350:183-193, 2009) and PCR products were sequenced. In some cases, identification of functional light chain was complicated by abundant non-functional kappa light chain cDNA from the fusion myeloma cell line, and to resolve this a previously described technique was employed, adding excess primer specific for the non-functional chain CDR3 to force truncation of the aberrant chain product (Yuan et al. J Immunol Methods. 294:39553-61, 2005).
[0429] Variable domain sequences were assessed using the NCBI IgBlast tool to determine the location of the CDRs.
Example 2. Binding to Soluble Human and Cynomolgus BTLA
[0430] The binding affinity and kinetics of the BTLA agonist antibodies of the present invention (2.8.6 and 11.5.1) to human or cynomolgus BTLA were determined by surface plasmon resonance using the Biacore T200 (GE Healthcare). Mouse antibody capture kit (GE Healthcare) was used to coat a Series S CMS Sensor Chip (GE Healthcare) with polyclonal anti-mouse IgG. Anti-BTLA antibody was then captured onto the biosensor surface and a negative control antibody (clone Mopc21; Biolegend) captured in the reference channel. Various concentrations of monomeric soluble human BTLA extracellular domain (BTLA.sup.K31-R151) (from SEQ ID NO: 225) or soluble cynomolgus macaque BTLA extracellular domain (BTLA.sup.K31-R151) (from SEQ ID NO: 226) were then injected over the immobilized antibodies in the buffer 10 mM Hepes, 150 mM NaCl, 0.005% v/v Surfactant P20, pH 7.4 (HBS-P) at 37° C., in a single cycle kinetics analysis (
Example 3. Binding to BTLA on Cells
[0431] The ability of the BTLA agonist antibodies of the present invention (2.8.6 and 11.5.1) to bind to human or cynomolgus BTLA expressed on the cell surface was assessed by flow cytometry. A lentiviral transfection system was used to express full length human or cynomolgus BTLA in a Jurkat T cell line. 1×10.sup.5 cells per well were plated in 96 well U-bottom plates. BTLA antibody binding versus mIgG1 isotype control (clone MOPC-21, Biolegend #400165) was assessed at twelve concentrations by 1 in 3 serial dilution in FACS buffer (PBS, 2% FCS, 0.05% sodium azide), starting at a concentration of 90 μg/ml. Non-specific antibody binding was prevented by addition of Fc block (Biolegend #101319). Antibodies were incubated with cells for 30 minutes on ice, then cells were washed twice with FACS buffer prior to staining with an AF647 conjugated anti-mIgG1 secondary antibody (Biolegend #406618). Secondary antibody was incubated for 30 minutes on ice, then cells were washed and resuspended in FACS buffer for analysis on a flow cytometer. The geometric mean fluorescent intensity of secondary antibody was plotted for each concentration and the EC50 for receptor binding calculated by non-linear curve fitting using GraphPad Prism software. Clone 11.5.1 bound to human BTLA expressing cells with an EC50 of 0.016 nM and cynomolgus BTLA expressing cells with an EC50 of 0.0057 nM. Clone 2.8.6 bound to human BTLA expressing cells with an EC50 of 0.085 nM and cynomolgus BTLA expressing cells with an EC50 of 0.16 nM (
Example 4. Competition with the Natural Ligand HVEM for Binding to BTLA
[0432] The ability of the BTLA agonist antibodies of the present invention (2.8.6 and 11.5.1) to block natural ligand binding to BTLA was assessed by surface plasmon resonance using the Biacore T200 (GE Healthcare). Human BTLA extracellular domain (BTLA.sup.31K-151R) was covalently coupled to a CMS Sensor chip using amine coupling. Human HVEM extracellular domain, fused to mouse IgG1 Fc, was then injected over the immobilized hBTLA in HBS-P buffer at 37° C., and allowed to fully dissociate. A saturating amount of anti-BTLA antibody (2.8.6 or 11.5.1) was then injected, followed immediately by a second injection of human HVEM-mFc at the same concentration as the initial injection (
Example 5. Binding Epitope of Antibody 11.5.1 on Human BTLA
[0433] The functional epitope of the antibody 11.5.1 on human BTLA was determined by flow cytometry assessment of binding to a panel of single residue mutants of the receptor expressed on the cell surface. Constructs encoding the human extracellular region of BTLA with the transmembrane and intracellular regions of murine CD28 were cloned into the bi-cistronic mammalian expression vector pGFP2-n2 (BioSignal Packard Ltd), which also encodes GFP. Mutant constructs varying by one amino acid were prepared using the “drastic” mutagenesis approach (Davis et al. Proc Natl Acad Sci USA. 95, 5490-4 (1998)). Plasmids (2 μg/well) were transfected into HEK-293T cells in 6 well plates using Genejuice transfection reagent (Novagen; 6 μl/well). Mock and no-transfection controls were included with each experiment. Cells were harvested at 48 hours and stained with fluorochrome-conjugated anti-BTLA antibody at 10 μg/ml, alongside a Live/Dead marker, in PBS, 0.05% azide, 2% FCS (FACS buffer) for 1 h at 4° C. Cells were washed, pelleted and resuspended in 200 μl FACS buffer before being analysed on a BD FACSCanto flow cytometer. GFP-positive (transfected) viable cells were gated and analysed for binding of anti-BTLA antibodies (an example of the binding analysis for clone 11.5.1 is shown in
Example 6. Crystal Structure of the Fab′ Fragment of 2.8.6 in Complex with Human BTLA
[0434] The structural epitope of antibody 2.8.6 on human BTLA was determined by solving the crystal structure of antibody Fab in complex with human BTLA extracellular domain. The heavy and light variable domains of antibody 2.8.6 were cloned into the pOPINVH and pOPINVL expression vectors (Addgene), which encode the first constant domain of the mouse IgG1 heavy chain (with a 6×Histidine tag) and the constant domain of the mouse Ig kappa chain, respectively. These vectors were transiently co-transfected into HEK293T cells to produce the Fab′ fragment of anti-BTLA 2.8.6, which was purified by Ni-NTA purification. Human BTLA Ig-V set domain (BTLA.sup.S33-D135) was cloned into the pGMT7 vector and expressed in BL21(DE3)pLysS E. coli cells (Novagen) to produce inclusion bodies. The inclusion bodies were isolated from the cell pellet by sonication and washed repeatedly with a wash solution containing 0.5% Triton X-100. The purified BTLA inclusion bodies were solubilized in a denaturant solution containing 6 M guanidine hydrochloride. The solubilized protein solution was diluted slowly in refolding buffer [0.1 M Tris-HCl (pH 8.0), 0.6 M L-arginine, 2 mM ethylenediaminetetraacetic acid, 3.73 mM cystamine, and 6.73 mM cysteamine] to a final protein concentration of 1-2 μM and then stirred for 48 h at 4° C. The refolded mixture of BTLA was then concentrated with a VIVA FLOW50 system (Sartorius). BTLA was purified by gel filtration on a Superdex 75 column (GE Healthcare).
[0435] The purified BTLA and Fab′ were mixed and purified as a complex by size exclusion chromatography. The crystal suitable for data collection was obtained in 0.2 M calcium acetate, 0.1 M imidazole pH 8.0, 10% (w/v) PEG 8000 at 293° K by the hanging drop vapor-diffusion method. The final dataset was collected at the Photon Factory, and the structure was determined by molecular replacement using the structure of BTLA (PDB ID; 2AW2 chain A) and anti-PD1-Fab (PDB ID: SGGS chain C, D) as search probes.
[0436] The residues on BTLA at the interface with antibody 2.8.6 are A50, G51, D52, P53, E83, D84, R85, Q86, E103, P104, V105, L106, P107, N108, D135.
Example 7. Development of Humanised BTLA Mice
[0437] To provide a platform to assess anti human-BTLA antibodies in mouse models, a knock-in strain of C57Bl/6 mice was developed expressing a chimeric form of BTLA with the human extracellular region and the murine transmembrane and signaling regions. A section of human genomic DNA from the beginning of exon 2 to the end of exon 3 was inserted into the mouse locus replacing the mouse sequence from the beginning of exon 2 to the end of exon 4. The sequences at the exon-intron junction at the beginning of mouse exon 2 and end of mouse exon 4 were left intact to ensure proper splicing (
Example 8. Inhibition of Antigen-Specific T Cell Proliferation In Vivo
[0438] The ability of the BTLA agonist antibodies of the present invention (2.8.6 and 11.5.1) to inhibit antigen specific T cell proliferation in vivo was assessed using a sensitive T-cell transfer assay (
Example 9. Inhibition of T Cell Proliferation in a Mixed Lymphocyte Reaction
[0439] The ability of the BTLA agonist antibodies of the present invention (2.8.6 and 11.5.1) to inhibit proliferation of primary T cells from the humanised mice in vitro was assessed using a mixed lymphocyte reaction (MLR). Splenocytes from Balb/c mice were treated with Mitomycin C for 30 mins at 37° C. then washed and used as stimulator cells. T cells were purified from the spleens of humanised BTLA mice, by negative selection using magnetic-activated cell sorting (Mojosort Mouse CD3 T cell isolation kit, Biolegend #480023), and stained with CellTrace Violet Cell Proliferation Kit (ThermoFisher) to use as responder cells. 4×10.sup.5 stimulator cells and 2×10.sup.5 responder cells per well were mixed in 96-well U-bottom plates with various concentrations of anti-BTLA or isotype control antibody (clone MOPC-21, Biolegend #400165). Serial 1 in 3 dilutions of antibody were assessed starting at a concentration of 1 μg/ml for a total of 10 concentrations. Polyclonal anti-mHVEM antibody (R&D systems #AF2516) was also added to all wells at 1 μg/ml to block any baseline signaling through the BTLA pathway and accentuate the effects of agonist antibodies. After 96 hours, dilution of CellTrace Violet in responder cells was assessed by flow cytometry as a marker of proliferation. Proliferation in the presence of anti-BTLA antibody or isotype control was compared to proliferation in the absence of antibody. CD4.sup.+ and CD8.sup.+ populations were gated out and analysed separately. Both antibodies 2.8.6 and 11.5.1 reduced proliferation of human-BTLA expressing T cells, indicating that they induce inhibitory signaling through the human BTLA receptor. Clone 2.8.6 inhibited CD4 T cells with an IC50 of 0.029 nM and had a maximal effect of 42% inhibition of proliferation (
Example 10. Inhibition of NFkB Signalling in Human BTLA or Cynomolgus BTLA Transfected Jurkat T Cell Lines
[0440] The ability of the BTLA agonist antibodies of the present invention (2.8.6 and 11.5.1) to inhibit NFkB signalling was assessed using a BTLA transfected reporter T cell line. A Jurkat T cell line stably transfected with an expression cassette that includes NF-κB-responsive transcriptional elements upstream of a minimal CMV promoter (mCMV)-GFP cassette (Source BioSciences #TR850A-1) was used as a reporter cell line for NFkB signalling. A lentiviral transfection system was used to express full length human or cynomolgus BTLA in this reporter cell line. These cells were mixed with a stimulator cell line comprised of bw5147 cells expressing an anti-CD3 ScFv construct on their surface as described by Leitner et al. J Immunol Methods. 2010 Oct. 31; 362(1-2):131-41. The stimulator cell line was also transfected with murine FcγR2B to provide Fc receptors for presentation of the agonist BTLA antibodies. 5×10.sup.4 reporter cells per well were mixed in 96 well U-bottom plates with 5×10.sup.4 stimulator cells in the presence of various concentrations of BTLA antibody or isotype control (clone MOPC-21, Biolegend #400165). After 24 hours incubation at 37° C., cells were pelleted and stained for flow cytometry with a viability dye (Zombie Aqua, Biolegend #423101) and a mouse CD45 antibody (Pe-Cy7 conjugated clone 104, Biolegend #109830) to separate stimulator (murine) from responder (human) cells. Geometric mean of GFP expression was assessed for each antibody concentration and normalized to GFP expression in the absence of antibody. Clone 2.8.6 inhibited human BTLA transfected cells with an IC50 of 0.06 nM and cynomolgus BTLA transfected cells with an IC50 of 0.22 nM. Clone 11.5.1 inhibited human BTLA transfected cells with an IC50 of 0.033 nM and cynomolgus BTLA transfected cells with an IC50 of 0.14 nM.
Example 11. Treatment of a T Cell Driven Mouse Model of Colitis by Antibody 2.8.6
[0441] The ability of the BTLA agonist antibody 2.8.6 to ameliorate a T cell driven model of colitis was assessed using the humanised mice. This T cell transfer model has previously been described as a murine model of inflammatory bowel disease (Ostanin et al., Am J Physiol Gastrointest Liver Physiol. 296:G135-46, 2009). CD45RB.sup.hiCD25-CD4+ T cells sorted from spleens and lymph nodes of humanised BTLA mice were injected intraperitoneally into Rag1 KO recipients, (Rag1.sup.tm1Mom; The Jackson Laboratory), at a dose of 5×10.sup.5 cells per mouse. The transferred T cells cause an inflammatory colitis that develops after approximately 3 weeks and leads to diarrhea and weight loss. Rag1 KO cagemates that did not receive transferred T cells serve as non-diseased controls. On days 7, 21 and 35 after T cell transfer the recipient mice were injected intraperitoneally with 200 μg of 2.8.6 or isotype control antibody. All mice were weighed regularly, and at 8 weeks colons were weighed and measured and inflammatory infiltration assessed by histology, as well as by cell counting and flow cytometry of extracted lamina propria leucocytes. Antibody 2.8.6 prevented weight loss (
Example 12. Treatment of a Mouse Model of Graft-Versus-Host Disease (GVHD)
[0442] The effects of the anti-BTLA agonist antibodies were assessed in a non-lethal parent-into-F1 model of GVHD. Bone marrow cells (BMCs) and splenocytes were harvested from humanised BTLA donor mice (C57BL/6 background; H2B). 2×10.sup.7 BMCs and 107 splenocytes were injected intravenously into CB6F1 (H2B.sup./d) recipients that had been lethally irradiated with 9 Gy total body irradiation. Irradiated CB6F1 mice reconstituted with syngeneic BMCs and splenocytes served as non-diseased controls. On the day of immune cell transfer mice were injected intraperitoneally with 200 μg anti-BTLA antibody or isotype control. Mice were weighed regularly and GVHD was monitored by calculating relative loss of body weight and by clinical observation. Mice were culled 5 weeks after immune cell transfer or when they reached a humane endpoint (which included >20% weight loss relative to starting weight in the first 14 days, or >15% weight loss at any other time). At the time of death colons were weighed and measured and a colon weight:length ratio calculated as a marker of colon inflammation, which is a prominent clinical feature of GVHD. Both antibodies 2.8.6 and 11.5.1 significantly reduced weight loss, leading to increased survival (
Example 13. Agonist Activity of Antibody 11.5.1 is Dependent on Fc Receptor Binding
[0443] Antibody 11.5.1 was recombinantly expressed as a mIgG1k containing a D265A mutation which has previously been described as significantly reducing Fc receptor binding (Clynes et al., Nat Med. 6:443-446, 2000). This mutated antibody was assessed in the T cell transfer assay described in Example 8. The parental 11.5.1 antibody inhibited proliferation of humanised T cells as its net effect is agonism of the BTLA receptor. The FcR-null D265A mutation, however, led to enhanced proliferation of humanised T cells suggesting that the FcR-null mutation removes the antibody's agonistic effect, leaving only the effect of receptor blockade (
[0444] The D265A mutated 11.5.1 antibody was also assessed in the in vitro MLR assay described in Example 9. Again, the parental 11.5.1 antibody inhibited proliferation of humanised T cells as its net effect is agonism of the BTLA receptor. The FcR-null D265A mutation removes the antibody's agonistic effect, so this antibody showed no effect in this assay (
Example 14. Antibodies 2.8.6 and 11.5.1 do not Fix Complement In Vitro
[0445] Splenocytes from humanised mice were incubated with 10% baby rabbit complement (BioRad) and anti-BTLA antibodies (or an isotype control or a positive control depleting anti-CD20 antibody; clone SA271G2 from Biolegend) at 20 μg/ml for 15 min at 37° C. Whilst anti-CD20 antibody depleted the majority of B220.sup.+ B cells, anti-BTLA antibodies did not deplete either B220.sup.+ or CD4.sup.+ cells (
Example 15. Antibodies 2.8.6 and 11.5.1 do not Induce ADCC In Vitro
[0446] Whole splenocytes (including myeloid effector cells) from humanised mice were incubated with anti-BTLA antibodies (or isotype control or depleting anti-CD20 antibody SA271G2) at 20 μg/ml for 24 hours at 37° C. Whilst anti-CD20 antibody depleted the majority of B220.sup.+ cells, anti-BTLA antibodies did not deplete either B220.sup.+ or CD4.sup.+ cells (
Example 16. Antibodies 2.8.6 and 11.5.1 do not Deplete BTLA Expressing Cells In Vivo
[0447] Humanised BTLA mice were injected intraperitoneally with 200 μg anti-BTLA antibody or isotype control. At 24 hours spleens were harvested and the frequency of different cell populations identified by flow cytometry. Anti-BTLA antibody had no effect on the frequency or absolute number of B or T cells in the spleen or on the number of B cell precursors in the bone marrow (
Example 17. Antibodies 2.8.6 and 11.5.1 Stabilize Expression of BTLA on Immune Cells In Vivo
[0448] Humanised mice were injected intraperitoneally with 10 mg/kg of antibody 2.8.6 or 11.5.1. Six days after injection mice were humanely sacrificed and spleens harvested and processed to single cell suspension for assessment by flow cytometry. Cells were stained with a cocktail of antibodies to identify immune cell subsets and with fluorescently conjugated anti-BTLA antibody that had a non-competing epitope with the antibody that had been injected. The geometric mean of BTLA staining following in vivo incubation with anti-BTLA antibody was normalized to the geometric mean of BTLA staining (using the same staining antibody) following incubation with isotype control. BTLA expression was significantly higher on B cells and CD4 T cells from mice that had been injected with either clone 2.8.6 or 11.5.1, compared to mice that had been injected with isotype control (
Example 18. Tolerability and Side Effects in Animal Models
[0449] There were no tolerability issues or side effects noted in any animal studies with antibodies 2.8.6 or 11.5.1.
Example 19. Characterisation of Exemplary BTLA Antibodies
[0450] Described in this example is characterisation of exemplary mIgG1 BTLA antibodies provided herein in addition to 2.8.6 and 11.5.1. Various clones listed in Tables 1 and 2 were evaluated for their binding affinity to BTLA and inhibition efficiency of lymphocytes (Table 3).
TABLE-US-00003 TABLE 1 Exemplary BTLA Agonistic Antibodies SEQ ID NOs Clone Scheme CDR H1 CDR H2 CDR H3 CDR L1 CDR L2 CDR L3 VH VL 10B1 Kabat 45 46 47 33 34 35 51 52 12F11 Kabat 53 54 55 56 57 58 59 60 14D4 Kabat 61 62 63 64 65 66 67 68 15B6 Kabat 61 69 70 71 72 73 74 75 15C6 Kabat 76 77 78 79 80 81 82 83 16E1 Kabat 45 46 84 33 34 85 86 87 16F10 Kabat 88 89 90 91 65 92 93 94 16H2 Kabat 95 96 97 98 99 100 101 102 1H6 Kabat 103 104 105 106 107 108 109 110 21C7 Kabat 76 111 112 113 114 115 116 117 24H7 Kabat 118 119 120 121 122 123 124 125 26B1 Kabat 126 127 128 79 129 130 131 132 26F3 Kabat 133 134 135 106 107 136 137 138 27G9 Kabat 103 134 139 106 107 136 141 138 3A9 Kabat 143 144 145 146 147 148 149 142 4B1 Kabat 151 152 153 154 155 156 157 158 4D3 Kabat 159 160 161 4 12 164 165 166 4D5 Kabat 167 168 169 170 171 172 173 174 4E8 Kabat 45 46 47 170 171 172 175 174 4H4 Kabat 45 46 177 154 155 178 179 180 6G8 Kabat 181 182 183 184 185 186 187 188 7A1 Kabat 76 77 78 79 80 189 82 190 8B4 Kabat 45 191 192 154 155 193 194 195 8C4 Kabat 196 197 198 199 200 201 202 203 11.5.1 Kabat 204 205 206 207 208 209 210 211 831 Kabat 212 213 214 215 34 216 217 218 6.2 Kabat 1 2 3 4 5 6 219 220 2.8.6 Kabat 20 163 22 23 176 25 221 222 3E8 Kabat 30 48 32 33 34 35 223 150
TABLE-US-00004 TABLE 2 Humanised and engineered antibodies SEQ ID Nos. Heavy Light Clone CDR H1 CDR H2 CDR H3 CDR L1 CDR L2 CDR L3 VH VL chain chain humanised 6.2 1 2 3 4 5 6 7 8 9 10 Engineered 1 11 3 4 12 6 13 14 15 16 humanised 6.2 (Variant A) Engineered 1 11 3 4 5 6 13 8 15 10 humanised 6.2 (Variant B) Engineered 1 17 3 4 12 6 18 14 19 16 humanised 6.2 (Variant C) Humanised 20 21 22 23 24 25 26 27 28 29 2.8.6 Humanised 3E8 30 31 32 33 34 35 36 37 38 39 Engineered 30 40 32 33 34 35 41 37 42 39 humanised 3E8 (Variant A) Engineered 30 31 32 33 34 35 36 43 38 44 humanised 3E8 (Variant B)
[0451] For each antibody, the association rate (“on rate”) and dissociation rate (“off rate”) for binding human BTLA, and KD for binding human or cynomolgus BTLA were measured according to the method described in Example 2, fitting curves for injection of BTLA extracellular domain at a single concentration. Inhibition efficiency of individual antibodies on T cells was also evaluated at a single concentration of 10 μg/ml. MLR assay was performed for each individual antibody according to the method as described in Example 9 (two biological repeats as shown in Table 4); anti-CD3 assay was performed according to the method described below (two biological repeats, Table 4); and inhibition of NFkB signalling in human BTLA transfected Jurkat T cell line by each antibody was determined according to the method as described in Example 10 (Table 4). The average inhibition of T cells relative to isotype control in various in vitro stimulation assays for each exemplary antibody was calculated as a mean of the percentage inhibition of all assay results (Table 3 and Table 4).
TABLE-US-00005 TABLE 3 Characterisation of binding affinity and inhibitory effect of exemplary antibodies Human Human Human Cyno Average BTLA BTLA BTLA BTLA inhibitory Ligand On rate Off rate KD KD effect Clone Blocking (1/Ms) (1/s) (nM) (nM) in vitro Epitope 2.8.6 No 6.46E+05 4.23E−04 0.65 7.89 39% 1 24H7 No 2.43E+05 1.60E−04 0.66 — 30% 4 11.5.1 Yes 6.03E+05 4.49E−04 0.75 0.99 30% 2 14D4 Yes 2.54E+05 3.77E−04 1.49 1.83 33% 2 6.2 No 6.30E+05 1.07E−03 1.70 9.71 35% 1 4B1 No 5.77E+05 1.85E−03 3.21 — 29% 4 8B4 No 5.38E+05 4.40E−03 8.17 — 29% 4 16H2 No 3.97E+05 3.27E−03 8.25 160.1 34% 1 1H6 Yes 7.72E+05 6.90E−03 8.94 6.08 31% 2 8C4 Yes 3.63E+05 5.76E−03 15.89 161.48 19% 2 26B1 Yes 3.23E+05 9.70E−03 30.03 167.66 21% 3 7A1 No 4.13E+05 1.66E−02 40.17 — 24% 1 21C7 No 9.30E+05 4.06E−02 43.65 — 18% 5 16F10 No 5.81E+05 2.83E−02 48.78 — — 1 6G8 No 3.18E+05 1.67E−02 52.42 — — 1 3E8 No 5.43E+05 6.08E−02 111.98 607.46 41% 1 4E8 No 1.75E+05 3.14E−02 180.00 — — 1 27G9 Yes 1.92E+05 8.38E−02 436.86 653.63 16% 2 15C6 No 1.93E+05 1.38E−01 718.44 — — 1 12F11 No 2.15E+05 1.55E−01 722.33 — 24% 1 10B1 No 4.22E+05 5.21E−01 1233.36 — 21% 1 15B6 No 4.47E+05 5.76E−01 1287.18 — 14% 1 4D3 No 1.52E+05 2.51E−01 1651.32 — — 1 4H4 No 2.03E+05 3.47E−01 1708.23 — 26% 4 26F3 Yes 9.21E+05 2.02E+00 2195.81 809.75 9% 2 16E1 No 7.30E+05 2.13E+00 2923.69 — 15% 1 4D5 No 2.70E+05 7.90E−01 2929.18 — — 1 3A9 No 4.06E+05 1.63E+00 4006.90 — 19% 1
TABLE-US-00006 TABLE 4 Inhibitory effect assay results of exemplary antibodies T cell MLR AntiCD3/CD28 AntiCD3/CD28 reporter (CD4 T cell proliferation) (CD4 T cell proliferation) (CD69+ CD4 T cells) (NFκB Clone repeat 1 repeat 2 repeat 1 repeat 2 repeat 1 repeat 2 signaling) Average 2.8.6 30% 36% 23% 35% 58% 67% 22% 39% 24H7 23% 31% 13% 23% 52% 44% 22% 30% 6.2 31% 35% 19% 21% 53% 61% 26% 35% 11.5.1 23% 18% 21% 28% 50% 47% 19% 30% 11.5.1 D265A −3% 1% −3% −9% −47% −26% −13% −14% 4B1 33% 30% 14% 18% 47% 41% 23% 29% 14D4 39% 26% 24% 29% 43% 52% 16% 33% 831 25% 34% 10% 8% 50% 53% 24% 29% 16H2 40% 26% 11% 23% 51% 60% 29% 34% 1H6 31% 16% 26% 19% 47% 53% 26% 31% 8B4 33% 23% 20% 4% 51% 47% 24% 29% 21C7 8% 17% 10% −4% 39% 35% 23% 18% 3E8 43% 35% 27% 35% 52% 64% 30% 41% 7A1 23% 29% 14% 17% 28% 38% 20% 24% 26B1 12% 10% 11% 19% 35% 30% 29% 21% 8C4 42% −2% 12% 4% 29% 29% 21% 19% 27G9 9% 8% 10% 13% 24% 22% 24% 16% 12F11 28% 23% 5% 9% 30% 40% 30% 24% 15C6 19% 8% 2% −2% 12% 19% 9% 10% 26F3 9% −5% 4% 0% 19% 17% 20% 9% 4D3 12% 9% −4% −2% 6% 2% 26% 7% 10B1 16% 25% 8% 14% 24% 36% 27% 21% 16E1 33% 8% 4% 8% 9% 23% 22% 15% 15B6 7% 13% 9% 16% 13% 20% 21% 14% 3A9 7% 24% 9% 9% 22% 34% 27% 19% 4H4 10% 17% 14% 22% 43% 52% 25% 26% No antibody 3% −3% 1% −6% 2% −9% 2% −1%
[0452] The ability of the BTLA agonist antibodies to inhibit anti-CD3 and anti-CD28 induced T cell activation was assessed as follows. Splenocytes from humanised BTLA mice were processed to single cell suspension and treated with ACK buffer to lyse red blood cells. Cells were stained with CFSE (Biolegend Cat #423801) to enable tracking of cell proliferation. 2×10.sup.5 cells per well were plated in 96 well U-bottom plates with soluble anti-CD3 antibody (clone 145.2C11; Biolegend #100339) and anti-CD28 (clone 37.51; Biolegend #102115) each at a concentration of 50 ng/ml, and soluble anti-BTLA antibody or isotype control at a concentration of 10 μg/ml. After 72 hours cells were analysed by flow cytometry to assess proliferation (“antiCD3/CD28 (CD4 T cell proliferation)”) and T cell activation by staining of surface expressed activation markers (“antiCD3/CD28 (CD69+CD4 T cells)”). For each BTLA antibody the percentage inhibition compared to isotype control antibody was calculated.
[0453] Further, for each BTLA antibody, their ligand blocking capability, e.g., competition with HVEM for binding to BTLA, was assessed according to the method as described in Example 4, and the results are presented as “Yes” for more than 90% inhibition of HVEM-BTLA binding, and “No” for less than 10% inhibition of HVEM-BTLA binding. Functional epitope of each BTLA antibody was also determined according to the method as described in Example 5. The “epitope” column in Table 3 summarizes the epitope group that each individual BTLA antibody binds to. Antibodies 2.8.6, 6.2, 831, 16H2, 7A1, 16F10, 6G8, 3E8, 4E8, 15C6, 12F11, 10B1, 15B6, 4D3, 16E1, 4D5 and 3A9 all bind to a first epitope (named “epitope 1” in the table) comprising at least one critical residue selected from the list: D52, P53, E55, E57, E83, Q86, E103, L106 and E92 (position according to SEQ ID NO:225). Antibodies binding to epitope 1 do not compete with the ligand HVEM for binding to BTLA. Antibodies 11.5.1, 14D4, 1H6, 8C4, 27G9, 26F3 all bind to a different second epitope (“epitope 2”) comprising at least one critical residue selected from the list: Y39, K41, R42, Q43, E45 and S47. Antibodies binding to epitope 2 do compete with the ligand HVEM for binding to BTLA. Antibody 26B1 binds to a third epitope (“epitope 3”) comprising at least one critical residue selected from the list: D35, T78, K81, S121 and L123. Antibodies binding to epitope 3 do compete with the ligand HVEM for binding to BTLA.
[0454] Antibodies 24H7, 4B1, 8B4, 4H4 all bind to a different fourth epitope (“epitope 4”) comprising the critical residue H68. Antibodies binding to epitope 4 do not compete with the ligand HVEM for binding to BTLA. Antibody 21C7 binds to a different fifth epitope (“epitope 5”) comprising at least one critical residue selected from the list: N65 and A64. Antibodies binding to epitope 5 do not compete with the ligand HVEM for binding to BTLA.
Example 20. Humanisation and CDR Engineering of BTLA Antibodies 6.2, 2.8.2 and 3E8
[0455] Antibody 2.8.6 was humanised by CDR grafting on to homologous human germline framework regions (See SEQ ID NO: 26, 27). IGHV2-5*08 was used for the heavy chain and IGKV3-11*01 for the light chain. After humanisation, binding to BTLA was assessed by SPR. Humanised 2.8.6 bound to monomeric BTLA with a K.sub.D of 0.73 nM.
[0456] The variable domains of 6.2 and 3E8 were humanised by germlining to homologous human germline framework regions (Seq ID No. 7, 8 and 36, 37). For 3E8 the acceptor frameworks selected were VH1-1-08 and JH6 for the heavy chain and VK3-L6 and JK2 for the light chain. For 6.2 the acceptor frameworks selected were VH3-3-21 and JH6 for the heavy chain and VK2-A19 and JK4 for the light chain.
[0457] It is sometimes possible to substitute certain residues in the CDRs or variable domain framework regions of an antibody to remove undesirable characteristics without significantly impacting target binding. The CDRH2 of the humanised antibody 6.2 was modified with N56Q alone (SEQ ID NO: 17) or N56Q and D54E substitutions (Seq ID NO: 11) to remove deamidation potential and isomerisation potential respectively. The CDRL2 of humanized 6.2 was modified with a D61E substitution to reduce predicted immunogenicity as determined by Lonza's Epibase analysis (Seq ID NO: 12). Outside of the CDRs, an S77T substitution was introduced into the heavy variable framework region of humanized 6.2 to reduce predicted immunogenicity and a Q51K substitution was introduced into the light variable framework region to reduce immunogenicity. Three engineered variants of humanized 6.2 containing different combinations of these substitutions were created (Engineered humanized 6.2 “Variant A”, “Variant B” and “Variant C”). Table 2 describes the constituent CDRs and variable domains for each of these variants. An engineered variant of antibody 6.2 containing a CDRH2 with just the N56Q and not the D54E substitution (e.g. engineered humanised 6.2 variant C) is not disclosed in PCT/GB2019/053569.
[0458] Similarly, the CDRH2 of the humanised antibody 3E8 was modified with an N57Q substitution to remove deamidation potential and a K63S substitution to reduce predicted immunogenicity (Seq ID No. 40). Outside of the CDRs, G42D and A61S substitutions were introduced into the light chain variable framework of 3E8, to reduce predicted immunogenicity. Furthermore, P15L and P81A substitutions were introduced into the light chain variable framework to revert these positions to the murine sequence instead of introducing prolines that can have an impact on the local conformation. The sequence of the engineered 3E8 light chain variable domain contain all four of these substitutions is given in Seq ID No. 43. Table 2 describes the constituent CDRs and variable domains for engineered variants of humanized 3E8.
Example 21. Binding of Humanised Anti-BTLA Antibodies to Soluble Human and Cynomolgus BTLA
[0459] The binding affinity and kinetics of humanised BTLA agonist antibodies to human or cynomolgus BTLA were determined by surface plasmon resonance using the Biacore 8K (GE Healthcare). Human antibody capture kit (GE Healthcare cat #29234600) was used to coat a Series S CMS Sensor Chip (GE Healthcare) with polyclonal anti-human IgG. Anti-BTLA antibody was then captured onto the biosensor surface and a negative control antibody (human IgG1k isotype control; Sino Biological cat #HG1K) captured in the reference channel. Various concentrations of monomeric soluble human BTLA extracellular domain (BTLA.sup.K31-R151, produced recombinantly in house) or soluble cynomolgus macaque BTLA extracellular domain (BTLA.sup.K31-R151, produced recombinantly in house) were then injected over the immobilized antibodies in the buffer HBS-EP (GE Healthcare, cat #BR100669), pH 7.4 (HBS-P) at 37° C., in a single cycle kinetics analysis. For human BTLA concentrations from 673 nM to 164 pM in serial four-fold dilutions were used. For cyno BTLA concentration from 1351 nM to 330 pM in serial four-fold dilutions were used. Association and dissociation rates were fitted using BiaEvaluation Software (GE Healthcare) after reference and blank subtractions, and dissociation constants were calculated (Table 5). Humanised 2.8.6 binds human BTLA with a KD of 2.33 nM and cynomolgus BTLA with a KD of 147 nM. Humanised 3E8 variant B (3E8_var_B) binds human BTLA with a KD of 141 nM and cynomolgus BTLA with a KD of 1520 nM. The humanised 6.2 variant A, which contains both D54E and N56Q substitutions in its CDRH2 to remove isomerisation and deamidation potential respectively, binds to human BTLA with a KD of 10.9 nM and cynomolgus BTLA with a KD of 695 nM. This binding represents a significant reduction in affinity from the parent clone 6.2 antibody, which binds to human BTLA with a KD of 1.7 nM, and cynomolgus BTLA with a KD of 9.71 nM (Table 5). A humanised variant of 6.2 that contains just the N56Q substitution but not the D54E substitution in CDRH2, termed Humanised 6.2 variant C (or 6.2_var_C), binds human BTLA with a KD of 1.25 nM and cynomolgus BTLA with a KD of 15.4 nM therefore retaining affinity much closer to the parent clone.
TABLE-US-00007 TABLE 5 Binding kinetics and affinity for antibodies binding to soluble human or cynomolgus BTLA, as determined by surface plasmon resonance at 37° C. Human BTLA Cyno BTLA Antibody ka (1/Ms) kd (1/s) KD (M) ka (1/Ms) kd (1/s) KD (M) Humanised 6.73 × 10.sup.5 1.57 × 10.sup.−3 2.33 × 10.sup.−9 1.7 × 10.sup.5 2.5 × 10.sup.−2 1.47 × 10.sup.−7 2.8.6 Humanised 6.94 × 10.sup.5 7.56 × 10.sup.−3 1.09 × 10.sup.−8 7.29 × 10.sup.4 5.07 × 10.sup.−2 6.95 × 10.sup.−7 6.2_var_A Humanised 1.06 × 10.sup.6 1.33 × 10.sup.−3 1.25 × 10.sup.−9 2.29 × 10.sup.5 3.52 × 10.sup.−3 1.54 × 10.sup.−8 6.2_var_C Humanised 8.65 × 10.sup.5 1.22 × 10.sup.−1 1.41 × 10.sup.−7 2.87 × 10.sup.5 4.31 × 10.sup.−1 1.52 × 10.sup.−6 3E8_var_B
Example 22. Binding of Humanised Anti-BTLA Antibodies to BTLA on Cells
[0460] The ability of the BTLA agonist antibodies of the present invention to bind to human or cynomolgus BTLA expressed on the cell surface was assessed by flow cytometry. A lentiviral transfection system was used to express full length human or cynomolgus BTLA in a Jurkat T cell line. 1×10.sup.5 cells per well were plated in 96 well U-bottom plates. BTLA antibody binding versus hIgG1k P238D isotype control (clone MOPC-21, produced recombinantly by Absolute Antibody; Heavy chain SEQ ID NO: 230, light chain SEQ ID NO: 231) was assessed at twelve concentrations by 1 in 3 serial dilution in FACS buffer (PBS, 2% FCS, 0.05% sodium azide), starting at a concentration of 30 μg/ml. Non-specific antibody binding was prevented by addition of Fc block (Biolegend #101319). Antibodies were incubated with cells for 60 minutes on ice, then cells were washed twice with FACS buffer prior to staining with an AF647 conjugated anti-hIgG secondary antibody (Clone HP6017; BioLegend cat #409320). Secondary antibody was incubated for 30 minutes on ice, then cells were washed and resuspended in FACS buffer for analysis on a flow cytometer. The geometric mean fluorescent intensity of secondary antibody was plotted for each concentration and the EC50 for receptor binding calculated by non-linear curve fitting using GraphPad Prism software. Humanised 2.8.6 binds to human BTLA expressing cells with an EC50 of 0.066 nM (
Example 23. Binding Affinities of Fc Variant Antibodies to Human Fc Receptors
[0461] In Example 13 it was demonstrated surprisingly that the agonist function of BTLA antibodies may be dependent on Fc receptor engagement by the Fc portion of the antibody. In humans there is one inhibitory Fc gamma receptor (FcγR2B) whilst the other Fc gamma receptors all deliver immune activating signals (FcγR1A, FcγR2A, FcγR3A and FcγR3B). For a BTLA agonist antibody to be effective at suppressing immune responses without eliciting inflammatory FcR signalling we propose it might require selective Fc binding to FcγR2B. Furthermore, selective binding to FcγR2B would promote bidirectional inhibitory signalling through BTLA on the BTLA expressing cell and through FcγR2B on the FcγR2B expression cell, which would strengthen the immunosuppressive effect of the antibody. This would be desirable in a therapeutic antibody intended for the treatment of diseases of immune overactivation. Conversely, very high affinity for FcγR2B can adversely impact antibody half-life due to turnover of the receptor in liver sinusoidal epithelial cells (Ganesan et al. The Journal of Immunology 189(10): 4981-88, 2012) as demonstrated by the FcγR2B enhanced IgG1 antibody XmAb7195 which binds to FcγR2B with a KD of 7.74 nM (Chu et al. Journal of Allergy and Clinical Immunology 129(4): 1102-15, 2012; https://linkinghub.elsevier.com/retrieve/pii/S0091674911018343 (May 13, 2020) and was reported by Xencor to have an average in vivo half-life of 3.9 days in a phase 1a trial (American Thoracic Society (ATS) 2016 International Conference in San Francisco, Calif.—A6476: Poster Board Number 407), compared to an average half-life of around 21 days for a wildtype IgG1 (Morell, Terry, and Waldmann. Journal of Clinical Investigation 49(4): 673-80, 1970; http://www.jci.org/articles/view/106279 (May 16, 2020)). Therefore, whilst selectivity for FcγR2B and sufficient binding to support agonism might be desirable for a BTLA agonist antibody, excessively high affinity for FcγR2B might be undesirable in a therapeutic as the consequently shortened half-life would likely necessitate more frequent dosing.
[0462] A range of Fc mutated antibody variants were recombinantly produced (containing the variable domains of humanised 2.8.6) and their binding to the different human Fc gamma receptors assessed by surface plasmon resonance (at 37° C. in buffer HBS-EP+ at pH7.4). Fc variants were recombinantly produced on either a hIgG1 or a hIgG4 backbone with substitutions known to impact FcR binding or likely to do so based on their position in the Fc-FcR binding interface (hIgG1 G236D, hIgG1 G237D, hIgG1 P238D, hIgG1 D265A, hIgG1 S267E, hIgG1 P271G, hIgG1 A330R, hIgG1 K322A, hIgG1 N297A, hIgG4 P238D, hIgG4 G237D, hIgG4 P271G, hIgG4 S330R, hIgG4 F234A, hIgG4 L235A). These mutations were assessed as single substitutions or in combinations. Variants containing sections of sequence switched from hIgG2 as described by Armour et al. (Molecular Immunology 40(9): 585-93, 2003) were also assessed (termed delta b, delta c, delta ab and delta ac). The binding of mIgG1 and mIgG1 D265A to human FcRs was also assessed.
[0463] For the low affinity FcγRs (FcγR2A, FcγR2B, FcγR3A and FcγR3B) the interactions were assessed by surface plasmon resonance with the recombinantly expressed FcRs (extracellular domains only) as analyte. Briefly, recombinant human BTLA extracellular domain (BTLA.sup.K31-R151) was covalently immobilised to both flow cells of all channels of a CMS Series S sensor chip using the GE Healthcare Amine coupling kit. The 2.8.6 Fc variant to be assessed was then captured (approx. 500-1000 response units) in flow cell 2 of each channel. Steady state affinity analysis was then performed by injecting varying concentrations of FcR in multiple cycles and measuring equilibrium binding. Double referencing was used (subtracting the signal in the reference Fc1 and also subtracting the signal from a blank zero concentration injection). KDs were calculated from the Langmuir curves (plotting equilibrium binding against analyte concentration to determine the concentration required for half maximal binding).
[0464] For the high affinity FcR interactions (FcγR1A, and also FcRn assessed at pH6.0) the binding was assessed in a kinetic analysis with antibody as analyte. Briefly, biotinylated FcR (Sino Biological, cat #10256-H085-B for FcγR1A or cat #CT009-H08H-B for FcRn) was captured in flow cell 2 on a streptavidin chip (Series S Sensor Chip SA-BR-1005-31) as per the provided protocol. Reference flow cell 1 was left empty in all channels. Purified antibody was then injected at a single concentration and on/off rates calculated by curve fitting on BiaEvaluation software. FcRn interaction at pH6.0 does not cause inflammatory signalling but is required for maintained antibody half-life in vivo and so this interaction is desirable for a therapeutic antibody. IgG Fc has two binding sites for FcRn so this assessment performed with FcRn immobilised at high density provides an avidity estimate for the interaction rather than a true KD.
[0465] The KD values for each of the Fc variants binding to each of the human Fc receptors where they were assessed are provided in Table 6. The presence of the P238D mutation significantly enhanced selectively for FcγR2B (by slightly increasing affinity to FcγR2B whilst drastically reducing affinity to other FcγRs). A previously described combination of mutations including P238D (P238D G237D P271G A330R), termed V9 (Mimoto et al. Protein Engineering, Design and Selection 26(10): 589-98, 2013), significantly increased binding affinity to FcγR2B but also retained significant binding to the 131R polymorphic variant of FcγR2A. The same effect of increasing FcγR2B selectivity was seen when the P238D single or combination substitutions were introduced into a hIgG4 backbone.
TABLE-US-00008 TABLE 6 Binding affinities (KDs) for Fc variants binding to human FcRs as assessed by SPR at 37 C. n/a = not assessed, NB = no binding detected. KD (μM) FcRn pH6 FcγR2A FcγR2A FcγR3A FcγR3A (avidity FcγR1A 131R 131H FcγR2B 158F 158V FcγR3B in nM) hIgG1 0.00375 1.57 1.98 8.65 9.37 2.78 22.9 4.58 hIgG4 0.026 4.08 8.89 6.39 228 89.3 1100 13 hIgG1 P238D 0.465 28.9 76.5 4.78 2480 7010 1580 1.43 hIgG1 P238D 0.697 1.84 26.2 0.173 216 321 6090 1.98 G237D P271G A330R (V9) hIgG4PAA 1.34 21.4 39.1 41 NB 15 NB n/a hIgG4 P238D n/a 47.9 312 17.5 NB NB NB n/a hIgG4 P238D n/a 2.01 30.5 0.574 933 NB NB n/a G237D P271G S330R hIgG1 D265A 0.497 48.3 40.1 193 1490 NB 5200 12 hIgG1 D265A NB 91.4 NB 748 NB NB NB 14 G236D hIgG1 D265A n/a 67 39.4 347 NB NB 9100 n/a A330R hIgG1 D265A 0.415 26.7 173 157 NB NB NB 11.9 S267E hIgG1 D265A NB 61.8 NB 113 NB 1650 NB 18.7 G236D S267E hIgG1 D265A NB 91.8 91.3 398 NB NB NB 14 G236D A330R hIgG1 D265A n/a 22 113 80.1 NB NB NB n/a S267E A330R hIgG1 D265A NB 54.4 NB 104 NB NB NB n/a G236D S267E A330R hIgG1 D265A n/a 348 4590 684 NB NB NB n/a P238D hIgG4 D265A n/a 191 280 1000 NB NB NB n/a hIgG4 D265A n/a 680 NB NB NB NB NB n/a P238D hIgG4 D265A n/a 167 2430 114 NB NB NB n/a G236D S267E A330R hIgG1 delta ab NB 9.56 4.86 82.5 722 494 2820 14.8 hIgG1 delta ab NB — 771 876 NB NB NB n/a P238D hIgG1 delta ac 2.85 79.3 82.2 131 344 96.4 NB 11.9 hIgG1 delta ac NB 399 NB 1170 NB 1410 NB 14.2 P238D hIgG4 delta b n/a 6.2 5.67 46.2 1860 350 3240 12.1 hIgG4 delta b n/a 994 NB NB NB NB NB 17.3 P238D hIgG4 delta c NB 53.5 73.7 64.4 697 127 2290 14.5 hIgG4 delta c NB 502 NB 826 NB 4460 NB 18.7 P238D hIgG1 K322A 0.0052 n/a 4.6 11 5.75 2.18 11.9 n/a hIgG1 N297A 8.7 447 2150 1080 NB NB NB 9.41 mouse IgG1 NB 35.1 648 2510 NB NB NB 18.1 D265A mouse IgG1 NB 0.127 2.3 5.77 273 1860 NB n/a
Example 24. Inhibition of T Cell Activation by Humanised BTLA Agonists in an NFkB Reporter Assay is Dependent on Fc Receptor Binding
[0466] BTLA is an inhibitory receptor expressed on T cells and so agonist antibodies against BTLA might be expected to inhibit T cell activation by inducing inhibitory signalling through the receptor. The ability of selected humanised BTLA agonist antibodies to inhibit T cell activation was assessed using a BTLA transfected reporter T cell line. A Jurkat T cell line stably transfected with an expression cassette that includes NF-κB-responsive transcriptional elements upstream of a minimal CMV promoter (mCMV)-GFP cassette (Source BioSciences #TR850A-1) was used as a reporter cell line for NFkB signalling. A lentiviral transfection system was used to express full length human BTLA in this reporter cell line. These cells were mixed with a stimulator cell line comprised of bw5147 cells expressing an anti-CD3 ScFv construct on their surface as described by Leitner et al. (J Immunol Methods. 362(1-2):131-41, 2010). The stimulator cell line was also transfected with human FcγR2B to provide Fc receptors for presentation of the agonist BTLA antibodies. 5×10.sup.4 reporter cells per well were mixed in 96 well U-bottom plates with 5×10.sup.4 stimulator cells in the presence of various concentrations of BTLA antibody or hIgG1k isotype control antibody (Sino Biologicals cat #HG1K). After 24 hours incubation at 37° C., cells were pelleted and stained for flow cytometry with a viability dye (Zombie Aqua, Biolegend #423101) and a mouse CD45 antibody (Pe-Cy7 conjugated clone 104, Biolegend #109830) to separate stimulator (murine) from responder (human) cells. Geometric mean of GFP expression was assessed for each antibody concentration and normalized to GFP expression in the absence of antibody.
[0467] Humanised 2.8.6 was tested on a hIgG4 isotype, as well as a hIgG1 P238D isotype and a hIgG1 V9 (P238D G237D P271G A330R) isotype. 2.8.6 hIgG1 P238D led to more effective inhibition of NFkB signal than the 2.8.6 hIgG4, and 2.8.6 hIgG1 V9 led to more effective inhibition still (
[0468] Humanised 2.8.6, 6.2_var_C and 3E8_var_B were all produced on a hIgG1 P238D isotype and compared in the T cell reporter assay described above. They were also compared against the prior art BTLA agonist 22B3 (expressed on a hIgG4PAA isotype) as described in WO 2018/213113 and a fusion protein of the natural BTLA ligand HVEM fused to a mIgG1 Fc region (hHVEM-mFc, produced recombinantly in house; hHVEM-mFc fusion protein including signal peptide and C-terminal His-tag has the sequence disclosed in SEQ ID NO: 229). All three of the humanised P238D variant antibodies demonstrated significantly greater inhibition of NFkB signal compared to 22B3 or hHVEM-mFc (
Example 25. Inhibition of Primary Human T Cell Proliferation in a Mixed Lymphocyte Reaction by Humanised BTLA Agonists
[0469] The ability of selected BTLA agonist antibodies to inhibit human T cell proliferation was assessed in the context of a mixed lymphocyte reaction (MLR). Briefly, human primary T cells were isolated from healthy donor peripheral blood mononuclear cells (PBMCs) using human Pan T cell isolation kit (Miltenyi Biotec cat #130-096-535) and stained with a cell proliferation tracking dye, Tag-it Violet (Biolegend cat #425101). Allogeneic monocyte-derived dendritic cells (DC) were generated by culturing CD14+ monocytes isolated from PBMCs using a CD14+ isolation kit (Miltenyi Biotec cat #130-050-201). CD14+ monocytes were treated with human recombinant IL-4 (Peprotech cat #200-04) and GM-CSF (Biolegend cat #572904) for 7 days. DC maturation was then induced by adding human recombinant TNF-α (Biolegend cat #717904) for an additional 2 days. Mature dendritic cells express both activating and inhibitory FcγRs (Guilliams et al. Nature Reviews Immunology 14(2): 94-108, 2014. http://www.nature.com/articles/nri3582 (May 18, 2020)).
[0470] MLR was then performed by co-culturing 1×10.sup.5 total T cells with allogeneic mature DCs at a ratio of 4:1 (T:DC) in flat-bottom 96-well plates. T cells and DCs were incubated for 5 days with no antibody or in the presence of different doses of BTLA agonist antibody (2.8.6 hIgG1 P238D, 2.8.6 hIgG1 V9, 2.8.6 IgG4), a hIgG1k isotype control antibody (Sino Biologicals cat #HG1K), or the prior art BTLA agonist 22B3 hIgG4PAA. After 5 days T cell proliferation was evaluated by flow cytometry. T cells were harvested and stained with anti-CD3 antibody (PerCP/Cy5.5 conjugated clone OKT3, Biolegend cat #317336), anti-CD4 antibody (BB515 conjugated clone RPA-T4, BD Horizon cat #564419), anti-CD8 antibody (BV510 conjugated clone SK1, BD Horizon cat #563919) together with a viability dye (Zombie NIR, Biolegend cat #423105) and acquired on a BD FACSCelesta instrument. CD4 proliferation (measured as the percentage of CTV low cells) in the presence of antibody was normalised to the average proliferation in the absence of antibody.
[0471] The 22B3 hIgG4PAA also had no inhibitory effect in the mixed lymphocyte reaction, and in fact trended towards increasing proliferation of CD4 T cells, which could be explained by the antibody blocking the natural inhibitory signalling through BTLA by interfering with its interaction with the ligand HVEM. The antibodies 6.2, 3E8 and 286 bind to an epitope on BTLA that does not overlap with the HVEM binding interface and so these antibodies do not block the BTLA-HVEM interaction (Example 19).
Example 26. Inhibition of Primary Human B Cell Activation by BTLA Agonists
[0472] The ability of BTLA agonist antibodies to inhibit primary human B cell activation was evaluated. B cells express high levels of both BTLA and FcγR2B.
[0473] Human primary B cells were isolated from healthy donor peripheral blood mononuclear cells using human B cell isolation kit (Miltenyi Biotec cat #130-050-301) and stained with a cell proliferation tracking dye, Tag-it Violet™ (Biolegend cat #425101). 1×10.sup.5 B cells per well of a 96 well flat bottom plate were then stimulated with 0.01 μM of the TLR9 agonist ODN2006 (InvivoGen cat #tlrl-2006-1), in the presence or absence of different doses of isotype control antibody or selected BTLA agonist antibodies. BTLA agonist 2.8.6, 6.2_var_C and 3E8_var_B (all hIgG1 P238D isotype) were tested and compared against the prior art BTLA agonist 22B3 hIgG4PAA. A recombinant HVEM fusion protein (hHVEM-mFc, produced in house) was used as a positive control. After 5 days of incubation at 37° C., B cells were harvested and stained with anti-CD20 antibody (PE-CF594 conjugated clone 2H7, BD Horizon #562295) together with a viability dye (Zombie NIR, Biolegend #423105) to evaluate their proliferation by flow cytometry. In addition, culture supernatant was collected to assess by ELISA the production of IL-6 (rndsystems cat #DY206) and IL-10 (rndsystems cat #DY217B).
[0474] Following procedures essentially as described above, BTLA agonist antibodies were able to inhibit B cell proliferation as efficiently as the hHVEM-mFc positive control. In addition, all three antibody variants demonstrated significantly greater inhibition of B cell proliferation compared to 22B3. Furthermore, the P238D BTLA agonists impaired the production of IL-10 (
Example 27. Treatment of a Xenogeneic Model of Graft-Versus-Host Disease (GVHD)
[0475] Prevention of human PBMC-driven graft vs. host disease (GvHD) was determined in vivo. Briefly, female NSG mice (JAX Labs, Stock #05557), approximately 8-10 weeks old (n=10 mice per treatment group) were irradiated with 2.4Gy total body irradiation. Human peripheral blood mononuclear cells (PBMCs) were isolated from a leukopak (a HemaCare product ordered via Tissue Solutions) and resuspended at 50×10.sup.6 cells per ml of PBS. Mice are injected with 200 μl cell suspension (10×10.sup.6 PBMCs) intravenously (IV) by tail injection 1 day after irradiation. The following day mice are treated with 10 mg/kg of test antibody by intraperitoneal injection. Mice are weighed regularly and euthanised when they have lost 15% body weight or after 28 days. At study termination infiltration of human PBMCs into lung, liver and spleen is quantified by flow cytometry using markers for hCD45, hCD4, hCD8, hCD20, hCD25 and FOXP3.
[0476] Following procedures as described above humanised 2.8.6 hIgG1 P238D, 6.2_var_C hIgG1 P238D and 3E8_var_B hIgG1 P238D all significantly reduced weight loss compared to hIgG1 P238D isotype control (
Example 28. In Vivo Half-Life of P238D Mutated hIgG1 Antibody in Cynomolgus Macaques and Prediction of Human Half Life
[0477] The in vivo half-life of 6.2_var_C on a hIgG1 P238D isotype in cynomolgus macaques was evaluated. 2 female macaques were injected IV with 3 mg/kg of the antibody and 2 female macaques were injected with 10 mg/kg of the antibody. Macaques were bled before antibody injection, and at 1 hour, 6 hours, 24 hours, 48 hours, 72 hours, 168 hours, 240 hours, 336 hours, 432 hours and 504 hours after antibody injection. The concentration of 6.2_var_C in serum at each of these time points was assessed by target capture ELISA. A 96 well microplate (Thermoscientific Cat #439454) was coated overnight at 4° C. with 100 μl of human BTLA extracellular domain at 1 ug/ml in PBS. The plate was then washed 3 times with wash buffer (PBS with 0.05% Tween 20 (ThermoScientific Cat #28320)), and wells were blocked for 1 hour at room temperature with 300 μl SuperBlock buffer (Thermoscientific Cat #37515), followed by again washing 3 times with wash buffer. 100 μl of serum samples diluted in ELISA buffer (PBS, 1% Bovine Serum Albumin, 0.05% Tween 20) were then added and incubated for 1 hour at room temperature. An 11-point standard curve of 6.2_var_C at known concentrations in ELISA buffer was performed in duplicate and duplicate wells containing only ELISA buffer used as blanks. Following incubation, wells were washed 3 times with wash buffer, then HRP-conjugated anti-human detection antibody (Abcam Cat #ab98624) diluted 1 in 20,000 in ELISA buffer was added and incubated for 1 hour at room temperature. Wells were again washed 3 times with wash buffer then 100 μL of Ultra TMB-ELISA Substrate Solution (ThermoScientific Cat #34028) was added per well. Incubated for 90 seconds with a covering of foil to ensure the plate was not in direct light then 50 μL of stop solution (ThermoScientific Cat #SSO4) added per well. Absorbance at 450 nm then read on a Thermo MultiSkan FC. Concentrations interpolated from standard curve using GraphPad Prism software.
[0478] Using the serum antibody concentrations at each time point, pharmacokinetics in each monkey was fitted with a 2-compartment model (Dirks et al. Clin. Pharmacokinet 49(10):633-659, 2010). The average terminal half-life in macaques was calculated as 5.4 days (130 hours). The model parameters (the volumes of distribution V1 and V2, clearance C1 and inter-compartmental exchange coefficient Q) were then scaled to human using allometric scaling. With allometric scaling the parameter.sub.1 for a species with a body weight BW.sub.1 is estimated from the parameter.sub.2 from another species with body weight BW.sub.2 with the equation:
[0479] where β is the scaling coefficient for the given parameter. This approach is well documented and has been shown to provide adequate predictions in human from preclinical species (Dong et al Clin Pharmacokinet, 50(2):131-142, 2011) and (Wang et al. Biopharmaceutics & drug disposition, 31:253-263, 2010).
[0480] For humans, a body weight of 70 kg was assumed. For cynomolgus monkeys, a reference body weight of 3 kg was used. Theoretical scaling exponents for large molecules were used: β=1 for V1 and V2, β3=0.75 for C1 (as described in Kleiber et al. Hilgardia 6(11): 315-333. 1932) and β=2/3 for Q. For the scaling of the inter-compartmental exchange coefficient Q, it was assumed that the rate of exchange of the compound depended on the surface area of the vascular endothelium. This assumption was based on the implementation of the inter-compartmental exchange which is written as:
Q.Math.(c.sub.p−c.sub.t)=P.Math.S(c.sub.p−c.sub.t)
where c.sub.p−c.sub.t is the concentration difference across the vascular boundary, P is the vascular permeability coefficient with units (m/s) and S is the surface area in units (m.sup.2) of the vasculature that is involved in the exchange. It was assumed that the vascular permeability P is a property of the molecule, and that it is independent of the species. The only difference between species is the vascular surface which is scaled with a coefficient of 2/3 with body weight. With these arguments, the assumed scaling value for Q is 2/3.
[0481] The predicted terminal half-life in human was then computed from the scaled parameters using a 2-compartment model. The average predicted half-life in humans was calculated as 12.5 days (300 hours).
Certain Embodiments of the Invention
[0482] 1. An isolated antibody that specifically binds to human BTLA, wherein said antibody comprises a heavy chain and a light chain, wherein said heavy chain comprises an Fc region that comprises a substitution that results in increased binding to FcγR2B compared to a parent molecule that lacks the substitution.
2. The antibody according to embodiment 1, wherein the antibody has selectivity for binding FcγR2B over FcγR2A compared to a parent molecule that lacks the substitution.
3. The antibody according to embodiment 1 or 2, wherein the antibody has:
(i) enhanced FcγR2B binding activity and maintained or decreased binding activities towards FcγR2A (type R) and/or FcγR2A (type H) in comparison with a parent polypeptide; and/or
(ii) a value of [KD value of polypeptide variant for FcγR2A (type R)]/[KD value of polypeptide variant for FcγR2B] of 2 or more, such as 3, 4, 5, 6, 7, 8, 9, 10 or more; and/or
(iii) a value of [KD value of polypeptide variant for FcγR2A (type H)]/[KD value of polypeptide variant for FcγR2B] of 2 or more, such as 3, 4, 5, 6, 7, 8, 9, 10 or more; and/or
(iv) enhanced FcγR2B binding activity and maintained or decreased binding activities towards FcγR1A in comparison with a parent polypeptide; and/or
(v) a value of [KD value of polypeptide variant for FcγR1A]/[KD value of polypeptide variant for FcγR2B] of 2 or more, such as 3, 4, 5, 6, 7, 8, 9, 10 or more.
4. The antibody of any of embodiments 1 to 3, wherein the antibody binds a residue of human BTLA selected from: [0483] (i) D52, P53, E55, E57, E83, Q86, E103, L106 and E92 (position according to SEQ ID NO:225); or [0484] (ii) Y39, K41, R42, Q43, E45 and S47; or [0485] (iii) D35, T78, K81, S121 and L123; or [0486] (iv) H68; or [0487] (v) N65 and A64;
wherein each position is in relation to the amino acid sequence disclosed in SEQ ID NO:225.
5. An antibody that specifically binds to human BTLA, wherein said antibody comprises a heavy chain and a light chain, wherein said heavy chain comprises an Fc region that comprises one or more of the following amino acids: alanine (A) at position 234, alanine (A) at position 235, aspartic acid (D) at position 236, aspartic acid (D) at position 237 aspartic acid (D) at position 238, alanine (A) at position 265, glutamic acid (E) at position 267, glycine (G) at position 271, arginine (R) at position 330, alanine (A) at position 332, or alanine (A) at position 297 (numbering according to EU Index).
6. Then antibody of embodiment 5, wherein said heavy chain comprises an Fc region that comprises an aspartic acid at position 238 (EU Index).
7. The antibody of any one of the preceding embodiments, which is an agonistic antibody.
8. The antibody of embodiments 6, wherein said antibody binds to FcγR2B with a higher affinity relative to a comparable control antibody that comprises an Fc region that comprises a proline at position 238 (EU Index).
9. The antibody of any one of the preceding embodiments, wherein said antibody binds to FcγR2B with an affinity of from about 5 μM to 0.1 μM, as determined by surface plasmon resonance (SPR).
10. The antibody of any one of the preceding embodiments, wherein said antibody binds to FcγR2B with an affinity of at most 5 μM, as determined by surface plasmon resonance (SPR).
11. The antibody of any one of embodiments 6 to 10, wherein said antibody binds to FcγR2A (131R allotype) with a lower or equal affinity relative to a comparable control antibody that comprises an Fc region that comprises a proline at position 238 (EU Index).
12. The antibody of any one of the preceding embodiments, wherein said antibody binds to FcγR2A (131R allotype) with a K.sub.D of at least 20 μM, as determined by surface plasmon resonance (SPR).
13. The antibody of any one of embodiments 6 to 12, wherein said antibody binds to FcγR2A (131H allotype) with a lower or equal affinity relative to a comparable control antibody that comprises an Fc region that comprises a proline at position 238 (EU Index).
14. The antibody of any preceding embodiment, wherein said antibody binds to FcγR2A (131H allotype) with a K.sub.D of at least 50 μM, as determined by surface plasmon resonance (SPR).
15. The antibody of any one of the preceding embodiments, wherein said antibody exhibits increased agonism of human BTLA expressed on the surface of a human immune cell as measured by a BTLA agonist assay selected from a T cell activation assay such as that described in example 24, a mixed lymphocyte reaction such as that described in example 25 or a B cell activation assay such as that described in example 26.
16. An isolated antibody that specifically binds to human BTLA, wherein said antibody comprises a heavy chain and a light chain, wherein: the heavy chain comprises an Fc region and a heavy chain variable region comprising three complementarity determining regions (CDRs): CDRH1, CDRH2 and CDRH3 and the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2, and CDRL3, wherein (1) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 17, and SEQ ID NO: 3, respectively, with from 0 to 3 amino acid modification, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 4, SEQ ID NO: 12, and SEQ ID NO: 6, respectively, with from 0 to 3 amino acid modifications; or (2) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively, with from 0 to 3 amino acid modification, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, respectively, with from 0 to 3 amino acid modifications; or (3) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32, respectively, with from 0 to 3 amino acid modification, and CDRL1, CDRL2, and CDRL3 have an amino acid sequence as set forth in SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35, respectively, with from 0 to 3 amino acid modifications, and wherein the Fc region comprises an aspartic acid at position 238 (EU Index).
17. An isolated antibody that specifically binds BTLA, comprising a heavy chain and a light chain, wherein (1) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 18, or a sequence with at least 90% identity thereto and an Fc region comprising an aspartic acid at position 238 (EU Index) and the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 14, or a sequence with at least 90% identity thereto; or (2) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 26, or a sequence with at least 90% identity thereto and an Fc region comprising an aspartic acid at position 238 (EU Index) and the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 27, or a sequence with at least 90% identity thereto; or (3) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 36, or a sequence with at least 90% identity thereto and an Fc region comprising an aspartic acid at position 238 (EU Index) and the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 43, or a sequence with at least 90% identity thereto.
18. An isolated antibody that specifically binds BTLA, comprising a heavy chain and a light chain, wherein (1) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 19, or a sequence with at least 90% sequence identity thereto, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 16, or a sequence with at least 90% identity thereto; (2) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 28, or a sequence with at least 90% sequence identity thereto, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 29, or a sequence with at least 90% identity thereto; or (3) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 38, or a sequence with at least 90% sequence identity thereto, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 44, or a sequence with at least 90% identity thereto; and wherein for each of 1) (2) and (3) the heavy chain comprises an aspartic acid at position 238 (EU Index).
19. The antibody of any one of the preceding embodiments, which is an IgG1, IgG2 or IgG4 antibody.
20. The antibody of any one of the preceding embodiments, which is selected from the group consisting of: a human antibody, a humanised antibody, a chimeric antibody and a multispecific antibody (such as a bispecific antibody).
21. The antibody of any one of the preceding embodiments, which is monoclonal.
22. The antibody of any one of the preceding embodiments, wherein said antibody agonizes human BTLA expressed on the surface of an immune cell, wherein said immune cell is optionally a T cell.
23. The antibody of any one of the preceding embodiments, wherein binding of said antibody to human BTLA expressed on the surface of an immune cell decreases proliferation of said cell relative to a comparable immune cell not bound by said antibody, and wherein said cell is optionally a T cell.
24. The antibody of embodiment 23, wherein said decrease in cell proliferation is at least about 10%, 15%, 20%, 25%, 30%, 40%, or 50%.
25. The antibody of embodiment 23, wherein said decrease in cell proliferation is from about 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 10% to 15%, 20% to 50%, 20% to 40%, or 20% to 30%.
26. The antibody of any one of the preceding embodiments, wherein said antibody comprises a domain that binds to an Fc receptor.
27. The antibody of any one of the preceding embodiments, wherein said Fc receptor is expressed on the surface of an immune cell.
28. The antibody of embodiment 27, wherein said immune cell is an antigen presenting cell.
29. The antibody of embodiment 28, wherein said antigen presenting cell is a dendritic cell, macrophage, monocyte, or neutrophil.
30. The antibody of any one of the preceding embodiments, wherein said antibody binds to human BTLA expressed on the surface of a T cell.
31. The antibody of any one of embodiments 26 to 30, wherein said Fc receptor is FcγR2B.
32. The antibody of any one of the preceding embodiments, wherein binding of said antibody to human BTLA expressed on the surface of an immune cell decreases NFκB signaling of said immune cell relative to a comparable immune cell not bound by said antibody, and wherein said immune cell is optionally a T cell.
33. The antibody of embodiment 32, wherein said decrease in NFκB signaling of said immune cell is measured by an assay described in Example 10.
33. The antibody of embodiment 32 or 33, wherein said decrease in NFκB signaling of said immune cell is at least about 10%, 15%, 20%, 25%, 30%, or 40%.
34. The antibody of embodiment 32 or 33, wherein said decrease in NFκB signaling of said immune cell is from about 10% to 40%, 10% to 30%, 10% to 20%, 20% to 40%, or 20% to 30%.
35. The antibody of any one of the preceding embodiments, wherein binding of said antibody to human BTLA expressed on the surface of an immune cell decreases dephosphorylation of a cytoplasmic domain of said human BTLA.
36. The antibody of embodiment 35, wherein said dephosphorylation is mediated by CD45 expressed on the surface of said immune cell.
37. The antibody of any one of the preceding embodiments, wherein said antibody specifically binds human B and T Lymphocyte Attenuator (BTLA) with a K.sub.D of less than 10 nM, each as determined by surface plasmon resonance (SPR) at 37° C., and wherein said antibody binds cynomolgus BTLA with a K.sub.D of less than 20 nM, as determined by surface plasmon resonance (SPR) at 37° C.; does not inhibit binding of BTLA to herpes virus entry mediator (HVEM); and inhibits proliferation of T cells in vitro, as determined by a mixed lymphocyte reaction assay.
38. The antibody of embodiment 37, wherein said antibody binds human B and T Lymphocyte Attenuator (BTLA) with an on rate of at least 5.0×10.sup.5 (l/Ms) at 37° C.
39. The antibody of embodiment 37 or 38, wherein said antibody binds human B and T Lymphocyte Attenuator (BTLA) with an off rate of less than 3.0×10.sup.−4 (l/s) at 37° C.
40. The antibody of any one of embodiments 37 to 39, wherein said antibody binds human B and T Lymphocyte Attenuator (BTLA) with an off rate from 3.0×10.sup.−4 (l/s) to 1.0×10.sup.−3 (l/s).
41. The antibody of any one of the preceding embodiments, wherein said antibody specifically binds human B and T Lymphocyte Attenuator (BTLA) with an on rate of at least 5.0×10.sup.5 (l/Ms), as determined by surface plasmon resonance (SPR) at 37° C., wherein said antibody does not inhibit binding of BTLA to herpes virus entry mediator (HVEM); and wherein said antibody inhibits proliferation of T cells in vitro, as determined by a mixed lymphocyte reaction assay.
42. The antibody of any one of the preceding embodiments, wherein said antibody binds human B and T Lymphocyte Attenuator (BTLA) with a K.sub.D of less than 10 nM, as determined by surface plasmon resonance (SPR) at 37° C.
43. The antibody of any one of the preceding embodiments, wherein said antibody binds cynomolgus BTLA with a K.sub.D of less than 20 nM, as determined by surface plasmon resonance (SPR) at 37° C.
44. The antibody of any one of the preceding embodiments, wherein said antibody specifically binds human B and T Lymphocyte Attenuator (BTLA) with an off rate from 3.0×10.sup.−4 (l/Ms) to 1.0×10.sup.−3 (l/Ms) as measured by surface plasmon resonance (SPR) at 37° C., wherein said antibody does not inhibit binding of BTLA to herpes virus entry mediator (HVEM); and wherein said antibody inhibits proliferation of T cells in vitro, as determined by a mixed lymphocyte reaction assay.
45. The antibody of any one of the preceding embodiments, wherein said antibody specifically binds human B and T Lymphocyte Attenuator (BTLA) with an off rate of less than 1.0×10.sup.−3 (l/Ms) and an on rate of at least 5.0×10.sup.5 (l/Ms), each as measured by surface plasmon resonance (SPR) at 37° C., wherein said antibody does not inhibit binding of BTLA to herpes virus entry mediator (HVEM); and wherein said antibody inhibits proliferation of T cells in vitro, as determined by a mixed lymphocyte reaction assay.
46. The antibody of any one of the preceding embodiments, wherein said antibody specifically binds human B and T Lymphocyte Attenuator (BTLA) with a K.sub.D of less than 2 nM, as determined by surface plasmon resonance (SPR) at 37° C., wherein said antibody inhibits binding of BTLA to herpes virus entry mediator (HVEM); and inhibits proliferation of T cells in vitro, as determined by a mixed lymphocyte reaction assay.
47. The antibody of any one of the preceding embodiments, wherein said antibody specifically binds human B and T Lymphocyte Attenuator (BTLA), wherein said antibody binds cynomolgus BTLA with a K.sub.D of at least 5 nM, as determined by surface plasmon resonance (SPR) at 37° C.; and wherein said antibody inhibits binding of BTLA to herpes virus entry mediator (HVEM); and inhibits proliferation of T cells in vitro, as determined by a mixed lymphocyte reaction assay.
48. The antibody of any one of the preceding embodiments, wherein said antibody specifically binds human B and T Lymphocyte Attenuator (BTLA), wherein said antibody binds cynomolgus BTLA with a K.sub.D of at least than 50 nM, as determined by surface plasmon resonance (SPR) at 37° C.; and wherein said antibody does not inhibit binding of BTLA to herpes virus entry mediator (HVEM); and inhibits proliferation of T cells in vitro, as determined by a mixed lymphocyte reaction assay.
49. The antibody of any one of the preceding embodiments, which has an in vivo half-life of at least 7 days in the human body.
50. A nucleic acid which comprises one or more nucleotide sequences encoding polypeptides capable of forming an antibody of any of embodiments 1 to 49.
51. An expression vector comprising the nucleic acid molecule of embodiment 50.
52. A host cell comprising the nucleic acid sequence of embodiment 50 or 51.
53. A method of producing an antibody (or BTLA binding molecule) that binds to BTLA, comprising the step of culturing the host cell of embodiment 52 under conditions for production of said antibody, optionally further comprising isolating and/or purifying said antibody.
54. A method for preparing a human antibody (or BTLA binding molecule) that specifically binds BTLA, the method comprising the steps of: [0488] (i) providing a host cell comprising one or more nucleic acid molecules encoding the amino acid sequence of a heavy chain and a light chain which when expressed are capable of combining to create an antibody of any one of embodiments 1 to 49; [0489] (ii) culturing the host cell expressing the encoded amino acid sequence; and [0490] (iii) isolating the antibody.
55. A pharmaceutical composition comprising a therapeutically effective amount of the antibody of any one of embodiments 1 to 49 and at least one pharmaceutically acceptable excipient.
56. An antibody in accordance with any one of embodiments 1 to 49, or the pharmaceutical composition in accordance with embodiment 55, for use in therapy.
57. An antibody in accordance with any one of embodiments 1 to 49, or the pharmaceutical composition in accordance with embodiment 55, for use in the treatment or prevention of inflammatory or autoimmune diseases, and disorders of excessive immune cell proliferation.
58. The antibody for use according to embodiment 56, wherein the inflammatory or autoimmune disease is selected from Addison's disease, allergy, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, anti-phospholipid syndrome, asthma (including allergic asthma), autoimmune haemolytic anaemia, autoimmune hepatitis, autoimmune pancreatitis, autoimmune polyendocrine syndrome, Behcet's disease, bullous pemphigoid, cerebral malaria, chronic inflammatory demyelinating polyneuropathy, coeliac disease, Crohn's disease, Cushing's Syndrome, dermatomyositis, diabetes mellitus type 1, eosinophilic granulomatosis with polyangiitis, graft versus host disease, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, Hidradenitis Suppurativa, inflammatory fibrosis (e.g., scleroderma, lung fibrosis, and cirrhosis), juvenile arthritis, Kawasaki disease, leukemia, lymphoma, lymphoproliferative disorders, multiple sclerosis, myasthenia gravis, myeloma, neuromyelitis optica, pemphigus, polymyositis, primary biliary cholangitis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, rheumatoid arthritis, sarcoidosis, Sjögren's syndrome, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis, transplant rejection, transverse myelitis, ulcerative colitis, uveitis, vasculitis, vitiligo and Vogt-Koyanagi-Harada Disease.
59. The antibody for use according to embodiment 57, wherein the disorder of excessive immune cell proliferation is selected from lymphoma, leukemia, systemic mastocytosis, myeloma, or a lymphoproliferative disorder.
60. An isolated antibody that specifically binds B and T lymphocyte attenuator (BTLA), comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2 and CDRH3, wherein (i) CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 17, and SEQ ID NO: 3, respectively, and wherein the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2 and CDRL3, wherein CDRL1 has an amino acid sequence as set forth in SEQ ID NO: 4, CDRL2 has an amino acid sequence as set forth in SEQ ID NO: 12, and CDRL3 has an amino acid sequence as set forth in SEQ ID NO: 6; and wherein said heavy chain comprises an aspartic acid at position 238 (EU Index).
61. An isolated antibody that specifically binds BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 18, and wherein the heavy chain comprises an aspartic acid at position 238 (EU Index).
62. The isolated antibody according to embodiment 61, wherein the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 14, or a sequence with at least 90% identity thereto.
63. The isolated antibody according to any one of embodiments 60 to 62, wherein the heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 19 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 16.
64. An isolated antibody that specifically binds human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2 and CDRH3, wherein CDRH1 has an amino acid sequence as set forth in SEQ ID NO: 20, CDRH2 has an amino acid sequence as set forth in SEQ ID NO: 21, and CDRH3 has an amino acid sequence as set forth in SEQ ID NO: 22, and the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2 and CDRL3, wherein CDRL1 has an amino acid sequence as set forth in SEQ ID NO: 23, CDRL2 has an amino acid sequence as set forth in SEQ ID NO: 24, and CDRL3 has an amino acid sequence as set forth in SEQ ID NO: 25; and wherein said heavy chain comprises an aspartic acid at position 238 (EU Index).
65. The isolated antibody according to embodiment 64, wherein the heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 28 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 29.
66. An isolated antibody that specifically binds human BTLA, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2 and CDRH3, wherein CDRH1 has an amino acid sequence as set forth in SEQ ID NO: 30, CDRH2 has an amino acid sequence as set forth in SEQ ID NO: 31, and CDRH3 has an amino acid sequence as set forth in SEQ ID NO: 32, and the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2 and CDRL3, wherein CDRL1 has an amino acid sequence as set forth in SEQ ID NO: 33, CDRL2 has an amino acid sequence as set forth in SEQ ID NO: 34, and CDRL3 has an amino acid sequence as set forth in SEQ ID NO: 35; and wherein said heavy chain comprises an aspartic acid at position 238 (EU Index).
67. The isolated antibody according to embodiment 66, wherein the heavy chain comprises the amino acid sequence as set forth in SEQ ID NO: 38 and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 39.
68. The antibody of any one of embodiments 60 to 67 or 82-84, which is an IgG1, IgG2 or IgG4 antibody.
69. The antibody of any one of embodiments 60 to 68 or 82 to 84, which is selected from the group consisting of: a human antibody, a humanised antibody, a chimeric antibody and a multispecific antibody (such as a bispecific antibody).
70. The antibody of any one of embodiments 60 to 69 or 82 to 84, which is an antigen-binding fragment moiety selected from the group consisting of: scFv, sc(Fv)2, dsFv, Fab, Fab′, (Fab′)2 and a diabody.
71. The antibody of any one of embodiments 60 to 70 or 82 to 84, which is monoclonal.
72. The antibody of any one of embodiments 60 to 71 or 82 to 84, wherein said antibody agonizes human BTLA expressed on the surface of an immune cell, wherein said immune cell is optionally a T cell.
73. The antibody of any one of embodiments 60 to 72 or 82 to 84, wherein binding of said antibody to human BTLA expressed on the surface of an immune cell decreases proliferation of said cell relative to a comparable immune cell not bound by said antibody, and wherein said cell is optionally a T cell.
74. An isolated nucleic acid which comprises one or more nucleotide sequences encoding polypeptides capable of forming an antibody in any of embodiments 60 to 73 or 82 to 84.
75. A host cell comprising the nucleic acid sequence according to embodiment 74.
76. A method of producing an antibody that binds to BTLA, comprising the step of culturing the host cell of embodiment 75, under conditions for production of said antibody, optionally further comprising isolating and/or purifying said antibody.
77. A method for preparing a human antibody that specifically binds BTLA, the method comprising the steps of: [0491] i) providing a host cell comprising one or more nucleic acid molecules encoding the amino acid sequence of a heavy chain and a light chain which when expressed are capable of combining to create an antibody of any of embodiments 60 to 73 or 82 to 84; [0492] ii) culturing the host cell expressing the encoded amino acid sequence; and [0493] iii) isolating the antibody.
78. A pharmaceutical composition comprising a therapeutically effective amount of the antibody of any of embodiments 60 to 73 or 82 to 84 and at least one pharmaceutically acceptable excipient.
79. An antibody in accordance with any one of embodiments 60 to 73 or 82 to 84, or the pharmaceutical composition in accordance with embodiment 78, for use in therapy.
80. An antibody in accordance with any one of embodiments 60 to 73 or 82 to 84, or the pharmaceutical composition in accordance with embodiment 78, for use in the treatment or prevention of inflammatory or autoimmune diseases, and disorders of excessive immune cell proliferation
81. The antibody for use according to claim 80, wherein the inflammatory or autoimmune disease is selected from Addison's disease, allergy, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, anti-phospholipid syndrome, asthma (including allergic asthma), autoimmune haemolytic anaemia, autoimmune hepatitis, autoimmune pancreatitis, autoimmune polyendocrine syndrome, Behcet's disease, bullous pemphigoid, cerebral malaria, chronic inflammatory demyelinating polyneuropathy, coeliac disease, Crohn's disease, Cushing's Syndrome, dermatomyositis, diabetes mellitus type 1, eosinophilic granulomatosis with polyangiitis, graft versus host disease, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, Hidradenitis Suppurativa, inflammatory fibrosis (e.g., scleroderma, lung fibrosis, and cirrhosis), juvenile arthritis, Kawasaki disease, leukemia, lymphoma, lymphoproliferative disorders, multiple sclerosis, myasthenia gravis, myeloma, neuromyelitis optica, pemphigus, polymyositis, primary biliary cholangitis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, rheumatoid arthritis, sarcoidosis, Sjögren's syndrome, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis, transplant rejection, transverse myelitis, ulcerative colitis, uveitis, vasculitis, vitiligo and Vogt-Koyanagi-Harada Disease.
82. An isolated antibody that specifically binds BTLA, comprising a heavy chain and a light chain, wherein (1) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 18, or a sequence with at least 90% identity thereto and the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 14, or a sequence with at least 90% identity thereto.
83. An isolated antibody that specifically binds BTLA, comprising a heavy chain and a light chain, wherein (1) the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 19, or a sequence with at least 90% sequence identity thereto and light chain comprises an amino acid sequence as set forth in SEQ ID NO: 16, or a sequence with at least 90% identity thereto.
84. An isolated human antibody that specifically binds B and T lymphocyte attenuator (BTLA), comprising a heavy chain and a light chain, wherein
(a) the heavy chain comprises a heavy chain variable region comprising three CDRs: CDRH1, CDRH2 and CDRH3, wherein CDRH1, CDRH2, CDRH3 have an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 17, and SEQ ID NO: 3, respectively, and wherein the light chain comprises a light chain variable region comprising three CDRs: CDRL1, CDRL2 and CDRL3, wherein CDRL1 has an amino acid sequence as set forth in SEQ ID NO: 4, CDRL2 has an amino acid sequence as set forth in SEQ ID NO: 12, and CDRL3 has an amino acid sequence as set forth in SEQ ID NO: 6; and/or
(b) the heavy chain comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 18; or a sequence with at least 90% identity thereto; and/or (c) the light chain comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 14, or a sequence with at least 90% identity thereto;
optionally wherein the antibody is an IgG1, IgG2 or IgG4 antibody.
Sequences:
[0494]
TABLE-US-00009 TABLE 7 Exemplary CDR Sequences SEQ SEQ ID Amino Acid ID Amino Acid NOs Sequences NOs Sequences 45 SYGIS 136 WQGTHFPQT 46 EIYPRSGNTY 139 TYYGSSQYYF YNEKFKG DY 47 NYGSSYPFAY 143 DYYIN 33 SASSSVSSSY 144 RIYPGSGNTY LH YNEKFKG 34 RTSNLAS 145 GYGNSDY 35 QQWSGYPFT 146 RASQSIGTRI H 53 DYYMN 147 YASESIS 54 DINPNNGGTS 148 QQSNSWPYT YNQKFKG 55 WRQLRSDY 30 SYAIR 56 LASQTIGTWL 48 EIYPRSGNTY A YNENFKG 57 AATSLAD 32 SGGASYTMDY 58 QQLYSTPLT 151 SYGLI 61 SYWMH 152 EIYPRSGSTY YNEWFKG 62 MIHPNNGIPN 153 RRGTGDGFDY YNEKFKS 63 EGYYGSEGYF 154 SASQGISNYL DV N 64 SASSSISYIH 155 YTSSLHS 65 DTSKLAS 156 QQYIELPFT 66 HQRSTYPYT 159 DYYMH 69 MIHPNSGSTN 160 YIYPNNGGNG YNEKFKS YNQKFKG 70 KRGGLGDY 161 GDYYGSLRLT FAY 71 RASKSVSTSG 4 KSSQSLLYSS YSYMH NQKNYLA 72 LASNLES 12 WASTRES 73 QHSRELPYT 164 QQYYSYPLT 76 SSWMN 167 TYGVS 77 RIYPGDGDTN 168 WINTYSGVPT YNGKFKG YADDFKG 78 RGYGYLAY 169 VTTILHWYFD V 79 KASQDVSTAV 170 RASQEISGYL A S 80 SASYRYT 171 AASTLDS 81 QQHYSTPYT 172 LQYASYPFT 84 GYGSSYGFAY 177 RRGAGDGFDY 85 QQWSGYPWT 178 QQYSKLPFT 88 SGYYWN 181 DHTIH 89 YISYDGSNNY 182 YIYPRDGSTK NPSLKN YNEKFKG 90 IYGNYYAMDY 183 SNWNFDY 91 SASSSVSYMH 184 KASQDVGTAV A 92 QQWSSNPPT 185 WASTRRT 95 DYYMI 186 QQYSSYPLT 96 NINPNNGGTT 189 QQHYSTPWT YNQKFKG 97 GGLRPLYFDY 191 EIYPRSGTTY YNEKFKG 98 KASENVDTYV 192 RISSGSGVDY S 99 GASNRYT 193 QQYSELPWT 100 GQSYSYPLT 196 SGYDWH 103 NTYMH 197 YISYSGSTNY NPSLKS 104 RIDPANGNTK 198 GTPVVAEDYF YDPKFQG DY 105 TYYGSSQHYF 199 RSSTGAVTTS DY NYAN 106 KSSQSLLDSD 200 ATNNRAP GKTYLN 107 LVSKLDS 201 ALWYSNHLV 108 WQDTHFPQT 20 TYGVH 111 RIYPGDGDAN 21 VMWPGGRTSY YNGKFKG NPSLKS 112 EGHYYGSGYR 22 GDYEYDYYAM WYLDV DY 113 RASENIYSNL 23 RASSSVSYMH A 114 AATNLAD 24 ATSNRAT 115 QHFRGAPFT 25 HQWSSNPYT 118 DYEIH 204 SAYWN 119 PIDPDTGNTA 205 YISYSGSTYF YNQNLKG NPSLKS 120 GGYDSDWGFA 206 SHYYGYYFDY Y 121 RSSKSLLHSN 207 RASETIDSYG GNTFLF DSLMH 163 VMWPGGRTSY 208 RASNLES NPAPMS 176 ATSNLAS 209 QQTDEDPYT 122 RMSDLAS 1 SYGMS 123 MQHLEYPFT 2 SIRSDGNTYY PDSVKG 126 DYYLN 3 GGYYGSSPYY 127 LIDPYNGGSS 5 WASTRDS CNQKFKG 128 GNAMDY 6 QQYYNYLT 129 WASTRHT 212 SGYSWH 130 QQHYIIPYM 213 YIHYSGSTNY NPSLKS 133 NTYMY 214 GPHRYDGVWF AY 134 RIDPANGNTK 215 SASSSISSNY YAPKFQG LH 135 LYYGSSYDYF 216 QQGTNIPLT DY 31 EIYPRSGNTY 40 EIYPRSGQTY YAQKFQG YAQSFQG 11 SIRSEGQTYY 17 SIRSDGQTYY PDSVKG PDSVKG
TABLE-US-00010 TABLE 8 Exemplary VH and VL Sequences SEQ ID NOs Amino Acid Sequences 51 QVQLQQSGAELARPGASVKLSCKASGYTFTSYGISWVKQRTGQGLEWIGEIYP RSGNTYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCARNYGSSYPF AYWGQGTLVTVSA 59 EVQLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVKQSHGKSLEWIGDINP NNGGTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARWRQLRSD YWGQGTTLTVSS 67 QVQLQQPGAELVKPRASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGMI HPNNGIPNYNEKFKSKATLTVDKSSTTAYMQLSSLTSEDSAVYHCAREGYYGS EGYFDVWGTGTTVTVSS 74 QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGMI HPNSGSTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARKRGGLG DYWGQGTSVTVSS 82 QVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIY PGDGDTNYNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCARRGYGYL AYWGQGTLVTVSA 86 QVQLQQSGAELARPGASVKLSCKASGYTFTSYGISWVKQRTGQGLEWIGEIYP RSGNTYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCARGYGSSYGF AYWGQGTLVTVSA 93 DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYIS YDGSNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCASIYGNYYAM DYWGQGTSVTVSS 101 EVQLQQSGPELVQPGASVKISCKASGYTFTDYYMIWVKQSHGKSLEWIGNINP NNGGTTYNQKFKGKATLTVDKSSSTAYMGLPSLTSEDSAVYYCARGGLRPLY FDYWGQGTTLTVSS 109 EVQLQQSVAELVRPGASVKLSCTASGFNIKNTYMHWVKQRPEQGLEWIGRIDP ANGNTKYDPKFQGKATITADTSSNTAYVQLSSLTSEDTAIYYCALTYYGSSQH YFDYWGQGTTLTVSS 116 QIQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKKRPGKGLEWIGRIYP GDGDANYNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCAGEGHYYGS GYRWYLDVWGTGTTVTVSS 124 QVQLQQSGAELVRPGASVTLSCKASGYTFTDYEIHWVKQTLVHGLEWIGPIDP DTGNTAYNQNLKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRGGYDSDW GFAYWGQGTLVTVSA 131 EVQLQQSGPVLVKPGASVKMSCKASGYTFTDYYLNWVKQSHGKSLEWIGLID PYNGGSSCNQKFKGKATLTVDKSSSTAYMDLNSLTSEDSAVYYCARGNAMD YWGQGTSVTVSS 137 EVQLQQSVAELVRPGASVKLSCTASGFNIKNTYMYWVKQRPEQGLEWIGRIDP ANGNTKYAPKFQGKATITADTSSNTAYLQLSSLTSEDTAIYYCALLYYGSSYD YFDYWGQGTTLTVSS 141 EVQLQQSVAELVRPGASVKLSCTASGFNIKNTYMHWVKQRPEQGLEWIGRIDP ANGNTKYAPKFQGKATITADTSSNTAYLQLSSLTSEDTAIYYCALTYYGSSQY YFDYWGQGTTLTVSS 149 QVQLKQSGAELVRPGASVKLSCKASGYTFTDYYINWVKQRPGQGLEWIARIYP GSGNTYYNEKFKGKATLTAEKSSSTAYMQLSSLTSEDSAVYFCARGYGNSDY WGQGTTLTVSS 223 QVQLQQSGAELARPGASVRLSCKASGYTFTSYAIRWVKQRTGQGLEWIGEIYP RSGNTYYNENFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCARSGGASYT MDYWGQGTSVTVSS 157 QVQLQQSGAELARPGASVRLSCKASGYTFTSYGLIWLKQRTGQGLEWIGEIYP RSGSTYYNEWFKGKATLTADKSSNTAFMELRSLTSEDSAVYFCARRRGTGDG FDYWGQGTILTVSS 165 EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMHWVKQSHGKSLEWIGYIY PNNGGNGYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCAIGDYYGS LRLTFAYWGQGTLVTVSA 173 QIQLVQSGPELKKPGETVKISCKASGYTFTTYGVSWVKQAPGKVLKWMGWIN TYSGVPTYADDFKGRFAFSLETSASTAYLQISNLKNEDTATYFCAPVTTILHWY FDVWGTGTTVTVSS 175 QVQLQQSGAELARPGASVRLSCKASGYTFTSYGISWVKQRTGQGLEWIGEIYP RSGNTYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCARNYGSSYPF AYWGQGTLVTVSA 179 QVQLQQSGAELARPGASVKLSCKASGYTFTSYGISWVKQRTGQGLEWIGEIYP RSGNTYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCARRRGAGDG FDYWGQGTTLTVSS 187 QDQLQQSDAELVKPGASVKISCKVSGYTFTDHTIHWMKQRPEQGLEWIGYIYP RDGSTKYNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCASSNWNFDY WGQGTTLTVSS 194 QVQLQQSGAELARPGASVKLPCKASGYTFTSYGISWVKQRTGQGLEWIGEIYP RSGTTYYNEKFKGKATLTADKSSSTAYMELRSLTSEDSAVYFCARRISSGSGV DYWGQGTTLTVSS 202 DVQLQESGPGMVKPSQSLSLTCTVTGYSITSGYDWHWIRHFPGNKLEWMGYIS YSGSTNYNPSLKSRISITHDTSKNHFFLKLNSVTTEDTATYYCARGTPVVAEDY FDYWGQGTTLTVSS 219 EVKLVESGGGLVKPGGSLKLSCAASGFTLSSYGMSWVRQIPEKRLEWVASIRS DGNTYYPDSVKGRFIISRDNARNILYLQMSSLRSEDTAMYYCTRGGYYGSSPY YWGQGTTLTVSS 217 DVQLQESGPDLVKPSQSLSVTCTVTGYSITSGYSWHWIRQFPGNKLEWMGYIH YSGSTNYNPSLKSRISITRDTSKNQFFLQLSSVTTEDTATYYCASGPHRYDGVW FAYWGQGTLVTVSS 52 ENVLTQSPAIMAASLGQKVTMTCSASSSVSSSYLHWYQQKSGASPKPLIHRTS NLASGVPARFSGSGSGTSYSLTISSVEAEDDATYYCQQWSGYPFTFGGGTKLEI K 60 DIQMTQSPASQSASLGESVTITCLASQTIGTWLAWYQQKPGKSPQLLIYAATSL ADGVPSRFSGSGSGTKFSFKISSLQAEDFVSYYCQQLYSTPLTFGAGTKLELK 68 QIVLTQSPAIMSASPGEKVTMTCSASSSISYIHWYQQKPGTSPKRWIYDTSKLAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRSTYPYTFGGGTKLEIK 75 DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYL ASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPYTFGGGTKL EIK 83 DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASY RYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPYTFGGGTKLEIK 87 ENVLTQSPAIMAASLGQKVTMTCSASSSVSSSYLHWYQQKSGASPKPLIHRTS NLASGVPARFSGSGSGTSYSLTISSVEAEDDATYYCQQWSGYPWTFGGGTKLE IK 94 QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKL ASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPTFGSGTKLEIK 102 NIVMTQSPKSMSMSVGERVTLSCKASENVDTYVSWYQQKPEQSPKLLIYGASN RYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQSYSYPLTFGAGTKLELI 110 DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIY LVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQDTHFPQTFGGGT KLEIK 117 DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYAATN LADGVPSRFSGSGSGTQYSLKINSLQSEDFGSYYCQHFRGAPFTFGSGTKLEIK 125 DIVMTQATPSVPVTPGESVSISCRSSKSLLHSNGNTFLFWFLQRPGQSPQLLIYR MSDLASGVPDRFSGSGSGTAFTLRISRVEAEDVGIYYCMQHLEYPFTFGSGTKL EIK 132 DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQEKPGQSPKLLIYWAST RHTGVPDRFTGSGSGTDYILNISSVQAEDLALYYCQQHYIIPYMFGGGTKLEIK 138 DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIY LVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGT KLEIK 142 DILLTQSPAILSVSPGERVSFSCRASQSIGTRIHWYQQRTNGSPRLLIKYASESI SGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPYTFGGGTKLEIK 150 ENVLTQSPAIMAASLGQKVTMTCSASSSVSSSYLHWYQQKSGASPKPLIHRTS NLASGVPARFSGSGSGTSYSLTISSVEAEDDATYYCQQWSGYPFTFGSGTKLEI K 158 DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSL HSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYIELPFTFGSGTKLEIK 166 DIVMSQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLL IYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAG TKLELK 174 DIQMTQSPSSLSASLGERVSLTCRASQEISGYLSWLQQKPDGTIKRLIYAASTLD SGVPKRFRGSRSGSDYSLTISSLESEDFADYYCLQYASYPFTFGSGTKLEIK 180 DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSL HSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPFTFGSGTKLEIK 188 DIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKLLIYWAS TRRTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPLTFGAGTKLEL K 190 DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASY RYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPWTFGGGTKLEIK 195 DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSL HSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSELPWTFGGGTKLEIK 203 QAVVTQESALSTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGAT NNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHLVFGGGTKL TVLG 220 DIVMSQSPSSLPVSVGEKISMTCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLI YWASTRDSGVPDRFIGSGSGTDFTLTINSVKAEDLAVYYCQQYYNYLTFGAGT KLELK 218 EIVLTQSPTTMAASPGEKITITCSASSSISSNYLHWYQQKPGFSPKLLIYRTSNLA SGVPARFSGSGSGTSYSLTIGTMEAEDVATYYCQQGTNIPLTFGAGTKLEIK 36 QVQLVQSGAELKKPGASVKVSCKASGYTFTSYAIRWVRQATGQGLEWMGEIY PRSGNTYYAQKFQGRATLTADKSISTAYMELSSLRSEDTAVYFCARSGGASYT MDYWGQGTTVTVSS 37 ENVLTQSPATLSLSPGERATLSCSASSSVSSSYLHWYQQKPGQSPRPLIHRTSNL ASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSGYPFTFGSGTKLEIK 7 EVQLVESGGGLVKPGGSLRLSCAASGFTLSSYGMSWVRQAPGKGLEWVASIR SDGNTYYPDSVKGRFTISRDNAKNSLYLQMSSLRAEDTAVYYCTRGGYYGSSP YYWGQGTTVTVSS 8 DIVMTQSPLSLPVTPGEPASISCKSSQSLLYSSNQKNYLAWYQQKPGQSPQLLIY WASTRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQYYNYLTFGGGT KVEIK 13 EVQLVESGGGLVKPGGSLRLSCAASGFTLSSYGMSWVRQAPGKGLEWVASIR SEGQTYYPDSVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCTRGGYYGSSP YYWGQGTTVTVSS 14 DIVMTQSPLSLPVTPGEPASISCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIY WASTRESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQYYNYLTFGGGTK VEIK 18 EVQLVESGGGLVKPGGSLRLSCAASGFTLSSYGMSWVRQAPGKGLEWVASIR SDGQTYYPDSVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCTRGGYYGSSP YYWGQGTTVTVSS 26 QVTLKESGPALVKPTQTLTLTCTVSGFSLTTYGVHWIRQPPGKALEWLGVMW PGGRTSYNPSLKSRLTITKDNSKSQVVLTMTNMDPVDTATYYCVRGDYEYDY YAMDYWGQGTLVTVSS 27 EIVLTQSPATLSLSPGERATLSCRASSSVSYMHWYQQKPGQAPRPLIYATSNRA TGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCHQWSSNPYTFGQGTKLEIK 41 QVQLVQSGAELKKPGASVKVSCKASGYTFTSYAIRWVRQATGQGLEWMGEIY PRSGQTYYAQSFQGRATLTADKSTSTAYMELSSLRSEDTAVYFCARSGGASYT MDYWGQGTTVTVSS 43 ENVLTQSPATLSLSLGERATLSCSASSSVSSSYLHWYQQKPDQSPRPLIHRTSNL ASGIPSRFSGSGSGTDYTLTISSLEAEDFAVYYCQQWSGYPFTFGSGTKLEIK 210 EVQLQESGPSLVKPSQTLSLTCSVTGDSITSAYWNWIRKFPGNKLEYMGYISYS GSTYFNPSLKSRISITRNTSKNQYYLQLNSVTTEDTATYYCARSHYYGYYFDY WGHGTTLTVSS 211 DIVLTQSPASLAVSLGQRATISCRASETIDSYGDSLMHWYQQKAGQPPKLLIYR ASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQTDEDPYTFGGGTKL EIK 221 QVQLKESGPGLVAPSQSLSITCTVSGFSLTTYGVHWVRQSPGKGLEWLGVMW PGGRTSYNPAPMSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCVRGDYEYDY YAMDYWGQGTSVTVSS 222 QIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLA SGVPARFSGSGSGTSYSLTISRMEAEDAATYYCHQWSSNPYTFGGGTKLEIK
TABLE-US-00011 TABLE 9 Exemplary heavy chain and light chain Sequences SEQ ID NOs Amino Acid Sequences 9 EVQLVESGGGLVKPGGSLRLSCAASGFTLSSYGMSWVRQAPGKGLEWVASIR SDGNTYYPDSVKGRFTISRDNAKNSLYLQMSSLRAEDTAVYYCTRGGYYGSS PYYWGQGTTVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 10 DIVMTQSPLSLPVTPGEPASISCKSSQSLLYSSNQKNYLAWYQQKPGQSPQLLI YWASTRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQYYNYLTFGGG TKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC 15 EVQLVESGGGLVKPGGSLRLSCAASGFTLSSYGMSWVRQAPGKGLEWVASIR SEGQTYYPDSVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCTRGGYYGSS PYYWGQGTTVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 16 DIVMTQSPLSLPVTPGEPASISCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLI YWASTRESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQYYNYLTFGGG TKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC 19 EVQLVESGGGLVKPGGSLRLSCAASGFTLSSYGMSWVRQAPGKGLEWVASIR SDGQTYYPDSVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCTRGGYYGSS PYYWGQGTTVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 28 QVTLKESGPALVKPTQTLTLTCTVSGFSLTTYGVHWIRQPPGKALEWLGVMW PGGRTSYNPSLKSRLTITKDNSKSQVVLTMTNMDPVDTATYYCVRGDYEYDY YAMDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 29 EIVLTQSPATLSLSPGERATLSCRASSSVSYMHWYQQKPGQAPRPLIYATSNRA TGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCHQWSSNPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC 38 QVQLVQSGAELKKPGASVKVSCKASGYTFTSYAIRWVRQATGQGLEWMGEI YPRSGNTYYAQKFQGRATLTADKSISTAYMELSSLRSEDTAVYFCARSGGAS YTMDYWGQGTTVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 39 ENVLTQSPATLSLSPGERATLSCSASSSVSSSYLHWYQQKPGQSPRPLIHRTSN LASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQWSGYPFTFGSGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC 42 QVQLVQSGAELKKPGASVKVSCKASGYTFTSYAIRWVRQATGQGLEWMGEI YPRSGQTYYAQSFQGRATLTADKSTSTAYMELSSLRSEDTAVYFCARSGGAS YTMDYWGQGTTVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGDSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 44 ENVLTQSPATLSLSLGERATLSCSASSSVSSSYLHWYQQKPDQSPRPLIHRTSN LASGIPSRFSGSGSGTDYTLTISSLEAEDFAVYYCQQWSGYPFTFGSGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
TABLE-US-00012 Human (Homo sapiens) BTLA polypeptide. Positions 1-30 is signal sequence, 31-151 is extracellular region, 152-178 is transmembrane region and 179 to end is intracellular region SEQ ID NO: 225 MKTLPAMLGTGKLFWVFFLIPYLDIWNIHGKESCD VQLYIKRQSEHSILAGDPFELECPVKYCANRPHVT WCKLNGTTCVKLEDRQTSWKEEKNISFFILHFEPV LPNDNGSYRCSANFQSNLIESHSTTLYVTDVKSAS ERPSKDEMASRPWLLYRLLPLGGLPLLITTCFCLF CCLRRHQGKQNELSDTAGREINLVDAHLKSEQTEA STRQNSQVLLSETGIYDNDPDLCFRMQEGSEVYSN PCLEENKPGIVYASLNHSVIGPNSRLARNVKEAPT EYASICVRS cynomolgus monkey (Macaca fascicularis) BTLA polypeptide. SEQ ID NO: 226 MKTLPAMLGSGRLFWVVFLIPYLDIWNIHGKESCD VQLYIKRQSYHSIFAGDPFKLECPVKYCAHRPQVT WCKLNGTTCVKLEGRHTSWKQEKNLSFFILHFEPV LPSDNGSYRCSANFLSAIIESHSTTLYVTDVKSAS ERPSKDEMASRPWLLYSLLPLGGLPLLITTCFCLF CFLRRHQGKQNELSDTTGREITLVDVPFKSEQTEA STRQNSQVLLSETGIYDNEPDFCFRMQEGSEVYSN PCLEENKPGIIYASLNHSIIGLNSRQARNVKEAPT EYASICVRS hIgG1 const region with 238D SEQ ID NO: 227 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGDSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK hkappa const region SEQ ID NO: 228 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC hHVEM-mFc fusion protein (including signal peptide and C-terminal His- tag) SEQ ID NO: 229 MEPPGDWGPPPWRSTPRTDVLRLVLYLTFLGAPCY APALPSCKEDEYPVGSECCPKCSPGYRVKEACGEL TGTVCEPCPPGTYIAHLNGLSKCLQCQMCDPAMGL RASRNCSRTENAVCGCSPGHFCIVQDGDHCAACRA YATSSPGQRVQKGGTESQDTLCQNCPPGTFSPNGT LEECQHQTKCSWLVTKAGAGTSSSHLVPRGSGSKP SISTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVV DISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNST FRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEK TISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMI TDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSY FVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEK SLSHSPGKHHHHHH Mopc21 hIgG1 P238D isotype control heavy chain SEQ ID NO: 230 DVQLVESGGGLVQPGGSRKLSCAASGFTFSSFGMH WVRQAPEKGLEWVAYISSGSSTLHYADTVKGRFTI SRDNPKNTLFLQMTSLRSEDTAMYYCARWGNYPYY AMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGDSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK Mopc21 hIgG1 P238D isotype control light chain SEQ ID NO: 231 NIVMTQSPKSMSMSVGERVTLTCKASENVVTYVSW YQQKPEQSPKLLIYGASNRYTGVPDRFTGSGSATD FTLTISSVQAEDLADYHCGQGYSYPYTFGGGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTISKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC SEQ ID NO: 232 GGGGS SEQ ID NO: 233 KESGSVSSEQLAQFRSLD SEQ ID NO: 234 EGKSSGSGSESKST Reference IgG4 constant sequence containing a P238D and also a S228P substitution. SEQ ID NO: 235 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGDSVFLFPPKPKDTLMISRTPEVT CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLGK