IP Library › Patent Application 10952493

Patent Application

App. No. 10/952,493

Novel variants of CD40L protein

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Patent No.: US None
App. No.: 10/952,493

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Abstract

The present invention relates to soluble, recombinant CD40L variant proteins that may be expressed solubly in E. coli . The variants of the present invention may substantially reduced binding to alpha IIb-beta3 integrin, act as CD40L antagonists or agonists, and methods for generating the same.

Claims (287)

1 . A composition comprising a variant CD40L protein having the formula:

Fx (45-115)-Vb (116)-Vb (117)-Vb(118)-Vb(119)-Vb(120)-Vb(121)-Fx(122)-Vb(123)-Fx(124)-Vb(125)-Fx(126)-Vb(127)-Vb(128)-Vb(129)-Vb(130)-Fx(131)-Vb(132)-Vb(133)-Vb(134)-Fx(135-136)-Vb(137)-Fx(138-139)-Vb(140)-Vb(141)-Vb(142)-Vb(143)-Vb(144)-Vb(145)-Vb(146)-Vb(147)-Vb(148)-Vb(149)-Fx(150)-Vb(151)-Vb(152)-Fx(153-154)-Vb(155)-Fx(156)-Vb(157)-Fx(158-165)-Vb(166)-Vb(167)-Vb(168)-Vb(169)-Vb(170)-Fx(171)-Vb(172)-Vb(173)-Vb(174)-Fx(175)-Vb(176)-Vb(177)-Vb(178)-Vb(179)-Fx(180)-Vb(181)-Vb(182)-Vb(183)-Vb(184)-Vb(185)-Vb(186)-Vb(187)-Vb(188)-Vb(189)-Vb(190)-Vb(191)-Vb(192)-Vb(193)-Vb(194)-Vb(195)-Vb(196)-Vb(197)-Vb(198)-Vb(199)-Vb(200)-Vb(201)-Vb(202)-Vb(203)-Vb(204)-Vb(205)-Vb(206)-Vb(207)-Vb(208)-Vb(209)-Vb(210)-Vb(211)-Fx(212)-Vb(213)-Vb(214)-Vb(215)-Vb(216)-Vb(217)-Vb(218)-Vb(219)-Vb(220)-Vb(221)-Vb(222)-Vb(223)-Vb(224)-Vb(225)-Vb(226)-Vb(227)-Vb(228)-Vb(229)-Vb(230)-Vb(231)-Vb(232)-Vb(233)-Fx(234-241)-Vb(242)-Vb(243)-Vb(244)-Vb(245)-Vb(246)-Vb(247)-Vb(248)-Vb(249)-Vb(250)-Vb(251)-Vb(252)-Vb(253)-Vb(254)-Vb(255)-Fx(256-258)-Vb(259)-Vb(260)-Vb(261)

wherein

Fx(45-115) is optionally present, and if present, comprises the human amino acid sequence of CD40L at positions 45-115;

Vb(116) is selected from the group consisting of G and C;

Vb(117) is selected from the group consisting of D and C;

Vb(118) is selected from the group consisting of Q, C, H, E, N, A, T, S, W, F, V, G, K, R, Y, D, and M;

Vb(119) is selected from the group consisting of N, H, Q, L, N, R, D, A, S, M, K, G, and E;

Vb(120) is selected from the group consisting of P, C, R, Q, N, E, D, K, G, and S;

Vb(121) is selected from the group consisting of Q, H, T, V, Y, M, C, D, A, E, I, N, F, L, S, and G;

Fx(122) comprises the human amino acid sequence of CD40L at position 122;

Vb(123) is selected from the group consisting of A and E;

Fx(124) comprises the human amino acid sequence of CD40L at position 124;

Vb(125) is selected from the group consisting of H, R, N, and M;

Fx(126) is selected from the group consisting of V, D, and A;

Vb(127) is selected from the group consisting of H, Q, E, C, S, D, and I;

Vb(128) is selected from the group consisting of S and R;

Vb(129) is selected from the group consisting of E and G;

Vb(130) is selected from the group consisting of P, Q, K, M, N, C, R, E, D, and A;

Fx(131) comprises the human amino acid sequence of CD40L at position 131;

Vb(132) is selected from the group consisting of C and S;

Vb(133) is selected from the group consisting of C and K;

Vb(134) is selected from the group consisting of C, Q, E, D, R, N, K, S, G, and T;

Fx(135-136) comprises the human amino acid sequence of CD40L at positions 135-136;

Vb(137) is selected from the group consisting of R, K, S, and V;

Fx(138-139) comprises the human amino acid sequence of CD40L at positions 138-139;

Vb(140) is selected from the group consisting of W, C, G, R, and a deletion;

Vb(141) comprises any amino acid;

Vb(142) is selected from the group consisting of M, V, C, I, E, and a deletion;

Vb(143) is selected from the group consisting of C, E, K, and a deletion;

Vb(144) is selected from the group consisting of Q, M, E, H, K, R, and G;

Vb(145) is selected from the group consisting of P, E, N, C, D, H, Y, and a deletion;

Vb(146) is selected from the group consisting of S, C, and N;

Vb(147) is selected from the group consisting of T, V, I, and N;

Vb(148) is selected from the group consisting of M and K;

Vb(149) is selected from the group consisting of S and A;

Fx(150) comprises the human amino acid sequence of CD40L at position 150;

Vb(151) comprises any amino acid;

Vb(152) is selected from the group consisting of L, E, and Q;

Fx(153-154) comprises the human amino acid sequence of CD40L at positions 153-154;

Vb(155) is selected from the group consisting of L and P;

Fx(156) comprises the human amino acid sequence of CD40L at position 156;

Vb(157) comprises any amino acid;

Fx(158-165) comprises the human amino acid sequence of CD40L at positions 158-165;

Vb(166) is selected from the group consisting of Q, S, G, C, D, Q, E, A, and N;

Vb(167) comprises any amino acid;

Vb(168) is selected from the group consisting of C, P, and L;

Vb(169) is selected from the group consisting of Y and D;

Vb(170) is selected from the group consisting of Y, C, and P;

Fx(171) comprises the human amino acid sequence of CD40L at position 171;

Vb(172) comprises any amino acid;

Vb(173) is selected from the group consisting of A and D;

Vb(174) is selected from the group consisting of Q, R, I, V, L, H, and K;

Fx(175) comprises the human amino acid sequence of CD40L at position 175;

Vb(176) is selected from the group consisting of T, G, I, A, S, and G;

Vb(177) comprises any amino acid;

Vb(178) is selected from the group consisting of C, G, S, D, E, H, Q, N, K, Y, M, and R;

Vb(179) comprises any amino acid;

Fx(180) comprises the human amino acid sequence of CD40L at position 180;

Vb(181) is selected from the group consisting of R, G, S, C, and A;

Vb(182) is selected from the group consisting of E and C;

Vb(183) is selected from the group consisting of A, Q, E, R, D, N, K, S, G, and an insertion;

Vb(184) is selected from the group consisting of S, C, L, R, M, and deletion;

Vb(185) is selected from the group consisting of S, R, H, N, K, H, T, A, and Q;

Vb(186) is selected from the group consisting of Q, T, I, V, L, A, C, H, K, R, N, M, E, D, S, and deletion;

Vb(187) is selected from the group consisting of A, E, H, W, Q, M, D, L, R, N, and K;

Vb(188) is selected from the group consisting of P, D, N, H, E, M, C, Q, K, T, A, V, S, and G;

Vb(189) comprises any amino acid;

Vb(190) is selected from the group consisting of I, Y, A, C, S, E, and V;

Vb(191) comprises any amino acid;

Vb(192) comprises any amino acid;

Vb(193) is selected from the group consisting of L and an insertion;

Vb(194) is selected from the group consisting of C, A, Q, K, and M;

Vb(195) is selected from the group consisting of L and P;

Vb(196) comprises any amino acid;

Vb(197) comprises any amino acid;

Vb(198) is selected from the group consisting of C, E, D, N, Q, and P;

Vb(199) comprises any amino acid;

Vb(200) is selected from the group consisting of R, D, M, S, Q, H, N, and a deletion;

Vb(201) is selected from the group consisting of F, R, H, E, N, Q, D, K, S, T, and G;

Vb(202) is selected from the group consisting of E and a deletion;

Vb(203) is selected from the group consisting of R, Y, F, T, W, A, S, D, C, I, E, N, H, and Q;

Vb(204) is selected from the group consisting of I, E, Q, S, V, P, L, M, T, Q, and C;

Vb(205) comprises any amino acid;

Vb(206) is selected from the group consisting of L, Q, M, and E;

Vb(207) is selected from the group consisting of R, C, E, and N;

Vb(208) is selected from the group consisting of A and D;

Vb(209) is selected from the group consisting of C, S, and A;

Vb(210) is selected from the group consisting of N, Q, C, and E;

Vb(211) is selected from the group consisting of C, D, Y, and E;

Fx(212) comprises the human amino acid sequence of CD40L at position 212;

Vb(213) is selected from the group consisting of S, D, and A;

Vb(214) is selected from the group consisting of S, P, R, D, N, A, E, and Q;

Vb(215) is selected from the group consisting of C, G, A, and S;

Vb(216) is selected from the group consisting of K and D;

Vb(217) is selected from the group consisting of P, D, E, Q, S, N, G, Y, H, F, and A;

Vb(218) is selected from the group consisting of C, P, D, G, and a deletion;

Vb(219) comprises any amino acid;

Vb(220) is selected from the group consisting of Q, P, C, T, E, D, M, N, and a deletion;

Vb(221) is selected from the group consisting of Q, L, Y, M, E, S, D, F, and a deletion;

Vb(222) is selected from the group consisting of S, A, T, and C;

Vb(223) is selected from the group consisting of P, E, D, S, Q, N, R, and G;

Vb(224) is selected from the group consisting of H, Y, and F;

Vb(225) comprises any amino acid;

Vb(226) is selected from the group consisting of G and A;

Vb(227) is selected from the group consisting of G and V;

Vb(228) comprises any amino acid;

Vb(229) comprises any amino acid;

Vb(230) is selected from the group consisting of E, P, H, M, and Q;

Vb(231) is selected from the group consisting of L and S;

Vb(232) is selected from the group consisting of Q, D, E, H, N, Y, V, S, and a deletion;

Vb(233) comprises any amino acid;

Fx(234-241) comprises the human amino acid sequence of CD40L at positions 234-241;

Vb(242) comprises any amino acid;

Vb(243) comprises any amino acid;

Vb(244) comprises any amino acid;

Vb(245) is selected from the group consisting of S, R, P, H, M, V, L, Y, and I;

Vb(246) comprises any amino acid;

Vb(247) comprises any amino acid;

Vb(248) is selected from the group consisting of S and C;

Vb(249) is selected from the group consisting of H and D;

Vb(250) comprises any amino acid;

Vb(251) is selected from the group consisting of T, E, Q, M, D, P, H, I, L, R, V, N, A, S, and G;

Vb(252) comprises any amino acid;

Vb(253) is selected from the group consisting of F, E, D, H, Y, and M;

Vb(254) is selected from the group consisting of T and M;

Vb(255) is selected from the group consisting of S, U, C, T, and A;

Fx(256-258) comprises the human amino acid sequence of CD40L at positions 256-258;

Vb(259) comprises any amino acid;

Vb(260) is selected from the group consisting of K, S, A, C, Q, M, N, and E;

Vb(261) is selected from the group consisting of L, N, S, D, V, I, T, C, and A.

wherein at least one of said amino acids at at least one of said positions is the not the wild-type amino acid.

2 . A composition according to claim 1 wherein said variant comprises a polymer.

3 . A composition according to claim 2 wherein said polymer comprises polyethylene glycol (PEG).

4 . A composition according to claim 3 wherein said PEG is attached at an amino acid position selected from the group consisting of 116, 117, 118, 120, 121, 130, 132, 133, 134, 143, 145, 151, 157, 168, 182, 183, 185, 186, 187, 198, 199, 201, 203, 209, 211, 213, 215, 217, 218, 220, 224, 228, 230, 233, 245, 251, 252 and 261.

5 . A composition according to claims 1 to 4 wherein said composition comprises a TNF-a trimer.

6 . A composition according to claim 5 wherein said trimer comprises three variant monomers according to said formula.

7 . A composition according to claim 5 wherein said trimer comprises three identical variant monomers.

8 . A composition according to claim 5 wherein said trimer comprises at least one variant monomer and at least one wild-type monomer.

9 . A composition according to claims 1 to 4 comprising two of said variant monomers covalently attached via a linker.

10 . A composition according to claim 9 wherein said linker is a polypeptide linker.

11 . A composition according to claim 10 wherein said polypeptide linker is attached to the N-terminus of a first monomer and the C-terminus of a second monomer.

12 . A composition according to claim 1 wherein Vb positions 141, 151, 157, 167, 172, 177, 179, 189, 191, 192, 196, 197, 199, 205, 219, 225, 228, 229, 233, 242, 243, 244, 246, 247, 250, 252 and 259 are human wild-type amino acids.

13 . A composition according to claim 1 further comprising a pharmaceutical carrier.

14 . A method of treating a CD40L related disorder comprising administering to said patient a composition according to claim 1 .

15 . A method according to claim 14 wherein said composition comprises a CD40L trimer.

16 . A method according to claim 15 wherein said trimer comprises three variant monomers according to said formula.

17 . A method according to claim 16 wherein said trimer comprises three identical variant monomers.

18 . A method according to claim 15 wherein said trimer comprises at least one variant monomer and at least one wild-type monomer.

19 . A method according to claim 14 comprising two of said variant monomers covalently attached via a linker.

20 . A method according to claim 14 wherein said variant comprises at least one PEG molecule.

21 . A composition comprising a PEGylated variant CD40L protein having the formula:

wherein

Fx(45-115) is optionally present, and if present, comprises the human amino acid sequence of CD40L at positions 45-115;

Vb(116) is selected from the group consisting of G and C;

Vb(117) is selected from the group consisting of D and C;

Vb(118) is selected from the group consisting of Q, C, H, E, N, A, T, S, W, F, V, G, K, R, Y, D, and M;

Vb(119) is selected from the group consisting of N, H, Q, L, N, R, D, A, S, M, K, G, and E;

Vb(120) is selected from the group consisting of P, C, R, Q, N, E, D, K, G, and S;

Vb(121) is selected from the group consisting of Q, H, T, V, Y, M, C, D, A, E, I, N, F, L, S, and G;

Fx(122) comprises the human amino acid sequence of CD40L at position 122;

Vb(123) is selected from the group consisting of A and E;

Fx(124) comprises the human amino acid sequence of CD40L at position 124;

Vb(125) is selected from the group consisting of H, R, N, and M;

Fx(126) is selected from the group consisting of V, D, and A;

Vb(127) is selected from the group consisting of H, Q, E, C, S, D, and I;

Vb(128) is selected from the group consisting of S and R;

Vb(129) is selected from the group consisting of E and G;

Vb(130) is selected from the group consisting of P, Q, K, M, N, C, R, E, D, and A;

Fx(131) comprises the human amino acid sequence of CD40L at position 131;

Vb(132) is selected from the group consisting of C and S;

Vb(133) is selected from the group consisting of C and K;

Vb(134) is selected from the group consisting of C, Q, E, D, R, N, K, S, G, and T;

Fx(135-136) comprises the human amino acid sequence of CD40L at positions 135-136;

Vb(137) is selected from the group consisting of R, K, S, and V;

Fx(138-139) comprises the human amino acid sequence of CD40L at positions 138-139;

Vb(140) is selected from the group consisting of W, C, G, R, and a deletion;

Vb(141) comprises any amino acid;

Vb(142) is selected from the group consisting of M, V, C, I, E, and a deletion;

Vb(143) is selected from the group consisting of C, E, K, and a deletion;

Vb(144) is selected from the group consisting of Q, M, E, H, K, R, and G;

Vb(145) is selected from the group consisting of P, E, N, C, D, H, Y, and a deletion;

Vb(146) is selected from the group consisting of S, C, and N;

Vb(147) is selected from the group consisting of T, V, I, and N;

Vb(148) is selected from the group consisting of M and K;

Vb(149) is selected from the group consisting of S and A;

Fx(150) comprises the human amino acid sequence of CD40L at position 150;

Vb(151) comprises any amino acid;

Vb(152) is selected from the group consisting of L, E, and Q;

Fx(153-154) comprises the human amino acid sequence of CD40L at positions 153-154;

Vb(155) is selected from the group consisting of L and P;

Fx(156) comprises the human amino acid sequence of CD40L at position 156;

Vb(157) comprises any amino acid;

Fx(158-165) comprises the human amino acid sequence of CD40L at positions 158-165;

Vb(166) is selected from the group consisting of Q, S, G, C, D, Q, E, A, and N;

Vb(167) comprises any amino acid;

Vb(168) is selected from the group consisting of C, P, and L;

Vb(169) is selected from the group consisting of Y and D;

Vb(170) is selected from the group consisting of Y, C, and P;

Fx(171) comprises the human amino acid sequence of CD40L at position 171;

Vb(172) comprises any amino acid;

Vb(173) is selected from the group consisting of A and D;

Vb(174) is selected from the group consisting of Q, R, I, V, L, H, and K;

Fx(175) comprises the human amino acid sequence of CD40L at position 175;

Vb(176) is selected from the group consisting of T, G, I, A, S, and G;

Vb(177) comprises any amino acid;

Vb(178) is selected from the group consisting of C, G, S, D, E, H, Q, N, K, Y, M, and R;

Vb(179) comprises any amino acid;

Fx(180) comprises the human amino acid sequence of CD40L at position 180;

Vb(181) is selected from the group consisting of R, G, S, C, and A;

Vb(182) is selected from the group consisting of E and C;

Vb(183) is selected from the group consisting of A, Q, E, R, D, N, K, S, G, and an insertion;

Vb(184) is selected from the group consisting of S, C, L, R, M, and deletion;

Vb(185) is selected from the group consisting of S, R, H, N, K, H, T, A, and Q;

Vb(186) is selected from the group consisting of Q, T, I, V, L, A, C, H, K, R, N, M, E, D, S, and deletion;

Vb(187) is selected from the group consisting of A, E, H, W, Q, M, D, L, R, N, and K;

Vb(188) is selected from the group consisting of P, D, N, H, E, M, C, Q, K, T, A, V, S, and G;

Vb(189) comprises any amino acid;

Vb(190) is selected from the group consisting of I, Y, A, C, S, E, and V;

Vb(191) comprises any amino acid;

Vb(192) comprises any amino acid;

Vb(193) is selected from the group consisting of L and an insertion;

Vb(194) is selected from the group consisting of C, A, Q, K, and M;

Vb(195) is selected from the group consisting of L and P;

Vb(196) comprises any amino acid;

Vb(197) comprises any amino acid;

Vb(198) is selected from the group consisting of C, E, D, N, Q, and P;

Vb(199) comprises any amino acid;

Vb(200) is selected from the group consisting of R, D, M, S, Q, H, N, and a deletion;

Vb(201) is selected from the group consisting of F, R, H, E, N, Q, D, K, S, T, and G;

Vb(202) is selected from the group consisting of E and a deletion;

Vb(203) is selected from the group consisting of R, Y, F, T, W, A, S, D, C, I, E, N, H, and Q;

Vb(204) is selected from the group consisting of I, E, Q, S, V, P, L, M, T, Q, and C;

Vb(205) comprises any amino acid;

Vb(206) is selected from the group consisting of L, Q, M, and E;

Vb(207) is selected from the group consisting of R, C, E, and N;

Vb(208) is selected from the group consisting of A and D;

Vb(209) is selected from the group consisting of C, S, and A;

Vb(210) is selected from the group consisting of N, Q, C, and E;

Vb(211) is selected from the group consisting of C, D, Y, and E;

Fx(212) comprises the human amino acid sequence of CD40L at position 212;

Vb(213) is selected from the group consisting of S, D, and A;

Vb(214) is selected from the group consisting of S, P, R, D, N, A, E, and Q;

Vb(215) is selected from the group consisting of C, G, A, and S;

Vb(216) is selected from the group consisting of K and D;

Vb(217) is selected from the group consisting of P, D, E, Q, S, N, G, Y, H, F, and A;

Vb(218) is selected from the group consisting of C, P, D, G, and a deletion;

Vb(219) comprises any amino acid;

Vb(220) is selected from the group consisting of Q, P, C, T, E, D, M, N, and a deletion;

Vb(221) is selected from the group consisting of Q, L, Y, M, E, S, D, F, and a deletion;

Vb(222) is selected from the group consisting of S, A, T, and C;

Vb(223) is selected from the group consisting of P, E, D, S, Q, N, R, and G;

Vb(224) is selected from the group consisting of H, Y, and F;

Vb(225) comprises any amino acid;

Vb(226) is selected from the group consisting of G and A;

Vb(227) is selected from the group consisting of G and V;

Vb(228) comprises any amino acid;

Vb(229) comprises any amino acid;

Vb(230) is selected from the group consisting of E, P, H, M, and Q;

Vb(231) is selected from the group consisting of L and S;

Vb(232) is selected from the group consisting of Q, D, E, H, N, Y, V, S, and a deletion;

Vb(233) comprises any amino acid;

Fx(234-241) comprises the human amino acid sequence of CD40L at positions 234-241;

Vb(242) comprises any amino acid;

Vb(243) comprises any amino acid;

Vb(244) comprises any amino acid;

Vb(245) is selected from the group consisting of S, R, P, H, M, V, L, Y, and I;

Vb(246) comprises any amino acid;

Vb(247) comprises any amino acid;

Vb(248) is selected from the group consisting of S and C;

Vb(249) is selected from the group consisting of H and D;

Vb(250) comprises any amino acid;

Vb(251) is selected from the group consisting of T, E, Q, M, D, P, H, I, L, R, V, N, A, S, and G;

Vb(252) comprises any amino acid;

Vb(253) is selected from the group consisting of F, E, D, H, Y, and M;

Vb(254) is selected from the group consisting of T and M;

Vb(255) is selected from the group consisting of S, U, C, T, and A;

Fx(256-258) comprises the human amino acid sequence of CD40L at positions 256-258;

Vb(259) comprises any amino acid;

Vb(260) is selected from the group consisting of K, S, A, C, Q, M, N, and E;

Vb(261) is selected from the group consisting of L, N, S, D, V, I, T, C, and A.

wherein at least one PEG molecule is attached at an amino acid position selected from the group consisting of 116, 117, 118, 120, 121, 130, 132, 133, 134, 143, 145, 151, 157, 168, 182, 183, 185, 186, 187, 198, 199, 201, 203, 209, 211, 213, 215, 217, 218, 220, 224, 228, 230, 233, 245, 251, 252 and 261.

22 . A method of making a PEGylated CD40L molecule comprising:

a) substituting a cysteine at at least one position selected from the group consisting of 116, 117, 118, 120, 121, 130, 132, 133, 134, 143, 145, 151, 157, 168, 182, 183, 185, 186, 187, 198, 199, 201, 203, 209, 211, 213, 215, 217, 218, 220, 224, 228, 230, 233, 245, 251, 252 and 261 ; and

b) adding a PEG molecule to said position.

23 . A composition according to claim 6 wherein said trimer comprises three identical variant monomers.

Assignments (1)

ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Apr 15, 2005

From: CHAMBERLAIN, AARON KEITH; DESJARLAIS, JOHN R.; MOORE, GREGORY L.

To: XENCOR, INC.

Reel/Frame 016465/0671 →