# Patent

## Title
Mutant antibodies and conjugation thereof

## Abstract

The present invention relates to a polypeptide comprising 7 β-strands A, B, C, D, E, F, and G sequentially connected together by connecting chains of amino acids, and a first α-helix sequentially located on the EF chain between β-strands E and F, wherein the β-strands are arranged so as to form a first β-sheet comprising β-strands A, B, D, and E, and a second β-sheet comprising β-strands C, F and G, said first and second β-sheets being covalently bonded together so as to form a first Ig domain; wherein the EF chain between β-strands E and F comprises the sequence X1-X2-X3-X4-K5H6 (SEQ ID NO:98), and X1, X3 and X4 are each independently any amino acid residue, characterized in that X2 is selected from the group consisting of A, G, I, V, L, R, S, T, Q, P, N, M, H, W, and pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, and prodrugs thereof.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 13/736,029, filed Jan. 7, 2013, which claims the benefit of U.S. Provisional Patent Application No. 61/584,675, filed Jan. 9, 2012. The entire contents of each of the above-referenced patent applications are hereby incorporated by reference in their entirety.


BACKGROUND
The development of bifunctional therapeutics has great potential to augment combination therapy strategies. A bifunctional therapeutic can provide the benefit of a combination therapy by modulating 2 different pathways with one therapeutic entity. In addition, bifunctional therapeutics may also benefit from synergies between pathways and demonstrate increased activity compared to mono-functional agents. Furthermore, bifunctional therapeutics can provide benefits in terms of reduced manufacturing, storage, and shipping costs, as well as reducing the number of therapies given to the patient and simplifying dosage regimes.
The reference to any art in this specification is not, and should not be taken as, an acknowledgement of any form or suggestion that the referenced art forms part of the common general knowledge.
REFERENCE TO SEQUENCE LISTING
This application is being filed electronically via EFS-Web and includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled “PC71862B_Sequence_Listing_ST25.txt” created on Jan. 29, 2021 and having a size of 265 kb. The sequence listing contained in this .txt file is part of the specification and is herein incorporated by reference in its entirety.
SUMMARY OF THE INVENTION
The present invention provides a polypeptide comprising an antibody constant domain, the antibody constant domain comprising residues K and H at positions corresponding to positions 80 and 81 of SEQ ID NO:6 when said antibody constant domain is aligned with the sequence of SEQ ID NO:6, and characterized in that the antibody constant domain further comprises a residue selected from the group consisting of A, G, I, V, L, R, S, T, Q, P, N, M, H, W at a position corresponding to position 77 of SEQ ID NO:6.
In some aspects, the invention provides an antibody constant domain comprising SEQ ID NO:98 (and all sequences herein described that fall within the scope of SEQ ID NO:98), wherein the position of SEQ ID NO:98 on said constant domain corresponds to residues 76-81 of SEQ ID NO:6 when the constant domain sequence is aligned with SEQ ID NO:6.
Sequences may be aligned by structural alignment, where the structure of the two polypeptides are known, or by sequence alignment; when sequence alignment is used, the method is preferably augmented using structural knowledge of homologous polypeptides whose structures are known.
The present invention provides a polypeptide comprising an antibody constant light domain, the antibody constant domain comprising residues K188 and H181 according to Kabat numbering, and characterized in that the antibody constant domain further comprises a residue selected from the group consisting of A, G, I, V, L, R, S, T, Q, P, N, M, H, W at a position corresponding to position 185 according to Kabat numbering.
The present invention provides a polypeptide comprising an immunoglobulin domain comprising 7 β-strands A, B, C, D, E, F, and G sequentially connected together by chains of connecting amino acids, wherein the β-strands are arranged so as to form a first β-sheet comprising β-strands A, B, D, and E, and a second β-sheet comprising β-strands C, F and G, said first and second β-sheets being covalently bonded together; wherein β-strands E and F are connected together by an EF chain, and said EF chain comprises the sequence, X1-X2-X3-X4-K5-H6 (SEQ ID NO:98), and wherein X1, X3 and X4 are each independently any amino acid residue, and characterized in that X2 is selected from the group consisting of A, G, I, V, L, R, S, T, Q, P, N, M, H, W, and pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, and prodrugs thereof.
X2 may be selected from the group consisting of A, G, I, L, R, S, T, P, N, and M (SEQ ID NO:99). X2 may be selected from the group consisting of A, G, I, L, S, T, P, and M (SEQ ID NO:100). In some aspects, the EF chain comprises a sequence selected from the group consisting of sequence SEQ ID NO:101, SEQ ID NO:102, and SEQ ID NO:103. X2 may be selected from the group consisting of A, G, I, V, L, R, S, T, Q, N, P, and M (SEQ ID NO:123). X2 may be selected from the group consisting of A, G, I, V, L, R, S, T, P, and M (SEQ ID NO:124). X2 may be selected from the group consisting of A, G, I, V, L, S, T, and M (SEQ ID NO:125). X2 may be selected from the group consisting of A, G, I, L, S, T, and M (SEQ ID NO:126). X2 may be S or T. X2 may be A or G. X2 may be I or L. The selections of X2 described herein may also be applied to antibody constant domains of the invention, wherein position X2 corresponds with residue 77 of SEQ ID NO:6, or residue 185 of a constant light domain according to Kabat numbering.
In some aspects, the EF chain comprises a sequence selected from the group consisting of SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, and SEQ ID NO:126, and may further be selected from the group consisting of SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, and SEQ ID NO:224.
In some aspects, there may be a single residue difference when compared against residues 75-79 of SEQ ID NO:10; or SEQ ID NO:6. In some aspects, there may be up to 2 differences when compared against residues 75-79 of SEQ ID NO:10; or SEQ ID NO:6. In some aspects, there may be up to 2 differences when compared against residues 75-79 of SEQ ID NO:10; or SEQ ID NO:6. In some aspects, there may be up to 4 differences when compared against residues 75-79 of SEQ ID NO:10; or SEQ ID NO:6. In some aspects, there may be up two non-sequential differences when compared against residues 75-79 of SEQ ID NO:10; or SEQ ID NO:6. In some aspects, there may be up to three non-sequential differences when compared against residues 75-79 of SEQ ID NO:10; or SEQ ID NO:6.
In some aspects, there may be a single residue difference when compared against residues 76-79 of SEQ ID NO:10; or SEQ ID NO:6. In some aspects, there may be up to 2 differences when compared against residues 76-79 of SEQ ID NO:10; or SEQ ID NO:6. In some aspects, there may be up to 3 differences when compared against residues 76-79 of SEQ ID NO:10; or SEQ ID NO:6. In some aspects, there may be up to 4 differences when compared against residues 76-79 of SEQ ID NO:10; or SEQ ID NO:6. In some aspects, there may be up two non-sequential differences when compared against residues 76-79 of SEQ ID NO:10; or SEQ ID NO:6.
The polypeptide may comprise an EF α-helix located on the EF chain. In some aspects, one or more of residues X1, X2, X3 and X4 of SEQ ID NO:98 comprise part of the EF α-helix. In some aspects, two or more of residues X1, X2, X3 and X4 of SEQ ID NO:98 comprise part of the EF α-helix. In some aspects, three or more of residues X1, X2, X3 and X4 of SEQ ID NO:98 comprise part of the EF α-helix. In some aspects, all residues X1, X2, X3 and X4 of SEQ ID NO:98 comprise part of the EF α-helix. In some aspects, residues K5 and H6 of SEQ ID NO:98 do not form part of an α-helix. The selections of X1, X2, X3, X4, K5, and H6 described herein may also be applied to antibody constant domains of the invention, wherein positions X1, X2, X3, X4, K5, and H6 correspond with residues 76, 77, 78, 79, 800 and 81 of SEQ ID NO:6, or positions 184, 185, 186, 187, 188 and 189 of a constant light domain according to Kabat numbering.
In some aspects, the EF chain comprises a sequence selected from the group consisting of SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, and SEQ ID NO:224.
In some aspects of the invention, and with particular reference to all sequences in the application with variability at a residue corresponding to X1, X1 according to the above sequences may be selected from the group consisting of A, I, V, L, G, P, F, W, Y, S, T, C, M, N, Q, K, R, H, E, and D. In some aspects, X1 may be selected from the group consisting of A, I, V, L, G, F, W, Y, S, T, C, M, N, Q, K, R, H, E, and D. In some aspects, X1 may be selected from the group consisting of A, I, V, L, F, W, Y, S, T, C, M, N, Q, K, R, H, E, and D. In some aspects, X1 may be selected from the group consisting of A, I, V, L, F, W, Y, S, T, M, N, Q, K, R, H, E, and D. In some aspects, X1 may be selected from the group consisting of A, I, V, L, S, T, M, N, Q, K, R, H, E, and D. In some aspects, X1 may be selected from the group consisting of A, I, V, L, S, T, M, N, Q, R, H, E, and D. X1 may be selected from the group consisting of A, I, V, L, S, T, M, N, Q, E, and D. X1 may be selected from the group consisting of A, I, V, L, S, T, M, N, Q, E, and D. The selections of X1 described herein may also be applied to antibody constant domains of the invention, wherein position X1 corresponds with residue 76 of SEQ ID NO:6, or residue 184 of a constant light domain according to Kabat numbering.
In some aspects of the invention, and with particular reference to all sequences in the application with variability at a residue corresponding to X3, X3 according to the above sequences may be selected from the group consisting of A, I, V, L, G, P, F, W, Y, S, T, C, M, N, Q, K, R, H, E, and D. X3 may be selected from the group consisting of A, I, V, L, G, F, W, Y, S, T, C, M, N, Q, K, R, H, E, and D. X3 may be selected from the group consisting of A, I, V, L, F, W, Y, S, T, C, M, N, Q, K, R, H, E, and D. X3 may be selected from the group consisting of A, I, V, L, F, W, Y, S, T, M, N, Q, K, R, H, E, and D. X3 may be selected from the group consisting of I, L, F, W, Y, S, T, M, N, Q, K, R, H, E, and D. X3 may be selected from the group consisting of I, L, F, W, Y, M, N, Q, K, R, H, E, and D. X3 may be selected from the group consisting of I, L, F, W, Y, N, Q, E, and D. X3 may be selected from the group consisting of I, L, F, W, and Y. X3 may be selected from the group consisting of F, W, and Y. X3 may be selected from the group consisting of W and Y. The selections of X3 described herein may also be applied to antibody constant domains of the invention, wherein position X3 corresponds with residue 78 of SEQ ID NO:6, or residue 186 of a constant light domain according to Kabat numbering.
In some aspects of the invention, and with particular reference to all sequences in the application with variability at a residue corresponding to X4, X4 according to the above sequences may be selected from the group consisting of A, I, V, L, G, P, F, W, Y, S, T, C, M, N, Q, K, R, H, E, and D. X4 may be selected from the group consisting of A, I, V, L, G, P, F, W, Y, S, T, M, N, Q, K, R, H, E, and D. X4 may be selected from the group consisting of A, I, V, L, G, F, W, Y, S, T, M, N, Q, K, R, H, E, and D. X4 may be selected from the group consisting of A, I, V, L, F, W, Y, S, T, M, N, Q, K, R, H, E, and D. X4 may be selected from the group consisting of A, I, V, L, G, S, T, M, N, Q, K, R, H, E, and D. X4 may be selected from the group consisting of A, I, V, L, S, T, M, N, Q, K, R, H, E, and D. X4 may be selected from the group consisting of I, L, S, T, M, N, Q, K, R, E, and D. X4 may be selected from the group consisting of S, T, M, N, Q, K, R, E, and D. X4 may be selected from the group consisting of S, T, N, Q, K, R, E, and D. X4 may be selected from the group consisting of N, Q, K, R, E, and D. X4 may be selected from the group consisting of N, Q, K, R, and E. X4 may be selected from the group consisting of, Q, K, and E. The selections of X4 described herein may also be applied to antibody constant domains of the invention, wherein position X4 corresponds with residue 79 of SEQ ID NO:6, or residue 187 of a constant light domain according to Kabat numbering.
In some aspects, the EF chain is between 6 and 12 residues long. In some aspects, the EF chain is between 7 and 12 residues long. In some aspects, the EF chain is between 8 and 12 residues long. In some aspects, the EF chain is between 9 and 12 residues long. In some aspects, the EF chain is between 6 and 11 residues long. In some aspects, the EF chain is between 6 and 10 residues long. In some aspects, the EF chain is between 6 and 9 residues long. In some aspects, the EF chain is between 7 and 11 residues long. In some aspects, the EF chain is between 7 and 10 residues long. In some aspects, the EF chain is between 8 and 10 residues long.
The EF chain may comprise an α-helix (the EF α-helix). The first residue of the EF α-helix may be located within the first 3 residues of the EF chain. The first residue of the EF α-helix may be located within the first 2 residues of the EF chain. The first residue of the EF α-helix may be located at residue of the EF chain. The EF α-helix may comprise at least residues X1 and X2 of SEQ ID NO:98 or one of the corresponding sequences herein that fall within the scope of SEQ ID NO:98. In some aspects, residues K5 and H6 corresponding to SEQ ID NO:98 are not within the α-helix. In some aspects, residues KH that correspond to positions 80 and 81 of SEQ ID NO:6 are not within the α-helix. In some aspects of the invention relating to antibody constant domains, the residue corresponding to position 77 of SEQ ID NO:6 falls within an α-helix.
In some aspects, the immunoglobulin domain of the invention, which may be a CL domain, further comprises the residue D, E, Q or N on the connecting chain between β-strands C and D; the CD chain. In some aspects, the CL domain further comprises the residue D, E, Q or N on the CD chain, the residue being positioned so as to allow its amino acid side chain interact with at least one of the side chains of K5 or H6 of SEQ ID NO:98. In some aspects, the polypeptide comprises a D, E, Q, or N residue located at the position corresponding to position 43 of SEQ ID NO:10 or SEQ ID NO:6, in some aspects, according to a BLAST sequence alignment. In some aspects, the residue is D, E or N. In some aspects, the residue is D or E. In some aspects the residue is D or N.
In some aspects, the β-strands C and D are connected together by a CD chain, comprising a CD motif C1-C2-C3-C4 (SEQ ID NO:255) wherein each of C1, C2, C3 and C4 may be any amino acid, or further specified as set forth below. The CD motif may be selected from the group consisting of SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, and SEQ ID NO:253, said CD motif beginning at the first or second residue of said CD chain. In some aspects, the CD motif begins at the first residue of the CD chain. In some aspects, the CD motif begins at the second residue of the CD chain. Favourably, the CD motif may not form part of an α-helix.
In some aspects, the residue C1 of the CD motif may be selected from the group consisting of A, I, L, G, V, M, P, S, T, F, Y, W, N, Q, D, and E. In some aspects, the residue C1 of the CD motif may be selected from the group consisting of I, L, V, M, P, F, Y, and W. In some aspects, the residue C1 of the CD motif may be selected from the group consisting of I, L, V, P, F, Y, and W. In some aspects, the residue C1 of the CD motif may be selected from the group consisting of I, L, V, P, F, and W. In some aspects, the residue C1 of the CD motif may be selected from the group consisting of I, L, V, F, and W In some aspects, the residue C1 of the CD motif may be selected from the group consisting of I, L, and V. In some aspects, the residue C1 of the CD motif may be selected from the group consisting of L and V. In some aspects, the residue C1 of the CD motif may be V. The selections of C1 of the CD motif described herein may also be applied to antibody constant domains of the invention, wherein position C1 of the CD motif corresponds with residue 42 of SEQ ID NO:6, or residue 150 of a constant light domain according to Kabat numbering.
In some aspects, the residue C2 of the CD motif may be selected from the group consisting of A, I, L, G, V, M, P, S, T, F, Y, W, N, Q, D, and E. In some aspects, the residue C2 of the CD motif may be selected from the group consisting of A, I, L, G, V, M, P, S, T, F, Y, W, N, Q, D, and E. In some aspects, the residue C2 of the CD motif may be selected from the group consisting of A, I, L, G, V, M, P, S, T, N, Q, D, and E. In some aspects, the residue C2 of the CD motif may be selected from the group consisting of A, I, L, V, M, P, S, T, N, Q, D, and E. In some aspects, the residue C2 of the CD motif may be selected from the group consisting of M, P, S, T, N, Q, D, and E. In some aspects, the residue C2 of the CD motif may be selected from the group consisting of S, T, N, Q, D, and E. In some aspects, the residue C2 of the CD motif may be selected from the group consisting of N, Q, D, and E. In some aspects, the residue C2 of the CD motif may be D. The selections of C2 of the CD motif described herein may also be applied to antibody constant domains of the invention, wherein position C2 of the CD motif corresponds with residue 43 of SEQ ID NO:6, or residue 151 of a constant light domain according to Kabat numbering.
In some aspects, the residue C3 of the CD motif may be selected from the group consisting of A, I, L, G, V, M, P, S, T, F, Y, W, N, Q, D, and E. In some aspects, the residue C3 of the CD motif may be selected from the group consisting of A, I, L, G, V, M, P, S, T, F, Y, W, N, Q, D, and E. In some aspects, the residue C3 of the CD motif may be selected from the group consisting of A, I, L, G, V, M, P, S, T, N, Q, D, and E. In some aspects, the residue C3 of the CD motif may be selected from the group consisting of A, I, L, V, M, P, S, T, N, Q, D, and E. In some aspects, the residue C3 of the CD motif may be selected from the group consisting of M, P, S, T, N, Q, D, and E. In some aspects, the residue C3 of the CD motif may be selected from the group consisting of S, T, N, Q, D, and E. In some aspects, the residue C3 of the CD motif may be selected from the group consisting of N, Q, D, and E. In some aspects, the residue C3 of the CD motif may be N. The selections of C3 of the CD motif described herein may also be applied to antibody constant domains of the invention, wherein position C3 of the CD motif corresponds with residue 44 of SEQ ID NO:6, or residue 152 of a constant light domain according to Kabat numbering.
In some aspects, the residue C4 of the CD motif may be selected from the group consisting of A, I, L, G, V, M, P, S, T, F, Y, W, N, Q, D, and E. In some aspects, the residue C4 of the CD motif may be selected from the group consisting of A, I, L, G, V, M, P, S, T, F, Y, W, N, Q, D, and E. In some aspects, the residue C4 of the CD motif may be selected from the group consisting of A, I, L, G, V, M, P, S, T, N, Q, D, E, K, R, and H. In some aspects, the residue C4 of the CD motif may be selected from the group consisting of A, I, L, V, G, M, P, S, T, N, Q, D, and E. In some aspects, the residue C4 of the CD motif may be selected from the group consisting of A, I, L, V, G, S, T, N, Q, D, and E. In some aspects, the residue C4 of the CD motif may be selected from the group consisting of A, V, Q, and S. In some aspects, the residue C4 of the CD motif may be selected from the group consisting of A and S. In some aspects, the residue C4 of the CD motif may be A. The selections of C4 of the CD motif described herein may also be applied to antibody constant domains of the invention, wherein position C4 of the CD motif corresponds with residue 45 of SEQ ID NO:6, or residue 153 of a constant light domain according to Kabat numbering.
In some aspects, the CD chain is between 6 and 12 residues long. In some aspects, the CD chain is between 7 and 12 residues long. In some aspects, the CD chain is between 8 and 12 residues long. In some aspects, the CD chain is between 9 and 12 residues long. In some aspects, the CD chain is between 6 and 11 residues long. In some aspects, the CD chain is between 6 and 10 residues long. In some aspects, the CD chain is between 6 and 9 residues long. In some aspects, the CD chain is between 7 and 11 residues long. In some aspects, the CD chain is between 7 and 10 residues long. In some aspects, the CD chain is between 8 and 10 residues long.
The immunoglobulin domain may be an antibody domain. The antibody domain may be an antibody constant domain. The antibody constant domain may be a constant heavy (CH) domain or constant light (CL) domain. Antibody CH domains may be selected from the group consisting of CHα1, CHα2, CHα3, CHδ1, CHδ2, CHδ3, CHε1, CHε2, CHε3, CHε4, CHγ1, CHγ2, CHγ3, CHμ1, CHμ2, CHμ3, and CHμ4.
In some aspects, the immunoglobulin domains of the invention are mammalian in origin (notwithstanding the method used to generate any artificially mutated or otherwise engineered versions). The mammalian species may be human, mouse, rabbit, rat, rodent, pig, cow, sheep, goat, donkey, horse, camel, primate, monkey, dog, or cat. The immunoglobulin domains of the invention, and other proteins such as antibodies to which they comprise or attached may be humanized.
In some aspects, the invention comprises mutant immunoglobulin domains, wherein a mutant is defined as sequence that has been engineered or altered to a sequence other than its natural canonical sequence, such that certain embodiments of polypeptides of the invention specifically excludes naturally occurring sequences that fall within the scope of the definition. In some aspects, therefore, the present invention relates to polypeptides of the invention comprising an EF chain that differs from their naturally occurring corresponding sequence.
The antibody domains of the invention may specifically exclude one or more natural IgA constant heavy domains (CHα1, CHα2, CHα3) from one or more species selected from the group consisting of Bornean orangutan and Pongo pygmaeus, and/or one or more natural IgM constant heavy domains (CHμ1, CHμ2, CHμ3, and CHμ4) from one or more species selected from the group consisting of mouse, rat, horse, Equus caballus, Heterocephalus glaber, bat, Eptesicus fuscus, and/or one or more natural IgE constant heavy domains (CHε1, CHε2, CHε3, and CHε4) from one or more species selected from the group consisting of human, chimp, monkey, Erythrocebus patas, mouse, rat, bat, Cynopterus sphinx, sheep, Ovis aries, echidna, and Tachyglossus aculeatus. 
In some aspects, one or more constant heavy domains selected from the group consisting of CHα1, CHα2, CHα3, CHδ1, CHδ2, CHδ3, CHε1, CHε2, CHε3, CHε4, CHγ1, CHγ2, CHγ3, CHμ1, CHμ2, CHμ3, and CHμ4 from one or more of the species selected from the group consisting of human, mouse, rabbit, rat, rodent, pig, cow, sheep, goat, donkey, horse, camel, primate, monkey, dog, or cat are specifically excluded.
In some aspects, the invention provides an immunoglobulin domain, that may be a constant light chain (CL) domain, comprising 7 β-strands A, B, C, D, E, F, and G sequentially connected together by chains of amino acids, wherein the β-strands are arranged so as to form a first β-sheet comprising β-strands A, B, D, and E, and a second β-sheet comprising β-strands C, F and G, said first and second β-sheets being covalently bonded together; wherein the chain between β-strands E and F comprises the sequence X1-X2-X3-X4-K5-H6 (SEQ ID NO:98), and X1, X3 and X4 are each independently any amino acid residue, and characterized in that X2 is selected from the group consisting of A, G, I, V, L, R, S, T, Q, P, N, M, H, and W, and pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, and prodrugs thereof. The invention also provides for pharmaceutical compositions and samples comprising CL domains of the invention. In some aspects, an α-helix is sequentially located on the connecting chain between β-strands E and F.
In part, aspects of the invention are based on the surprising discovery that site directed conjugation to a reactive KH group located on the EF chain of an immunoglobulin domain, that may be a constant light chain (CL) domain, is improved by a mutation 3 amino acid residues upstream that eliminates the presence of an acidic residue such as D or E, and that avoids introducing the aromatic residues F or Y, or other potential conjugation sites such as K or C.
Grafting a sequence of the invention onto the EF chain can impart increased specificity of conjugation on immunoglobulin domains, in particular, CL domains. This can be useful when conjugating Linkers and/or Effector Moieties onto immunoglobulin domains, and CL domains in general, and antibody and antigen-binding portions thereof in particular. In some aspects, therefore, the invention relates to a novel class of Multifunctional Antibody Conjugates (MACs), comprising an antibody, or antigen binding portion thereof, covalently conjugated to an Linker and/or Effector Moiety via a linker, characterized in that the antibody or antigen binding portion thereof comprises a polypeptide of the invention, and the linker is covalently bonded to the ε-amino group of the side chain of K5 of SEQ ID NO:98.
In some aspects, the immunoglobulin domain of the invention, that may be a CL domain, is connected to a variable light chain (VL) domain. Together, these may comprise an antibody light chain.
In some aspects, the covalent bond between the first and second β-sheets is a disulfide bond. In some aspects, the disulfide bond is between β-strands B and F.
The CL domain may be a constant light chain kappa (CLκ), and may be of rat, mouse, monkey, rabbit, goat, sheep, cow, pig, horse, donkey, dog, cat, or human origin. In some aspects, the CLκ comprises a sequence selected from the group consisting of SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, and SEQ ID NO:122.
In some aspects, the the CLκ comprises an N-terminal portion defined by SEQ ID NO:225 and a C′ terminal portion defined by SEQ ID NO:226 contiguously connected together by an intermediate sequence selected from the group consisting of SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, and SEQ ID NO:224.
The immunoglobulin domain may be a CLλ domain, and may comprise a sequence selected from the group consisting of SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:141, SEQ ID NO:144, SEQ ID NO:143, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242, SEQ ID NO:243, and SEQ ID NO:244. In some aspects, the CLλ domain comprises a sequence selected from the group consisting of SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:143, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242, SEQ ID NO:243, and SEQ ID NO:244.
The CLλ may comprise an N′ terminal portion defined by one of SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, or SEQ ID NO:233 contiguously connected together by an intermediate sequence to a C′ terminal portion defined by either of SEQ ID NO:234 or SEQ ID NO:235, the intermediate sequence being selected from the group consisting of SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, and SEQ ID NO:224.
In some embodiments where the immunoglobulin domain comprises a CLλ domain, the domain may further comprise a CD motif as described herein.
In some embodiments where the immunoglobulin domain comprises a CHγ1 domain, the domain may further comprise a CD motif as described herein.
In some embodiments where the immunoglobulin domain comprises a CHγ2 domain, the domain may further comprise a CD motif as described herein.
In some embodiments where the immunoglobulin domain comprises a CHγ3 domain, residue X2 of the EF chain may not be R; in some aspects, the EF chain sequence may be selected from the group consisting of SEQ ID NO:100, SEQ ID NO:103, SEQ ID NO:117, SEQ ID NO:125, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, and SEQ ID NO:224. IN some aspects, the CD chain of a CHγ3 domain of the invention may further comprise a CD motif as described herein.
When conjugated to a linker suitable for conjugation to an Effector Moiety, the ε-amino group of the side chain of K5 of SEQ ID NO:98 may be covalently attached to the linker.
In part, the invention is based on the surprising discovery that mutating CLκ-D77 to one of A, G, I, V, L, R, S, T, Q, P, N, M, H, or W provides a significant increase in the degree of specificity of conjugation to CLκ-K80.
Reaction of the Effector Moiety with the constant light domain of an antibody is particularly desirable to minimize, or prevent, any interference with binding of the Fc portion of the antibody to Fc receptors (such as FcγR and FcRn) or binding of the antibody to its respective target. Conversely, conjugation of the respective Effector Moiety to the Fc portion of an antibody may decrease the antibody half-life in vivo and/or its capacity to interact with the immune system (effector function). Conjugation of the Effector Moiety in the variable heavy chain (VH) or variable light chain (VL) region of the antibody carry a risk of diminishing the binding of the antibody to its cognate.
Preferential conjugation of the Effector Moiety to the CLκ or the constant light chain lambda (CLλ) domain simplifies the creation of MAC isotypes by allowing isotypic switches of the constant heavy chain (CH) domains of the antibody without affecting the conjugation sites of the Effector Moiety to the antibody.
The Linker and/or Effector Moiety may be covalently attached to the side chain of CLκ-K80 (for example, a sequence selected from the group consisting of SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, and SEQ ID NO:122.). The CL is located away from key regions of a typical antibody upon which it would form a part of, such as paratope region, FcRn binding domain, hinge, FcR binding domains; this provides the advantage that preferentially linking at these sites limits the amount of interference to antibody-antigen interaction when the MAC is conjugated to the Effector Moiety.
In some aspect, the CLκ region comprises at least residues 62-103 of SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, or SEQ ID NO:109, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, or SEQ ID NO:122. In some aspects, CLκ-x82 may be any amino acid. In some aspects, CLκ-x82 may be selected from the group consisting of K, R, G, A, V, L, I, S, T, C, M, N, Q, D, E, H, F, W and Y. In some aspects, CLκ-x82 may be G, A, V, L, or I. In some aspects, CLκ-x82 may be K, R, N, or Q. In some aspects, CLκ-x82 may be D, or E. In some aspects, CLκ-x82 may be K, R, G, A, V, L, I, N, or Q. In some aspects, CLκ-x82 may be D, or E. In some aspects, CLκ-x82 may be K, R, G, A, V, L, I, N, Q, D or E. In some aspects, CLκ-x82 may be D, or E. In some aspects, CLκ-x82 may be H, F, W or Y. In some aspects CLκ-x82 is not proline. In some aspects, CLκ-x82 is K. In some aspects, CLκ-x82 is R.
In some aspects, antibodies of the invention, or antigen-binding portions thereof, comprise an Effector Moiety conjugated to K5 of SEQ ID NO:98 on both light chains. In some aspects, the Effector Moiety is conjugated to K5 of SEQ ID NO:98 on one light chain only. In some aspects, the Effector Moiety is only conjugated to K5 of SEQ ID NO:98. In some aspects, the Effector Moiety is conjugated at K5 of SEQ ID NO:98 on one light chain and one other location on the antibody, or antigen-binding portions thereof. In some aspects, the Effector Moiety is conjugated at K5 of SEQ ID NO:98 on one light chain and 2 other locations on the antibody, or antigen-binding portions thereof. In some aspects, the Effector Moiety is conjugated to K5 of SEQ ID NO:98 on one light chain and 3 other locations on the antibody, or antigen-binding portions thereof. In some aspects, the Effector Moiety is conjugated to K5 of SEQ ID NO:98 on both light chains, and at one other location. In some aspects, the Effector Moiety is conjugated at K5 of SEQ ID NO:98 on both light chains, and at 2 other locations. In some aspects, the Effector Moiety is conjugated at K5 of SEQ ID NO:98 on both light chains, and at 3 other locations.
In some aspects, the invention relates to a mutated Immunoglobulin (Ig) domain, comprising a substituted residue within the mutated Ig domain that corresponds to position 77 of SEQ ID NO:10; or SEQ ID NO:6, characterized in that the substituted residue is selected from the group consisting of A, G, I, V, L, R, S, T, M, Q, N, P, H, and W, provided that the mutated Ig domain further comprises residues K and H at positions corresponding to positions 80 and 81 respectively of SEQ ID NO:10; or SEQ ID NO:6. In some aspects, the mutated Ig domain comprises a sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40, 41, 42, 44, 46, 47, 49, 50, 51, 52, 54, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 115, 116, 117, 119, 120, 121, 122, 123, 124, 125, and 126.
Deposits
In some aspects, the invention provides for vectors and nucleic acids as desposited with the ATCC, polypeptides encoded by said vectors and nucleic acids, compositions comprising polypeptides encoded by said vectors and nucleic acids, and polypeptides expressed by said nucleic acids and vectors. The following materials have been deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, USA (ATCC):














SEQ 
ATCC 
Date of 


Material
ID NO:
Accession No.
Deposit


















hCLk-Km(3)-D77A
37
PTA-13394
Dec. 12, 2012


h38C2-[LC-D185A]
254
PTA-13395
Dec. 12, 2012









Vector hCLk-Km (3)-D77A is a TA cloning vector with a polynucleotide DNA insert encoding the human constant light chain kappa (Km(3)) domain with a D77A mutation, as set forth in SEQ ID NO:37, and vector h38C2-[LC-D185A] is a polynucleotide DNA insert encoding the humanized 38C2 light chain with a D77A mutation, as set forth in SEQ ID NO:254.
In some aspects, the invention provides for an isolated host cell that recombinantly produces an immunoglobulin domain of the present invention, or immunoglobulin domain-comprising protein or antibody of the present invention. The present invention provides for an isolated polynucleotide comprising a nucleotide sequence encoding proteins, domains and antibodies of the present invention, and vectors comprising said polynucleotides. Vectors of the present invention may comprise ATCC deposit sequences. In some aspects, the invention provides for a method of producing an antibody, immunoglobulin domain, or protein, comprising culturing a host cell under conditions that result in production of the antibody, immunoglobulin domain, or protein, and isolating the antibody, immunoglobulin domain, or protein, from the host cell or culture.
In some aspects, the present invention provide a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, and SEQ ID NO:254, or a polypeptide that is at least about 85%, or at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identical to one or more of the aforementioned sequences.
Samples and Compositions of the Invention
In some aspects, the invention provides for a composition or sample of an antibody or antigen binding portion thereof comprising a CL domain of the invention covalently conjugated to an Effector Moiety, wherein at least about 50% of the Effector Moiety in the composition or sample is conjugated to K5 of SEQ ID NO:98. In some aspects, it is at least about 60%. In some aspects, it is at least about 70%. In some aspects, it is at least about 80%. In some aspects, it is at least about 90%.
In some aspects, the invention provides for a composition (or sample) of a antibody or antigen binding portion thereof comprising a CL domain of the invention, wherein at least about 50% of the antibody comprises an Effector Moiety covalently attached to K5 of SEQ ID NO:98 on at least one light chain. In some aspects, it is at least about 60%. In some aspects, it is at least about 70%. In some aspects, it is at least about 80%. In some aspects, it is at least about 90%. In some aspects, the Effector Moiety is covalently conjugated to K5 of SEQ ID NO:98 on both light chain constant regions.
In some aspects, the invention provides for a composition (or sample) of a antibody or antigen binding portion thereof comprising a CL domain of the invention covalently conjugated to an Effector Moiety, wherein at least about 30% of the sample comprises Effector Moieties conjugated at about 2 locations per antibody, and wherein at least one Effector Moiety conjugation site is K5 of SEQ ID NO:98. In some aspects, the amount is about 40%. In some aspects, the amount is about 50%. In some aspects, the amount is about 60%. In some aspects, the amount is about 70%. In some aspects, the amount is about 80%. In some aspects, the amount is about 90%. In some aspects, the amount is about 95%. In some aspects, the amount is about 99%.
In some aspects, the invention provides for a composition (or sample) of a antibody or antigen binding portion thereof comprising a CL domain of the invention covalently conjugated to an Effector Moiety, wherein at least about 30% of the sample comprises Effector Moieties conjugated at about 3 locations per antibody, and wherein at least 2 Effector Moiety conjugation sites are K5 of SEQ ID NO:98 on each light chain. In some aspects, the amount is about 40%. In some aspects, the amount is about 50%. In some aspects, the amount is about 60%. In some aspects, the amount is about 70%. In some aspects, the amount is about 80%. In some aspects, the amount is about 90%. In some aspects, the amount is about 95%. In some aspects, the amount is about 99%.
In some aspects, the invention provides for a composition (or sample) of a antibody or antigen binding portion thereof comprising a CL domain of the invention, wherein at least 50% of the light chain molecules are conjugated with at least one Effector Moiety at K5 of SEQ ID NO:98. In some aspects, it is at least about 60%. In some aspects, it is at least about 65%. In some aspects, it is at least about 70%. In some aspects, it is at least about 75%. In some aspects, it is at least about 80%. In some aspects, it is at least about 85%. In some aspects, it is at least about 90%. In some aspects, it is at least about 95%.
In some aspects, the invention provides for a composition (or sample) of a antibody or antigen binding portion thereof comprising a CL domain of the invention conjugated to an Effector Moiety at K5 of SEQ ID NO:98, wherein at least about 70% of the heavy chain molecules are unconjugated with the Effector Moiety. In some aspects, the amount is about 75%. In some aspects, the amount is about 80%. In some aspects, the amount is about 85%. In some aspects, the amount is about 90%. In some aspects, the amount is about 95%. In some aspects, the amount is about 99%. In some aspects, substantially all of the heavy chain molecules are unconjugated with the Effector Moiety.
In some aspects, the amount of individual light chain fragments that are unconjugated has a lower limit selected from the group consisting of about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, and 55%, and an upper limit selected from the group consisting of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60%. In some aspects, the amount of individual light chain fragments that are conjugated at one location has a lower limit selected from the group consisting of about 25, 30, 35, 40, 45, 50, and 55%, and an upper limit selected from the group consisting of about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95%. In some aspects, the amount of individual light chain fragments that are conjugated at 2 locations has a lower limit selected from the group consisting of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 5, 10, 15, 20, and 25%, and an upper limit selected from the group consisting of about 5, 16, 7, 8, 9, 5, 10, 15, 20, 25, 30, 35, and 40%.
In some aspects, the amount of individual heavy chain fragments that are unconjugated has a lower limit selected from the group consisting of about 50, 55, 60, 65, 70, 75, and 80% and an upper limit selected from the group consisting of about 60, 65, 70, 75, 80, 85, 90, 95, and 99%. In some aspects, the amount of individual heavy chain fragments that are conjugated at one location has a lower limit selected from the group consisting of about 1, 2, 5, 10, 15, 20, and 25% and an upper limit selected from the group consisting of about 5, 10, 15, 20, 25, 30, 35, 40, and 50%. In some aspects, the amount of individual heavy chain fragments that are conjugated at 2 locations has a lower limit selected from the group consisting of about 0, 1, 2, 3, 4, 5, 10, and 15% and an upper limit selected from the group consisting of about 2, 3, 4, 5, 10, 15 and 20%.
In some aspects the number of conjugations per antibody in a sample or composition of the invention has a lower limit selected from the group consisting of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.55, 1.6, 1.65, 1.7, 1.75, 1.8, 1.85, 1.9, 1.95 and 2, and an upper limit selected from the group consisting of about 1.6, 1.7, 1.75 1.8, 1.85, 1.9, 1.95, 2.0, 2.05, 2.1, 2.15, 2.2, 2.25, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, 4.0, 4.5 and 5. In some aspects the number of conjugations per antibody in a sample or composition of the invention is between about 1.5 and about 2.5. In some aspects the number of conjugations per antibody in a sample or composition of the invention is between about 1.6 and about 2.4. In some aspects the number of conjugations per antibody in a sample or composition of the invention is between about 1.7 and about 2.3. In some aspects the number of conjugations per antibody in a sample or composition of the invention is between about 1.8 and about 2.2. In some aspects the number of conjugations per antibody in a sample or composition of the invention is an amount selected from the group consisting of about 1.5, about 1.55, about 1.6, about 1.65, about 1.7, about 1.75, about 1.8, about 1.85, about 1.9, about 1.95, about 2.0, about 2.05, about 2.1, about 2.15, about 2.2, about 2.25, about 2.3, about 2.4 and about 2.5. In some aspects, the amount is about 1.7. In some aspects, the amount is about 1.8. In some aspects, the amount is about 1.9. In some aspects, the amount is about 2. In some aspects, the amount is about 2.1. In some aspects, the amount is about 2.1. In some aspects, the amount is about 2.3.
In some aspects of the invention, the number of conjugations per antibody is less than 2, with at least 50% of the antibody population having only a single conjugation per antibody. These samples are advantageous as they allow additional conjugation reactions to be targeted at the remaining CLκ site. In some aspects the number of conjugations per antibody in a sample or composition of the invention is between about 0.5 and about 1.5. In some aspects the number of conjugations per antibody in a sample or composition of the invention is between about 0.6 and about 1.4. In some aspects the number of conjugations per antibody in a sample or composition of the invention is between about 0.7 and about 1.3. In some aspects the number of conjugations per antibody in a sample or composition of the invention is between about 0.8 and about 1.2. In some aspects the number of conjugations per antibody in a sample or composition of the invention is between about 0.9 and about 1.1.
One of the advantages of the invention is that depending on the reagents and reaction conditions (especially the leaving group ester and molar ratio of linker:antibody), compositions and samples of the invention can be generated with a defined number of Effector Moieties relative to a defined number of antibodies. This can be especially useful when balancing the relative reactivities and therapeutic windows of the Effector Moiety and antibody. Moreover, in some situations, increasing the number of peptides or other Active Moieties per antibody beyond a certain threshold may not result in increased target binding or therapeutic effect. It is useful, therefore, to be able to control the number of peptides conjugated per antibody, and in doing so, direct the location of conjugation so as to minimize Fc or combining site interference. In some situations, therefore, aspects of the invention that allow for reduced conjugation, preferentially decorating only a single CLκ-K80 can be advantageous. Furthermore, whereas conjugation to CLκ-K80 is reliable and robust, conjugation to other antibody surface lysines, each of slightly different reactivity and pl can result in an heterogeneous sample of conjugated antibodies that can release conjugated molecules at inopportune or irregular times, such as during circulation and prior to delivery of the Effector Moiety to the target by antibody recognition (or delivery of the antibody to the target, by recognition with the Effector Moiety). This can be particularly undesirable with toxins (i.e. a cytotoxic agent with potential utility in killing tumors and tumor cells).
In some aspects, the toxin is an auristatin; a derivative of the natural product dolastatin 10 (MMAD). Representative auristatins include MMAE (N-methylvaline-valine-dolaisoleuine-dolaproline-norephedrine) and MMAF (N-methylvaline-valine-dolaisoleuine-dolaproline-phenylalanine).
In some aspects, the antibody targets a different target within the same pathway as the Effector Moiety. In some aspects, the antibody targets a different target to the Effector Moiety.
In some aspects, the VH and VL of antibody used for conjugation may be useful in the field of oncology. Suitable antibodies include; Rituximab, (RituxanTM), a chimeric, IgG1κ, anti-CD20 antibody, used to treat cancer and in particular non Hodgkin's lymphoma and also rheumatoid arthritis; Cetuximab (ErbituxTM) a chimeric, IgG1κ, anti-EGF receptor antibody, used to treat cancer, and in particular colon, head & neck cancer.
In some aspects, the antibody used for conjugation may be useful in the field of auto-immune and other immunological disorders. Suitable antibodies include Infliximab (RemicadeTM) a chimeric, IgG1κ, anti-TNFα antibody, used to treat rheumatoid arthritis, ulcerative colitis, Crohn's disease, psoriasis, psoriatic arthritis, and ankylosing spondylitis; Adalimumab (HumiraTM) a human, IgG1κ, anti-TNFα antibody, used to treat rheumatoid arthritis, Crohn's disease, psoriasis, psoriatic arthritis, juvenile idiopathic arthritis and ankylosing spondylitis; Natalizumab (TysabriTM) a humanized, IgG4κ, anti-α4-integrin antibody used to treat multiple sclerosis, rheumatoid arthritis, psoriasis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease; Omalizumab (XolairTM) a humanized, IgG1κ, anti-IgE antibody used to treat allergic asthma; Ranibizumab (LucentisTM) a humanized, IgG1κ, anti-VEGF antibody, used to treat wet AMD; and Palivizumab (SynagisTM) a humanized, IgG1κ, anti-RSV antibody, used to treat infective diseases, including respiratory syncytical virus.
In some aspect, compounds and compositions of the invention may be used to treat the above mentioned conditions.
Effector Moieties
The Effector Moiety may be a therapeutic agent, protein, peptide, nucleic acid, aptamer, small molecule, protein agonist, protein antagonist, metabolic regulator, hormone, toxin, growth factor or other regulatory protein, or may be a diagnostic agent, such as an enzyme that may be easily detected or visualized, such as horseradish peroxidase.
In some aspects, the Effector Moiety may be a protein or peptide, and may be connected to the linker through a peptide-linking residue. The protein or peptide may comprise one or both of an amino-terminal capping group R1 and a carboxyl-terminal capping group R2. R1 may be CH3, C(O)CH3, C(O)CH3, C(O)CH2CH3, C(O)CH2CH2CH3, C(O)CH(CH3)CH3, C(O)CH2CH2CH2CH3, C(O)CH(CH3)CH2CH3, C(O)C6H5, C(O)CH2CH2(CH2CH2O)1-5 Me, dichlorobenzoyl (DCB), difluorobenzoyl (DFB), pyridinyl carboxlate (PyC) or amido-2-PEG, an amino protecting group, a lipid fatty acid group or a carbohydrate. R2 may be OH, NH2, NH(CH3), NHCH2CH3, NHCH2CH2CH3, NHCH(CH3)CH3, NHCH2CH2CH2CH3, NHCH(CH3)CH2CH3, NHC6H5, NHCH2CH2OCH3, NHOCH3, NHOCH2CH3, a carboxy protecting group, a lipid fatty acid group or a carbohydrate.
The protein or peptide linking residue may be K, KSH, lysine homologs, Dap, Dab, Orn, R, C, thiol containing residues, S, T, Y, D, E, N or Q. The protein or peptide may be connected to the linker through the amino terminus of the N-terminal amino acid. The protein or peptide may be connected to the linker through the carboxyl terminus of the C-terminal amino acid. An additional amino acid residue may be added to the N- or C-terminus in order to function as a linking residue, whether by connection through the amino acid side chain, or the amino or carboxyl terminus.
Linkers
In aspects of the invention relating to conjugates with Effector Moieties, in particular methods of preparing conjugates and MACs, it will be understood that the invention equally applies to conjugates with Linkers in the absence of Effector Moieties. An example of the utility of such conjugates would be as intermediates that may usefully used to prepate Effector Moiety-linker-polypeptide conjugates of the invention.
The Effector Moiety of the invention (such as a small molecule, aptamer, nucleic acid, protein, or peptide) may be covalently attached to the antibody or antigen binding portion thereof by a linker. The linker may be covalently attached to the peptide by an amino group of the side chain of the peptide-linking residue. This may be a lysine residue. In some embodiments, the linking residue is a thiol bearing residue, such as Cys or KSH and the linker is covalently attached to the peptide via the terminal thiol group of the linking residue.
The linker may be linear or branched (to allow for conjugation to more than one Effector Moiety per Conjugation Addition (CA)), and optionally includes one or more carbocyclic or heterocyclic groups. Linker length may be viewed in terms of the number of linear atoms between the Effector Moiety and Antibody, with cyclic moieties such as aromatic rings and the like to be counted by taking the shortest route around the ring. In some embodiments, the linker has a linear stretch of between 5-15 atoms, in other embodiments 15-30 atoms, in still other embodiments 30-50 atoms, in still other embodiments 50-100 atoms, and in still other embodiments 100-200 atoms. In some embodiments, the length of the linker is a range with a lower limit selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, and an upper limit selected from the group consisting of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200.
Other linker considerations include the effect on physical or pharmacokinetic properties of the resulting compound, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (more or less stable as well as planned degradation), rigidity, flexibility, immunogenicity, modulation of antibody binding, the ability to be incorporated into a micelle or liposome, and the like.
The linker may be a peptidyl linker. In some embodiments, the peptidyl linker may be between 3-20 amino acids long, such as repeats of a single amino acid residue (e.g. polyglycine) or combinations of amino acid residues to give a peptide linker which imparts favorable presentation of the Effector Moiety or pharmacokinetics. Peptidyl linkers that would be most compatible with the presence of activating groups may lack lysine and histidine residues. SEQ ID NO:79 is an exemplary peptidyl linker.
Alternatively, the linker may be a non-peptidyl linker. Typical examples of these types of linker would be those based on straight or branched chain hydrocarbons or polyethylene glycols of varying lengths. These may incorporate other groups to affect solubility, rigidity, isoelectric point, such as aromatic or non-aromatic rings, halogens, ketones, aldehydes, esters, sulfonyls, phosphate groups, and so on.
In some aspects of the invention, the linker may comprise the formula: —X1-Y1-Z—; wherein X1 is the attachment group to the Effector Moiety (for example, via a peptide-linking residue), Y1 is a spacer region, and Z is an attachment moiety to the side chain of a lysine residue on an antibody (for example, an anti-IGF1R antibody). In some aspects, the linker may be of the formula X1Y1Z* when unbound to the antibody, where Z* is a leaving group, such that when conjugated to the antibody, the leaving group Z* reacts with the conjugation site of the antibody to form the conjugated linker X1Y1Z.
X1 may be selected so as to enable a specific directional covalent linking strategy to the Effector Moiety (for example, via the peptide-linking residue). In some aspects, X1 may be selected from the group consisting of COOH, isocyanate, isothiocyanate, acyl azide, sulfonic acid, sulfonyl halide, aldehyde, ketone, epoxide, carbonate, arylating reagent, imidoester, amine group, and a malemide group. For example, where the peptide-linking residue comprises a nucleophilic group, X1 may be an electrophilic group and vice versa. For example, if the peptide-linking residue side chain comprises an amine group, such as K, H, Ornithine, Dap, or Dab, X1 may be COOH, or other similarly reactive electrophile, for example, an isocyanate, isothiocyanate, acyl azide, sulfonic acid or sulfonyl halide, aldehyde or ketone, epoxide, carbonate, arylating reagent or imidoester. If the peptide-linking residue is D or E, X1 may comprise a nucleophilic group, such as an amine group. Either of these strategies permits a covalent bond to be formed between the X1 group and the peptide-linking residue by amide bond formation strategies. For example, when X1 is COOH, it may be activated as a pentafluorophenyl ester. In this case, reaction with an amine group on the peptide-linking peptide leads to amide bond formation, while the pentafluorophenol is a leaving group (which may be termed X1*).




The arrow indicates the point of attachment to the peptide-linking residue and the parallel line represents the point of attachment to the Y1 group of the linker.





Where the peptide-linking group is C, homologs of C, or other thiol-group containing residues (such as KSH), X1 may comprise a malemide group, permitting a thiol-malemide addition reaction strategy to covalently link the X1 group to the peptide-linking residue. In some aspects, X1 may be maleimide:




wherein the arrow indicates the point of attachment to the peptide linking residue and the parallel line represents to attachment to the Y1 group of the linker. For ease of nomenclature, linkers described herein that have been constructed using maleimide groups are described as maleimide-containing linkers, and may be titled MAL to indicate this, even though following construction of the linker, the maleimide group is generally converted to a succinimide ring.





In some aspects, the linking residue is KSH, and the X1 group is maleimide. In some aspects, X1 may comprise a pentafluorophenyl ester activated carboxyl function which may form an amide with the lysine side chain on the peptide.




In some aspects, X1 may comprise a thiol group, allowing a disulphide bridge to be formed between the peptide-linking residue and X1 group.
In some embodiments, Y1 is a biologically compatible connecting chain including any atom selected from the group consisting of C, H, N, O, P, S, F, Cl, Br, and I, and may comprise one or more amino acids, polymer or block co-polymer. Y1 may be selected so as to provide an overall length of the linker of between 2-100 atoms. Y1 may be selected so that the overall length of the linker is between 5 and 30 atoms. Y1 may be selected so that the overall length of linker is 15-25 atoms. Y1 may be selected so that the overall length of linker is between about 17 and about 19 atoms.
In some aspects, Y1 may be an amino alkanoic acid, such as:




where n=0 to 20 in some aspects 1-10, in some aspects, 1-5, and in some aspects, 1 and in some aspects, 2.

In some aspects, Y1 may be an alkanoic diacid, such as:




where n=0 to 20 in some aspects 1-10, in some aspects, 1-5, and in some aspects, 1 and in some aspects, 2.

In some aspects, Y1 may be a polyglycine, such as:




where n=0 to 10, in some aspects 1-10, in some aspects, 1-5, and in some aspects, 1 and in some aspects, 2.

In some aspects, Y1, X1—Y1, Y1—Z, and X1—Y1-Z may be selected from the group consisting of:







where m, n, j and k are each independently 0 to 30. In some aspects n=1-10, in some aspects, n=1-5. In some aspects, the lower limit of the range of values for n is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and the upper limit for the range of values for n is selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. N may be 1. N may be 2. N may be 3. N may be 4. N may be 5. N may be 6. In some aspects m=1-10, in some aspects, m=1-5. In some aspects, the lower limit of the range of values for m is selected form the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and the upper limit for the range of values for m is selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. M may be 1. M may be 2. M may be 3. M may be 4. M may be 5. M may be 6. In some aspects j=1-10, in some aspects, j=1-5. In some aspects, the lower limit of the range of values for j is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and the upper limit for the range of values for j is selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. J may be 1. J may be 2. J may be 3. J may be 4. J may be 5. J may be 6. In some aspects k=1-10, in some aspects, k=1-5. In some aspects, the lower limit of the range of values for k is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and the upper limit for the range of values for k is selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. K may be 1. K may be 2. K may be 3. K may be 4. K may be 5. K may be 6. In some aspects, the overall length of Y1 does not exceed 200 atoms. In some aspects, the overall length of Y1 does not exceed 150 atoms. In some aspects, the overall length of Y1 does not exceed 100 atoms. In some aspects, the overall length of Y1 does not exceed 50 atoms. In some aspects, the range of overall chain length of Y1 in numbers of atoms may have a lower limit selected from the group consisting of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60, and an upper limit selected from the group consisting of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100. In some aspects, the X1Y1Z linker may be identical to the above Y1 groups. In some aspects, the wavy line connects to the X1 group. In some aspects, the parallel lines connect to the X1 group. In some aspects, the wavy line connects to the Z group. In some aspects, the parallel lines connect to the Z group. In some aspects, the wavy line connects to the side chain of CLκ-K80. In some aspects, the parallel lines connect to the side chain of CLκ-K80. In some aspects, the wavy line connects to the Effector Moiety. In some aspects, the parallel lines connect to Effector Moiety. In some aspects, one of the wavy or parallel lines are points of attachment to a cleavable portion of the linker (Φ).

Leaving Groups

Z* may be selected so as to enable a specific directional covalent linking strategy to a lysine side chain on the antibody. For example, Z may be COOH, or another similarly reactive electrophile to react with the ε-amino of the surface lysine side chains using one of a number of possible amide bond formation strategies.
In some aspects, Z* may be used to form an active ester. Active esters connect to amines, and can thus conjugate to the ε-amino of a lysine side chain of the antibody. The Z carboxyl function to enable the formation of the active ester will be present at the terminus of Y group. The alcoholic or phenolic function of the active ester acts as a leaving group Z* during the conjugation reaction, enabling connection with the lysine side chain on the antibody via generation of an amide.
In some embodiments, the Z* group comprises a structure of the formula:




where R′ is an aliphatic or aromatic group.

In some embodiments, the Z* group is of the formula:




where R′=any of F, Cl, Br or I, nitro, cyano, trifluoromethyl, alone or in combination, and may be present in an amount of between 1 and 5. In some embodiments, R1 may be a halogen, and 4 or 5 halogen atoms may be present. In some embodiments, there may be 4 R1 atoms. In some embodiments, there may be 5 R1 atoms. In some embodiments, Z* may be tetrafluorophenyl. In some embodiments, Z* may comprise the formula:





wherein the parallel line represents the point of attachment to the Y1 portion of the linker.

In some embodiments, Z* may comprise the formula:




wherein R3, R4, R5, R6 and R7 are each independently selected from the group consisting of F, CL, H and the formula CR8R9R10, such that no more than two of R3, R4, R5, R6 and R7 are H, and one of R3, R4, R5, R6 and R7 is CR8R9R10, and R8, R9, and R10 are each independently selected from the group consisting of F, Cl and H such that no more than one of R8, R9 and R10 may be H, and wherein the parallel line represents the point of attachment to the Y1 portion of the linker. In some aspects, the group CR8R9R10 is located at one of R4, R5 or R6. In some aspects, the group CR8R9R10 is located at R5 

In some aspects, R3, R4, R5, R6 and R7 are each independently selected from the group consisting of F, Cl, H and the formula CR8R9R10, such that no more than one of R3, R4, R5, R6 and R7 is H, and one of R3, R4, R5, R6 and R7 is CR8R9R10. In some aspects, R3, R4, R5, R6 and R7 are each independently selected from the group consisting of F, Cl, and the formula CR8R9R10, and one is CR8R9R10.
In some aspects, R3, R4, R6 and R7 are each F. In some aspects, R8, R9, and R10 are each independently selected from the group consisting of F and Cl. In some aspects, R8, R9, and R10 are each F.
In some embodiments, Z* may comprise the formula:




2,3,5,6-tetrafluoro-4-(trifluoromethyl)phenyl wherein the parallel line represents the point of attachment to the Y1 portion of the linker.

In some aspects, the Z* group is of the formula:




where R′=any of F, Cl, Br or I, nitro, cyano, trifluoromethyl, alone or in combination, and h=1, 2, 3, 4, or 5. In some embodiments, R1 may be a halogen. In some embodiments, R1 is F or Cl, and h=4 or 5. In some embodiments, R1 is F or Cl, and h=5. In some embodiments, R1 is F, and h=2, 3, 4 or 5. In some embodiments, R1 is F, and h=3, 4 or 5. In some embodiments, R1 is F, and h=4 or 5. In some embodiments, R1 is F, and h=5. In some aspects, Z* may be selected from the group consisting of:





In some aspects, Z* may be selected from the group consisting of:





In some aspects, Z* may be selected from the group consisting of:





In some aspects, Z* may be selected from the group consisting of:





For such active esters, the leaving group is Z* and the Z group itself is the carbonyl attached to the Y1 group. When reacted with the antibody, the Z* group forms an amide, as shown below,




In some embodiments, Z is




In some embodiments, the Z* group comprises a squarate ester such as




wherein R=aliphatic group or substituted aromatic and may be selected from the group consisting of:





In some embodiments, the Z group comprises a Maleimide group:




In some aspects, the X1*Y1Z* linker comprises a Maleimide-PEG-PFP ester of the structure:




where n=1 to 12. In some aspects, n=1 to 5. In some aspects n=2. In some aspects n=1.

In some aspects, the X1*Y1Z* linker comprises a structure selected from the group consisting of:







where m, n and j are each independently 0 to 30, R1 is F and h=2, 3, 4, or 5. In some aspects n=1-10, in some aspects, n=1-5. In some aspects, the lower limit of the range of values for n is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and the upper limit for the range of values for n is selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. N may be 1. N may be 2. N may be 3. N may be 4. N may be 5. N may be 6. In some aspects m=1-10, in some aspects, m=1-5. In some aspects, the lower limit of the range of values for m is selected form the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and the upper limit for the range of values for m is selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. M may be 1. M may be 2. M may be 3. M may be 4. M may be 5. M may be 6. In some aspects j=1-10, in some aspects, j=1-5. In some aspects, the lower limit of the range of values for j is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and the upper limit for the range of values for j is selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. J may be 1. J may be 2. J may be 3. J may be 4. J may be 5. J may be 6. In some aspects k=1-10, in some aspects, k=1-5. In some aspects, the lower limit of the range of values for k is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and the upper limit for the range of values for k is selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. K may be 1. K may be 2. K may be 3. K may be 4. K may be 5. K may be 6. In some aspects, the overall length of Y1 does not exceed 200 atoms. In some aspects, the overall length of Y1 does not exceed 150 atoms. In some aspects, the overall length of Y1 does not exceed 100 atoms. In some aspects, the overall length of Y1 does not exceed 50 atoms. In some aspects, the range of overall chain length of Y1 in numbers of atoms may have a lower limit selected from the group consisting of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60, and an upper limit selected from the group consisting of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100.

In some aspects the MAC comprises a X1Y1Z linker of the formula:




In some aspects, the X1*Y1Z* linker comprises a PEG-bis-pentafluorophenyl ester of the formula:




where n=1 to 25. In some aspects n=1 to 20. In some aspects n=1 to 10. In some aspects n=4. N=3, In some aspects n=1.

In some aspects the MAC comprises 2 peptides conjugated per antibody. In some aspects, one peptide is conjugated at each of the 2 CLκ-K80 residues of the antibody or antigen binding fragment thereof.
In some aspects, the polypeptide of the invention comprises a formula selected from the group consisting of:




wherein —KH— is a covalent link to the side chain of K5 of SEQ ID NO:98, Effector Moiety-LR is a covalent link to the Effector Moiety, and m, n and j are each independently 0-30. In some aspects n=1-10, in some aspects, n=1-5. In some aspects, the lower limit of the range of values for n is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and the upper limit for the range of values for n is selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. N may be 1. N may be 2. N may be 3. N may be 4. N may be 5. N may be 6. In some aspects m=1-10, in some aspects, m=1-5. In some aspects, the lower limit of the range of values for m is selected form the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and the upper limit for the range of values for m is selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. M may be 1. M may be 2. M may be 3. M may be 4. M may be 5. M may be 6. In some aspects j=1-10, in some aspects, j=1-5. In some aspects, the lower limit of the range of values for j is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and the upper limit for the range of values for j is selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. J may be 1. J may be 2. J may be 3. J may be 4. J may be 5. J may be 6. In some aspects, the overall length of Y1 does not exceed 200 atoms. In some aspects, the overall length of Y1 does not exceed 150 atoms. In some aspects, the overall length of Y1 does not exceed 100 atoms. In some aspects, the overall length of Y1 does not exceed 50 atoms. In some aspects, the range of overall chain length of Y1 in numbers of atoms may have a lower limit selected from the group consisting of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60, and an upper limit selected from the group consisting of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100.

In some aspects, the linker is selected from the group consisting of










Cleavable Linkers

In some aspects, the invention provides for MACs as described herein comprising “non-cleavable” linkers. In other aspects, the invention provides for MACs comprising “cleavable” linkers. The term “cleavable linker” is used herein to describe a rapidly cleaved linker that is designed to be degraded by intracellular or extracellular enzymes or when subjected to changes in pH or redox environment so as to release the cargo at the desired location. For example, cleavable linkers may be preferentially stable in plasma, blood or serum, and less stable in intracellular environments.
Cleavable linkers can be formed by adding a cleavable portion (Φ) to the Y1 portion of the linker (or P1 portion, where the linker is for a catalytic antibody combining site). Accordingly, the linkers would take the formula X1-Φ-Y1-Z, X1-Φ-Y1-Z*, and P1-Φ-Q1-W1.
A representative example of a cleavable portion of a linker is valine-citrulline p-aminobenzyl carbamate (VitCitABC) that is cleaved by intracellular proteases such as cathepsin B.




where the wavy line typically indicates the point of attachment to the Y1 (or Q1) portion of the linker, and the parallel line represents the point of attachment to the X1 (or P1) portion of the linker, or even to the Effector Moiety itself. In some aspects, of course, the wavy line may indicate the point of attachment to the X1 or P1 linker portion (or Effector Moiety), and the parallel line may indicate the point of attachment to the Y1 or Q1 portion of the linker.

Accordingly, in some aspects, the invention provides for linker of the formula:







where m, n and j are each independently 0 to 30, R1 is F and h=2, 3, 4, or 5. In some aspects n=1-10, in some aspects, n=1-5. In some aspects, the lower limit of the range of values for n is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and the upper limit for the range of values for n is selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. N may be 1. N may be 2. N may be 3. N may be 4. N may be 5. N may be 6. In some aspects m=1-10, in some aspects, m=1-5. In some aspects, the lower limit of the range of values for m is selected form the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and the upper limit for the range of values for m is selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. M may be 1. M may be 2. M may be 3. M may be 4. M may be 5. M may be 6. In some aspects j=1-10, in some aspects, j=1-5. In some aspects, the lower limit of the range of values for j is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and the upper limit for the range of values for j is selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. J may be 1. J may be 2. J may be 3. J may be 4. J may be 5. J may be 6. In some aspects k=1-10, in some aspects, k=1-5. In some aspects, the lower limit of the range of values for k is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and the upper limit for the range of values fork is selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30. K may be 1. K may be 2. K may be 3. K may be 4. K may be 5. K may be 6. In some aspects, the overall length of Y1 does not exceed 200 atoms. In some aspects, the overall length of Y1 does not exceed 150 atoms. In some aspects, the overall length of Y1 does not exceed 100 atoms. In some aspects, the overall length of Y1 does not exceed 50 atoms. In some aspects, the range of overall chain length of Y1 in numbers of atoms may have a lower limit selected from the group consisting of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60, and an upper limit selected from the group consisting of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100.

Use of Auristatin-Based Payloads

Auristatin-based effector moieties are also useful in connection with the targeted conjugation technology of the present invention when used in conjunction with the appropriate linker technology. Specifically, useful payloads include those disclosed in PCT/162012/056224 including all pharmaceutically acceptable salts, hydrates and free base forms.
Accordingly, the following effector moieties and linkers may be used in aspects of the invention:




or a pharmaceutically acceptable salt or solvate thereof, wherein, independently for each occurrence,

W2 is





    
    
        R11 is
    
    






    
    
        Y12 is —C2-C20 alkylene-, —C2-C20 heteroalkylene-; —C3-C8 carbocyclo-, -arylene-, —C3-C8heterocyclo-, —C1-C10 alkylene-arylene-, -arylene-C1-C10alkylene-, —C1-C10alkylene-(C3-C8carbocyclo)-, —(C3-C8carbocyclo)-C1-C10alkylene-, —C1-C10alkylene-(C3-C8heterocyclo)- or —(C3-C8 heterocyclo)-C1-C10alkylene-;
        Z2 is
    
    






    
    
        R12 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl;
        R13A and R13B are either of the following:
        (i) R13A is hydrogen, C1-C8alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and
        
            R13B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl or halogen; or
        
        
        (ii) R13A and R13B taken together are C2-C8 alkylene or C1-C8 heteroalkylene;
        R14A and R14B are either of the following:
        (i) R14A is hydrogen, C1-C8alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and
        
            R14B is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, heterocyclyl, aryl, heteroaralkyl or aralkyl; or
        
        
        (ii) R14A and R14B taken together are C2-C8 alkylene or C1-C8 heteroalkylene;

R15 is

    
    






C1-C10 heterocyclyl, C3-C8 carbocycly and C6-C14 aryl optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from the group consisting of —C1-C8 alkyl, —C1-C8 alkyl-N(R′)2, —C1-C8 alkyl-C(O)R′, —C1-C8 alkyl-C(O)OR′—O—(C1-C8 alkyl), —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)N(R′)2, —NHC(O)R′, —S(O)2R′, —S(O)R′, —OH, halogen, —N3, —N(R′)2, —CN, —NHC(═NH)NH2, —NHCONH2, —S(═O)2R′ and —SR′, wherein each R′ is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl, or two R′ can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl;

or R15 is





optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C8 alkyl, —C1-C8 alkyl-N(R′)2, —C1-C8 alkyl-C(O)R′, —C1-C8 alkyl-C(O)OR′, —O—(C1-C8 alkyl), —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)N(R′)2, —NHC(O)R′, —S(O)2R′, —S(O)R′, —OH, halogen, —N3, —N(R′)2, —CN, —NHC(═NH)NH2, —NHCONH2, —S(═O)2R′, —SR′ and arylene-R′, wherein each R′ is independently selected from the group consisting of hydrogen, C1-C8 alkyl, C1-C8heterocyclyl, C1-C10alkylene-C3-C8heterocyclyl and aryl, or two R′ can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl;

    
    
        R16 is hydrogen, —C1-C8 alkyl, —C2-C8 alkenyl, —C2-C8 alkynyl or —C1-C8 haloalkyl;
        R22 is hydrogen, C1-C4 alkyl, C1-C10 heterocyclyl or C6-C14 aryl;
        R23 is C1-C10 heterocyclyl; and
        R17 is independently selected for each occurrence from the group consisting of F, Cl, I and Br;
        R20 is -aryl, —C1-C10alkylene-aryl, where aryl on R10 comprising aryl is substituted with [R17]h;
        h is 5; and
        X is O or S;
        provided that when R13A is hydrogen X is S.
    
    


In some aspects, the effector moieties may be selected from Table 73. In some aspects, the effector moiety is Toxin #54. In some aspects, the effector moiety is Toxin #115. In some aspects, the effector moiety is Toxin #69.
In some aspects, the Effector Moiety when conjugated to a linker of the invention comprises a formula selected from the group consisting of




Effector Moieties conjugated to linkers useful in connection with the present invention include auristatin-based toxin-linkers such as those disclosed in PCT/162012/056224. In some aspects, the toxin-linkers of the invention may be selected from the group consisting of




Methods of Conjugation

In some aspects, the invention provides for a method of preparing a multifunctional antibody conjugate (MAC) comprising an antibody or antigen binding portion, the antibody being covalently conjugated to at least one Effector Moiety through a linker attached to a side chain of K5 of SEQ ID NO:98 (or SEQ ID herein disclosed falling within the scope of SEQ ID NO:98), or to a side chain of residue K on an antibody constant domain wherein the position of K corresponds with residue 77 of SEQ ID NO:6, or residue 185 of a constant light domain according to Kabat numbering;
said method comprising: covalently attaching the Effector Moiety to a linker terminating in a leaving group Z* of the formula:




where R1 is any of F, Cl, Br or I, nitro, cyano, trifluoromethyl, alone or in combination, and h=1, 2, 3, 4, or 5, and reacting the Effector Moiety-linker-leaving group complex so formed with the antibody at a molar ratio of between about 3.5:1 to about 4.5:1 of Effector Moiety:antibody. In some aspects, the molar ratio is about 3.7:1 to about 4.3:1. In some embodiments, R1 may be a halogen. In some embodiments, R1 is F or Cl, and h=4 or 5. In some embodiments, R1 is F or Cl, and h=5. In some embodiments, R1 is F, and h=2, 3, 4 or 5. In some embodiments, R1 is F, and h=3, 4 or 5. In some embodiments, R1 is F, and h=4 or 5. In some embodiments, R1 is F, and h=5. In some aspects, Z* may be selected from the group consisting of:





R1 may be present in an amount of between 3 and 5. There may be 3 R1 groups. R1 may be present in an amount of between 4 and 5. There may be 4 R1 groups. There may be R1 groups. R1 may be fluorine. R1 may be chlorine. R1 may be bromine. The leaving group may comprise the formula:





In some aspects, the invention provides for methods of producing a MAC, wherein the MAC comprises an antibody, or fragment thereof, covalently linked to at least one Effector Moiety that binds an additional target (such as peptide, small molecule, aptamer, nucleic acid molecule, or protein), characterised in that Effector Moiety comprises a linker with a PFP leaving group capable of reacting with the ε-amino of surface lysine residues of the antibody. In some aspects, the invention provides for a process for conjugating an Effector Moiety (such as a peptide) to a CL domain of the invention comprising SEQ ID NO:98, comprising conjugating the Effector Moiety with a linker comprising a leaving group of the formula:




where R1 is any of F, Cl, Br or I, nitro, cyano, trifluoromethyl, alone or in combination, and may be present in an amount of between 1 and 5 and reacting the leaving group with the side chain of K5 of SEQ ID NO:98.

In some aspects, the method comprises combining an antibody or antigen binding portion thereof with an Effector Moiety, wherein the Effector Moiety is covalently attached to a linker comprising a PFP leaving group.
In some aspects, the molar ratio of Effector Moiety: antibody is between about 2.5 and about 4.6:1. In some aspects of the invention, the molar ratio is about 3.7:1, and about 4.3:1. In some aspects of the invention, the molar ratio of Effector Moiety:antibody is about 4:1. In some aspects, the molar ratio is between about 2:1 and about 7:1. In some aspects, the molar ratio is between about 3:1 and about 6:1. In some aspects, the molar ratio is between about 3:1 and about 7:1. In some aspects, the molar ratio is between about 3:1 and about 5:1.
In aspects of the invention where it is desirable to have less than 1.5 conjugations per antibody (such as where a single Effector Moiety is required) the molar ratio may be between about 1:1 and about 6:1, wherein the buffer comprises HEPES at a concentration of at least 0.02M. The concentration of HEPES may be between about 0.1M and about 1M. The concentration of HEPES may between about 0.1M and about 0.5M. In aspects of the invention where it is desirable to have less than 1.5 conjugations per antibody (such as where a single Effector Moiety is required) the molar ratio may be between about 1:1 and about 3:1.
In some aspects, the preferred molar ratio is a range with a lower limit selected from the group consisting of about 1, about 1.2, about 1.4, about 1.5, about 1.6, about 1.8, about 2, about 2.2, about 2.4, about 2.5, about 2.6, about 2.8, about 3, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4. about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5, about 5.2, about 5.4, about 5.5, about 5.6, about 5.8, about 6, about 6.2, about 6.4, about 6.5, about 6.6, about 6.8, about 7, about 7.3, about 7.5, about 7.7, about 8, about 8.5, about 9, about 9.5, and about 10 to 1, and an upper limit selected from the group consisting of about 1.5, about 1.6, about 1.8, about 2, about 2.2, about 2.4, about 2.5, about 2.6, about 2.8, about 3, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4. about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5, about 5.2, about 5.4, about 5.5, about 5.6, about 5.8, about 6, about 6.2, about 6.4, about 6.5, about 6.6, about 6.8, about 7, about 7.3, about 7.5, about 7.7, about 8, about 8.5, about 9, about 9.5, about 10, and about 15 to 1.
In some aspects, the invention further comprises conjugating the Effector Moiety and protein together for at least about 30 mins. In some aspects, the duration is at least about 60 mins. In some aspects, the duration is at least about 2 hrs. In some aspect, the invention further comprises conjugating the Effector Moiety and antibody at between about 4° C. and about 40° C. In some aspect, the invention further comprises conjugating the Effector Moiety and antibody at between about 10° C. and about 30° C. In some aspect, the invention further comprises conjugating the Effector Moiety and antibody at between about 15° C. and about 30° C. In some aspects, the reaction is conducted at about 18° C. to about 25° C. In some aspects, the reaction is conducted at about 22° C. In some aspects, the reaction is conducted at about room temperature.
In some aspects, the conjugation reaction takes place at between about pH 6.5 and about pH 8.0. In some aspects, the conjugation reaction takes place at between about pH 6.75 and about pH 8.0. In some aspects, the conjugation reaction takes place at about pH 7.7. In some aspects, the conjugation reaction takes place at about pH 7. In some aspects, the conjugation reaction takes place at about pH 7.2. In some aspects, the conjugation reaction takes place at about pH 7.5. In some aspects, the conjugation reaction takes place at between a range of pH values, whose lower limit is selected from the group consisting of 5.5, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 and 8, and whose upper limit is selected from the group consisting of 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.5, and 9.
In some aspects, the pH may be below 6.5; this may be particularly useful in applications were less than about 1.5 conjugations per antibody are required. In some aspects, the pH is between about 5.5 and about 6.5.
In some aspects, the salt concentration may be below about 0.2M. The salt may be a halide salt (F, Cl, Br, I) and may comprise a metal such as Li, Na, K, Be, Mg, Ca. The salt may be NaCl. The salt may be KCl. Salt concentrations of above about 0.1M may be used to limit the rate and/or number of conjugations per antibody. The salt concentration may be between about 0 and about 0.1M. The salt concentration may be between about 0 and about 0.5M. The salt concentration may be between about 0 and about 0.3M.
In some aspects, the method of the invention comprises formulating the antibody or antigen binding portion thereof in a formulation buffer at about pH 5.5. The formulation buffer may be sodium acetate and trehalose buffer. This buffer has the advantage of not containing any primary amines, and lends itself well to pH adjustment. The antibody may be present in an amount of about 15 to about 25 mg·ml−1. In some aspects, the antibody may be present at an amount of 20 mg·ml−1.
The pH of the formulation buffer may be adjusted to about pH 7.2 to about pH 8.0; in some embodiments, the formulation buffer may be adjusted to pH 7.7. The pH of the formulation buffer may be adjusted with a phosphate buffer. The phosphate buffer may be at a concentration of between about 40 mM and about 80 mM. The phosphate buffer may be at a concentration of between about 10 mM and about 200 mM.
In some aspects, the concentration of antibody during the conjugation reaction with the Effector Moiety/linker and leaving group Z* may be in a range where the lower limit of the range is selected from about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 30, and about 40 mg·ml−1, and the upper limit of the range is selected form the group consisting of about 7, about 8, about 9, about 10, about 15, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 500 mg·ml−1.
The Effector Moiety may be reconstituted at a concentration of at least about 2 mg·ml−1. The Effector Moiety may be reconstituted at a concentration of about 5 to about 20 mg·ml−1 in diluted propylene glycol prior to use and, in some embodiments, may be at a concentration of 10 mg·ml−1.
The conjugation reaction may be performed by combining the antibody or antigen binding portion thereof and the Effector Moiety at a molar ratio of 4 moles Effector Moiety to 1 mole of antibody and incubated at about 18° C. to about 25° C. for about 2 to about 24 hrs. In some embodiments, the conjugation reaction between antibody and Effector Moiety is at room temperature for 2 hrs. In some embodiments, the conjugation reaction is for at least about 2 hrs. In some embodiments, the conjugation reaction is for at least about 30 mins.
The reaction may be quenched and adjusted to about pH 5.0 to about pH 6.0. In some embodiments, the quenched reaction may be adjusted to pH 5.5. This may be accomplished using a succinate and glycine buffer at, for example, about pH 4.0. This buffer has advantages over other more common buffers such as TRIS, or other amino-acid buffers. The succinate assists in limiting aggregation and precipitation during diafiltration, which can be stressful on the conjugated molecule, and glycine contains an additional primary amine.
The reaction may be concentrated and unreacted Effector Moiety, related species (such as peptide where the linker was hydrolyzed by reaction with water solvent) and other unreacted elements of the reaction mixture (such as PFP) may be removed by diafiltration, for example, using a 50 kDa membrane or size exclusion chromatography into a succinate, glycine, sodium chloride, and trehalose buffer, pH 5.5 at 30 mg·ml−1.
In some aspects, the method may comprise conjugating an Effector Moiety to CLκ-K80. In some aspects, the invention comprises conjugating a Effector Moiety to an Ig domain, comprising mutating the CLλ so as to comprise a SEQ ID NO:98 on the EF connecting chain loop between β-strands E and F, attaching to the Effector Moiety a linker comprising a leaving group Z* as herein defined, and reacting said Effector Moiety-linker-leaving group complex with the side chain of K5 of SEQ ID NO:98.
Pharmaceutical Compositions of the Invention
The invention provides a pharmaceutical composition comprising the MAC and a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof, and may include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable substances such as wetting or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody or antibody portion.
The compositions of this invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with antibodies in general. The preferred mode of administration is parenteral (e.g. intravenous, subcutaneous, intraperitoneal, intramuscular). In a preferred embodiment, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intramuscular or subcutaneous injection.
The pharmaceutical composition may further comprise another component, such as an anti-tumour agent or an imaging reagent. Another aspect of the present invention provides kits comprising MACs of the invention and pharmaceutical compositions comprising these antibodies. A kit may include, in addition to the MAC or pharmaceutical composition, diagnostic or therapeutic agents. A kit may also include instructions for use in a diagnostic or therapeutic method. In some embodiments, the kit includes the antibody or a pharmaceutical composition thereof and a diagnostic agent. In other embodiments, the kit includes the antibody or a pharmaceutical composition thereof and one or more therapeutic agents, such as an additional antineoplastic agent, anti-tumour agent or chemotherapeutic agent.
These agents and compounds of the invention can be combined with pharmaceutically acceptable vehicles such as saline, Ringer's solution, dextrose solution, and the like. The particular dosage regimen, i.e., dose, timing and repetition, will depend on the particular individual and that individual's medical history.
Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and may comprise buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or Igs; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG).
Liposomes containing compounds of the invention are prepared by methods known in the art, such as described in U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington, The Science and Practice of Pharmacy, 20th Ed., Mack Publishing (2000).
Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or ‘poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(−)-3-hydroxybutyric acid.
The formulations to be used for in vivo administration must be sterile. This is readily accomplished by, for example, filtration through sterile filtration membranes. Therapeutic compounds of the invention are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
Suitable emulsions may be prepared using commercially available fat emulsions, such as IntralipidTM, LiposynTM, InfonutrolTM, LipofundinTM and LipiphysanTM. The active ingredient may be either dissolved in a pre-mixed emulsion composition or alternatively it may be dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and an emulsion formed upon mixing with a phospholipid (e.g., egg phospholipids, soybean phospholipids or soybean lecithin) and water. It will be appreciated that other ingredients may be added, for example glycerol or glucose, to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20% oil, for example, between 5 and 20%. The fat emulsion can comprise fat droplets between 0.1 and 1.0 μm, particularly 0.1 and 0.5 μm, and have a pH in the range of 5.5 to 8.0.
The emulsion compositions can be those prepared by mixing a compound of the invention with IntralipidTM or the components thereof (soybean oil, egg phospholipids, glycerol and water).
Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as set out above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions in preferably sterile pharmaceutically acceptable solvents may be nebulised by use of gases. Nebulised solutions may be breathed directly from the nebulising device or the nebulising device may be attached to a face mask, tent or intermittent positive pressure breathing machine. Solution, suspension or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.
Compounds and compositions of the invention may be used in conjunction with established treatments for the relevant indication. Examples include 5-Flurouracil, irinotecan, oxilaplatin, cetuximab, sunitinib, and rituximab for the treatment of angiogenic disorders in particular, especially cancer. Other examples include ranibizumab, infliximab, adalimumab, natalizumab, omalizumab, and palivizumab.
Therapeutic Methods of the Invention
Therapeutic methods are also provided by the invention. A therapeutic method comprises administering a compound or composition of the invention to a subject in need thereof.
The invention provides for the use of compounds of the invention or pharmaceutical compositions of the invention in a method of inhibiting or reducing angiogenesis or for treating or preventing a disease or symptom associated with an angiogenic disorder. The invention provides methods of inhibiting or reducing angiogenesis or treating or preventing a disease or symptom associated with an angiogenic disorder comprising administering to a patient a therapeutically effective dose of compounds and compositions of the invention. Also provided are methods of delivering or administering compounds and compositions of the invention and methods of treatment using compounds and compositions of the invention. Also provided are methods of treating cancer comprising administering to the subject a therapeutically effective amount of a compound or pharmaceutical composition according to the invention. As used herein, an angiogenesis-mediated condition is a condition that is caused by abnormal angiogenesis activity or one in which compounds that modulate angiogenesis activity have therapeutic use. Diseases and conditions that may be treated and/or diagnosed with compounds and compositions of the invention include cancer, arthritis, hypertension, kidney disease, psoriasis, angiogenesis of the eye associated with ocular disorder, infection or surgical intervention, macular degeneration, diabetic retinopathy, and the like.
More specifically, examples of “cancer” when used herein in connection with the present invention include cancers of the lung (NSCLC and SCLC), the head or neck, the ovary, the colon, the rectum, the prostate, the anal region, the stomach, the breast, the kidney or ureter, the renal pelvis, the thyroid gland, the bladder, the brain, renal cell carcinoma, carcinoma of, neoplasms of the central nervous system (CNS), primary CNS lymphoma, non-Hodgkin's lymphoma, spinal axis tumours, carcinomas of the, oropharynx, hypopharynx, esophagus, pancreas, liver, gallbladder and bile ducts, small intestine, urinary tract; or lymphoma or a combination of one or more of the foregoing cancers. Still more specifically, examples of “cancer” when used herein in connection with the present invention include cancer selected from lung cancer (NSCLC and SCLC), breast cancer, ovarian cancer, colon cancer, rectal cancer, prostate cancer, cancer of the anal region, or a combination of one or more of the foregoing cancers.
In other embodiments, pharmaceutical compositions of the invention relate to non-cancerous hyperproliferative disorders such as, without limitation, age-related macular degeneration, restenosis after angioplasty and psoriasis. In another embodiment, the invention relates to pharmaceutical compositions for the treatment of a mammal that requires activation of IGF1R and/or Ang2, wherein the pharmaceutical composition comprises a therapeutically effective amount of an activating antibody of the invention and a pharmaceutically acceptable carrier. Pharmaceutical compositions of the invention may be used to treat osteoporosis, frailty or disorders in which the mammal secretes too little active growth hormone or is unable to respond to growth hormone.
As used herein, an “effective dosage” or “effective amount” of drug, compound, or pharmaceutical composition is an amount sufficient to affect any one or more beneficial or desired results. For prophylactic use, beneficial or desired results include eliminating or reducing the risk, lessening the severity, or delaying the outset of the disease, including biochemical, histological and/or behavioural symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. For therapeutic use, beneficial or desired results include clinical results such as reducing tumour size, spread, vasculature of tumours, or one or more symptoms of cancer or other diseases associated with increased angiogenesis, decreasing the dose of other medications required to treat the disease, enhancing the effect of another medication, and/or delaying the progression of the disease of patients. An effective dosage can be administered in one or more administrations. For purposes of this invention, an effective dosage of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an “effective dosage” may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.
An “individual” or a “subject” is a mammal, more preferably, a human. Mammals also include, but are not limited to, farm animals, sport animals, pets, primates, and horses.
Advantageously, therapeutic administration of compounds of the invention results in decrease in angiogenesis and/or in the case of cancers, stabilized or reduced tumour volume. Preferably, tumour volume is at least about 10% or about 15% lower than before administration of a MAC of the invention. More preferably, tumour volume is at least about 20% lower than before administration of the MAC. Yet more preferably, tumour volume is at least 30% lower than before administration of the MAC. Advantageously, tumour volume is at least 40% lower than before administration of the MAC. More advantageously, tumour volume is at least 50% lower than before administration of the MAC. Very preferably, tumour volume is at least 60% lower than before administration of the MAC. Most preferably, tumour volume is at least 70% lower than before administration of the MAC.
Administration of compounds of the invention in accordance with the method in the present invention can be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of a compound of the invention may be essentially continuous over a preselected period of time or may be in a series of spaced doses.
Antibodies
The immunoglobulin (Ig) domain is a type of protein domain that consists of a 2-layer sandwich of between 7 and 9 antiparallel β-strands arranged in two β-sheets with a Greek key topology. A β-strand is a stretch of polypeptide chain typically 3 to 10 amino acids long with backbone in an almost fully extended conformation. B sheets consist of β-strands connected laterally by at least two or three backbone hydrogen bonds, forming a generally twisted, pleated sheet. The backbone switches repeatedly between the two β-sheets. Typically, the pattern is (N-terminal β-hairpin in sheet 1)-(β-hairpin in sheet 2)-(β-strand in sheet 1)-(C-terminal β-hairpin in sheet 2). The cross-overs between sheets form an “X”, so that the N- and C-terminal hairpins are facing each other. Members of the Ig superfamily are found in hundreds of proteins of different functions. Examples include antibodies, the giant muscle kinase titin and receptor tyrosine kinases. Ig-like domains may be involved in protein-protein and protein-ligand interactions
The α-helix is a right-handed coiled or spiral conformation of amino acids, in which every backbone N—H group donates a hydrogen bond to the backbone C═O group of the amino acid four residues earlier. This secondary structure is also sometimes called a classic Pauling-Corey-Branson α-helix. Among types of local structure in proteins, the α-helix is the most regular and the most predictable from sequence, as well as the most prevalent.
An immunoglobulin (Ig) is a tetrameric molecule. In a naturally occurring Ig, each tetramer is composed of 2 identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa).
The amino-terminal portion of each chain includes a variable region, of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as κ and λ light chains. Heavy chains are classified as α, δ, ε, γ, and μ, and define the antibody's isotype as IgA, IgD, IgE, IgG, IgM, respectively. Within light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. The variable regions of each light/heavy chain pair form the antibody binding site such that an intact Ig has 2 binding sites.
Each domain in an antibody molecule has a similar structure of two β-sheets packed tightly against each other in a compressed antiparallel β-barrel. This conserved structure is termed the immunoglobulin (Ig) fold. The Ig fold of constant domains contains a 3-stranded β sheet packed against a 4-stranded β sheet, with each sheet separated by chains; these chains typically comprise α-helices, loops, turns, and short, sharp turns between two β-sheets called β-hairpins.
Ig chains exhibit the same general structure of relatively conserved framework regions (FR) joined by 3 hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the 2 chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminus to C-terminus, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)).
The identity of the amino acid residues in a particular antibody that make up a CDR can be determined using methods well known in the art. For example, antibody CDRs may be identified as the hypervariable regions originally defined by Kabat et al (Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C.). The positions of the CDRs may also be identified as the structural loop structures originally described by Chothia and others (Chothia et al., 1989, Nature 342:877-883). Other approaches to CDR identification include the “AbM definition,” which is a compromise between Kabat and Chothia and is derived using Oxford Molecular's AbM antibody modeling software (now Accelrys®), or the “contact definition” of CDRs based on observed antigen contacts, set forth in MacCallum et al., 1996, J. Mol. Biol., 262:732-745. In another approach, referred to herein as the “conformational definition” of CDRs, the positions of the CDRs may be identified as the residues that make enthalpic contributions to antigen binding (Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166). Still other CDR boundary definitions may not strictly follow one of the above approaches, but will nonetheless overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. As used herein, a CDR may refer to CDRs defined by any approach known in the art, including combinations of approaches. The methods used herein may utilize CDRs defined according to any of these approaches. For any given embodiment containing more than one CDR, the CDRs (or other residue of the antibody) may be defined in accordance with any of Kabat, Chothia, extended, AbM, contact, and/or conformational definitions.
The numbering of residues of the CLκ and CLλ domains can vary. For example, the numbering of the CLκ can begin at either LC-R108 according to Kabat numbering (for example, R108 of SEQ ID NO:2), or LC-T109 according to Kabat numbering (for example, T109 of SEQ ID NO:2). The numbering convention used herein is that provided by the Swiss-Prot group, a part of the Swiss Institute of Bioinformatics, and begins at LC-T109. It will be appreciated that where a different numbering system is preferred, the numbering of specified residues of the invention may be adjusted accordingly. LC refers to Light Chain.
An “antibody” refers to an intact Ig or to an antigen binding portion thereof that competes with the intact antibody for specific binding. Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding portions include, inter alia, Fab, Fab′, F(ab′)2, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies and polypeptides that contain at least a portion of an Ig that is sufficient to confer specific antigen binding to the polypeptide. A Fab fragment is a monovalent fragment consisting of the VL, VH, CL and CH I domains; a F(ab′)2 fragment is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consists of the VH and CH1 domains; an Fv fragment consists of the VL and VH domains of a single arm of an antibody; and a dAb fragment consists of a VH domain or a VL domain (e.g. human, camelid, or shark).
In general, references to antibodies are to be construed as also referring to antigen binding portions thereof, and in particular, may include antigen binding portions thereof that comprise SEQ ID NO:98 between their E and F β-strands.
A single-chain antibody (scFv) is an antibody in which a VL and VH regions are paired to form a monovalent molecules via a synthetic linker that enables them to be made as a single protein chain. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the 2 domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating 2 antigen binding sites. One or more CDRs may be incorporated into a molecule either covalently or noncovalently to make it an immunoadhesin. An immunoadhesin may incorporate the CDR (s) as part of a larger polypeptide chain, may covalently link the CDR (s) to another polypeptide chain, or may incorporate the CDR(s) noncovalently. The CDRs permit the immunoadhesin to specifically bind to a particular antigen of interest.
Mammalian light chains are of two types, K and A, and in any given antibody molecule only one type occurs. Approximately twice as many K as A molecules are produced in humans but in other mammals this ratio can vary. Each free light chain molecule contains approximately 220 amino acids in a single polypeptide chain that is folded to form the constant and variable region domains.
During B cell development, a recombination event at the DNA level joins a single variable (V) segment with a joining (J) segment; the constant (C) segment is later joined by splicing at the RNA level. Recombination of many different V segments with several J segments provides a wide range of antigen recognition. Additional diversity is attained by junctional diversity, resulting from the random additional of nucleotides by terminal deoxynucleotidyltransferase, and by somatic hypermutation, which occurs during B cell maturation in the spleen and lymph nodes. Constant kappa (CLκ) regions are encoded by a single gene, whereas lambda constant (CLλ) regions are encoded by multiple genes, and undergo splicing. Several markers associated with particular polymorphic species of CLλ are known: IgCLλ1 (Mcg marker); IGLC2-IgCLλ2 (Kern-Oz-marker); IgCLλ 3 (Kern-Oz+ marker), and IgCLλ7, for example. The skilled person can easily establish all of the polymorphisms so far identified in human CLλ chains. SEQ ID NO:93 incorporates many of the presently identified polymorphisms. The sequences of the present invention encompass other known polymorphisms of the CLκ and CLλ, and antibodies in general. Two polymorphic loci have been identified in the CLκ; CLκ-V/A45 and CLκ-L/V83. The three polymorphisms so far identified are: Km(1): CLκ-V45/L83; Km(1,2): CLκ-A45/L83; and Km(3): CLκ-A45/V83.
An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be different. For instance, a naturally-occurring Ig has 2 identical binding sites, a single-chain antibody or Fab fragment has one binding site, while a “bispecific” or “bifunctional” antibody has 2 different binding sites.
An “isolated antibody” is an antibody that (1) is not associated with naturally-associated components, including other naturally-associated antibodies, that accompany it in its native state, (2) is free of other proteins from the same species, (3) is expressed by a cell that does not naturally express the antibody, or is expressed by a cell from a different species, or (4) does not occur in nature.
The term “human antibody” includes all antibodies that have one or more variable and constant regions derived from human Ig sequences. In some embodiments of the present invention, all of the variable and constant domains of the anti-IGF1R antibody are derived from human Ig sequences (a fully human antibody). A humanized antibody is an antibody that is derived from a non-human species, in which certain amino acids in the framework and constant domains of the heavy and light chains have been mutated so as to avoid or abrogate an immune response in humans. Alternatively, a humanized antibody may be produced by fusing the constant domains from a human antibody to the variable domains of a non-human species.
The term “chimeric antibody” refers to an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies.
The term “epitope” includes any protein determinant capable of specific binding to an Ig or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific 3 dimensional structural characteristics, as well as specific charge characteristics. An antibody is said to specifically bind an antigen when the dissociation constant is <1 uM, preferably <100 nM and more preferably: <10 nM.
The term multifunctional antibody conjugate, or MAC, refers to an antibody as defined herein, or antigen binding portion thereof, covalently conjugated to at least one Effector Moiety that binds to a target. The Effector Moiety may be a peptide, small molecule, protein, nucleic acid molecule, toxin, aptamer, or antigen binding antibody or fragment thereof. References to conjugation of peptides and the like referred to throughout the specification generally applies to conjugation to proteins and (antigen binding) antibodies or fragments thereof.
Fully human antibodies are expected to minimize the immunogenic and allergic responses intrinsic to mouse or mouse-derivatized monoclonal antibodies (Mabs) and thus to increase the efficacy and safety of the administered antibodies. The use of fully human antibodies can be expected to provide a substantial advantage in the treatment of chronic and recurring human diseases, such as inflammation and cancer, which may require repeated antibody administrations. In another embodiment, the invention provides a MAC comprising an antibody that does not bind complement.
In addition, fusion antibodies can be created in which 2 (or more) single-chain antibodies are linked to one another. This is useful if one wants to create a divalent or polyvalent antibody on a single polypeptide chain, or if one wants to create a bispecific antibody.
One type of derivatized antibody is produced by crosslinking 2 or more antibodies (of the same type or of different types; e. g. to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, having 2 distinctly reactive groups separated by an appropriate spacer (e. g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e. g. disuccinimidyl suberate).
Another type of derivatized antibody is a labelled antibody. Useful detection agents with which an antibody or antibody portion of the invention may be derivatized include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors and the like. An antibody may also be labelled with enzymes that are useful for detection, such as horseradish peroxidase, galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like. When an antibody is labelled with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a reaction product that can be discerned. For example, when the agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is detectable. An antibody may also be labelled with biotin, and detected through indirect measurement of avidin or streptavidin binding. An antibody may be labelled with a magnetic agent, such as gadolinium. An antibody may also be labelled with a predetermined polypeptide epitope recognized by a secondary reporter (e. g. leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
The antibody may also be derivatized with a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate group. These groups may be useful to improve the biological characteristics of the antibody, e.g. to increase serum half-life or to increase tissue binding.
Antibody Specificity
In some embodiments comprising antigen binding domains, the antigen binding domain (for example, but not limited to, an antibody variable region having all 6 CDRs, or an equivalent region that is at least 90 percent identical to an antibody variable region) is chosen from: abagovomab, abatacept (ORENCIA®), abciximab (REOPRO®, c7E3 Fab), adalimumab (HUMIRA®), adecatumumab, alemtuzumab (CAMPATH®, MabCampath or Campath-1H), altumomab, afelimomab, anatumomab mafenatox, anetumumab, anrukizumab, apolizumab, arcitumomab, aselizumab, atlizumab, atorolimumab, bapineuzumab, basiliximab (SIMULECT®), bavituximab, bectumomab (LYMPHOSCAN®), belimumab (LYMPHO-STAT-B®), bertilimumab, besilesomab, βcept (ENBREL®), bevacizumab (AVASTIN®), biciromab brallobarbital, bivatuzumab mertansine, brentuximab vedotin (ADCETRIS®), canakinumab (ACZ885), cantuzumab mertansine, capromab (PROSTASCINT®), catumaxomab (REMOV ABC)), cedelizumab (CIMZIA®), certolizumab pegol, cetuximab (ERBITUX®), clenoliximab, dacetuzumab, dacliximab, daclizumab (ZENAP AX(®), denosumab (AMG 162), detumomab, dorlimomab aritox, dorlixizumab, duntumumab, durimulumab, durmulumab, ecromeximab, eculizumab (SOLIRIS®), edobacomab, edrecolomab (Mab17-1A, PANOREX®), efalizumab (RAPTIVA®), efungumab (MYCOGRAB®), elsilimomab, enlimomab pegol, epitumomab cituxetan, efalizumab, epitumomab, epratuzumab, erlizumab, ertumaxomab (REXOMUN®), etaracizumab (etaratuzumab, VITAXIN®, ABEGRINTM), exbivirumab, fanolesomab (NEUTROSPEC®), faralimomab, felvizumab, fontolizumab (HUZAF®), galiximab, gantenerumab, gavilimomab (ABX-CBL®), gemtuzumab ozogamicin (MYLOTARG®), golimumab (CNTO 148), gomiliximab, ibalizumab (TNX-355), ibritumomab tiuxetan (ZEVALIN®), igovomab, imciromab, infliximab (REMICAD E®), inolimomab, inotuzumab ozogamicin, ipilimumab (YERVOY®, MDX-010), iratumumab, keliximab, labetuzumab, lemalesomab, lebrilizumab, lerdelimumab, lexatumumab (HGS-ETR2, ETR2-ST01), lexitumumab, libivirumab, lintuzumab, lucatumumab, lumiliximab, mapatumumab (HGS-ETRI, TRM-I), maslimomab, matuzumab (EMD72000), mepolizumab (BOSATRIA®), metelimumab, milatuzumab, minretumomab, mitumomab, morolimumab, motavizumab (NUMAXTM), muromonab (OKT3), nacolomab tafenatox, naptumomab estafenatox, natalizumab (TYSABRI®, ANTEGREN®), nebacumab, nerelimomab, nimotuzumab (THERACIM hR3®, THERA-CIM-hR3®, THERALOC®), nofetumomab merpentan (VERLUMA®), ocrelizumab, odulimomab, ofatumumab, omalizumab (XOLAIR®), oregovomab (OVAREX®), otelixizumab, pagibaximab, palivizumab (SYNAGIS®), panitumumab (ABX-EGF, VECTIBIX®), pascolizumab, pemtumomab (THERAGYN®), pertuzumab (2C4, OMNITARG®), pexelizumab, pintumomab, ponezumab, priliximab, pritumumab, ranibizumab (LUCENTIS®), raxibacumab, regavirumab, reslizumab, rituximab (RITUXAN®, MabTHERA®), rovelizumab, ruplizumab, satumomab, sevirumab, sibrotuzumab, siplizumab (MEDI-507), sontuzumab, stamulumab (Myo-029), sulesomab (LEUKOSCAN®), tacatuzumab tetraxetan, tadocizumab, talizumab, taplitumomab paptox, tefibazumab (AUREXIS®), telimomab aritox, teneliximab, teplizumab, ticilimumab, tocilizumab (ACTEMRA®), toralizumab, tositumomab, trastuzumab (HERCEPTIN®), tremelimumab (CP-675,206), tucotuzumab celmoleukin, tuvirumab, urtoxazumab, ustekinumab (CNTO 1275), vapaliximab, veltuzumab, vepalimomab, visilizumab (NUVION®), volociximab (M200), votumumab (HUMASPECT®), zalutumumab, zanolimumab (HuMAX-CD4), ziralimumab, or zolimomab aritox.
In some embodiments comprising antigen binding domains, the antigen binding domain comprise a heavy and light chain variable domain having six CDRs, and/or compete for binding with an antibody selected from the preceding list. In some embodiments comprising antigen binding domains, the antigen binding domain binds the same epitope as the antibodies in the preceding list. In some embodiments comprising antigen binding domains, the antigen binding domain comprises a heavy and light chain variable domain having six total CDRs, and binds to the same antigen as the antibodies in the preceding list.
In some embodiments comprising antigen binding domains, the antigen binding domain comprises a heavy and light chain variable domain having six (6) total CDRs, and specifically binds to an antigen selected from: PDGFRα, PDGFRβ, PDGF, VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, VEGFR1, VEGFR2, VEGFR3, FGF, FGF2, HGF, KDR, flt-1, FLK-1, Ang-2, Ang-1, PLGF, CEA, CXCL13, Baff, IL-21, CCL21, TNF-α, CXCL12, SDF-I, bFGF, MAC-I, IL23p19, FPR, IGFBP4, CXCR3, TLR4, CXCR2, EphA2, EphA4, EphrinB2, EGFR(ErbBl), HER2(ErbB2 or pI85neu), HER3(ErbB3), HER4 ErbB4 or tyro2), SCI, LRP5, LRP6, RAGE, s100A8, s100A9, Nav1.7, GLPI, RSV, RSV F protein, Influenza HA protein, Influenza NA protein, HMGBI, CD16, CD19, CD20, CD21, CD28, CD32, CD32b, CD64, CD79, CD22, ICAM-I, FGFRI, FGFR2, HDGF, EphB4, GITR, β-amyloid, hMPV, PIV-I, PIV-2, OX40L, IGFBP3, cMet, PD-I, PLGF, Neprolysin, CTD, IL-18, IL-6, CXCL-13, IL-IRI, IL-15, IL-4R, IgE, PAI-I, NGF, EphA2, uPARt, DLL-4, αvβ5, αvβ6, α5β1, α3β1, interferon receptor type I and type II, CD 19, ICOS, IL-17, Factor II, Hsp90, IGF, IGF-I, IGF-II, CD 19, GM-CSFR, PIV-3, CMV, IL-13, IL-9, and EBV.
In some embodiments comprising antigen binding domains, the antigen binding domain specifically binds to a member (receptor or ligand) of the TNF superfamily. Various molecules include, but are not limited to Tumor Necrosis Factor-α (“TNF-α”), Tumor Necrosis Factor-β (“TNF-β”), Lymphotoxin-α (“LT-α”), CD30 ligand, CD27 ligand, CD40 ligand, 4-1 BB ligand, Apo-1 ligand (also referred to as Fas ligand or CD95 ligand), Apo-2 ligand (also referred to as TRAIL), Apo-3 ligand (also referred to as TWEAK), osteoprotegerin (OPG), APRIL, RANK ligand (also referred to as TRANCE), TALL-I (also referred to as BlyS, BAFF or THANK), DR4, DR5 (also known as Apo-2, TRAIL-R2, TR6, Tango-63, hAPO8, TRICK2, or KILLER), DR6, DcRI, DcR2, DcR3 (also known as TR6 or M68), CART, HVEM (also known as ATAR or TR2), GITR, ZTNFR-5, NTR-I, TNFLI, CD30, LTBr, 4-1BB receptor and TR9.
In some embodiments comprising antigen binding domains, the antigen binding domain is capable of binding one or more targets chosen from 5T4, ABL, ABCB5, ABCFI, ACVRI, ACVRIB, ACVR2, ACVR2B, ACVRLI, ADORA2A, Aggrecan, AGR2, AICDA, AIFI, AIGI, AKAPI, AKAP2, AMH, AMHR2, angiogenin (ANG), ANGPTI, ANGPT2, ANGPTL3, ANGPTL4, Annexin A2, ANPEP, APC, APOCI, AR, aromatase, ATX, AXI, AZGPI (zinc-a-glycoprotein), B7.1, B7.2, B7-H1, BAD, BAFF, BAGI, BAII, BCR, BCL2, BCL6, BDNF, BLNK, BLRI (MDR15), BlyS, BMP1, BMP2, BMP3B (GDFIO), BMP4, BMP6, BMP7, BMP8, BMP9, BMP11, BMP12, BMPR1A, BMPR1B, BMPR2, BPAGI (plectin), BRCAI, C19orf10 (IL27w), C3, C4A, C5, C5R1, CANTI, CASPI, CASP4, CAVI, CCBP2 (D6/JAB61), CCLI (1-309), CCLI 1 (eotaxin), CCL13 (MCP-4), CCL15 (MIP-Id), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19 (MIP-3b), CCL2 (MCP-1), MCAF, CCL20 (MIP-3a), CCL21 (MEP-2), SLC, exodus-2, CCL22(MDC/STC-I), CCL23 (MPIF-I), CCL24 (MPIF-2/eotaxin-2), CCL25 (TECK), CCL26(eotaxin-3), CCL27 (CTACK/ILC), CCL28, CCL3 (MIP-la), CCL4 (MIP-Ib), CCL5(RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CCNAI, CCNA2, CCNDI, CCNEI, CCNE2, CCRI (CKRI/HM145), CCR2 (mcp-IRB/RA), CCR3 (CKR3/CMKBR3), CCR4, CCR5(CMKBR5/ChemR13), CCR6 (CMKBR6/CKR-L3/STRL22/DRY6), CCR7 (CKR7/EBI1), CCR8 (CMKBR8/TERI/CKR-LI), CCR9 (GPR-9-6), CCRLI (VSHKI), CCRL2 (L-CCR), CD164, CD19, CDIC, CD20, CD200, CD-22, CD24, CD28, CD3, CD33, CD35, CD37, CD38, CD3E, CD3G, CD3Z, CD4, CD40, CD40L, CD44, CD45RB, CD46, CD52, CD69, CD72, CD74, CD79A, CD79B, CD8, CD80, CD81, CD83, CD86, CD105, CD137, CDHI (E-cadherin), CDCP1CDH10, CDH12, CDH13, CDH18, CDH19, CDH20, CDH5, CDH7, CDH8, CDH9, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK9, CDKNIA (p21Wapl/Cipl), CDKNIB (p27Kipl), CDKNIC, CDKN2A (p16INK4a), CDKN2B, CDKN2C, CDKN3, CEBPB, CERI, CHGA, CHGB, Chitinase, CHSTIO, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSF8, CLDN3, CLDN7 (claudin-7), CLN3, CLU (clusterin), CMKLRI, CMKORI (RDCI), CNRI, COLI 8A1, COL1A1.COL4A3, COL6A1, CR2, Cripto, CRP, CSFI (M-CSF), CSF2 (GM-CSF), CSF3 (GCSF), CTLA4, CTL8, CTNNBI (b-catenin), CTSB (cathepsin B), CX3CL1 (SCYDI), CX3CR1 (V28), CXCLI(GROI), CXCLIO (IP-IO), CXCLII (I-TAC/IP-9), CXCL12 (SDFI), CXCL13, CXCL 14, CXCL 16, CXCL2 (GRO2), CXCL3 (GRO3), CXCL5 (ENA-78/LIX), CXCL6 (GCP-2), CXCL9 (MIG), CXCR3 (GPR9/CKR-L2), CXCR4, CXCR6 (TYMSTR/STRL33/Bonzo), CYB5, CYCI, Cyr61, CYSLTRI, c-Met, DAB2IP, DES, DKFZp451J0118, DNCLI, DPP4, E2F1, ECGFI5EDGI, EFNAI, EFNA3, EFNB2, EGF, ELAC2, ENG, endoglin, ENOI, EN02, EN03, EPHAI, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHAIO, EPHBI, EPHB2, EPHB3, EPHB4, EPHB5, EPHB6, EPHRIN-AI, EPHRIN-A2, EPHRIN-A3, EPHRIN-A4, EPHRIN-A5, EPHRIN-A6, EPHRIN-BI, EPHRIN-B2, EPHRTN-B3, EPHB4, EPG, ERBB2 (Her-2), EREG, ERK8, Estrogen receptor, ESRI, ESR2, F3 (TF), FADD, farnesyltransferase, FasL, FASNf, FCER1A, FCER2, FCGR3A, FGF, FGFI (aFGF), FGFIO, FGFI 1, FGF12, FGF12B, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2 (bFGF), FGF20, FGF21 (such as mimAb1), FGF22, FGF23, FGF3 (int-2), FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF8, FGF9, FGFR3, FIGF (VEGFD), FILI(EPSILON), FBLI (ZETA), FLJ12584, FLJ25530, FLRTI (fibronectin), FLTI, FLT-3, FOS, FOSLI(FRA-I), FY (DARC), GABRP (GABAa), GAGEBI, GAGECI, GALNAC4S-65T, GATA3, GD2, GD3, GDF5, GDF8, GFII, GGTI, GM-CSF, GNASI, GNRHI, GPR2 (CCRIO), GPR31, GPR44, GPR81 (FKSG80), GRCCIO (CIO), gremlin, GRP, GSN (Gelsolin), GSTPI, HAVCR2, HDAC, HDAC4, HDAC5, HDAC7A, HDAC9, Hedgehog, HGF, HIFIA, HIPI, histamine and histamine receptors, HLA-A, HLA-DRA, HM74, HMOXI, HSP90, HUMCYT2A, ICEBERG, ICOSL, ID2, IFN-α, IFNAI, IFNA2, IFNA4, IFNA5, EFNA6, BFNA7, IFNBI, IFNgamma, IFNWI, IGBPI, IGFI, IGFIR, IGF2, IGFBP2, IGFBP3, IGFBP6, DL-I, ILIO, ILIORA, ILIORB, IL-1, ILIRI (CD121a), ILIR2(CD121b), IL-IRA, IL-2, IL2RA (CD25), IL2RB(CD122), IL2RG(CD132), IL-4, IL-4R(CD123), IL-5, IL5RA(CD125), IL3RB(CD131), IL-6, IL6RA (CD126), IR6RB(CD130), IL-7, IL7RA(CD127), IL-8, CXCRI (IL8RA), CXCR2 (IL8RB/CD128), IL-9, IL9R (CD129), IL-10, IL10RA(CD210), IL10RB(CDW210B), IL-11, ILI IRA, IL-12, IL-12A, IL-12B, IL-12RB1, IL-12RB2, IL-13, IL13RA1, IL13RA2, IL14, IL15, IL15RA, 1L16, IL17, IL17A, IL17B, IL17C, IL17R, IL18, IL18BP, IL18R1, IL18RAP, IL19, ILIA, ILIB, ILIFIO, IL1F5, IL1F6, IL1F7, IL1F8, DL1F9, ILIHYI, ILIRI, IL1R2, ILIRAP, ILIRAPLI, IL1RAPL2, ILIRLI, IL1RL2, ILIRN, IL2, IL20, IL20RA, IL21R, IL22, IL22R, IL22RA2, IL23, DL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL2RA, IL2RB, IL2RG, IL3, IL30, IL3RA, IL4, IL4R, IL6ST (glycoprotein 130), ILK, INHA, INHBA, INSL3, INSL4, IRAKI, IRAK2, ITGA1, ITGA2, ITGA3, ITGA6 (α 6 integrin), ITGAV, ITGB3, ITGB4 (β 4 integrin), JAKI, JAK3, JTB, JUN, K6HF, KAII, KDR, KIM-1, KITLG, KLF5 (GC Box BP), KLF6, KLKIO, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, KRTI, KRT19 (Keratin 19), KRT2A, KRTHB6 (hair-specific type II keratin), LAMAS, LEP (leptin), Lingo-p75, Lingo-Troy, LPS, LRP5, LRP6, LTA (TNF-b), LTB, LTB4R (GPR16), LTB4R2, LTBR, MACMARCKS, MAG or Omgp, MAP2K7 (c-Jun), MCP-I, MDK, MIBI, midkine, MIF, MISRII, MJP-2, MK, MKI67 (Ki-67), MMP2, MMP9, MS4A1, MSMB, MT3 (metallothionectin-Ui), mTOR, MTSSI, MUCI (mucin), MYC, MYD88, NCK2, neurocan, neuregulin-1, neuropilin-1, NFKBI, NFKB2, NGFB (NGF), NGFR, NgR-Lingo, NgR-Nogo66 (Nogo), NgR-p75, NgR-Troy, NMEI (NM23A), NOTCH, NOTCHI, NOX5, NPPB, NROBI, NROB2, NRIDI, NR1D2, NR1H2, NR1H3, NR1H4, NR1I2, NR1I3, NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR2F6, NR3C1, NR3C2, NR4A1, NR4A2, NR4A3, NR5A1, NR5A2, NR6A1, NRPI, NRP2, NT5E, NTN4, OCT-1, ODZ1, OPN1, OPN2, OPRDI, P2RX7, PAP, PARTI, PATE, PAWR, PCA3, PCDGF, PCNA, PDGFA, PDGFB, PDGFRA, PDGFRB, PECAMI, peg-asparaginase, PF4 (CXCL4), Plexin B2 (PLXNB2), PGF, PGR, phosphacan, PIAS2, PI3 Kinase, PIK3CG, PLAU (uPA), PLG5PLXDCI, PKC, PKC-β, PPBP (CXCL7), PPID, PRI, PRKCQ, PRKDI, PRL, PROC, PROK2, pro-NGF, prosaposin, PSAP, PSCA, PTAFR, PTEN, PTGS2 (COX-2), PTN, RAC2 (P21Rac2), RANK, RANK ligand, RARB, RGSI, RGS13, RGS3, RNFI10 (ZNF144), Ron, R0B02, RXR, selectin, S100A2, S100A8, S100A9, SCGB 1D2 (lipophilin B), SCGB2A1 (mammaglobin 2), SCGB2A2 (mammaglobin 1), SCYEI (endothelial Monocyte-activating cytokine), SDF2, SERPENA1, SERPINA3, SERPINBS (maspin), SERPINEI (PAI-I), SERPINFI, SHIP-I, SHIP-2, SHBI, SHB2, SHBG, SfcAZ, SLC2A2, SLC33A1, SLC43A1, SLIT2, SPPI, SPRRIB (SprI), ST6GAL1, STABI, STATE, STEAP, STEAP2, SULF-1, Sulf-2, TB4R2, TBX21, TCPIO, TDGFI, TEK, TGFA, TGFBI, TGFBIII, TGFB2, TGFB3, TGFBI, TGFBRI, TGFBR2, TGFBR3, THIL, THBSI (thrombospondin-1), THBS2/THBS4, THPO, TIE (Tie-1), TIMP3, tissue factor, TIKI2, TLR10, TLR2, TLR3, TLR4, TLR5, TLR6JLR7, TLR8, TLR9, TM4SF1, TNF, TNF-a, TNFAIP2 (B94), TNFAIP3, TNFRSFIIA, TNFRSFIA, TNFRSFIB, TNFRSF21, TNFRSF5, TNFRSF6 (Fas), TNFRSF7, TNFRSF8, TNFRSF9, TNFSFIO (TRAIL), TNFSFI 1 (TRANCE), TNFSF12 (APO3L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFSF15 (VEGI), TNFSF 18, TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TOLLIP, Toll-like receptors, TLR2, TLR4, TLR9, T0P2A (topoisomerase Iia), TP53, TPMI, TPM2, TRADD, TRAFI, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TRKA, TREMI, TREM2, TRPC6, TROY, TSLP, TWEAK, Tyrosinase, uPAR, VEGF, VEGFB, VEGFC, versican, VHL C5, VLA-4, Wnt-1, XCLI (lymphotactin), XCL2 (SCM-Ib), XCRI (GPR5/CCXCRI), YYI, and ZFPM2.
Catalytic Antibodies
In some aspects of the invention, the MAC comprises a catalytic antibody, or antigen binding portion thereof. In some aspects, the antibody may be an aldolase antibody.
The contents of US2006205670 are incorporated herein by reference—in particular paragraphs [0153]-[0233], describing antibodies, useful fragments and variants and modifications thereof, combining sites and CDRs, antibody preparation, expression, humanization, amino acid modification, glycosylation, ADCC, CDC, increasing serum half life of antibodies, expression vectors, mammalian host systems, and folding, amongst other elements of antibody technology.
“Combining site”, as used herein, (also known as the antibody binding site) refers to the region of the Ig or Ig domains that combine (or can combine) with the determinant of an appropriate antigen (or a structurally similar protein). The term generally includes the CDRs and the adjacent framework residues that are involved in antigen binding.
“Aldolase antibodies” as used herein, refers to antibodies containing combining site portions that, when unencumbered (for example by conjugation), catalyze an aldol addition reaction between an aliphatic ketone donor and an aldehyde acceptor. Aldolase antibodies are capable of being generated by immunization of an immune-responsive animal with an immunogen that includes a 1,3 diketone hapten of the formula:




coupled to a carrier protein, and further characterized by having a lysine with a reactive ε-amino group in the combining site of the antibody. Aldolase antibodies are further characterized by their catalytic activity being subject to inhibition with the 1,3-diketone hapten by formation of a complex between the 1,3-diketone hapten and the ε-amino group of the lysine of the catalytic antibody.

As discussed, in certain embodiments, certain antibodies that can be used to make MACs, compositions and samples of the invention may comprise a reactive side chain in the antibody combining site. A reactive side chain may be present naturally or may be placed in an antibody by mutation. The reactive residue of the antibody combining site may be associated with the antibody, such as when the residue is encoded by nucleic acid present in the lymphoid cell first identified to make the antibody. Alternatively, the amino acid residue may arise by purposely mutating the DNA so as to encode the particular residue. The reactive residue may be a non-natural residue arising, for example, by biosynthetic incorporation using a unique codon, tRNA, and aminoacyl-tRNA as discussed herein. In another approach, the amino acid residue or its reactive functional groups (e.g., a nucleophilic amino group or sulfhydryl group) may be attached to an amino acid residue in the antibody combining site. Thus, covalent linkage with the antibody occurring “through an amino acid residue in a combining site of an antibody” as used herein means that linkage can be directly to an amino acid residue of an antibody combining site or through a chemical moiety that is linked to a side chain of an amino acid residue of an antibody combining site. In some embodiments, the amino acid is cysteine, and the reactive group of the side chain is a sulfhydryl group. In other embodiments, the amino acid residue is lysine, and the reactive group of the side chain is the ε-amino group. In some embodiments, the amino acid is K93 on the heavy chain according to Kabat numbering. In some embodiments, the amino acid is on HC-K99 of h38C2 according to the numbering of SEQ ID NOs: 65 and 66.
Catalytic antibodies are one source of antibodies with suitable combining sites that comprise one or more reactive amino acid side chains. Such antibodies include aldolase antibodies, β lactamase antibodies, esterase antibodies, and amidase antibodies.
One embodiment comprises an aldolase antibody such as the mouse monoclonal antibodies mAb 33F12 and mAb 38C2 (whose VL and VH comprise SEQ ID NO:68 and 69), as well as suitably chimeric and humanized versions of such antibodies (e.g. h38C2IgG1: SEQ ID NOs:64 and 65 and h38C2-IgG2: SEQ ID NOs:64 and 66). In favourable aspects, a heavy chain such as SEQ ID NO:65 or SEQ ID NO:66 is used in conjunction with the h38C2 VL (SEQ ID NO:67) fused to one of the CL domains of the invention comprising SEQ ID NO:98.
Mouse mAb 38C2 (and h38C2) has a reactive lysine near to but outside HCDR3, and is the prototype of a new class of catalytic antibodies that were generated by reactive immunization and mechanistically mimic natural aldolase enzymes. Other aldolase catalytic antibodies that may be used include the antibodies produced by the hybridoma 85A2, having ATCC accession number PTA-1015; hybridoma 85C7, having ATCC accession number PTA-1014; hybridoma 92F9, having ATCC accession number PTA-1017; hybridoma 93F3, having ATCC accession number PTA-823; hybridoma 84G3, having ATCC accession number PTA-824; hybridoma 84G11, having ATCC accession number PTA-1018; hybridoma 84H9, having ATCC accession number PTA-1019; hybridoma 85H6, having ATCC accession number PTA-825; hybridoma 90G8, having ATCC accession number PTA-1016. Through a reactive lysine, these antibodies catalyze aldol and retro-aldol reactions using the enamine mechanism of natural aldolases.
Compounds of the invention may also be formed by linking a targeting agent to a reactive cysteine, such as those found in the combining sites of thioesterase and esterase catalytic antibodies. Reactive amino acid-containing antibodies may be prepared by means well known in the art, including mutating an antibody combining site residue to encode for the reactive amino acid or chemically derivatizing an amino acid side chain in an antibody combining site with a linker that contains the reactive group.
The antibody may be a humanized antibody. Where compounds of the invention are covalently linked to the combining site of an antibody, and such antibodies are humanized, it is important that such antibodies be humanized with retention of high linking affinity for the W group. Various forms of humanized murine aldolase antibodies are contemplated. One embodiment uses the humanized aldolase catalytic antibody h38c2 IgG1 or h38c2 Fab with human constant domains CLκ and CHγ11. Human germline VLk gene DPK-9 and human Jk gene JK4 were used as frameworks for the humanization of the kappa light chain variable domain of m38c2, and human germline gene DP-47 and human JH gene JH4 were used as frameworks for the humanization of the heavy chain variable domain of m38c2. FIG. 8A illustrates a sequence alignment between the variable light and heavy chains in m38c2, h38c2, and human germlines. h38c2 may utilize IgG1, IgG2, IgG3, or IgG4 constant domains, including any of the allotypes thereof. Another embodiment uses a chimeric antibody comprising the variable domains (VL and VH) of h38c2 (SEQ ID NOs: 67 and 68) and the constant domains from an IgG1, IgG2, IgG3, or IgG4 antibody that comprises a polypeptide of the invention comprising SEQ ID NO:98 between β-sheets E and F. The LC may comprise SEQ ID NO:254. The antibody may be a full-length antibody, Fab, Fab′, F(ab′)2, VH, VL, diabody, or minibody comprising VH and VL domains from h38c2. The antibody may be an antibody comprising the VL and VH domains from h38c2 and a constant domain selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. The antibody may be a humanized version of a murine aldolase antibody comprising a constant region from a human IgG, IgA, IgM, IgD, or IgE antibody. In another embodiment, the antibody is a chimeric antibody comprising the VL and VH region from a murine aldolase antibody (e.g. SEQ ID NO:69 and 70) and a constant region from a human IgG, IgA, IgM, IgD, or IgE antibody, comprising a polypeptide of the invention comprising SEQ ID NO:98 between β-sheets E and F. In further embodiments, the antibody is a fully human version of a murine aldolase antibody comprising a polypeptide sequence from natural or native human IgG, IgA, IgM, IgD, or IgE antibody.
One embodiment uses h38c2 F(ab′)2. h38c2 F(ab′)2 may be produced by the proteolytic digestion of h38c2 IgG1.
As used herein, “pharmacokinetics” refers to the concentration of an administered compound in the serum over time. Pharmacodynamics refers to the concentration of an administered compound in target and nontarget tissues over time and the effects on the target tissue (e.g., efficacy) and the non-target tissue (e.g., toxicity). Improvements in, for example, pharmacokinetics or pharmacodynamics can be designed for a particular targeting agent or biological agent, such as by using labile linkages or by modifying the chemical nature of any linker (e.g., changing solubility, charge, and the like). The term “Koff” refers to the off rate constant for dissociation of an antibody from the antibody/antigen complex. The term“Kd” refers to the dissociation constant of a particular antibody-antigen interaction.
In some aspects, the invention provides for pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, and prodrugs of compounds, samples, compositions and pharmaceutical compositions of the invention.
Catalytic Antibody Linkers
Certain linkers suitable for connecting targeting agents (TA) to the combining site of catalytic antibodies (Catalytic Antibody Linkers: CAb-linkers) are disclosed in US2009098130, the contents of which are incorporated herein by reference. The term “targeting agents” is used herein to distinguish from the term “Effector Moiety” but it is apparent that the types of molecules attached at the end of a CAb-linker as a TA, or attached to the end of a MAC-linker as an Effector Moiety may be interchangable. In particular, aspects of US2009098130 pertaining to the general formulae describing (CAb-linkers, specific CAb-linker structure, synthesis of Cab-linkers and combinations of different elements of P1, Q1 and W1, (therein classified as X, Y and Z groups respectively) as specifically and generally described therein are herein included.
The CAb-linker may be linear or branched, and optionally includes one or more carbocyclic or heterocyclic groups. CAb-linker length may be viewed in terms of the number of linear atoms, with cyclic moieties such as aromatic rings and the like to be counted by taking the shortest route around the ring. In some embodiments, the CAb-linker has a linear stretch of between 5-15 atoms, in other embodiments 15-30 atoms, in still other embodiments 30-50 atoms, in still other embodiments 50-100 atoms, and in still other embodiments 100-200 atoms. Other CAb-linker considerations include the effect on physical or pharmacokinetic properties of the resulting compound, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (more or less stable as well as planned degradation), rigidity, flexibility, immunogenicity, and modulation of antibody binding, the ability to be incorporated into a micelle or liposome, and the like.
In some aspects the CAb-linker may be covalently linked to the side chain of the TA-linking residue. The linker may comprise the formula: P1-Q1-W1; wherein P1 is a biologically compatible connecting chain including any atom selected from the group consisting of C, H, N, O, P, S, F, Cl, Br, and I, and may comprise a polymer or block co-polymer, and is covalently linked to the linking residue (through side chain, amino terminus, or carboxyl terminus as appropriate) where the linker is linear, Q1 is an optionally present recognition group comprising at least a ring structure; and W1 is an attachment moiety comprising a covalent link to an amino acid side chain in a combining site of an antibody.
When present, Q1 may have the optionally substituted structure:




wherein a, b, c, d, and e are independently carbon or nitrogen; f is carbon, nitrogen, oxygen, or sulfur; Q1 is attached to P1 and W1 independently at any 2 ring positions of sufficient valence; and no more than 4 of a, b, c, d, e, or f are simultaneously nitrogen and preferably a, b, c, d, and e in the ring structure are each carbon. In some aspects, Q1 may be phenyl. Although not wishing to be bound by any theory, it is believed that the Q1 group can assist in positioning the reactive group into a suitable antibody combining site so that the W1 group can react with a reactive amino acid side chain.

The CAb-linker may be designed such that it contains a reactive group capable of covalently or non-covalently forming a bond with a macromolecule, such as an antibody, protein, or fragment thereof. The reactive group is chosen for use with a reactive residue in a particular combining site. For example, a chemical moiety for modification by an aldolase antibody may be a ketone, diketone, β lactam, active ester haloketone, lactone, anhydride, maleimide, α-haloacetamide, cyclohexyl diketone, epoxide, aldehyde, amidine, guanidine, imine, enamine, phosphate, phosphonate, epoxide, aziridine, thioepoxide, masked or protected diketone (ketal for example), lactam, haloketone, aldehyde, and the like.
In some embodiments, W1, prior to conjugation with the side-chain of a residue in the combining site of an antibody, includes one or more C═O groups arranged to form an azetidinone, diketone, an acyl β-lactam, an active ester, a haloketone, a cyclohexyl diketone group, an aldehyde, a maleimide, an activated alkene, an activated alkyne or, in general, a molecule comprising a leaving group susceptible to nucleophilic or electrophilic displacement. Other groups may include a lactone, an anhydride, an α-haloacetamide, an imine, a hydrazide, or an epoxide. Exemplary linker electrophilic reactive groups that can covalently bond to a reactive nucleophilic group (e.g., a lysine or cysteine side chain) in a combining site of antibody include acyl β-lactam, simple diketone, succinimide active ester, maleimide, haloacetamide with linker, haloketone, cyclohexyl diketone, aldehyde, amidine, guanidine, imine, enamine, phosphate, phosphonate, epoxide, aziridine, thioepoxide, a masked or protected diketone (a ketal, for example), lactam, sulfonate, and the like, masked C═O groups such as imines, ketals, acetals, and any other known electrophilic group. In certain embodiments, the reactive group includes one or more C═O groups arranged to form an acyl β-lactam, simple diketone, succinimide active ester, maleimide, haloacetamide with linker, haloketone, cyclohexyl diketone, or aldehyde. W1 may be a substituted alkyl, substituted cycloalkyl, substituted aryl, substituted arylalkyl, substituted heterocyclyl, or substituted heterocyclylalkyl, wherein at least one substituent is a 1,3-diketone moiety, an acyl lactam, an active ester, an α-haloketone, an aldehyde, a maleimide, a lactone, an anhydride, an α-haloacetamide, an amine, a hydrazide, or an epoxide. In some aspects, the W1 group is covalently linked to a macromolecule scaffold that can provide increased half-life to the peptides of the invention. In some aspects, the W1 group if present is covalently linked to the combining site of an antibody.
In some aspects, prior to conjugation (for example, with the combining site of an antibody), W1 has the structure:




wherein q=0-5. q may be 1 or 2. q may be 1. In other aspects, q may be 2.

In some aspects, following conjugation with the antibody combining site, W1 has the structure:





wherein q=0-5 and Antibody-N— is a covalent bond to a side chain in a combining site of an antibody. q may be 1 or 2. q may be 1. In other aspects, q may be 2.

P1 may be a group comprising three components; P1p-P1s-P1y, wherein P1p is a group specifically adapted to be combinable with the targeting agent, P1s is a spacer region of the P1 group, and P1y is a group adapted to bind to the W1 group. In some aspects, P1y is selected from an amide bond, an enamine bond, or a guanidinium bond. P1y may be selected so as to provide a hydrogen molecule adjacent (within two atoms) to the Q1 group. While not wishing to be bound by theory, it is believed that the H atom can assist the Q1 group recognition of a hydrophobic pocket through H-bond interaction, particularly in respect of the hydrophobic pocket of the binding cleft of a catalytic antibody, such as h38C2. Thus the amide bond, for example, may be orientated such that the NH group is directly bonded to the Q1 group, providing the H of the NH group for hydrogen bonding. Alternatively, the C═O group of an amide may be bonded to the Q1 group, with the H of the NH group about 2 atoms adjacent to the Q1 group, but still available for H-bonding. In some embodiments, P1y is absent. In some embodiments the P1y group has the formula:





In some aspects, P1s is selected such that P1s does not provide any overly reactive groups. P1s may be selected so as to provide an overall length of the P1 groups of between 2-15 atoms. P1s may be selected so that the overall length of the P1 group is between 2 and 10 atoms. X1 groups may be selected so that the overall length of P1 group is 4-8 atoms. P1 groups may be selected so that the overall length of the P1 group is 5 atoms. P1 groups may be selected so that the overall length of P1 group is 6 atoms. In some aspects, P1 groups may comprise one of the following formulae:




where n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and m is present or absent; n may be 1, 2, 3, 4, 5, or 6; n may be 1, 2, 3, or 4; n may be 1; n may be 2; n may be 3; n may be 4.

P1p ideally is selected so as to enable a specific directional covalent linking strategy to the linking residue of a targeting molecule (TA-linking residue), such as a peptide, protein, small molecule, nucleic acid or aptamer. For example, where the TA-linking residue comprises a nucleophilic group, P1p may be an electrophilic group and vice versa. For example, if the TA-linking residue side chain comprises an amine group, such as K, H, Y, orthinine, Dap, or Dab, Xp may be COOH, or other similarly reactive electrophile. If the TA-linking residue is D or E, P1p may comprise a nucleophilic group, such as an amine group. Either of these strategies permits a covalent bond to be formed between the P1p group and the TA-linking residue by amide bond formation strategies. Where the TA-linking group is an amine group, P1p may comprise the formula:




P1 may be an optionally present biologically compatible polymer or block copolymer. P1 may be of the structure:




wherein p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 32, 43, 44, or 45; w, r, and s are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; and Rb at each occurrence is independently hydrogen, substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C3-7 cycloalkyl-C1-6 alkyl, or substituted or unsubstituted aryl-C1-6 alkyl.

Where the TA-linking residue is C, homologs of C, or other thiol-group containing residues, P1p may comprise a maleimide group (or similar) permitting a thiol-maleimide addition reaction strategy to covalently link the P1p group to the TA-linking residue. In some aspects, P1p may also comprise a thiol group, allowing a disulphide bridge to be formed between the TA-linking residue and P1p group. In some aspects, P1p may be be maleimide:




wherein the arrow indicates the point of attachment to the targeting molecule and the parallel line represents to attachment to the Q1 group of the linker. Where the point of attachment to the targeting molecule comprises a cysteine residue, or other thiol bearing side chain, the mechanism of conjugation may be as follows:





In some aspects, the P1p group comprises a substituted maleimide:




In some aspects, P1 is





wherein v and w are selected such that the backbone length of X1 is 6-12 atoms;

In some aspects, the TA-linker is of the formula:




wherein n=1, or 2, or 3, or 4, 5, 6, 7, 8, 9, or 10; n may be 1, 2, 3, 4, 5, or 6; n may be 1; n may be 2; n may be 3; n may be 4. M may be absent. M may be present.

In some aspects, TA-linker is of the formula:





wherein n=1, or 2, or 3, or 4, 5, 6, 7, 8, 9, or 10; n may be 1, 2, 3, 4, 5, or 6; n may be 1; n may be 2; n may be 3; n may be 4. M may be absent. M may be present.

In some aspects, the P1 portion of CA-linkers may be used as the Y1, X1-Y1, Y1-Z and X1-Y1-Z, portion of linkers for a MAC of the invention.
Peptides and Proteins
Acyl lysine, or







Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. As used herein, the 20 natural, or conventional, amino acids and their abbreviations follow IUPAC single letter and three letter codes. “Polypeptide,” “peptide,” and “protein” are used interchangeably to refer to a polymer of amino acid residues. As used herein, these terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analog of a corresponding naturally occurring amino acid. These terms also apply to naturally occurring amino acid polymers. Amino acids can be in the L-form or D-form as long as the binding and other desired characteristics of the peptide are maintained. A polypeptide may be monomeric or polymeric.
Unless indicated otherwise by a “D” prefix, e.g., D-Ala or N-Me-D-Ile, or written in lower case format, e.g., a, i, l, (D versions of Ala, Ile, Leu), the stereochemistry of the α-carbon of the amino acids and aminoacyl residues in peptides described in this specification and the appended claims is the natural or “L” configuration.
All peptide sequences are written according to the generally accepted convention whereby the α-N-terminal amino acid residue is on the left and the α-C-terminal amino acid residue is on the right. As used herein, the term “N-terminus” refers to the free α-amino group of an amino acid in a peptide, and the term “C-terminus” refers to the free α-carboxylic acid terminus of an amino acid in a peptide. A peptide which is N-terminated with a group refers to a peptide bearing a group on the α-amino nitrogen of the N-terminal amino acid residue. An amino acid which is N-terminated with a group refers to an amino acid bearing a group on the α-amino nitrogen.
As used herein, “halo,” “halogen” or “halide” refers to F, Cl, Br or I.
As used herein, “biological activity” refers to the in vivo activities of a compound, composition, or other mixture, or physiological responses that result upon in vivo administration of a compound, composition or other mixture. Biological activity thus encompasses therapeutic effects, diagnostic effects and pharmaceutical activity of such compounds, compositions, and mixtures.
The term “biologically compatible” as used herein means something that is biologically inert or non reactive with intracellular and extra cellular biological molecules, and non toxic.
The term “alkyl” by itself or as part of another term refers to a straight chain or branched, saturated hydrocarbon having the indicated number of carbon atoms (e.g. “C1-C8” alkyl refer to an alkyl group having from 1 to 8 carbon atoms). When the number of carbon atoms is not indicated, the alkyl group has from 1 to 8 carbon atoms. Representative straight chain C1-C8 alkyls include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl and n-octyl; while branched C1-C8 alkyls include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and -2-methylbutyl; unsaturated C2-C8 alkyls include, but are not limited to, vinyl, allyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl, acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl and 3-methyl-1-butynyl.
The phrase “substituted alkyl” refers to an alkyl group in which one or more bonds to a carbon(s) or hydrogen(s) are replaced by a bond to non-hydrogen and non-carbon atoms such as, but not limited to, a halogen atom in halides such as F, Cl, Br, and I; an oxygen atom in groups such as hydroxyl groups, alkoxy groups, aryloxy groups, and ester groups; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfone groups, sulfonyl groups, and sulfoxide groups; a nitrogen atom in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; a silicon atom in groups such as in trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilyl groups; and other heteroatoms in various other groups. Substituted alkyl groups also include groups in which one or more bonds to a carbon(s) or hydrogen(s) atom is replaced by a bond to a heteroatom such as oxygen in carbonyl, carboxyl, and ester groups; nitrogen in groups such as imines, oximes, hydrazones, and nitriles. Substituted alkyl groups include, among others, alkyl groups in which one or more bonds to a carbon or hydrogen atom is/are replaced by one or more bonds to fluorine atoms. One example of a substituted alkyl group is the trifluoromethyl group and other alkyl groups that contain the trifluoromethyl group. Other alkyl groups include those in which one or more bonds to a carbon or hydrogen atom is replaced by a bond to an oxygen atom such that the substituted alkyl group contains a hydroxyl, alkoxy, aryloxy group, or heterocyclyloxy group. Still other alkyl groups include alkyl groups that have an amine, alkylamine, dialkylamine, arylamine, (alkyl)(aryl)amine, diarylamine, heterocyclylamine, (alkyl)(heterocyclyl)amine, (aryl)(heterocyclyl)amine, or diheterocyclylamine group.
The phrase “unsubstituted alkyl” refers to a divalent unsubstituted alkyl group as defined above. Thus methylene, ethylene, and propylene are each examples of unsubstituted alkylenes. The phrase “substituted alkyl” refers to a divalent substituted alkyl group as defined above. Substituted or unsubstituted lower alkylene groups have from 1 to about 6 carbons.
The phrase “unsubstituted cycloalkyl” refers to cyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl and such rings substituted with straight and branched chain alkyl groups as defined above. The phrase also includes polycyclic alkyl groups such as, but not limited to, adamantyl norbornyl, and bicyclo[2.2.2]octyl and the like, as well as such rings substituted with straight and branched chain alkyl groups as defined above. Thus, the phrase would include methylcylcohexyl groups among others. The phrase does not include cyclic alkyl groups containing heteroatoms. Unsubstituted cycloalkyl groups may be bonded to one or more carbon atom(s), oxygen atom(s), nitrogen atom(s), and/or sulfur atom(s) in the parent compound. In some embodiments unsubstituted cycloalkyl groups have from 3 to 20 carbon atoms. In other embodiments, such unsubstituted alkyl groups have from 3 to 8 carbon atoms while in others, such groups have from 3 to 7 carbon atoms.
The phrase “substituted cycloalkyl” has the same meaning with respect to unsubstituted cycloalkyl groups that substituted alkyl groups have with respect to unsubstituted alkyl groups. Thus, the phrase includes, but is not limited to, oxocyclohexyl, chlorocyclohexyl, hydroxycyclopentyl, and chloromethylcyclohexyl groups.
The term “aryl,” by itself or an part of another term, means a substituted or unsubstituted monovalent carbocyclic aromatic hydrocarbon radical of 6-20 carbon atoms derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. Typical aryl groups include, but are not limited to, radicals derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, and the like. A substituted carbocyclic aromatic group (e.g., an aryl group) can be substituted with one or more, preferably 1 to 5, of the following groups: C1-C8 alkyl, —O—(C1-C8 alkyl), —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH2, —C(O)NHR′, —C(O)N(R′)2, —NHC(O)R′, —S(O)2R′, —S(O)R′, —OH, halogen, —N3, —NH2, —NH(R′), —N(R′)2 and —CN; wherein each R′ is independently selected from —H, C1-C8 alkyl and unsubstituted aryl. In some embodiments, a substituted carbocyclic aromatic group can further include one or more of: —NHC(═NH)NH2, —NHCONH2, —S(═O)2R′ and —SR′. “Arylene” is the corresponding divalent moiety.
The term “substituted alkyl” means an alkyl in which one or more hydrogen atoms are each independently replaced with a substituent. Typical substituents include, but are not limited to, —X, —R, —O—, —OR, —SR, —S−, —NR2, —NR3, ═NR, —CX3, —CN, —OCN, —SCN, —N═C═O, —NCS, —NO, —NO2, ═N2, —N3, —NRC(═O)R, —C(═O)NR2, —SO3−, —SO3H, —S(═O)2R, —OS(═O)2OR, —S(═O)2NR, —S(═O)R, —OP(═O)(OR)2, —P(═O)(OR)2, —PO32−, PO3H2, —AsO2H2, —C(═O)R, —C(═O)X, —C(═S)R, —CO2R, —CO2−, —C(═S)OR, —C(═O)SR, —C(═S)SR, —C(═O)NR2, —C(═S)NR2, or —C(═NR)NR2, where each X is independently a halogen: —F, —Cl, —Br, or —I; and each R is independently —H, C1-C20 alkyl, C1-C20 heteroalkyl, C8-C20 aryl, C3-C8 heterocyclyl, a protecting group or a prodrug moiety. Aryl, alkylene and heteroalkylene groups as described above may also be similarly substituted.
The term “aralkyl” by itself or part of another term, means an alkyl group, as defined above, substituted with an aryl group, as defined above.
The term “alkylene,” by itself of as part of another term, refers to a saturated, branched or straight chain or cyclic hydrocarbon radical of the stated number of carbon atoms, typically 1-18 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane. Typical alkylene radicals include, but are not limited to: methylene (—CH2—), 1,2-ethylene-CH2CH2—), 1,3-propylene (—CH2CH2CH2—), 1,4-butylene (—CH2CH2CH2CH2—), and the like. A “C1-C10” straight chain alkylene is a straight chain, saturated hydrocarbon group of the formula —(CH2)1-10—. Examples of a C1-C10 alkylene include methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, ocytylene, nonylene and decalene.
The term “heteroalkylene” by itself or as part of another substituent means a divalent group derived from heteroalkyl (as discussed above). For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini.
The term “C3-C8 heterocyclyl” by itself or as part of another term, refers to a monovalent substituted or unsubstituted aromatic or non-aromatic monocyclic, bicyclic or tricyclic ring system having from 3 to 8 carbon atoms (also referred to as ring members) and one to four heteroatom ring members independently selected from N, O, P or S, and derived by removal of one hydrogen atom from a ring atom of a parent ring system. One or more N, C or S atoms in the heterocyclyl can be oxidized. The ring that includes the heteroatom can be aromatic or nonaromatic. Unless otherwise noted, the heterocyclyl is attached to its pendant group at any heteroatom or carbon atom that results in a stable structure. Representative examples of a C3-C8 heterocyclyl include, but are not limited to, tetrahyrofuranyl, oxetanyl, pyranyl, pyrrolidinyl, piperidinyl, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, pyrrolyl, thiophenyl (thiopene), furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl and tetrazolyl. A C3-C8 heterocyclyl can be substituted with up to seven groups including, but not limited to, C1-C8 alkyl, C1-C8 heteroalkyl, —OR′, aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH2, —C(O)NHR′, —C(O)N(R′)2, —NHC(O)R′, —S(═O)2R′, —S(O)R′, halogen, —N3, —NH2, —NH(R′), —N(R′)2 and —CN; wherein each R′ is independently selected from —H, C1-C8 alkyl, C1-C8 heteroalkyl and aryl. In some embodiments, a substituted heterocyclyl can also include one or more of: —NHC(═NH)NH2, —NHCONH2, —S(═O)2R′ and —SR′. “Heterocyclo” is the corresponding divalent moiety.
The term “C3-C8 carbocyclyl” by itself or as part of another term, is a 3-, 4-, 5-, 6-, 7- or 8-membered monovalent, substituted or unsubstituted, saturated or unsaturated non-aromatic monocyclic or bicyclic carbocyclic ring derived by the removal of one hydrogen atom from a ring atom of a parent ring system. Representative C3-C8 carbocyclyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, cycloheptyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl, cyclooctyl, cyclooctadienyl, bicyclo(111)pentane, and bicyclo(222)octane. A C3-C8 carbocyclyl group can be unsubstituted or substituted with up to seven groups including, but not limited to, C1-C8 alkyl, C1-C8 heteroalkyl, —OR′, aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH2, —C(O)NHR′, —C(O)N(R′)2, —NHC(O)R′, —S(═O)2R′, —S(═O)R′, —OH, -halogen, —N3, —NH2, —NH(R′), —N(R1)2 and —CN; where each R′ is independently selected from —H, C1-C8 alkyl, C1-C8 heteroalkyl and aryl. “C3-C8 carbocyclo” is the corresponding divalent moiety.
The term “heteroaralkyl” by itself or part of another term, means an alkyl group, as defined above, substituted with an aromatic heterocyclyl group, as defined above. Heteroaralclo is the corresponding divalent moiety.
“About” or “approximately,” when used in connection with a measurable numerical variable, refers to the indicated value of the variable and to all values of the variable that are within the experimental error of the indicated value (e.g., within the 95% confidence interval for the mean) or within 10 percent of the indicated value, whichever is greater.
“Connecting chain”, or “chain” as herein used refers to the sequences of amino acids in any tertiary structural form other than a β-strand that connect the individual β-strands of an immunoglobulin domain. The terms encompass the structural motifs of α-helices, turns, loops, and β-hairpins. The terms “α-helices”, “turns”, “loops”, and “β-hairpins” have the meaning commonly ascribed to them in the art so as to be able to distinguish between the four distinct three dimensional structural motifs.
The term “identity,” as known in the art, refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or nucleic acid molecule sequences, as the case may be, as determined by the match between strings of nucleotide or amino acid sequences. “Identity” measures the percent of identical matches between two or more sequences with gap alignments addressed by a particular mathematical model of computer programs (i. e. “algorithms”).
The term “similarity” is a related concept, but in contrast to “identity”, refers to a measure of similarity which includes both identical matches and conservative substitution matches. Since conservative substitutions apply to polypeptides and not nucleic acid molecules, similarity only deals with polypeptide sequence comparisons. If two polypeptide sequences have, for example, 10 out of 20 identical amino acids, and the remainder are all nonconservative substitutions, then the percent identity and similarity would both be 50%. If in the same example, there are 5 more positions where there are conservative substitutions, then the percent identity remains 50%, but the percent similarity would be 75% (15 out of 20). Therefore, in cases where there are conservative substitutions, the degree of similarity between two polypeptide sequences will be higher than the percent identity between those two sequences.
The term “conservative amino acid substitution” refers to a substitution of a native amino acid residue with a nonnative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. For example, a conservative substitution results from the replacement of a non-polar residue in a polypeptide with any other non-polar residue.
Structural Alignment
Structural alignments, which are usually specific to protein and sometimes RNA sequences, use information about the secondary and tertiary structure of the protein or RNA molecule to aid in aligning the sequences. These methods are used for two or more sequences and typically produce local alignments; however, because they depend on the availability of structural information, they can only be used for sequences whose corresponding structures are known (usually through X-ray crystallography or NMR spectroscopy). Because both protein and RNA structure is more evolutionarily conserved than sequence, structural alignments can be more reliable between sequences that are very distantly related and that have diverged so extensively that sequence comparison cannot reliably detect their similarity. Where there is no available structural data on one of the proteins, a comparison can still be made if structural data is available on one or preferably more closely related proteins, such as immunoglobulins across species, and in particular antibody constant domains across species and subtype.
Structural alignments are used as the “gold standard” in evaluating alignments for homology-based protein structure prediction because they explicitly align regions of the protein sequence that are structurally similar rather than relying exclusively on sequence information.
The DALI method, or distance matrix alignment, is a fragment-based method for constructing structural alignments based on contact similarity patterns between successive hexapeptides in the query sequences. It can generate pairwise or multiple alignments and identify a query sequence's structural neighbors in the Protein Data Bank (PDB). It has been used to construct the FSSP structural alignment database (Fold classification based on Structure-Structure alignment of Proteins, or Families of Structurally Similar Proteins). A DALI webserver can be accessed at EBI DALI and the FSSP is located at The Dali Database.
SSAP (sequential structure alignment program) is a dynamic programming-based method of structural alignment that uses atom-to-atom vectors in structure space as comparison points. It has been extended since its original description to include multiple as well as pairwise alignments, and has been used in the construction of the CATH (Class, Architecture, Topology, Homology) hierarchical database classification of protein folds. The CATH database can be accessed at CATH Protein Structure Classification.
The combinatorial extension method of structural alignment generates a pairwise structural alignment by using local geometry to align short fragments of the two proteins being analyzed and then assembles these fragments into a larger alignment. Based on measures such as rigid-body root mean square distance, residue distances, local secondary structure, and surrounding environmental features such as residue neighbor hydrophobicity, local alignments called “aligned fragment pairs” are generated and used to build a similarity matrix representing all possible structural alignments within predefined cutoff criteria. A path from one protein structure state to the other is then traced through the matrix by extending the growing alignment one fragment at a time. The optimal such path defines the combinatorial-extension alignment. A web-based server implementing the method and providing a database of pairwise alignments of structures in the Protein Data Bank is located at the Combinatorial Extension website.
Sequence Alignment
Where structural alignment with sequences of the invention is not possible, for example due to an absence of target sequence NMR or crystal structure data, sequence alignment may be used. The skilled person is familiar with sequence alignment tools (such as BLAST, CLUSTAL and others known to the skilled person, such as those described herein), and is able to align sequences, particularly antibody constant domain sequences according to known structural motifs, especially due to the large number of exemplary structural studies already existent for immunoglobulin domains, antibodies and antibody constant domains in particular, across subtype and species.
Computational approaches to sequence alignment generally fall into two categories: global alignments and local alignments. Calculating a global alignment is a form of global optimization that “forces” the alignment to span the entire length of all query sequences. By contrast, local alignments identify regions of similarity within long sequences that are often widely divergent overall. Local alignments are often preferable, but can be more difficult to calculate because of the additional challenge of identifying the regions of similarity. A variety of computational algorithms have been applied to the sequence alignment problem. These include slow but formally correct methods like dynamic programming and also efficient, heuristic algorithms or probabilistic methods designed for large-scale database search, that do not guarantee to find best matches.
Global alignments, which attempt to align every residue in every sequence, are most useful when the sequences in the query set are similar and of roughly equal size. A general global alignment technique is the Needleman-Wunsch algorithm, which is based on dynamic programming. Local alignments are more useful for dissimilar sequences that are suspected to contain regions of similarity or similar sequence motifs within their larger sequence context. The Smith-Waterman algorithm is a general local alignment method also based on dynamic programming.
Pairwise sequence alignment methods are used to find the best-matching piecewise (local) or global alignments of two query sequences. The three primary methods of producing pairwise alignments are dot-matrix methods, dynamic programming, and word methods; however, multiple sequence alignment techniques can also align pairs of sequences. Although each method has its individual strengths and weaknesses, all three pairwise methods have difficulty with highly repetitive sequences of low information content—especially where the number of repetitions differ in the two sequences to be aligned. One way of quantifying the utility of a given pairwise alignment is the ‘maximum unique match’ (MUM), or the longest subsequence that occurs in both query sequence. Longer MUM sequences typically reflect closer relatedness. Preferred methods to determine identity and/or similarity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package, including GAP (Devereux et al., Nuc. Acids Res. 12: 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, WI), BLASTP, BLASTN, and FASTA (Atschul et al., J. Mol. Biol. 215: 403-10 (1990)). The BLAST X program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (Altschul et al., BLAST Manual (NCB NLM NIH, Bethesda, MD); Altschul et al., 1990, supra). The well-known Smith Waterman algorithm may also be used to determine identity.
By way of example, using the computer algorithm GAP (Genetics Computer Group), two polypeptides for which the percent sequence identity is to be determined are aligned for optimal matching of their respective amino acids (the “matched span”, as determined by the algorithm). A gap opening penalty (which is calculated as 3× the average diagonal; the “average diagonal” is the average of the diagonal of the comparison matrix being used; the “diagonal” is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 0.1× the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48: 443-53 (1970). Comparison matrix: BLOSUM 62 from Henikoff et al., Proc. Natl. Acad. Sci. U.S.A. 89: 10915-19 (1992).
Other exemplary algorithms, gap opening penalties, gap extension penalties, comparison matrices, thresholds of similarity, etc. may be used by those of skill in the art, including those set forth in the Program Manual, WisconsinPackage, Version 9, September, 1997. The particular choices to be made will depend on the specific comparison to be made, such as DNA to DNA, protein to protein, protein to DNA; and additionally, whether the comparison is between given pairs of sequences (in which case GAP or BestFit are generally preferred) or between one sequence and a large database of sequences (in which case FASTA or BLASTA are preferred).










SEQUENCE LIST









SEQ
Description
Sequence












1
Heavy Chain
QVQLVESGGG LVKPGGSLRL SCAASGFTFS DYYMSWIRQA PGKGLEWVSY



2.12.1.fx
ISSSGSTRDY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCVRDG



CDRs underlined
VETTFYYYYY GMDVWGQGTT VTVSSASTKG PSVFPLAPCS RSTSESTAAL




GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSVVTVPSSN




FGTQTYTCNV DHKPSNTKVD KTVERKCCVE CPPCPAPPVA GPSVFLFPPK




PKDTLMISRT PEVTCVVVDV SHEDPEVQFN WYVDGVEVHN AKTKPREEQF




NSTFRVVSVL TVVHQDWLNG KEYKCKVSNK GLPAPIEKTI SKTKGQPREP




QVYTLPPSRE EMTENQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP




MLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPGK


 


2
Light Chain
DIQMTQSPSS LSASVGDRVT ITCRASQDIR RDLGWYQQKP GKAPKRLIYA



2.12.1.fx
ASRLQSGVPS RFSGSGSGTE FTLTISSLQP EDFATYYCLQ HNNYPRTFGQ



CDRs underlined
GTKLVIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV




DNALQSGNSQ ESVTEQDSED STYSLSSTLT LSKADYEKHK VYACEVTHQG




LSSPVTKSFN RGEC


 


3
HC variable
QVQLVESGGG LVKPGGSLRL SCAASGFTFS DYYMSWIRQA PGKGLEWVSY



2.12.1.fx
ISSSGSTRDY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCVRDG




VETTFYYYYY GMDVWGQGTT VT


 


4
HC constant
VSSASTKG PSVFPLAPCS RSTSESTAAL GCLVKDYFPE PVTVSWNSGA



2.12.1.fx
LTSGVHTFPA VLQSSGLYSL SSVVTVPSSN FGTQTYTCNV DHKPSNTKVD




KTVERKCCVE CPPCPAPPVA GPSVFLFPPK PKDTLMISRT PEVTCVVVDV




SHEDPEVQFN WYVDGVEVHN AKTKPREEQF NSTFRVVSVL TVVHQDWLNG




KEYKCKVSNK GLPAPIEKTI SKTKGQPREP QVYTLPPSRE EMTKNQVSLT




CLVKGFYPSD IAVEWESNGQ PENNYKTTPP MLDSDGSFFL YSKLTVDKSR




WQQGNVFSCS VMHEALHNHY TQKSLSLSPGK


 


5
LC variable
DIQMTQSPSS LSASVGDRVT ITCRASQDIR RDLGWYQQKP GKAPKRLIYA



2.12.1.fx
ASRLQSGVPS RFSGSGSGTE FTLTISSLQP EDFATYYCLQ HNNYPRTFGQ




GTKLVIKR


 


6
Human CLκ1-106
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN



(Km(3) X45 = A/X83 = V
SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS



K80 bold & underlined
FNRGEC



2.12.1.fx



 


7
Human CLκ 1-106
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN



Km(1,2) X45 = A/X83 = L
SQESVTEQDS KDSTYSLSST LTLSKADYEK HKLYACEVTH QGLSSPVTKS



K80 bold & underlined
FNRGEC


 


8
Human CLκ 1-106
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNVLQSGN



(Km(1, 2) X45 = A/X83 = L
SQESVTEQDS KDSTYSLSST LTLSKADYEK HKLYACEVTH QGLSSPVTKS



K80 bold & underlined
FNRGEC


 


9
hCLκ 1-106
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNxLQSGN



X82 = any AA
SQESVTEQDS KDSTYSLSST LTLSKADYEK HxxYACEVTH QGLSSPVTKS



X45 = V/A X83 = L/V
FNRGEC


 


10
Human CLκ 1-106
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNxLQSGN



X45 = V/A X83 = L/V
SQESVTEQDS KDSTYSLSST LTLSKADYEK HKxYACEVTH QGLSSPVTKS




FNRGEC


 


11
Human CLκ 1-106
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN



X82 = any aa
SQESVTEQDS KDSTYSLSST LTLSKADYEK HxVYACEVTH QGLSSPVTKS




FNRGEC


 


12
Human CLk.K80R
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKADYER HKVYACEVTH QGLSSPVTKS




FNRGEC


 


13
Human CLk.K82R
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKADYEK HRVYACEVTH QGLSSPVTKS




FNRGEC


 


14
Human CLk.K80R/K82R
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKADYER HRVYACEVTH QGLSSPVTKS




FNRGEC


 


15
Human CLk.D43A
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVANALQSGN




SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


16
Human CLk.K80A
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKADYEA HKVYACEVTH QGLSSPVTKS




FNRGEC


 


17
Human CLk.H81A
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKADYEK AKVYACEVTH QGLSSPVTKS




FNRGEC


 


18
Human CLk.K82A
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKADYEK HAVYACEVTH QGLSSPVTKS




FNRGEC


 


19
Human CLk.D43A/H81A
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVANALQSGN




SQESVTEQDS KDSTYSLSST LTLSKADYEK AKVYACEVTH QGLSSPVTKS




FNRGEC


 


20
Human CLk.K41A
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW AVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


21
Human CLk.V42A
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KADNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


22
Human CLk.N44A
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDAALQSGN




SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


23
Human CLk.L46A
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNAAQSGN




SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


24
Human CLk.Q47A
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALASGN




SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


25
Human CLk.S48A
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQAGN




SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


26
Human CLk.N50A
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGA




SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


27
ANG 2 binding
xQxYQPLDEx DKTLYDQFML QQGx



peptide




X1 = COCH3




X3 and x10 = Kac




X24 = NH2



 


28
Human CLk.L73A
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTASKADYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


29
Human CLk.S74A
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLAKADYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


30
Human CLk.K75A
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSAADYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


31
Human CLk.Y78A
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKADAEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


32
Human CLk.E79A
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKADYAK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


33
Human CLk.H81A
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKADYEK AKVYACEVTH QGLSSPVTKS




FNRGEC


 


34
Human CLk.V83A
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKADYEK HKAYACEVTH QGLSSPVTKS




FNRGEC


 


35
Human CLk.Y84A
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVAACEVTH QGLSSPVTKS




FNRGEC


 


36
Human CLk.R103A
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS




FNAGEC


 


37
Human CLk.D77A
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKAAYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


38
Human CLk.D77G
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKAGYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


39
Human CLk.D77V
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKAVYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


40
Human CLk.D77L
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKALYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


41
Human CLk.D77I
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKAIYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


42
Human CLk.D77P
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKAPYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


43
Human CLk.D77F
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKAFYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


44
Human CLk.D77W
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKAWYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


45
Human CLk.D77Y
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKAYYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


46
Human CLk.D77H
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKAHYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


47
Human CLk.D77M
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKAMYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


48
Human CLk.D77C
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKACYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


49
Human CLk.D77S
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKASYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


50
Human CLk.D77T
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKATYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


51
Human CLk.D77Q
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKAQYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


52
Human CLk.D77N
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKANYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


53
Human CLk.D77E
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKAEYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


54
Human CLk.D77R
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKARYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


55
Human CLk.D77K
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKAKYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


56
Human CLk.D43A
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVANALQSGN




SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


57
Human CLλ
GQPKAAPSVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKADSSPVK




AGVETTTPSK QSNNKYAASS YLSLTPEQWK SHRSYSCQVT HEGSTVEKTV




APTEC


 


58
mCLκ
ADAAPTVSIF PPSSEQLTSG GASVVCFLNN FYPRDINVKW KIDGSERQNG




VLNSWTDQDS KDSTYSMSST LTLTKDEYER HNSYTCEATH KTSTSPIVKS




FNRNEC


 


59
hAbλTest LC Light
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVET



chain constant
TTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS



region (lambda)



 


60
hAbλTest-λκ
RTVAAPSVFIEPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVENALQSGNSQES




VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC


 


61
hAbλTest-λκJ
KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG




NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG




EC


 


62
hAbλTest S81H/H82S
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVET




TTPSKQSNNKYAASSYLSLTPEQWKHSRSYSCQVTHEGSTVEKTVAPTECS


 


63
2.12.1.fx Fab HC
QVQLVESGGG LVKPGGSLRL SCAASGFTFS DYYMSWIRQA PGKGLEWVSY




ISSSGSTRDY ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCVRDG




VETTFYYYYY GMDVWGQGTT VTVSSASTKG PSVFPLAPCS RSTSESTAAL




GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSVVTVPSSN




FGTQTYTCNV DHKPSNTKVD KTVERKCCVE


 


64
h38C2-IgG1 LC
ELQMTQSPSS LSASVGDRVT ITCRSSQSLL HTYGSPYLNW YLQKPGQSPK




LLIYKVSNRF SGVPSRFSGS GSGTDFTLTI SSLQPEDFAV YFCSQGTHLP




YTFGGGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK




VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE




VTHQGLSSPV TKSFNRGEC


 


65
h38C2-IgG1 HC
EVQLVESGGG LVQPGGSLRL SCAASGFTFS NYWMSWVRQS PEKGLEWVSE




IRLRSDNYAT HYAESVKGRF TISRDNSKNT LYLQMNSLRA EDTGIYYCKT




YFYSFSYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY




FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI




CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC RAPELLGGPS VFLFPPKPKD




TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST




YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY




TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD




SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK


 


66
h38C2-IgG2 HC
EVQLVESGGG LVQPGGSLRL SCAASGFTFS NYWMSWVRQS PEKGLEWVSE




IRLRSDNYAT HYAESVKGRF TISRDNSKNT LYLQMNSLRA EDTGIYYCKT




YFYSFSYWGQ GTLVTVSSAS TKGPSVFPLA PCSRSTSEST AALGCLVKDY




FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVT SSNFGTQTYT




CNVDHKPSNT KVDKTVERKC CVECPPCPAP PVAGPSVFLF PPKPKDTLMI




SRTPEVTCVV VDVSHEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTFRVV




SVLTVVHQDW LNGKEYKCKV SNKGLPSSIE KTISKTKGQP REPQVYTLPP




SREEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPMLDSDGS




FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK


 


67
VL h38C2
ELQMTQSPSS LSASVGDRVT ITCRSSQSLL HTYGSPYLNW YLQKPGQSPK




LLIYKVSNRF SGVPSRFSGS GSGTDFTLTI SSLQPEDFAV YFCSQGTHLP




YTFGGGTKVE IKR


 


68
VH h38C2
EVQLVESGGG LVQPGGSLRL SCAASGFTFS NYWMSWVRQS PEKGLEWVSE




IRLRSDNYAT HYAESVKGRF TISRDNSKNT LYLQMNSLRA EDTGIYYCKT




YFYSFSYWGQ GTLVTVSS


 


69
VL m38C2
DVVMTQTPLS LPVRLGDQAS ISCRSSQSLL HTYGSPYLNW YLQKPGQSPK




LLIYKVSNRF SGVPDRFSGS GSGTDFTLRI SRVEAEDLGV YFCSQGTHLP




YTFGGGTKLE IK


 


70
VH m38C2
EVKLVESGGG LVQPGGTMKL SCEISGLTFR NYWMSWVRQS PEKGLEWVAE




IRLRSDNYAT HYAESVKGKF TISRDDSKSR LYLQMNSLRT EDTGIYYCKY




YFYSFSYWGQ GTLVTVSA


 


71
Trastuzumab HC
EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR




IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG




GDGFYAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK




DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT




YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP




KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN




STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ




VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV




LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK


 


72
Trastuzumab VH
EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR




IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG




GDGFYAMDYW GQGTLVTVSS


 


73
Trastuzumab CH
ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV




HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP




KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS




HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK




EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREE MTKNQVSLTC




LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW




QQGNVFSCSV MHEALHNHYT QKSLSLSPGK


 


74
Trastuzumab LC
DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS




ASFLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ




GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV




DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG




LSSPVTKSFN RGEC


 


75
Trastuzumab VL
DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS




ASFLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ




GTKVEIKR


 


76
Trastuzumab CL
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


77
Trastuzumab D77A CL
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKAAYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


78
Trastuzumab D77x CL
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNxLQSGN



X45 = V/A; X77 = A, G,
SQESVTEQDS KDSTYSLSST LTLSKAxYEK HKxYACEVTH QGLSSPVTKS



I, V, L, R, S, T, Q,
FNRGEC



P, N, M, H, W;




X83 = V/L



 


79
(Gly4 Ser)3
GGGGSGGGG SGGGGS


 


80
Test peptide-1
GRGDSPK


 


81
Test peptide-2
DVPKSDQFVG LM


 


82
Cleavage example
SKADYEK HKVYACEVTH QGLSSPVTKS


 


83
Trypsin cleavage
ADYEK HK



product 1



 


84
Trypsin cleavage
HKVYACEVTH QGLSSPVTK



product 2



 


85
2.12.1.fx.LC.FOR
CAA CAA GAT CTG CCA CCA TGG ACA TGA GGG T


 


86
2.12.1.fx.LC.REV
CAA CAG CTA GCC TAA CAC TCT CCC CTG TTG A


 


87
L181A.FOR
CAA CAG GTC TCG GCC AGC AAA GCA GAC TAC GAG AA


 


88
L181A.REV
CAA CAG GTC TCC TGG CCG TCA GGG TGC TGC TGA G


 


89
TRAST.VL.FOR
CAA CAG GTC TCA GAT CTG CCA CCA TGG GAT GGA GC


 


90
TRAST.VL.REV
CAA CAG GTC TCA TCC GCT TGA TTT CCA CCT TG


 


91
TRAST.CL.D185A.FOR
CAA CAG GTC TCA CGG ACC GTG GCC GCT CC


 


92
TRAST.CL.D185A.REV
CAA CAG CTA GCC TAT CAG CAC TCG CCC CG


 


93
CLλ consensus
GQPKAxPxVT LFPPSSEELQ ANxATLVCLI SDFYPGxVxV AWKADxSPxx



X6 = N/A; X8 = S/T;
xGVETTxPSK QSNNxYAASS YLSLTPEQWx SHxSYSCxVT HEGSTVEKTV



x23 = K/S/R/Q/E;
APxECS



x37 = A/V; X39 = T/K;




X46 = S/G; x49 = V/A/I;




x50 = K/E; x51 = A/T;




x57 = T/K/A;




x65 = K/R/N; x80 = K/Q:




x83 = K/R; x88 = Q/L;




x103 = A/T



 


94
CLλ consensus
GQPKAxPxVT LFPPSSEELQ ANxATLVCLI SDFYPGxVxV AWKADxSPxx



S81K
xGVETTxPSK QSNNxYAASS YLSLTPEQWx KHxSYSCxVT HEGSTVEKTV



X6 = N/A; X8 = S/T;
APxECS



x23 = K/S/R/Q/E;




x37 = A/V; X39 = T/K;




X46 = S/G; x49 = V/A/I;




x50 = K/E; x51 = A/T;




x57 = T/K/A;




x65 = K/R/N; x80 = K/Q:




x83 = K/R; x88 = Q/L;




x103 = A/T



 


95
CLλ consensus;
GQPKAxPxVT LFPPSSEELQ ANxATLVCLI SDFYPGxVxV AWKADxSPxx



K80X/S81K
xGVETTxPSK QSNNxYAASS YLSLTPEQWx KHxSYSCxVT HEGSTVEKTV



X6 = N/A; X8 = S/T;
APxECS



x23 = K/S/R/Q/E;




x37 = A/V; X39 = T/K;




X46 = S/G; x49 = V/A/I;




x50 = K/E; x51 = A/T;




x57 = T/K/A;




x65 = K/R/N; x80 = G,




A, I, L, V, S, T, M,




N, Q, F, Y, W, D, or




E x83 = K/R; x88 = Q/L;




x103 = A/T



 


96
CLλ consensus
GQPKAxPxVT LFPPSSEELQ ANxATLVCLI SDFYPGxVxV AWKADxSPxx



E77x/S81Δ
xGVETTxPSK QSNNxYAASS YLSLTPxQWK -HxSYSCxVT HEGSTVEKTV



X6 = N/A; X8 = S/T;
APxECS



x23 = K/S/R/Q/E;




x37 = A/V; X39 = T/K;




X46 = S/G; x49 = V/A/I;




x50 = K/E; x51 = A/T;




x57 = T/K/A;




x65 = K/R/N; x77 = 




R/L/S/G/Q/P/N/V/I/T/




M; x83 = K/R; x88 = Q/L;




x103 = A/T



 


97
CLλ consensus;
GQPKAxPxVT LFPPSSEELQ ANxATLVCLI SDFYPGxVxV AWKADxSPxx



E77x/K80X/S81K
xGVETTxPSK QSNNxYAASS YLSLTPxQWx KHxSYSCxVT HEGSTVEKTV



X6 = N/A; X8 = S/T;
APxECS



x23 = K/S/R/Q/E;




x37 = A/V; X39 = T/K;




X46 = S/G; x49 = V/A/I;




x50 = K/E; x51 = A/T;




x57 = T/K/A;




x65 = K/R/N; x77 = R,




L, S, G, Q, P, N, V,




I, T, and M




x80 = G, A, I, L, V,




S, T, M, N, Q, F, Y,




W, D, or E x83 = K/R;




x88 = Q/L; x103 = A/T



 


98
X1 = any aa; X2 = A,
xxxxKH



G, I, V, L, R, S, T,




Q, P, N, M, H, W; X3 =




any aa; X4 = any aa.



 


99
X1 = any aa; X2 = A,
xxxxKH



G, I, L, R, S, T, P,




N, M; X3 = any aa; X4 =




any aa.



 


100
X1 = any aa; X2 = A,
xxxxKH



G, I, L, S, T, P, M;




X3 = any aa; X4 = any




aa.



 


101
X3 = A, G, I, V, L,
KAxYEKH



R, S, T, Q, P, N, M,




H, W.



 


102
X3 = A, G, I, L, R,
KAxYEKH



S, T, P, N, M.



 


103
X3 = A, G, I, L, S,
KAxYEKH



T, P, M.



 


104
hCLκ 1-106 X45 = V/A
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNxLQSGN



x77 = A, G, I, V, L,
SQESVTEQDS KDSTYSLSST LTLSKAxYEK HxxYACEVTH QGLSSPVTKS



R, S, T, Q, P, N, M,
FNRGEC



H, W; X82 = any AA;




X83 = L/V



 


105
hCLκ 1-106 X45 = V/A
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNxLQSGN



x77 = A, G, I, L, R,
SQESVTEQDS KDSTYSLSST LTLSKAxYEK HxxYACEVTH QGLSSPVTKS



S, T, P, N, M; X82 = 
FNRGEC



any AA X83 = L/V



 


106
hCLκ 1-106 X45 = V/A
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNxLQSGN



x77 = A, G, I, L, S,
SQESVTEQDS KDSTYSLSST LTLSKAxYEK HxxYACEVTH QGLSSPVTKS



T, P, M; X82 = any AA
FNRGEC



X83 = L/V



 


107
Human CLk.D43E
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVENALQSGN




SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


108
Human CLk.D43N
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVNNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


109
Human CLk.D43L
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVLNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


110
Human CLk.H81N
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKADYEK NKVYACEVTH QGLSSPVTKS




FNRGEC


 


111
Human CLk.H81Q
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKADYEK QKVYACEVTH QGLSSPVTKS




FNRGEC


 


112
Human CLk.H81Y
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKADYEK YKVYACEVTH QGLSSPVTKS




FNRGEC


 


113
Human CLk.H81W
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKADYEK WKVYACEVTH QGLSSPVTKS




FNRGEC


 


114
Human CLk.H81F
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKADYEK FKVYACEVTH QGLSSPVTKS




FNRGEC


 


115
X1 = any aa; X2 = any
xxxxxKH



aa; X3 = A, G, I, V,




L, R, S, T, Q, P, N,




M, H, W; X4 = any




aa; X5 = any aa.



 


116
X1 = any aa; X2 = any
xxxxxKH



aa; X3 = A, G, I, L,




R, S, T, P, N, M; X4 =




any aa; X5 = any aa.



 


117
X1 = any aa; X2 = any
xxxxxKH



aa; X3 = A, G, I, L,




S, T, P, M; X4 = any




aa; X5 = any aa.



 


118
X1 = any aa; X2 = any
xxxxxKH



aa; X3 = A, G, I, V,




L, R, S, T, Q, P, N,




M, H, W; X4 = any




aromatic amino acid;




X5 = any aa.



 


119
hCLκ 1-106 x43 = D, E,
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNxLQSGN



N, Q; X45 = V/A; x76 =
SQESVTEQDS KDSTYSLSST LTLSKxxxxK HxxYACEVTH QGLSSPVTKS



any aa; x77 = A, G,
FNRGEC



I, V, L, R, S, T, Q,




P, N, M, H, W; x78 =




any aa; x79 = any aa;




X82 = any AA; X83 = L/V



 


120
hCLκ 1-106 x43 = D, E,
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNxLQSGN



N, Q; X45 = V/A; x76 =
SQESVTEQDS KDSTYSLSST LTLSKxxxxK HxxYACEVTH QGLSSPVTKS



any aa; x77 = A, G,
FNRGEC



I, V, L, R, S, T, Q,




P, N, M, H, W; x78 =




any aromatic aa;




x79 = any aa; X82 = any




AA; X83 = L/V



 


121
hCLκ 1-106 x43 = D, E,
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNxLQSGN



N, Q; X45 = V/A; x76 =
SQESVTEQDS KDSTYSLSST LTLSKxxxxK HxxYACEVTHQGLSSPVTKS



any aa; x77 = A, G,
FNRGEC



I, L, S, T, P, M;




x78 = any aa; x79 = any




aa; X82 = any AA;




X83 = L/V



 


122
hCLκ 1-106 x43 = D, E,
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNxLQSGN



N, Q; X45 = V/A; x76 =
SQESVTEQDS KDSTYSLSST LTLSKxxxxK HxxYACEVTH QGLSSPVTKS



any aa; x77 = A, G,
FNRGEC



I, L, S, T, P, M;




x78 = any aromatic




aa; x79 = any aa; X82 =




any AA; X83 = L/V



 


123
X1 = any aa; X2 = A,
xxxxKH



G, I, V, L, R, S, T,




Q, P, N, M; X3 = any




aa; X4 = any aa.



 


124
X1 = any aa; X2 = A,
xxxxKH



G, I, V, L, R, S, T,




P, M; X3 = any aa;




X4 = any aa.



 


125
X1 = any aa; X2 = A,
xxxxKH



G, I, V, L, S, T, M;




X3 = any aa; X4 = any




aa.



 


126
X1 = any aa; X2 = A,
xxxxKH



G, I, L, S, T, M; X3 =




any aa; X4 = any aa.



 


127
kappa light chain
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVANALQSGN



D43A/D77A
SQESVTEQDS KDSTYSLSST LTLSKAAYEK HKVYACEVTH QGLSSPVTKS




FNRGEC


 


128
Human CLk D77A/H81A
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN




SQESVTEQDS KDSTYSLSST LTLSKAAYEK AKVYACEVTH QGLSSPVTKS




FNRGEC


 


129
Human CLk
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVANALQSGN



D43A/D77A/H81A
SQESVTEQDS KDSTYSLSST LTLSKAAYEK AKVYACEVTH QGLSSPVTKS




FNRGEC


 


130
Rabbit CLκ
RDPVAPTVLI FPPAADQVAT GTVTIVCVAN KYFPDVTVTW EVDGTTQTTG




IENSKTPQNS ADCTYNLSST LTLTSTQYNS HKEYTCKVTQ GTTSVVQSFN




RGDC


 


131
Rabbit Constant
GQPKAPSVFP LAPCCGDTPS STVTLGCLVK GYLPEPVTVT WNSGTLTNGV



Heavy Chain
RTFPSVRQSS GLYSLSSVVS VTSSSQPVTC NVAHPATNTK VDKTVAPSTC



rCH
SKPTCPPPEL LGGPSVFIFP PKPKDTLMIS RTPEVTCVVV DVSQDDPEVQ




FTWYINNEQV RTARPPLREQ QFNSTIRVVS TLPIAHQDWL RGKEFKCKVH




NKALPAPIEK TISKARGQPL EPKVYTMGPP REELSSRSVS LTCMINGFYP




SDISVEWEKN GKAEDNYKTT PAVLDSDGSY FLYSKLSVPT SEWQRGDVFT




CSVMHEALHN HYTQKSISRS PGK


 


132
Trastuzumab -rabbit
DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GRAPKLLIYS



k1 chimera (variable
ASFLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ



domain)
GTKVEIKRDP VAPTVLIFPP AADQVATGTV TIVCVANKYF PDVTVTWEVD



rTrast-LC
GTTQTTGIEN SKTPQNSADC TYNLSSTLTL TSTQYNSHKE YTCKVTQGTT




SVVQSFNRGD C


 


133
Trastuzumab rabbit
EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR



IgG chimera
IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG



(variable domain)
GDGFYAMDYW GQGTLVTVSS GQPKAPSVFP LAPCCGDTPS STVTLGCLVK



rTrast-HC
GYLPEPVTVT WNSGTLTNGV RTFPSVRQSS GLYSLSSVVS VTSSSQPVTC




NVAHPATNTK VDKTVAPSTC SKPTCPPPEL LGGPSVFIFP PKPKDTLMIS




RTPEVTCVVV DVSQDDPEVQ FTWYINNEQV RTARPPLREQ QFNSTIRVVS




TLPIAHQDWL RGKEFKCKVH NKALPAPIEK TISKARGQPL EPKVYTMGPP




REELSSRSVS LTCMINGFYP SDISVEWEKN GKAEDNYKTT PAVLDSDGSY




FLYSKLSVPT SEWQRGDVFT CSVMHEALHN HYTQKSISRS PGK


 


134
Rabbit CLκ-S80K
RDPVAPTVLI FPPAADQVAT GTVTIVCVAN KYFPDVTVTW EVDGTTQTTG




IENSKTPQNS ADCTYNLSST LTLTSTQYNK HKEYTCKVTQ GTTSVVQSFN




RGDC


 


135
Trastuzumab -rabbit
DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GRAPKLLIYS



k1 chimera
ASFLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ



S188K(variable
GTKVEIKRDP VAPTVLIFPP AADQVATGTV TIVCVANKYF PDVTVTWEVD



domain)
GTTQTTGIEN SKTPQNSADC TYNLSSTLTL TSTQYNKHKE YTCKVTQGTT



rTrastLC-[rCLk-S80K]
SVVQSFNRGD C


 


136
hIL22 LC
QAVLTQPPSV SGAPGQRVTI SCTGSSSNIG AGYGVHWYQQ LPGTAPKLLI




YGDSNRPSGV PDRFSGSKSG TSASLAITGL QAEDEADYYC QSYDNSLSGY




VFGGGTQLTV LGQPKAAPSV TLFPPSSEEL QANKATLVCL ISDFYPGAVT




VAWKADSSPV KAGVETTTPS KQSNNKYAAS SYLSLTPEQW KSHRSYSCQV




THEGSTVEKT VAPTECS


 


137
hIL22 HC
QVQLVQSGAE VKKPGASVKV SCKASGYTFT NYYMHWVRQA PGQGLEWVGW




INPYTGSAFY AQKFRGRVTM TRDTSISTAY MELSRLRSDD TAVYYCAREP




EKFDSDDSDV WGRGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV




KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ




TYICNVNHKP SNTKVDKKVE PKSCDKTHTC PPCPAPELLG GPSVFLFPPK




PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY




NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP




QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP




VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG




K


 


138
hIL22 VL
QAVLTQPPSV SGAPGQRVTI SCTGSSSNIG AGYGVHWYQQ LPGTAPKLLI




YGDSNRPSGV PDRFSGSKSG TSASLAITGL QAEDEADYYC QSYDNSLSGY




VFGGGTQLTV L


 


139
hIL22 lambda
GQPKAAPSVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKADSSPVK



constant
AGVETTTPSK QSNNKYAASS YLSLTPEQWK SHRSYSCQVT HEGSTVEKTV




APTECS


 


140
hIL22-[CLλ-S81K]
GQPKA APSVTLFPPS SEELQANKAT LVCLISDFYP GAVTVAWKAD




SSPVKAGVET TTPSKQSNNK YAASSYLSLT PEQWKKHRSY SCQVTHEGST




VEKTVAPTEC S


 


141
hIL22-
GQPKA APSVTLFPPS SEELQANKAT LVCLISDFYP GAVTVAWKAD



[CLλ-Q78A/S81K
SSPVKAGVET TTPSKQSNNK YAASSYLSLT PEAWKKHRSY SCQVTHEGST




VEKTVAPTEC S


 


142
hIL22-[CLλA44V/S81K]
GQPKA APSVTLFPPS SEELQANKAT LVCLISDFYP GAVTVAWKVD




SSPVKAGVET TTPSKQSNNK YAASSYLSLT PEQWKKHRSY SCQVTHEGST




VEKTVAPTEC S


 


143
hIL22-
GQPKA APSVTLFPPS SEELQANKAT LVCLISDFYP GAVTVAWKVD



[CLλ-A44V/Q78A/S81K]
SSPVKAGVET TTPSKQSNNK YAASSYLSLT PEAWKKHRSY SCQVTHEGST




VEKTVAPTEC S


 


144
hIL22-
GQPKA APSVTLFPPS SEELQANKAT LVCLISDFYP GAVTVAWKAD



[CLλ-λ76-84/145]
SSPVKAGVET TTPSKQSNNK YAASSYLSLT KAAYEKHKVY SCQVTHEGST




VEKTVAPTEC S


 


145
CLK 9-aa loop
KAAYEKHKV


 


146
Trastuzumab LC K188A
DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS



(variable domain)
ASFLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ




GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV




DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEAHK VYACEVTHQG




LSSPVTKSFN RGEC


 


147
hIGg1-CH1
ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV




HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKV


 


148
hIGgq-CH1-m1
ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV




HTFPAVLQSS GLYSLSSVVT VPSSSEKHKV YICNVNHKPS NTKVDKKV


 


149
hIGgq-CH1-m1-D44
ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSDGALTSG




VHTFPAVLQS SGLYSLSSVV TVPSSSEKHK VYICNVNHKP SNTKVDKKV


 


150
hIGg1-CH1-m2
ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV




HTFPAVLQSS GLYSLSSVVT VPSSSYEKHK VYICNVNHKP SNTKVDKKV


 


151
hIGg1-CH1-m2-D44
ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSDGALTSG




VHTFPAVLQS SGLYSLSSVV TVPSSSYEKH KVYICNVNHK PSNTKVDKKV


 


152
hIGg1-CH1 Δ fragment
LGTQT


 


153
M1 insert fragment
EKHKV


 


154
M2 insert fragment
YEKHKV


 


155
hIGg1-CH2
APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD




GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA




PIEKTISKAK


 


156
hIGg1-CH2m
APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD




GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLKHKEYK CKVSNKALPA




PIEKTISKAK


 


157
hIGg1-H3
GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN




YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS




LSLSPGK


 


158
hIGg1-CH3m
GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN




YKTTPPVLDS DGSFFLYSKL TVDKSRWQKH NVFSCSVMHE ALHNHYTQKS




LSLSPGK


 


159
Trastuzumab CH1-m2
EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR




IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG




GDGFYAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK




DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSYEKHK




VYICNVNHKP SNTKVDKKVE PKSCDKTHTC PPCPAPELLG GPSVFLFPPK




PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY




NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP




QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP




VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG




K


 


160
Trastuzumab CH1-m1-
EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR



D44
IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG




GDGFYAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK




DYFPEPVTVS WNSDGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSEKHK




VYICNVNHKP SNTKVDKKVE PKSCDKTHTC PPCPAPELLG GPSVFLFPPK




PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY




NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP




QVYTLPPSRE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP




VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG




K


 


161
Trastuzumab CH1-m2-
EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR



D44
IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG




GDGFYAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK




DYFPEPVTVS WNSDGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSYEKH




KVYICNVNHK PSNTKVDKKV EPKSCDKTHT CPPCPAPELL GGPSVFLFPP




KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ




YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE




PQVYTLPPSR EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP




PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP




GK


 


162
Trastuzumab CH2m
EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR




IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG




GDGFYAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK




DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT




YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP




KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN




STYRVVSVLT VLHQDWLKHK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ




VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV




LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK


 


163
Trastuzumab CH3m
EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR




IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG




GDGFYAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK




DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT




YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP




KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN




STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ




VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV




LDSDGSFFLY SKLTVDKSRW QKHNVFSCSV MHEALHNHYT QKSLSLSPGK


 


164
Ex4 homologue x2 is
HxEGTFTSDL SKQxEEEAVR LFIEWLKNGG PSSGAPPPS



Aib




X14 = KSH



 


165
hIgG1-CH1-
ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WKVDNALATS



T78K/Q79K/CD loop
GVHTFPAVLQ SSGLYSLSSV VTVPSSSEKH KVYICNVNHK PSNTKVDKKV



swap



 


166
hIgG1-CH1-CD insert
KVDNALA


 


167
hIGg1-CH2m-
APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD



D82A/N85K/G86H
GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QAWLKHKEYK CKVSNKALPA




PIEKTISKAK


 


168
hIGg1-CH3m-CD1/EF
GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WEVDGQPENN




YKTTPPVLDS DGSFFLYSKL TVDKSAWQKH NVFSCSVMHE ALHNHYTQKS




LSLSPGK


 


169
hIGg1-CH3m-CD2/EF
GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WELEGQPENN




YKTTPPVLDS DGSFFLYSKL TVDKSAWQKH NVFSCSVMHE ALHNHYTQKS




LSLSPGK


 


170
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not cys



K, R, H, D, E, G, P.




X2 = A, G, I, V, L,




R, S, T, Q, P, N, M,




H, W.




X3 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E, G, P;




X4 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E, G, P;



 


171
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not Cys, Pro



K, R, H, D, E, G.




X2 = A, G, I, V, L,




R, S, T, Q, P, N, M,




H, W.




X3 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E, G;




X4 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E, G;



 


172
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 no Cis, Pro, Gly



K, R, H, D, E.




X2 = A, G, I, V, L,




R, S, T, Q, P, N, M,




H, W.




X3 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;




X4 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;



 


173
X1 =10 A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C, P, G



K, R, H, D, E.
X3 is not A or V



X2 = A, G, I, V, L,




R, S, T, Q, P, N, M,




H, W.




X3 = L, I, F, W, Y,




S, T, M, N, Q, K, R,




H, D, E;




X4 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;



 


174
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C, P, G



K, R, H, D, E.
X3 is not A or V, S or T



X2 = A, G, I, V, L,




R, S, T, Q, P, N, M,




H, W.




X3 = L, I, F, W, Y,




M, N, Q, K, R, H, D,




E;




X4 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;



 


175
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C, P, G



K, R, H, D, E.
X3 is not A, V, S or T, M, K, R, H



X2 = A, G, I, V, L,




R, S, T, Q, P, N, M,




H, W.




X3 = L, I, F, W, Y,




N, Q, D, E;




X4 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;



 


176
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C, P, G



K, R, H, D, E.
X3 is not C, G, P, A, V, S or T, M, K, R, H, N, Q, D,



X2 = A, G, I, V, L,
or E;



R, S, T, Q, P, N, M,




H, W.




X3 = L, I, F, W, Y,




X4 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;



 


177
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C, P, G



K, R, H, D, E.
X3 is Y or W



X2 = A, G, I, V, L,




R, S, T, Q, P, N, M,




H, W.




X3 = L, I, F, W, Y,




X4 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;



 


178
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C, P, G



K, R, H, D, E.
X4 is not F, Y, W



X2 = A, G, I, V, L,




R, S, T, Q, P, N, M,




H, W.




X3 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;




X4 = A, V, L, I, S,




T, M, N, Q, K, R, H,




D, E;



 


179
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C, P, G



K, R, H, D, E.
X4 is not F, W, Y, H



X2 = A, G, I, V, L,




R, S, T, Q, P, N, M,




H, W.




X3 = A, V, L, I, S,




T, M, N, Q, K, R, D,




E;




X4 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;



 


180
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C, P, G



K, R, H, D, E.
X3 is not A or V



X2 = A, G, I, V, L,
X4 is not F, Y, W



R, S, T, Q, P, N, M,




H, W.




X3 = L, I, F, W, Y,




S, T, M, N, Q, K, R,




H, D, E;




X4 = A, V, L, I, S,




T, M, N, Q, K, R, H,




D, E;



 


181
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C, P, G



K, R, H, D, E.
X3 is not A or V, S or T



X2 = A, G, I, V, L,
X4 is not F, Y, W



R, S, T, Q, P, N, M,




H, W.




X3 = L, I, F, W, Y,




M, N, Q, K, R, H, D,




E;




X4 = A, V, L, I, S,




T, M, N, Q, K, R, H,




D, E;



 


182
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C, P, G



K, R, H, D, E.
X3 is not A, V, S or T, M, K, R, H



X2 = A, G, I, V, L,
X4 is not F, Y, W, or H



R, S, T, Q, P, N, M,




H, W.




X3 = L, I, F, W, Y,




N, Q, D, E;




X4 = A, V, L, I, S,




T, M, N, Q, K, R, D,




E;



 


183
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C, P, G



K, R, H, D, E.
X3 is not C, G, P, A, V, S or T, M, K, R, H, N, Q, D,



X2 = A, G, I, V, L,
or E;



R, S, T, Q, P, N, M,
X4 is not F, Y, W, or H



H, W.




X3 = L, I, F, W, Y,




X4 = A, V, L, I, S,




T, M, N, Q, K, R, D,




E;



 


184
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C, P, G



K, R, H, D, E.
X3 is Y or W



X2 = A, G, I, V, L,
X4 is E, K, D, R, N, or Q.



R, S, T, Q, P, N, M,




H, W.




X3 = L, I, F, W, Y,




X4 = N, Q, K, R, D,




E;



 


185
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C, P, G



K, R, H, D, E.
X4 is E, K, D, R, N, or Q.



X2 = A, G, I, V, L,




R, S, T, Q, P, N, M,




H, W.




X3 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;




X4 = N, Q, K, R, D,




E;



 


186
X1 = A, V, L, I, S,
xxxxKH



T, M, N, Q, K, R, H,
X1 X3 X4 not C, P, G



D, E.
X1 is not aromatic



X2 = A, G, I, V, L,




R, S, T, Q, P, N, M,




H, W.




X3 - A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;




X4 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;



 


187
X1 = A, V, L, I, S,
xxxxKH



T, M, N, Q, R, H, D,
X1 X3 X4 not C, P, G



E.
X1 is not aromatic or K



X2 = A, G, I, V, L,




R, S, T, Q, P, N, M,




H, W.




X3 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;




X4 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;



 


188
X1 = A, V, L, I, S,
xxxxKH



T, M, N.
X1 X3 X4 no c, p, g



X2 = A, G, I, V, L,
X1 is not aromatic or K, or charged



R, S, T, Q, P, N, M,




H, W.




X3 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;




X4 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;



 


189
X1 = A, V, L, I, M.
xxxxKH



X2 = A, G, I, V, L,
X1 X3 X4 not C, P, G



R, S, T, Q, P, N, M,
X1 is hydrophilic



H, W.




X3 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;




X4 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;



 


190
X1 = A, V, L, I, S,
xxxxKH



T, M, N, Q, K, R, H,
X1 X3 X4 not C, P, G



D, E.
X1 is not aromatic



X2 = A, G, I, V, L,
X4 is not F, Y, W



R, S, T, Q, P, N, M,




H, W.




X3 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;




X4 = A, V, L, I, S,




T, M, N, Q, K, R, H,




D, E;



 


191
X1 = A, V, L, I, S,
xxxxKH



T, M, N, Q, K, R, H,
X1 X3 X4 not C, P, G



D, E.
X1 is not aromatic



X2 = A, G, I, V, L,
X4 is not F, W, Y, H



R, S, T, Q, P, N, M,




H, W.




X3 = A, V, L, I, S,




T, M, N, Q, K, R, D,




E;




X4 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;



 


192
X1 = A, V, L, I, S,
xxxxKH



T, M, N, Q, K, R, H,
X1 X3 X4 not C, P, G



D, E.
X1 is not aromatic



X2 = A, G, I, V, L,
X3 is not A or V



R, S, T, Q, P, N, M,
X4 is not F, Y, W



H, W.




X3 = L, I, F, W, Y,




S, T, M, N, Q, K, R,




H, D, E;




X4 = A, V, L, I, S,




T, M, N, Q, K, R, H,




D, E;



 


193
X1 = A, V, L, I, S,
xxxxKH



T, M, N, Q, R, H, D,
X1 X3 X4 not C, P, G



E.
X1 is not aromatic or K



X2 = A, G, I, V, L,
X3 is not A or V, S or T



R, S, T, Q, P, N, M,
X4 is not F, Y, W



H, W.




X3 = L, I, F, W, Y,




M, N, Q, K, R, H, D,




E;




X4 = A, V, L, I, S,




T, M, N, Q, K, R, H,




D, E;



 


194
X1 = A, V, L, I, S,
xxxxKH



T, M, N, Q.
X1 X3 X4 not C, P, G



X2 = A, G, I, V, L,
X1 is not aromatic, K or charged



R, S, T, Q, P, N, M,
X3 is not A, V, S or T, M, K, R, H



H, W.
X4 is not F, Y, W, or H



X3 = L, I, F, W, Y,




N, Q, D, E;




X4 = A, V, L, I, S,




T, M, N, Q, K, R, D,




E;



 


195
X1 = A, V, L, I.
xxxxKH



X2 = A, G, I, V, L,
X1 X3 X4 not C, P, G



R, S, T, Q, P, N, M,
X1 is hydrophilic



H, W.
X3 is not C, G, P, A, V, S or T, M, K, R, H, N, Q, D,



X3 = L, I, F, W, Y,
or E;



X4 = A, V, L, I, S,
X4 is not F, Y, W, or H



T, M, N, Q, K, R, D,




E;



 


196
X1 = A, V, L, I.
xxxxKH



X2 = A, G, I, V, L,
X1 X3 X4 not C, P, G



R, S, T, Q, P, N, M,
X1 is hydrophilic



H, W.
X3 is Y or W



X3 = L, I, F, W, Y,
X4 is E, K, D, R, N, or Q.



X4 = N, Q, K, R, D,




E;



 


197
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C, P, G



K, R, H, D, E.
X2 = is not W



X2 = A, G, I, V, L,
X4 is not F, Y, W



R, S, T, Q, P, N, M,




H.




X3 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;




X4 = A, V, L, I, S,




T, M, N, Q, K, R, H,




D, E;



 


198
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C, P, G



K, R, H, D, E.
X2 is not W



X2 = A, G, I, V, L,
X4 is not F, W, Y, H



R, S, T, Q, P, N, M,




H, W.




X3 = A, V, L, I, S,




T, M, N, Q, K, R, D,




E;




X4 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;



 


199
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C, P, G



K, R, H, D, E, G, P.
X2 is not W



X2 = A, G, I, V, L,




R, S, T, Q, P, N, M,




H, W.




X3 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E, G, P;




X4 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E, G, P;



 


200
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C, P, G



K, R, H, D, E, G.
X2 is not W



X2 = A, G, I, V, L,




R, S, T, Q, P, N, M,




H, W.




X3 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E, G;




X4 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E, G;



 


201
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C P, G



K, R, H, D, E.
X2 is not W



X2 = A, G, I, V, L,




R, S, T, Q, P, N, M,




H, W.




X3 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;




X4 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;



 


202
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C, P, G



K, R, H, D, E.
X2 is not W



X2 = A, G, I, V, L,
X3 is not A or V



R, S, T, Q, P, N, M,




H, W.




X3 = L, I, F, W, Y,




S, T, M, N, Q, K, R,




H, D, E;




X4 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;



 


203
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C, P, G



K, R, H, D, E.
X2 is not W, I, L, R



X2 = A, G, V, S, T,
X3 is not A or V, S or T



Q, P, N, M, H.




X3 = L, I, F, W, Y,




M, N, Q, K, R, H, D,




E;




X4 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;



 


204
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C, P, G



K, R, H, D, E.
X2 is not W, I, L, R



X2 = A, G, V, S, T,
X3 is not A, V, S or T, M, K, R, H



Q, P, N, M, H.




X3 = L, I, F, W, Y,




N, Q, D, E;




X4 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;



 


205
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C, P, G



K, R, H, D, E.
X2 is A, G, P, S, T, M



X2 = A, G, S, T, P,
X3 is not C, G, P, A, V, S or T, M, K, R, H, N, Q, D,



M.
or E;



X3 = L, I, F, W, Y,




X4 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;



 


206
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C, P, G



K, R, H, D, E.
X2 is A, G, P, S, T, M



X2 = A, G, S, T, P,
X3 is Y or W



M.




X3 = L, I, F, W, Y,




X4 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;



 


207
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C, P, G



K, R, H, D, E.
X2 is A, G, P, S, T, M



X2 = A, G, S, T, P,
X3 is not A or V



M.
X4 is not F, Y, W



X3 = L, I, F, W, Y,




S, T, M, N, Q, K, R,




H, D, E;




X4 = A, V, L, I, S,




T, M, N, Q, K, R, H,




D, E;



 


208
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C, P, G



K, R, H, D, E.
X2 is A, G, P, S, T, M



X2 = A, G, S, T, P,
X3 is not A or V, S or T



M.
X4 is not F, Y, W



X3 = L, I, F, W, Y,




M, N, Q, K, R, H, D,




E;




X4 = A, V, L, I, S,




T, M, N, Q, K, R, H,




D, E;



 


209
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C, P, G



K, R, H, D, E.
X2 is A, G, P, S, T, M



X2 = A, G, S, T, P,
X3 is not A, V, S or T, M, K, R, H



M.
X4 is not F, Y, W, or H



X3 = L, I, F, W, Y,




N, Q, D, E;




X4 = A, V, L, I, S,




T, M, N, Q, K, R, D,




E;



 


210
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C, P, G



K, R, H, D, E.
X2 is A, G, P, M, S, T



X2 = A, G, S, T, P,
X3 is not C, G, P, A, V, S or T, M, K, R, H, N, Q, D,



M.
or E;



X3 = L, I, F, W, Y,
X4 is not F, Y, W, or H



X4 = A, V, L, I, S,




T, M, N, Q, K, R, D,




E;



 


211
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C, P, G



K, R, H, D, E.
X2 is A, G, P, M, S, T



X2 = A, G, S, T, P,
X3 is Y or W



M.
X4 is E, K, D, R, N, or Q.



X3 = L, I, F, W, Y,




X4 = N, Q, K, R, D,




E;



 


212
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C, P, G



K, R, H, D, E.
X2 is not W or R,



X2 = A, G, I, V, L,
X4 is E, K, D, R, N, or Q.



S, T, Q, P, N, M, H.




X3 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;




X4 = N, Q, K, R, D,




E;



 


213
X1 = A, V, L, I, S,
xxxxKH



T, M, N, Q, K, R, H,
X1 X3 X4 not C, P, G



D, E.
W2 is not W or R



X2 = A, G, I, V, L,
X1 is not aromatic



S, T, Q, P, N, M, H.




X3 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;




X4 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;



 


214
X1 = A, V, L, I, S,
xxxxKH



T, M, N, Q, R, H, D,
X1 X3 X4 not C, P, G



E.
X1 is not aromatic or K



X2 = A, G, I, V, L,
X2 is not W or R



S, T, Q, P, N, M, H.




X3 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;




X4 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;



 


215
X1 = A, V, L, I, S,
xxxxKH



T, M, N.
X1 X3 X4 not C, P, G



X2 = A, G, I, V, L,
X1 is not aromatic or K, or charged



S, T, Q, P, N, M, H.
X2 = is not W or R



X3 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;




X4 = A V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;



 


216
X1 = A, V, L, I, M.
xxxxKH



X2 = A, G, I, V, L,
X1 X3 X4 not C, P, G



S, T, Q, P, N, M, H.
X1 is hydrophilic



X3 = A, V, L, I, F,
X2 is not W or R



W, Y, S, T, M, N, Q,




K, R, H, D, E;




X4 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;



 


217
X1 = A, V, L, I, S,
xxxxKH



T, M, N, Q, K, R, H,
X1 X3 X4 not C, P, G



D, E.
X1 is not aromatic



X2 = A, G, I, V, L,
X2 is not W or R



S, T, Q, P, N, M, H.
X4 is not F, Y, W



X3 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;




X4 = A, V, L, I, S,




T, M, N, Q, K, R, H,




D, E;



 


218
X1 = A, V, L, I, S,
xxxxKH



T, M, N, Q, K, R, H,
X1 X3 X4 not C, P, G



D, E.
X1 is not aromatic



X2 = A, G, I, V, L,
X2 is not W, or R



S, T, Q, P, N, M, H.
X4 is not F, W, Y, H



X3 = A, V, L, I, S,




T, M, N, Q, K, R, D,




E;




X4 = A, V, L, I, F,




W, Y, S, T, M, N, Q,




K, R, H, D, E;



 


219
X1 = A, V, L, I, S,
xxxxKH



T, M, N, Q, K, R, H,
X1 X3 X4 not C, P, G



D, E.
X1 is not aromatic



X2 = A, G, I, V, L,
Is not W or R



S, T, Q, P, N, M, H.
X3 is not A or V



X3 = L, I, F, W, Y,
X4 is not F, Y, W



S, T, M, N, Q, K, R,




H, D, E;




X4 = A, V, L, I, S,




T, M, N, Q, K, R, H,




D, E;



 


220
X1 = A, V, L, I, S,
xxxxKH



T, M, N, Q, R, H, D,
X1 X3 X4 not C, P, G



E.
X1 is not aromatic or K



X2 = A, G, I, V, L,
X2 is not W or R



S, T, Q, P, N, M, H.
X3 is not A or V, S or T



X3 = L, I, F, W, Y,
X4 is not F, Y, W



M, N, Q, K, R, H, D,




E;




X4 = A, V, L, I, S,




T, M, N, Q, K, R, H,




D, E;



 


221
X1 = A, V, L, I, S,
xxxxKH



T, M, N, Q.
X1 X3 X4 not C, P, G



X2 = A, G, S, T, M.
X1 is not aromatic, K or charged



X3 = L, I F, W, Y,
X2 is A, G, P, S, T, M



N, Q, D, E;
X3 is not A, V, S or T, M, K, R, H



X4 = A, V, L, I, S,
X4 is not F, Y, W, or H



T, M, N, Q, K, R, D,




E;



 


222
X1 = A, V, L, I.
xxxxKH



X2 = A, G, S, T, M.
X1 X3 X4 not C, P, G



X3 = L, I, F, W, Y,
X1 is hydrophilic



X4 = A, V, L, I, S,
X2 is A, G, P, S, T, M



T, M, N, Q, K, R, D,
X3 is not C, G, P, A, V, S or T, M, K, R, H, N, Q, D,



E;
or E;




X4 is not F, Y, W, or H


 


223
X1 = A, V, L, I.
xxxxKH



X2 = A, G, S, T, M.
X1 X3 X4 not C, P, G



X3 = L, I, F, W, Y,
X2 is A, G, P, S, T, M



X4 = N, Q, K, R, D,
X1 is hydrophilic



E;
X3 is Y or W




X4 is E, K, D, R, N, or Q.


 


224
X1 = A, V, L, I, F,
xxxxKH



W, Y, S, T, M, N, Q,
X1 X3 X4 not C, P, G



K, R, H, D, E.
X2 is A, G, P, S, T, M



X2 = A, G, S, T, M.
X3 is not A, V, S or T, M, K, R, H



X3 = L, I, F, W, Y,
X4 is not F, Y, W, or H



N, Q, D, E;




X4 = A, V, L, I, S,




T, M, N, Q, K, R, D,




E;



 


225
hCLκ 1-75 x42-V, I,
TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KxxNxLQSGN



L, x43 = D, E, N, Q;
SQESVTEQDS KDSTYSLSST LTLSx



X45 = V/A; x75-any




amino acid



 


226
hCLK 81-106 X1 = any
xxYACEVTH QGLSSPVTKS FNRGEC



AA; X2 = L/V



 


227
CLλ consensus
GQPKAxPxVT LFPPSSEELQ ANxATLVCLI SDFYPGxVxV AWKADxSPxx



1-76
xGVETTxPSK QSNNxYAASS YLSLTP



X6 = N/A; X8 = S/T;




x23 = K/S/R/Q/E;




x37 = A/V; X39 = T/K;




X46 = S/G; x49 = V/A/I;




x50 = K/E; x51 = A/T;




x57 = T/K/A;




x65 = K/R/N;



 


228
CLλ consensus
GQPKAxPxVT LFPPSSEELQ ANxATLVCLI SDFYPGxVxV AWKxDxSPxx



1-76, x44 inc A
xGVETTxPSK QSNNxYAASS YLSLTP



X6 = N/A; X8 = S/T;




x23 = K/S/R/Q/E;




x37 = A/V; X39 = T/K;




X44 is A, V, I, L




X46 = S/G; x49 = V/A/I;




x50 = K/E; x51 = A/T;




x57 = T/K/A;




x65 = K/R/N;



 


229
CLλ consensus
GQPKAxPxVT LFPPSSEELQ ANxATLVCLI SDFYPGxVxV AWKxDxSPxx



1-76, X44 no A
xGVETTxPSK QSNNxYAASS YLSLTP



X6 = N/A; X8 = S/T;




x23 = K/S/R/Q/E;




x37 = A/V; X39 = T/K;




x44 = V, I L




X46 = S/G; x49 = V/A/I;




x50 = K/E; x51 = A/T;




x57 = T/K/A;




x65 = K/R/N;



 


230
CLλ consensus
GQPKAxPxVT LFPPSSEELQ ANxATLVCLI SDFYPGxVxV AWKVDxSPxx



1-76, X44 = V
xGVETTxPSK QSNNxYAASS YLSLTP



X6 = N/A; X8 = S/T;




x23 = K/S/R/Q/E;




x37 = A/V; X39 = T/K;




X46 = S/G; x49 = V/A/I;




x50 = K/E; x51 = A/T;




x57 = T/K/A;




x65 = K/R/N;



 


231
CLλ 1-76
GQPKAAPSVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKxDSSPVK



X44 is A, V, I, L
AGVETTTPSK QSNNKYAASS YLSLTP


 


232
CLλ 1-76
GQPKAAPSVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKxDSSPVK



X44 is V, I, L
AGVETTTPSK QSNNKYAASS YLSLTP


 


233
CLλ 1-76
GQPKAAPSVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKVDSSPVK



X44 is V
AGVETTTPSK QSNNKYAASS YLSLTP


 


234
CLλ consensus 83-103
xSYSCxVTHE GSTVEKTVAP xECS



x1 = K/R; x6 = Q/L;




x21 = A/T



 


235
CLλ 83-103
RSYSCQVTHE GSTVEKTVAP TECS


 


236
CLλ consensus
GQPKAxPxVT LFPPSSEELQ ANxATLVCLI SDFYPGxVxV AWKxDxSPxx



S81K, x44 = AVIL,
xGVETTxPSK QSNNxYAASS YLSLTPExWx KHxSYSCxVT HEGSTVEKTV



X6 = N/A; X8 = S/T;
APxECS



x23 = K/S/R/Q/E;




x37 = A/V; X39 = T/K;




x44 A, V, I, L




X46 = S/G; x49 = V/A/I;




x50 = K/E; x51 = A/T;




x57 = T/K/A;




x65 = K/R/N; Q78 = A/Q




x80 = K/Q; x83 = K/R;




x88 = Q/L; x103 = A/T



 


237
CLλ consensus
GQPKAxPxVT LFPPSSEELQ ANxATLVCLI SDFYPGxVxV AWKxDxSPxx



S81K, X44 = VIL,
xGVETTxPSK QSNNxYAASS YLSLTPExWx KHxSYSCxVT HEGSTVEKTV



X6 = N/A; X8 = S/T;
APxECS



x23 = K/S/R/Q/E;




x37 = A/V; X39 = T/K;




x44 V, I, L




X46 = S/G; x49 = V/A/I;




x50 = K/E; x51 = A/T;




x57 = T/K/A;




x65 = K/R/N; X78 = A/Q




x80 = K/Q; x83 = K/R;




x88 = Q/L; x103 = A/T



 


238
CLλ consensus
GQPKAxPxVT LFPPSSEELQ ANxATLVCLI SDFYPGxVxV AWKVDxSPxx



S81K, x44 = V,
xGVETTxPSK QSNNxYAASS YLSLTPExWx KHxSYSCxVT HEGSTVEKTV



X6 = N/A; X8 = S/T;
APxECS



x23 = K/S/R/Q/E;




x37 = A/V; X39 = T/K;




X46 = S/G; x49 = V/A/I;




x50 = K/E; x51 = A/T;




x57 = T/K/A;




x65 = K/R/N; x78 = A/Q




x80 = K/Q; x83 = K/R;




x88 = Q/L; x103 = A/T



 


239
CLλ consensus
GQPKAxPxVT LFPPSSEELQ ANxATLVCLI SDFYPGxVxV AWKxDxSPxx



S81K, x44 = AVIL, A78
xGVETTxPSK QSNNxYAASS YLSLTPEAWx KHxSYSCxVT HEGSTVEKTV



X6 = N/A; X8 = S/T;
APxECS



x23 = K/S/R/Q/E;




x37 = A/V; X39 = T/K;




x44 A, V, I, L




X46 = S/G; x49 = V/A/I;




x50 = K/E; x51 = A/T;




x57 = T/K/A;




x65 = K/R/N;




x80 = K/Q; x83 = K/R;




x88 = Q/L; x103 = A/T



 


240
CLλ consensus
GQPKAxPxVT LFPPSSEELQ ANxATLVCLI SDFYPGxVxV AWKxDxSPxx



S81K, X44 = VIL, A78
xGVETTxPSK QSNNxYAASS YLSLTPEAWx KHxSYSCxVT HEGSTVEKTV



X6 = N/A; X8 = S/T;
APxECS



x23 = K/S/R/Q/E;




x37 = A/V; X39 = T/K;




x44 V, I, L




X46 = S/G; x49 = V/A/I;




x50 = K/E; x51 = A/T;




x57 = T/K/A;




x65 = K/R/N; x80 = K/Q:




x83 = K/R; x88 = Q/L;




x103 = A/T



 


241
CLλ consensus
GQPKAxPxVT LFPPSSEELQ ANxATLVCLI SDFYPGxVxV AWKVDxSPxx



S81K, x44 = V, A78
xGVETTxPSK QSNNxYAASS YLSLTPEAWx KHxSYSCxVT HEGSTVEKTV



X6 = N/A; X8 = S/T;
APxECS



x23 = K/S/R/Q/E;




x37 = A/V; X39 = T/K;




X46 = S/G; x49 = V/A/I;




x50 = K/E; x51 = A/T;




x57 = T/K/A;




x65 = K/R/N; x80 = K/Q:




x83 = K/R; x88 = Q/L;




x103 = A/T



 


242
CLλ
GQPKAAPSVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKxDSSPVK



X44 is A, V, I, L
AGVETTTPSK QSNNKYAASS YLSLTPExWK KHRSYSCQVT HEGSTVEKTV



X78 is A.
APTECS


 


243
CLλ
GQPKAAPSVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKxDSSPVK



X44 is V, I, L
AGVETTTPSK QSNNKYAASS YLSLTPExWK KHRSYSCQVT HEGSTVEKTV



X78 is A
APTECS


 


244
CLλ
GQPKAAPSVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKVDSSPVK



X44 is V
AGVETTTPSK QSNNKYAASS YLSLTPExWK KHRSYSCQVT HEGSTVEKTV



X78 is A
APTECS


 


245
CD motif
xxxx



X1 is V, I or L




X2 is D, N, Q, E




X3, x4 is any AA



 


246
CD motif
xxxx



X1 is V or I




X2 is D, N, Q, E




X3, x4 is any AA



 


247
CD motif
Vxxx



X2 is D, N, Q, E X3,




x4 is any AA



 


248
CD motif
xxxx



X1 is V, I or L




X2 is D, or N




X3, x4 is any AA



 


249
CD motif
xxxx



X1 is V, I or L




X2 is Q or E




X3, x4 is any AA



 


250
CD motif
xDxx



X1 is V, I or L




X3, x4 is any AA



 


251
CD motif
VDxx



X3, x4 is any AA



 


252
CD motif
LExx



X3, x4 is any AA



 


253
CD motif
IExx



X3, x4 is any AA



 


254
h38C2-[LC-D185A]
ELQMTQSPSS LSASVGDRVT ITCRSSQSLL HTYGSPYLNW YLQKPGQSPK




LLIYKVSNPF SGVPSRFSGS GSGTDFTLTI SSLQPEDFAV YFCSQGTHLP




YTFGGGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK




VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAA YEKHKVYACE




VTHQGLSSPV TKSFNRGEC


 


255
CD motif
xxxx



X1, x2, x3, x4 = any




amino acid












DETAILED DESCRIPTION OF FIGURES
FIGS. 1A, 1B, 1C, and 1D: Intact molecular weight analysis of MAC by mass spectrometry demonstrates that multiple peptides are attached to the anti-IGF1R antibody 2.12.1.fx. FIG. 1A: mass spectrometry data of anti-IGF1R antibody 2.12.1.fx. FIG. 1B-1D: mass spectrometry data of MAC-2, showing replicate experiments of 3 individual lots.
FIGS. 2A, 2B, 2C, 2D, 2E, 2F, 2G, and 2H: Mass spectrometry data of 2.12.1.fx (IGF1R) and 3 lots of MAC-2 (MAC) where the disulfide bonds have been reduced. FIG. 2A: Mass spectrometry data of 2.12.1.fx (IGF1R), light chain. FIG. 2B: Mass spectrometry data of 2.12.1.fx (IGF1R), heavy chain. FIG. 2C: mass spectrometry data of light chain of MAC-2, lot-1. FIG. 2D: mass spectrometry data of heavy chain of MAC-2, lot-1. FIG. 2E: mass spectrometry data of light chain of MAC-2, lot-2. FIG. 2F: mass spectrometry data of heavy chain of MAC-2, lot-2. FIG. 2G: mass spectrometry data of light chain of MAC-2, lot-3. FIG. 2H: mass spectrometry data of heavy chain of MAC-2, lot-3.
FIG. 3A: Amino acid sequence of light chain of antibody 2.12.1.fx with chymotrypsin cleavage sites noted with bullets. Chymotryptic fragments that contain a Lys residue (site of potential conjugation) are labeled by number from the N-terminus. The Y15 fragment of the light chain is underlined. FIG. 3B: Amino acid sequence of heavy chain of antibody 2.12.1.fx with chymotrypsin cleavage sites noted with bullets. Chymotryptic fragments that contain a Lys residue (site of potential conjugation) are labeled by number from the N-terminus.
FIG. 4A: Mass spectrometry data of a conjugated lysine-containing peptide: light chain Y15, showing mass spectrometry data for unconjugated anti-IGF1R antibody 2.12.1.fx (IGF1r) and MAC-2 (MAC), as well as a representation of the Y15 fragment. FIG. 4B: Mass spectrometry data of un-conjugated light chain Y15 fragment, showing mass spectrometry data for unconjugated anti-IGF1R antibody 2.12.1.fx (IGF1r) and MAC-2 (MAC), as well as a representation of the Y15 fragment.
FIG. 5A: The selected ion LCMS chromatogram data for the tryptic fragment of 2.12.1.fx. FIG. 5B: The selected ion LCMS chromatogram data for the tryptic fragment when LC-K188 is modified with ABP of MAC-2.
FIG. 6A: The selected ion LCMS chromatogram data for the tryptic fragment of 2.12.1.fx. FIG. 6B: The selected ion LCMS chromatogram data for the tryptic peptide when LC-K190 is modified with ABP of MAC-2.
FIG. 7A: Mass spectra of intact MAC-2. FIG. 7B: Mass spectra of reduced heavy chain for MAC-2. FIG. 7C: Mass spectra of reduced light chain for MAC-2.
FIG. 8A: Amino acid sequence alignment of the variable domains of m38c2, h38c2, and human germlines. Framework regions (FR) and complementarity determining regions (CDR) are defined according to Kabat et al. Asterisks mark differences between m38c2 and h38c2 or between h38c2 and the human germlines.
FIG. 8B: Amino acid sequence alignment of murine constant light chain kappa region (mCLκ), human constant light chain kappa region (hCLκ), and human constant light chain lambda region (hCLλ). Differences between mCLκ and hCLκ; and between hCLκ and hCLλ; are shown as asterisks, and conserved substitutions are shown as crosses. β-strands A-G are underlined. The turn between β-strands A and B and the α-helix between β-strands E and F are each indicated in italics. Di-sulfide bond-forming cysteines between the first β-sheet (made up of β-strands ABDE; single underline) and the second β-sheet (made up of β-strands CGF, double underline) are indicated by §. Known polymorphic loci in the human sequences are indicated in bold.
FIG. 9A: Binding ELISA data for HER2 receptor binding of trastuzumab and trastuzumab-[CLκ-D185A] conjugation products to [PEG5-K11-SEQ:27]. FIG. 9B: Binding ELISA data for HER2 receptor binding of trastuzumab and trastuzumab-[CLκ-D185A] conjugation products to MMAD toxin.
FIG. 10A: representation of a constant Ig domain showing the 7 β-strands forming the two β-sheets. FIG. 10B: close up of the α-helix between β-sheets E and F.
FIG. 11. Crystal structure-based minimized ribbon representation of CLκ, showing the halo-phenyl ester reactive ‘binding site’ (small jacks) within the overall steric ‘binding pocket’ created by the 3D structure. B-strands are labeled.
FIG. 12. Crystal structure-based minimized ribbon representation of the CLκ ‘binding pocket’, showing CLκ-D77 and CLκ-D43 (as a stick model) in the hydrogen bond with CLκ-H81 Nε or Nδ, and atomic distances in Å.
FIGS. 13A and 13B. Crystal structure-based minimized ribbon representation of the CLκ and CLκ-D77A mutant ‘binding pockets’. The distance between carbonyl oxygen of CLκ-D43 and Nδ of CLκ-H81 differs by 1 Å between the CLκ and CLκ-D77A mutant, pointing to the predominance of catalytically active CLκ-H81 tautomer Nδ in the CLκ-D77A mutant. In addition, the modeling identifies a clear increase in the overall size of the pocket in CLκ-D77A mutant, which is represented by the figure. FIG. 13C. Crystal structure-based ribbon representation of the CLκ (grey) and CLκ-D77A mutant (black) ‘binding pockets’ superimposed over each other. In the CLκ-D77A mutant, CLκ-H81 is shifted toward CLκ-D43 by the hydrogen bond interactions, as CLκ-D77A is unable to form a hydrogen bond with Nε of CLκ-H81.
FIG. 14. Crystal structure-based minimized ribbon representation of the CLκ ‘binding pocket’. The binding site is depicted as a small jacks. The π electron stacking interactions with CLκ-H81 are shown, maintaining the imidazole ring at the optimum position in relation to the incoming halo-phenyl ester substrate.
FIGS. 15A and 15B. Crystal structure-based minimized ribbon representation of the CLκ and CLλ ‘binding pockets. FIG. 15A shows the 7-electron interactions between CLκ-V42 and CLκ-H81, assisting in maintaining the CLκ-H81 imidazole ring and the Nε electron pair at the plane needed for nucleophilic attack during catalytic reaction. The distance between the center of CLκ-H81 imidazole ring and each of the hydrogen atoms on the CLκ-V42 are 2.8 Å allowing for strong interactions. In FIG. 15B, CLλ-A49 is shown at a distance of 4.2 Å from CLλ-H82 (identified as H81 in the figure for the purposes of clarity of comparison). This distance is modeled as likely too far to have a significant influence on the position or tautomeric form of CLλ-H82.
FIG. 16. Sequence alignment of hCHλ1, hCHλ2, hCHλ3, hCLκ and hCLλ. β-strands of the CLκ are indicated as underlined regions. α-helices are indicated in italics.
FIG. 17. Crystal structure based alignment of sequences of hCHλ1, hCHλ2, hCHλ3, hCLκ and hCLλ according to minimized 3D homology. β-strands are indicated as boxed regions, α-helices are indicated within wavy scrolls. Key residues corresponding to CLκ-V42, CLκ-D43, CLκ-D77, CLκ-K80, and CLκ-H81 are identified with rectangular dotted-line boxes extending vertically between sequences. The crystal structure modeling of the domains that generated this alignment suggested a short break in the D β-strand in the hCHλ1, hCHλ2, hCHλ3, and hCLλ domains. Other modeling and crystal structure analysis indicates that most, if not all antibody constant domains comprise 7 β-strands, and the D β-strand is contiguous. The two D β-strands modeled herein have accordingly been indicated as D′ and D′′. FIGS. 18B-31B, depicting CLλ, and CHγ domains, are orientated such that the D β-strand is on the lower left of the structure pointing downwards, and lying against the E β-strand. The D′ and D′′ β-strands together can be seen to occupy approximately the same relative position as the CLκ D β-strand (β-strands D and D′, D′′ labelled in FIG. 18 as a point of reference).
FIGS. 18A and 18B. Crystal structure-based minimized ribbon representation of the hCLκ (A) and hCLλ (B) domains.
FIGS. 19A and 19B. Crystal structure-based minimized ribbon representation of the hCLκ (A) and WT-hCHγ1 (B) domains.
FIGS. 20A and 20B. Crystal structure-based minimized ribbon representation of the hCLκ (A) and WT-hCHγ1 (B) domains, showing the sidechain location of significant residues.
FIGS. 21A and 21B. Crystal structure-based minimized ribbon representation of the hCLκ (A) and hCHγ1-m1-D44 mutant (B) domains, showing the sidechain location of significant residues.
FIGS. 22A and 22B. Crystal structure-based minimized ribbon representation of the hCLκ (A) and hCHγ1-T78K/Q79H/CD loop swap mutant (B) domains, showing the sidechain location of significant residues.
FIGS. 23A and 23B. Crystal structure-based minimized ribbon representation of the hCLκ (A) and WT-hCHγ2 (B) domains.
FIGS. 24A and 24B. Crystal structure-based minimized ribbon representation of the hCLκ (A) and WT-hCHγ2 (B) domains, showing the sidechain location of significant residues.
FIG. 25A and FIG. 25B. Crystal structure-based minimized ribbon representation of the hCLκ (A) and hCHγ2m mutant (B) domains, showing the sidechain location of significant residues.
FIGS. 26A and 26B. Crystal structure-based minimized ribbon representation of the hCLκ (A) and hCHγ2m-D82A mutant (B) domains, showing the sidechain location of significant residues.
FIGS. 27A and 27B. Crystal structure-based minimized ribbon representation of the hCLκ (A) and WT-hCHγ3 (B) domains.
FIGS. 28A and 28B. Crystal structure-based minimized ribbon representation of the hCLκ (A) and WT-hCHγ3 (B) domains, showing the sidechain location of significant residues.
FIGS. 29A and 29B. Crystal structure-based minimized ribbon representation of the hCLκ (A) and hCHγ3m mutant (B) domains, showing the sidechain location of significant residues.
FIGS. 30A and 30B. Crystal structure-based minimized ribbon representation of the hCLκ (A) and hCHγ3m CD/EF mutant (B) domains, showing the sidechain location of significant residues.
FIGS. 31A and 31B. Crystal structure-based minimized ribbon representation of the hCLκ (A) and hCHγ3m CD/EF-m2 mutant (B) domains, showing the sidechain location of significant residues.
FIGS. 32A and 32B. Alignment of CHγ1, CHγ2, and CHγ3 with their respective mutants and proposed mutant, together with CLκ and CLλ.



EXAMPLES
In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner.
Description of Conjugation Additions (CA), Average CA
Conjugation additions (CA) are measured on an antibody scaffold using intact mass measurement by mass spectrometry. Upon conjugation, the overall mass of the intact product increases by the mass and number of additions of the conjugated peptide, toxin, etc. If multiple additions occur then a distribution of conjugate forms is observed in a mass spectra and the observed signal intensity of each conjugate form gives a quantitative measurement. This analysis is routinely presented in a table by listing each CA form as a percentage of all the observed CA forms. Average CA (e.g.: the overall number of CA present on a scaffold) is an additional value that describes the average conjugate load. An example is provided below of 2 conjugated drug products. Example 1 has an even distribution of CA with an average CA=2.00. Example 2 has a distribution that heavily favors the presence of 2 CA with a minimal amount of other conjugation forms. The average CA is similar between these examples (2.13 vs. 2.00); however, Example 1 is a more heterogeneous product comprised of more conjugation forms, while Example 2 is a more homogeneous product that contains mostly 2 CA.














TABLE 1












Average



0 CA %
1 CA %
2 CA %
3 CA %
4 CA %
CA






















Example 1
11
22
33
22
11
2.00


Example 2
2
4
77
12
5
2.13









A similar analytical treatment is also possible on these antibody scaffolds after disulfide bonds have been reduced to generate free light chains and heavy chains. Measurement of the intact mass of conjugated light/heavy chains can provide information about the location of CA on these respective subunits.
Explanation of Directional Conjugation to CLκ-K80 
To determine site specific attachment of drug conjugates to an antibody scaffold, a peptide map is produced. Peptide maps are the analysis of a protein sequence in detail to characterize peptide produced following a proteolytic digestion of the conjugated drug product. Once the protein is digested then the resulting peptides are analyzed by reversed phase liquid chromatography with mass spectrometry detection (RPLC/MS). The presence of conjugate additions on discreet amino acid residues is observed as a corresponding mass shift compared to the un-conjugated peptide. This process has been repeated on multiple antibodies conjugated with multiple conjugate additions using PFP reactive esters to target lysine residues. (Data is presented below, and also in WO2012007896, whose contents are herein incorporated entirely). In these studies, the following observations were consistent: 1—conjugate additions were observed more frequently on the LC than the HC, 2-CLκ-K80 is the specific preferred residue that is modified, 3—multiple other locations are also modified on both the LC and HC; however, each alternative site is modified at a low level. To summarize, halo-phenyl ester conjugation results in preferred modification of CLκ-K80 and additional conjugation is distributed at a low level across multiple residues. For this reason, the conjugation process is generally optimized to result in high % CA values for 2 conjugate additions because this promotes a product that is fully conjugated at a single location on each LC. While elevated % CA levels of 0-1 conjugates result in preferable CLκ-K80 modification, these conjugate forms also represent a significant amount of un-reacted scaffold. Products that display average CA values significantly greater than 2 suggest the presence of conjugate additions that are not targeted at discreet residues. When HC and LC are reduced and analysed separately, the % value of 1 CA indicates the % conjugation on single LC species, and is thus reliable indicator of the efficiency of conjugation to the CLκ-K80 residue.
Example 1 Exemplary Synthesis of Peptides Used in the Invention




Synthesis of Peptide-Thiol-Linker Compounds





An Ang2-binding peptide (ABP; SEQ ID NO:27) (284 mg, 0.1 mmol) was dissolved in dimethylformamide (0.5 ml) with stirring. Separately, S-Trityl-mercaptopropionic acid (MPA, 62 mg, approx 0.125 mmol), HBTU (48 mg, 0.125 mmol) and N-methylmorpholine (0.025 ml, 0.25 mmol) were stirred in DMF (0.5 ml) for 5 min until dissolved. The ABP solution and activated MPA solutions were mixed together for 2 hrs. Progress of the reaction was monitored by LCMS. After 2 hrs, the solution was slowly added to ice-cold ether (40 ml) to precipitate the ABP-S-trityl-MPA product. The white precipitate was collected by filtration then dried. The solid residue was then dissolved in a solution of trifluoroacetic acid in dichloromethane (1:10, 10 ml), with triisopropylsilane (TIPS) added (0.050 ml) and stirred for 1 hr. The solution was evaporated under reduced pressure to a light-yellow oil then the crude thiol peptide precipitated by the addition of ice-cold ether. The product was collected by centrifugation and dried in vacuo. The residue was dissolved in 50% aqueous acetonitrile then lyophilized to yield the crude thiol peptide (approx 80% pure by HPLC analysis). The crude thiol peptide was purified by semi-preparative HPLC to yield 145 mg of SEQ ID NO:27-KSH11.
Generation of Ang-2-Binding-Peptide-Thiol Intermediates
Peptide chain assembly was conducted on a 0.1 mmol scale. The resin used was Fmoc-Rink-PL resin (150 mg, 0.67 mmol/g substitution). Standard Fmoc chemistry protocols were used to assemble the peptide. Fmoc removal was with 20% piperidine/DMF for 3×5 min. and all resin washing steps used DMF. To incorporate the amino acids, a single coupling step was employed for each residue, using HBTU/HOBt/NMM activation, for a 2 hr period. The Linking Residue (KSH) was incorporated as Fmoc-Lys(Nε-mercaptopropionate-S-Trt)-OH. Upon chain assembly, the N-terminal Fmoc group was removed and the peptidoresin capped by acetylation. The final resin was washed with DCM and dried overnight in vacuo.
Acidolytic removal of protecting groups and cleavage of the peptide from the resin was achieved using a cocktail of TFA/water/dithiothreitol/triisopropylsilane (ratio 90:4:4:2, 5 ml) for 2 hrs. The solution was filtered from the resin and the resin washed with another 5 ml of neat TFA. The combined filtrates were evaporated to a syrup then addition of ice-cold ether precipitated a white powder. The powder was collected by centrifugation then dissolved in 50% aqueous acetonitrile (20 ml), frozen and lyophilized overnight.
A preparative HPLC column was pre-equilibrated with dilute aqueous TFA and acetonitrile. The crude ABP-thiol intermediates (i.e. ABP with KSH as linking residue) was dissolved in DMF (3 ml), then adsorbed onto the column and eluted by applying a gradient of acetonitrile in dilute TFA. Fractions were collected automatically by mass (M=1465). Elution from the column was monitored by UV, the fractions obtained were analyzed by analytical RP-HPLC.
Example 2 Conjugation Strategies
5 different conjugation strategies were considered for conjugating peptides to antibodies (exemplary structures are shown using SEQ ID NO:27-KSH11 and 2.12.1.fx) (full details are provided in the Examples of PCT/US2011/053092, filed 11 Jul. 2011, whose contents are hereby incorporated entirely). Briefly, NHS esters, maleimide, squarate esters, AZD and halo-phenyl esters were all investigated as potential mechanisms to develop directional conjugation to antibodies.




NHS esters suffered from the problems of slowly converting to a free acid form, where the NHS ester is converted to an inactive carboxyl. It was concluded that although some success was obtained with NHS esters, it appeared that the aqueous lability of the resulting NHS-ester may limit their application in subsequent conjugation reactions. Further tests of NHS-PEG2-MAL are shown below (comprising Z* group Z13).
The ethyl squarates conjugate well to free thiols but poorly to free amines on proteins and antibodies unless the pH is above 9.
In general, the maleimide-activated peptides did not conjugate well to proteins or antibodies which lack either an endogenous thiol (derived from a free cysteine side chain) or a thiol introduced by other chemical means, e.g. via Traut's reagent.
AZD reacted slowly with antibody amino groups, and attempts to increase the pH to 7-9 yielded low levels of conjugation and high levels of AZD hydrolysis (in order to increase the nucleophilic tendency of the antibody surface lysines by decreasing their charge, as the pKa of surface lysines is about 9.1 to 11.2).
Example 3 Synthesis of Pentafluorophenyl Esters (PFP)




The present invention also provides for the use of pentafluorophenyl (PFP; Z*=Z1) esters to form relatively stable Effector Moiety-linker complexes. This method has several advantages over other approaches in that the PFP group can be introduced in solution easily from a stable activated peptide product, which itself can be purified using standard HPLC methods with little PFP ester hydrolysis observed.
The present invention provides a synthetic route whereby an activated ester group, such as PFP, can be coupled directly to a side chain lysine on the peptide by either a chemoselective reaction (using thiol/maleimide chemistry) or by using a bis-active ester reagent, which forms an amide with the peptide side chain but leaves the other end as the active ester.
In some embodiments, the strategy may be a bis-acid PEG with each acid activated as a PFP ester. In organic solutions, with some base present, the end of the bis-PFP linker reacted with the N-ε-amino side chain of lysine in the required tether position to form a stable amide linkage, while the other end maintained the other PFP group. One potential problem with this strategy is the possibility of forming peptide dimers, where a peptide would add to each of the PFP moieties present at each end of the linker. In some aspects, the present invention overcomes this additional problem by altering the stoichiometry and addition of the respective peptide and bis-PEG-PFP linker. One solution provided by the invention is to have an excess of the bis-Pfp linker in solution and slowly add the peptide in solution, such that an excess of linker over peptide is always present. By having a ratio of between about 3.7:1 to about 4.3:1, or in some embodiments, a ratio of about 4:1, of linker over peptide, the required PFP-activated peptide can be synthesized with no dimer present. The synthesis scheme for [PFP-PEG5-K11-SEQ:27] is shown below in Scheme 3.
Synthesis of [PFP-PEG5-K11-SEQ:27]




Bis-dPEG5-acid (1 mmol, 338 mg) was dissolved in anhydrous dichloromethane (5 ml) then pentafluorophenol (2 mmol, 368 mg) was added, along with dicyclohexycarbodiimide (1 mmol, 208 mg). The solution was stirred overnight at RT. After this time, the fine white dicyclohexylurea side-product was filtered off and the filtrate evaporated to dryness to give a pale yellow light oil. Analysis by TLC and HPLC indicated a pure product with correct MS=670. The product was used in the next step without further purification. The product is stable for several months at −20° C.
SEQ ID NO:27 (730 mg) was dissolved in anhydrous dimethylformamide (8 ml) and N-methylmorpholine (0.05 ml) added. An aliquot of neat bis-dPEG5-OPfp reagent (0.5 ml) was placed in a glass vial (20 ml). With vigorous stirring, the SEQ ID NO:27/NMM solution was added in 4×2 ml aliquots to the bis-dPEG5-OPfp reagent over 2 hr, then the final mixture stirred for a further 1 hr. Progress of the conversion to [PFP-PEG5-K11-SEQ:27] product was monitored by analytical HPLC. At the end of the reaction, the solution was filtered and directly purified by semi-preparative HPLC on a 1′′ C8 column. The purest fractions (>95% by analytical HPLC) were combined and lyophilized to give 400 mg (48% yield) of final [PFP-PEG5-K11-SEQ:27] peptide-linker product. A similar mechanism can be used to generate [PFP-PEG2-MAL-KSH11-SEQ:27] (see Scheme 4).
Synthesis of [PFP-PEG2-KSH11-SEQ:27]




Maleimide-dPEG2-acid (328 mg, 1 mmol, Quanta Biodesign), pentafluorophenol (0.103 ml, 1 mmol, PFP) and dicyclohexylcarbodiimide (206 mg, 1 mmol, DCC) were dissolved in dry DCM (10 ml) and stirred for 1 hr at RT. The fine white precipitate (DCU side-product) that formed was removed by filtration and the filtrate evaporated to dryness in vacuo. The product was obtained as a fine white powder in high yield (490 mg, quantitative). Purity was >95% by analytical HPLC; MS showed [M+H]+=495.
A sample (30-40 mg) of SEQ ID NO:27-KSH11 was dissolved in anhydrous DMF (2 ml). Maleimide-PEG2-PFP (20 mg) was added along with N-methylmorpholine (5 mL). The reaction was stirred and monitored at RT by HPLC to follow the time-course of product formation. The complete conversion of starting peptide to PFP-activated product was observed within the first 2 hrs. The solution was filtered and the product peak directly isolated by semi-preparative HPLC. In each case, the product was isolated in approximately 40% yield after lyophilization.
Example 4 Antibody Conjugation
The MAC-1 and MAC-2 exemplary antibody-Effector Moiety conjugates were made by conjugating the antibody 2.12.1.fx (SEQ ID NO:1 and SEQ ID NO:2) with an Ang2 binding peptide (SEQ ID NO:27). MAC-1 comprises 2.12.1.fx conjugated to [PFP-PEG2-MAL-KSH11-SEQ:27] to yield 2.12.1.fx-[PEG2-MAL-KSH11-SEQ:27] and MAC-2 comprises 2.12.1.fx conjugated to [PFP-PEG5-K11-SEQ:27] to yield 2.12.1.fx-[PEG5-K11-SEQ:27].
Generation of MAC-1




Generation of MAC-2





The number of peptide conjugations per 2.12.1.fx antibody in a sample of each MAC was calculated (see Table 2).







TABLE 2







Conjugation profile of MAC-1 and MAC-2.










Conjugation Additions (CA) (%)















0
1
2
3
4
Avg CA



















MAC-1
3
26
42
25
3
1.97



MAC-2
2
20
47
26
5
2.12










Example 5 Optimizing Conditions for PFP-Based Conjugation
A series of assays were run to establish optimal reaction conditions for directed conjugation. At the end of each reaction conjugation, the reaction was quenched with a succinate and glycine buffer, lowering the pH to approximately 5.5 and quenching any free peptide or peptide/linker. MAC-2 analysis was conducted by measuring the intact molecular weight (MW) of the MAC using electrospray time-of-flight mass spectrometry detection following protein separation from salts and excipients through a size exclusion chromatography column.
Temperature
2.12.1.fx antibody was adjusted to 18 mg·ml−1 at pH 7.7 with a phosphate buffer to a final concentration of 0.06M sodium phosphate. [PFP-PEG5-K11-SEQ:27] was reconstituted in a propylene glycol solution to 10 mg·ml−1. [PFP-PEG5-K11-SEQ:27] was added to 2.12.1.fx at a molar ratio of 4.3:1 and allowed to react for 2 hrs at 18, 22, or 25° C. Results are presented in Table 3.







TABLE 3







Reaction temperature in 0.06M phosphate


at 4.3:1 peptide:antibody.










CA (%)














Temp
0
1
2
3
4
Avg CA
















18 C.
1
16
51
23
8
2.21


22 C.
3
15
57
21
5
2.11


25 C.
2
12
53
25
7
2.24









Reaction pH

2.12.1.fx antibody was adjusted to 18 mg·ml−1 at pH 6.5, 6.75, 7.0, 7.25, 7.5, 7.75, or 8.0 with a phosphate buffer to a final concentration of 0.06M sodium phosphate. [PFP-PEG5-K11-SEQ:27] was reconstituted in a propylene glycol solution to 10 mg·ml−1. [PFP-PEG5-K11-SEQ:27] was added to 2.12.1.fx at a molar ratio of 4.3:1 and allowed to react for 2 hrs at RT. The results are presented in Table 4.







TABLE 4







pH in 0.06M sodium phosphate buffer at 4.3:1 peptide:antibody.










CA (%)














pH
0
1
2
3
4
Avg CA
















6.5
7
42
41
 9
0
1.51


6.75
3
31
52
12
3
1.83


7.0
3
24
53
16
4
1.94


7.25
2
18
54
22
5
2.12


7.5
2
12
57
23
7
2.23


7.75
3
15
55
22
6
2.15


8.0
1
14
52
29
4
2.21









2.12.1.fx was adjusted to 2 mg·ml−1 at pH 7.0, 7.5 and 8.0 with a HEPES buffer to a final concentration of 0.02M. [PFP-PEG5-K11-SEQ:27] was reconstituted in DMSO to 10 mg·ml−1. [PFP-PEG5-K11-SEQ:27] was added to 2.12.1.fx at a molar ratio of 5:1 and allowed to react overnight at RT. The results are presented in Table 5. The level of conjugation decreased above pH 8.0







TABLE 5







pH in 0.02M HEPES Buffer at 5:1 peptide:antibody.










ABP Additions (%)














pH
0
1
2
3
4
Avg CA
















7
2
21
41
28
4
2.03


7.5
3
22
44
26
5
2.08


8
9
30
42
17
2
1.73









Duration of Conjugation Reaction

2.12.1.fx was adjusted to 18 mg·ml−1 at pH 7.7 with a phosphate buffer to a final concentration of 0.06M sodium phosphate. [PFP-PEG5-K11-SEQ:27] was reconstituted in a propylene glycol solution to 10 mg·ml−1. [PFP-PEG5-K11-SEQ:27] was added to 2.12.1.fx at a molar ratio of 4.3:1 and allowed react for 30, 60, 120, 180, 240, 300, or 2400 mins at room temperature (Table 6).







TABLE 6 







Duration of conjugation reaction in 0.06M


sodium phosphate at 4.3:1 peptide:antibody.









Time
CA (%)














(mins)
0
1
2
3
4
Avg CA
















30
6
38
44
13
0
1.64


60
1
22
52
21
3
2.02


120
0
15
50
29
6
2.24


180
1
12
51
31
5
2.28


240
1
9
51
33
5
2.33


300
1
9
50
35
5
2.35


2400
1
10
48
35
6
2.35









Molar Ratio of Peptide to Protein

2.12.1.fx was adjusted 18 mg·ml−1 to pH 7.5 with a HEPES buffer to a final concentration of 0.2M HEPES. [PFP-PEG5-K11-SEQ:27] was reconstituted in a propylene glycol solution to 10 mg·ml−1. [PFP-PEG5-K11-SEQ:27] was added to 2.12.1.fx at a molar ratio of 1, 2, 3, 4, and 5:1 (Table 7), and reacted for at least 2 hrs at RT, but the high concentration of HEPES buffer resulted in decreased conjugation.







TABLE 7







Molar ratio of peptide to protein 1:1-5:1 in 0.2M HEPES.










CA (%)

















Peptide:2.12.1.fx
0
1
2
3
4
5
6
7
Avg CA



















1:1
80
20
0
0
0
0
0
0
0.20


2:1
60
35
5
0
0
0
0
0
0.45


3:1
39
49
12
0
0
0
0
0
0.73


4:1
27
51
19
3
0
0
0
0
0.98


5:1
11
47
37
5
0
0
0
0
1.36









2.12.1.fx was adjusted 18 mg·ml−1 to pH 7.7 with a phosphate buffer to a final concentration of 0.06M sodium phosphate. [PFP-PEG5-K11-SEQ:27] was reconstituted in a propylene glycol solution to 10 mg·ml−1. [PFP-PEG5-K11-SEQ:27] was added to 2.12.1.fx at a molar ratio of 5, 7, 10, 12, and 15:1 (Table 8) and allowed to react for 2 hrs at RT to generate a MAC with a higher level of conjugation.







TABLE 8







Molar ratio of peptide to protein


7:1-15:1 in 0.06M sodium phosphate.










CA (%)

















Peptide:2.12.1.fx
0
1
2
3
4
5
6
7
Avg CA



















 7:1
1
1
29
39
17
10
2
0
3.06


10:1
1
1
18
33
25
19
3
0
3.49


12:1
3
1
11
22
26
26
8
3
3.92


15:1
1
2
9
19
23
32
12
3
4.22









To further optimize the molar ratio of 2.12.1.fx and [PFP-PEG5-K11-SEQ:27], 2.12.1.fx was adjusted 18 mg·ml−1 to pH 7.7 with a phosphate buffer to a final concentration of 0.06 M sodium phosphate. [PFP-PEG5-K11-SEQ:27] was reconstituted in a propylene glycol solution to 10 mg·ml−1. [PFP-PEG5-K11-SEQ:27] was added to 2.12.1.fx antibody at a molar ratio of 2.5, 2.8, 3.1, 3.4, 3.7, 4.0, 4.3, or 4.6:1 (Table 9) and allowed to react for 2 hrs at RT.







TABLE 9







Molar ratio of peptide to protein 2.5:1-


4.6:1 in 0.06M sodium phosphate.










CA (%)

















Peptide:2.12.1.fx
0
1
2
3
4
5
6
7
Avg CA



















2.5:1
14
53
30
4
0
0
0
0
1.25


2.8:1
10
45
37
8
0
0
0
0
1.43


3.1:1
7
39
45
8
0
0
0
0
1.53


3.4:1
5
40
44
11
0
0
0
0
1.61


3.7:1
4
25
51
15
5
0
0
0
1.92


4.0:1
2
26
55
15
2
0
0
0
1.89


4.3:1
1
24
55
16
4
0
0
0
1.98


4.6:1
2
19
56
19
5
0
0
0
2.08









2.12.1.fx was adjusted to 2 mg·ml−1 at pH 7.0 with a HEPES buffer to a final concentration of 0.02M. [PFP-PEG5-K11-SEQ:27] was reconstituted in DMSO to 10 mg·ml−1. [PFP-PEG5-K11-SEQ:27] was added to 2.12.1.fx at a molar ratio of 5, 6, 7, 8, 10:1 and allowed to react overnight at RT. The results are presented in Table 10.







TABLE 10







Molar ratio of peptide to protein 5:1-10:1 in 0.02M HEPES.










CA (%)

















Peptide:2.12.1.fx
0
1
2
3
4
5
6
7
Avg CA



















5:1
2
21
49
24
4
0
0
0
2.07


6:1
2
15
42
32
9
0
0
0
2.31


7:1
1
11
34
42
13
0
0
0
2.57


8:1
0
9
32
42
16
1
0
0
2.68


10:1 
0
4
21
47
25
4
0
0
3.07









Conjugation Profile of 2.12.1.Fx at Various Protein Concentrations

The conjugation profiles of 2.12.1.fx with [PFP-PEG5-K11-SEQ:27] at various concentrations were analyzed. 2.12.1.fx was concentrated to >50 mg/mL, diluted to the desired concentration with 20 mM sodium acetate, 200 mM trehalose pH 5.5, and spiked with 60 mM sodium phosphate pH 7.7. [PFP-PEG5-K11-SEQ:27] was resuspended with 50% propylene glycol and mixed with the antibody at a 4.3:1 molar ratio and allowed to react overnight at RT. All samples were diluted to 2 mg/ml and analyzed as an intact conjugated protein by size exclusion chromatography-mass spectrometry (SEC-MS) to determine the number and quantitation of conjugate forms of the protein. This technique measures the molecular weight of each protein form; multiple conjugation sites are observed as distinct signals separated by the mass difference of a peptide. Relative quantitation of multiple conjugation species is performed by measuring the signal magnitude. Table 11 shows the conjugation profile of 2.12.1.fx with peptide at various concentrations of antibody. At antibody concentrations 10 mg/mL to 50 mg/mL, the conjugation occurs at a distribution between 0-5 addition with an average of 1.8 or greater additions. At antibody concentrations 0.5 to 5 mg/mL, the conjugation occurs at a distribution between 0-3 additions with an average of 1.5 or less additions.







TABLE 11 







Effect of antibody concentration.









Antibody




Concentration
CA (%)














(mg/ml)
0
1
2
3
4
5
Avg CA

















0.5
65
32
3
—
—
—
0.37


1
44
44
12
—
—
—
0.67


5
10
41
40
8
—
—
1.45


10
3
30
47
17
2
1
1.87


15
1
24
51
20
3
1
2.02


20
1
16
57
22
2
1
2.11


30
2
20
55
20
3
1
2.04


40
2
21
53
22
2
0
2.04


50
2
19
50
24
4
1
2.11









Reaction Buffer Selection

2.12.1.fx was adjusted to 18 mg·ml−1 at pH 7.7 with a sodium carbonate, sodium borate, or sodium phosphate buffer to a final concentration of 0.05M sodium phosphate. [PFP-PEG5-K11-SEQ:27] was reconstituted in a propylene glycol solution to 10 mg·ml−1. [PFP-PEG5-K11-SEQ:27] was added to 2.12.1.fx at a molar ratio of 1, 2, 3, 4, or 5:1 and allowed to react for 2 hrs at RT. The low reaction pH resulted in the reduced level of conjugation (Table 12).







TABLE 12 







Buffer and pH alterations.










CA (%)














Buffer
0
1
2
3
4
Avg CA
















50 mM sodium carbonate pH 7.4
2
24
48
26
0
1.98


50 mM sodium borate pH 7.0
1
17
45
31
5
2.20


50 mM sodium phosphate pH 7.0
10
48
38
4
0
1.36









2.12.1.fx was adjusted to 18 mg·ml−1 at pH 7.5, 7.7 and 8.0 with a sodium borate and sodium phosphate buffer to a final concentration of 0.04 M. [PFP-PEG5-K11-SEQ:27] was reconstituted in a propylene glycol solution to 10 mg·ml−1 and added to 2.12.1.fx at a molar ratio of 4.3:1, and reacted for 2 hrs at RT (Table 13).







TABLE 13







Buffer and pH alterations.










CA (%)














Buffer
0
1
2
3
4
Avg CA
















Phosphate, pH 7.5
1
21
53
21
3
2.02


Phosphate, pH 7.7
0
15
50
29
6
2.26


Phosphate, pH 8.0
1
14
52
29
4
2.21


Borate, pH 7.5
46
44
10
0
0
0.64


Borate, pH 7.7
22
51
23
4
0
1.09


Borate, pH 8.0
1
17
48
30
4
2.19









2.12.1.fx was adjusted to 18 mg·ml−1 at pH 7.7 with a phosphate buffer to a final concentration of 0.04 M, 0.06 M, or 0.08 M sodium phosphate. [PFP-PEG5-K11-SEQ:27] was reconstituted in a propylene glycol solution to 10 mg·ml−1. [PFP-PEG5-K11-SEQ:27] was added to 2.12.1.fx at a molar ratio of 4.3:1 and allowed to react for 2 hrs at RT. The results are presented in Table 14.







TABLE 14







Concentration of phosphate.









Concentration (mM) of
CA (%)














phosphate at pH 7.7
0
1
2
3
4
Avg CA
















40
2
23
54
16
4
1.95


60
2
28
51
15
4
1.91


80
2
29
51
13
4
1.86









Effect of Buffer Constituents on Conjugation

Propylene glycol: 2.12.1.fx was adjusted to 18 mg·ml−1 at pH 7.7 with a phosphate buffer to a final concentration of 0.06 M sodium phosphate. [PFP-PEG5-K11-SEQ:27] was reconstituted in a propylene glycol solution to 20 mg·ml−1 (5% propylene glycol in the conjugation reaction). [PFP-PEG5-K11-SEQ:27] was added to 2.12.1.fx at a molar ratio of 4.3:1 and spiked with an additional 0 to 15% propylene glycol (final propylene glycol percentage of 5, 10, 15, and 20%) and allowed to react for 2 hrs at RT. The results are presented in Table 15.







TABLE 15







Percent of propylene glycol in 0.06M sodium phosphate.










CA (%)














Percent (%) Propylene Glycol
0
1
2
3
4
Avg CA
















5
2
18
55
20
5
2.08


10
2
20
53
21
5
2.09


15
2
23
49
20
5
2.01


20
4
23
50
19
4
1.96









NaCl: 2.12.1.fx was adjusted to 2 mg·ml−1 at pH 7.0 with a HEPES buffer to a final concentration of 0.02M in the presence and absence of 0.14M NaCl. [PFP-PEG5-K11-SEQ:27] was reconstituted in DMSO to 10 mg·ml−1. [PFP-PEG5-K11-SEQ:27] was added to 2.12.1.fx at a molar ratio of 5:1 and allowed to react overnight at RT. The level of conjugation decreases in the presence of NaCl (Table 16).







TABLE 16







Concentration of sodium chloride in 0.02M HEPES.









Concentration of sodium
ABP Additions (%)














chloride (mM)
0
1
2
3
4
Avg CA
















0
2
21
41
28
4
2.03


0.14
9
34
42
14
1
1.64









HEPES: 2.12.1.fx was adjusted to 2 mg·ml−1 at pH 7.0 with a HEPES buffer to a final concentration of 0.2 M and 0.02 M. [PFP-PEG5-K11-SEQ:27] was reconstituted in 50% propylene glycol to 10 mg·ml−1. [PFP-PEG5-K11-SEQ:27] was added to 2.12.1.fx at a molar ratio of 5:1 and allowed to react 2 hrs at RT. The results are presented in Table 17. The level of conjugation is reduced at 0.2M HEPES buffer.







TABLE 17







HEPES concentration.










ABP Additions (%)














Concentration of HEPES (mM)
0
1
2
3
4
Avg CA
















0.02
2
35
47
16
0
1.77


0.2
21
49
26
4
0
1.13









DMSO: 2.12.1.fx was adjusted to 15 mg·ml−1 at pH 7.7 with sodium phosphate buffer to a final concentration of 0.06 M and DMSO was added to a final concentration of 30%. [PFP-PEG5-K11-SEQ:27] was reconstituted in a propylene glycol solution to 10 mg·ml−1. [PFP-PEG5-K11-SEQ:27] was added to 2.12.1.fx at a molar ratio of 4:1 and allowed to react for 2 hrs at RT. The results are presented in Table 18.







TABLE 18







DMSO in 0.06M sodium phosphate.










ABP Additions (%)














Percent of DMSO
0
1
2
3
4
Avg CA
















0
3
28
49
14
6
1.92


30
8
28
32
22
10
1.98









Discussion of Conjugation Reaction Parameters

When the molar ratio of Effector Moiety (in this example, a peptide) to antibody is reduced below about 3.5:1, the level of conjugation is decreased, as seen in Table 9. Alternatively, Table 10 shows that increasing the molar ratio results in an increased level of conjugation. Increasing the number of peptides per antibody generally decreases the binding efficiency of the antibody (in this case 2.12.1 fx) to its antigen (in this case the IGF1R receptor), therefore the molar ratio of peptide to antibody was optimized to maximise both antibody-antigen, and peptide-cognate binding.
It was also found that varying the conjugation buffer can alter the conjugation pattern. Amine-containing excipients are less preferable in general as they can react with the PFP group. Buffers such as carbonate and borate can be used for conjugation but were avoided as their pKa (boric acid with a pKa  ̃9 and carbonate with two pKa of  ̃6 and  ̃11) were far from the conjugation pH of 7.7 that was identified as optimal for MAC-1 and MAC-2 (Table 12). The level of conjugation is not only dependent on the chemical conditions of the reaction but also based on time. After 2 hrs, most of the PFP-activated peptide had reacted with the antibody or the PFP Z* has hydrolyzed (Table 6).
The PFP-activated peptide/linker reacted quickly with lysine side chain amino groups. Conjugation was performed at pH 6.5 to 8 in phosphate buffer to increase the nucleophilic tendency of the antibody surface lysines by decreasing their charge (the pKa of lysines on the surface proteins is about 9.1 to 11.2) as shown in Tables 4 and 5.
Optimal conditions for conjugation of MAC-1 and MAC-2 are described as follows: 2.12.1.fx antibody was adjusted to pH 7.7 with a phosphate buffer to a final concentration of 0.06M sodium phosphate. [PFP-PEG5-K11-SEQ:27] was reconstituted in a propylene glycol solution to 10 mg·ml−1 (final propylene glycol concentration in reaction is 10%). [PFP-PEG5-K11-SEQ:27] was added to 2.12.1.fx antibody at a molar ratio of 4.3:1 and allowed to react for 2 hrs at RT. The reaction was quenched with a succinate and glycine buffer, lowering the pH to approximately 6.0 and quenching any free peptide. In some aspects, the reaction may be concentrated and peptide-related species (such as peptides where the linker was hydrolyzed by reaction with water solvent) and other elements of the reaction mixture (such as PFP) may be removed by diafiltration, for example, using a 50 kDa membrane or size exclusion chromatography into a succinate, glycine, sodium chloride, and trehalose buffer, pH 5.5 at 30 mg/ml.
The conjugation conditions listed above were varied to determine the range of each process parameter. Parameter ranges were set based on variability that may occur during the conjugation and/or were expanded until greater than 10% change in species population was observed. Table 19 summarizes the parameters that result in similar conjugation profiles for MAC-2.







TABLE 19







Process parameters optimized for MAC-2.










Parameters tested
Range tested
Desirable range
Optimum





Temperature
18-25° C.
18-25° C.
RT


Reaction pH
6.5 to 8.0
pH 7.25-8.0
7.7


Reaction duration
30-2400 mins
180-2400 mins
>about 2 hrs


Molar ratio of Peptide to 
2.5 to 4.6
3.7:1 to 4.3:1
4.3:1


Antibody





2.12.1.fx concentration 
0.5 to 50 mg/mL
10 to 50 mg/mL
20 mg/mL


added to the reaction





Phosphate concentration 
40 to 80 mM
40-80 mM
60 mM


in the reaction buffer





Final propylene glycol 
5 to 20%
5-20%
10%


concentration









Example 6 Location of Conjugated Peptides on Antibody
The MAC-2 drug product molecule consists of a distribution of 1-4 [PEG5-K11-SEQ:27] molecules attached to the 2.12.1.fx antibody. This was determined by measuring the intact molecular weight (MW) of MAC-2 using electrospray, time-of-flight mass spectrometry detection following protein separation from salts and excipients through a size exclusion chromatography column. Mass spectrometry data that demonstrated the MW of the 2.12.1.fx and 3 lots of MAC-2 are shown in FIG. 2. FIG. 1A shows 2.12.1.fx before conjugation. This is a uniform molecule that displays a single MW. The MAC-2 lots display a distribution of conjugated peptides to 2.12.1.fx; between 1-4 conjugation additions (CA) are observed. The relative amount of each form is consistent between lots and the most common form in each lot has 2 peptides (SEQ ID NO:27) attached to each individual 2.12.1.fx antibody.
By reducing disulfide bonds in the 2.12.1.fx antibody, light and heavy chains are observed separately. Disulfide reduction is performed by treating the intact 2.12.1.fx antibody with 20 mM tris(2-carboxyethyl) phosphine (TCEP). The resulting mixture of heavy and light chains is analyzed for intact molecular weight as described above. The data shown in FIG. 2 provides evidence toward the location of the ABP on 2.12.1.fx. The majority of light chain (>65%) in the MAC-2 lots are conjugated. Most of the conjugated light chain contains 1CA. 2CA is also observed at a lower level. Almost all observed heavy chain (>90%) is unmodified, which suggests that very few of the conjugated peptides are located on the heavy chain.
Peptide mapping was used to determine the precise location of conjugation. The procedure was as follows: an aliquot of MAC-2 was denatured with 8M Guanidine-hydrochloride, disulfide bonds were reduced with TCEP, and the resulting cysteine sulphydryls were alkylated with Iodoacetamide. This treated protein sample was then digested with the protease chymotrypsin (1:125 protease:MAC ratio by weight). The resulting chymotryptic peptides were then detected individually by mass spectrometry after separation through a C8 liquid chromatography column. With this technique, MAC-2 was digested by chymotrypsin on the heavy and light chains into fragments at the locations noted in the sequence (with bullets) in FIG. 3. Liquid chromatography-mass spectrometry (LC-MS) detection of the MW of each peptide was then used to determine which Lysine residues are modified by a conjugated peptide. If a fragment was modified by attachment of conjugated peptide, its MW was shifted accordingly.
Fragments Y1, Y6, Y9, Y10, Y20, Y25, Y26, Y29, Y32, Y33, Y34, Y37, Y40 and Y43 of the heavy chain contain Lys residues. Of these, peptide conjugation was detected at Y6, Y10, Y25, Y33, and Y37. Fragments Y3, Y10, Y11, Y12, Y13, Y14, Y15, and Y16 of the light chain contain Lys residues. Of these, conjugation was detected at Y3, Y13, and Y15.
The light chain fragment referred to as Y15 (the 15th chymotryptic fragment on the light chain from the N-terminus) was found to be conjugated based on the data shown in FIG. 4. The MW of the modified Y15 fragment in MAC was clearly detected. In the un-conjugated 2.12.1.fx sample, there was no evidence of modified Y15 fragment. The unmodified Y15 fragment was observed in both MAC-2 and 2.12.1.fx. The magnitude of this fragment is higher in the 2.12.1.fx sample because this entire fragment is present in the un-modified form. As this fragment is conjugated in MAC-2, the observed level of un-modified Y15 decreases, which is seen in FIG. 4 as a peak with a smaller area.
The amount of conjugation of [SEQ:27-K11-PEG5] observed on light chain fragment Y15 in MAC-2 is estimated by measuring the decreased peak area of un-modified Y15. After normalizing the signal intensity such that unconjugated 2.12.1.fx showed 100%, 3 independent lots of MAC-2 showed 17%, 27% and 22% unconjugated Y15 fragments respectively.
The observed magnitude of Y15 in the MAC samples was normalized to the magnitude of Y15 in the 2.12.1.fx sample. Between 75-85% of the Y15 fragments are determined as modified in MAC-2. Considering that MAC-2 contains mostly 1-2 conjugation additions, this suggests that most of the conjugation in MAC-2 is located at one of the 2 K residues of light chain fragment Y15 (LC-K188 or LC-K190). The location of fragment Y15 in relation to the sequence of 2.12.1.fx is shown in FIG. 3.
Trypsin enzymatic digestion was used to discriminate between LC-K188 and LC-K190 (trypsin has specificity for the C-terminus of K and R). As trypsin does not digest conjugated K residues, the enzymatic digestion generates different peptide lengths, depending on which K residue is conjugated. Examination of LCMS data from MAC-2 that was digested with trypsin provides evidence that the peptide attaches specifically to LC-K188. No evidence of modified LC-K190 was observed.
MAC-2 was reduced with TCEP and denatured with guanidine hydrochloride as described above. The protein concentration was adjusted to 2 mg/ml and the pH to 7.8 with Tris digestion buffer. Purified trypsin was added at a 1:125 protease:MAC ratio by weight and incubated at 30° C. for 4 hrs. Samples were stored at −20° C. until analyzed by LCMS. Fragment samples were separated on a C18 reversed phase column using water/acetonitrile+0.1% TFA mobile phases. Detection of fragments was monitored both by UV 214 nm and ESI-TOF mass spectrometry. All data analysis was performed using MassLynx software.
The formation of fragments upon trypsin digestion of MAC-2 depends on the site of peptide conjugation. Lysines are the targeted residue for conjugation. Data shown in FIGS. 1-4 indicates that the predominant site of peptide binding is either LC-K188 or LC-K190. The scheme below shows the trypsin digestion reactions that would occur upon conjugation at either 2.12.1.fx-[LC-K188] or 2.12.1.fx-[LC-K190].




The chemical structures of the two potential digestion fragments in question are as follows:




FIG. 5 shows the selected ion LCMS chromatogram data for the trypsin peptide when LC-K188 is conjugated to the peptide. FIG. 6 shows the selected ion LCMS chromatogram data for the trypsin fragment when LC-K190 is modified with a conjugated peptide. These data suggest that only LC-K188 alone is conjugated; this situation results in a significant signal that is detected in MAC-2 but is absent in the 2.12.1.fx control experiment. The results from modification at LC-K190 do not provide any data that is unique compared to the negative control.
In contrast to what may be expected, the peptide/linker appears to preferentially decorate LC-K188 of the light chain of 2.12.1.fx. This has the surprising advantage that the Fc portion of the 2.12.1.fx antibody is unaffected. Tests show that the resulting PK of MAC-2 is approximately equal to the PK of unconjugated 2.12.1.fx. Promiscuous, non-specific conjugation to multiple sites on an antibody can result in a product with lower PK. The directional conjugation of the invention, exemplified by MAC-1 and MAC-2, provide the advantage of minimizing some of the possible deleterious effects that can be caused by promiscuous, non-specific conjugation, including lower PK. LC-K188 is the same residue as CLκ-K80 (i.e. K80 of SEQ ID NO:6), as the Light Chain (LC) comprises the variable region as well as the constant light kappa chain (CLκ).
To establish the reproducibility of the process, the experiment was repeated. MAC-2 was diluted to 2 mg/ml and analyzed as an intact conjugated protein by size exclusion chromatography-mass spectrometry (SEC-MS) to determine the number and quantitation of conjugate forms of the protein. This technique measures the molecular weight of each protein form; multiple conjugation sites are observed as distinct signals separated by the mass difference of a conjugated peptide/linker. Relative quantitation of multiple conjugation species is performed by measuring the signal magnitude. FIG. 7A shows a representative spectrum of MAC-2; the calculations used for quantitation are shown in Table 20. The average conjugation addition for the intact MAC-2 is calculated as 2.11 using the following formula: SUMPRODUCT (Number of Conjugation Additions (CA), Percent per CA). This example demonstrates conjugation of peptides occurring as a distribution between 0-4 peptide additions with the largest form being 2 peptide additions and the average number of peptide additions is 2.11. Replicate analysis by multiple individuals demonstrates that the profile of conjugation is consistent and reproducible.







TABLE 20







Weighted average of conjugation additions: 2.11.










Conjugation additions
Predicted mass
Intensity
Percent













0
149210
1615
 1%


1
152350
20533
17%


2
155490
69395
56%


3
158630
27708
22%


4
161770
4818
 4%




124069
100% 









The extent of peptide conjugation was examined separately on the light and heavy chains of 2.12.1.fx. MAC-2 was denatured and disulfide bonds were reduced using guanidine hydrochloride and dithiothreitol. The resulting free light and heavy chains were analyzed using LCMS to determine the conjugation profile on each. FIGS. 7B and 7C show a representative spectrum of each chain; the calculation used for quantitation are shown in Table 21. The average conjugation additions (Avg CA) for the reduced heavy chain MAC-2 is calculated as 0.14 and the Avg CA for the reduced light chain MAC-2 is calculated at 0.86 using the following formula: SUMPRODUCT (Number of Conjugation Additions (CA), Percent per CA). These data demonstrate that the location of conjugation is higher on the light chain; the most abundant form on the light chain contains one peptide addition and the light chain contains an average of 0.86 peptide additions. Conjugation on the heavy chain is observed at a significantly lower level. Replicate analysis of this experiment by multiple individuals demonstrates that the profile of conjugation is consistent and reproducible.







TABLE 21







Peptide mapping characterization of MAC-2 identifying


specific location of conjugation.












Mass
Conj.






(Da)
Additions
Species
Intensity
Percent
Avg CA















51020
0
HC
102093
86%



54165
1
HC + (1x) ABP-1
16204
14%




Total HC
118297
100% 
0.14


23584
0
LC
19752
21%


26729
1
LC + (1x) ABP-1
68757
72%


29874
2
LC + (2x) ABP-2
6561
 7%




Total LC
95070
100% 
0.86









MAC-2 was reduced with dithiothreitol and cysteine residues were alkylated by carboxymethylation with iodoacetamide. Chymotrypsin was used for proteolytic digestion. Digested fragments in solution were analyzed using liquid chromatography mass spectrometry (LCMS). Individual fragments were separated over a C18 HPLC column and their accurate mass is measured in a Quadrupole Time-of-Flight (Q-ToF) mass spectrometer. The resulting fragment mass was used to identify unmodified fragments or fragments modified with a conjugated peptide. This experiment was interpreted by focusing on chymotryptic fragments that contain a lysine residue, as these were possible sites for peptide conjugation. Table 22 shows a listing of all such fragments. Blank entries are fragments that are not detected using this technique. Detected fragments that are observed with a peptide modifier are considered potential sites of conjugation.
The table entries for Table 17 are explained below:
Fragment number: Chymotrypsin fragment numbering from the N-terminus; joined fragments (i.e. Y1-2) indicate a missed cleavage site.
Start/End: Numbering of the fragment location from the N-terminus.
Peptide Mass (Da): Theoretical mass of the fragment listed in Daltons.
Retention Time (Control/Analyte): Time of chromatographic retention/elution in the LCMS fragment mapping experiment.
MS Signal Intensity (Control/Analyte): Magnitude of observed signal observed by MS.
Mass Error-ppm (Control/Analyte): Comparison of theoretical vs. observed mass of the fragment; values >10, and especially closer to zero (0) demonstrate better mass accuracy.
Modifiers: Potential covalent additions to the fragment; peptide-antibody binding fragment of Lys residue, CAM-carboxymethylation of Cysteine residue.
Asterisks indicate the modified (e.g. conjugated) version of the respective fragment. Pep indicates a conjugated peptide.
Directional conjugation of a peptide to the Y15 fragment is demonstrated by quantitating the conjugation level. The following analysis was performed on each of the peptide fragments that were observed having conjugation during the peptide mapping experiment of the 2.12.1.fx reference product. The ratio of observed signal intensity for the unmodified peptide in the non-conjugated control (2.12.1.fx antibody scaffold—no conjugation) compared to the conjugated reference product (MAC-2) is shown in Table 23. The unmodified signal is used because a direct comparison of the same peptide signal is possible in each sample. For example, an unconjugated peptide would be expected to have the same observed signal intensity in the control vs. product samples resulting in a ratio of one (1). Conjugation would result in a decrease in the observed amount of unmodified peptide in the product sample which would be indicated by a ratio greater than one (1). The data in Table 23 was further normalized to correct for sample and experimental variation between the control and product. Table 23 demonstrates that light chain peptide Y15 is conjugated at a significantly higher level than each of the other conjugated peptides. This suggests that conjugation occurs in a directional manner and is not randomly distributed across K residues.







TABLE 22







Peptide mapping characterization of MAC-2 heavy chain reference product.










Peptide













Fragment
Mass
Retention Time
MS Signal Intensity
Mass Error (ppm)


















Number
Start
End
(Da)
Control
Analyte
Control
Analyte
Control
Analyte
Modifiers




















Y1
1
27
2617.3533









Y1-2
1
29
2865.4695


Y5-6
34
47
1657.8398


Y6
37
47
1253.688
19.2
19.2
516640
583534
1.9
−1.1


Y6-7
37
50
1602.8518
22.1
22.1
26537
37988
−1.6
−2.2


Y6-7*
37
50
3295.7017

21.8

6316

−19.4
Pep(1)


Y8-9
51
68
1931.9337
16.5
16.5
60894
85742
−2.2
0.4


Y9
61
68
878.461
11.3
11.3
376224
412997
0
−1


Y9-10
61
80
2241.1501


Y10
69
80
1380.6997
13.3
13.3
261813
299847
−1.1
0.7


Y10*
69
80
3073.5498

23.4

6350

−8.7
Pep (1)


Y10-011
69
94
2972.4661


Y19-20
111
157
4748.2773


Y20
116
157
4160.0405


Y20-21
116
166
5202.5527


Y20-21*
116
166
5316.5957
34.1

6445

0.5

CAM(2)


Y24-25
202
245
4702.2109


Y25
207
245
4151.9722


Y25*
207
245
4437.0796
20.9
20.9
1495322
1800079
1.1
−3.1
CAM(5)


Y25*
207
245
6129.9297

24.4

6652

−4.5
CAM(5) Pep(1)


Y25-26
207
279
7985.9092


Y26
246
279
3851.9478


Y26-27
246
281
4152.0698


Y28-29
282
300
2245.1128


Y29
283
300
2082.0493
14.6
14.6
20665
16662
−0.6
−3.8


Y29-30
283
304
2531.2405


Y31-32
305
323
2241.1907


Y32
318
323
722.3599
7.9
7.9
93966
96639
0.1
2.6


Y32
318
323
722.3599
17.7
18.4
37943
12802
11.4
30.6


Y32
318
323
722.3599
18.4

11761

23.8


Y32-33
318
353
4028.188


Y33
324
353
3323.8386
20

5422

3.1


Y33*
324
353
3380.8601
19.7
19.7
2196329
2497507
−2.5
−3.1
CAM(1)


Y33*
324
353
5073.71

24

5973

1.3
CAM(1) Pep(1)


Y33-34
324
376
5883.1577


Y34
354
376
2577.3293


Y34-35
354
385
3637.8159


Y34-35*
354
385
3694.8374
33
32.9
10095
20682
1.9
−2.4
CAM(1)


Y36-37
386
408
2527.0808


Y37
396
408
1394.6388
19.6
19.6
62942
71902
−0.9
−0.4


Y37-38
396
409
1541.7072
25.1
25.1
827336
878570
0
−1.9


Y37-38*
396
409
3234.5571

29.7

7749

−5.3
Pep(1)


Y39-40
410
421
1494.8195


Y40
412
421
1218.672
15.8
15.8
77917
88243
−0.3
−1.6


Y40-41
412
427
1891.9905
20.3
20.3
107513
149676
0.2
−2


Y42-43
428
450
2525.1792


Y43
441
450
1016.5502


Y2-3
36
49
1688.9725
16.2
16.2
145374
170451
−1.7
−2.6


Y2-3*
36
49
3381.8225

24.2

7192

−9.2
Pep(1)


Y3
37
49
1525.9093
15.5
15.5
331068
393638
−2.7
−2.9


Y3*
37
49
3218.7593

24

28193

−9
Pep(1)


Y3-4
37
62
2882.6355


Y9-10
88
116
3244.729


Y10
99
116
1871.0992


Y10-11
99
139
4331.335


Y11
117
139
2478.2463

22.8

47035

−5.9


Y11-12
117
148
3635.8445


Y12
140
148
1175.6088


Y12-13
140
173
3886.8245


Y13
149
173
2729.2263
13.1
13.1
1140556
1218022
−1.1
0.1


Y13*
149
173
4422.0762

21.4

8424

−6.5
Pep(1)


Y13-14
149
186
4095.9243


Y14
174
186
1384.7086


Y14-15
174
192
2169.1318


Y15
187
192
802.4337
7.5
7.5
275639
62720
−1.9
−0.2


Y15*
187
192
2495.2837

20.9

936267

−9.8
Pep(1)


Y15-16
187
209
2574.29


Y16
193
209
1789.8668
18.7

5400

4.4


Y16*
193
209
1846.8883
18.1
18.1
169490
235914
−1.7
−2.5
CAM(1)


Y16-17
193
214
2349.0842
17.8

9211

0.1
















TABLE 23







Directional conjugation of peptide


to Y15 fragment on the light chain.











Unmodified Intensity




Ratio: Control/Analyte-



Fragment
normalized














Light Y3 
1.000



Light Y13
1.112



Light Y15
5.218



Heavy Y6 
0.831



Heavy Y10
1.038



Heavy Y25
0.988



Heavy Y33
1.045



Heavy Y37
1.120










Example 7 Demonstration of Potency of MAC Products
Full details of in vitro and in vivo assays of MAC-1 and MAC-2 are provided in the Examples of PCT/US2011/053092 (WO2012/007896). Ang2-h38C2-IgG1 was used as a control in certain examples. The generation and structure of the Ang2-h38C2 is fully described as compound 43 in WO2008056346, whose contents is incorporated herein, with particular reference to aspects referring to the generation of compound 43. Briefly, the structure is as follows:




wherein the linker is covalently attached to the ε-amino group of HC-K99 (K93 according to Kabat numbering) of the combining site of the antibody and the antibody is h38C2-IgG1 (SEQ ID NO:64 and 65) (SEQ ID NO:189 and SEQ ID NO:190 of WO2008/056346).

In summary, MAC-1 and MAC-2 were able to bind Ang2 and prevent its binding to Tie2 as shown in an Ang2 competition assay, and both MAC-1 and MAC-2 have similar activity as the parental anti-IGF1R antibody (2.12.1.fx) for competing with IGF1 for IGF1R binding (Table 24). Surprisingly, in comparison with Ang2-h38c2, MAC-1 and MAC-2 both showed an increase in ability to competitively bind Ang2. Therefore, conjugation of limited Ang2 peptides does not appear to change the innate binding and inhibition of the antibody, and may in some cases improve the Effector Moiety activity.
The MACs were tested for the ability to downregulate IGF1R levels on a human colon carcinoma cell line Colo205. Cells were treated for 3 hrs in culture with titration of MAC compounds. Cells were collected and IGF1R surface expression determined by flow cytometry. The percentage of IGF1R downregulated as compared to negative control hIgG2 was determined (Table 23).







TABLE 24







Ability of MAC-1 and MAC-2 to


bind & modulate IGF1R and Ang2.














IGF1R
% IGF1R



Ang2
IGF1R
phosphorylation
down-



IC50 (nM)
IC50 (nM)
IC50 (nM)
regulated















MAC-1
0.092 ± 0.049
5.1 ± 1.1
150.7 ± 59.6
43 ± 5


MAC-2
0.057 ± 0.022
6.1 ± 1.1
 91.4 ± 40.2
50 ± 5


2.12.1.fx
nd
3.8 ± 0.8
 48.7 ± 14.0
48 ± 3


antibody


Ang2-h38c2-
0.582 ± 0.242
nd
nd
nd


IgG1









It was also demonstrated that conjugating 2 peptides per antibody was ideal in terms of effecting IGF1R autophosphorylation and downregulation and that conjugating more or less than 2 peptides per antibody lessens the ability of the MAC to effect these functions.
To assess the effect of the number of peptides per antibody on the ability of 2.12.1.fx to modulate IGF1R activity, 2 samples of MAC-1 were prepared where the reaction conditions were set to provide either reduced conjugation (MAC-1 low) or increased conjugation (MAC-1 high) (Table 25). The samples were analysed for the ability to downregulate and phosphorylate IGF1R (Table 25). There is a significant difference in the ability of the MAC-1 high as compared with MAC-1 low to effectively modulate the IGF1R pathway. Conjugation of greater than about 2 peptides per antibody limits the functional activity of the MAC to both inhibit IGF1R autophosphorylation and induce IGF1R downregulation, compared to conjugation of about 2 or less peptides per antibody. Therefore, in order to efficiently modulate 2 different biological pathways in one bifunctional entity, conjugation of about 2 peptides per antibody may be ideal (depending on peptide's and target's pharmacokinetic profile).







TABLE 25







Analysis of MAC-1-High and MAC-1 Low.




















Phosphory-










Ang2
% IGF1R
lation




















IC50
down-
IGF1R
CA (%)
Avg


















(nM)
regulated
IC50 (nM)
0
1
2
3
4
5
CA




















MAC-1 
0.103
32 ± 1
12.8
14
42
32
12
0
0
1.42


Low












MAC-1 
0.035
 9 ± 2
>300
0
4
19
41
32
5
3.18


High












2.12.1.fx
nd
36 ± 3
3.5









Ang2-
0.252
nd
nd









h38c2-












IgG1









Example 8 In Vivo Pharmacokinetics
PK studies were conducted using male Swiss Webster mice and 2 male Cynomolgus monkeys (Macaca fascicularis). Full details of PK studies are provided in the Examples of PCT/US2011/053092. In mouse, MAC-1 and MAC-2 demonstrated similar residence time as the parental anti-IGF1R antibody with 13 phase half-lives of 383-397 hrs. The MAC-1 and MAC-2 Ang2 binding capability demonstrated similar residence time as Ang2-h38c2 with T1⁄2 of 105-120 hrs in mouse in single dose IV studies. In cynomolgus monkey, MAC-2 demonstrated a slightly shorter residence time as the parental anti-IGF1R antibody with T1⁄2 of 100.4 hrs. The MAC-2 Ang2 binding capability demonstrated similar residence time as Ang2-h38c2 with T1⁄2 of 97.8 hrs.







TABLE 26







Single-dose PK of IV administered MACs at 10 mg/mkg


in mouse and cynomolgus monkey. α-IGF1R antibody


dosed at 10 mg/kg in mouse, and 5 mg/kg in monkey.










Mouse β-T 1⁄2 (hr)
Monkey T 1⁄2 (hr)











Compound (mg · Kg−1)
Ang2
IGF1R
Ang2
IGF1R














Ang2-h38c2, (10)
95.2
—
95.3
—


α-IGF1R antibody, (10), (5)
—
390
—
146.4


MAC-1, (10)
105
383
NT
NT


MAC-2, (10)
120
397
97.8
100.4





NT: not tested.






Example 9 In Vivo Pharmacology
The anti-tumour activity of MAC-2 was evaluated in the Colo205 (human colon adenocarcinoma) or MDA-MB-435 (melanoma) xenograft model. Full details of tumour studies are provided in the Examples of PCT/US2011/053092 (WO2012/007896). Weekly administration of Ang2-h38c2 or anti-IGF1R antibody (2.12.1.fx) inhibited Colo205 tumour growth. Combination of weekly administered Ang2-h38c2 and anti-IGF1R antibody showed an additive benefit on inhibiting Colo205 tumour growth. Weekly administration of MAC-2 alone showed similar benefit as the combination. In a separate study, MAC-2 dose-dependently inhibited Colo205 tumour growth and final tumour weights.
At day 28, tumour microvessel density after compound treated was significantly reduced ( ̃42%) by MAC-2 (10 mg/kg, once weekly) in comparison with the Vehicle-treated group confirming the anti-angiogenic activity of the MAC-2 treatment.
To investigate whether MAC-2 targets both Ang2 and IGF1R in vivo, the effects of MAC-2 on Ang2 and IGF1R expression levels were assessed in 2 independent Colo205 xenograft tumors treated with Vehicle, Ang2-h38c2, IGF1R antibody (2.12.1.fx) or MAC-2 (dose response ranging from 0.3 mg/kg to 10 mg/kg). The results showed that Ang2 and IGF1R immunoreactivity was significantly reduced by MAC-2 treatment in a dose-dependent manner (1, 3 and 10 mg/kg) in comparison with the Vehicle-treated group. The effect of MAC-2 on IGF1R levels was similar to that observed for an IGF1R antagonizing antibody. In addition, the levels of phosphorylated IGF1R were reduced in tumours from MAC-2 treated animals. These data demonstrate that MAC-2 treatment affects both Ang2 and IGF1R pathways in Colo205 xenograft model. MAC-2 treatment did not affect body weight gain and mice appeared to be in good health throughout the studies. The anti-tumor efficacy of MAC-2 was also evaluated in an MDA-MB-435 melanoma xenograft model. Weekly administration of MAC-2 (3 and 20 mg/kg IP) resulted in a significant 40% reduction (day 67) in tumor growth in the MDA-MB-435 model. Thus, MAC-2 demonstrates significant anti-tumor efficacy in 2 different human xenograft tumor models.
Example 10 Peptide Conjugation Profile of Various Antibodies
The conjugation profiles of several different antibodies with peptides were analyzed, using SEQ ID NO:27 and PEG5 as an exemplary peptide and linker respectively. All antibodies tested were human or fully humanized IgG antibodies with well defined and characterized antigen interactions. hAbλTest comprises a CLλ (hIL22: SEQ ID NOs:136 and 137), whereas 2.12.1.fx, mAbκTest1 (an IgG2 anti-Alk1 antibody, as disclosed in U.S. Pat. No. 7,537,762, incorporated herein by reference), h38C2-IgG1 (SEQ ID NO:64 and 65) and h38C2-IgG2 (SEQ ID NO:64 and 66) each comprise CLκ. Each of the antibodies were buffer exchanged into 20 mM HEPES, pH 7.0 and concentrated to 5-20 mg/mL. [PFP-PEG5-K11-SEQ:27] was resuspended with 50% propylene glycol and mixed with the relevant antibody at a 4.3:1 molar ratio and allowed to react for at least 2 hrs at RT. All samples were diluted to 2 mg/ml and analyzed as an intact conjugated protein by size exclusion chromatography-mass spectrometry (SEC-MS) to determine the number and quantitation of conjugate forms of the protein. This technique measures the molecular weight of each protein form; multiple peptide conjugation sites are observed as distinct signals separated by the mass difference of a bound peptide. Relative quantitation of multiple peptide conjugation species is performed by measuring the signal magnitude. Table 22 shows the peptide conjugation profile of various antibodies
For antibodies containing a CLκ, peptide conjugation occurs at a distribution between 0-4 peptide additions with the largest form being 2 to 3 peptide additions. In contrast, for the CLλ comprising antibody, hAbλTest, conjugation of the peptide occurs at a distribution between 0-4 peptides additions with the largest form being 1 to 2 peptide additions.
The extent of peptide conjugation was examined separately on the light and heavy chains. Each sample was denatured and disulfide bonds were reduced using guanidine hydrochloride and dithiothreitol. The resulting free light and heavy chains were analyzed using LCMS to determine the conjugation profile on each. The peptide conjugation profile on the light and heavy chain of various antibodies is shown in Table 27. On 2.12.1.fx and hAbκTest1, the data demonstrate that the location of conjugation is higher on the light chain; the most abundant form on the light chain contains 1 peptide addition. Conjugation on the heavy chain is observed at a significantly lower level. On h38C2-IgG1 and h38C2-IgG2, comparable levels of conjugation are observed on the light and heavy chain, with a slight conjugation preference on the light chain. On a CLλ containing antibody (hAbλTest; comprising SEQ ID NOs:136 and 137)), the majority of the conjugation occurs on the heavy chain with a low level of conjugation observed on the light chain.







TABLE 27







Conjugation profile of various antibodies.






















Light Chain
Heavy chain 












CA (%)
Avg
% CA
% CA



















Antibody
0
1
2
3
4
CA
0
1
2
0
1
2






















2.12.1.fx
1
15
53
26
5
2.2
20
70
9
84
16
0


hAbλTest
10
37
37
11
6
1.66
95
5
0
74
22
4


hAbκTest1
7
10
35
27
14
2.55
11
74
14
87
13
0


h38C2 IgG1
1
3
28
55
13
2.75
49
46
4
70
30
0


h38C2 IgG2

4
6
31
44
2.6
61
35
4
73
27
0









Each of the antibodies 2.12.1.fx, hAbλTest and hAbκTest1 was assessed after the conjugation process to determine the effect of the conjugation additions on the ability of the antibody scaffold to retain its receptor binding (compared to native mAb) (Table 28). The results show that the directional conjugation of peptides to the test antibodies did not appear to alter the antibody binding.








TABLE 28







Antibody binding to respective native


antigen before and after conjugation.










Antigen binding










Antibody
Native (IC50, nM)
After conjugation (IC50, nM)












2.12.1.fx
3.2
5.7


hAbλTest
0.4
1.7


hAbκTest1
59
53









Example 11 Peptide Conjugation Profile of an IgG2-κ Antibody
The conjugation profile of an IgG2 κ antibody (hABκTest2) with a 39-mer peptide was analyzed (SEQ ID NO:164). The antibody was concentrated to 8 mg/mL and buffered exchanged into 40 mM HEPES pH 8.0. The peptide was resuspended with 100% DMSO and mixed with the antibody at a 5.0:1 molar ratio and allowed to react overnight at room temperature. All samples were diluted to 2 mg/ml and analyzed as an intact conjugated protein by size exclusion chromatography-mass spectrometry (SEC-MS) to determine the number and quantitation of conjugate forms of the protein. This technique measures the molecular weight of each protein form; multiple peptide conjugation sites are observed as distinct signals separated by the mass difference of a peptide. Relative quantitation of multiple peptide conjugation species is performed by measuring the signal magnitude. Table 29 shows the peptide conjugation profile of hAbκTest2 with the 39-mer peptide. The conjugation of peptide occurs at a distribution between 0-4 CA with an average of 2.03 CA, and is consistent with directional conjugation on the CLκ-K80.







TABLE 29







Conjugation profile of 39-mer peptide and hAbκTest2.










Antibody

% CA
Avg














scaffold
Binding Peptide
0
1
2
3
4
CA

















hAbκTest2
39-mer peptide
1
22
53
18
5
2.03









In a separate experiment, the 39-mer peptide was conjugated to h38C2-IgG2 with MAL-PEG2-PFP as described above, at different molar concentrations. In addition, binding of the cognate receptor for the 39-mer peptide was assayed. The results (Table 30) shown are consistent with directional conjugation at CLκ-K80. Moreover, increasing the average number of peptides per antibody did not substantially increase overall binding to the target. This demonstrates that in certain scenarios, increasing the conjugation per antibody may not increase target binding, demonstrating one of the advantages of the invention; control of the number of peptides conjugating per antibody can help achieve the maximum target binding per unit peptide.







TABLE 30







Conjugation profile of 39-mer peptide and H38C2-IgG2.










39-mer peptide:
CA (%)
Avg #
Peptide target:













h38C2-IgG2 mole ratio
0
1
2
3
Conjugates
EC50 (nM)
















  2:1
57
32
10
0
0.52
0.99


2.5:1
19
56
25
0
1.06
1.06


  4:1
20
25
35
20
1.55
1.01


  5:1
0
16
45
40
2.26
0.82









Example 12 Conjugation of Biotin to 2.12.1.fx Fab
The conjugation profile of the Fab region of 2.12.1.fx (SEQ ID NOs:4 and 64) with PFP-Biotin was analyzed. The antibody Fab was concentrated to 20 mg/mL and buffered exchanged into 20 mM sodium acetate+200 mM trehalose, pH 5.5 and spiked with 60 mM sodium phosphate pH 7.7. PFP-Biotin was resuspended with 100% DMSO and mixed with the antibody at successive molar ratios and allowed to react overnight at room temperature. All samples were diluted to 2 mg/ml and analyzed as an intact conjugated protein by size exclusion chromatography-mass spectrometry (SEC-MS) to determine the number and quantitation of conjugate forms. This technique measures the molecular weight of each protein form; multiple conjugation sites are observed as distinct signals separated by the mass difference of a conjugated peptide. Relative quantitation of multiple conjugation species is performed by measuring the signal magnitude. Table 31 shows the conjugation profile of 2.12.1.fx Fab with PFP-Biotin at molar ratios. The conjugation of occurs at a distribution between 0-2 additions as the molar ratio increases. The lower number of molecules per antibody was consistent with earlier results, based on the molar ratio used. This demonstrates the flexibility of the process to control the amount of conjugation by altering reaction parameters.







TABLE 31







Conjugation profile of Biotin to 2.12.1.fx Fab.








 


Biotin-PFP


 










Binding
Peptide:Antibody
% CA
Avg













Peptide
Molar Ratio
0
1
2
3
CA





Biotin-PFP
1:1
54
46
—
—
0.46


Biotin-PFP
1.5:1  
42
51
 7
—
0.65


Biotin-PFP
2:1
34
55
10
—
0.76


Biotin-PFP
3:1
28
55
17
—
0.88


Biotin-PFP
4:1
21
46
26
8
1.21









Example 12 Conjugation of Biotin to h38C2-IgG1
The antibody h38C2-IgG1 was adjusted to 20 mg/mL with HEPES buffer pH 7.5 to a final concentration of 0.02 M. Biotin-PFP was reconstituted in water to 10 mg/mL and added to h38C2-IgG1 at a molar ratio of 5:1 and allowed to react at room temperature for 2 hrs. The unreacted PFP-Biotin was removed by size exclusion chromatography and buffer exchanged into a histidine, glycine, and sucrose buffer pH 6.5. The samples were diluted to 2 mg/ml and analyzed as an intact conjugated protein by size exclusion chromatography-mass spectrometry (SEC-MS) to determine the number and quantitation of conjugate forms of the protein. Table 32 shows the conjugation profile of h38C2-IgG1 with Biotin-PFP. Conjugation of h38C2-IgG1 occurs at a distribution between 0-3 CA with an average of 1.1 conjugations. Increased conjugation would be possible following optimization of the reaction conditions. The reactivity of VH-K99 (K93 according to Kabat numbering) on h38C2-IgG1 was confirmed to be >95% when reacted with the catalytic antibody test compound CATC-1, and analyzed via reversed phase chromatography.







TABLE 32







Conjugation of Biotin and h38C2-IgG1.








 


CATC-1


 












Antibody
0
1
2
3
Avg CA





h38C2-IgG1
16
61
20
3
1.1









Example 13 Conjugation Profile of 2.12.1.fx and CLκ-K80, CLκ-K82 Mutants
Based on peptide mapping, there are 2 Lys in Y15 fragment. In order to distinguish the active conjugation site, CLκ-K80 and CLκ-K82 were mutated to R respectively or in combination. Mutants of the test antibody, 2.12.1.fx, were generated following protocols described in QuickChange site-directed mutagenesis kit (Stratagene®). Mutations were introduced by oligonucleotide primers and confirmed by DNA sequencing. The mutated mAbs were transiently expressed in HEK 293 cells, and purified using Protein A affinity column. The purified mAbs were characterized using MS. SEQ ID NOs:12, 13 and 14 show the mutant CLκ sequences.
The antibody was buffer exchanged to 0.02M HEPES buffer pH 7.5 or 6.5 at 2 mg/mL. If the pH was 6.5, the antibody was then spiked with 60 mM sodium phosphate pH 7.7. [PFP-PEG5-K11-SEQ:27] was resuspended with 50% propylene glycol and mixed with the protein at a 4.3:1 molar ratio and allowed to react overnight at RT. All samples were diluted to 2 mg/ml and analyzed as an intact conjugated protein by size exclusion chromatography—mass spectrometry (SEC-MS) to determine the number and quantitation of conjugate forms of the protein. This technique measures the molecular weight of each protein form; multiple conjugation sites are observed as distinct signals separated by the mass difference of a conjugated protein. Relative quantitation of multiple protein conjugation species is performed by measuring the signal magnitude. Table 33 shows the conjugation profile of unmodified 2.12.1.fx, 2.12.1.fx-[CLκ-K80R] (CLκ: SEQ ID NO:12), 2.12.1.fx-[CLκ-K82R] (CLκ: SEQ ID NO:13), and 2.12.1.fx-[CLκ-K80R-K82R] (CLκ: SEQ ID NO:14). CLκ-K80R mutant showed reduced conjugation. CLκ-K82R had similar conjugation as the unconjugated 2.12.1.fx. The conjugation of MAC-2 was lower than observed in other assays due using a combination HEPES/phosphate buffer.







TABLE 33







Conjugation profile of 2.12.1.fx, K80 and R82 mutants.












CA (%)















LC SEQ ID NO:
Mutants
0
1
2
3
4
Avg CA

















 6
MAC-2
14
49
31
5
1
1.29


12
K80R
82
14
4
0
0
0.22


13
K82R
11
46
36
6
0
1.37


14
K80R/K82R
51
37
9
3
0
0.63









Example 14 Elucidation of Directional Conjugation Mechanism on K80 
CLκ-H81 side chain is very close to the ε-amino group of CLκ-K80. Since His is often involved in proton transfer reactions, CLκ-H81 is very likely required for CLκ-K80 conjugation. In order to study the role of CLκ-H81 in CLκ-K80 site specific conjugation, the imidazole ring was eliminated by a CLκ-H81A mutation. CLκ-D43A and CLκ-D43A/H81A mutants were made to study the role of CLκ-D43 in site specific conjugation and the combined effect of CLκ-D43 and CLκ-H81.
Mutants were generated following protocols described in QuickChange site-directed mutagenesis kit (Stratagene®). Mutations were introduced by oligonucleotide primers and confirmed by DNA sequencing. The mutated mAbs were transiently expressed in HEK 293 cells, and purified using protein A affinity column. The purified mAbs were characterized using MS. The following 2.12.1.fx I CLκ mutants were generated: CLκ-D43A (SEQ ID NO:15), CLκ-K80A (SEQ ID NO:16), CLκ-H81A (SEQ ID NO:17), CLκ-K82A (SEQ ID NO:18) and CLκ-D43A/H81A (SEQ ID NO:19).
Each of the antibodies was buffer exchanged to 20 mM sodium acetate, 200m trehalose pH 5.5 at 20 mg/ml. The proteins were then spiked with 60 mM sodium phosphate pH 7.7. [PFP-PEG5-K11-SEQ:27] was resuspended with 50% propylene glycol and mixed with the antibody at a 4.3:1 molar ratio and allowed to react overnight at room temperature. All samples were diluted to 2 mg/ml and analyzed as an intact conjugated protein by size exclusion chromatography-mass spectrometry (SEC-MS) to determine the number and quantitation of conjugate forms of the protein. This technique measures the molecular weight of each protein form; multiple conjugation sites are observed as distinct signals separated by the mass difference of a conjugated peptide. Relative quantitation of multiple conjugation species is performed by measuring the signal magnitude.
Table 34 shows the conjugation profile of 2.12.1.fx, 2.12.1.fx-[CLκ-D43A], 2.12.1.fx-[CLκ-K80A], 2.12.1.fx-[CLκ-H81A], 2.12.1.fx-[CLκ-K82A], and 2.12.1.fx-[CLκ-D43A/H81A] mutants. All the mutants showed reduced average conjugation level compared to the unmodified 2.12.1.fx antibody, except for CLκ-K80A, which maintained directional conjugation.
The extent of conjugation was examined separately on the light and heavy chains. Each sample was denatured and disulfide bonds were reduced using guanidine hydrochloride and dithiothreitol. The resulting free light and heavy chains were analyzed using LCMS to determine the conjugation profile on each. The conjugation profile on the light and heavy chain of 2.12.1.fx and mutants are shown in Table 34. All the mutants listed in the table showed reduced conjugation level on light chain compared to the unmodified 2.12.1.fx except CLκ-K80A. The heavy chain conjugation level of the mutants was at the similar level as the unmodified 2.12.1.fx. The % of 1-LC % relative to the respective WT run is shown in the right column, as described in Table 34.







TABLE 34







Conjugation profile of MAC-2 and K80A, D43 and H81 mutants.















LC SEQ

CA (%)
Avg
LC CA%
LC Avg
HC CA %
HC Avg
1 LC























ID NO:
Mutants
0
1
2
3
4
CA
0
1
2
CA-LC
0
1
2
CA-HC
WT %


























 6
MAC-2
1
15
53
26
5
2.2
23
69
8
0.85
86
14
0
0.14



15
D43A
17
38
31
14
0
1.41
68
30
1
0.33
79
21
0
0.21
43


16
K80A
56
31
10
4
0
0.61
89
11
0
0.11
91
9
0
0.09
16


17
H81A
34
44
17
6
0
0.95
89
11
0
0.11
78
22
0
0.22
16


18
K82A
9
7
31
37
16
2.42
8
77
15
1.06
83
17
0
0.17
111


19
D43A/
34
39
18
9
0
1.02
83
17
0
0.17
87
13
0
0.13
25



H81A









Example 15 Lambda/Kappa Substitution
The CLλ in hAbλTest1 (SEQ ID NOs:136 and 137) was substituted with CLκ□ to determine whether this increased the level, directionality and/or control of CL-specific conjugation. The CLλ/CLκ□ domain substitution hybrid constructs were generated using overlap PCR. The VLλ and CLκ were PCR amplified using hAbλTest and a κ mAb light chain as templates separately. These 2 PCR products were mixed as templates; hAbλTest1 forward primer and LCLκ□ reverse primer were used in overlap PCR reaction to amplify the full length hAbλTestVL/CLκ□ DNA. The hybrid antibody constructs were transiently expressed in HEK 293 cells, and purified using Protein A affinity column. The purified antibodies were characterized using MS. The hAbλTest CLκ□ hybrid bound to its cognate ligand similarly to the native mAb (hAbλTest) (Table 35). SEQ ID NOs:59, 60 and 61 are the light chain constant regions from hAbλTest, hAbλTest-λκ (with λJ), and hAbλTest-λκJ (with κJ).







TABLE 35







Antibody: Antigen binding of lambda/Kappa substitution.










LC SEQ
Inhibition of IL22 binding to


hAbλTest1 Mutants
ID NO:
antigen (IC50, nM)





hAbλTest (CONTROL)
59
0.4


hAλTest-λκ
60
0.3


hAbλTest-λκJ
61
0.3









Example 16 hAbλTest1 Mutants: Motif Modification
To establish whether the short motif “KH” was sufficient for MAC formation in the corresponding region of the CLλ, a mutant with simple sequence switch of residues CLλ81/82 in hAbλTest to place a histidine beside K80 was made, hence “K80S81H82” became “K80H81S82”. Mutants were generated following protocols described in QuickChange site-directed mutagenesis kit (Stratagene). Mutations were introduced by oligonucleotide primers and confirmed by DNA sequencing. The mutated antibody constructs were transiently expressed in HEK 293 cells, and purified using Protein A affinity column. The purified antibodies were characterized using MS. The hAbλTest-[CLλ-S81H/H82S] (CL: SEQ ID NO:62) mutant bound to its ligand as well as the parent hAbλTest antibody did (Table 36).







TABLE 36







hAbλTest-S81H/H82S.









hAbλTest1 Mutants
LC SEQ ID NO:
Ligand binding (IC50, nM)





hAbλTest (CONTROL)
59
0.3


hAbλTest-S81H/H82S
62
0.4









Example 17 Conjugation Profile of hAbλTest1 Mutants
Each antibody (hAbλTest, hAbλTest-AK, hAbλTest-λκJ and hAbλTest-[CLλ-S81H/H82S]) was buffer exchanged to 20 mM sodium acetate, 200m trehalose pH 5.5 at 20 mg/ml. The proteins were then spiked with 60 mM sodium phosphate pH 7.7. [PFP-PEG5-K11-SEQ:27] was resuspended with 50% propylene glycol and mixed with the antibody at a 4.3:1 molar ratio and allowed to react overnight at room temperature. All samples were diluted to 2 mg/ml and analyzed as an intact conjugated protein by size exclusion chromatography-mass spectrometry (SEC-MS) to determine the number and quantitation of conjugate forms of the protein. This technique measures the molecular weight of each protein form; multiple peptide conjugation sites are observed as distinct signals separated by the mass difference of a peptide. Relative quantitation of multiple peptide conjugation species is performed by measuring the signal magnitude. Table 37 shows the overall level of conjugation has been increased in the 2 LC-switched hybrids (λκ and λκJ—the former includes a λ J fragment, the latter includes a κ J fragment). The conjugation level increases over the hAbλTest control's average CA, going from 1.66 to 2.19 (λκ) and 2.53 (λκJ) respectively. The mutant had little effect compared to the native sequence, suggesting that “KH” motif alone is not sufficient for MAC formation.
The extent of peptide conjugation was examined separately on the light and heavy chains (Table 37). Each sample was denatured and disulfide bonds were reduced using guanidine hydrochloride and dithiothreitol. The resulting free light and heavy chains were analyzed using LCMS to determine the conjugation profile on each. In the reduced analyses, the LC of native hAbλTest has only 5% 1CA but this jumps dramatically to 58% 1CA for hAbλTest-λκ and 63% 1CA for hAbλTest-λκJ. The LC switch had little effect on the level of HC conjugation, which remained fairly constant (except for λκJ, where HC conjugation increased moderately). Again, the mutant had little effect compared to the native sequence, suggesting that “KH” motif alone is not sufficient for MAC formation. The % of 1-LC % relative to the respective WT run is shown in the right column, as described in Table 37.







TABLE 37







Conjugation profile of hAbATest mutants.
















LC 










SEQ



Avg

Avg



hAbλTest
ID
CA (%)
Avg
LC CA%
CA-
HC CA %
CA-
1LC























Mutants
NO:
0
1
2
3
4
CA
0
1
2
LC
0
1
2
HC
WT %


























hAbλTest
59
10
37
37
11
6
1.66
95
5
0
0.05
74
22
4
0.3



hAbλTest-
60
3
18
43
29
7
2.19
42
58
0
0.58
78
22
0
0.22
1160


λκ


















hAbλTest-
61
2
11
34
36
17
2.53
33
63
4
0.71
64
36
0
0.36
1260


λκJ


















hAbλTest-
62
7
34
37
16
6
1.79
82
18
0
0.18
79
21
0
0.21
360


S81H/H82S









The receptor binding attributes of these conjugated forms was also assessed to determine the effect of conjugation with [PFP-PEG5-K11-SEQ:27] on the ability of the conjugated antibodies to still bind to their ligand (Table 38).







TABLE 38







Antibody: Antigen binding of lambda at antibodies.









SEQ ID NO: 27 conjugated
LC SEQ
Inhibition of IL22 binding to


hAbλTest1 Mutants
ID NO:
antigen (IC50, nM)





hAbλTest
59
1.7


hAbλTest-λκ
60
1.5


hAbλTest-λκJ
61
1.6


hAbλTest1-S81H/H82S
62
1.6









Example 18 MAC Generation Using Different Leaving Groups
To investigate if the degree of activation and/or structure of the active ester leaving group was important in defining the directional conjugation effect, a series of alternatively activated ester analogs of [PFP-PEG2-MAL-KSH11-SEQ:27] were synthesized. The distribution of the conjugate product was examined by MS of the intact conjugates, and the degree of peptide addition to both the light and heavy chains were also determined by MS following reduction of the intact conjugate and separation of the light and heavy chains.
The structure and designations of the alternatively activated esters are shown below. The alternatively activated peptides were synthesized using the same strategies and methods shown above. Briefly, each activated group was incorporated into a MAL-PEG2-Z* linker, where Z* represented the new leaving group replacing PFP. To synthesize the above compounds, a sample (30-40 mg) of the purified ABP-thiol peptide (i.e. ABP with KSH as linking residue) was dissolved in anhydrous DMF (2 ml). MAL-PEG2-Z* (20 mg) was added along with N-methylmorpholine (5 mL). The reaction was stirred and monitored at RT by HPLC to follow the time-course of product formation. The complete conversion of starting peptide to activate-ester linked ABP product was observed within 2-6 hrs. The solution was filtered and the product peak directly isolated by semi-preparative HPLC. The products were isolated in yields ranging from approximately 30-50%, after lyophilization.
The conjugation reactions were carried out under the standard conditions. Briefly, the 2.12.1.fx antibody solution was prepared by diluting the 2.12.1.fx solution with sodium phosphate, pH 7.7 to a final concentration of 0.06M. Separately, the peptide solution was prepared by dissolving the peptide to 20 mg/ml in propylene glycol, then diluting this solution to 10 mg/ml with water. For the conjugation reaction, the peptide and antibody solutions were mixed at a 4:1 molar ratio for the prescribed period. For the time-course studies, samples of the conjugation solution were quenched at various time points by mixing a sample of the conjugation reaction with a solution of 40 mM succinic acid, 200 mM glycine, pH 4.0 (1:1, v/v). Time-course of the conjugation reactions were followed by HPLC. SEQ ID NO:27 was used as an exemplary peptide.







TABLE 39







Reactive esters - intact conjugation at 24 hrs.








 


Z*—PEG2—MAL—KSH11—SEQ: 27


 



























Z8
Z9







Z2
Z3
Z4
Z5
Z6
Z7
NH5
2HI

Z11
Z12



Z1
2,3,4
2,3,6
2,3,6
2,6
2,4
5,7
NB2, 3
1,3
Z10
2,6-
1


CA
PFP
TFP
TFP
TCP
DCP
DCN
DCQ
DCI
D
4NP
DFP
NAP





0
3
32
17
100
81
38
73
34
20
41
50
100


1
34
45
43
0
19
45
25
40
36
42
39
0


2
51
20
30
0
0
16
2
18
31
15
11
0


3
12
3
11
0
0
2
0
5
12
3
0
0









Table 39 shows the final product distribution of the intact conjugates 24 hrs after initiation of the conjugation reaction. The results show that some of esters did not react at all (Z4, Z12), others reacted sluggishly (e.g. Z5), while several gave profiles approaching that of PFP (Z1) (e.g. Z3).
Conjugation Kinetics
The rates of addition over time for each of the final conjugates are shown in Tables 39, 40, 41, and 42. OCA represents underivatized 2.12.1.fx antibody, whereas 1, 2 or 3CA represents additions of 1, 2 or 3 peptides to the 2.12.1.fx antibody at each of the time periods examined.







TABLE 40







Conjugation kinetics of different Z* groups yielding 0 CA.



























Z8






0CA

Z2
Z3
Z4
Z5
Z6
Z7
NH5






time
Z1
2,3,4 
2,3,6
2,3,6
2,6
2,4
5,7
NB2,3
Z9
Z10
Z11
Z12


(hr)
PFP
TFP
TFP
TCP
DCP
DCN
DCQ
DCI
2HI 1,3 D
4NP
2,6-DFP
1 nap






















 0
84
97
94
100
100
100
100
95
95
96
100
100


 1
5
83
58
100
100
95
96
43
24
79
93
100


 2
4
75
40
100
100
89
93
42
20
67
88
100


 4
4
62
27
100
96
81
88
40
20
54
79
100


24
3
32
17
100
81
38
73
34
20
41
50
100
















TABLE 41







Conjugation kinetics of different Z* groups yielding 1 CA.





















Z2
Z3
Z4
Z5
Z6
Z7
Z8
Z9

Z11



1CA
Z1
2,3,4
2,3,6
2,3,6 
2,6
2,4
5,7
NH5
2HI
Z10
2,6-
Z12


time (hr)
PFP
TFP
TFP
TCP
DCP
DCN
DCQ
NB2,3 DCI
1,3 D
4NP
DFP
1 nap






















 0
16
3
6
0
0
0
0
5
5
5
0
0


 1
38
17
36
0
0
5
4
39
39
21
8
0


 2
37
25
45
0
0
11
7
39
38
29
12
0


 4
33
34
43
0
4
19
12
42
39
37
21
0


24
34
45
43
0
19
45
25
40
36
42
39
0
















TABLE 42







Conjugtion kinetics of different Z* arouos yielding 2 CA.



























Z8








Z2
Z3
Z4
Z5
Z6
Z7
NH5
Z9





2CA
Z1
2,3,4
2,3,6
2,3,6
2,6
2,4
5,7
NB2,3
2HI
Z10
Z11
Z12


time (hr)
PFP
TFP
TFP
TCP
DCP
DCN
DCQ
DCI
1,3 D
4NP
2,6-DFP
1 nap






















 0
0
0
0
0
0
0
0
0
0
0
0
0


 1
49
0
6
0
0
0
0
15
27
0
0
0


 2
50
0
14
0
0
0
0
16
30
4
0
0


 4
52
4
25
0
0
0
0
15
29
9
0
0


24
51
20
30
0
0
16
2
18
31
15
11
0
















TABLE 43







Conjugation kinetics of different Z* groups yielding 3 CA.



























Z8








Z2
Z3
Z4
Z5
Z6
Z7
NH5
Z9





3CA
Z1
2,3,4
2,3,6
2,3,6
2,6
2,4
5,7
NB2,3
2HI
Z10
Z11
Z12


time (hr)
PFP
TFP
TFP
TCP
DCP
DCN
DCQ
DCI
1,3 D
4NP
2,6-DFP
1 nap






















 0
0
0
0
0
0
0
0
0
0
0
0
0


 1
8
0
0
0
0
0
0
3
11
0
0
0


 2
10
0
2
0
0
0
0
3
12
0
0
0


 4
12
0
5
0
0
0
0
4
12
0
0
0


24
12
3
11
0
0
2
0
5
12
3
0
0









Light and Heavy Chain Distribution

The extent of peptide conjugation for each of the alternatively activated esters was examined separately on the light and heavy chains. Each sample was denatured and disulfide bonds were reduced using guanidine hydrochloride and dithiothreitol. The resulting free light and heavy chains were analyzed using LCMS to determine the conjugation profile on each. The peptide conjugation profile on the light and heavy chain of 2.12.1.fx and mutants are shown in Table 44. Almost all of the activated peptides listed in the table showed reduced conjugation level on light chain compared to the compound using PFP (Z1), except 2,3,6-trifluorophenyl (Z3), which showed a similar level of conjugation. Activated esters derived from N-hydroxysuccinimide (NHS), i.e. N-Hydroxyl-5-norbornene-2,3-dicarboxylic acid imide and 2-hydroxyl-isoindoline-1,3-dione (Z8 and Z9) showed a greater propensity for heavy chain derivatization.







TABLE 44







Summary of activated ester results.











Z*
Z*
Z*
Time course of conjugation adducts
Reduced conjugation


#
Name
Structure
[separate 24 hr expt in bold]
at 24 hr
























 1
Penta Fluoro Phenyl




CA 0 1 2 3
 0  84  16  0  0
 1  5  38  49  8
 2  4  37  50  10
 4  4  33  52  12
 24  3  34  51  12
LC 30
LC + 1CA 64
L + 2CA 7
HC  94
HC + 1CA  6
HC +  2CA —


 


 2
2,3,4- trifluoro- phenyl




CA 0 1 2 3
 0  97  3  0  0
 1  83  17  0  0
 2  75  25  0  0
 4  62  34  4  0
 24  32  45  20  3
LC 59
LC + 1CA 41
L + 2CA —
HC  94
HC + 1CA  6
HC +  2CA —


 


 3
2,3,6- trifluoro- phenyl




CA 0 1 2 3
 0  94  6  0  0
 1  58  36  6  0
 2  40  45  14  2
 4  27  43  25  5
 24  17  43  30  11
LC 30
LC + 1CA 64
L + 2CA 7
HC  90
HC + 1CA 10
HC +  2CA —


 


 4
2,3,6- trichloro- phenyl




CA 0 1 2 3
 0 100  0  0  0
 1 100  0  0  0
 2 100  0  0  0
 4 100  0  0  0
 24 100  0  0  0
LC 95
LC + 1CA  5
L + 2CA —
HC 100
HC + 1CA —
HC +  2CA —


 


 5
2,6 dichloro- phenyl




CA 0 1 2 3
 0 100  0  0  0
 1 100  0  0  0
 2 100  0  0  0
 4  96  4  0  0
 24  81  19  0  0
LC 89
LC + 1CA 11
L + 2CA —
HC 100
HC + 1CA —
HC +  2CA —


 


 6
2,4 DiCl Napthalene




CA 0 1 2 3
 0 100  0  0  0
 1  95  5  0  0
 2  89  11  0  0
 4  81  19  0  0
 24  38  45  16  2
LC 66
LC + 1CA 34
L + 2CA —
HC  95
HC + 1CA  5
HC +  2CA —


 


 7
5,7- dichloro- quinolin- 8-yl




CA 0 1 2 3
 0 100  0  0  0
 1  96  4  0  0
 2  93  7  0  0
 4  88  12  0  0
 24  73  25  2  0
LC 92
LC + 1CA  8
L + 2CA —
HC  95
HC + 1CA  5
HC +  2CA —


 


 8
N- Hydroxyl- 5- norbornene- 2,3- dicarboxylic




CA 0 1 2 3
 0  95  5  0  0
 1  43  39  15  3
 2  42  39  16  3
 4  40  42  15  4
 24  38  40  18  5
LC 77
LC + 1CA 23
L + 2CA —
HC  82
HC + 1CA 18
HC +  2CA —



acid imide















 


 9
2- hydroxyl- isoindoline- 1,3-dione




CA 0 1 2 3
 0  95  5  0  0
 1  24  39  27  11
 2  20  38  30  12
 4  20  39  29  12
 24  20  36  31  12
LC 70
LC + 1CA 30
L + 2CA —
HC  50
HC + 1CA 50
HC +  2CA —


 


10
4-nitro- phenyl




CA 0 1 2
 0  96  5  0
 1  79  21  0
 2  67  29  4
 4  54  37  9
 24  41  42  15
LC 68
LC + 1CA 32
L + 2CA —
HC  92
HC + 1CA  8
HC +  2CA —





3
 0
 0
 0
 0
 3








 


11
2,6- difluoro- phenyl




CA 0 1 2 3
 0 100  0  0  0
 1  93  8  0  0
 2  88  12  0  0
 4  79  29  0  0
 24  50  39  11  0








 


12
1- naphthyl




CA 0 1 2 3
 0 100  0  0  0
 1 100  0  0  0
 2 100  0  0  0
 4 100  0  0  0
 24 100  0  0  0









Example 19
Further examples of alternatively activated esters are shown in Table 45. The time-course of conjugation of several analogs of PFP esters were examined. By decreasing the number and position of the fluorine groups in PFP, less reactive active ester forms can be synthesized and investigated. 2,3,5,6-tetrafluorophenyl ester and 2,4,6-trifluorophenyl ester were both tested after conjugation to [PEG2-MAL-KSH11-SEQ:27]. 1-hydroxyl-pyrrolidine-2,5-dione (NHS) was conjugated to [PEG5-K11-SEQ:27].




After 2 hrs conjugation, these less activated forms gave lower overall conjugation to 2.12.1.fx than PFP. NHS group also showed lower overall conjugation. NHS and PFP-containing peptides were conjugated to 2.12.1.fx. The reduced forms were analyzed to see the distribution at 2 hrs. PFP showed a much greater propensity for light chain derivatization (77% overall to LC, only 6% to heavy) compared to 1-hydroxyl-pyrrolidine-2,5-dione (NHS) (31% overall to LC, but 34% overall to heavy).







TABLE 45







Alternatively activated esters - further examples.















Active esters—reduced analysis of



Name
Structure
CA at 2 hr
conjugation at 2 hr




















 1
Penta Fluoro Phenyl




CA 0 1 2 3 4
2 hr  3 40 42 14  1
LC 23
LC + 1CA 72
LC + 2CA 5
HC 94
HC + 1CA  6
HC + 2CA 0


 


13
1- hydroxyl- pyrrolidine- 2,5-dione (NHS)




CA 0 1 2 3 4
2 hr 18 44 24 12  3
LC 70
LC + 1CA 28
LC + 2CA 3
HC 66
HC + 1CA 31
HC + 2CA 3


 


14
2,3,5,6- tetra- fluoro- phenyl




CA 0 1 2 3 4
2 hr 21 44 29  5  2








 


15
2,4,6- trifluoro phenyl




CA 0 1 2 3 4
2 hr 80 27  2  0  0









Compounds Z1-Z15 represent a variety of different structural types of active ester. It is enlightening to consider the series of fluorinated aromatic active esters, which have a different number and pattern of substitution of fluorine atoms around the aromatic ring (compounds Z1, Z2, Z3, Z11, Z14 and Z15) and consider how their structure influences their reactivity and propensity for protein derivatization. The kinetics of the antibody-conjugation of these derivatives can be conveniently compared at the 2 hr time-point, when the pentafluorophenyl (Z1) reaction has gone to completion. With an increasing level of fluorine substitution around the ring, there is an increasing level of overall conjugation and a concomitant decrease in unreacted antibody. The rate of reaction is directly related to the pKa of the fluorinated phenol leaving group, with the most acidic phenols giving higher reaction rates. The rates of conjugation are Z1>Z14>Z3>Z15>Z2>Z11. The subtle effects of the fluorine substitution patterns can be seen by comparing compounds Z2, Z3 and Z15.
The structure of the active ester also significantly affected the directionality of the conjugation reaction. In general, the fluorinated aromatic esters showed a marked propensity towards light chain derivatization (principally CLκ-K80 as previously mentioned). In contrast, several esters based on N-hydroxysuccinimide derivatives (Z8, Z9 and Z13) showed less preference, with often greater levels of heavy chain derivatization observed.
Example 20
The rate of conjugation between MAC-1 (PEG2-MAL-mercaptopropionyl linker between the peptide and PFP activating group) and MAC-2 (straight-chained PEG5 linker between the peptide and PFP activating group) was assessed. Table 46 compares these activated peptides to 2.12.1x. The results show that the activated peptides behave very similarly in terms of the rate and extent of derivatization, despite their slightly different linker structures.







TABLE 46







Comparison of conjugation between MAC-1 and MAC-2.










MAC-2
MAC-1

















Intact time (min)
OCA
1CA
2CA
3CA
4CA
OCA
1CA
2CA
3CA
4CA




















  0
72
27
1
0
0
82
18
1
0
0


 10
26
56
17
1
0
29
49
20
2
0


 20
13
53
29
5
0
15
47
33
5
0


 30
9
51
32
8
1
9
43
40
8
0


 40
7
45
39
9
1
8
41
41
8
2


 50
6
43
39
11
1
7
41
42
9
2


 60
5
41
40
11
2
6
36
45
11
2


 70
4
40
40
14
2
6
35
46
11
2


 80
3
38
44
14
2
5
36
47
10
2


 90
4
37
45
13
1
6
35
46
12
2


100
4
40
41
13
2
6
35
46
11
2


110
3
40
42
14
1
6
34
46
12
3


120
4
37
44
13
1
5
35
46
12
2









Example 21 Effect of Linker Length
The effect on the final conjugate distribution profile of having different lengths of linker was examined. Compounds were synthesized with different PEG length linkers joining the peptide to the PFP group. The results for the addition to 2.12.1.fx of 0, 1, 2, 3 and 4 peptides are summarized in Table 47. Overall, changing the length of the PEG linker had generally little effect on the distribution of conjugates obtained.







TABLE 47







Effect of linker length.








Y1 =



 





  
CA (%)












n
0
1
2
3
4















2
8
39
44
8
0


3
6
34
47
10
2


5
4
37
44
13
1


7
4
35
49
11
0


9
3
28
49
19
2


13
3
32
54
10
0


17
6
37
51
7
0


21
4
43
45
5
2


25
11
44
38
7
0















Example 22 Conjugation of Alternative Peptide Sequences
To confirm the applicability of the invention across other peptide sequences, SEQ ID NO:80 and SEQ ID NO:81 (Test-peptides-1, and -2) were conjugated. SEQ ID NOs:80 and 81 were conjugated with [PFP-PEG5] and then the 2.12.1.fx under conditions previously optimized for reaction with [PFP-PEG5-K11-SEQ:27]. The results of analysis of the conjugation profile and LC/HC conjugation are shown in Table 48. SEQ ID NO:80 and SEQ ID NO:81 both showed directional conjugation to the light chain. On further analysis of the LC/HC distributions, similar profiles to that of MAC-2 were observed, with around 70% LC derivatization and less than 10% on the HC.







TABLE 48







Conjugation profile of SEQ ID NO 80 and SEQ ID NO: 81.












LC % CA
HC % CA














SEQ
% CA

LC
LC

HC
HC


















ID NO:
0
1
2
3
4
LC
+1
+2
HC
+1
+2





















27
2
24
55
17
3
24
65
11
91
9
—


80
11
39
43
8
0
32
68
—
95
5
—


81
8
35
48
10
0
29
71
—
94
6
—








 












Example 23 Summary of Peptide Conjugation Analysis
Peptide mapping experiments were performed on a range of protein/conjugate combinations for the purpose of confirming the important parameters that lead to directional conjugation at CLκ-K80 on antibody light chains. Table 49 lists the results of the peptide mapping experiments performed. For each study parameter, the peptide mapping procedure described earlier was used. “***” indicates a high level of directional conjugation to CLκ-K80. “**” and to a lesser extent, “*”, indicates directional conjugation is still observed, but may show differences, such as slower reaction conditions, less overall conjugation, or averaging at one light chain only, and so may be more suitable to special circumstances, such as generating MACs with between 0.5 and 1.5 peptide per antibody (for example). “-” indicates that these reaction conditions did not appear favorable towards directional conjugation at CLκ-K80.
As CLκ-K80 was observed in MAC-2 to be the location of directional conjugation, peptide mapping studies on alternative parameters focused on this location. Detailed peptide mapping data for each study parameter is not included, but significant conjugation levels at other K residues was not observed, and observations of other MACs were consistent with directional conjugation at CLκ-K80.
CLκ-K80R and CLκ-K80A mutations of 2.12.1.fx resulted in the loss of directional conjugation at this site; suggesting an essential role for this specific residue. CLκ-K82R, and CLκ-K82A mutations did not hinder directional conjugation to CLκ-K80, and may even enhance it. Of the other study parameters examined, at least a portion of the sub-type of light chain constant region was observed to have a significant impact on directional conjugation; at least a portion of the light chain sub-type kappa was determined to be necessary. Conjugation onto a CLλ sub-type (using an exemplary A containing antibody, hAbλTest1), did not demonstrate directional conjugation. When the CLλ of hAbλTest1 was mutated to a CLκ, directional conjugation at K80 was recovered.







TABLE 49







Summary of directional conjugation at CLK-K80.















Mutations/








Differences



Directional


Antibody
LC
Vs MAC1/2
SEQ ID NO
Linker
Z*
conjugation





2.12.1.fx
κ

27
PEG2-MAL
PFP
* * *


2.12.1.fx
κ

27
PEG5
PFP
* * *


2.12.1.fx Fab
κ

27
PEG5
PFP
* * *


h38C2-IgG1
κ

27
PEG5
PFP
* * *


h38C2-IgG2
κ

27
PEG5
PFP
* * *


hAbλTest
λ
K80SH
27
PEG5
PFP
—


hAbκTest1
κ

27
PEG5
PFP
* * *


hAbκTest3
κ

39-mer
PEG5
PFP
* * *


hAbλTest
λκ

27
PEG5
PFP
* * *


hAbλTest
λκJ

27
PEG5
PFP
* * *


2.12.1.fx
κ
K80R
27
PEG5
PFP
—


2.12.1.fx
κ
K82R
27
PEG5
PFP
* * *


2.12.1.fx
κ
K80R/R82R
27
PEG5
PFP
—


2.12.1.fx
κ
D43A
27
PEG5
PFP
* *


2.12.1.fx
κ
K80A
27
PEG5
PFP
—


2.12.1.fx
κ
H81A
27
PEG5
PFP
—


2.12.1.fx
κ
H82A
27
PEG5
PFP
* * *


2.12.1.fx
κ
D43A/H81A
27
PEG5
PFP
—


hAbλTest1
λ
S81H/H82S
27
PEG5
PFP
—


2.12.1.fx
κ

39-mer
PEG5
PFP
* * *


2.12.1.fx
κ

80
PEG5
PFP
* * *


2.12.1.fx
κ

81
PEG5
PFP
* * *


h38C2-IgG2
κ

39-mer
PEG5
PFP
* * *


2.12.1.fx Fab
κ

biotin
PEG5
PFP
* * *


2.12.1.fx
κ

27
PEG2-MAL
PFP
* * *


2.12.1.fx
κ

27
PEG2-MAL
2,3,4 TFP (2)
* *


2.12.1.fx
κ

27
PEG2-MAL
2,3,6 TFP (3)
* *


2.12.1.fx
κ

27
PEG2-MAL
2,3,6 TCP (4)
—


2.12.1.fx
κ

27
PEG2-MAL
2,6 DCP (5)
—


2.12.1.fx
κ

27
PEG2-MAL
2,4 DCN (6)
*


2.12.1.fx
κ

27
PEG2-MAL
5,7 DCQ (7)
—


2.12.1.fx
κ

27
PEG2-MAL
NH-5-N2,3DI (8)
*


2.12.1.fx
κ

27
PEG2-MAL
2Hi1,3 DIO (9)
*


2.12.1.fx
κ

27
PEG2-MAL
4NP (10)
* *


2.12.1.fx
κ

27
PEG2-MAL
2,6 DFP (11)
* *


2.12.1.fx
κ

27
PEG2-MAL
NAP (12)
—


2.12.1.fx
κ

27
PEG2-MAL
1HP 2,5D (13)
*


2.12.1.fx
κ

27
PEG2-MAL
2,3,5,6 TFP (14)
* *


2.12.1.fx
κ

27
PEG2-MAL
2,4,6 TFP (15)
* *


2.12.1.fx
κ

27
PEG2-MAL
Squarate
*


2.12.1.fx
κ

27
PEG2-MAL
AZD
*


2.12.1.fx
κ

27
PEG2-17
PFP
* * *


2.12.1.fx
κ

27
PEG17-21
PFP
* *


2.12.1.fx
κ

27
PEG25
PFP
* *









Example 24 Examination of CLκ-D77 
Residues geographically close to the CLκ-K80 in the 3-D structure were examined. Initial Crystal structure analysis suggested the possibility that CLκ-D77 could form a salt bridge with CLκ-K80, which could have an impact on the CLκ-K80 directional conjugation. In order to study the effect of CLκ-D77 on conjugation to CLκ-K80, CLκ-D77 was mutated to CLκ-A77 on the 2.12.1.fx antibody to create 2.12.1.fx-[CLκ-D77A] (CLκ of SEQ ID NO:37). The CLκ-D77A mutation was generated on an antibody light chain following protocols described in QuickChange site-directed mutagenesis kit (Stratagene®). The mutation was introduced by oligonucleotide primers and confirmed by DNA sequencing. 2.12.1.fx-[CLκ-D77A] was transiently expressed in HEK 293 cells, and purified using Protein A affinity column. The purified mAbs were characterized using MS.
2.12.1.fx and 2.12.1.fx-[CLκ-D77A] (1 mg reaction size) were adjusted to 18 mg/ml to pH 7.7 with a phosphate buffer to a final concentration of 0.06M sodium phosphate. The exemplary test peptide-linker pair [PFP-PEG5-K11-SEQ:27] was reconstituted in a propylene glycol solution to 10 mg/ml. [PFP-PEG5-K11-SEQ:27] was added to antibody at a molar ratio of 4.3:1 and allowed to react for 2 hrs at RT. The conjugated product was diluted to 2 mg/ml and analyzed as an intact conjugated protein by SEC-MS to determine the number and quantitation of conjugate forms of the protein. Relative quantitation of multiple peptide-linker conjugation species was performed by measuring the signal magnitude.
Table 50 compares the conjugation profile of [2.12.1.fx]-[PEG5-K11-SEQ:27] and [2.12.1.fx-[CLκ-D77A]-[PEG5-K11-SEQ:27]. The conjugation profile of [2.12.1.fx]-[PEG5-K11-SEQ:27] occurs as a distribution between 0-4 peptide additions with the largest form being 2 peptide additions and the average number of peptide additions is 2.16. The profile changes when the residue CLκ-D77 is mutated to CLκ-A77 in the scaffold protein; the average number of peptide additions rises to 2.38 and significantly less single peptide addition is observed. This result suggests that the single point mutation CLκ-D77A has the effect of increasing the overall conjugation to the scaffold. In both conditions replicate analysis (n=3) demonstrates that the conjugations profiles observed are reproducible.
The extent of peptide conjugation was examined separately on the light and heavy chains of 2.12.1.fx and 2.12.1.fx-[CLκ-D77A]. The produced MACs were denatured and disulfide bonds were reduced using guanidine hydrochloride and dithiothreitol. The resulting free light and heavy chains were analyzed using LCMS to determine the conjugation profile on each. Table 50 demonstrates that the average conjugation is higher on the light chain of 2.12.1.fx-[CLκ-D77A] than 2.12.1.fx; the average conjugate addition value for 2.12.1.fx-[CLκ-D77A] is 1.15 compared to 0.85 for 2.12.1.fx. In addition, unconjugated light chain is undetected in 2.12.1.fx-[CLκ-D77A]. Conjugation on the heavy chain is observed at a significantly lower level. The majority of observed heavy chain for both 2.12.1.fx and 2.12.1.fx-[CLκ-D77A] is unconjugated; this is especially true in the case of 2.12.1.fx-[CLκ-D77A] heavy chain. These results suggest that the CLκ-D77A mutation alters the light chain to make it significantly more susceptible to conjugation. Replicate analysis of this experiment by multiple scientists is shown in Table 50 which demonstrates that the profile of conjugation is consistent and reproducible.







TABLE 50







Analysis of conjugation of [PEG5-K11-SEQ: 27] to Abs 2.12.1.fx (WT) and 


2.12.1.fx-[CLK-D77A]. Rep = replicate. AvReps is the average of the results of the


three replicate experiments, with the standard deviation shown beneath (StdDev).


Ab % CA shows % conjugations additions per antibody, followed by the average 


CA per antibody. Reduced light and heavy chain analysis also shown, 


with respective average CA per chain. CLκ-D77A shows 123% 


rate of LC % 1CA species compared to native CLκ.














Ab % CA
Avg.
LC % CA
Avg.
HC % CA
Avg.




















Ab 
0
1
2
3
4
CA
0
1
2
CA
0
1
CA























WT















Rep1
1
17
56
22
4
2.11
21
72
7
0.86
86
14
0.14


Rep2
2
14
54
25
5
2.18
25
67
8
0.83
85
15
0.15


Rep3
2
13
55
25
6
2.2
23
69
8
0.85
85
15
0.15


Av.Rep
2
15
55
24
5
2.16
23
69
8
0.85
85
15
0.15


StdDev
1
2
1
2
1
0.05
2
3
1
0.02
1
1
0.01


D77A















Rep1
5
4
54
30
8
2.32
0
84
16
1.16
94
6
0.06


Rep2
6
4
43
36
12
2.44
0
85
15
1.15
94
6
0.06


Rep3
3
3
56
31
7
2.37
0
86
14
1.14
94
6
0.06


AvRep
5
4
51
32
9
2.38
0
85
15
1.15
94
6
0.06


StdDev
2
1
7
3
3
0.06
0
1
1
0.01
0
0
0.00









Example 25 Peptide Mapping Characterization of 2.12.1.Fx-[CLκ-D77A]-[SEQ:27-K11-PEG5] Heavy and Light Chain Reference Product
2.12.1.fx-[CLκ-D77A]-[PEG5-K11-SEQ:27] conjugated antibody was reduced with dithiothreitol and cysteine residues were alkylated by carboxymethylation with iodoacetamide. Chymotrypsin was used for proteolytic digestion. Digested fragments in solution were analyzed using LCMS. Individual fragments were separated over a C18 HPLC column and their accurate mass was measured in a Q-Tof mass spectrometer. The resulting fragment mass was used to identify unmodified fragments or fragments modified with a [PEG5-K11-SEQ:27] conjugation group. This experiment was interpreted by focusing on chymotryptic fragments that contain a lysine residue and are therefore possible sites for peptide conjugation. Tables 51 and 52 list of all such fragments on the heavy chain and light chains respectively. Blank entries are fragments that were not detected using this technique. Detected fragments that were observed with a [PEG5-K11-SEQ:27] modifier are considered potential sites of peptide conjugation.
2 [PEG5-K11-SEQ:27]-conjugated fragments were detected using LCMS peptide mapping of the 2.12.1.fx-[CLκ-D77A]-[PEG5-K11-SEQ:27] product. Both of these conjugated fragments were present on the light chain of the 2.12.1.fx-[CLκ-D77A] antibody. In comparison, 8 fragments conjugated to [PEG5-K11-SEQ:27] were detected in 2.12.1.fx-[PEG5-K11-SEQ:27].
Overall, these results suggest that conjugation levels in the CLκ-D77A mutant are elevated at fewer conjugation sites, possibly suggesting increased conjugation specificity relative to the unmutated antibody. Further, structural analysis has shown that the CLκ-D77 residue is in close proximity (<10 Ås) to the identified major conjugation site CLκ-K80. It was speculated that an electrostatic interaction, possibly a salt bridge, could exist between the carboxylic acid of CLκ-D77 and the primary amine of CLκ-K80. The CLκ-D77A mutation would disrupt such an electrostatic interaction, resulting in the reactive amine on CLκ-K80 being more exposed and susceptible to conjugation with the reactive esters of the invention. Although subsequent analysis coupled with sophisticated modeling helped build a more complete picture of the reaction site, and indicated that CLκ-D77 exerted its effect primarily through its interaction with CLκ-H81, the initial hypothesis of an interaction between CLκ-D77 and CLκ-K80 was helpful in underlining the significance of the CLκ-D77 residue.
The observed conjugation sites in the 2.12.1.fx-[CLκ-D77A]-[PEG5-K11-SEQ:27] product are light chain chymotrypsin fragments Y3 and Y15. Analysis of the signal intensities for these fragments suggests that fragment Y15, which carries the CLκ-K80 residue, is the primary conjugation site. Fragment Y15 is only observed as an [PEG5-K11-SEQ:27]-modified fragment at a very high signal intensity (1118572 counts, Table 52), whilst the unmodified form of Y15 is not observed, suggesting that all or nearly all of fragment Y15 exists in the modified form. Fragment Y3 is observed in both the [PEG5-K11-SEQ:27]-modified and unmodified forms; unmodified Y3 signal intensities in 2.12.1.fx-[PEG5-K11-SEQ:27] and 2.12.1.fx-[CLκ-D77A]-[PEG5-K11-SEQ:27] are within 15%. [PEG5-K11-SEQ:27]-modified Y3 is observed at a relatively low level (9737 counts, Table 52).
The table entries for Tables 51 and 52 are explained below:
Fragment number: Chymotrypsin fragments numbering from the N-terminus; joined fragments (ie—Y1-2) indicate a missed cleavage site.
Start/End: Numbering of the fragment location from the N-terminus.
Fragment Mass (Da): Theoretical mass of the fragment listed in Daltons.
Retention Time (Control/Analyte): Time of chromatographic retention/elution in the LCMS peptide mapping experiment.
MS Signal Intensity (Control/Analyte): Magnitude of observed signal observed by MS.
Mass Error-ppm (Control/Analyte): Comparison of theoretical vs. observed mass of the peptide fragment; values closer to zero (0) demonstrate better mass accuracy. The control protein for Retention Time, MS signal intensity and Mass Error is 2.12.1.fx-[CLκ-D77A] and the analyte protein in each case is 2.12.1.fx-[CLκ-D77A]+[PEG5-K11-SEQ:27].

Modifiers: Potential covalent additions to the fragment; [PEG5-K11-SEQ:27]-antibody binding peptide of Lysine residue, CAM-carboxymethylation of Cysteine residue.








TABLE 51







Peptide mapping characterization of 2.12.1.fx-[CLκ-D77A]-[PEG5-K11-SEQ: 27]


heavy chain referenceproduct.














Fragment


Fragment
Retention Time
MS Signal Intensity
Mass Error (ppm)


















Number
Start
End
Mass (Da)
Control
Analyte
Control
Analyte
Control
Analyte
Modifiers




















Y1
1
27
2617.3533









Y1-2
1
29
2865.4695









Y5-6
34
47
1657.8398









Y6
37
47
1253.688
19.2
19.2
516640
548267
1.9
0



Y6-7
37
50
1602.8518
22.1
22.1
26537
31229
−1.6
−2



Y8-9
51
68
1931.9337
16.5
16.5
60894
82459
−2.2
0.5



Y9
61
68
878.461
11.3
11.4
376224
403402
0
−0.3



Y9-10
61
80
2241.1501









Y10
69
80
1380.6997
13.3
13.3
261813
286406
−1.1
0.3



Y10-011
69
94
2972.4661









Y19-20
111
157
4748.2773









Y20
116
157
4160.0405









Y20-21
116
166
5202.5527









Y20-21*
116
166
5316.5957
34.1
34.1
6445
8275
0.5
−5.7
CAM(2)


Y24-25
202
245
4702.2109









Y25
207
245
4151.9722









Y25*
207
245
4437.0796
20.9
20.9
1495322
1771622
1.1
0.7
CAM(5)


Y25-26
207
279
7985.9092









Y26
246
279
3851.9478









Y26-27
246
281
4152.0698









Y28-29
282
300
2245.1128









Y29
283
300
2082.0493
14.6
14.6
20665
18618
−0.6
−0.2



Y29-30
283
304
2531.2405









Y31-32
305
323
2241.1907









Y32
318
323
722.3599
7.9
7.9
93966
81618
0.1
3



Y32
318
323
722.3599
17.7
18.4
37943
11371
11.4
27.1



Y32
318
323
722.3599
18.4

11761

23.8




Y32-33
318
353
4028.188









Y33
324
353
3323.8386
20

5422

3.1




Y33*
324
353
3380.8601
19.7
19.7
2196329
2374835
−2.5
−3.8
CAM(1)


Y33-34
324
376
5883.1577









Y34
354
376
2577.3293









Y34-35
354
385
3637.8159









Y34-35*
354
385
3694.8374
33
33
10095
10026
1.9
−2
CAM(1)


Y36-37
386
408
2527.0808









Y37
396
408
1394.6388
19.6
19.6
62942
65871
−0.9
−1.6



Y37-38
396
409
1541.7072
25.1
25.1
827336
874876
0
−3.8



Y39-40
410
421
1494.8195









Y40
412
421
1218.672
15.8
15.8
77917
78774
−0.3
0.2



Y40-41
412
427
1891.9905
20.3
20.3
107513
150305
0.2
0.1



Y42-43
428
450
2525.1792









Y43
441
450
1016.5502
















TABLE 52







Peptide mapping characterization of 2.12.1.fx-[CLκ-D77A]-


[PEG5-K11-SEQ: 27] light chain reference product.




















Peptide 





















Fragment


Mass
Retention Time
MS Signal Intensity
Mass Error (ppm)


















Number
Start
End
(Da)
Control
Analyte
Control
Analyte
Control
Analyte
Modifiers




















Y2-3
36
49
1688.9725
16.2
16.2
145374
172582
−1.7
−1.1



Y3
37
49
1525.9093
15.5
15.5
331068
390175
−2.7
−2.4



Y3*
37
49
3218.7593

24

9737

−6.8
[SEQ: 27-K11-












PEG5](1)


Y3-4
37
62
2882.6355









Y9-10
88
116
3244.729









Y10
99
116
1871.0992









Y10-11
99
139
4331.335









Y11
117
139
2478.2463

22.8

61217

−1.7



Y11*
117
139
2535.2678

21.5

5127

−16.8
CAM(1)


Y11-12
117
148
3635.8445









Y12
140
148
1175.6088









Y12-13
140
173
3886.8245









Y13
149
173
2729.2263
13.1
13.1
1140556
1153543
−1.1
−1.8



Y13-14
149
186
4051.9346









Y14
174
186
1340.7188









Y14-15
174
192
2125.1418









Y15
187
192
802.4337
7.5

275639

−1.9




Y15*
187
192
2495.2837

20.9

1118572

−6.3
[SEQ: 27-K11-












PEG5](1)


Y15-16
187
209
2574.29









Y16
193
209
1789.8668
18.7

5400

4.4




Y16*
193
209
1846.8883
18.1
18.1
169490
246823
−1.7
−2.2
CAM(1)


Y16-17
193
214
2349.0842
17.8

9211

0.1









Example 26 Examination of CLκ-D77 Mutations
CLκ-D77 residue of 2.12.1.fx antibody was mutated to each of the other 18 amino acids in addition to the CLκ-D77A mutation. The CLκ-D77G (SEQ ID NO:38), CLκ-D77L (SEQ ID NO:40), CLκ-D77S (SEQ ID NO:49), CLκ-D77E (SEQ ID NO:53), and CLκ-D77R (SEQ ID NO:54) and mutants were generated following protocols described in QuickChange site-directed mutagenesis kit (Stratagene®). Mutations were introduced by oligonucleotide primers and confirmed by DNA sequencing. The other 13 mutants on the CLκ-D77 site (CLκ-D77V (SEQ ID NO:39), CLκ-D771 (SEQ ID NO:41), CLκ-D77P (SEQ ID NO:42), CLκ-D77F (SEQ ID NO:43), CLκ-D77W (SEQ ID NO:44), CLκ-D77Y (SEQ ID NO:45), CLκ-D77H (SEQ ID NO:46), CLκ-D77M (SEQ ID NO:47), CLκ-D77C (SEQ ID NO:48), CLκ-D77T (SEQ ID NO:50), CLκ-D77Q (SEQ ID NO:51), CLκ-D77N (SEQ ID NO:52), CLκ-D77K (SEQ ID NO:55)) were generated following protocols described in Quick PCR Cloning Kit (BPS Bioscience). Mutations were introduced by oligonucleotide primers and cloned to a modified p2.12.1.fxP4 vector (Invitrogen) cut with BglII and NheI. Insert DNA were confirmed by DNA sequencing. The mutated mAbs were transiently expressed in HEK 293 cells, and purified using protein A affinity column. The purified mAbs were characterized using MS.
2.12.1.fx and 2.12.1.fx mutants were adjusted 18 mg/ml to pH 7.7 with a phosphate buffer to a final concentration of 0.06 M sodium phosphate. [PFP-PEG5-K11-SEQ:27] was reconstituted in a propylene glycol solution to 10 mg/ml. The peptide/linker was added to antibody at a molar ratio of 4.3:1 and allowed to react for 2 hrs at RT.
Table 53 describes the overall conjugation profile of the CLκ-D77 mutants. CLκ-D77C aggregated due to an introduction of a free cysteine, and the results were not interpretable. Mutations CLκ-D77W, CLκ-D77M, CLκ-D77H, CLκ-D77Q, CLκ-D77N, and CLκ-D77V did not change the overall conjugation profile compared to wild-type 2.12.1.fx. Mutations CLκ-D77F, CLκ-CLκ-D77K, CLκ-D77Y, and CLκ-D77E decreased the overall level of conjugation. Mutations CLκ-D77P, CLκ-D771, CLκ-D77T, CLκ-D77R, CLκ-D77L, CLκ-D77S, and CLκ-D77G increased the level of conjugation.
Analysis of the reduced LC and HC showed that only the mutations CLκ-D77F, CLκ-D77K, CLκ-D77Y, CLκ-D77E, and CLκ-D77C resulted in reduced levels of conjugation on the light chain. The levels of conjugation on the light chain increased (and conjugation on the heavy slightly decreased) for CLκ-D77M, CLκ-D77H, CLκ-D77Q, CLκ-D77N, CLκ-D77W and CLκ-D77V. Mutations CLκ-D77P, CLκ-D77I, CLκ-D77T, CLκ-D77R, CLκ-D77L, CLκ-D77S, and CLκ-D77G increased the level of conjugation on the light chain by reducing the level of unconjugated light chain. CLκ-D77K increased the level of 2 conjugates on the light chain due to the introduction of another lysine, a potential conjugation site.







TABLE 53







Conjugation analysis of CLκ-D77 mutants to alternative amino acids


(data is separated within the table according to the protein amount used during the


conjugation reaction, or because the conjugation reaction was set up at a different


time). The decreased level of conjugation for the tests run at 0.5 and 0.25 mg/ml was


due to the low levels of the antibody. Ab % CA shows % conjugations additions per


antibody, followed by the average CA per antibody. Reduced light and heavy chain


analysis also shown, with respective average CA per chain. The % of 1-LC % relative


to the respective WT run is shown in the right column: for example, D77Q, 1-LC %


value of 81 is 126% of the respective WT 1-LC % of 69 for that experimental run. All


samples were tested with 1 mg Ab, except the run including D77M, D77F and D77H


(0.5 mg), and the run including D77W and D77C (0.25 mg).















Ab % CA
Avg.
LC % CA
Avg.
HC % CA
Avg.
1LC





















Ab
0
1
2
3
4
CA
0
1
2
CA
0
1
CA
WT %
























WT
1
16
54
24
4
2.14
19
69
13
0.94
88
12
0.12



D77R
6
12
41
26
15
2.33
8
87
5
0.96
94
6
0.06
126


D77L
5
5
53
28
10
2.33
1
88
11
1.11
91
9
0.09
127


D77E
8
27
39
18
8
1.90
32
57
10
0.78
84
16
0.78
82


D77S
5
6
47
29
12
2.36
0
92
8
1.08
94
6
0.06
133


D77G
7
7
44
28
13
2.34
0
91
9
1.09
92
8
0.08
131


WT
2
24
50
21
3
1.99
24
66
10
0.85
90
10
0.1



D77Q
2
10
78
10

1.96
15
81
4
0.88
96
4
0.04
122


D77P
3
4
63
24
7
2.26
5
88
7
1.02
96
4
0.04
133


D77K
9
30
36
17
8
1.84
34
45
21
0.86
93
7
0.07
68


D77N
3
8
79
11

1.98
11
83
6
0.94
96
4
0.04
125


D77Y
28
41
19
11

1.14
54
42
4
0.51
89
11
0.11
63


D77V
6
10
63
21

1.98
13
79
9
0.96
95
5
0.05
119


D77I
3
4
63
24
7
2.27
6
85
9
1.03
93
7
0.07
128


D77T
3
11
59
18
9
2.17
8
88
4
0.97
95
5
0.05
133


WT
4
30
44
20
3
1.88
28
63
10
0.82
89
11
0.11



D77M
4
15
67
14

1.90
16
81
3
0.87
96
4
0.04
132


D77F
21
42
24
13

1.29
54
42
4
0.49
86
14
0.14
66


D77H
5
16
60
19

1.92
16
75
9
0.93
94
6
0.06
119


WT
5
36
47
12

1.66
30
60
10
0.8
90
10
0.1



D77W
8
29
46
18

1.73
20
75
4
0.84
91
9
0.09
125


D77C






60
37
2
0.42
95
5
0.05
61









Example 27 Effects of Other Mutations to the CHκ Region on Conjugation
In addition to CLκ-D77A, other residues within 10A distance to CLκ-K80 were mutated to alanine: CLκ-K41A (SEQ ID NO:20), V42A (SEQ ID NO:21), CLκ-D43A (SEQ ID NO:56), CLκ-N44A (SEQ ID NO:22), CLκ-L48A (SEQ ID NO:23), CLκ-Q47A (SEQ ID NO:24), CLκ-S48A (SEQ ID NO:25), CLκ-N80A (SEQ ID NO:26), CLκ-L73A (SEQ ID NO:28), CLκ-S74A (SEQ ID NO:29), CLκ-K75A (SEQ ID NO:30), CLκ-Y78A (SEQ ID NO:31), CLκ-E79A (SEQ ID NO:32), CLκ-H81A (SEQ ID NO:33), CLκ-V83A (SEQ ID NO:34), CLκ-Y84A (SEQ ID NO:35), and CLκ-R103A (SEQ ID NO:36) were also mutated to Ala. The data of CLκ-D43A and CLκ-H81A are discussed in Example 14.
The L73A mutant was introduced to 2.12.1.fx CLκ using the three way ligation method. A primer specific to the 5′ end of 2.12.1.fx-LC (2.12.1.fx.LC.FOR: SEQ ID NO:85) and a reverse primer containing the desired L73A mutation (L181A.REV: SEQ ID NO:88) were used to PCR the first half of the 2.12.1.fx-LC using 2.12.1.fx-LC DNA as the PCR template. This PCR fragment was then digested using restriction enzymes BglII and BsaI. A forward primer containing CLκ-L73A mutation (L181A.FOR: SEQ ID NO:87) paired with the reverse primer specific to the 3′ end of 2.12.1.fx-LC (2.12.1.fx.LC.REV: SEQ ID NO:86) were used to PCR amplify the second half of 2.12.1.fx-LC DNA fragments carrying mutation using 2.12.1.fx-LC DNA as the PCR template. This PCR fragment was then digested using restriction enzymes BsaI and NheI. The two restriction enzyme digested PCR fragments were ligated with a modified p2.12.1.fxP4 plasmid (Invitrogen®) cut with BglII and NheI. The insert sequence was confirmed by DNA sequencing. 2.12.1.fx-[CLκ-L73A] (i.e. comprising SEQ ID NO:28) was transiently expressed in HEK 293 cells, and purified using protein A affinity column. The purified mAbs were characterized using MS.
The CLκ-V42A and CLκ-K75A mutants were generated by overlap PCR. Mutations were introduced by oligonucleotide primers. Primer specific to the 5′ end of 2.12.1.fx-LC (2.12.1.fx.LC.FOR) paired a reverse primer carrying the desired mutation, and a forward primer carrying the desired mutation paired with the reverse primer specific to the 3′ end of 2.12.1.fx light chain (2.12.1.fx.LC.FOR) were used to PCR amplify 2.12.1.fx-LC DNA fragments using 2.12.1.fx-LC as template. These two PCR products were mixed as templates; 2.12.1.fx-LC forward primer and reverse primer were used in overlap PCR reaction to amplify the full length 2.12.1.fx-LC DNA with desired mutation. The PCR was then digested with restriction enzyme BglII and NheI. The digested PCR was ligated with a modified p2.12.1.fxP4 plasmid (Invitrogen®) cut with BglII and NheI. The insert sequence was confirmed by DNA sequencing. The mutated mAbs were transiently expressed in HEK 293 cells, and purified using protein A affinity column. The purified mAbs were characterized using MS.
The other mutants were generated on 2.12.1.fx-LC following protocols described in QuickChange site-directed mutagenesis kit (Stratagene®). Mutations were introduced by oligonucleotide primers and confirmed by DNA sequencing. The mutated mAbs were transiently expressed in HEK 293 cells, and purified using protein A affinity column. The purified mAbs were characterized using MS.
2.12.1.fx and 2.12.1.fx mutants (1 mg reaction size) were adjusted 18 mg/ml to pH 7.7 with a phosphate buffer to a final concentration of 0.06 M sodium phosphate. [PFP-PEG5-K11-SEQ:27] was reconstituted in a propylene glycol solution to 10 mg/ml. The peptide/linker was added to the antibody at a molar ratio of 4.3:1 and allowed to react for 2 hrs at room temperature.
Table 54 compares the conjugation profile of 2.12.1.fx-[PEG5-K11-SEQ:27] with 2.12.1.fx-[CLκ-mutants]-[PEG5-K11-SEQ:27]. The conjugation profile of 2.12.1.fx-[PEG5-K11-SEQ:27] occurs as a distribution between 0-4 peptide additions with the largest form being 2 peptide additions. The profile changes when the residues are mutated to Ala in the scaffold protein; the average number of [PEG5-K11-SEQ:27] additions either decreased (CLκ-V42A, CLκ-L46A, CLκ-S74A, CLκ-Y78A and CLκ-Y84A) or increased (CLκ-Q47A, CLκ-N50A and CLκ-D77A/E79A double mutants) compared to their corresponding 2.12.1.fx-[PEG5-K11-SEQ:27] controls. When comparing the conjugation profile of CLκ-D77A/E79A with CLκ-E79A, the significant increase of average [PEG5-K11-SEQ:27] additions to the antibody is mainly contributed by the CLκ-D77A mutation.
The extent of [PEG5-K11-SEQ:27] conjugation was examined separately on the light and heavy chains of 2.12.1.fx and 2.12.1.fx-[CLκ-mutants]. The MACs were denatured and disulfide bonds were reduced using guanidine hydrochloride and dithiothreitol. The resulting free light and heavy chains were analyzed using LCMS to determine the conjugation profile on each. Table 54 demonstrates that the 1CA on the light chain of 2.12.1.fx-[CLκ-Q47A] and -[CLκ-N50A] are higher than 2.12.1.fx. The average CAs are 0.84 and 0.85 compared to 0.78 of the 2.12.1.fx antibody. Both CLκ-Q47A and CLκ-N80A mutants have over 70% 1CA compared to the 59% 1CA of the 2.12.1.fx wild type antibody. In addition, the unconjugated light chain levels of these two mutants were reduced from 31% of the wild type antibody to 22% and 19%. The V42A had reduced level of light chain conjugation. The average light chain CA is 0.45 with 59% unconjugated light chain and 37% 1CA. The % of 1-LC % relative to the respective WT run is shown in the right column, as described in Table 54.
Conjugation of CLκ-V42A, CLκ-Q47A and CLκ-N80A to [PEG5-K11-SEQ:27] were repeated, and the results are shown at the bottom of the table. The elevated levels of light chain conjugation in CLκ-Q47A and CLκ-N80A and reduced light chain conjugation in CLκ-V42A were confirmed by both intact and reduced LC-MS analysis. Overall, the conjugation data suggests that CLκ-V42, CLκ-D43 and CLκ-H81 all have an impact on for PFP directional conjugation at CLκ-K80.







TABLE 54







Conjugation analysis of 2.12.1.fx variants conjugated to [SEQ: 27-K11-


PEG5], showing amino acid mutants within10 Ås of light chain K80. Ab % CA shows


% conjugations additions per antibody, followed by the average CA per antibody.


Reduced light and heavy chain analysis also shown, with respective average CA per


chain. The % of 1-LC % relative to the respective WT run is shown in the right


column, as described in Table 53.















AB % CA
Avg.
LC % CA
Avg.
HC % CA
Avg.
1LC





















Ab
0
1
2
3
4
CA
0
1
2
CA
0
1
CA
WT %
























WT
5
40
43
10
2
1.63
31
59
9
0.78
90
10
0.1



K41A
6
32
49
10
2
1.70
28
65
7
0.78
90
10
0.1
110


V42A
26
45
21
8

1.11
59
37
4
0.45
90
10
0.1
63


N44A
9
37
40
10
4
1.6
31
63
6
0.75
93
7
0.07
107


L46A
14
42
35
9

1.4
35
62
3
0.68
95
5
0.05
105


Q47A
6
27
53
9
4
1.78
22
73
5
0.84
95
5
0.05
124


S48A
6
34
46
9
4
1.72
34
59
7
0.73
93
7
0.07
100


N50A
4
23
56
13
4
1.89
19
76
4
0.85
95
5
0.05
129


L73A
12
36
33
13
7
1.69
41
55
3
0.62
87
13
0.13
93


S74A
11
42
39
7

1.43
35
61
5
0.7
96
4
0.04
103


K75A
12
38
41
9

1.48
35
62
3
0.68
93
7
0.07
105


D77A-
3
7
71
15
4
2.1
5
87
8
1.04
95
5
0.05
147


E79A
















V83A-
9
35
39
11
6
1.7
33
61
6
0.73
86
14
0.14
103


WT
2
23
49
22
4
2.03
34
60
5
0.71
81
19
0



E79A
6
13
51
24
6
2.12
21
73
6
0.86
85
15
0
122


R103A
4
18
51
20
6
2.06
23
70
7
0.83
82
18
0
117


WT
1
17
51
25
5
2.16
14
77
9
0.96
84
16
0.16



D77A
4
6
60
23
7
2.23
0
86
13
1.13
92
8
0.08
112


V42A
13
28
29
21
9
1.86
49
42
9
0.61
86
14
0.14
55


Q47A
6
7
58
21
9
2.2
8
85
7
0.99
91
9
0.09
110


N50A
7
8
51
25
9
2.2
10
81
9
0.99
90
10
0.1
105









Example 28 Analysis of CLκ-D43A and CLκ-H81A Mutants
In order to determine whether the charge, hydrogen bond or the size of CLκ-D43 are important to the CLκ-K80 directional conjugation, CLκ-D43 was mutated to CLκ-D43E (SEQ ID NO:107), CLκ-D43N (SEQ ID NO:108) and CLκ-D43L (SEQ ID NO:109) respectively. The mutants were generated on 2.12.1.fx antibody light chain following protocols described in QuickChange site-directed mutagenesis kit (Stratagene®). Mutations were introduced by oligonucleotide primers and confirmed by DNA sequencing. The mutated mAbs were transiently expressed in HEK 293 cells, and purified using protein A affinity column. The purified mAbs were characterized using MS.
Similarly, in order to assess the role of CLκ-H81, the following mutant versions of the test antibody 2.12.1.fx were assessed: CLκ-H81N (SEQ ID NO:110), CLκ-H81Q (SEQ ID NO:111), CLκ-H81Y (SEQ ID NO:112), CLκ-H81W (SEQ ID NO:113) and CLκ-H81F (SEQ ID NO:114).
2.12.1.fx antibody and 2.12.1.fx-[CLκ-mutant]antibodies (1 mg reaction size) were adjusted 18 mg/ml to pH 7.7 with a phosphate buffer to a final concentration of 0.06 M sodium phosphate. [PFP-PEG5-K11-SEQ:27] was reconstituted in a propylene glycol solution to 10 mg/ml. The peptide/linker was added to antibody at a molar ratio of 4.3:1 and allowed to react for 2 hrs at RT.
CLκ-D43N has the similar overall conjugation and light chain levels (Table 55) compared to the wild type antibody. CLκ-D43E and CLκ-D43L showed reduced overall conjugation level light chain conjugation level.
CLκ-H81N, CLκ-H81Q, CLκ-H81Y, CLκ-H81W and CLκ-H81F mutants showed reduced overall conjugation level light chain conjugation level, suggesting that the imidazole ring is required for the PFP directional conjugation. The conjugation reaction does not involve the 7-stacking interaction nor the H-bonds formed with Nε2 or Nδ11 of the imidazole ring.







TABLE 55







2.12.1.fx variants conjugated to [PEG5-K11-SEQ: 27] 


(WT is 2.12.1.1fx). Ab % CA shows % conjugations additions per antibody, 


followed by the average CA per antibody. Reduced light and heavy chain analysis 


also shown, with respective average CA per chain. The % of 1-LC % relative to the 


respective WT run is shown in the right column, as described in Table 53.















Ab % CA
Avg
LC % CA
Avg
HC % CA
Avg
1LC






















Ab
0
1
2
3
4
CA
0
1
2
CA
0
1
2
CA
WT %

























WT
2
23
49
22
4
2.03
34
60
5
0.71
81
19
0
0.19



D43N
7
24
39
21
8
2.00
34
59
7
0.73
84
16
0
0.16
98


D43L
14
33
34
19
0
1.57
55
40
5
0.50
74
26
0
0.26
67


D43E
9
33
37
17
4
1.75
44
48
8
0.63
74
26
4
0.34
80


H81N
32
41
20
7
0
1.04
84
16
0
0.16
70
26
5
0.35
27


H81Q
29
40
21
11
0
1.14
82
17
1
0.19
73
27
0
0.27
28


H81Y
27
40
24
8
0
1.14
80
20
0
0.20
70
26
4
0.35
33


H81W
29
45
19
8
0
1.05
85
15
0
0.15
69
26
5
0.36
25


H81F
13
41
30
15
0
1.48
79
21
0
0.21
70
30
0
0.30
35









Example 29 2.12.1.Fx-[CLκ-DnA] Conjugation Using Different Reactive Esters
2.12.1.fx and 2.12.1.fx-[CLκ-D77A] were conjugated to [PEG5-K11-SEQ:27] using different reactive esters (see Examples 18 and 19) (results shown in Table 56). For all of the different activated esters, the 2.12.1.fx-[CLκ-D77A] mutant gave a higher level of intact average CA upon conjugation compared to the wt 2.12.1.fx. Another clear trend was that the level of 0 and 1 CA in the wild type 2.12.1.fx was markedly decreased in the 2.12.1.fx-[CLκ-D77A] mutant for each of the activated esters, and that the level of 2 CA was increased in each case for most activated esters, except for Z9.
The results of the reduced LC/HC analyses showed a further obvious trend comparing the 2.12.1.fx and 2.12.1.fx-[CLκ-D77A] conjugation results. In each case, the degree of underivatized LC decreased, substantially in some cases. This was accompanied by a concomitant increase in the level of 1 CA on the LC, again for each different active ester, so that overall the average amount of derivatization on the LC increased. The general trend for the LC was that the amount of 1CA increased by the amount that OCA decreased, as the amount of 2CA present in each case was essentially unchanged.
In considering the HC, another trend was apparent in that the already low amount of 1CA derivatization for each active ester was further decreased. The outlier in this trend was Z9, the only non-phenolic ester. This ester shows little of the directional conjugation effect towards CLκ-K80 compared to the other phenolic esters and the levels of both LC and HC derivatization are similar, with only a minor improvement in directionality imparted by the 2.12.1.fx-[CLκ-D77A] mutant. Overall, the 2.12.1.fx-[CLκ-D77A] mutant provides clear evidence of improved directional LC conjugation compared to native 2.12.1.fx fora range of activated esters







TABLE 56







Analysis of 2.12.1.fx.-[CLκ-D77A] conjugation to [PEG5-K11-SEQ: 27]


using different reactive esters (see Examples 26 and 27). Ab % CA shows


the overall % of conjugation additions per antibody, with reduced light chain and


heavy chain analysis also shown (LC % CAN, HC % CA). and Δ indicates the


difference between the WT and D185A mutant results for Ab % CA. The % of 1-LC %


relative to the respective WT run is shown in the right column, as described in Table 53.




















Ab % CA
Avg

LC %
Avg.



Avg.
1LC
























Z#
Ab
0
1
2
3
4
CA
Δ
0
1
2
CA
0
1
2
CA
WT %



























1
WT
1
17
51
25
5
2.16
0.07
14
77
9
0.96
84
16

0.16




D77A
4
6
60
23
7
2.23

0
86
13
1.13
92
8

0.08
112


2
WT
15
43
33
9

1.36
0.72
49
49
3
0.54
84
16

0.16




D77A
7
14
53
19
8
2.09

16
81
3
0.87
90
10

0.1
33


10
WT
23
44
26
7

1.18
0.73
60
38
2
0.42
81
19

0.19




D77A
6
27
43
18
6
1.91

29
67
4
0.74
89
11

0.11
176


3
WT
7
32
39
18
5
1.82
0.43
44
48
7
0.63
79
21

0.21




D77A
6
11
46
25
12
2.25

12
79
9
0.97
90
10

0.1
165


6
WT
15
46
30
9

1.34
0.71
41
52
7
0.66
88
12

0.12




D77A
6
21
44
20
9
2.05

18
73
8
0.9
93
7

0.07
140


9
WT
7
29
35
21
8
1.94
0.26
57
34
9
0.51
56
34
10
0.54




D77A
10
19
29
27
15
2.20

47
44
9
0.63
61
32
7
0.46
129









Example 30 Trastuzumab (Herceptin®) Conjugation
In order to confirm that the improved directional conjugation to CLκ-K80 caused by CLκ-D77 mutation can be applied to other antibodies comprising CLκ, D77A mutation was also inserted to the CLκ of trastuzumab (hTrast). Trastuzumab light chain and heavy chain DNA were synthesized based on the amino acid sequences on Drug Bank, Accession Number DB00072 (BIOD00098, BTD00098).
hTrast-[CLκ-D77A] mutant was generated in two steps. First, D77A mutation was generated on an antibody light chain following protocols described in QuickChange site-directed mutagenesis kit (Stratagene®). Mutations were introduced by oligonucleotide primers and confirmed by DNA sequencing. The VL of trastuzumab was ligated with the CL of the antibody with D77A mutation. Primer pair TRAST.VL.FOR (SEQ ID NO:89) and TRAST.VL.REV (SEQ ID NO:90) were used to amplify trastuzumab VL. The PCR fragment was digested with BglII and BsaI. Primer pair TRAST.CL.D185A.FOR (SEQ ID NO:91) and TRAST.CL.D185.A.REV (SEQ ID NO:92) were used to amplify CL with D77A mutation. The resulting PCR fragment was digested with BsaI and NheI. Restriction enzyme digested PCR fragments were ligated with a modified p2.12.1.fxP4 plasmid (Invitrogen®) cut with BglII and NheI. The insert sequence was confirmed by DNA sequencing. The mutated mAb was transiently expressed in HEK 293 cells, and purified using protein A affinity column. The purified mAb was characterized using MS.
Trastuzumab and hTrast-[CLκ-D77A] (1 mg reaction size) were adjusted 18 mg/ml to pH 7.7 with a phosphate buffer to a final concentration of 0.06 M sodium phosphate. [PFP-PEG5-K11-SEQ:27] was reconstituted in a propylene glycol solution to 10 mg/ml. The peptide/linker was added to antibody at a molar ratio of 4.3:1 and allowed to react for 2 hrs at RT.
Table 57 compares the conjugation profile of trastuzumab-[PEG5-K11-SEQ:27] with hTrast-[CLκ-D77A]-[PEG5-K11-SEQ:27]. The conjugation profile of trastuzumab-[PEG5-K11-SEQ:27] occurs as a distribution between 0-4 peptide additions with the average number of peptide additions being 1.75. The profile changes following the D77A mutation; the average number of peptide additions rises to 2.18 and significantly less overall levels of 0 and 1 peptide addition is observed. This result suggests that the single point mutation CLκ-D77A has the effect of increasing the overall conjugation to the scaffold, as seen in the test antibody 2.12.1.fx.
The reduced light and heavy chain analysis demonstrates that the average conjugation is higher on the light chain of hTrast-[CLκ-D77A] than unmodified trastuzumab; the average light chain conjugate addition value for hTrast-[CLκ-D77A] is 1.01 compared to 0.70 for trastuzumab. In addition, unconjugated light chain is significantly reduced in hTrast-[CLκ-D77A]. Conjugation on the heavy chain is observed at a significantly lower level. The majority of observed heavy chain for both trastuzumab and hTrast-[CLκ-D77A] is unconjugated; this is especially true in the case of hTrast-[CLκ-D77A] heavy chain. These results suggest that the CLκ-D77A mutation alters the light chain to make it significantly more susceptible to conjugation.







TABLE 57







Analysis of conjugation of [PEG5-K11-SEQ: 27] to Abs trastuzumab


(WT) and hTrast-[CLκ-D77A]. Rep = replicate. AvReps is the average of the results 


of the three replicate experiments, with the standard deviation shown beneath


(StdDev). Ab % CA shows % conjugations additions per antibody, followed by the


average CA per antibody. Reduced light and heavy chain analysis also shown.













Ab
Ab % CA
Avg.
LC % CA
Avg.
HC % CA
Avg.




















WT
0
1
2
3
4
CA
0
1
2
CA
0
1
CA























Rep1
6
33
42
15
5
1.80
35
65
ND
0.65
85
15
0.15


Rep2
7
34
39
14
6
1.79
26
74
ND
0.74
84
16
0.16


Rep3
9
38
38
11
5
1.65
30
70
ND
0.7
82
18
0.18


Av. Rep
7
35
40
13
5
1.75
30
70

0.70
84
16
0.16


StdDev
2
3
2
2
1
0.08
5
5

0.05
2
2
0.02


D77A






5
92
3
0.99
94
6
0.06


Rep1
4
5
66
20
6
2.19
1
94
5
1.04
91
9
0.09


Rep2
3
5
65
21
6
2.22
3
95
2
0.99
94
6
0.06


Rep3
3
8
69
17
4
2.12
3
94
3
1.01
93
7
0.07


AvRep
3
6
67
19
5
2.18
2
2
2
0.03
2
2
0.02


StdDev
1
2
2
2
1
0.05
5
92
3
0.99
94
6
0.06









Example 31 Conjugation of Trastuzumab with MMAD
Table 58 compares the conjugation profile of trastuzumab-[PEG5-MMAD] (Auristatin derivative) with hTrast-[CLκ-D77A]-[PEG5-MMAD].




The conjugation profile of trastuzumab-[PEG5-MMAD] occurs as a distribution between 0-4 conjugations per antibody with the largest form being 2 conjugations and the average number of conjugations is 1.65. When CLκ-D77A is mutated, the average number of conjugations rises to 2.00 and significantly less overall levels of 0 and 1 MMAD addition is observed. This result suggests that the single point mutation CLκ-D77A has the effect of increasing the overall conjugation to the scaffold and that this technology is applicable to an antibody toxin conjugation model.
Reduced heavy and light chain analysis demonstrates that the average conjugation is higher on the light chain of hTrast-[CLκ-D77A] than unmodified trastuzumab; the average light chain conjugate addition value for hTrast-[CLκ-D77A] is 0.88 compared to 0.56 for trastuzumab. In addition, unconjugated light chain is significantly reduced in hTrast-[CLκ-D77A]. Conjugation on the heavy chain is observed at a significantly lower level. The majority of observed heavy chain for both trastuzumab and hTrast-[CLκ-D77A] is unconjugated; this is especially true in the case of hTrast-[CLκ-D77A] heavy chain. These results suggest that the CLκ-D77A mutation alters the light chain to make it significantly more susceptible to conjugation.







TABLE 58







Analysis of [PEG5-MMAD] conjugation to trastuzumab (WT) and


hTrast-[CLκ-D77A], also showing reduced LC and HC analysis. Ab % CA shows %


conjugations additions per antibody, followed by the average CA per antibody.


Reduced light and heavy chain analysis also shown. The % of 1-LC % relative to the


respective WT run is shown in the right column, as described in Table 53.





















hTrast




























conjugated
Percent CA
Avg.
LC % CA
Avg.
HC % CA
Avg.
1LC





















to MMAD
0
1
2
3
4
CA
0
1
2
CA
0
1
CA
WT %
























WT
8
35
42
13
2
1.65
45
54
1
0.56
76
23
0.23



D77A
1
16
65
16
1
2.00
11
88

0.88
84
14
0.14
163









Example 32 Ability of Conjugated Trastuzumab to Bind Targets
The ability of trastuzumab and hTrast-[CLκ-D77A], unconjugated and conjugated to either [PEG5-K11-SEQ:27] or [PEG5-MMAD] and to bind to the Her2 receptor was studied using a Her2 binding ELISA assay. Half well ELISA plates were coated with 1 ug/ml of Fc-ErbB2 fusion protein in PBS and incubated at 4° C. overnight. Plates were washed 3 with KPL wash buffer and subsequently blocked with Superblock for 1 hr at RT. 10× serial dilutions of samples were prepared in Superblock, with a top concentration of 100 μg/ml. Samples were added to wells and plates were incubated for 1 hr at RT. Plates were washed 3× with KPL wash buffer. Bound samples were detected by incubating with a 1:1000 dilution of anti-human Fab-HRP secondary antibody for 1 hr at RT. Plates were again washed 3× with KPL wash buffer and HRP was detected with TMB substrate. The reaction was stopped with 2M H2SO4 and OD was measured at 450 nm on a Spectramax plate reader.
FIGS. 9A and 9B demonstrate that commercial trastuzumab, trastuzumab generated from the available sequence, and hTrast-[CLκ-D77A], as well as trastuzumab and hTrast-[CLκ-D77A] when bound to either of [PEG5-K11-SEQ:27] or [PEG5-MMAD] each display similar Her2 binding characteristics. These result suggest that conjugation, primarily at CLκ-K80, does not significantly interfere with the receptor binding function of the native antibody.
Example 33 Comparison of PFP and NHS Conjugation Strategies
Trastuzumab was conjugated to [PEG5-MMAD] using two separate strategies: directional conjugation to CLκ-K80 using PFP ester (Z1) as the Z* group (generating trastuzumab-[5PEG-MMAD]), or NHS (Z13, generating trastuzumab-[MMAD]n), which resulted in a wider conjugation pattern across the antibody, and dosed to rats to compare the tolerability of the antibody drug conjugates. Both conjugates were given as 10, 30 and 100 mg/kg single bolus doses. All animals dosed at 10, and 30 mg/kg doses of both conjugates during the one week study period survived without significant body weight loss. However, the 100 mg/kg dose group showed a clear difference between random conjugation (Z13) and site selective conjugation to CLκ-K80 (Z1). Greater than 50% of the animals in 100 mg/kg dose of the random conjugate (NHS conjugation) died within the one week study period while all animals in the 100 mg/kg dose of the site selective conjugate (PFP conjugation) survived without significant body weight loss (Table 59). This may suggest that preferential conjugation at CLκ-K80 may provide a more reliable mechanism for conjugation of Effector Moieties then traditional ‘random’ approaches, as conjugation on multiple surface lysine residues may give rise to Effector Moieties that have less reliable cleavage and degradation patterns.







TABLE 59







Site selective conjugation of toxin improves the tolerability


of the antibody drug conjugates. Comparison of tolerability


of trastuzumab -[PEG5-MMAD] in rats after conjugation


using Z1 and Z13 as Z* groups.











Conjugation
Dose
%


Conjugate
type
(mg/Kg)
Survival













Trastuzumab-[PEG5-
PFP
10
100


MMAD]

30
100




100
100


Trastuzumab-[MMAD]n
NHS
10
100




30
100




100
50









Example 34 h38C2 Conjugated with Toxin and Cleavable Linker
A targeting peptide was conjugated to the combining site of a CLκ-D77A mutated version of catalytic antibody h38C2 (HC=SEQ ID NO:65 and LC=SEQ ID NOs:37 and 67) using a linker of the formula P-Q-W as herein described, with a β-lactam group as the W group to form a covalent attachment with the side chain of K99 of SEQ ID NO:65. This conjugated antibody was then further conjugated with the PFP-activated ester of an exemplary Auristatin-based toxin attached to a valine-citrulline p-aminobenzyl carbamate cleavable linker ([PFP-PEG2-ValCitABC-TOXIN]).




A distribution of conjugates was observed with primarily 2-3 toxins per antibody scaffold (Table 60). In vitro cytotoxicity assays of this conjugate demonstrated potent anti-proliferative effects in AU565 cell lines (IC50=0.4 nM) and OVCAR5 cell lines (IC50=0.2 nM).







TABLE 60







Conjugation profile of h38C2 conjugated to 


[PFP-PEG2-ValCitABC-TOXIN].










Conjugation Additions 




(CA) (%)
















0
1
2
3
4
5
Avg CA

















PFP-PEG2-ValCitABC-TOXIN
4
8
31
29
18
10
2.8









Example 35
A structural analog of PFP, with a trifluoromethyl group replacing the para-fluorine atom, was used to make a derivative Z* group; 2,3,5,6-tetrafluoro-4-(trifluoromethyl)phenyl (Z16):




This was used to generate a Z16-PEG5 linker, which was conjugated to the test peptide SEQ ID NO:27, and tested in conjugation with the test antibody 2.12.1.fx. In contrast to other Z* groups tested, this derivative gave a conjugate with a slightly higher level of conjugation for the native 2.12.1.fx compared to 2.12.1.fx-[CLκ-D77A], but both levels of intact conjugation were higher for the conjugate using the Z16 group than the corresponding PFP (Z1) analogs (around 10% increase in overall average CA compared to Z1). Using Z16, both native 2.12.1.fx and 2.12.1.fx-[CLκ-D77A] conjugations showed an equivalent level of 1CA, and this was lower than that observed for native 2.12.1.fx conjugated with [PFP-PEG5-K11-SEQ:27]: for other conjugations, the level of 1CA is typically reduced in the D77A compared to the corresponding native antibody. Overall, the results suggest that the leaving group D16 is more reactive than Z1 (PFP) (Table 61).

The Z16 leaving group shows roughly equivalent derivatization for both the native 2.12.1.fx and 2.12.1.fx-[CLκ-D77A] antibodies and the amount of underivatized LC is small in both cases. Again the overall level of LC and HC derivatization is increased using Z*16 compared to Z1. The leaving group Z16 appears a more reactive ester than PFP, but it is possible that the CF3 group is providing an additional interaction near the CLκ-K80 region that is also driving reactivity and preferential derivatization of the LC







TABLE 61







Analysis of 2.12.1.fx and 2.12.1.fx.-[CLκ-D77A] conjugation to


[PEG5-K11-SEQ: 27] using different reactive Z* groups Z1 and Z16. Ab % CA shows


the overall % of conjugation additions per antibody, with reduced light chain and


heavy chain analysis also shown (LC % CA, HC % CA). Δ indicates the difference


between the WT and D185A mutant results for Ab % CA. The % of 1-LC % relative to


the respective WT run is shown in the right column, as described in Table 53.


































Avg
HC %
Avg



















Ab % CA
Avg

LC % CA
CA-
CA
CA-
1LC























Z*
2.12.1.fx
0
1
2
3
4
CA
Δ
0
1
2
LC
0
1
HC
WT %


























1
WT
1
17
51
25
5
2.16

14
77
9
0.96
84
16
0.16



1
D77A
4
6
60
23
7
2.23
0.07
0
86
13
1.13
92
8
0.08
112


16
WT
4
4
44
36
12
2.48

5
83
12
1.07
81
19
0.19



16
D77A
9
9
31
33
18
2.43
0.05
1
79
20
1.2
89
11
0.11
95









Example 36 Synthesis of Toxin 0101
Experimental for Toxin 0101 (#54 in the Schematic)
Preparation of 2-Methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (#54)




Step 1. Synthesis of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-2-methylalanyl-N-R3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (#53). According to general procedure D (below), from #32 (2.05 g, 2.83 mmol, 1 eq.) in dichloromethane (20 mL, 0.1 M) and N,N-dimethylformamide (3 mL), the amine #19 ((2R,3R)-3-methoxy-2-methyl-N-[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]-3-[(2S)-pyrrolidin-2-yl]propanamide, trifluoroacetic acid salt) (2.5 g, 3.4 mmol, 1.2 eq.), HATU (1.29 g, 3.38 mmol, 1.2 eq.) and triethylamine (1.57 mL, 11.3 mmol, 4 eq.) was synthesized the crude desired material, which was purified by silica gel chromatography (Gradient: 0% to 55% acetone in heptane), producing #53 (2.42 g, 74%) as a solid. LC-MS: m/z 965.7 [M+H+], 987.6 [M+Na+], retention time=1.04 mins; HPLC (Protocol A): m/z 965.4 [M+H+], retention time=11.344 mins (purity >97%), 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signals: δ 7.86-7.91 (m, 2H), [7.77 (d, J=3.3 Hz) and 7.79 (d, J=3.2 Hz), total 1H], 7.67-7.74 (m, 2H), [7.63 (d, J=3.2 Hz) and 7.65 (d, J=3.2 Hz), total 1H], 7.38-7.44 (m, 2H), 7.30-7.36 (m, 2H), 7.11-7.30 (m, 5H), [5.39 (ddd, J=11.4, 8.4, 4.1 Hz) and 5.52 (ddd, J=11.7, 8.8, 4.2 Hz), total 1H], [4.49 (dd, J=8.6, 7.6 Hz) and 4.59 (dd, J=8.6, 6.8 Hz), total 1H], 3.13, 3.17, 3.18 and 3.24 (4 s, total 6H), 2.90 and 3.00 (2 br s, total 3H), 1.31 and 1.36 (2 br s, total 6H), [1.05 (d, J=6.7 Hz) and 1.09 (d, J=6.7 Hz), total 3H]. Step 2. Synthesis of 2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (#54)
According to general procedure A (below), from #53 (701 mg, 0.726 mmol) in dichloromethane (10 mL, 0.07 M) was synthesized the crude desired material, which was purified by silica gel chromatography (Gradient: 0% to 10% methanol in dichloromethane). The residue was diluted with diethyl ether and heptane and was concentrated in vacuo to afford #54 (406 mg, 75%) as a white solid. LC-MS: m/z 743.6 [M+H+], retention time=0.70 minutes; HPLC (Protocol A): m/z 743.4 [M+H+], retention time=6.903 minutes, (purity >97%); 1H NMR (400 MHz, DMSO-d6), presumed to be a mixture of rotamers, characteristic signals: δ [8.64 (br d, J=8.5 Hz) and 8.86 (br d, J=8.7 Hz), total 1H], [8.04 (br d, J=9.3 Hz) and 8.08 (br d, J=9.3 Hz), total 1H], [7.77 (d, J=3.3 Hz) and 7.80 (d, J=3.2 Hz), total 1H], [7.63 (d, J=3.3 Hz) and 7.66 (d, J=3.2 Hz), total 1H], 7.13-7.31 (m, 5H), [5.39 (ddd, J=11, 8.5, 4 Hz) and 5.53 (ddd, J=12, 9, 4 Hz), total 1H], [4.49 (dd, J=9, 8 Hz) and 4.60 (dd, J=9, 7 Hz), total 1H], 3.16, 3.20, 3.21 and 3.25 (4 s, total 6H), 2.93 and 3.02 (2 br s, total 3H), 1.21 (s, 3H), 1.13 and 1.13 (2 s, total 3H), [1.05 (d, J=6.7 Hz) and 1.10 (d, J=6.7 Hz), total 3H], 0.73-0.80 (m, 3H).
General Procedure A: Fmoc removal using diethylamine. To a solution of the Fmoc-containing compound in dichloromethane was added an equal volume of diethylamine. Reaction progress was monitored by LC-MS (or HPLC or TLC); the reaction was usually completed within three hours. Solvents were removed in vacuo, and the residue was azeotroped three times with heptane. The residue was then diluted with dichloromethane and a small amount of methanol before being reduced down onto silica and purified by chromatography on silica gel, eluting with methanol in dichloromethane to afford the desired material.
General Procedure D: coupling with O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU). To a stirring solution of the amine (1 eq.) and acid (1.1 eq.) in dichloromethane: N,N-dimethylformamide (4:1, 0.3 M in amine) was added HATU (1.2 eq.) followed by Et3N (3 eq.). Reaction progress was monitored by LC-MS (or HPLC or TLC), the reaction was usually completed within three hours. Solvents were removed in vacuo. The residue was azeotroped three times with heptane and was diluted with a small amount of ethyl acetate before being reduced down onto silica and purified by silica gel or reverse phase chromatography.
Example 37 Preparation of MAC Comprising mAb Hu08 and Toxin 0101
Hu08 is a human anti-IL-13Rα2 antibody, and is described fully in U.S. 61/723,545, whose contents are herein incorporated by reference.
A mutant version of hu08, comprising the CLκ-D77A mutation was generated according to standard protocols (hu08-[CLκ-D77A]). Toxin-0101 (#54: Example 36) was conjugated with a cleavable linker to form the structure:




and then the toxin-linker was conjugated to hu08-[CLκ-D77A] according to the techniques described herein. The compound hu08-vc-0101 was generated, resulting in non-specific conjugation of multiples of Toxin-0101 on the antibody hu08 via the linker-Toxin-0101 species:





In Vitro Cytotoxicity Assay

Cell lines expressing the IL-13Rα2 antigen and a negative control cell line were cultured with increasing concentrations of hu08-[CLκ-D77A]. After four days, viability of cultures were assessed. IC50 values were calculated by logistic non-linear regression and are presented as ng Ab/mL.
The data demonstrate that hu08-vc0101 and hu08-[CLκ40, -D77A] were both effective against both of the IL-13Rα2 positive cell lines tested (PC3MM2 and A375), having an IC50 ranging from 2.5 to 7.9 ng Ab/mL (Table 62). Neither hu08-vc0101 nor hu08-[CLκ-D77A] were active against the IL-13Rα2 negative cell line, H460, and the non-IL-13Rα2 binding control, hIgG8.84-vc0101, was not active against any of the cell lines tested.







TABLE 62







In vitro cytotoxicity assay of hu08-[CLκ-D77A] -0101.









IC50 (ng Ab/mL)











ADC
Toxin: Ab
PC3MM2
A375
H460














hu08-vc-0101
3.2
2.5
3.8
>400000


hu08-[CLκ-D77A] -0101
1.9
4.9
7.9
>400000


hIgG8.8-vc-0101
3.7
>400000
>400000
>400000









Subcutaneous Xenograft Models of Cys Mutant ADCs

Female, athymic (nude) mice were injected s.c. with PC3MM2 tumor cells. Mice with staged tumors, approximately 0.1 to 0.3 g (n=8 to 10 mice/treatment group) were administered intravenously q4d×4 with normal saline (vehicle) or MAC-0001. Compounds were dosed based on Ab content. Tumors were measured at least once a week and their size (mm2+/−□SEM) is calculated as mm3=0.5×(tumor width2)×(tumor length). The data in Table 63 indicate that hu08-[CLκ-D77A]-0101 inhibits the growth of PC3MM2 xenografts.









TABLE 63








Dose
PC3MM2 xenograft, tumor volume (mm3 ± SEM)



















single
Day
Day
Day
Day
Day
Day
Day
Day
Day
Day


ADC 
dose
0
5
8
12
15
20
30
41
55
77





Vehicle
0
325 ±
590 ±
782 ±
1140 ±
GT
GT
GT
GT
GT
GT




9
41
79
142








hu08-[CLκ-
1.5
328 ±
393 ±
352 ±
432 ±
556 ±
732 ±
GT
GT
GT
GT


D77A]-0101

49
96
106
124
236
305






hu08-vc-
1.5
333 ±
431 ±
281 ±
299 ±
362 ±
450 ±
956 ±
GT
GT
GT


0101

12
40
25
32
47
58
166





GT = group terminated due to large tumor size






Example 38 Trastuzumab MMAD Conjugate Activities
Three trastuzumab conjugates were made: trastuzumab-[5PEG-MMAD], hTrast-[CLκ-D77A]-[5PEG-MMAD], and trastuzumab-(MMAD)n, where MMAD was connected to a 5PEG linker with Z13 (NHS) as leaving group, and conjugated to trastuzumab without directional conjugation techniques, resulting in non-specific conjugation of MMAD to trastuzumab surface lysines (see Examples 30-33)




The three mAb conjugates were evaluated in an exploratory toxicity study in rats in which animals received single intravenous bolus doses of each ADC at 0 (vehicle), 10, 30, and 100 mg/kg (5 male rats/group) and were then observed for 14 days. Toxicology evaluation included daily clinical observations, weekly body weight measurements and clinical pathology evaluation on the day of necropsy. Animals were euthanized on day 15 and selected tissues were collected for microscopic examination. In addition, blood samples were collected from all animals at approximately 0.0833 (5 min), 6, 24 (day 2), 48 (day 3), 72 (day 4), 96 (day 5), 168 (day 8), and 312 (day 14) hrs post-dose and analyzed for antibody-conjugate and trastuzumab antibody concentrations.
Results
The plasma exposures (based on AUC) were overall similar for all 3 conjugates at any given dose. The AUC(0-312) of hTrast-[CLκ-D77A]-[5PEG-MMAD], trastuzumab-[5PEG-MMAD] and trastuzumab-(MMAD)n at 100 mg/kg were 177000, 174000 and 139000 ng·h/mL, respectively. hTrast-[CLκ-D77A]-[5PEG-MMAD] and trastuzumab-[5PEG-MMAD] were clinically well tolerated at all doses. However, trastuzumab-(MMAD)n administration at 100 mg/kg was associated with marked clinical signs and premature euthanasia of 1⁄5 rats on Day 8. Other rats from this group had decreased skin turgor and decreased body weight gain.
Clinical pathology changes were overall similar with hTrast-[CLκ-D77A]-[5PEG-MMAD] and trastuzumab-[5PEG-MMAD] and included in particular mild decreases in red blood cell (RBC) mass (RBC count, hemoglobin and/or hematocrit) at ≥10 or ≥30 mg/kg and minimal increases in aspartate aminotransferase (AST) at 100 mg/kg. The RBC mass changes were more pronounced with trastuzumab-(MMAD)n and were associated with decreased erythroid cellularity in the bone marrow. Other noteworthy trastuzumab-(MMAD)n-related clinical pathology changes at 100 mg/kg included moderate decreases in platelet counts and mild increases in ALT, AST, ALP and total bilirubin.
Microscopic findings were overall similar for hTrast-[CLκ-D77A]-[5PEG-MMAD] and trastuzumab-[5PEG-MMAD] and included alveolar histiocytosis/inflammation in the lung, degeneration of small bile ducts in the liver and increased tingible body macrophages (containing cell debris) in the bone marrow. Increased mitoses in several tissues and single cell necrosis in the cornea were considered pharmacologically mediated effects of tubulin inhibition resulting in mitotic arrest and apoptosis. By contrast, trastuzumab-(MMAD)n administration was associated with more pronounced microscopic tissue alterations, which included tubular degeneration/necrosis and glomerulopathy in the kidney; single cell necrosis, bile duct degeneration and hyperplasia and centrilobular necrosis/fibrosis in the liver; alveolar histiocytosis/inflammation in the lung; increased tangible body macrophages, degeneration/decreased numbers of hematopoietic cells and osteolysis in the bone marrow; decreased marginal zone cellularity in the spleen. Of special note, centrilobular fibrosis in the liver in association with disruption of the normal lobular architecture was consistent with a reparative change suggesting earlier, more extensive treatment-related hepatocellular damage. In addition, pharmacologically mediated increased mitoses and/or single cell necrosis were observed in several tissues.
In summary, hTrast-[CLκ-D77A]-[5PEG-MMAD] and trastuzumab-[5PEG-MMAD] were well tolerated at all doses (10, 30, and 100 mg/kg) and demonstrated overall similar toxicity profiles. Trastuzumab-(MMAD)n administration led to premature mortality at 100 mg/kg and was associated with significant target organ toxicities in the liver, kidney, lung and bone marrow in particular.
Example 39 Modeling of CLκ and CLλ
FIG. 10A depicts an Ig fold of a constant light domain containing a 3-stranded β-sheet packed against a 4-stranded β-sheet. The fold is stabilized by hydrogen bonding between the β-strands of each β-sheet, by hydrophobic bonding between residues of opposite β-sheets in the interior, and by a disulfide bond between the β-sheets. The 3-stranded β-sheet comprises β-strands C, F, and G, and the 4-stranded β-sheet has β-strands A, B, E, and D. The letters A through G denote the sequential positions of the β-strands along the amino acid sequence of the Ig fold. Linking each β-strand with the subsequent β-strand is an amino acid connecting chain that may or may not comprise a turn (A/B) or α-helix (E/F) (FIG. 10B).
FIG. 8B plots the secondary structures along the primary sequence of the mouse and human CLκ, and the human CLλ. The EF connecting chain between β-strands E and F of the CLκ and CLλ have identical secondary structure and consist of a 5-6 residue α-helical region (CLκ-K75-K80, and CLλ-P78-S81), followed by a 2-3 amino-acid turn (CLκ-H81-K82 and CLλ-H82-S84) (FIGS. 8B and 10). The CD connecting chain brings the side chain of structurally equivalent aspartic acids (CLκ-D53, CLλ-D45) to the vicinity of the EF chain (approximately 3.5 Å), and allows CLκ-D53 interact with the imidazole ring of CLκ-H81.
Modeling of the CLλ and comparison with the CLκ suggests that CLλ-D45 cooperates with the imidazole ring of CLλ-H82 in the same manner, and that mutating CLλ-S81 to CLλ-K81 recreates a similar local environment as found on the CLκ, and so allows directional conjugation to the CLλ.
Accordingly, the present invention also provides for CLλ domains comprising one of the following mutations: CLλ-S81K, and CLλ-K80x/S81K, wherein x is any amino acid except P, K, R or H, wherein the numbering is according to SEQ ID NO:93. In some aspects, the invention provides for novel CLλ domains comprising K81, or x80/K81, wherein x is one of G, A, I, L, V, S, T, M, N, Q, F, Y, W, D, or E. In some aspects, the invention also provides for a CLλ domain comprising a sequence selected from the group consisting of SEQ ID NO:94, and SEQ ID NO:95.
Modeling also suggests that CLλ-E77 would be available to form a salt bridge with either of CLλ-K80 or CLλ-K81 in the same manner as CLκ-D77 appears to form a salt bridge to CLκ-K80. Thus, mutating CLλ-E77 to any of R, L, S, G, Q, P, N, V, I, T, and M is likely to facilitate directional conjugation at either of CLλ-K80CLλ when S81Δ (i.e. deletion of S81), or CLλ-K81. Accordingly, the present invention also provides for a CLλ domain comprising a sequence selected from the group consisting of SEQ ID NO:96 and SEQ ID NO:97.
Example 40 Modeling of CLκ-K80 Conjugation Mechanism
Structure and Sequence Description
Crystal structures of the Fab domain of 2.12.1.fx and h38C2-[CLκ-D77A] were used to model the specificity of CLκ-K80 reactivity towards a halo-phenol (such as PFP)/ester-mediated conjugation. Experience with conjugations across multiple and varied antibodies, as well as early modeling analysis, indicated that modeling only needed be focused on the CLκ, as the remainder of the antibody appeared to exert very little, if any, influence on the mechanism of conjugation.
Computational Approach
The goal of the computational calculations was to clarify the critical attributes of CLκ-K80 and interacting residues that preferentially bias this site towards the PFP-ester conjugation reaction. After the 3-D coordinates of CLκ and CLκ-D77A were selected from the crystal structure (based on those coordinates that contained those residues already identified as being relevant to directional conjugation), the coordinates were subjected to standard computational protocols, such as protein preparation, attachment of hydrogen atoms, and force field parameters assignment.
Hydrogen atoms were assigned to all amino acid atoms on the respective 3D CLκ domain according to the calculated pKa values and pI (protein ionization potential). pKa is the value of a protonation state of a given titratable amino acid at the neutral pH (negative logarithm of hydrogen concentration) taking in to account the influence of amino acids in the protein chain (Spassov; A fast and accurate computational approach to protein ionization. Protein Science 2008, 17, 1955-1970). The results of these calculations are shown in Table 64.







TABLE 64







pKa values of amino acid residues in CLκ











Titratable residues
Calculated pKa
Standard pKa















CLκ-D14
3.315
3.65



CLκ-D43
3.324
3.65



CLκ-D59
3.667
3.65



CLκ-D62
4.411
3.65



CLκ-D77
3.307
3.65



CLκ-E15
3.93
4.25



CLκ-E35
3.906
4.25



CLκ-E53
4.604
4.25



CLκ-E57
4.284
4.25



CLκ-E79
3.636
4.25



CLκ-E89
4.244
4.25



CLκ-Y32
10.276
10.07



CLκ-Y65
11.847
10.07



CLκ-Y78
11.844
10.07



CLκ-Y84
14
10.07



CLκ-H81
7.22
6.0



CLκ-H90
6.126
6.0



CLκ-K18
11.057
10.53



CLκ-K37
10.761
10.53



CLκ-K40
10.505
10.53



CLκ-K61
10.852
10.53



CLκ-K75
10.952
10.53



CLκ-K80
11.119
10.53



CLκ-K82
10.451
10.53



CLκ-K99
10.706
10.53



CLκ-R34
13.428
12.48



CLκ-R103
14
12.48










Hydrogen atoms are added accordingly to the pKa value for all titratable amino acids and for the remaining amino acids, according to the atomic valence number. The calculations indicated that CLκ-H81 is unprotonated at pH 7.0-7.4 (physiological pH). This observation is consistent with proposed mechanism for catalytic reaction, where CLκ-H81 acts as a nucleophilic catalyst. In general, conjugation would be expected to decrease with as the pH goes below about pH 6.5-7.0, as CLκ-H81 would be protonated. As the pH increases above about 7.4, a greater overall level of conjugation would be predicted, as other residues (especially lysines) become more reactive and contribute to the overall conjugation reaction, consequently we would expect that the directional effect would be increasingly lost as the pH rises. This correlates with what was observed in the pH study shown in Example 4, Table 4.
Minimization
CHARMm [Chemistry at HARvard Macromolecular Mechanics] is an energy minimization technique, and was used to bring the 3-D structures to the equilibrium position and find the best geometrical position for its atomic structure. CHARMm was used at the first step with the SMART Minimizer with 1000 steps of Steepest Descent minimization with a RMS gradient tolerance of 3[Kcal/(mol*Å)], followed by Conjugate Gradient minimization with a RMS gradient of 0.01. For the energy change, a tolerance of 3[Kcal/(mol*Å)] was applied to the average gradient during a cycle of minimization. The Steepest Descent method takes the molecule to the nearest minimum and the Conjugated Gradient improves the final conformation obtained. Momany Rone charges were used, (as described in Momany & Rone; Validation of the general purpose QUANTA 3.2/CHARMm force field. Comp. Chem. 1992, 13, 888-900.) The minimized structure of CLκ differed by 1 Å when compared to the un-minimized structure.
Complex Between CLκ Domains and PFP-PEG2 
The respective complexes between a PFP ester and the CLκ and CLκ-D77A domains was built in silico to better understand each interaction. Structural analysis of the 3D CLκ domains by the Accelrys protocol Define and Edit Binding Site (Discovery Studio software version DS3.5) revealed a region that could be termed a ‘binding pocket’, located between the CD and EF connecting chains, and underneath CLκ-K80, as shown in FIG. 11. This pocket is facilitated by the amino acids located at CLκ-K75-E79 (EF α-helix on the EF connecting chain), CLκ-K80-K82 (EF loop on the EF connecting chain) and CLκ-V42 (CD loop on the CD connecting chain) giving the shape and the electrostatic properties required by the catalytic reaction.
To measure the pocket size, the Define and Edit Binding Site protocol was used on the previously minimized CLκ structure. This protocol requires defining the CLκ domain as a receptor to use on it the space filling method to calculate the cavity size and assess its suitability for binding of a molecule with a particular size, such as PFP. The binding site of a receptor can be represented in many ways, for example a sphere or a list of residues surrounding this sphere. To define a binding site, the receptor is first mapped to a grid. Grid points within a given distance of the receptor atoms are marked as occupied by the receptor, and thus undesirable as locations for ligand atoms. Two methods exist to identify a binding site. The first uses an “eraser” algorithm to identify sites based on the shape of the receptor. The second uses the volume occupied by a known ligand already positioned in an active site (Venkatachalam et al Flt: a novel method for the shape-directed rapid docking of ligands to protein active sites. J. Mol. Graph. Model 2003, 21, 289-307).
As a result of protocol execution, binding sites are identified as a set of points located on a grid that encompasses the molecule under consideration. This definition permits measurement of the size and shape of the binding site, which allows for its qualification as a binding site. PFP-PEG2 was docked manually to the site surrounded by amino acids experimentally identified as important for the catalytic reaction. These amino acids were CLκ-K80, CLκ-H81, CLκ-D43 and other amino acids within 10 Å distance from CLκ-K80 (shown in Table 54).
The initial complex between each CLκ domain and a PFP ester was minimized using the QM/MM hybrid method, applying the CHARMm force field for the CLκ domain and QM calculations for the PFP ester. The PFP ester was minimized prior to the placement using first CHARMm then the QM/MM approach in which the PFP ester is treated as a QM system. The structure was minimized using the CHARMm minimization protocol described above.
For the minimizations of the complex, a QM/MM hybrid protocol was used in which quantum mechanical (QM) calculations gave information about the electron densities change upon interaction with WT or mutated protein allowing us to capture the influence of the surrounding environment on the PFP ester electron densities and its susceptibility to the conjugation reaction. The QM/MM protocol is a hybrid method where the molecular system is divided into two regions: first, the central region PFP ester, to be treated by a QM calculation and second, the outer region CLκ domain, treated by molecular mechanics (MM) methods.
QM treatment brings a higher level of theory, enabling the modeling of additional phenomena compared to traditional forcefield techniques, for example, where a chemical reaction occurs or polarization effects play an important role. The remaining bulk of the structure is described using a forcefield CLκ. The way in which the two regions are allowed to interact and how the total QM/MM energy is evaluated define the specific QM/MM protocol employed. Energy calculated in this method is composed from three basic parts: the QM energy of the PFP ester (EQM), CD-kappa (EMM), and the interaction energy between these two systems (EQMMM). The formula Etot=EQM+EMM+EQM/MM is used.
As far as the coulomb interaction between the electronic density of the QM region and the forcefield point charges is concerned, QM/MM methods in this application always employ electronic embedding. This means that the forcefield atom's partial charges enter the QM calculation as an external potential, thereby polarizing the QM electronic density relative to a QM calculation in vacuo, and giving rise to an electrostatic interaction energy between the QM density and the point charges. Contrary to the handling of the electrostatic interactions in QM/MM methods, the van der Waals interactions are treated entirely at the classical level. This means that appropriate forcefield parameters must be determined for all atoms in the simulation. The van der Waals QM/MM interaction energy (and forces) is a part of the CHARMm simulation server energy and is listed as a separate term in the output file.
Calculations
The total effects observed from in vitro experiments during and after the conjugation can be modeled by computational techniques using stepwise approximations to describe such phenomenon as:

    
    
        1. PFP binding pocket size—binding of the PFP ester and direction.
        2. Protein stability—especially, the stability of a PFP binding pocket.
        3. Tautomerization of the imidazole ring of CLκ-H81.
        4. Directional PFP placement.
        5. Initial interaction between the PFP ester and CLκ.
        6. Reactivity of each of the catalytic amino acids.

Modeling Results

    
    


Identification of the binding pocket in the CLκ domain permitted modeling and docking-placement of the PFP ester. After the PFP ester was modeled as docked into the respective CLκ binding site, the 3-D structure of the complex was minimized using a hybrid method QM/MM. In this approach, the PFP ester was defined as a QM system and the CLκ domain was treated by a MM method. After minimization of the complex, a network of intermolecular interactions between the CLκ and the PFP ester was revealed. This network was formed by intermolecular hydrogen bonds, hydrophobic interactions, and 7-electron stacking. All of these forces together appear to be involved in formation of a network responsible for the directional placement and conjugation of the PFP ester to CLκ-K80. The minimized structure of the complex indicates that CLκ-H81 is an important catalytic amino acid.
It is known that the imidazole moiety in histidine sidechains can serve as a catalytic amino acid in enzymatic reactions, with the imidazole acting as a nucleophile and forming an acyl imidazole during the catalytic transition state [J Phys Chem B. 2011 Oct. 20; 115(41):11895-901. Epub 2011 Sep. 23]. It is also known that the imidazole ring of histidine can undergo tautomerization, depending on whether the Nδ or Nε atom bonds with H:




Spatial positioning of CLκ-H81 in the PFP binding pocket points to the requirement of Nδ tautomeric form of imidazole ring CLκ-H81 for nucleophilic attack on carbonyl carbon of the PFP ester. It also indicates a requirement for the electron lone pair at Nε to be in the same plane as the carbonyl carbon of PFP ester group. This is possible when the imidazole ring of CLκ-H81 is in the Nδ tautomeric form (see Scheme II). The tautomeric equilibrium can be controlled by the hydrogen bond interactions with the neighboring hydrogen acceptor amino acids. There are two aspartic acid residues, CLκ-D77 and CLκ-D43, in the vicinity of CLκ-H81 and both of them appear to control the tautomerization state of the CLκ-H81 imidazole ring.




FIG. 12 depicts the 3-D arrangement of CLκ-H81, CLκ-D43 and CLκ-D77, accompanied by the distances between the oxygen atoms of the aspartic acids and nitrogen atoms of the imidazole ring. Hydrogen bonding between CLκ-D43 and CLκ-H81 promote and stabilize the catalytically-active tautomeric form NO, while the hydrogen bond with CLκ-D77 will promote the catalytically-inactive form of CLκ-H81 NE. In addition to the hydrogen bonds formed between CLκ-H81 Nδ and CLκ-D77 Oδ (negatively charged), CLκ-H81 Nε and CLκ-D77 Oδ, CLκ-H81 Nδ can form a hydrogen bond with the carbonyl oxygen of the CLκ-D77 backbone. This last interaction stabilizes the catalytically-active conformation of CLκ-K81 NO, rendering it capable of catalysis. It is known from the mutational analysis and computational work discussed herein that the optimal conformation for CLκ-K81 reactivity involves hydrogen bonding with CLκ-D43 via Nδ, as this stabilizes an active conformation capable of the hydrogen Hε transfer from CLκ-K80 Nε. The two other CLκ-K81 tautomeric forms will therefore most likely be less active or inactive in the catalytic reaction.
Modeling the Effect of CLκ-D77A Mutation
As predicted from the WT model, in the [CLκ-D77A] domain, CLκ-H81 forms a hydrogen bond with CLκ-D43 via the imidazole Nδ atom, exposing electron pair at NE for the nucleophilic attack on carbonyl carbon of the ester group in the PFP-PEG2 molecule (Scheme III). FIGS. 13A, 13B and 13C depict a comparison of crystal structure modeled CLκ and CLκ-D77A domains, and illustrate the shift in spatial position of CLκ-H81 in the CLκ-D77A mutant.




Mutations to CLκ-D77 

Modeling suggests that CLκ-D77 mutations would have a significant impact on the conjugation rate of CLκ-K80. A series of QM/MM calculations were conducted on the CLκ domain complexed with PFP, where CLκ-D77 was mutated to all other natural amino acids (CLκ-D77C was excluded).
Modeling and experimental analysis suggests that CLκ-D77 may bind to PFP and change the unfavorable electron density on the ester atoms of PFP moiety involved in the catalytic reaction. The CLκ-D77A mutation would then improve the conjugation rate by allowing better distribution of the electron densities on the reacting atoms of PFP ester.
Analysis of the calculation results performed on the mutants of CLκ-D77 indicate that the best performing mutants (in terms of conjugation efficiency) are likely to be those small hydrophobic amino acids unable to form hydrogen bonds to the PFP moiety. Modeling data therefore suggests the mutations CLκ-D77G, CLκ-D77P, CLκ-D77M, CLκ-D77L, CLκ-D77I, CLκ-D77A, and CLκ-D77V would improve directional conjugation. As seen in Example 26, this is borne out by experimental testing. All CLκ 77 mutants with hydrophobic amino acids have no impact on the tautomeric equilibrium of CLκ-H81, thus CLκ-H81 remains in the Nδ tautomeric form and consequently conjugation rates improve.
Modeling also suggest that most hydrophilic amino acids are likely to result in higher protein stability than CLκ-D77 according to by QM/MM calculations; and points to the mutations CLκ-D77S and CLκ-D77T in particular, and also CLκ-D77Q, CLκ-D77N, CLκ-D77H and CLκ-D77R as potentially improving directional conjugation. As seen in Example 26, this is also borne out by experimental testing.
In addition to the interaction energies and the modifications of the charge distribution, the strongest interaction and the lower conjugation rates were observed for CLκ-D77F and CLκ-D77Y mutants. The model indicates that the side chains of these aromatic amino acids include directly into the binding pocket, and are likely to be involved in 7-electron stacking interactions with the PFP group and therefore change the directional placement of PFP moiety. In addition, aromatic amino acids can sterically hinder PFP, preventing a similar binding mode to that possible in the WT protein thus adversely impacting the directional placement of the PFP moiety and resulting in a decreased conjugation rate. Interestingly, the model suggests that the side chain of CLκ-D77W occupies a slightly different spatial position to the aromatic side chains of CLκ-D77F and CLκ-D77Y, with the CLκ-D77W indole group sitting outside the binding pocket and not predicted to interfere with the spatial access of a halo-phenyl ester to the binding pocket. The model also predicts that a conservative mutation, such as CLκ-D77E is likely to have a similar effect on the tautomeric form of CLκ-H81, but due to the larger side-chain, create additional steric interference in the binding pocket. Mutations to CLκ-D77C or CLκ-D77K are also predicted to interfere with the reactivity of the site.
CLκ-D77 favors the presence of the hydrogen atom on Nε of the imidazole ring, while CLκ-D77A favors maintaining the hydrogen at the position Nδ. Spatial distribution of amino acids in the site indicates the important function Nδ tautomeric form of CLκ-H81 in the catalytic reaction enabling nucleophilic attack by Nε at the carbonyl carbon of the ester group, further facilitating directional conjugation.
Example 41 Predictive Modeling Based on Model of CLκ and CLκ-D77A
The impact of mutations on protein stability and interaction strength with the PFP ester were analyzed to identify the most relevant and reactive mutants of CLκ. For this computational experiment, key amino acids within a 10A distance from Cα carbon of CLκ-K80 were selected and analysed for (except CLκ-K80 and CLκ-H81, given the unique requirement for each of the residues).
For many or most of the mutations modeled for PFP conjugation with the CLκ or CLκ-D77A, it will be understood that in certain applications, it may appropriate to substitute or retain a residue that would reduce PFP conjugation in the CLκ or CLκ-D77A, as it may be desirable to reduce or increase the pocket size; such as where larger or smaller halo-phenols are used as Z1 groups, or where the precise geometry of a specific immunoglobulin domain appears to merit such a feature.
CLκ-K75 
This residue does not appear to have any direct impact on the conjugation reactivity of CLκ-K80 in the native or CLκ-D77A mutant by QM/MM calculations.
CLκ-A76 
CLκ-A76 is located at the beginning of the α-helix, and lies and above the plane that contains the hydrogen-bonded carboxyl group of CLκ-D77 and the hydroxyl group of CLκ-S74. This location allows amino acids with a large sidechain to interact with CLκ-D77 and they may have a positive impact of the conjugation reaction, especially those with hydrophilic groups capable of hydrogen bond formation. Most amino acid substitutions at this residue would be expected to have little effect on conjugation, specifically CLκ-S74A, CLκ-S74D, CLκ-S74E, CLκ-S74I, CLκ-S74L, CLκ-CLκ-S74F, CLκ-S74W and CLκ-S74V. Other residues that could provide hydrogen bonding opportunities would be expected to enhance conjugation, namely CLκ-S74R, CLκ-S74N, CLκ-S74Q, CLκ-S74H, and CLκ-S74K, and to a lesser degree CLκ-S74S, CLκ-S74T and CLκ-S74Y. While residues that disrupt the α-helix may have a somewhat negative effect on directional conjugation to CLκ-K80, such as CLκ-S74G and CLκ-S74P, directional conjugation is unlikely to be abrogated, merely reduced. Introduction of a cysteine would present a risk of the potential to form aggregates in expression.
CLκ-Y78 
CLκ-Y78 is located on the α-helix, facing the opposite direction to the binding pocket. CLκ-Y78 makes a number of hydrophobic interactions with surrounding amino acids and supports the CLκ structure. Accordingly, smaller sidechains (Ala, Ser, Thr and Val) or those that affect the stability or formation of the α-helix (Gly, Pro) will be more likely to adversely affect the conjugation reaction to CLκ-K80, although as these mutations are not predicted to directly interfere with the CLκ-K80 reactivity, such mutations may not necessarily abrogate directional conjugation. Additionally, CLκ-Y78 appear to interact with the sidechain of CLκ-R103, thus hydrophobic or negatively-charged sidechains would be expected to favor conjugation to facilitate this interaction (Asn, Asp, Gln, Glu, Phe and Trp). Other sidechains would not be expected to effect the conjugation reaction (Arg, His, Ile, Leu, Lys and Met).
CLκ-E79 
Modeling suggests that the side chain of CLκ-E79 is pointing in the opposite direction from the binding site. In addition, CLκ-E79 appears to form a salt bridge with sidechain of CLκ-K75. Based on the modelling, it is postulated that most amino acid substitutions in this position would have little effect on the conjugation reaction (Asn, Asp, Gln, His, Met, Phe, Ser, Thr, Tyr and Trp). Small hydrophilic or charged residues would likely favour the conjugation reaction (Ala, Arg, Ile, Leu, Lys and Val), while those that affect the stability or formation of the α-helix (Gly, Pro) may adversely affect the conjugation reaction to CLκ-K80, without necessarily abrogating directional conjugation.
CLκ-V83 
This residue does not appear to have a direct impact on the conjugation reaction with CLκ-K80 by experimental data and QM/MM calculations.







TABLE 65







Summary of effect of mutations on certain CLκ residues


on directional conjugation to CLκ-K80.












A76
D77
Y78
E79

















G
0
+++
−−
−



P
0
+++
−−
−



A
WT
++
−
+



V
0
+
−
+



L
0
++
0
+



I
0
++
0
+



M
0
+++
0
0



F
0
−−−
+
0



W
0
+
+
0



Y
+
−−−
WT
0



T
+
+++
−
0



S
+
+++
−
0



N
++
++
+
0



Q
++
++
+
0



D
0
WT
+
0



E
0
−−
+
WT



H
++
++
0
0



R
++
++
0
+



K
++
−−
0
+










Additional Modeling

CLκ-V42 

CLκ-V42 is positioned at the end of β-strand C and the beginning of the CD loop. From a careful examination of the structure, it was observed that CLκ-V42 is located underneath the imidazole ring of CLκ-H81 (FIGS. 14 and 15). To help determine the nature of the interactions between CLκ-H81 and CLκ-V42, the x,y,z coordinates of the centroid for the imidazole ring were calculated, followed by measurement of the distance between the calculated centroid and the hydrogen atom Hδ of the CLκ-V42. This distance of 2.9 Å indicates direct interactions between the π-electrons on the imidazole ring and the hydrogen Hδ of CLκ-V42. This interaction therefore appears to strongly influence the optimal positioning of CLκ-H81 in the pocket and for the tautomeric equilibrium shift to the Nδ tautomer. Thus, while not essential, the presence of CLκ-V42 exerts a positive influence on directional conjugation. These analyses are borne out by experimental data, where it was found that CLκ-V42A mutation caused a decrease in the conjugation rate (Table 54). Modeling suggests that CLκ-V421 is likely to be able to assist in positioning the CLκ-H81 imidazole ring to favour directional conjugation, although the slightly larger side chain will reduce the overall binding pocket size. In many circumstances, this reduction of pocket size may not have an appreciable effect on the directional conjugation mechanics. CLκ-V42L may also be able to assist with positioning the CLκ-H81 imidazole ring.
CLκ-D43 
CLκ-D43 is located on the CD loop. In native CLκ, CLκ-D43 appears to interact with the backbone of CLκ-H81 and CLκ-K82, contributing to protein stability. No hydrogen bonds between Hδ of CLκ-H81 and the carboxylic group of CLκ-D43 were identified on the crystal structure of CLκ. This may suggest that CLκ-D43 exerts only a minimal influence on the tautomeric equilibrium of the Nδ catalytically active form of CLκ-H81. However, experimental analysis with 2.12.1.fx-[CLκ-D43A] mutants showed that mutating this residue had a significant inhibitory effect on directional conjugation. Taken together with the overall model, it is likely that CLκ-H81 alternates between the catalytically active and inactive form, forming H-bonds between CLκ-D43 and CLκ-D77 as it does so, and that removing the CLκ-D43 residue eliminates one of the forces pushing CLκ-H81 towards the active Nδ tautomer.
Analyses of the CLκ-D77A crystal structure revealed a well-defined hydrogen bond between hydrogen Nδ and the CLκ-D43 carboxylic group. This suggests that one of the most significant effects of the CLκ-D77A mutant is in stabilization of CLκ-H81 catalytically favorable tautomeric form NO, as shown in FIGS. 13 and 14. Finally, proximity of CLκ-D43 to CLκ-H81 would allow CLκ-D43 to participate in the catalytic reaction as a hydrogen atom recipient in the last step of the putative reaction mechanism (Scheme III). Thus the role of CLκ-D43 in the native CLκ and CLκ-D77A mutant, appears to be in stabilization of catalytically active Nδ tautomeric form of CLκ-H81 and participation in the catalytic reaction as the hydrogen atom recipient.
The modeling analyses strongly suggests that binding of the PFP molecule in native CLκ is controlled by the tautomeric forms of CLκ-H81, and the affinity of this binding is in turn controlled by the state of the tautomeric equilibrium constant. The presence of Nδ tautomeric form allows the lone pair on Nε to nucleophilic attack the PFP carbonyl (Scheme III).
CLκ-D43 is involved in the catalytic reaction by being a hydrogen ion acceptor (Scheme III). CLκ-D43 was mutated in silico to all 19 amino acids and in the final series of calculated mutants, the WT protein (comprising CLκ-D43) creates the highest interaction energy with the CLκ-PFP ester complex, with CLκ-D43N also predicted to be of similar chain length and be able to form a hydrogen bond to the CLκ-H81 NO, thus aiding the catalytically-active tautomeric form Nδ of CLκ-H81. Other residues likely to be acceptable substitutions are CLκ-D43E, CLκ-D43Q and CLκ-D43S, these being able to form the desired H-bond with CLκ-H81, but likely to have chain lengths either slightly too large or small to be of optimal size for CLκ conjugation with PFP, but may be better suited to either optimizing a non-CLκ immunoglobulin domain for directional conjugation with PFP, or optimizing an Ig domain for conjugation with a different halo-phenyl ester.
CLκ-N44 
CLκ-N44 is located on the CD loop. Its sidechain is pointing outwards, away from the PFP binding pocket, and appears not to have any role in, nor influence on, the conjugation reaction. QM/MM calculations predicted that polar amino acids and those with a negatively-charged sidechain may enhance protein stability due to the putative interactions with CLκ-K41, also located outside of the pocket.
CLκ-L46 
A CLκ-L46A mutation had a relatively neutral impact on the conjugation rate, based on experimental data, as well as from our QM/MM calculations. CLκ-L46A is located outside of the PFP binding pocket, but it may facilitate the pocket shape due to the size of its sidechain. Being located outside of the pocket, it can interact with amino acids located outside of the pocket as well. Mutations to large and hydrophilic sidechains may increase protein stability due to the interaction with CLκ-Q39 and support the shape of the PFP pocket according to QM/MM calculations.
CLκ-Q47 
A CLκ-Q47A mutation improved the conjugation rate. This mutation is likely exerting an effect by increasing the size of the PFP binding site, thus impacting complex formation and protein stability in a positive way according to modeling calculations. Amino acids with larger sidechains will likely impact conjugation in a slightly negative way, according to QM/MM calculations. Thus, mutations to CLκ-Q47A, CLκ-Q47G, CLκ-Q47V, CLκ-Q471, CLκ-Q47L, CLκ-Q47T, CLκ-Q47S, CLκ-Q47N, CLκ-Q47D, CLκ-Q47H, CLκ-Q47P, or CLκ-Q47E will likely be beneficial or neutral; whereas mutations to CLκ-Q47W, CLκ-Q47F, CLκ-Q47Y, or CLκ-Q47K may have a somewhat negative impact on PFP conjugation to a CLκ domain. As before, it will be understood that in certain applications, it may be desirable to reduce the pocket size; such as where smaller halo-phenols are used as Z1 groups, or where the precise geometry of a specific immunoglobulin domain appears to merit such a feature.
CLκ-S48 
Mutation of CLκ-S48A improved the conjugation rates according to experimental data: modeling suggest that the reason for this is most likely due to the change of electrostatic properties of the pocket. Hydrophobic amino acids at this position will therefore likely have a positive impact on the directional conjugation. Consequently, the following mutations may be especially favoured: CLκ-S48A, CLκ-S48G, CLκ-S48V, CLκ-S48I, CLκ-S48L, CLκ-S48P, and CLκ-S48M. Other mutations are likely to be tolerated.
Example 42 Double and Triple Mutations of CLκ-D77A
Antibody 2.12.1.fx was used to test the effect of further mutations to the CLκ region. As before, numbering of residues is according to their location within the CLκ (SEQ ID NO:6, for example). CLκ-D43 and CLκ-H81 were each mutated to Ala in on 2.12.1.fx-[CLκ-D77A] antibody to understand the conjugation mechanisms. Mutations were generated on 2.12.1.fx light chain following protocols described in QuickChange site-directed mutagenesis kit (Stratagene). The antibodies 2.12.1.fx, 2.12.1.fx-[CLκ-D77A] (CLκ comprising SEQ ID NO:37), 2.12.1.fx-[CLκ-D43A] (CLκ comprising SEQ ID NO:15), 2.12.1.fx, 2.12.1.fx-[CLκ-D43A/D77A] (CLκ comprising SEQ ID NO:127), 2.12.1.fx-[CLκ-D77A/H81A] (CLκ comprising SEQ ID NO:128), and 2.12.1.fx-[CLκ-D43A/D77A/H81A] (CLκ comprising SEQ ID NO:129), were transiently expressed in HEK 293 cells, and purified using protein A affinity column. The purified mAbs were characterized using MS.
The expressed antibodies were buffer exchanged to 20 mM sodium acetate, 200 mM trehalose pH 5.5 at 20 mg/ml. The antibody solutions were then spiked with 60 mM sodium phosphate pH 7.7. [PEG5-K11-SEQ:27] (ABP) was resuspended with 50% propylene glycol and mixed with the protein at a 4.3:1 molar ratio and allowed to react overnight at room temperature. All samples were diluted to 2 mg/ml and analyzed as an intact conjugated protein by size exclusion chromatography-mass spectrometry (SEC-MS) to determine the number and quantitation of conjugate forms of the protein. This technique measures the molecular weight of each protein form; multiple ABP conjugation sites are observed as distinct signals separated by the mass difference of an ABP. Relative quantitation of multiple ABP conjugation species is performed by measuring the signal magnitude. Results of % CA are shown in Table 66.







TABLE 66







Intact conjugation analysis of 2.12.1.fx-[CLκ-mutants] with ABP.











CLκ
% CA
Avg 















SEQ ID
0
1
2
3
4
CA

















2.12.1.fx
6
1
15
53
26
5
2.2


D77A
37
2
3
56
32
7
2.39


D43A
15
17
38
31
14
0
1.41


D43A/D77A
127
4
21
46
21
7
2.06


D77A/H81A
128
33
38
20
10
0
1.07


D43A/D77A/H81A
129
25
40
28
7
0
1.18









The extent of ABP conjugation was examined separately on the light and heavy chains of 2.12.1.fx and 2.12.1.fx-[CLκ mutants]. MACs were denatured and disulfide bonds were reduced using guanidine hydrochloride and dithiothreitol. The resulting free light and heavy chains were analyzed using LCMS to determine the conjugation profile on each (Table 67).







TABLE 67







Reduced heavy, light chain conjugation analysis of 2.12 1.fx-[CLκ-


mutants] with ABP. The % of 1-LC % relative to the respective WT 


run is shown in the right column, as described in Table 67.

















CLκ



Avg 


Avg 















SEQ
LC % CA
CA-
HC % CA
CA-
1LC 

















ID
0
1
2
LC
0
1
HC
WT %



















2.12.1.fx
6
23
69
8
0.85
86
14
0.14



D77A
37
1
92
7
1.06
97
3
0.03
133


D43A
15
68
30
1
0.33
79
21
0.21
43


D43A/D77A
127
24
64
12
0.88
90
10
0.1
93


D77A/H81A
128
72
28
0
0.28
85
15
0.15
41


D43A/D77A/H81A
129
76
20
4
0.28
87
13
0.13
29









These results are consistent with the His tautomer hypothesis. In the CLκ-D77A mutant, CLκ-D43 stabilizes the catalytically active tautomer of CLκ-H81, which in turn allows CLκ-K80 to be more receptive and reactive to a PFP-ester. Conversely, in the CLκ-D43A mutant, CLκ-D77 stabilizes the inactive tautomer of CLκ-H81, therefore leading to a reduction in the directional conjugation observed at CLκ-K80. In the double mutant CLκ-D43A/D77A, there are no interactions between CLκ-H81 and either of CLκ-D43 or CLκ-D77, and accordingly, the double mutant acts more like the WT CLκ.
Example 43 Rabbit CLκ Analysis
Rabbit antibody light chain kappa region (rCLκ) has the same 3D structure as that of other immunoglobulins. rCLκ has Asp at position 151 (kabat number), Ser at position 188, and His at 189, (rCLκ-D43, rCLκ-S80, rCLκ-H81). It was postulated that a rCLκ-S80K mutant may create the reaction site for PFP directional conjugation. To validate this hypothesis, two trastuzumab rabbit chimera antibodies were constructed. mAb “rTrast” (rabbit trastuzumab) comprises the VL and VH domains of trastuzumab (SEQ ID NOs:75 and 72 respectively) fused to the rCLκ and rabbit constant heavy chain (rCH) (SEQ ID NOs:130 and 131 respectively), to generate the full length rTrast-LC (SEQ ID NO:132) and rTrast-HC (SEQ ID NO:133).
rTrast-[rCLκ-S80K] comprises the VL and VH domains of trastuzumab (SEQ ID NOs:75 and 72 respectively) fused to the rCLκ-S80K (SEQ ID NO:134) and rabbit constant heavy chain (rCH) (SEQ ID NO:131), to generate the full length rTrast-LC-[rCLκ-S80K] (SEQ ID NO:135) and rTrast-HC (SEQ ID NO:133).
Rabbit IgG heavy chain and kappa1 light chain were PCRed from plasmids pFUSE-CHIg-rG and pFUSE2ss-CLIg-rk1 (Invivogen) respectively with ends overlapping with trastuzumab variable domains and vector. Trastuzumab VH and VL were PCRed from synthetic genes with ends overlapping with vector and rabbit constant domains. PCRs were mixed with a modified pCEP4 vector (Invitrogen) cut with BglII and NheI following protocols described in Quick PCR Cloning Kit (BPS Bioscience). Insert DNA were confirmed by DNA sequencing. rCLκ-S80K mutation was generated following protocols described in Quick PCR Cloning Kit (BPS Bioscience). Mutation was introduced by oligonucleotide primers and cloned to a modified pCEP4 vector (Invitrogen) cut with BglII and NheI. Insert DNA were confirmed by DNA sequencing.
The chimeric mAbs were transiently expressed in HEK 293 cells, and purified using protein A affinity column. The purified mAbs were characterized using MS. Each of the antibodies was buffer exchanged to 20 mM sodium acetate, 200 mM trehalose pH 5.5 at 20 mg/ml, and then spiked with 60 mM sodium phosphate pH 7.7. [PEG5-K11-SEQ:27] (ABP) was resuspended with 50% propylene glycol and mixed with the protein at a 4.3:1 molar ratio and allowed to react overnight at room temperature. All samples were diluted to 2 mg/ml and analyzed as an intact conjugated protein by size exclusion chromatography-mass spectrometry (SEC-MS) to determine the number and quantitation of conjugate forms of the protein. This technique measures the molecular weight of each protein form; multiple ABP conjugation sites are observed as distinct signals separated by the mass difference of an ABP. Relative quantitation of multiple ABP conjugation species is performed by measuring the signal magnitude.
The extent of ABP conjugation was examined on the light of antibodies. MACs were denatured and disulfide bonds were reduced using guanidine hydrochloride and dithiothreitol. The resulting free light and heavy chains were analyzed using LCMS to determine the conjugation profile on each (Table 68).







TABLE 68







Intact conjugation and reduced light chain analysis 


of rabbit chimera conjugated with ABP.













% CA
Avg
LC % CA
Avg 
1LC


















0
1
2
3
CA
0
1
2
CA-LC
WT %




















rTrast
58
24
19
0
0.61
56
38
6
0.5



rTrast-[rCLκ-S80K]
10
25
54
11
1.657
18
73
8
0.89
192









The rCLκ-S80K mutation significantly increased total conjugation (0.61 to 1.657) and 1CA on the light chain (38% to 73%). This result suggests that a directional conjugation site can be created on CLκ chains from species other than human, as long as the critical residues, CLκ-K80H81, are present on the immunoglobulin. The % of 1-LC % relative to the respective WT run is shown in the right column, as described in Table 68.
Example 44 Lambda Chain
As demonstrated in Example 10 above, hCLλ does not demonstrate directional conjugation with PFP esters. hCLλ shares sequence identity with hCLκ at hCLκ-D43 (CLλ-D48) and hCLκ-H81 (CLλ-H82), and has serine in place of hCLκ-K80 (CLλ-S81). It was postulated that a CLλ-S81K mutant may enable PFP conjugation to at the CLλ-S81K residue.
Comparing the crystal structures of CLκ and CLλ (FIGS. 17 & 18), CLκ-V42 is located at the bottom of the putative PFP binding pocket, whereas the corresponding residue in CLλ is CLλ-A44. As discussed above, CLκ-V42A mutations have a negative impact on the directional conjugation, likely owing to the ability of CLκ-V42 to stabilize the orientation of the imidazole ring of CLκ-H81. Modeling suggested that a CLλ-A44V mutation should exert a similar effect on CLλ-H82.
As in Example 10, and Examples 15-17, a monoclonal anti-human IL22 antibody (hIL22) was used as an exemplary CLλ comprising antibody. hIL22 comprised SEQ ID NOs:136 and 137 (hIL22-LC and hIL22-HC respectively), and variable light chain comprising SEQ ID NO:138 (hIL22-CLλ-VL).
A number of mutant versions of hIL22 were generated, to assess the effects of various CLλ mutations on directional conjugation. All hIL22 mutant antibodies comprised SEQ ID NO:137 (hIL22-HC), and SEQ ID NO:138 (hIL22-CLλ-VL).
hIL22-[LKJ] comprised the A/K swap as described in Example 15, and comprised a CL of SEQ ID NO:61. hIL22-[CLλ-S81K] comprised the single residue swap of CLλ-S81K, and comprised SEQ ID NO:140. hIL22-[CLλ-Q78A/S81K] comprised a double mutation in the loop, and comprised SEQ ID NO:141. hIL22-[CLλ-A44V/S81K] comprised the CLλ-S81K residue swap, and also a CLλ-A44V mutation at the bottom of the “binding pocket”, and comprised SEQ ID NO:142. hIL22-[CLλ-A44V/Q78A/S81K] comprised both loop mutations CLλ-Q78A and CLλ-S81 K, as well as the “binding pocket” CLλ-A44V mutation, and comprised SEQ ID NO:143. All the point mutations were generated on hIL22-LC following protocols described in QuickChange site-directed mutagenesis kit (Stratagene). Mutations were introduced by oligonucleotide primers and confirmed by DNA sequencing.
hIL22-[CLλ-λ76-84/145] comprised inserting SEQ ID NO:145 in place of the CLλ E-F loop, located from CLλ-P76 inclusive through to CLλ-S83 inclusive. SEQ ID NO:145 comprises the sequence KAAYEKHKV, which corresponds to the [CLκ-D77A] E-F loop (i.e. between β-strands E and F) from [CLκ-D77A]K75 inclusive through to [CLκ-D77A]K82 inclusive.
hIL22-[CLλ-λ76-84/145] was generated by overlap PCR. Mutations were introduced by oligonucleotide primers. Primer specific to the 5′ end of hIL22-LC paired with a reverse primer encoding SEQ ID NO:145, and a forward primer encoding SEQ ID NO:145, paired with the reverse primer specific to the 3′ end of 1L22-LC were used to PCR amplify DNA fragments carrying CLκ E-F loop using 1L22-LC as template. These two PCR products were mixed as templates; 1L22-LC forward primer and reverse primer were used in overlap PCR reaction to amplify the full length 1L22-LC DNA with SEQ ID NO:145. The PCR was then digested with restriction enzyme BglII and NheI. The digested PCR was ligated with a modified pCEP4 plasmid (Invitrogen) cut with BglII and NheI.
The mutated mAbs were transiently expressed in HEK 293 cells, and purified using protein A affinity column. The purified mAbs were characterized using MS.
Each of the antibodies was buffer exchanged to 20 mM sodium acetate, 200 mM trehalose pH 5.5 at 20 mg/mi, and then spiked with 60 mM sodium phosphate pH 7.7. [PEG5-K11-SEQ:27] (ABP) was resuspended with 50% propylene glycol and mixed with the protein at a 4.3:1 molar ratio and allowed to react overnight at room temperature. All samples were diluted to 2 mg/mi and the extent of ABP conjugation was examined separately on the light and heavy chains of antibodies. The MACs were denatured and disulfide bonds were reduced using guanidine hydrochloride and dithiothreitol. The resulting free light and heavy chains were analyzed using LCMS to determine the conjugation profile on each (Table 69).







TABLE 69







Reduced heavy, light chain conjugation analysis 


of lambda antibody and mutants.











CL
LC % CA
HC % CA

















SEQ



Ave 



Ave


Ab conjugated with ABP
ID
0
1
2
LC
0
1
2
HC



















2.12.1.fx
6
15
79
6
0.91
93
7
0
0.07


2.12.1.fx-CLκ-D77A
37
0
92
8
1.08
98
2
0
0.02


Trastuzumab-WT
76
32
66
2
0.7
83
17
0
0.17


Trastuzumab-CLκ-D77A
77
0
95
5
1.05
90
10
0
0.1


hIL22
136
100
0
0
0
76
22
2
0.26


hIL22-[LKJ]
61
55
45
0
0.45
79
21
0
0.21


hIL22-[CLλ-S81K]
140
69
31
0
0.31
80
20
0
0.2


hIL22-[CLλ-Q78A/S81K]
141
89
11
0
0.11
85
15
0
0.15


hIL22-[CLλ-A44V/S81K]
142
48
52
0
0.52
87
13
0
0.13


hIL22-[CLλ-A44V/Q78A/S81K]
143
30
70
0
0.7
82
18
0
0.18


hIL22-[CLλ-A76-84/145]
144
83
17
0
0.34
82
18
0
0.2









A single point mutation CLλ-S81K enabled PFP conjugation to the CLλ. The LC % 1CA increased from 0% to 31% on the CLλ-S81K mutant compared against the unmutated hIL22, with more conjugation additions seemingly occurring at the newly created conjugation site than at any other single site on the antibody. Peptide mapping studies confirmed that the conjugation events were occurring on CLλ-S81K (Table 70).







TABLE 70







Overview of peptide mapping studies on hIL22-[CLλ-S81K]-CL.











Average Δ




(% conju-




gation,


Protein Chain
Peptide
n = 3)





hIL22-[CLλ-S81K]-CL
SEQ ID NO: 140, residues 80-85
54 ± 10


hIL22-[CLλ-S81K]-HC
SEQ ID NO: 137, residues 96-103
21 ± 10


hIL22-[CLλ-S81K]-HC
SEQ ID NO: 137, residues 95-103
19 ± 10









Referring back to Table 69, combining mutations at CLλ-S81K and CLλ-A44V (hIL22-[CLλ-A44V/S81K]) further increased light chain 1CA to 52%.
Interestingly, while the CLλ-Q78A/CLλ-S81K double mutation did improve conjugation of the CLλ-S81K compared to WT CLλ (1CA from 0% to 11%), the improvement was less pronounced than that seen in the single CLλ-S81K mutant. This correlates well with the model: the effect of mutating CLκ-D77 is to remove the hydrogen bond between CLκ-D77 and CLκ-H81, enabling CLκ-H81 to revert to the catalytically tautomeric Nδ form. The corresponding position to CLκ-D77 is CLλ-Q78, which both modeling and mutational analysis suggest would not have a limiting effect on CLλ-H82. Taken together, this suggests that the diminishing effect of CLλ-Q78A on the directional conjugation is likely caused by an alteration to the size and shape of the binding pocket.
Most surprising, however, was the result of the triple mutation CLλ-A44V/Q78A/S81K. Directional conjugation as measured by the 1CA % increased to 70%; reaching levels typically seen in native CLκ domains.
These results and analysis were borne out by the similar level of directional conjugation seen in the loop swap (hIL22-[CLλ-λ76-84/145]).
Overall, these data suggested that the CLλ-Q78A mutation does improve directionality of CLλ-S81K conjugation, provided the size and shape of the binding pocket is adapted for the specific Z group used. Moreover, the results of CLλ mutants suggested that directional conjugation sites can be created on immunoglobulins other than CLκ, provided that the motif KH is present in the correct 3D location. Naturally, the binding pocket must be of suitable size and shape to accommodate the specific halo-phenyl ester employed, and, as demonstrated herein, additional features, such as the presence or absence of residues corresponding to CLκ-V42, CLκ-D43, and CLκ-D77 can have significant effects on the rate and optimization of directional conjugation of the KH motif relative to the specific immunoglobulin domain and halo-phenyl ester. While PFP was used in these examples, it will be apparent that other Z groups with acceptable levels of directional conjugation may be selected, and using rational modeling techniques, a balance may be obtained between the desired Z group, size of the binding pocket and the specific mutations required to maintain an active binding pocket.
Example 45 Recreating PFP Conjugation Sites on CH Domains
The CH domains of antibodies also comprise immunoglobulin structures. Prior to modeling the domains as described in Examples 40 and 41, it was postulated that moving the conjugation motif to the EF loop portion of the EF connecting chain of other CH domains may permit directional conjugation. A sequence alignment of the CHγ1 (SEQ ID NO:147), CHγ2 (SEQ ID NO:155), CHγ3 (SEQ ID NO:158), CLκ (SEQ ID NO:6) and CLλ (SEQ ID NO:57) domains is shown in FIG. 16.
Two mutant versions were made on the CH1 domain.
In hCHγ1-m1, the sequence LGTQT (SEQ ID NO:152), which corresponds to residues L76-T80 of SEQ ID NO:147, was removed, and replaced by EKHKV (SEQ ID NO:153), which corresponds to E79-V83 of CLκ. The resultant mutant, hCHγ1-m1, comprised SEQ ID NO:148.
In CHγ1-m2, the sequence LGTQT (SEQ ID NO:152), which corresponds to residues L76-T80 of SEQ ID NO:147, was removed, and replaced by YEKHKV (SEQ ID NO:154), which corresponds to Y78-V83 of CLκ-. The resultant mutant, hCHγ1-m2, comprised SEQ ID NO:150. The additional Y residue was incorporated to allow the hCHγ1-m2 sequence to better align with CLκ sequence.
Sequence alignment indicated that hCHγ1 lacks an Asp residue corresponding to CLκ-D43. Accordingly, two additional mutants were generated; where each of hCHγ1-m1 and hCHγ1-m2 were subjected to an additional insertional mutation of an Asp residue between CHγ1-S43 and CHγ1-G44, creating the two new mutants of hCHγ1-ml-D44 (SEQ ID NO:149) and hCHγ1-m2-D44 (SEQ ID NO:151).
A mutant version of hCHγ2 (SEQ ID NO:155) was generated, where residues N85G86 of SEQ ID NO:155 were substituted with KH to generate hCHγ2-m (SEQ ID NO:156). Sequence alignment suggested that hCHγ2 (SEQ ID NO:155) comprised an Asp residue (D50) at a location that may correspond to CLκ-D43.
A mutant version of hCHγ3 (SEQ ID NO:157) was generated, where residues Q79G80 of SEQ ID NO:157 were substituted with KH to generate hCHγ3-m (SEQ ID NO:158). Sequence alignment suggested that hCHγ3 (SEQ ID NO:155) comprised an Asn residue (N44) at a location corresponding to CLκ-D43.
Trastuzumab was used as a model Ab in the study. All the hTrast-CHγ mutants were expressed with a [CLκ-K80A] mutation, so that conjugation events would preferentially occur on the test CHγ domain. The hTrast-LC-[CLκ-K80A] (SEQ ID NO:146) mutation was generated following protocols described in QuickChange site-directed mutagenesis kit (Stratagene).
The mutations on CHγ domains were generated using overlap PCR. Mutations were introduced by oligonucleotide primers. Primer specific to the 5′ end of trastuzumab HC paired with a reverse primer carrying the desired mutation, and a forward primer carrying the desired mutation paired with the reverse primer specific to the 3′ end of trastuzumab HC were used to PCR amplify DNA fragments using trastuzumab HC as template. These two PCR products were mixed as templates; trastuzumab heavy chain forward primer and reverse primer were used in overlap PCR reaction to amplify the full length trastuzumab HC DNA with desired mutations. The PCR was then digested with restriction enzyme BglII and NheI. The digested PCR was ligated with a modified pCEP4 plasmid (Invitrogen) cut with BglII and NheI.
The trastuzumab antibody carrying mutations were transiently expressed in HEK 293 cells, and purified using protein A affinity column. The purified mAbs were characterized using MS.
The expressed antibody was buffer exchanged to 20 mM sodium acetate, 200 mM trehalose pH 5.5 at 20 mg/ml. The proteins were then spiked with 60 mM sodium phosphate pH 7.7. ABP was resuspended with 50% propylene glycol and mixed with the protein at a 4.3:1 molar ratio and allowed to react overnight at room temperature. All samples were diluted to 2 mg/ml and analyzed as an intact conjugated protein by size exclusion chromatography-mass spectrometry (SEC-MS) to determine the number and quantitation of conjugate forms of the protein. This technique measures the molecular weight of each protein form; multiple ABP conjugation sites are observed as distinct signals separated by the mass difference of an ABP. Relative quantitation of multiple ABP conjugation species is performed by measuring the signal magnitude.







TABLE 71







Intact conjugation analysis of hTrast


antibody and CHγ domain mutants.










% CA













Ab + ABP
0
1
2
3
Avg CA















hTrast
1
16
56
24
2.112


hTrast-[CLκ-D77A]
0
1
62
34
2.338


hTrast-[CLκ-K80A]
48
41
10
2
0.665


hTrast-[CLκ-K80A]/CHγ1-m1
23
48
24
5
1.107


hTrast-[CLκ-K80A]/CHγ1-m2
24
48
24
4
1.087


hTrast-[CLκ-K80A]/CHγ1-m1-D44
33
49
17
1
0.854


hTrast-[CLκ-K80A]/CHγ1-m2-D44
41
45
12
2
0.746


hTrast-[CLκ-K80A]/CHγ2m
45
44
9
3
0.694


hTrast-[CLκ-K80A]/CHγ3m
40
44
13
3
0.78









The extent of ABP conjugation was examined separately on the light and heavy chains of trastuzumab and trastuzumab mutants. MAC product was denatured and disulfide bonds were reduced using guanidine hydrochloride and dithiothreitol. The resulting free light and heavy chains were analyzed using LCMS to determine the conjugation profile on each chain (Table 72).







TABLE 72







Reduced heavy, light chain conjugation analysis of hTrast antibody and


CHγ domain mutants. The % of 1-HC % relative to the respective WT run 


is shown in the right column, as described in Table 53, although in this 


example, hTrast-[CLκ-K80A HC 1CA was taken as the WT FIGURE.











LC % CA
HC % CA





















Avg



Avg







CA-



CA-
1HC



0
1
2
LC
0
1
2
HC
WT %



















hTrast
32
66
2
0.7
83
17
0
0.17
N/A


hTrast-[CLκ-D77A]
0
95
5
1.05
90
10
0
0.1
N/A


hTrast-[CLκ-K80A]
98
2
0
0.02
79
21
0
0.21



hTrast-[CLκ-K80A]/CHγ1-m1
99
1
0
0.01
64
31
5
0.41
148


hTrast-[CLκ-K80A]/CHγ1-m2
98
2
0
0.02
62
32
6
0.44
152


hTrast-[CLκ-K80A]/CHγ1-m1-D44
97
3
0
0.03
74
24
2
0.28
114


hTrast-[CLκ-K80A]/CHγ1-m2-D44
97
3
0
0.03
73
24
3
0.3
114


hTrast-[CLκ-K80A]/CHγ2m
99
1
0
0.01
75
23
2
0.27
110


hTrast-[CLκ-K80A]/CHγ3m
100
0
0
0
70
27
3
0.33
129









The total conjugation on hTrast-[CLκ-K80A]/CHγ1-ml and hTrast-[CLκ-K80A]/CHγ1-m2 were increased compared to hTrast-[CLκ-K80A], from 0.66 CA to  ̃1CA; and the HC 1CA of these two mutants increased from 21% to 31%, and improvement of about 150% This supports the hypothesis that directional conjugation can be introduced to immunoglobulin domains other than CLκ and CLλ by the introduction of the KH motif.
The total conjugation on hTrast-[CLκ-K80A]/CHγ2m also increased, but to a lesser extent than that of the CHγ1 mutants. A comparison of the sequences of the CHγ1 and CHγ2 sequences shows that at the residue corresponding to CLκ-D77, CHγ1 comprises Ser, and CHγ2 comprises Asp. This could suggest that a mutation similar to the CLκ-D77 mutations in CHγ2 domains could improve the extent of conjugation.
In addition, the improvement in conjugation on the CHγ3 HC, while evident, was also modest in comparison to the CHγ1 domain mutants. The CHγ3 domain sequence appeared to comprise Arg at a position corresponding to CLκ-D77, and Ser at a position corresponding to CLκ-D43.
Furthermore, the conjugation of the CA % HC for both hTrast-[CLκ-K80A]/CHγ1-ml-D44 and hTrast-[CLκ-K80A]/CHγ1-m2-D44 showed a very surprising result, in that the apparent increase in conjugation seen in both hTrast-[CLκ-K80A]/CHγ1-ml and hTrast-[CLκ-K80A]/CHγ1-m2 went from approximately 150% of WT to only about 114% of WT.
An explanatory hypothesis suggested that the sequence alignment of FIG. 17 may not accurately align the respective residues according to where they are found on a 3D immunoglobulin structure.
Example 46 Modeling the Immunoglobulin Fold
Consequently, crystal structure coordinates for the hCHγ1, hCHγ2 and hCHγ3 domains were obtained from the “Protein Data Bank”, maintained by Rutgers, the State University of New Jersey, Center for Integrative Proteomics Research, the San Diego Supercomputer Center (SDSC) and Skaggs School of Pharmacy and Pharmaceutical Sciences, San Diego. The structure of hCHλ1 is based on X-ray structure of 3dv, available through the “Protein Data Bank”. The structure of hCHλ2 is based on the X-ray structure of 2dts available through the “Protein Data Bank”. The structure of hCHλ3 is based on the X-ray structure of 2dts available through the “Protein Data Bank”. The structure of hCLλ is based on the X-ray structure of 4fqh available through the “Protein Data Bank”.
An homology alignment was generated, which aligned the sequences according to structure (FIG. 17). Crystal structure comparisons of CLκ and hCHγ1-ml (FIG. 19) showed that the hCHγ-CD connecting chain comprised a short α-helix. Modeling suggested that this CD α-helix presents the native S43 and the hTrast-[CLκ-K80A]/CHγ1-m1-D44 Asp insertion with side chains extending away from the binding pocket (FIGS. 20 & 21), and that the addition of CHγ1-D44 increases the size of and extends the CD α-helix. In addition, the model suggests that the CHγ1-Q79H residue is not in the optimal planar orientation, reducing its ability to participate in the reaction between PFP and the adjacent CHγ1-T78K (FIG. 21).
A comparison of the CLκ and CHγ1-m1-D44 domains of FIG. 22 suggests that the pocket shape may be optimized by removing the CD α-helix on the CD connecting chain, and replacing with a loop structure. Accordingly, a mutant CHγ1 domain, SEQ ID NO:165, was modelled and minimized. SEQ ID NO:165 comprises the CHγ1-T84K/Q79H double mutation of the CHγ1-ml mutant, as well as substituting the CHγ1 CD connecting chain from CHγ1-N42 inclusive through to CHγ1-L46 inclusive with KVDNALA (SEQ ID NO:166); SEQ ID NO:166 corresponds to residues of the CLκ domain (SEQ ID NO:6), with an additional Ala residue added. Modeling results shown in FIG. 22 suggest that CHγ1-Q79H occupies a more planar orientation, assisted by the introduced Val residue on the CD connecting chain. Mutating this residue to Leu or Ile may provide further stabilization to the imidazole ring, owing to the longer side chains being able to close the increased gap to the H residue, relative to the distance in native CLκ. Significantly, the model places the adjacent Asp residue in a suitable orientation and distance from CHγ1-Q79H to favour the δ-tautomeric form, and promote increased reactivity of CHγ1-T78K.
Similar modeling was performed on the CHγ2 and CHγ3 domains. FIG. 24 compares key WT residues of CLκ and CHγ2. While CHγ2-D49 and CHγ2-D50 appear to be in the correct position and orientation to assist with directional conjugation, CHγ2-D82 occupies a position even closer to CHγ2-G86H in the 3D model, thereby favouring the ε-tautomeric form, and reducing the reactivity of CHγ2-N85K. This model suggests that the CHγ2-D82 residue may explain the relatively low increase in directional conjugation seen in the CHγ2m mutant. FIG. 26 illustrates a minimized model of a proposed mutant for CHγ2: comprising the mutations CHγ2-D82A/N85K/G86H (SEQ ID NO:167). An immediate difference is seen: the imidazole ring appears in a more planar orientation, and within a suitable distance to CHγ2-D50 to bias formation of the δ-tautomer, and the CHγ2-D82A mutation has opened up the binding pocket, likely leading to a reduction in steric inhibition of PFP access.
FIGS. 27-29 show the corresponding comparisons of CLκ with CHγ3 WT, and CHγ3m mutant. The model suggests that despite there being no residue biasing CHγ3-G80H towards the ε-tautomer form, the availability of CHγ3-Q79K to conjugate is likely sterically hindered by the long CHγ3-R76 side-chain. FIG. 30 illustrates a minimized model of a first proposed mutant for CHγ3: hCH3γm-CD1/EF, comprising the mutations CHγ3-S43V/N44D/R76A/Q79K/G80H (SEQ ID NO:168). The modeled difference can be easily observed in FIG. 30B: the availability of the binding pocket has opened up, and reduced steric interference on the reactive Lys.
From the sidechain distances in the modeled structure of FIG. 30B, it is unlikely that CHγ3-S43V or CHγ3-N44D interact with CHγ3-G80H to the same extent as the corresponding residues in CLκ. Accordingly, a second proposed mutant for CHγ3 was also minimized and modeled: hCH3γm-CD2/EF, comprising the mutations CHγ3S43L/N44E/R76A/Q79K/G80H (SEQ ID NO:169). The introduction of suitable residues on the CD connecting chain with longer side chains (CHγ3-S43L instead of CHγ3-S43V, and CHγ3-N44E instead of CHγ3-E44D) is modeled to have a binding pocket with greater structure similarities to that of CLκ (FIG. 31), and a greater likelihood of hydrogen bonding between CHγ3-N44E and CHγ3-G86H than between CHγ3-N44D and CHγ3-G80H.
Sequences of the CHγ domains and mutants were aligned with the CLκ and CLλ (FIG. 32).
The invention thus has been disclosed broadly and illustrated in reference to representative embodiments described above. Those skilled in the art will recognize that various modifications can be made to the present invention without departing from the spirit and scope thereof. All publications, patent applications, and issued patents, are herein incorporated by reference to the same extent as if each individual publication, patent application or issued patent were specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.
It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. In particular, any aspect of the invention described in the claims, alone or in combination with one or more additional claims and/or aspects of the description, is to be understood as being combinable with other aspects of the invention set out elsewhere in the claims and/or description and/or sequence listings and/or drawings
In so far as specific examples found herein do not fall within the scope of an invention, said specific example may be explicitly disclaimed.
The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternative are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” As used herein the specification, “a” or “an” may mean one or more, unless clearly indicated otherwise. As used herein in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one. As used herein “another” may mean at least a second or more. Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The words “comprises/comprising” and the words “having/including” when used herein with reference to the present invention are used to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.









TABLE 73





Toxin




#
IUPAC name
STRUCTURE







 #54
2-Methylalanyl-N-[(3R,4S,5S)-3- methoxy-1-{(2S)-2-[(1R,2R)-1- methoxy-2-methyl-3-oxo-3-{[(1S)- 2-phenyl-1-(1,3-thiazol-2- yl)ethyl]amino}propyl]pyrrolidin-1- yl}-5-methyl-1-oxoheptan-4-yl]-N- methyl-L-valinamide (#54






 


#226
N,2-dimethylalanyl-N-[(3R,4S,5S)- 3-methoxy-1-{(2S)-2-[(1R,2R)-1- methoxy-3-{[(2S)-1-methoxy-1- oxo-3-phenylpropan-2-yl]amino}-2- methyl-3-oxopropyl]pyrrolidin-1- yl}-5-methyl-1-oxoheptan-4-yl]-N- methyl-L-valinamide (#226)






 


#194
N,2-{[(3S)-3-fluoropyrrolidin-3- yl]carbonyl}-N-[(3R,4S,5S)-3- methoxy-1-{(2S)-2-[(1R,2R)-1- methoxy-2-methyl-3-oxo-3-{[(1S)- 2-phenyl-1-(1,3-thiazol-2- yl)ethyl]amino}propyl]pyrrolidin-1- yl}-5-methyl-1-oxoheptan-4-yl]-N- methyl-L-valinamide trifluoroacetic acid salt (#194)






 


#192
N,2-{[(3R)-3-fluoropyrrolidin-3- yl]carbonyl}-N-[(3R,45,5S)-3- methoxy-1-{(2S)-2-[(1R,2R)-1- methoxy-2-methyl-3-oxo-3-{[(1S)- 2-phenyl-1-(1,3-thiazol-2- yl)ethyl]amino}propyl]pyrrolidin-1- yl}-5-methyl-1-oxoheptan-4-yl]-N- methyl-L-valinamide trifluoroacetic acid salt (#192)






 


#201
1,2-dimethyl-D-prolyl-N- [(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3- {[(2S)-3-(4-aminophenyl)-1- methoxy-1-oxopropan-2-yl]amino}- 1-methoxy-2-methyl-3- oxopropyl]pyrrolidin-1-yl}-3- methoxy-5-methyl-1-oxoheptan-4- yl]-N-methyl-L-valinamide, formate salt (#201)






 


#158
N-{(2R,3R)-3-methoxy-3-[(2S)-1- {(3R,4S,5S)-3-methoxy-5-methyl-4- [methyl(N-{[(2S)-2- methylpiperidin-2-yl]carbonyl}-L- valyl)amino]heptanoyl}pyrrolidin- 2-yl]-2-methylpropanoyl}-L- phenylalaninate, trifluoroacetic acid salt (#158)






 


 #70
2-methylalanyl-N-[(3R,4S,5S)-3- methoxy-1-{(2S)-2-[(1R,2R)-1- methoxy-3-{[(2S)-1-methoxy-1- oxo-3-phenylpropan-2-yl]amino}- 2-methyl-3-oxopropyl]pyrrolidin-1- yl}-5-methyl-1-oxoheptan-4-yl]-N- methyl-L-valinamide (#70)






 


 #47
N2-[(1-Aminocyclopentyl)carbonyl]- N-[(3R,4S,5S)-3-methoxy-1-{(2S)- 2-[(1R,2R)-1-methoxy-2-methyl-3- oxo-3-{[(1S)-2-phenyl-1-(1,3- thiazol-2- yl)ethyl]amino}propyl]pyrrolidin-1- yl}-5-methyl-1-oxoheptan-4-yl]-N- methyl-L-valinamide (#47)






 


#130
N,2-dimethylalanyl-N-[(3R,4S,5S)- 1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-tert- butoxy-1-oxo-3-phenylpropan-2- yl]amino}-1-methoxy-2-methyl-3- oxopropyl]pyrrolidin-1-yl}-3- methoxy-5-methyl-1-oxoheptan-4- yl]-N-methyl-L-valinamide (#130)






 


#159
methyl N-{(2R,3R)-3-methoxy-3- [(2S)-1-{(3R,4S,5S)-3-methoxy-5- methyl-4-[methyl(N-{[(2R)-2- methylpiperidin-2-yl]carbonyl}-L- valyl)amino]heptanoyl}pyrrolidin- 2-yl]-2-methylpropanoyl}-L- phenylalaninate, trifluoroacetic acid salt (#159)






 


#209
1,2- dimethyl-L-prolyl-N-[(3R,4S,5S)- 3-methoxy-1-{(2S)-2-[(1R,2R)-1- methoxy-3-{[(2S)-1-methoxy-1- oxo-3-phenylpropan-2-yl]amino}-2- methyl-3-oxopropyl]pyrrolidin-1- yl}-5-methyl-1-oxoheptan-4-yl]-N- methyl-L-valinamide, trifluoroacetic acid salt (#209)






 


#131
2-methyl-D-prolyl-N-[(3R,4S,5S)-3- methoxy-1-{(2S)-2-[(1R,2R)-1- methoxy-3-{[(2S)-1-methoxy-1- oxo-3-phenylpropan-2-yl]amino}-2- methyl-3-oxopropyl]pyrrolidin-1- yl}-5-methyl-1-oxoheptan-4-yl]-N- methyl-L-valinamide, trifluoroacetic acid salt (#131)






 


#117
2-methyl-L-prolyl-N-[(3R,4S,5S)-3- methoxy-1-{(2S)-2-[(1R,2R)-1- methoxy-3-{[(2S)-1-methoxy-1- oxo-3-phenylpropan-2-yl]amino}-2- methyl-3 oxopropyl]pyrrolidin-1- yl}-5-methyl-1-oxoheptan-4-yl]-N- methyl-L-valinamide, trifluoroacetic acid salt (#117)






 


#115
N,2-dimethylalanyl-N-{(1S,2R)-4- {(2S)-2-[(1R,2R)-3-{[(1S)-1- carboxy-2-phenylethyl]amino}-1- methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl}-2-methoxy-1-[(1S)- 1-methylpropyI]-4-oxobutyl}-N- methyl-L-valinamide, trifluoroacetic acid salt (115)






 


 #69
2-Methylalanyl-N-[(3R,4S,5S)-1- {(2S)-2-[(1R,2R)-3-{[(1S)-1- carboxy-2-phenylethyl]amino}-1- methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl}-3-methoxy-5- methyl-1-oxoheptan-4-yl]-N- methyl-L-valinamide (#69)






 


#151
1,2-dimethyl-L-prolyl-N-{(1S,2R)- 4-{(2S)-2-[(1R,2R)-3-{[(1S)-1- carboxy-2-phenylethyl]amino}-1- methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl}-2-methoxy-1- [(1S)-1-methylpropyI]-4-oxobutyl}- N-methyl-L-valinamide (#151)






 


#162
N-{(2R,3R)-3-methoxy-3-[(2S)-1- {(3R,4S,5S)-3-methoxy-5-methyl-4- [methyl(N-{[(2S)-2- methylpiperidin-2-yl]carbonyl}-L- valyl)amino]heptanoyl}pyrrolidin- 2-yl]-2-methylpropanoyl}-L- phenylalanine, trifluoroacetic acid salt (#162)






 


#153
1,2-dimethyl-D-prolyl-N-{(1S,2R)- 4-{(2S)-2-[(1R,2R)-3-{[(1S)-1- carboxy-2-phenylethyl]amino}-1- methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl}-2-methoxy-1- [(1S)-1-methylpropyl]-4-oxobutyl}- N-methyl-L-valinamide (#153)






 


#118
2-methyl-L-prolyl-N-[(3R,4S,5S)-1- {(2S)-2-[(1R,2R)-3-{[(1S)-1- carboxy-2-phenylethyl]amino}-1- methoxy-2-methyl-3-oxopropyl] pyrrolidin-1-yl}-3-methoxy-5- methyl-1-oxoheptan-4-yl]-N- methyl-L-valinamide, trifluoroacetic acid salt (#118)






 


#163
N-{(2R,3R)-3-methoxy-3-[(2S)-1- {(3R,4S,5S)-3-methoxy-5-methyl- 4-[methyl(N-{[(2R)-2- methylpiperidin-2-yl]carbonyl}-L- valyl)amino]heptanoyl}pyrrolidin- 2-yl]-2-methylpropanoyl}-L- phenylalanine, trifluoroacetic acid salt. (#163)






 


#217
N-{(2R,3R)-3-[(2S)-1-{(3R,4S,5S)- 4-[(N-{[(3R)-3-fluoropyrrolidin-3- yl]carbonyl}-L- valyl)(methyl)amino]-3-methoxy-5- methylheptanoyl}pyrrolidin-2-yl]-3- methoxy-2-methylpropanoyl}-L- phenylalanine, trifluoroacetic acid salt (#217)






 


#112
2-methylalanyl-N-[(3R,4S,5S)-1- {(2S)-2-[(1R,2R)-3-{[(1S,2R)-1- hydroxy-1-phenylpropan-2- yl]amino}-1-methoxy-2-methyl-3- oxopropyl]pyrrolidin-1-yl}-3- methoxy-5-methyl-1-oxoheptan-4- yl]-N-methyl-L-valinamide (#112)






 



MMAD The wavy line indicates a typical location for linker attachment. In non-conjugated form, the wavy line is typically conneted to a H atom.






 



MMAE The wavy line indicates a typical location for linker attachment. In non-conjugated form, the wavy line is typically conneted to a H atom.






 



MMAF The wavy line indicates a typical location for linker attachment. In non-conjugated form, the wavy line is typically conneted to a H atom.

## Claims

1. An antibody or antigen binding portion thereof comprising a mammalian constant light domain;
wherein said mammalian constant light domain comprises a sequence selected from the group consisting of: SEQ ID NO: 37; SEQ ID NO: 38; SEQ ID NO: 41; SEQ ID NO: 40; SEQ ID NO: 54; SEQ ID NO: 49; SEQ ID NO: 50; and SEQ ID NO: 42;
wherein a K located at a position corresponding to residue 80 comprises a side chain with an ε-amino group covalently attached to a linker,
and pharmaceutically acceptable salts, stereoisomers, tautomers, and solvates, thereof.

2. The antibody or antigen binding portion thereof as claimed in claim 1, further comprising a residue selected from the group consisting of V, I and L at a position corresponding to residue 42 of.

3. The antibody or antigen binding portion thereof as claimed in claim 1, wherein the residues at positions corresponding to residues 42 and 43 of are not in α-helical formation.

4. The antibody or antigen binding portion thereof as claimed in claim 1, wherein the mammalian antibody constant domain is a humanized or human domain.

5. The antibody or antigen binding portion thereof as claimed in claim 1, wherein the constant domain is connected to an antibody variable domain.

6. The antibody or antigen binding portion thereof as claimed in claim 1, wherein the linker comprises a formula selected from the group consisting of X1-Y1-Z1, X1-Φ-Y1-Z1, and X1-Y1-Φ-Z, wherein Φ is a cleavable group, X1 is a group covalently connectable to at least one Effector Moiety, Y1 is a linear or branched connecting chain, and Z is a group covalently connected to the ε-amino group of the side chain of K that is located at a position corresponding to residue 80 of.

7. The antibody or antigen binding portion thereof as claimed in claim 6, wherein the cleavable group Φ is present, and is of the formula




wherein the wavy line and parallel line each indicate a point of attachment to either the X1, Y1 or Z1 group as appropriate.

8. The antibody or antigen binding portion thereof as claimed in claim 6, wherein the linker is selected from the group consisting of:













wherein m, n, j and k are each independently a range whose lower limits are selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and whose upper limit is selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, and wherein the overall length of the linker does not exceed 200 atoms.

9. The antibody or antigen binding portion thereof as claimed in claim 6, wherein the Effector Moiety is a therapeutic agent, protein, peptide, nucleic acid, aptamer, small molecule, protein agonist, protein antagonist, metabolic regulator, hormone, toxin, growth factor, or diagnostic agent.

10. The antibody or antigen binding portion thereof as claimed in claim 9, wherein the Effector Moiety is a toxin, and comprises the formula:




or a pharmaceutically acceptable salt or solvate thereof, wherein, independently for each occurrence,
W2 is





R11 is





Y2 is -C2-C20 alkylene-, —C2-C20 heteroalkylene-; —C3-C8 carbocyclo-, -arylene-, -C3-C8heterocyclo-, —C1-C10alkylene-arylene-, -arylene-C1-C10alkylene-, —C1-C10alkylene-(C3-C8carbocyclo)-, —(C3-C8carbocyclo)-C1-C10alkylene-, —C1-C10alkylene-(C3-C8heterocyclo)- or —(C3-C8 heterocyclo)-C1-C10alkylene-;
Z2 is





R12 is hydrogen, C1-C8 alkyl or C1-C8 haloalkyl;
R13A and R13B are either of the following:
(i) R13A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and

R13B is C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl or halogen; or
(ii) R13A and R13B taken together are C2-C8 alkylene or C1-C8 heteroalkylene;
R14A and R14B are either of the following:
(i) R14A is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; and

R14B is hydrogen, C1-C8 alkyl, C1-C8 haloalkyl, C3-C8 carbocyclyl, C1-C10 heterocyclyl, aryl, heteroaralkyl or aralkyl; or
(ii) R14A and R14B taken together are C2-C8 alkylene or C1-C8 heteroalkylene;

R15 is




 C1-C10 heterocyclyl, C3-C8 carbocycly and C6-C14 aryl optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from the group consisting of —C1-C8 alkyl, —C1-C8 alkyl-N(R′)2, —C1-C8 alkyl-C(O)R′, —C1-C8 alkyl-C(O)OR′—O—(C1-C8 alkyl), —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)N(R′)2, —NHC(O)R′, —S(O)2R′, —S(O)R′, —OH, halogen, —N3, —N(R′)2, —CN, —NHC(═NH)NH2, —NHCONH2, —S(═O)2R′ and —SR′, wherein each R′ is independently selected from the group consisting of hydrogen, C1-C8 alkyl and unsubstituted aryl, or two R′ can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl;
or R15 is




 optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C1-C8 alkyl, —C1-C8 alkyl-N(R′)2, —C1-C8 alkyl-C(O)R′, —C1-C8 alkyl-C(O)OR′, —O—(C1-C8 alkyl), —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)N(R′)2, —NHC(O)R′, —S(O)2R′, —S(O)R′, —OH, halogen, —N3, —N(R′)2, —CN, —NHC(═NH)NH2, —NHCONH2, —S(═O)2R′, —SR and arylene-R′, wherein each R′ is independently selected from the group consisting of hydrogen, C1-C8 alkyl, C1-C8heterocyclyl, C1-C10alkylene-C3-C8heterocyclyl and aryl, or two R′ can, together with the nitrogen to which they are attached, form a C1-C10 heterocyclyl;
R16 is hydrogen, —C1-C8 alkyl, —C2-C8 alkenyl, —C2-C8 alkynyl or -C1-C8 haloalkyl;
R22 is hydrogen, C1-C4 alkyl, C1-C10 heterocyclyl or C6-C14 aryl;
R23 is C1-C10 heterocyclyl; and
R17 is independently selected for each occurrence from the group consisting of F, Cl, I and Br;
R20 is -aryl, —C1-C10alkylene-aryl, where aryl on R10 comprising aryl is substituted with [R17]h;
h is 5; and
X is O or S;
provided that when R13A is hydrogen X is S.

11. The antibody or antigen binding portion thereof as claimed in claim 9, wherein the Effector Moiety is a toxin, and is selected from the group consisting of

12. The antibody as claimed in claim 1, wherein the constant light domain is a kappa domain (CLk).

13. The antibody as claimed in claim 1, wherein the constant light domain is a lambda domain (CLA).

14. A composition comprising multiples of the antibody or antigen binding portion thereof as claimed in claim 1, wherein at least about 70% of the antibodies or antigen binding portions thereof comprises the linker covalently attached to the ε-amine group of the side chain of K that is located at a position corresponding to residue 80.

15. The antibody or antigen binding portion thereof as claimed in claim 1, wherein the antibody is a full length antibody, Fab, Fab′, F(ab′)2, VH, diabody, or minibody.

16. The antibody or antigen binding portion thereof as claimed in claim 1, wherein the antibody comprises VH and VL domains from an antibody selected from the group consisting of h38C2, rituximab, cetuximab, infliximab, adalimumab, natalizumab, omalizumab, ranibizumab, trastuzumab and palivizumab.