US20130323234A1 - Mutant antibodies and conjugation thereof - Google Patents

Mutant antibodies and conjugation thereof Download PDF

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Publication number
US20130323234A1
US20130323234A1 US13/736,029 US201313736029A US2013323234A1 US 20130323234 A1 US20130323234 A1 US 20130323234A1 US 201313736029 A US201313736029 A US 201313736029A US 2013323234 A1 US2013323234 A1 US 2013323234A1
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seq
group
aspects
polypeptide
antibody
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Olivier Alexandre Laurent
Alice Lee
Richard Ryan Preston
David Tumelty
Wei Hong Yu
Abhijit Suresh Bhat
Anna Tempczyk-Russell
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Pfizer Healthcare Ireland
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Covx Technologies Ireland Ltd
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Priority to US13/736,029 priority Critical patent/US20130323234A1/en
Assigned to COVX TECHNOLOGIES IRELAND LIMITED reassignment COVX TECHNOLOGIES IRELAND LIMITED ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BHAT, ABHIJIT SURESH, LAURENT, OLIVIER ALEXANDRE, LEE, ALICE, PRESTON, RICHARD RYAN, TEMPCZYK-RUSSELL, ANNA, TUMELTY, DAVID, YU, WEI HONG
Publication of US20130323234A1 publication Critical patent/US20130323234A1/en
Assigned to PFIZER BIOLOGICS IRELAND HOLDINGS LIMITED reassignment PFIZER BIOLOGICS IRELAND HOLDINGS LIMITED ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: COVX TECHNOLOGIES IRELAND LIMITED
Assigned to PFIZER HEALTHCARE IRELAND reassignment PFIZER HEALTHCARE IRELAND ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: PFIZER BIOLOGICS IRELAND HOLDINGS LIMITED
Priority to US16/896,930 priority patent/US11780935B2/en
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/46—Hybrid immunoglobulins
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
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    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • A—HUMAN NECESSITIES
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0002—Antibodies with enzymatic activity, e.g. abzymes
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
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    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the .txt file contains a sequence listing entitled “PC71862ACorrectedSequenceListing.txt” created on Jan. 21, 2013 and having a size of 263 KB.
  • the sequence listing contained in this .txt file is part of the specification and is herein incorporated by reference in its entirety.
  • bifunctional therapeutics have 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.
  • bifunctional therapeutics may also benefit from synergies between pathways and demonstrate increased activity compared to mono-functional agents.
  • 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 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.
  • 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 ⁇ -strands A, B, C, D, E, F, and G sequentially connected together by chains of connecting amino acids, wherein the ⁇ -strands are arranged so as to form a first ⁇ -sheet comprising ⁇ -strands A, B, D, and E, and a second ⁇ -sheet comprising ⁇ -strands C, F and G, said first and second ⁇ -sheets being covalently bonded together; wherein ⁇ -strands E and F are connected together by an EF chain, and said EF chain comprises the sequence, X 1 -X 2 -X 3 -X 4 -K 5 ⁇ H 6 (SEQ ID NO:98), and wherein X 1 , X 3 and X 4 are each independently any amino acid residue, and characterized in that X 2 is selected from the group consisting of A, G, I, V, L, R, S, T, Q, P, N, M, H, W, and pharmaceutically
  • X 2 may be selected from the group consisting of A, G, I, L, R, S, T, P, N, and M (SEQ ID NO:99).
  • X 2 may be selected from the group consisting of A, G, I, L, S, T, P, and M (SEQ ID NO:100).
  • 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.
  • X 2 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).
  • X 2 may be selected from the group consisting of A, G, I, V, L, R, S, T, P, and M (SEQ ID NO:124). X 2 may be selected from the group consisting of A, G, I, V, L, S, T, and M (SEQ ID NO:125). X 2 may be selected from the group consisting of A, G, I, L, S, T, and M (SEQ ID NO:126). X 2 may be S or T. X 2 may be A or G. X 2 may be I or L.
  • the selections of X 2 described herein may also be applied to antibody constant domains of the invention, wherein position X 2 corresponds with residue 77 of SEQ ID NO:6, or residue 185 of a constant light domain according to Kabat numbering.
  • 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
  • the polypeptide may comprise an EF ⁇ -helix located on the EF chain.
  • one or more of residues X 1 , X 2 , X 3 and X 4 of SEQ ID NO:98 comprise part of the EF ⁇ -helix.
  • two or more of residues X 1 , X 2 , X 3 and X 4 of SEQ ID NO:98 comprise part of the EF ⁇ -helix.
  • three or more of residues X 1 , X 2 , X 3 and X 4 of SEQ ID NO:98 comprise part of the EF ⁇ -helix.
  • residues X 1 , X 2 , X 3 and X 4 of SEQ ID NO:98 comprise part of the EF ⁇ -helix. In some aspects, residues K 5 and H 6 of SEQ ID NO:98 do not form part of an ⁇ -helix.
  • X 1 , X 2 , X 3 , X 4 , K 5 , and H 6 may also be applied to antibody constant domains of the invention, wherein positions X 1 , X 2 , X 3 , X 4 , K 5 , and H 6 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.
  • 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:
  • X 1 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.
  • X 1 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.
  • X 1 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, X 1 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, X 1 may be selected from the group consisting of A, I, V, L, S, T, M, N, Q, K, R, H, E, and D.
  • X 1 may be selected from the group consisting of A, I, V, L, S, T, M, N, Q, R, H, E, and D.
  • X 1 may be selected from the group consisting of A, I, V, L, S, T, M, N, Q, E, and D.
  • X 1 may be selected from the group consisting of A, I, V, L, S, T, M, N, Q, E, and D.
  • the selections of X 1 described herein may also be applied to antibody constant domains of the invention, wherein position X 1 corresponds with residue 76 of SEQ ID NO:6, or residue 184 of a constant light domain according to Kabat numbering.
  • X 3 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.
  • X 3 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.
  • X 3 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.
  • X 3 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.
  • X 3 may be selected from the group consisting of I, L, F, W, Y, S, T, M, N, Q, K, R, H, E, and D.
  • X 3 may be selected from the group consisting of I, L, F, W, Y, M, N, Q, K, R, H, E, and D.
  • X 3 may be selected from the group consisting of I, L, F, W, Y, N, Q, E, and D.
  • X 3 may be selected from the group consisting of I, L, F, W, and Y.
  • X 3 may be selected from the group consisting of F, W, and Y.
  • X 3 may be selected from the group consisting of W and Y.
  • the selections of X 3 described herein may also be applied to antibody constant domains of the invention, wherein position X 3 corresponds with residue 78 of SEQ ID NO:6, or residue 186 of a constant light domain according to Kabat numbering.
  • X 4 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.
  • X 4 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.
  • X 4 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.
  • X 4 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.
  • X 4 may be selected from the group consisting of A, I, V, L, G, S, T, M, N, Q, K, R, H, E, and D.
  • X 4 may be selected from the group consisting of A, I, V, L, S, T, M, N, Q, K, R, H, E, and D.
  • X 4 may be selected from the group consisting of I, L, S, T, M, N, Q, K, R, E, and D. X 4 may be selected from the group consisting of S, T, M, N, Q, K, R, E, and D. X 4 may be selected from the group consisting of S, T, N, Q, K, R, E, and D. X 4 may be selected from the group consisting of N, Q, K, R, E, and D. X 4 may be selected from the group consisting of N, Q, K, R, and E. X 4 may be selected from the group consisting of, Q, K, and E. The selections of X 4 described herein may also be applied to antibody constant domains of the invention, wherein position X 4 corresponds with residue 79 of SEQ ID NO:6, or residue 187 of a constant light domain according to Kabat numbering.
  • 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 ⁇ -helix (the EF ⁇ -helix).
  • the first residue of the EF ⁇ -helix may be located within the first 3 residues of the EF chain.
  • the first residue of the EF ⁇ -helix may be located within the first 2 residues of the EF chain.
  • the first residue of the EF ⁇ -helix may be located at residue of the EF chain.
  • the EF ⁇ -helix may comprise at least residues X 1 and X 2 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 K 5 and H 6 corresponding to SEQ ID NO:98 are not within the ⁇ -helix.
  • residues KH that correspond to positions 80 and 81 of SEQ ID NO:6 are not within the ⁇ -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 ⁇ -helix.
  • 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 ⁇ -strands C and D; the CD chain.
  • 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 K 5 or H 6 of SEQ ID NO:98.
  • 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.
  • the residue is D, E or N.
  • the residue is D or E.
  • the residue is D or N.
  • the ⁇ -strands C and D are connected together by a CD chain, comprising a CD motif C 1 -C 2 -C 3 -C 4 (SEQ ID NO:255) wherein each of C 1 , C 2 , C 3 and C 4 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.
  • the CD motif begins at the first residue of the CD chain.
  • the CD motif begins at the second residue of the CD chain.
  • the CD motif may not form part of an ⁇ -helix.
  • the residue C 1 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 C 1 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 C 1 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 C 1 of the CD motif may be selected from the group consisting of I, L, V, P, F, and W.
  • the residue C 1 of the CD motif may be selected from the group consisting of I, L, V, F, and W. In some aspects, the residue C 1 of the CD motif may be selected from the group consisting of I, L, and V. In some aspects, the residue C 1 of the CD motif may be selected from the group consisting of L and V. In some aspects, the residue C 1 of the CD motif may be V.
  • the selections of C 1 of the CD motif described herein may also be applied to antibody constant domains of the invention, wherein position C 1 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.
  • the residue C 2 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 C 2 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 C 2 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.
  • the residue C 2 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 C 2 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 C 2 of the CD motif may be selected from the group consisting of S, T, N, Q, D, and E. In some aspects, the residue C 2 of the CD motif may be selected from the group consisting of N, Q, D, and E. In some aspects, the residue C 2 of the CD motif may be D.
  • C 2 of the CD motif described herein may also be applied to antibody constant domains of the invention, wherein position C 2 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.
  • the residue C 3 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 C 3 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 C 3 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.
  • the residue C 3 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 C 3 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 C 3 of the CD motif may be selected from the group consisting of S, T, N, Q, D, and E. In some aspects, the residue C 3 of the CD motif may be selected from the group consisting of N, Q, D, and E. In some aspects, the residue C 3 of the CD motif may be N.
  • C 3 of the CD motif described herein may also be applied to antibody constant domains of the invention, wherein position C 3 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.
  • the residue C 4 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 C 4 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 C 4 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.
  • the residue C 4 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 C 4 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 C 4 of the CD motif may be selected from the group consisting of A, V, Q, and S. In some aspects, the residue C 4 of the CD motif may be selected from the group consisting of A and S. In some aspects, the residue C 4 of the CD motif may be A.
  • C 4 of the CD motif described herein may also be applied to antibody constant domains of the invention, wherein position C 4 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.
  • 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 CH ⁇ 1, CH ⁇ 2, CH ⁇ 3, CH ⁇ 1, CH ⁇ 2, CH ⁇ 3, CH ⁇ 1, CH ⁇ 2, CH ⁇ 3, CH ⁇ 4, CH ⁇ 1, CH ⁇ 2, CH ⁇ 3, CH ⁇ 1, CH ⁇ 2, CH ⁇ 3, and CH ⁇ 4.
  • 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.
  • 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.
  • 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 (CH ⁇ 1, CH ⁇ 2, CH ⁇ 3) 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 (CH ⁇ 1, CH ⁇ 2, CH ⁇ 3, and CH ⁇ 4) 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 (CH ⁇ 1, CH ⁇ 2, CH ⁇ 3, and CH ⁇ 4) 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.
  • IgA constant heavy domains CH ⁇ 1,
  • the invention provides an immunoglobulin domain, that may be a constant light chain (CL) domain, comprising 7 ⁇ -strands A, B, C, D, E, F, and G sequentially connected together by chains of amino acids, wherein the ⁇ -strands are arranged so as to form a first ⁇ -sheet comprising ⁇ -strands A, B, D, and E, and a second ⁇ -sheet comprising ⁇ -strands C, F and G, said first and second ⁇ -sheets being covalently bonded together; wherein the chain between ⁇ -strands E and F comprises the sequence X 1 -X 2 -X 3 -X 4 -K 5 -H 6 (SEQ ID NO:98), and X 1 , X 3 and X 4 are each independently any amino acid residue, and characterized in that X 2 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,
  • 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.
  • a reactive KH group located on the EF chain of an immunoglobulin domain that may be a constant light chain (CL) domain
  • CL constant light chain
  • 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.
  • 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 ⁇ -amino group of the side chain of K 5 of SEQ ID NO:98.
  • MCS Multifunctional Antibody Conjugates
  • 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.
  • VL variable light chain
  • the covalent bond between the first and second ⁇ -sheets is a disulfide bond. In some aspects, the disulfide bond is between ⁇ -strands B and F.
  • the CL domain may be a constant light chain kappa (CL ⁇ ), and may be of rat, mouse, monkey, rabbit, goat, sheep, cow, pig, horse, donkey, dog, cat, or human origin.
  • the CL ⁇ 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.
  • the CL ⁇ 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:
  • the immunoglobulin domain may be a CL ⁇ 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.
  • the CL ⁇ 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 CL ⁇ 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, S
  • the immunoglobulin domain comprises a CL ⁇ domain
  • the domain may further comprise a CD motif as described herein.
  • the immunoglobulin domain comprises a CH ⁇ 1 domain
  • the domain may further comprise a CD motif as described herein.
  • the immunoglobulin domain comprises a CH ⁇ 2 domain
  • the domain may further comprise a CD motif as described herein.
  • residue X 2 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.
  • the ⁇ -amino group of the side chain of K 5 of SEQ ID NO:98 may be covalently attached to the linker.
  • the invention is based on the surprising discovery that mutating CL ⁇ -D 77 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 CL ⁇ -K 80 .
  • 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 Fc ⁇ R and FcRn) or binding of the antibody to its respective target.
  • Fc receptors such as Fc ⁇ R and FcRn
  • 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 CL ⁇ or the constant light chain lambda (CL ⁇ ) 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 CL ⁇ -K 80 (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.
  • the CL ⁇ 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.
  • CL ⁇ -x 82 may be any amino acid.
  • CL ⁇ -x 82 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.
  • CL ⁇ -x 82 may be G, A, V, L, or I.
  • CL ⁇ -x 82 may be K, R, N, or Q. In some aspects, CL ⁇ -x 82 may be D, or E. In some aspects, CL ⁇ -x 82 may be K, R, G, A, V, L, I, N, or Q. In some aspects, CL ⁇ -x 82 may be D, or E. In some aspects, CL ⁇ -x 82 may be K, R, G, A, V, L, I, N, Q, D or E. In some aspects, CL ⁇ -x 82 may be D, or E. In some aspects, CL ⁇ -x 82 may be H, F, W or Y. In some aspects CL ⁇ -x 82 is not proline. In some aspects, CL ⁇ -x 82 is K. In some aspects, CL ⁇ -x 82 is R.
  • antibodies of the invention, or antigen-binding portions thereof comprise an Effector Moiety conjugated to K 5 of SEQ ID NO:98 on both light chains.
  • the Effector Moiety is conjugated to K 5 of SEQ ID NO:98 on one light chain only. In some aspects, the Effector Moiety is only conjugated to K 5 of SEQ ID NO:98. In some aspects, the Effector Moiety is conjugated at K 5 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 K 5 of SEQ ID NO:98 on one light chain and 2 other locations on the antibody, or antigen-binding portions thereof.
  • the Effector Moiety is conjugated to K 5 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 K 5 of SEQ ID NO:98 on both light chains, and at one other location. In some aspects, the Effector Moiety is conjugated at K 5 of SEQ ID NO:98 on both light chains, and at 2 other locations. In some aspects, the Effector Moiety is conjugated at K 5 of SEQ ID NO:98 on both light chains, and at 3 other locations.
  • 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.
  • Ig Immunoglobulin
  • 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.
  • the invention provides for vectors and nucleic acids as deposited 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.
  • ATCC American Type Culture Collection
  • 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
  • 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.
  • 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.
  • 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.
  • 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
  • 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 K 5 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%.
  • 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 K 5 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 K 5 of SEQ ID NO:98 on both light chain constant regions.
  • 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 K 5 of SEQ ID NO:98.
  • 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%.
  • 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 K 5 of SEQ ID NO:98 on each light chain.
  • 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%.
  • 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 K 5 of SEQ ID NO:98.
  • 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%.
  • 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 K 5 of SEQ ID NO:98, wherein at least about 70% of the heavy chain molecules are unconjugated with the Effector Moiety.
  • 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.
  • 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%.
  • 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%.
  • 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%.
  • 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%.
  • 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%.
  • 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%.
  • 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.
  • 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.
  • 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.
  • the amount is about 1.7.
  • the amount is about 1.8.
  • the amount is about 1.9.
  • the amount is about 2.
  • the amount is about 2.1.
  • the amount is about 2.1.
  • the amount is about 2.3.
  • 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 CL ⁇ site.
  • 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.
  • 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.
  • aspects of the invention that allow for reduced conjugation, preferentially decorating only a single CL ⁇ -K 80 can be advantageous.
  • conjugation to CL ⁇ -K 80 is reliable and robust, conjugation to other antibody surface lysines, each of slightly different reactivity and pI 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).
  • the toxin is an auristatin; a derivative of the natural product dolastatin 10 (MMAD).
  • auristatins include MMAE (N-methylvaline-valine-dolaisoleuine-dolaproline-norephedrine) and MMAF (N-methylvaline-valine-dolaisoleuine-dolaproline-phenylalanine).
  • 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.
  • the VH and VL of antibody used for conjugation may be useful in the field of oncology.
  • Suitable antibodies include; Rituximab, (RituxanTM), a chimeric, IgG1 ⁇ , anti-CD20 antibody, used to treat cancer and in particular non Hodgkin's lymphoma and also rheumatoid arthritis; Cetuximab (ErbituxTM) a chimeric, IgG1 ⁇ , anti-EGF receptor antibody, used to treat cancer, and in particular colon, head & neck cancer.
  • 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, IgG1 ⁇ , anti-TNF ⁇ antibody, used to treat rheumatoid arthritis, ulcerative colitis, Crohn's disease, psoriasis, psoriatic arthritis, and ankylosing spondylitis; Adalimumab (HumiraTM) a human, IgG1 ⁇ , anti-TNF ⁇ antibody, used to treat rheumatoid arthritis, Crohn's disease, psoriasis, psoriatic arthritis, juvenile idiopathic arthritis and ankylosing spondylitis; Natalizumab (TysabriTM) a humanized, IgG4 ⁇ , anti- ⁇ 4-integrin antibody used to treat multiple sclerosis, rheumatoid arthritis, psoriasis, juvenile i
  • compounds and compositions of the invention may be used to treat the above mentioned conditions.
  • 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.
  • 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 R 1 and a carboxyl-terminal capping group R 2 .
  • R 1 may be CH 3 , C(O)CH 3 , C(O)CH 3 , C(O)CH 2 CH 3 , C(O)CH 2 CH 2 CH 3 , C(O)CH(CH 3 )CH 3 , C(O)CH 2 CH 2 CH 2 CH 3 , C(O)CH(CH 3 )CH 2 CH 3 , C(O)C 6 H 5 , C(O)CH 2 CH 2 (CH 2 CH 2 O) 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.
  • DCB dichlorobenzoyl
  • DFB difluorobenzoyl
  • PyC pyridinyl carboxlate
  • amido-2-PEG an amino protecting group, a lipid fatty acid group or a carbohydrate.
  • R 2 may be OH, NH 2 , NH(CH 3 ), NHCH 2 CH 3 , NHCH 2 CH 2 CH 3 , NHCH(CH 3 )CH 3 , NHCH 2 CH 2 CH 2 CH 3 , NHCH(CH 3 )CH 2 CH 3 , NHC 6 H 5 , NHCH 2 CH 2 OCH 3 , NHOCH 3 , NHOCH 2 CH 3 , a carboxy protecting group, a lipid fatty acid group or a carbohydrate.
  • the protein or peptide linking residue may be K, K SH , 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.
  • the Effector Moiety of the invention 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.
  • the linking residue is a thiol bearing residue, such as Cys or K SH 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the linker may comprise the formula: —X 1 —Y 1 —Z—; wherein X 1 is the attachment group to the Effector Moiety (for example, via a peptide-linking residue), Y 1 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).
  • the linker may be of the formula X 1 Y 1 Z* 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 X 1 Y 1 Z.
  • X 1 may be selected so as to enable a specific directional covalent linking strategy to the Effector Moiety (for example, via the peptide-linking residue).
  • X 1 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.
  • the peptide-linking residue comprises a nucleophilic group
  • X 1 may be an electrophilic group and vice versa.
  • X 1 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.
  • X 1 may comprise a nucleophilic group, such as an amine group.
  • amide bond formation strategies permit a covalent bond to be formed between the X 1 group and the peptide-linking residue by amide bond formation strategies.
  • X 1 when X 1 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 X 1 *).
  • the arrow indicates the point of attachment to the peptide-linking residue and the parallel line represents the point of attachment to the Y 1 group of the linker.
  • X 1 may comprise a malemide group, permitting a thiol-malemide addition reaction strategy to covalently link the X 1 group to the peptide-linking residue.
  • X 1 may be maleimide:
  • 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.
  • the linking residue is K SH
  • the X 1 group is maleimide
  • X 1 may comprise a pentafluorophenyl ester activated carboxyl function which may form an amide with the lysine side chain on the peptide.
  • X 1 may comprise a thiol group, allowing a disulphide bridge to be formed between the peptide-linking residue and X 1 group.
  • Y 1 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.
  • Y 1 may be selected so as to provide an overall length of the linker of between 2-100 atoms.
  • Y 1 may be selected so that the overall length of the linker is between 5 and 30 atoms.
  • Y 1 may be selected so that the overall length of linker is 15-25 atoms.
  • Y 1 may be selected so that the overall length of linker is between about 17 and about 19 atoms.
  • Y 1 may be an amino alkanoic acid, such as:
  • n 0 to 20 in some aspects 1-10, in some aspects, 1-5, and in some aspects, 1 and in some aspects, 2.
  • Y 1 may be an alkanoic diacid, such as:
  • n 0 to 20 in some aspects 1-10, in some aspects, 1-5, and in some aspects, 1 and in some aspects, 2.
  • Y 1 may be a polyglycine, such as:
  • n 0 to 10, in some aspects 1-10, in some aspects, 1-5, and in some aspects, 1 and in some aspects, 2.
  • Y 1 , X 1 —Y 1 , Y 1 —Z, and X 1 —Y 1 —Z may be selected from the group consisting of:
  • 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.
  • 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.
  • 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.
  • 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.
  • the overall length of Y 1 does not exceed 200 atoms. In some aspects, the overall length of Y 1 does not exceed 150 atoms. In some aspects, the overall length of Y 1 does not exceed 100 atoms.
  • the overall length of Y 1 does not exceed 50 atoms.
  • the range of overall chain length of Y 1 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.
  • the X 1 Y 1 Z linker may be identical to the above Y 1 groups.
  • the wavy line connects to the X 1 group.
  • the parallel lines connect to the X 1 group.
  • 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 CL ⁇ -K 80 . In some aspects, the parallel lines connect to the side chain of CL ⁇ -K 80 . 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 ( ⁇ ).
  • Z* may be selected so as to enable a specific directional covalent linking strategy to a lysine side chain on the antibody.
  • Z may be COOH, or another similarly reactive electrophile to react with the ⁇ -amino of the surface lysine side chains using one of a number of possible amide bond formation strategies.
  • Z* may be used to form an active ester.
  • Active esters connect to amines, and can thus conjugate to the ⁇ -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.
  • the Z* group comprises a structure of the formula:
  • R′ is an aliphatic or aromatic group.
  • the Z* group is of the formula:
  • 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.
  • R 1 may be a halogen, and 4 or 5 halogen atoms may be present.
  • Z* may be tetrafluorophenyl.
  • Z* may comprise the formula:
  • Z* may comprise the formula:
  • R 3 , R 4 , R 5 , R 6 and R 7 are each independently selected from the group consisting of F, CL, H and the formula CR 8 R 9 R 10 , such that no more than two of R 3 , R 4 , R 5 , R 6 and R 7 are H, and one of R 3 , R 4 , R 5 , R 6 and R 7 is CR 8 R 9 R 10 , and R 8 , R 9 , and R 10 are each independently selected from the group consisting of F, Cl and H such that no more than one of R 8 , R 9 and R 10 may be H, and wherein the parallel line represents the point of attachment to the Y 1 portion of the linker.
  • the group CR 8 R 9 R 10 is located at one of R 4 , R 5 or R 6 .
  • the group CR 8 R 9 R 10 is located at R 5
  • R 3 , R 4 , R 5 , R 6 and R 7 are each independently selected from the group consisting of F, Cl, H and the formula CR 8 R 9 R 10 , such that no more than one of R 3 , R 4 , R 5 , R 6 and R 7 is H, and one of R 3 , R 4 , R 5 , R 6 and R 7 is CR 8 R 9 R 10 .
  • R 3 , R 4 , R 5 , R 6 and R 7 are each independently selected from the group consisting of F, Cl, and the formula CR 8 R 9 R 10 , and one is CR 8 R 9 R 10 .
  • R 3 , R 4 , R 6 and R 7 are each F.
  • R 8 , R 9 , and R 10 are each independently selected from the group consisting of F and Cl. In some aspects, R 8 , R 9 , and R 10 are each F.
  • Z* may comprise the formula:
  • the Z* group is of the formula:
  • R 1 may be a halogen.
  • Z* may be selected from the group consisting of:
  • Z* may be selected from the group consisting of:
  • Z* may be selected from the group consisting of:
  • Z* may be selected from the group consisting of:
  • the leaving group is Z* and the Z group itself is the carbonyl attached to the Y 1 group.
  • the Z* group forms an amide, as shown below,
  • Z is
  • the Z* group comprises a squarate ester such as
  • R aliphatic group or substituted aromatic and may be selected from the group consisting of:
  • the Z group comprises a Maleimide group:
  • the X 1 *Y 1 Z* linker comprises a Maleimide-PEG-PFP ester of the structure:
  • the X 1 *Y 1 Z* linker comprises a structure selected from the group consisting of:
  • 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.
  • 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.
  • 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.
  • 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.
  • the overall length of Y 1 does not exceed 200 atoms. In some aspects, the overall length of Y 1 does not exceed 150 atoms. In some aspects, the overall length of Y 1 does not exceed 100 atoms.
  • the overall length of Y 1 does not exceed 50 atoms.
  • the range of overall chain length of Y 1 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.
  • the MAC comprises a X 1 Y 1 Z linker of the formula:
  • the X 1 *Y 1 Z* linker comprises a PEG-bis-pentafluorophenyl ester of the formula:
  • the MAC comprises 2 peptides conjugated per antibody. In some aspects, one peptide is conjugated at each of the 2 CL ⁇ -K 80 residues of the antibody or antigen binding fragment thereof.
  • polypeptide of the invention comprises a formula selected from the group consisting of:
  • 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.
  • 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.
  • 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.
  • the overall length of Y 1 does not exceed 200 atoms. In some aspects, the overall length of Y 1 does not exceed 150 atoms. In some aspects, the overall length of Y 1 does not exceed 100 atoms.
  • the overall length of Y 1 does not exceed 50 atoms.
  • the range of overall chain length of Y 1 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.
  • the linker is selected from the group consisting of
  • 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.
  • 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 ( ⁇ ) to the Y 1 portion of the linker (or P 1 portion, where the linker is for a catalytic antibody combining site). Accordingly, the linkers would take the formula X 1 - ⁇ -Y 1 —Z, X 1 - ⁇ -Y 1 —Z*, and P 1 - ⁇ -Q 1 -W 1 .
  • 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.
  • VitCitABC valine-citrulline p-aminobenzyl carbamate
  • the wavy line typically indicates the point of attachment to the Y 1 (or Q 1 ) portion of the linker
  • the parallel line represents the point of attachment to the X 1 (or P 1 ) portion of the linker, or even to the Effector Moiety itself.
  • the wavy line may indicate the point of attachment to the X 1 or P 1 linker portion (or Effector Moiety)
  • the parallel line may indicate the point of attachment to the Y 1 or Q 1 portion of the linker.
  • the invention provides for linker of the formula:
  • 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.
  • 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.
  • 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.
  • 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.
  • the overall length of Y 1 does not exceed 200 atoms. In some aspects, the overall length of Y 1 does not exceed 150 atoms. In some aspects, the overall length of Y 1 does not exceed 100 atoms.
  • the overall length of Y 1 does not exceed 50 atoms.
  • the range of overall chain length of Y 1 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.
  • 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.
  • useful payloads include those disclosed in PCT/IB2012/056224 including all pharmaceutically acceptable salts, hydrates and free base forms.
  • effector moieties and linkers may be used in aspects of the invention:
  • Y 2 is —C 2 -C 20 alkylene-, —C 2 -C 20 heteroalkylene-; —C 3 -C 8 carbocyclo-, -arylene-, —C 3 -C 8 heterocyclo-, —C 1 -C 10 alkylene-arylene-, -arylene-C 1 -C 10 alkylene-, —C 1 -C 10 alkylene-(C 3 -C 8 carbocyclo)-, -(C 3 -C 8 carbocyclo)-C 1 -C 10 alkylene-, —C 1 -C 10 alkylene-(C 3 -C 8 heterocyclo)- or —(C 3 -C 8 heterocyclo)-C 1 -C 10 alkylene-;
  • R 12 is hydrogen, C 1 -C 8 alkyl or C 1 -C 8 haloalkyl
  • R 13A and R 13B are either of the following:
  • R 14A and R 14B are either of the following:
  • each R′ is independently selected from the group consisting of C 1 -C 8 alkyl, —C 1 -C 8 alkyl-N(R′) 2 , —C 1 -C 8 alkyl-C(O)R′, —C 1 -C 8 alkyl-C(O)OR′, —O—(C 1 -C 8 alkyl), —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)N(R′) 2 , —NHC(O)R′, —S(O) 2 R′, —S(O)R′, —OH, halogen, —N 3 , —N(R′) 2 , —CN, —NHC( ⁇ NH)NH 2 , —NHCONH 2 , —S( ⁇ O) 2 R′, —SR′ and arylene-R′, wherein each R′ is independently selected from the group consisting of C 1 -
  • R 16 is hydrogen, —C 1 -C 8 alkyl, —C 2 -C 8 alkenyl, —C 2 -C 8 alkynyl or —C 1 -C 8 haloalkyl;
  • R 22 is hydrogen, C 1 -C 4 alkyl, C 1 -C 10 heterocyclyl or C 6 -C 14 aryl;
  • R 23 is C 1 -C 10 heterocyclyl
  • R 17 is independently selected for each occurrence from the group consisting of F, Cl, I and Br;
  • R 20 is -aryl, —C 1 -C 10 alkylene-aryl, where aryl on R 10 comprising aryl is substituted with [R 17 ] h ;
  • X is O or S
  • R 13A is hydrogen X is S.
  • the effector moieties may be selected from Table 73.
  • the effector moiety is Toxin #54.
  • the effector moiety is Toxin #115.
  • the effector moiety is Toxin #69.
  • 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/IB2012/056224.
  • the toxin-linkers of the invention may be selected from the group consisting of
  • 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 K 5 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;
  • MAC multifunctional antibody conjugate
  • said method comprising: covalently attaching the Effector Moiety to a linker terminating in a leaving group Z* of the formula:
  • R 1 may be a halogen.
  • R 1 may be present in an amount of between 3 and 5. There may be 3 R 1 groups. R 1 may be present in an amount of between 4 and 5. There may be 4 R 1 groups. There may be 5 R 1 groups. R 1 may be fluorine. R 1 may be chlorine. R 1 may be bromine.
  • the leaving group may comprise the formula:
  • 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 ⁇ -amino of surface lysine residues of the antibody.
  • 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:
  • R 1 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 K 5 of SEQ ID NO:98.
  • 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.
  • 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.
  • 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.
  • the molar ratio may be between about 1:1 and about 3:1.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the conjugation reaction between antibody and Effector Moiety is at room temperature for 2 hrs.
  • the conjugation reaction is for at least about 2 hrs.
  • 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 .
  • the method may comprise conjugating an Effector Moiety to CL ⁇ -K 80 .
  • the invention comprises conjugating a Effector Moiety to an Ig domain, comprising mutating the CL ⁇ so as to comprise a SEQ ID NO:98 on the EF connecting chain loop between ⁇ -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 K 5 of SEQ ID NO:98.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the MAC and a pharmaceutically acceptable carrier.
  • 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.
  • 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.
  • 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.
  • 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).
  • the antibody is administered by intravenous infusion or injection.
  • 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.
  • 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.
  • the kit includes the antibody or a pharmaceutical composition thereof and a diagnostic agent.
  • 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.
  • agents and compounds of the invention can be combined with pharmaceutically acceptable vehicles such as saline, Ringer's solution, dextrose solution, and the like.
  • 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, aspara
  • 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.
  • PEG-PE PEG-derivatized phosphatidylethanolamine
  • 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.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • 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.
  • copolymers of L-glutamic acid and 7 ethyl-L-glutamate 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.
  • LUPRON DEPOTTM injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate
  • sucrose acetate isobutyrate sucrose acetate isobutyrate
  • poly-D-( ⁇ )-3-hydroxybutyric acid poly-D-( ⁇ )-3-hydroxybutyric acid.
  • compositions 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.
  • an oil e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil
  • a phospholipid e.g., egg phospholipids, soybean phospholipids or soybean lecithin
  • other ingredients may be added, for example glycerol or glucose, to adjust the tonicity of the emul
  • 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 ⁇ m, particularly 0.1 and 0.5 ⁇ m, 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.
  • 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-Fluorouracil, 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.
  • 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.
  • methods of treating cancer comprising administering to the subject a therapeutically effective amount of a compound or pharmaceutical composition according to the invention.
  • 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.
  • cancers of the lung 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.
  • NNSCLC and SCLC cancers of the lung
  • SCLC head or neck
  • the ovary the colon
  • the rectum the prostate
  • the anal region the stomach
  • the breast the breast
  • kidney or ureter
  • 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.
  • compositions of the invention relate to non-cancerous hyperproliferative disorders such as, without limitation, age-related macular degeneration, restenosis after angioplasty and psoriasis.
  • 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.
  • 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.
  • 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.
  • 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.
  • an effective dosage of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly.
  • 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.
  • 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.
  • mammals are mammals, more preferably, a human. Mammals also include, but are not limited to, farm animals, sport animals, pets, primates, and horses.
  • 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.
  • 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.
  • the immunoglobulin (Ig) domain is a type of protein domain that consists of a 2-layer sandwich of between 7 and 9 antiparallel ⁇ -strands arranged in two ⁇ -sheets with a Greek key topology.
  • a ⁇ -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 ⁇ -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 ⁇ -sheets.
  • the pattern is (N-terminal ⁇ -hairpin in sheet 1)-( ⁇ -hairpin in sheet 2)-( ⁇ -strand in sheet 1)-(C-terminal ⁇ -hairpin in sheet 2).
  • 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 ⁇ -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 ⁇ -helix.
  • the ⁇ -helix is the most regular and the most predictable from sequence, as well as the most prevalent.
  • immunoglobulin is a tetrameric molecule.
  • 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).
  • 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 ⁇ and ⁇ light chains.
  • Heavy chains are classified as ⁇ , 67 , ⁇ , ⁇ , and ⁇ , and define the antibody's isotype as IgA, IgD, IgE, IgG, IgM, respectively.
  • 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.
  • Ig fold immunoglobulin fold
  • the Ig fold of constant domains contains a 3-stranded ⁇ sheet packed against a 4-stranded ⁇ sheet, with each sheet separated by chains; these chains typically comprise ⁇ -helices, loops, turns, and short, sharp turns between two ⁇ -sheets called ⁇ -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.
  • FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 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)).
  • 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).
  • CDR identification includes 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.
  • 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).
  • 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.
  • 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.
  • 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 CL ⁇ and CL ⁇ domains can vary.
  • the numbering of the CL ⁇ can begin at either LC-R 108 according to Kabat numbering (for example, R 108 of SEQ ID NO:2), or LC-T 109 according to Kabat numbering (for example, T 109 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-T 109 . 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;
  • a dAb fragment consists of a VH domain or a VL domain (e.g. human, camelid, or shark).
  • 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 ⁇ -strands .
  • a single-chain antibody 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, ⁇ and ⁇ , and in any given antibody molecule only one type occurs. Approximately twice as many ⁇ as ⁇ 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.
  • V variable
  • J joining
  • C constant
  • RNA level 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 (CL ⁇ ) regions are encoded by a single gene, whereas lambda constant (CL ⁇ ) regions are encoded by multiple genes, and undergo splicing.
  • IgCL ⁇ 1 Mcg marker
  • IGLC2-IgCL ⁇ 2 Kern-Oz ⁇ marker
  • IgCL ⁇ 3 Kern-Oz+ marker
  • IgCL ⁇ 7 IgCL ⁇ 7
  • SEQ ID NO:93 incorporates many of the presently identified polymorphisms.
  • the sequences of the present invention encompass other known polymorphisms of the CL ⁇ and CL ⁇ , and antibodies in general.
  • Two polymorphic loci have been identified in the CL ⁇ ; CL ⁇ -V/A 45 and CL ⁇ -L/V 83 .
  • the three polymorphisms so far identified are: Km(1): CL ⁇ -V 45 /L 83 ; Km(1,2): CL ⁇ -A 45 /L 83 ; and Km(3): CL ⁇ -A 45 /V 83 .
  • 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.
  • 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.
  • 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.
  • a humanized antibody may be produced by fusing the constant domains from a human antibody to the variable domains of a non-human species.
  • 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.
  • epitopic determinants 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.
  • multifunctional antibody conjugate 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.
  • the invention provides a MAC comprising an antibody that does not bind complement.
  • 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.
  • an antibody 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.
  • 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, atlizumabi, atorolimumab, bapineuzumab, basiliximab (SIMULECT®), bavituximab, bect
  • 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.
  • 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: PDGFR ⁇ , PDGFR ⁇ , 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- ⁇ , CXCL12, SDF-I, bFGF, MAC-I, IL23pl9, FPR, IGFBP4, CXCR3, TLR4, CXCR2, EphA2, EphA4, EphrinB2, EGFR (ErbBI), HER2 (ErbB2 or pl85neu), HER3 (ErbBI), HER2 (ErbB
  • the antigen binding domain specifically binds to a member (receptor or ligand) of the TNF superfamily.
  • TNF- ⁇ Tumor Necrosis Factor- ⁇
  • TNF- ⁇ Tumor Necrosis Factor- ⁇
  • LT- ⁇ Lymphotoxin- ⁇
  • 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,
  • the antigen binding domain is capable of binding one or more targets chosen from 5T4, ABL, ABCB5, ABCFI, ACVRI, ACVRIB, ACVR2, ACVR2B, ACVRLI, AD0RA2A, 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, B
  • the MAC comprises a catalytic antibody, or antigen binding portion thereof.
  • the antibody may be an aldolase antibody.
  • “Combining site”, as used herein, 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 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:
  • 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 ⁇ -amino group of the lysine of the catalytic antibody.
  • 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.
  • 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.
  • the amino acid residue or its reactive functional groups may be attached to an amino acid residue in the antibody combining site.
  • 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.
  • the amino acid is cysteine
  • the reactive group of the side chain is a sulfhydryl group.
  • the amino acid residue is lysine, and the reactive group of the side chain is the ⁇ -amino group.
  • the amino acid is K 93 on the heavy chain according to Kabat numbering.
  • the amino acid is on HC-K 99 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, ⁇ 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).
  • 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.
  • 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.
  • 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.
  • 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 CL ⁇ and CH ⁇ 1 1.
  • 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 ⁇ -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.
  • the antibody is a chimeric antibody comprising the VL and VH region from a murine aldolase antibody (e.g.
  • 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.
  • h38c2 F(ab′) 2 may be produced by the proteolytic digestion of h38c2 IgG1.
  • 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).
  • K off refers to the off rate constant for dissociation of an antibody from the antibody/antigen complex.
  • K d refers to the dissociation constant of a particular antibody-antigen interaction.
  • the invention provides for pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, and prodrugs of compounds, samples, compositions and pharmaceutical compositions of the invention.
  • Certain linkers suitable for connecting targeting agents (TA) to the combining site of catalytic antibodies 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.
  • 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.
  • 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.
  • 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.
  • the CAb-linker may be covalently linked to the side chain of the TA-linking residue.
  • the linker may comprise the formula: P 1 -Q 1 -W 1 ; wherein P 1 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, Q 1 is an optionally present recognition group comprising at least a ring structure; and W 1 is an attachment moiety comprising a covalent link to an amino acid side chain in a combining site of an antibody.
  • Q 1 may have the optionally substituted structure:
  • a, b, c, d, and e are independently carbon or nitrogen; f is carbon, nitrogen, oxygen, or sulfur; Q 1 is attached to P 1 and W 1 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.
  • Q 1 may be phenyl.
  • 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.
  • a chemical moiety for modification by an aldolase antibody may be a ketone, diketone, ⁇ lactam, active ester haloketone, lactone, anhydride, maleimide, ⁇ -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.
  • W 1 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 ⁇ -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.
  • 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 ⁇ -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
  • the reactive group includes one or more C ⁇ O groups arranged to form an acyl ⁇ -lactam, simple diketone, succinimide active ester, maleimide, haloacetamide with linker, haloketone, cyclohexyl diketone, or aldehyde.
  • W 1 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 ⁇ -haloketone, an aldehyde, a maleimide, a lactone, an anhydride, an ⁇ -haloacetamide, an amine, a hydrazide, or an epoxide.
  • the W 1 group is covalently linked to a macromolecule scaffold that can provide increased half-life to the peptides of the invention.
  • the W 1 group if present is covalently linked to the combining site of an antibody.
  • W 1 prior to conjugation (for example, with the combining site of an antibody), has the structure:
  • W 1 has the structure:
  • P 1 may be a group comprising three components; P 1 p-P 1 s-P 1 y, wherein P 1 p is a group specifically adapted to be combinable with the targeting agent, P 1 s is a spacer region of the P 1 group, and P 1 y is a group adapted to bind to the W 1 group.
  • P 1 y is selected from an amide bond, an enamine bond, or a guanidinium bond.
  • P 1 y may be selected so as to provide a hydrogen molecule adjacent (within two atoms) to the Q 1 group. While not wishing to be bound by theory, it is believed that the H atom can assist the Q 1 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 Q 1 group, providing the H of the NH group for hydrogen bonding.
  • the C ⁇ O group of an amide may be bonded to the Q 1 group, with the H of the NH group about 2 atoms adjacent to the Q 1 group, but still available for H-bonding.
  • P 1 y is absent.
  • the P 1 y group has the formula:
  • P 1 s is selected such that P 1 s does not provide any overly reactive groups.
  • P 1 s may be selected so as to provide an overall length of the P 1 groups of between 2-15 atoms.
  • P 1 s may be selected so that the overall length of the P 1 group is between 2 and 10 atoms.
  • X 1 groups may be selected so that the overall length of P 1 group is 4-8 atoms.
  • P 1 groups may be selected so that the overall length of the P 1 group is 5 atoms.
  • P 1 groups may be selected so that the overall length of P 1 group is 6 atoms.
  • P 1 groups may comprise one of the following formulae:
  • 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.
  • P 1 p 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.
  • TA-linking residue such as a peptide, protein, small molecule, nucleic acid or aptamer.
  • P 1 p may be an electrophilic group and vice versa.
  • 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.
  • P 1 p may comprise a nucleophilic group, such as an amine group. Either of these strategies permits a covalent bond to be formed between the P 1 p group and the TA-linking residue by amide bond formation strategies.
  • the TA-linking group is an amine group
  • P 1 p may comprise the formula:
  • P 1 may be an optionally present biologically compatible polymer or block copolymer.
  • P 1 may be of the structure:
  • 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 C 1-10 alkyl, substituted or unsubstituted C 3-7 cycloalkyl-C 1-6 alkyl, or substituted or unsubstituted aryl-C 1-6 alkyl.
  • P 1 p may comprise a maleimide group (or similar) permitting a thiol- maleimide addition reaction strategy to covalently link the P 1 p group to the TA-linking residue.
  • P 1 p may also comprise a thiol group, allowing a disulphide bridge to be formed between the TA-linking residue and P 1 p group.
  • P 1 p may be be maleimide:
  • the mechanism of conjugation may be as follows:
  • the P 1 p group comprises a substituted maleimide:
  • P 1 is
  • v and w are selected such that the backbone length of X 1 is 6-12 atoms;
  • the TA-linker is of the formula:
  • 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.
  • TA-linker is of the formula:
  • 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.
  • the P 1 portion of CA-linkers may be used as the Y 1 , X 1 —Y 1 , Y 1 —Z and X 1 —Y 1 —Z, portion of linkers for a MAC of the invention.
  • K SH refers to: Aib (2-aminoisobutyric acid):
  • 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.
  • N-terminus refers to the free ⁇ -amino group of an amino acid in a peptide
  • C-terminus refers to the free ⁇ -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 ⁇ -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 ⁇ -amino nitrogen.
  • halo refers to F, Cl, Br or I.
  • 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.
  • biologically compatible means something that is biologically inert or non reactive with intracellular and extra cellular biological molecules, and non toxic.
  • 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. “C 1 -C 8 ” 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 C 1 -C 8 alkyls include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl and n-octyl; while branched C 1 -C 8 alkyls include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and -2-methylbutyl; unsaturated C 2 -C 8 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-he
  • 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
  • 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.
  • 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.
  • unsubstituted alkyl refers to a divalent unsubstituted alkyl group as defined above.
  • methylene, ethylene, and propylene are each examples of unsubstituted alkylenes.
  • 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.
  • 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.
  • the phrase would include methylcylcohexyl groups among others.
  • 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.
  • substituted cycloalkyl has the same meaning with respect to unsubstituted cycloalkyl groups that substituted alkyl groups have with respect to unsubstituted alkyl groups.
  • the phrase includes, but is not limited to, oxocyclohexyl, chlorocyclohexyl, hydroxycyclopentyl, and chloromethylcyclohexyl groups.
  • 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: C 1 -C 8 alkyl, —O—(C 1 -C 8 alkyl), —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH 2 , —C(O)NHR′, —C(O)N(R′) 2 , —NHC(O)R′, —S(O) 2 R′, —S(O)R′, —OH, halogen, —N 3 , —NH 2 , —NH(R′), —N(R′) 2 and —CN; wherein each R′ is independently selected from —H, C 1 -C 8 alkyl and unsubstituted aryl.
  • a substituted carbocyclic aromatic group can further include one or more of: —NHC( ⁇ NH)NH 2 , —NHCONH 2 , —S( ⁇ O) 2 R′ and —SR′.
  • “Arylene” is the corresponding divalent moiety.
  • substituted alkyl means an alkyl in which one or more hydrogen atoms are each independently replaced with a substituent.
  • substituents include, but are not limited to, —X, —R, —O—, —OR, —SR, —S ⁇ , —NR 2 , —NR 3 , ⁇ NR, —CX 3 , —CN, —OCN, —SCN, —N ⁇ C ⁇ O, —NCS, —NO, —NO 2 , ⁇ N 2 , —N 3 , —NRC( ⁇ O)R, —C( ⁇ O)NR 2 , —SO 3 ⁇ , —SO 3 H, —S( ⁇ O) 2 R, —OS( ⁇ O) 2 OR, —S( ⁇ O) 2 NR, —S( ⁇ O)R, —OP( ⁇ O)(OR) 2 , —P( ⁇ O)(OR) 2 , —PO 3 2
  • aralkyl by itself or part of another term, means an alkyl group, as defined above, substituted with an aryl group, as defined above.
  • alkylene 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 (—CH 2 —), 1,2-ethylene —CH 2 CH 2 —), 1,3-propylene (—CH 2 CH 2 CH 2 —), 1,4-butylene (—CH 2 CH 2 CH 2 CH 2 —), and the like.
  • a “C 1 -C 10 ” straight chain alkylene is a straight chain, saturated hydrocarbon group of the formula —(CH 2 ) 1-10 —.
  • Examples of a C 1 -C 10 alkylene include methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, ocytylene, nonylene and decalene.
  • heteroalkylene by itself or as part of another substituent means a divalent group derived from heteroalkyl (as discussed above).
  • heteroatoms can also occupy either or both of the chain termini.
  • C 3 -C 8 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.
  • heterocyclyl is attached to its pendant group at any heteroatom or carbon atom that results in a stable structure.
  • Representative examples of a C 3 -C 8 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 C 3 -C 8 heterocyclyl can be substituted with up to seven groups including, but not limited to, C 1 -C 8 alkyl, C 1 -C 8 heteroalkyl, —OR′, aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH 2 , —C(O)NHR′, —C(O)N(R′) 2 , —NHC(O)R′, —S( ⁇ O) 2 R′, —S(O)R′, halogen, —N 3 , —NH 2 , —NH(R′), —N(R′) 2 and —CN; wherein each R′ is independently selected from —H, C 1 -C 8 alkyl, C 1 -C 8 heteroalkyl and aryl.
  • a substituted heterocyclyl can also include one or more of: —NHC( ⁇ NH)NH 2 , —NHCONH 2 , —S( ⁇ O) 2 R′ and —SR′.
  • “Heterocyclo” is the corresponding divalent moiety.
  • C 3 -C 8 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 C 3 -C 8 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 C 3 -C 8 carbocyclyl group can be unsubstituted or substituted with up to seven groups including, but not limited to, C 1 -C 8 alkyl, C 1 -C 8 heteroalkyl, —OR′, aryl, —C(O)R′, —OC(O)R′, —C(O)OR, —C(O)NH 2 , —C(O)NHR′, —C(O)N(R′) 2 , —NHC(O)R′, —S( ⁇ O) 2 R′, —S( ⁇ O)R′, —OH, -halogen, —N 3 , —NH 2 , —NH(R′), —N(R′) 2 and —CN; where each R′ is independently selected from —H, C 1 -C 8 alkyl, C 1 -C 8 heteroalkyl and aryl. “C 3 -C 8 carbocyclo” is the corresponding divalent mo
  • heteroarylkyl 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.
  • Connecting chain refers to the sequences of amino acids in any tertiary structural form other than a ⁇ -strand that connect the individual ⁇ -strands of an immunoglobulin domain.
  • the terms encompass the structural motifs of ⁇ -helices, turns, loops, and ⁇ -hairpins.
  • ⁇ -helices”, “turns”, “loops”, and “ ⁇ -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.
  • identity 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.
  • 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”).
  • 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.
  • conservative amino acid substitution refers to a substitution of a native amino acid residue with a normative 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 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 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
  • CATH Class, Architecture, Topology, Homology
  • 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 may be used.
  • sequence alignment tools such as BLAST, CLUSTAL and others known to the skilled person, such as those described herein
  • sequence alignment tools such as BLAST, CLUSTAL and others known to the skilled person, such as those described herein
  • sequence alignment tools such as BLAST, CLUSTAL and others known to the skilled person, such as those described herein
  • sequence alignment tools such as BLAST, CLUSTAL and others known to the skilled person, such as those described herein
  • sequence alignment tools such as BLAST, CLUSTAL and others known to the skilled person, such as those described herein
  • 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.
  • MUM maximum unique match
  • 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, Wis.), 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).
  • NCBI National Center for Biotechnology Information
  • the well-known Smith Waterman algorithm may also be used to determine identity.
  • 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).
  • gap opening penalties 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).
  • 101 X 3 A, G, I, V, L, KAxYEKH R, S, T, Q, P, N, M, H, W.
  • 102 X 3 A, G, I, L, R, KAxYEKH S, T, P, N, M.
  • X 3 A, G, I, L, S, KAxYEKH T, P, M.
  • X 82 any AA;
  • X 83 L/V 105 hCL ⁇ 1-106
  • X 1 any aa
  • X 2 any xxxxxKH aa
  • X 3 A, G, I, V, L, R, S, T, Q, P, N, M, H, W
  • X 4 any aromatic amino acid
  • X 5 any aa.
  • X 2 A, G, I, V, L, R, S, T, Q, P, N, M, H, W.
  • X 3 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E, G, P;
  • X 4 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E, G, P; 171
  • X 1 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.
  • X 2 A, G, I, V, L, R, S, T, Q, P, N, M, H, W.
  • X 3 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E, G;
  • X 4 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E, G; 172
  • X 1 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.
  • X 2 A, G, I, V, L, R, S, T, Q, P, N, M, H, W.
  • X 3 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E;
  • X 4 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E; 173
  • X 1 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.
  • X 3 L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E;
  • X 4 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E; 174
  • X 1 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.
  • X 3 L, I, F, W, Y, M, N, Q, K, R, H, D, E;
  • X 4 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E; 175
  • X 1 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
  • X 2 A, G, I, V, L, R, S, T, Q, P, N, M, H, W.
  • X 3 L, I, F, W, Y, N, Q, D, E;
  • X 4 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E; 176
  • X 1 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.
  • X 1 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.
  • X 1 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.
  • X 3 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E;
  • X 4 A, V, L, I, S, T, M, N, Q, K, R, H, D, E; 179
  • X 1 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
  • X 2 A, G, I, V, L, R, S, T, Q, P, N, M, H, W.
  • X 3 A, V, L, I, S, T, M, N, Q, K, R, D, E;
  • X 4 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E; 180
  • X 1 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.
  • X 3 L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E;
  • X3 is not A or V
  • S or T X 2 A, G, I, V, L, X4 is not F, Y, W R, S, T, Q, P, N, M, H, W.
  • X 3 L, I, F, W, Y, M, N, Q, K, R, H, D, E;
  • X 4 A, V, L, I, S, T, M, N, Q, K, R, H, D, E; 182
  • X 1 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
  • X 2 A, G, I, V, L, X4 is not F, Y, W, or H R, S, T, Q, P, N, M, H, W.
  • X 3 L, I, F, W, Y, N, Q, D, E;
  • X 4 A, V, L, I, S, T, M, N, Q, K, R, D, E; 183
  • X 1 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.
  • X 1 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.
  • X 2 A, G, I, V, L, R, S, T, Q, P, N, M, H, W.
  • X 3 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E;
  • X 4 N, Q, K, R, D, E; 186
  • X 1 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
  • X 2 A, G, I, V, L, R, S, T, Q, P, N, M, H, W.
  • X 3 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E;
  • X 4 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E; 187
  • X 1 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
  • K X 2 A, G, I, V, L, R, S, T, Q, P, N, M, H, W.
  • X 3 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E;
  • X 4 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E; 188
  • X 1 A, V, L, I, S, xxxxKH T, M, N.
  • X1 X3 X4 no c, p, g X 2 A, G, I, V, L, X1 is not aromatic or K, or charged R, S, T, Q, P, N, M, H, W.
  • X 3 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E;
  • X 4 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E; 189
  • X 1 A, V, L, I, M.
  • xxxxKH X 2 A, G, I, V, L, X1 X3 X4 not C, P, G R, S, T, Q, P, N, M, X1 is hydrophilic H, W.
  • X 3 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E;
  • X 4 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E; 190
  • X 1 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
  • X 2 A, G, I, V, L, X4 is not F, Y, W R, S, T, Q, P, N, M, H, W.
  • X 3 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E;
  • X 4 A, V, L, I, S, T, M, N, Q, K, R, H, D, E; 191
  • X 1 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
  • X 2 A, G, I, V, L, X4 is not F, W, Y, H R, S, T, Q, P, N, M, H, W.
  • X 3 A, V, L, I, S, T, M, N, Q, K, R, D, E;
  • X 4 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E; 192
  • X 1 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
  • X 2 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.
  • X 3 L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E;
  • X 4 A, V, L, I, S, T, M, N, Q, K, R, H, D, E; 193
  • X 1 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
  • X 2 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.
  • X 3 L, I, F, W, Y, M, N, Q, K, R, H, D, E;
  • X 4 A, V, L, I, S, T, M, N, Q, K, R, H, D, E; 194
  • X 1 A, V, L, I, S, xxxxKH T, M, N, Q.
  • X1 X3 X4 not C, P, G X 2 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
  • X 3 L, I, F, W, Y, N, Q, D, E
  • X 4 A, V, L, I, S, T, M, N, Q, K, R, D, E; 195
  • X 1 A, V, L, I.
  • X 2 A, G, I, V, L, X1 X3 X4 not C, P, G R, S, T, Q, P, N, M, X1 is hydrophilic H, W.
  • X 4 A, V, L, I, S, X4 is not F, Y, W, or H T, M, N, Q, K, R, D, E;
  • 196
  • X 1 A, V, L, I.
  • X 2 A, G, I, V, L, X1 X3 X4 not C, P, G R, S, T, Q, P, N, M, X1 is hydrophilic H, W.
  • X 4 N, Q, K, R, D, E; 197
  • X 1 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.
  • X 3 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E;
  • X 4 A, V, L, I, S, T, M, N, Q, K, R, H, D, E; 198
  • X 1 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
  • X 2 A, G, I, V, L, X4 is not F, W, Y, H R, S, T, Q, P, N, M, H, W.
  • X 3 A, V, L, I, S, T, M, N, Q, K, R, D, E;
  • X 4 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E; 199
  • X 1 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
  • X 2 A, G, I, V, L, R, S, T, Q, P, N, M, H, W.
  • X 3 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E, G, P;
  • X 4 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E, G, P; 200
  • X 1 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
  • X 2 A, G, I, V, L, R, S, T, Q, P, N, M, H, W.
  • X 3 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E, G;
  • X 4 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E, G;
  • 201 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
  • X 2 A, G, I, V, L, R, S, T, Q, P, N, M, H, W.
  • X 3 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E;
  • X 4 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E;
  • 202
  • X 1 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
  • X 2 A, G, I, V, L, X3 is not A or V R, S, T, Q, P, N, M, H, W.
  • X 3 L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E;
  • X 4 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E;
  • 203
  • X 1 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
  • X 2 A, G, V, S, T, X3 is not A or V, S or T Q, P, N, M, H.
  • X 3 L, I, F, W, Y, M, N, Q, K, R, H, D, E;
  • X 4 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E;
  • X 1 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.
  • X 3 L, I, F, W, Y, N, Q, D, E;
  • X 4 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E;
  • X 1 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
  • X 2 A, G, S, T, P, X3 is not C, G, P, A, V, S or T, M, K, R, H, N, Q, D, M.
  • X 1 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
  • X 2 A, G, S, T, P, X3 is Y or W M.
  • X2 is A, G, P, S, T, M
  • X 2 A, G, S, T, P, X3 is not A or V M.
  • X 4 A, V, L, I, S, T, M, N, Q, K, R, H, D, E;
  • X 1 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
  • X 2 A, G, S, T, P, X3 is not A or V, S or T M.
  • X 4 A, V, L, I, S, T, M, N, Q, K, R, H, D, E; 209
  • X 1 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
  • X 2 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
  • X 3 L, I, F, W, Y, N, Q, D, E
  • X 4 A, V, L, I, S, T, M, N, Q, K, R, D, E
  • X 1 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.
  • X 3 L, I, F, W, Y, X4 is not F, Y, W, or H
  • X 4 A, V, L, I, S, T, M, N, Q, K, R, D, E; 211
  • X 1 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.
  • X 1 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
  • X 2 A, G, I, V, L, X4 is E, K, D, R, N, or Q. S, T, Q, P, N, M, H.
  • X 3 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E;
  • X 4 N, Q, K, R, D, E; 213
  • X 1 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
  • X 2 A, G, I, V, L, X1 is not aromatic S, T, Q, P, N, M, H.
  • X 3 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E;
  • X 4 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E;
  • 214
  • X 1 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
  • X 2 A, G, I, V, L, X2 is not W or R S, T, Q, P, N, M, H.
  • X 3 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E;
  • X 4 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E; 215
  • X 1 A, V, L, I, S, xxxxKH T, M, N.
  • X1 is hydrophilic
  • X 3 A, V, L, I, F, X2 is not W or R W, Y, S, T, M, N, Q, K, R, H, D, E
  • X 4 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E
  • X1 is not aromatic
  • X 2 A, G, I, V, L, X2 is not W or R S, T, Q, P, N, M, H.
  • X 4 A, V, L, I, S, T, M, N, Q, K, R, H, D, E; 218
  • X 1 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
  • X 2 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
  • X 3 A, V, L, I, S, T, M, N, Q, K, R, D, E;
  • X 4 A, V, L, I, F, W, Y, S, T, M, N, Q, K, R, H, D, E; 219
  • X 1 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
  • X 2 A, G, I, V, L, Is not W or R S, T, Q, P, N, M, H.
  • X3 is not A or V
  • S or T X 3 L, I, F, W, Y, X4 is not F, Y, W M, N, Q, K, R, H, D, E
  • X 4 A, V, L, I, S, T, M, N, Q, K, R, H, D, E
  • X 1 A, V, L, I, S, xxxxKH T, M, N, Q.
  • X 4 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
  • X 1 A, V, L, I.
  • xxxxKH X 2 A, G, S, T, M.
  • APTECS 243 CL ⁇ GQPKAAPSVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKxDSSPVK X44 is V, I, L AGVETTTPSK QSNNKYAASS YLSLTPExWK K HRSYSCQVT HEGSTVEKTV X78 is A APTECS 244 CL ⁇ GQPKAAPSVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKVDSSPVK X44 is V AGVETTTPSK QSNNKYAASS YLSLTPExWK K HRSYSCQVT 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
  • FIGS. 1A , 1 B, 1 C, 1 D 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 , 2 B, 2 C, 2 D, 2 E, 2 F, 2 G, 2 H 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-K 188 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-K 190 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 (mCL ⁇ ), human constant light chain kappa region (hCL ⁇ ), and human constant light chain lambda region (hCL ⁇ ). Differences between mCL ⁇ and hCL ⁇ ; and between hCL ⁇ and hCL ⁇ ; are shown as asterisks, and conserved substitutions are shown as crosses. ⁇ -strands A-G are underlined. The turn between ⁇ -strands A and B and the ⁇ -helix between ⁇ -strands E and F are each indicated in italics.
  • Di-sulfide bond-forming cysteines between the first ⁇ -sheet (made up of ⁇ -strands ABDE; single underline) and the second ⁇ -sheet (made up of ⁇ -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-[CL ⁇ -D 185 A] conjugation products to [PEG 5 -K 11 -SEQ:27].
  • FIG. 9B Binding ELISA data for HER2 receptor binding of trastuzumab and trastuzumab-[CL ⁇ -D 185 A] conjugation products to MMAD toxin.
  • FIG. 10A representation of a constant Ig domain showing the 7 ⁇ -strands forming the two ⁇ -sheets.
  • FIG. 10B close up of the ⁇ -helix between ⁇ -sheets E and F.
  • FIG. 11 Crystal structure-based minimized ribbon representation of CL ⁇ , 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 CL ⁇ ‘binding pocket’, showing CL ⁇ -D 77 and CL ⁇ -D 43 (as a stick model) in the hydrogen bond with CL ⁇ -H 81 N ⁇ or N ⁇ , and atomic distances in ⁇ .
  • FIGS. 13A and 13B Crystal structure-based minimized ribbon representation of the CL ⁇ and CL ⁇ -D 77 A mutant ‘binding pockets’.
  • the distance between carbonyl oxygen of CL ⁇ -D 43 and N ⁇ of CL ⁇ -H 81 differs by 1 ⁇ between the CL ⁇ and CL ⁇ -D 77 A mutant, pointing to the predominance of catalytically active CL ⁇ -H 81 tautomer N ⁇ in the CL ⁇ -D 77 A mutant.
  • the modeling identifies a clear increase in the overall size of the pocket in CL ⁇ -D 77 A mutant, which is represented by the figure.
  • FIG. 13C Crystal structure-based minimized ribbon representation of the CL ⁇ and CL ⁇ -D 77 A mutant ‘binding pockets’.
  • the distance between carbonyl oxygen of CL ⁇ -D 43 and N ⁇ of CL ⁇ -H 81 differs by 1 ⁇ between the CL ⁇ and CL ⁇ -D 77 A mutant, pointing to the predominance of catalytically active CL ⁇ -H 81 tautomer
  • FIG. 14 Crystal structure-based minimized ribbon representation of the CL ⁇ ‘binding pocket’.
  • the binding site is depicted as a small jacks.
  • the ⁇ electron stacking interactions with CL ⁇ -H 81 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 CL ⁇ and CL ⁇ ‘binding pockets.
  • FIG. 15A shows the ⁇ -electron interactions between CL ⁇ -V 42 and CL ⁇ -H 81 , assisting in maintaining the CL ⁇ -H 81 imidazole ring and the N ⁇ electron pair at the plane needed for nucleophilic attack during catalytic reaction.
  • the distance between the center of CL ⁇ -H 81 imidazole ring and each of the hydrogen atoms on the CL ⁇ -V 42 are 2.8 ⁇ allowing for strong interactions.
  • CL ⁇ -A 49 is shown at a distance of 4.2 ⁇ from CL ⁇ -H 82 (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 CL ⁇ -H 82 .
  • FIG. 16 Sequence alignment of hCH ⁇ 1, hCH ⁇ 2, hCH ⁇ 3, hCL ⁇ and hCL ⁇ . ⁇ -strands of the CL ⁇ are indicated as underlined regions. ⁇ -helices are indicated in italics.
  • FIG. 17 Crystal structure based alignment of sequences of hCH ⁇ 1, hCH ⁇ 2, hCH ⁇ 3, hCL ⁇ and hCL ⁇ according to minimized 3D homology. ⁇ -strands are indicated as boxed regions, ⁇ -helices are indicated within wavy scrolls. Key residues corresponding to CL ⁇ -V 42 , CL ⁇ -D 43 , CL ⁇ -D 77 , CL ⁇ -K 80 , and CL ⁇ -H 81 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 ⁇ -strand in the hCH ⁇ 1, hCH ⁇ 2, hCH ⁇ 3, and hCL ⁇ domains.
  • FIGS. 18B-31B depicting CL ⁇ , and CH ⁇ domains, are orientated such that the D ⁇ -strand is on the lower left of the structure pointing downwards, and lying against the E ⁇ -strand.
  • the D′ and D′′ ⁇ -strands together can be seen to occupy approximately the same relative position as the CL ⁇ D ⁇ -strand ( ⁇ -strands D and D′, D′′ labelled in FIG. 18 as a point of reference).
  • FIG. 18 Crystal structure-based minimized ribbon representation of the hCL ⁇ (A) and hCL ⁇ (B) domains.
  • FIG. 19 Crystal structure-based minimized ribbon representation of the hCL ⁇ (A) and WT-hCH ⁇ 1 (B) domains.
  • FIG. 20 Crystal structure-based minimized ribbon representation of the hCL ⁇ (A) and WT-hCH ⁇ 1 (B) domains, showing the sidechain location of significant residues.
  • FIG. 21 Crystal structure-based minimized ribbon representation of the hCL ⁇ (A) and hCH ⁇ 1-m1-D44 mutant (B) domains, showing the sidechain location of significant residues.
  • FIG. 22 Crystal structure-based minimized ribbon representation of the hCL ⁇ (A) and hCH ⁇ 1-T78K/Q79H/CD loop swap mutant (B) domains, showing the sidechain location of significant residues.
  • FIG. 23 Crystal structure-based minimized ribbon representation of the hCL ⁇ (A) and WT-hCH ⁇ 2 (B) domains.
  • FIG. 24 Crystal structure-based minimized ribbon representation of the hCL ⁇ (A) and WT-hCH ⁇ 2 (B) domains, showing the sidechain location of significant residues.
  • FIG. 25 Crystal structure-based minimized ribbon representation of the hCL ⁇ (A) and hCH ⁇ 2m mutant (B) domains, showing the sidechain location of significant residues.
  • FIG. 26 Crystal structure-based minimized ribbon representation of the hCL ⁇ (A) and hCH ⁇ 2m-D 82 A mutant (B) domains, showing the sidechain location of significant residues.
  • FIG. 27 Crystal structure-based minimized ribbon representation of the hCL ⁇ (A) and WT-hCH ⁇ 3 (B) domains.
  • FIG. 28 Crystal structure-based minimized ribbon representation of the hCL ⁇ (A) and WT-hCH ⁇ 3 (B) domains, showing the sidechain location of significant residues.
  • FIG. 29 Crystal structure-based minimized ribbon representation of the hCL ⁇ (A) and hCH ⁇ 3m mutant (B) domains, showing the sidechain location of significant residues.
  • FIG. 30 Crystal structure-based minimized ribbon representation of the hCL ⁇ (A) and hCH ⁇ 3m CD/EF mutant (B) domains, showing the sidechain location of significant residues.
  • FIG. 31 Crystal structure-based minimized ribbon representation of the hCL ⁇ (A) and hCH ⁇ 3m CD/EF-m2 mutant (B) domains, showing the sidechain location of significant residues.
  • FIGS. 32A and 32B Alignment of CH ⁇ 1, CH ⁇ 2, and CH ⁇ 3 with their respective mutants and proposed mutant, together with CL ⁇ and CL ⁇ .
  • 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.
  • 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.
  • 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).
  • 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.
  • MPA S-Trityl-mercaptopropionic acid
  • HBTU 48 mg, 0.125 mmol
  • N-methylmorpholine 0.025 ml, 0.25 mmol
  • 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 (K SH ) was incorporated as Fmoc-Lys(N ⁇ -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 K SH as linking residue
  • DMF 3 ml
  • 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-PEG 2 -MAL are shown below (comprising Z* group Z13).
  • 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).
  • the present invention also provides for the use of pentafluorophenyl (PFP; Z* ⁇ Z1) esters to form relatively stable Effector Moiety-linker complexes.
  • PFP pentafluorophenyl
  • 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.
  • an activated ester group such as PFP
  • the strategy may be a bis-acid PEG with each acid activated as a PFP ester.
  • the end of the bis-PFP linker reacted with the N- ⁇ -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.
  • 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.
  • 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-PEG 5 -K 11 -SEQ:27] is shown below in Scheme 3.
  • 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-dPEG 5 -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-dPEG 5 -OPfp reagent over 2 hr, then the final mixture stirred for a further 1 hr. Progress of the conversion to [PFP-PEG 5 -K 11 -SEQ:27] product was monitored by analytical HPLC.
  • 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-PEG 2 -MAL-K SH 11 -SEQ:27] to yield 2.12.1.fx-[PEG 2 -MAL-K SH 11 -SEQ:27]
  • MAC-2 comprises 2.12.1.fx conjugated to [PFP-PEG 5 -K 11 -SEQ:27] to yield 2.12.1.fx-[PEG 5 -K 11 -SEQ:27].
  • 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.
  • 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-PEG 5 -K 11 -SEQ:27] was reconstituted in a propylene glycol solution to 10 mg.ml ⁇ 1 .
  • [PFP-PEG 5 -K 11 -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.
  • 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-PEG 5 -K 11 -SEQ:27] was reconstituted in a propylene glycol solution to 10 mg.ml ⁇ 1 .
  • [PFP-PEG 5 -K 11 -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.
  • 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-PEG 5 -K 11 -SEQ:27] was reconstituted in DMSO to 10 mg.ml ⁇ 1 .
  • [PFP-PEG 5 -K 11 -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
  • 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-PEG 5 -K 11 -SEQ:27] was reconstituted in a propylene glycol solution to 10 mg.ml ⁇ 1 .
  • [PFP-PEG 5 -K 11 -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).
  • 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-PEG 5 -K 11 -SEQ:27] was reconstituted in a propylene glycol solution to 10 mg.ml ⁇ 1 .
  • [PFP-PEG 5 -K 11 -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.
  • 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-PEG 5 -K 11 -SEQ:27] was reconstituted in a propylene glycol solution to 10 mg.ml ⁇ 1 .
  • [PFP-PEG 5 -K 11 -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.
  • 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-PEG 5 -K 11 -SEQ:27] was reconstituted in a propylene glycol solution to 10 mg.ml ⁇ 1 .
  • [PFP-PEG 5 -K 11 -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.
  • 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-PEG 5 -K 11 -SEQ:27] was reconstituted in DMSO to 10 mg.ml ⁇ 1 .
  • [PFP-PEG 5 -K 11 -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.
  • 2.12.1.fx The conjugation profiles of 2.12.1.fx with [PFP-PEG 5 -K 11 -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-PEG 5 -K 11 -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.
  • 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-PEG 5 -K 11 -SEQ:27] was reconstituted in a propylene glycol solution to 10 mg.ml ⁇ 1 .
  • [PFP-PEG 5 -K 11 -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).
  • 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-PEG 5 -K 11 -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).
  • 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-PEG 5 -K 11 -SEQ:27] was reconstituted in a propylene glycol solution to 10 mg.ml ⁇ 1 .
  • [PFP-PEG 5 -K 11 -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.
  • 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-PEG 5 -K 11 -SEQ:27] was reconstituted in a propylene glycol solution to 20 mg.ml ⁇ 1 (5% propylene glycol in the conjugation reaction).
  • [PFP-PEG 5 -K 11 -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.
  • 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-PEG 5 -K 11 -SEQ:27] was reconstituted in DMSO to 10 mg.ml ⁇ 1 .
  • [PFP-PEG 5 -K 11 -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).
  • 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-PEG 5 -K 11 -SEQ:27] was reconstituted in 50% propylene glycol to 10 mg.ml ⁇ 1 .
  • [PFP-PEG 5 -K 11 -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.
  • 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-PEG 5 -K 11 -SEQ:27] was reconstituted in a propylene glycol solution to 10 mg.ml ⁇ 1 .
  • [PFP-PEG 5 -K 11 -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.
  • 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-PEG 5 -K 11 -SEQ:27] was reconstituted in a propylene glycol solution to 10 mg.ml ⁇ 1 (final propylene glycol concentration in reaction is 10%). [PFP-PEG 5 -K 11 -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.
  • 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 MAC-2 drug product molecule consists of a distribution of 1-4 [PEG 5 -K 11 -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.
  • 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.
  • 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 15 th 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.
  • 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.
  • 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-K 11 -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-K 188 or LC-K 190 ). 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-K 188 and LC-K 190 (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-K 188 . No evidence of modified LC-K 190 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-K 188 or LC-K 190 .
  • the scheme below shows the trypsin digestion reactions that would occur upon conjugation at either 2.12.1.fx-[LC-K 188 ] or 2.12.1.fx-[LC-K 190 ].
  • FIG. 5 shows the selected ion LCMS chromatogram data for the trypsin peptide when LC-K 188 is conjugated to the peptide.
  • FIG. 6 shows the selected ion LCMS chromatogram data for the trypsin fragment when LC-K 190 is modified with a conjugated peptide.
  • the peptide/linker appears to preferentially decorate LC-K 188 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-K 188 is the same residue as CL ⁇ -K 80 (i.e. K 80 of SEQ ID NO:6), as the Light Chain (LC) comprises the variable region as well as the constant light kappa chain (CL ⁇ ).
  • 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.
  • SEC-MS size exclusion chromatography-mass spectrometry
  • 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).
  • SUMPRODUCT Number of Conjugation Additions (CA), Percent per CA.
  • 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).
  • 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.
  • 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.
  • 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:
  • linker is covalently attached to the ⁇ -amino group of HC-K 99 (K 93 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).
  • 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).
  • 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).
  • 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.
  • 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.
  • mouse MAC-1 and MAC-2 demonstrated similar residence time as the parental anti-IGF1R antibody with ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • MAC-2 demonstrates significant anti-tumor efficacy in 2 different human xenograft tumor models.
  • hAb ⁇ Test comprises a CL ⁇ (hIL22: SEQ ID NOs:136 and 137), whereas 2.12.1.fx, mAb ⁇ Test1 (an IgG2 anti-Alk1 antibody, as disclosed in US7537762, incorporated herein by reference), h38C2-IgG1 (SEQ ID NO:64 and 65) and h38C2-IgG2 (SEQ ID NO:64 and 66) each comprise CL ⁇ .
  • Each of the antibodies were buffer exchanged into 20 mM HEPES, pH 7.0 and concentrated to 5-20 mg/mL.
  • [PFP-PEG 5 -K 11 -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
  • peptide conjugation occurs at a distribution between 0-4 peptide additions with the largest form being 2 to 3 peptide additions.
  • conjugation of the peptide occurs at a distribution between 0-4 peptides additions with the largest form being 1 to 2 peptide additions.
  • 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.
  • hAb ⁇ Test 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.
  • the conjugation profile of an IgG2 ⁇ antibody (hABKTest2) 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.
  • SEC-MS size exclusion chromatography-mass spectrometry
  • 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 hAbKTest2 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 CL ⁇ -K 80 .
  • the 39-mer peptide was conjugated to h38C2-IgG2 with MAL-PEG2-PFP as described above, at different molar concentrations.
  • binding of the cognate receptor for the 39-mer peptide was assayed.
  • Table 30 The results (Table 30) shown are consistent with directional conjugation at CL ⁇ -K 80 .
  • 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.
  • 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.
  • SEC-MS size exclusion chromatography-mass spectrometry
  • 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.
  • 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.
  • SEC-MS size exclusion chromatography-mass spectrometry
  • 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-K 99 (K 93 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.
  • 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-PEG 5 -K 11 -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.
  • SEC-MS size exclusion chromatography - mass spectrometry
  • 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-[CL ⁇ -K 80 R] (CL ⁇ : SEQ ID NO:12), 2.12.1.fx-[CL ⁇ -K 82 R] (CL ⁇ : SEQ ID NO:13), and 2.12.1.fx-[CL ⁇ -K 80 R-K 82 R] (CL ⁇ : SEQ ID NO:14).
  • CL ⁇ -K 80 R mutant showed reduced conjugation.
  • CL ⁇ -K 82 R 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.
  • CL ⁇ -H 81 side chain is very close to the ⁇ -amino group of CL ⁇ -K 80 . Since His is often involved in proton transfer reactions, CL ⁇ -H 81 is very likely required for CL ⁇ -K 80 conjugation.
  • the imidazole ring was eliminated by a CL ⁇ -H 81 A mutation.
  • CL ⁇ -D 43 A and CL ⁇ -D 43 A/H 81 A mutants were made to study the role of CL ⁇ -D 43 in site specific conjugation and the combined effect of CL ⁇ -D 43 and CL ⁇ -H 81 .
  • 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 CL ⁇ mutants were generated: CL ⁇ -D 43 A (SEQ ID NO:15), CL ⁇ -K 80 A (SEQ ID NO:16), CL ⁇ -H 81 A (SEQ ID NO:17), CL ⁇ -K 82 A (SEQ ID NO:18) and CL ⁇ -D 43 A/H 81 A (SEQ ID NO:19).
  • Each of the antibodies was buffer exchanged to 20 mM sodium acetate, 200 m trehalose pH 5.5 at 20 mg/ml.
  • the proteins were then spiked with 60 mM sodium phosphate pH 7.7.
  • [PFP-PEG 5 -K 11 -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.
  • SEC-MS size exclusion chromatography-mass spectrometry
  • Table 34 shows the conjugation profile of 2.12.1.fx, 2.12.1.fx-[CL ⁇ -D 43 A],
  • 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 CL ⁇ -K 80 A.
  • 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.
  • the CL ⁇ in hAb ⁇ Test1 (SEQ ID NOs:136 and 137) was substituted with CL ⁇ to determine whether this increased the level, directionality and/or control of CL-specific conjugation.
  • the CL ⁇ /CL ⁇ domain substitution hybrid constructs were generated using overlap PCR.
  • the VL ⁇ and CL ⁇ were PCR amplified using hAb ⁇ Test and a K mAb light chain as templates separately. These 2 PCR products were mixed as templates; hAb ⁇ Test1 forward primer and LCL ⁇ reverse primer were used in overlap PCR reaction to amplify the full length hAb ⁇ TestVL/CL ⁇ 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.
  • hAb ⁇ Test CL ⁇ bound to its cognate ligand similarly to the native mAb (hAb ⁇ Test)(Table 35).
  • SEQ ID NOs:59, 60 and 61 are the light chain constant regions from hAb ⁇ Test, hAb ⁇ Test- ⁇ (with ⁇ J), and hAb ⁇ Test- ⁇ J (with ⁇ J).
  • Antibody Antigen binding of lambda/Kappa substitution. Inhibition of IL22 binding to hAb ⁇ Test1 Mutants LC SEQ ID NO: antigen (IC 50 , nM) hAb ⁇ Test (CONTROL) 59 0.4 hA ⁇ Test- ⁇ 60 0.3 hAb ⁇ Test- ⁇ J 61 0.3
  • Each antibody (hAb ⁇ Test, hAb ⁇ Test- ⁇ , hAb ⁇ Test- ⁇ J and hAb ⁇ Test-[CL ⁇ -S 81 H/H 82 S]) was buffer exchanged to 20 mM sodium acetate, 200 m trehalose pH 5.5 at 20 mg/ml. The proteins were then spiked with 60 mM sodium phosphate pH 7.7. [PFP-PEG 5 -K 11 -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.
  • the conjugation level increases over the hAb ⁇ Test control's average CA, going from 1.66 to 2.19 ( ⁇ ) and 2.53 ( ⁇ J) respectively.
  • the mutant had little effect compared to the native sequence, suggesting that “KH” motif alone is not sufficient for MAC formation.
  • Antibody Antigen binding of lambda at antibodies.
  • LC SEQ ID NO: antigen (IC 50 , nM) hAb ⁇ Test 59 1.7 hAb ⁇ Test- ⁇ 60 1.5 hAb ⁇ Test- ⁇ J 61 1.6 hAb ⁇ Test1-S 81 H/H 82 S 62 1.6
  • 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-PEG 2 -Z* linker, where Z* represented the new leaving group replacing PFP.
  • a sample (30-40 mg) of the purified ABP-thiol peptide (i.e. ABP with K SH as linking residue) was dissolved in anhydrous DMF (2 ml). MAL-PEG 2 -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 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.
  • 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).
  • 0CA represents underivatized 2.12.1.fx antibody
  • 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.
  • N-hydroxysuccinimide 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.
  • 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.
  • the fluorinated aromatic esters showed a marked propensity towards light chain derivatization (principally CL ⁇ -K 80 as previously mentioned).
  • several esters based on N-hydroxysuccinimide derivatives showed less preference, with often greater levels of heavy chain derivatization observed.
  • 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-PEG 5 ] and then the 2.12.1.fx under conditions previously optimized for reaction with [PFP-PEG 5 -K 11 -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.
  • Similar profiles to that of MAC-2 were observed, with around 70% LC derivatization and less than 10% on the HC.
  • the mutation was introduced by oligonucleotide primers and confirmed by DNA sequencing.
  • 2.12.1.fx-[CL ⁇ -D 77 A] 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-[CL ⁇ -D 77 A] (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-PEG 5 -K 11 -SEQ:27] was reconstituted in a propylene glycol solution to 10 mg/ml.
  • [PFP-PEG 5 -K 11 -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]-[PEG 5 -K 11 -SEQ:27] and [2.12.1.fx-[CL ⁇ -D 77 A]-[PEG 5 -K 11 -SEQ:27].
  • the conjugation profile of [2.12.1.fx]-[PEG 5 -K 11 -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.
  • [PEG 5 -K 11 -SEQ:27]-conjugated fragments were detected using LCMS peptide mapping of the 2.12.1.fx-[CL ⁇ -D 77 A]-[PEG 5 -K 11 -SEQ:27] product. Both of these conjugated fragments were present on the light chain of the 2.12.1.fx-[CL ⁇ -D 77 A] antibody. In comparison, 8 fragments conjugated to [PEG 5 -K 11 -SEQ:27] were detected in 2.12.1.fx -[PEG 5 -K 11 -SEQ:27].
  • the observed conjugation sites in the 2.12.1.fx-[CL ⁇ -D 77 A]-[PEG 5 -K 11 -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 CL ⁇ -K 80 residue, is the primary conjugation site. Fragment Y15 is only observed as an [PEG 5 -K 11 -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 [PEG 5 -K 11 -SEQ:27]-modified and unmodified forms; unmodified Y3 signal intensities in 2.12.1.fx-[PEG 5 -K 11 -SEQ:27] and 2.12.1.fx-[CL ⁇ -D 77 A]-[PEG 5 -K 11 -SEQ:27] are within 15%.
  • [PEG 5 -K 11 -SEQ:27]-modified Y3 is observed at a relatively low level (9737 counts, Table 52).
  • the control protein for Retention Time, MS signal intensity and Mass Error is 2.12.1.fx-[CL ⁇ -D 77 A] and the analyte protein in each case is 2.12.1.fx-[CL ⁇ -D 77 A]+[PEG 5 -K 11 -SEQ:27].
  • Modifiers Potential covalent additions to the fragment; [PEG 5 -K 11 -SEQ:27]- antibody binding peptide of Lysine residue, CAM- carboxymethylation of Cysteine residue.
  • CL ⁇ -D 77 residue of 2.12.1.fx antibody was mutated to each of the other 18 amino acids in addition to the CL ⁇ -D 77 A mutation.
  • the CL ⁇ -D 77 G (SEQ ID NO:38), CL ⁇ -D 77 L (SEQ ID NO:40), CL ⁇ -D 77 S (SEQ ID NO:49), CL ⁇ -D 77 E (SEQ ID NO:53), and CL ⁇ -D 77 R (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.
  • 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-PEG 5 -K 11 -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 CL ⁇ -D 77 mutants.
  • CL ⁇ -D 77 C aggregated due to an introduction of a free cysteine, and the results were not interpretable. Mutations CL ⁇ -D 77 W, CL ⁇ -D 77 M, CL ⁇ -D 77 H, CL ⁇ -D 77 Q, CL ⁇ -D 77 N, and CL ⁇ -D 77 V did not change the overall conjugation profile compared to wild-type 2.12.1.fx.
  • Mutations CL ⁇ -D 77 F, CL ⁇ - CL ⁇ -D 77 K, CL ⁇ -D 77 Y, and CL ⁇ -D 77 E decreased the overall level of conjugation. Mutations CL ⁇ -D 77 P, CL ⁇ -D 77 I, CL ⁇ -D 77 T, CL ⁇ -D 77 R, CL ⁇ -D 77 L, CL ⁇ -D 77 S, and CL ⁇ -D 77 G increased the level of conjugation.
  • 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, D 77 Q; 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 D 77 M, D 77 F and D 77 H (0.5 mg), and the run including D 77 W and D 77 C (0.25 mg).
  • the L 73 A mutant was introduced to 2.12.1.fx CL ⁇ 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 L 73 A 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 CL ⁇ -L 73 A 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-[CL ⁇ -L 73 A] (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 CL ⁇ -V 42 A and CL ⁇ -K 75 A 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.
  • 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.
  • 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-PEG 5 -K 11 -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-[PEG 5 -K 11 -SEQ:27] with 2.12.1.fx-[CL ⁇ -mutants]-[PEG 5 -K 11 -SEQ:27].
  • the conjugation profile of 2.12.1.fx-[PEG 5 -K 11 -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 [PEG 5 -K 11 -SEQ:27] additions either decreased (CL ⁇ -V 42 A, CL ⁇ -L 46 A, CL ⁇ -S 74 A, CL ⁇ -Y 78 A and CL ⁇ -Y 84 A) or increased (CL ⁇ -Q 47 A, CL ⁇ -N 50 A and CL ⁇ -D 77 A/E 79 A double mutants) compared to their corresponding 2.12.1.fx-[PEG 5 -K 11 -SEQ:27] controls.
  • Both CL ⁇ -Q 47 A and CL ⁇ -N 50 A mutants have over 70% 1 CA compared to the 59% 1 CA of the 2.12.1.fx wild type antibody.
  • the unconjugated light chain levels of these two mutants were reduced from 31% of the wild type antibody to 22% and 19%.
  • the V 42 A 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.
  • CL ⁇ -D 43 was mutated to CL ⁇ -D 43 E (SEQ ID NO:107), CL ⁇ -D 43 N (SEQ ID NO:108) and CL ⁇ -D 43 L (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.
  • 2.12.1.fx antibody and 2.12.1.fx-[CL ⁇ -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-PEG 5 -K 11 -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.
  • CL ⁇ -D 43 N has the similar overall conjugation and light chain levels (Table 55) compared to the wild type antibody.
  • CL ⁇ -D 43 E and CL ⁇ -D 43 L showed reduced overall conjugation level light chain conjugation level.
  • 2.12.1.fx and 2.12.1.fx-[CL ⁇ -D 77 A] were conjugated to [PEG 5 -K 11 -SEQ:27] using different reactive esters (see Examples 18 and 19) (results shown in Table 56).
  • the 2.12.1.fx-[CL ⁇ -D 77 A] mutant gave a higher level of intact average CA upon conjugation compared to the wt 2.12.1.fx.
  • 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 ⁇ indicates the difference between the WT and D 185 A 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.
  • D 77 A mutation was also inserted to the CL ⁇ 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-[CL ⁇ -D 77 A] mutant was generated in two steps.
  • D 77 A 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 D 77 A mutation.
  • Primer pair TRAST.VL.FOR (SEQ ID NO:89) and TRAST.VL.REV SEQ ID NO:90
  • the PCR fragment was digested with BglII and BsaI.
  • TRAST.CL.D185A.FOR SEQ ID NO:91
  • TRAST.CL.D185.A.REV SEQ ID NO:92
  • 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-[CL ⁇ -D 77 A] (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-PEG 5 -K 11 -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-[PEG 5 -K 11 -SEQ:27] with hTrast-[CL ⁇ -D 77 A]-[PEG 5 -K 11 -SEQ:27].
  • the conjugation profile of trastuzumab -[PEG 5 -K 11 -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 D 77 A 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 CL ⁇ -D 77 A 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-[CL ⁇ -D 77 A] than unmodified trastuzumab; the average light chain conjugate addition value for hTrast-[CL ⁇ -D 77 A] is 1.01 compared to 0.70 for trastuzumab.
  • unconjugated light chain is significantly reduced in hTrast-[CL ⁇ -D 77 A]. Conjugation on the heavy chain is observed at a significantly lower level.
  • Table 58 compares the conjugation profile of trastuzumab -[PEG 5 -MMAD] (Auristatin derivative) with hTrast-[CL ⁇ -D 77 A]-[PEG-MMAD].
  • the conjugation profile of trastuzumab-[PEG 5 -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.
  • CL ⁇ -D 77 A 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 CL ⁇ -D 77 A 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-[CL ⁇ -D 77 A] than unmodified trastuzumab; the average light chain conjugate addition value for hTrast-[CL ⁇ -D 77 A] is 0.88 compared to 0.56 for trastuzumab.
  • unconjugated light chain is significantly reduced in hTrast-[CL ⁇ -D 77 A]. Conjugation on the heavy chain is observed at a significantly lower level.
  • trastuzumab and hTrast-[CL ⁇ -D 77 A], unconjugated and conjugated to either [PEG 5 -K 11 -SEQ:27] or [PEG 5 -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 ⁇ g/ml.
  • FIGS. 9A and 9B demonstrate that commercial trastuzumab, trastuzumab generated from the available sequence, and hTrast-[CL ⁇ -D 77 A], as well as trastuzumab and hTrast-[CL ⁇ -D 77 A] when bound to either of [PEG 5 -K 11 -SEQ:27] or [PEG 5 -MMAD] each display similar Her2 binding characteristics. These result suggest that conjugation, primarily at CL ⁇ -K 80 , does not significantly interfere with the receptor binding function of the native antibody.
  • trastuzumab was conjugated to [PEG 5 -MMAD] using two separate strategies: directional conjugation to CL ⁇ -K 80 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.
  • 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-PEG 2 -ValCitABC-TOXIN]).
  • the Z16 leaving group shows roughly equivalent derivatization for both the native 2.12.1.fx and 2.12.1.fx-[CL ⁇ -D 77 A] 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 CF 3 group is providing an additional interaction near the CL ⁇ -K 80 region that is also driving reactivity and preferential derivatization of the LC.
  • Ab % CA Avg LC % CA CA- CA CA- WT * 1.fx 0 1 2 3 4 CA ⁇ 0 1 2 LC 0 1 HC % 1 WT 1 17 51 25 5 2.16 14 77 9 0.96 84 16 0.16 1 D 77 A 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 D 77 A 9 9 31 33 18 2.43 0.05 1 79 20 1.2 89 11 0.11 95
  • Ab % CA shows the overall % of conjugation additions per antibody, with reduced light chain and heavy chain analysis also shown (LC % CA, HC % CA). ⁇ indicates the difference between the WT and D 185 A 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.
  • HATU O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate
  • Hu08 is a human anti-IL-13R ⁇ 2 antibody, and is described fully in US61/723,545, whose contents are herein incorporated by reference.
  • a mutant version of hu08, comprising the CL ⁇ -D 77 A mutation was generated according to standard protocols (hu08-[CL ⁇ -D77A]).
  • Toxin-0101 (#54: Example 36) was conjugated with a cleavable linker to form the structure:
  • trastuzumab-[5PEG-MMAD], hTrast-[CL ⁇ -D 77 A]-[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.
  • the plasma exposures were overall similar for all 3 conjugates at any given dose.
  • the AUC (0-312) of hTrast-[CL ⁇ -D 77 A]-[5PEG-MMAD], trastuzumab-[5PEG-MMAD] and trastuzumab-(MMAD) n at 100 mg/kg were 177000, 174000 and 139000 ng ⁇ h/mL, respectively.
  • hTrast-[CL ⁇ -D 77 A]-[5PEG-MMAD] and trastuzumab-[5PEG-MMAD] were clinically well tolerated at all doses.
  • 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-[CL ⁇ -D 77 A]-[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 0 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.
  • 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.
  • 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
  • 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.
  • pharmacologically mediated increased mitoses and/or single cell necrosis were observed in several tissues.
  • hTrast-[CL ⁇ -D 77 A]-[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.
  • FIG. 10A depicts an Ig fold of a constant light domain containing a 3-stranded ⁇ -sheet packed against a 4-stranded ⁇ -sheet.
  • the fold is stabilized by hydrogen bonding between the ⁇ -strands of each ⁇ -sheet, by hydrophobic bonding between residues of opposite ⁇ -sheets in the interior, and by a disulfide bond between the ⁇ -sheets.
  • the 3-stranded ⁇ -sheet comprises ⁇ -strands C, F, and G, and the 4-stranded ⁇ -sheet has ⁇ -strands A, B, E, and D.
  • the letters A through G denote the sequential positions of the ⁇ -strands along the amino acid sequence of the Ig fold.
  • Linking each ⁇ -strand with the subsequent ⁇ -strand is an amino acid connecting chain that may or may not comprise a turn (NB) or ⁇ -helix (E/F) ( FIG. 10B ).
  • FIG. 8B plots the secondary structures along the primary sequence of the mouse and human CL ⁇ , and the human CL ⁇ .
  • the EF connecting chain between ⁇ -strands E and F of the CL ⁇ and CL ⁇ have identical secondary structure and consist of a 5-6 residue ⁇ -helical region (CL ⁇ -K 75 -K 80 , and CL ⁇ -P 76 -S 81 ), followed by a 2-3 amino-acid turn (CL ⁇ -H 81 -K 82 and CL ⁇ -H 82 -S 84 ) ( FIGS. 8B and 10 ).
  • the CD connecting chain brings the side chain of structurally equivalent aspartic acids (CL ⁇ -D 53 , CL ⁇ -D 45 ) to the vicinity of the EF chain (approximately 3.5 ⁇ ), and allows CL ⁇ -D 53 interact with the imidazole ring of CL ⁇ -H 81 .
  • the present invention also provides for CL ⁇ domains comprising one of the following mutations: CL ⁇ -S 81 K, and CL ⁇ -K 80 x/S 81 K, wherein x is any amino acid except P, K, R or H, wherein the numbering is according to SEQ ID NO:93.
  • the invention provides for novel CL ⁇ domains comprising K 81 , or x 80 /K 81 , wherein x is one of G, A, I, L, V, S, T, M, N, Q, F, Y, W, D, or E.
  • the invention also provides for a CL ⁇ domain comprising a sequence selected from the group consisting of SEQ ID NO:94, and SEQ ID NO:95.
  • Modeling also suggests that CL ⁇ -E 77 would be available to form a salt bridge with either of CL ⁇ -K 80 or CL ⁇ -K 81 in the same manner as CL ⁇ -D 77 appears to form a salt bridge to CL ⁇ -K 80 .
  • mutating CL ⁇ -E 77 to any of R, L, S, G, Q, P, N, V, I, T, and M is likely to facilitate directional conjugation at either of CL ⁇ -K 80 CL ⁇ when S 81 ⁇ (i.e. deletion of S 81 ), or CL ⁇ -K 81 .
  • the present invention also provides for a CL ⁇ domain comprising a sequence selected from the group consisting of SEQ ID NO:96 and SEQ ID NO:97.

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