WSGR Docket No. 52426-701201
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`MODIFED ANTIBODIES
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`CROSS-REFERENCE
`
`[0001] This application claims the benefit of US. Provisional Application No. 62/511,771 filed May
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`26, 2017 which is incorporated by reference herein in its entirety.
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`REFERENCE TO A SEQUENCE LISTING
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`[0002] The instant application contains a Sequence Listing which has been filed electronically in
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`ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on
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`May 24, 2018, is named 52426-701_201_SL.txt and is 73,393bytes in size.
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`SUMMARY OF THE INVENTION
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`[0003] Disclosed herein, in some embodiments, are modified antibodies comprising a formula: A-L-
`
`P wherein A is an antibody or antibody fragment that binds to a target antigen, P is a peptide that
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`reduces binding of A to the target antigen at physiological pH and that does not reduce binding of A
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`to the target antigen at acidic pH, and L is a linking moiety that connects A to P at physiological pH
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`and in a tumor microenvironment and L is bound to A outside an antigen binding site. In some
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`instances, at physiological pH P is reversibly bound to A through ionic, electrostatic, hydrophobic,
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`Pi-stacking, and H-bonding interactions, or a combination thereof. In some instances, at
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`physiological pH P is reversibly bound to A at or near the antigen binding site. In some instances, P
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`inhibits the binding of A to the target antigen at physiological pH and P does not inhibit the binding
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`of A to the target antigen at acidic pH. In some instances, in tissue other than the tumor
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`microenvironment, P sterically blocks A from binding to the target antigen. In some instances, at the
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`tumor microenvironment, P is removed from the antigen binding site, and the antigen binding site of
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`A is exposed. In some instances, the modified antibody has an increased binding affinity for the
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`target antigen in the tumor microenvironment compared to the binding affinity of the modified
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`antibody for the target antigen in a non-tumor microenvironment. In some instances, P comprises a
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`peptide sequence with at least one histidine. In some instances, the histidine forms a binding
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`interaction at or near the antigen binding site of A at physiological pH. In some instances, at acidic
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`pH P is reversibly bound to A in a region of A that is not the antigen binding site. In some instances,
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`at acidic pH P is reversibly bound to A in a region of A that is not the antigen binding site through
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`ionic, electrostatic, hydrophobic, Pi-stacking and H-bonding interactions, or a combination thereof.
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`In some instances, P is resistant to cleavage by a protease. In some instances, physiological pH is
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`about pH 7.4. In some instances, acidic pH is about pH 6.0 to about pH 7.0.
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`In some instances, P
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`comprises a peptide sequence of at least 6 amino acids in length. In some instances, P comprises a
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`peptide sequence of at least 10 amino acids in length. In some instances, P comprises a peptide
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`sequence of at least 6 to 20 amino acids in length. In some instances, P comprises a modified amino
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`acid, a non-natural amino acid, or a modified non-natural amino acids, or combination thereof. In
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`some instances, the modified amino acid or modified non-natural amino acid comprises a post-
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`translational modification. In some instances, at acidic pH P is reversibly bound to L. In some
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`instances, L comprises a peptide sequence with at least one aspartic acid or glutamic acid, or a
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`combination thereof. In some instances, the histidine of P forms an interaction with the aspartic acid
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`or glutamic acid of L. In some instances, L is a peptide sequence having at least 5 to no more than 50
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`amino acids. In some instances, L has a formula selected from the group consisting of: (GS)n,
`
`wherein n is an integer from 6 to 20 (SEQ ID NO: 1), (G2S)n, wherein n is an integer from 4 to 13
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`(SEQ ID NO: 2), (G3S)n, wherein n is an integer from 3 to 10 (SEQ ID NO: 3), and (G4S)n, wherein
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`n is an integer from 2 to 8 (SEQ ID NO: 4), and (G)n, wherein n is an integer from 12 to 40 (SEQ ID
`
`NO: 5). In some instances, L has a formula comprising (GGSGGD)n, wherein n is an integer from 2
`
`to 6 (SEQ ID NO: 8). In some instances, L has a formula comprising (GGSGGE)n, wherein n is an
`
`integer from 2 to 6 (SEQ ID NO: 9). In some instances, L has a formula comprising
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`(GGGSGSGGGGS) 11, wherein n is an integer from 1 to 3 (SEQ ID NO: 6). In some instances, L has
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`a formula comprising (GGGGGPGGGGP) 11, wherein n is an integer from 1 to 3 (SEQ ID NO: 7). In
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`some instances, L has a formula selected from (GX)n, wherein X is serine, aspartic acid, glutamic
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`acid, threonine, or proline and n is at least 20 (SEQ ID NO: 24), (GGX)n, wherein X is serine,
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`aspartic acid, glutamic acid, threonine, or proline and n is at least 13 (SEQ ID NO: 25), (GGGX)n,
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`wherein X is serine, aspartic acid, glutamic acid, threonine, or proline and n is at least 10 (SEQ ID
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`NO: 26), (GGGGX)n, wherein X is serine, aspartic acid, glutamic acid, threonine, or proline and n is
`
`at least 8 (SEQ ID NO: 27), (GZX)n, wherein X is serine, aspartic acid, glutamic acid, threonine, or
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`proline and n is at least 15, and z is between 1 and 20 (SEQ ID NO: 28). In some instances, L is
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`resistant to cleavage by a protease. In some instances, L comprises a modified amino acid. In some
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`instances, the modified amino acid comprises a post-translational modification. In some instances, L
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`comprises a non-natural amino acid or a modified non-natural amino acid, or combination thereof. In
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`some instances, the modified non-natural amino acid comprises a post-translational modification. In
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`some instances, the target antigen is selected from the group consisting of: 4-lBB, CTLA4, PD-l,
`
`and PD-Ll. In some instances, the target antigen is 4-lBB. In some instances, the target antigen is
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`CTLA4. In some instances, the target antigen is PD-l. In some instances, the target antigen is PD-
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`Ll. In some instances, A is a full length antibody, a single-chain antibody, an Fab fragment, an Fab’
`
`fragment, an (Fab’)2 fragment, an FV fragment, a divalent single chain antibody, bispecif1c antibody,
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`a trispecif1c antibody, a tetraspecif1c antibody, or an antibody drug conjugate. In some instances, A
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`is selected from the group consisting of utomilumab, urelumab, ipilimumab, tremelimumab,
`
`pembrolizumab, nivolumab, and atezolizumab. In some instances, A is utomilumab. In some
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`instances, A is urelumab. In some instances, A is ipilimumab. In some instances, A is
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`tremelimumab. In some instances, A is pembrolizumab. In some instances, A is nivolumab. In some
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`instances, A is atezolizumab. In some instances, P comprises an amino acid sequence selected from
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`the group consisting of Peptide l, Peptide 2, Peptide 5, Peptide 6, Peptide lO, Peptide l3, Peptide 26,
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`Peptide l4, Peptide 15, Peptide 20, Peptide 27, Peptide 2l, Peptide 22, Peptide 23, Peptide 28,
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`Peptide 29, Peptide 30, Peptide 3 l, and Peptide 24. In some instances, the target antigen is CTLA4
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`and P comprises an amino acid sequence selected from the group consisting of Peptide l, Peptide 2,
`
`Peptide 5, Peptide 6, Peptide lO, Peptide l3, Peptide 26, Peptide l4, and Peptide 15. In some
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`instances, the target antigen is CTLA4 and P comprises an amino acid sequence according to Peptide
`
`1. In some instances, the target antigen is CTLA4 and P comprises an amino acid sequence
`
`according to Peptide 2. In some instances, the target antigen is CTLA4 and P comprises an amino
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`acid sequence according to Peptide 5. In some instances, the target antigen is CTLA4 and P
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`comprises an amino acid sequence according to Peptide 6. In some instances, the target antigen is
`
`CTLA4 and P comprises an amino acid sequence according to Peptide 10. In some instances, the
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`target antigen is CTLA4 and P comprises an amino acid sequence according to Peptide 13. In some
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`instances, the target antigen is CTLA4 and P comprises an amino acid sequence according to Peptide
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`26. In some instances, the target antigen is CTLA4 and P comprises an amino acid sequence
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`according to Peptide 14. In some instances, the target antigen is CTLA4 and P comprises an amino
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`acid sequence according to Peptide 15. In some instances, the target antigen is CTLA4 and P
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`comprises an amino acid sequence selected from the group consisting of Peptide lO, Peptide l3,
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`Peptide 26, Peptide l4, and Peptide 15. In some instances, the target antigen is PD-Ll and P
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`comprises an amino acid sequence selected from the group consisting of Peptide 20, Peptide 27,
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`Peptide 2l, Peptide 22, Peptide 23, Peptide 28, Peptide 29, Peptide 30, Peptide 3 l, and Peptide 24.
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`In some instances, the target antigen is PD-Ll and P comprises an amino acid sequence according to
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`Peptide 20. In some instances, the target antigen is PD-Ll and P comprises an amino acid sequence
`
`according to Peptide 27. In some instances, the target antigen is PD-Ll and P comprises an amino
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`acid sequence according to Peptide 21. In some instances, the target antigen is PD-Ll and P
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`comprises an amino acid sequence according to Peptide 22. In some instances, the target antigen is
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`PD-Ll and P comprises an amino acid sequence according to Peptide 23. In some instances, the
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`target antigen is PD-Ll and P comprises an amino acid sequence according to Peptide 28. In some
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`instances, the target antigen is PD-Ll and P comprises an amino acid sequence according to Peptide
`
`29. In some instances, the target antigen is PD-Ll and P comprises an amino acid sequence
`
`according to Peptide 30. In some instances, the target antigen is PD-Ll and P comprises an amino
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`acid sequence according to Peptide 31. In some instances, the target antigen is PD-Ll and P
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`comprises an amino acid sequence according to Peptide 24. In some instances, the target antigen is
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`PD-Ll and P comprises an amino acid sequence selected from the group consisting of Peptide 27,
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`Peptide 22, Peptide 23, and Peptide 31. In some instances, A comprises a kappa light chain amino
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`acid sequence according to SEQ ID NO: 68. In some instances, A comprises a heavy chain amino
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`acid sequence according to SEQ ID NO: 70. In some instances, the modified antibody comprises an
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`amino acid sequence according to SEQ ID NO: 69. In some instances, P comprises an amino acid
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`sequence selected from the group consisting of Peptide 1, Peptide 2, Peptide 5, Peptide 6, Peptide
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`10, Peptide 13, Peptide 26, Peptide 14, Peptide 15, Peptide 20, Peptide 21, Peptide 22, Peptide 23,
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`Peptide 31, and Peptide 24. In some instances, P comprises a peptide sequence with at least two
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`histidines.
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`In some instances, P comprises an amino acid sequence selected from the group
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`consisting of Peptide 5, Peptide 13, Peptide 26, Peptide 14, Peptide 15, Peptide 31, and Peptide 24.
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`In some instances, P comprises a peptide sequence with at least two histidines and at least two
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`cysteines. In some instances, P comprises a peptide sequence with at least two charged amino acid
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`residues wherein the charged amino acid residues are selected from the group consisting of aspartate,
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`glutamate, and histidine.
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`In some instances, P comprises an amino acid sequence selected from the
`
`group consisting of Peptide 1, Peptide 2, Peptide 5, Peptide 6, Peptide 10, Peptide 13, Peptide 26,
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`Peptide 14, Peptide 15, Peptide 20, Peptide 27, Peptide 21, Peptide 22, Peptide 23, Peptide 28,
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`Peptide 29, Peptide 30, Peptide 31, and Peptide 24. In some instances, P comprises a peptide
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`sequence with at least three charged amino acid residues wherein the charged amino acid residues
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`are selected from the group consisting of aspartate, glutamate, and histidine. In some instances, P
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`comprises an amino acid sequence selected from the group consisting of Peptide 1, Peptide 2,
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`Peptide 5, Peptide 10, Peptide 13, Peptide 26, Peptide 14, Peptide 15, Peptide 21, Peptide 23, Peptide
`
`28, Peptide 31, and Peptide 24. In some instances, P comprises a peptide sequence with at least one
`
`histidine and at least two aspartates. In some instances, P comprises a peptide sequence with at least
`
`one cysteine. In some instances, P comprises an amino acid sequence selected from the group
`
`consisting of Peptide 5, Peptide 6, Peptide 10, Peptide 13, Peptide 26, Peptide 14, Peptide 15,
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`Peptide 22, Peptide 23, Peptide 29, Peptide 30, Peptide 31, and Peptide 24. In some instances, P
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`comprises a peptide sequence with at least two cysteine amino acid residues. In some instances, P
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`comprises an amino acid sequence selected from the group consisting of Peptide 6, Peptide 10,
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`Peptide 13, Peptide 26, Peptide 14, Peptide 22, Peptide 23, Peptide 29, Peptide 30, Peptide 31, and
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`Peptide 24. In some instances, P comprises a peptide sequence with at least two cysteines and at
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`least three charged amino acid residues wherein the charged amino acid residues are selected from
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`the group consisting of aspartate, glutamate, and histidine. In some instances, P comprises an amino
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`acid sequence of formula GGX, wherein X is cysteine, alanine, proline, methionine, histidine, or
`
`leucine. In some instances, P comprises an amino acid sequence selected from the group consisting
`
`of Peptide 5, Peptide 6, Peptide 10, Peptide 13, Peptide 26, Peptide 14, Peptide 15, Peptide 22,
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`Peptide 23, Peptide 29, Peptide 31, and Peptide 24. In some instances, P comprises an amino acid
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`sequence of GGC. In some instances, P comprises an amino acid sequence selected from the group
`
`consisting of Peptide 5, Peptide 6, Peptide 13, Peptide 26, Peptide 14, Peptide 31, and Peptide 24. In
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`some instances, P does not comprise a lysine or arginine. In some instances, P comprises at least one
`
`histidine and at least one aspartate. In some instances, P comprises at least one histidine and at least
`
`one glutamate. In some instances, P comprises at least one histidine and at least two glutamates. In
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`some instances, P comprises at least two histidines and at least one aspartate or at least one
`
`glutamate. In some instances, P comprises at least one histidine, wherein at least one hydrogen
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`bonding amino acid residue is within two amino acid positions to the histidine, wherein the hydrogen
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`bonding amino acid residue is selected from the group consisting of serine, threonine, tyrosine,
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`asparagine, and glutamine. In some instances, the hydrogen bonding amino acid residue is within
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`one amino acid position to the histidine. In some instances, the hydrogen bonding amino acid residue
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`is serine. In some instances, the hydrogen bonding amino acid residue is threonine. In some
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`instances, the hydrogen bonding amino acid residue is tyrosine. In some instances, the hydrogen
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`bonding amino acid residue is asparagine. In some instances, the hydrogen bonding amino acid
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`residue is glutamine. In some instances, A-L-P does not comprise a protease cleavage site that
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`releases A from P in a tumor microenVironment. In some instances, L comprises a peptide sequence
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`with at least one histidine.
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`In some instances, at acidic pH L is reversibly bound to P. In some
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`instances, P comprises a peptide sequence with at least one aspartic acid or glutamic acid, or a
`
`combination thereof. In some instances, the histidine of L forms an interaction with the aspartic acid
`
`or glutamic acid of P.
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`[0004] Disclosed herein, in some embodiments, are pharmaceutical compositions, comprising: (a) a
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`modified antibody according to any of the disclosures herein, and (b) a pharmaceutically acceptable
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`excipient.
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`BRIEF DESCRIPTION OF THE DRAWINGS
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`[0005] The novel features of the invention are set forth with particularity in the appended claims. A
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`better understanding of the features and advantages of the present invention will be obtained by
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`reference to the following detailed description that sets forth illustrative embodiments, in which the
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`principles of the invention are utilized, and the accompanying drawings of which:
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`[0006] Fig. 1 exemplifies an antibody that does not comprise a peptide modification. Such
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`antibodies bind to unique antigens that exist in abundance in tumor tissue. But, the unique antigens
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`are also found in some healthy tissues, which can trigger systemic immune activation in a subject,
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`and cause toxicity.
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`[0007] Fig. 2 shows an exemplary modified antibody. In this example, the modified antibody is
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`linked to a peptide which binds at or near the antigen binding site of the modified antibody at pH
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`7.4. This reduces binding of the modified antibody to its target antigen in healthy tissue. In tumor
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`tissue, the acidic tumor microenvironment disrupts the interaction of the peptide with the modified
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`antibody. The antigen binding site of the modified antibody is exposed, and the modified antibody
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`selectively binds to its target antigen in tumor tissue.
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`[0008] Fig. 3 shows an exemplary modified antibody. The peptide is linked to the antibody via a
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`linking moiety. The linking moiety creates a stable link between the antibody and peptide. The
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`peptide prevents the antibody from binding to its target antigen in physiological pH, non-diseased
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`tissue. A pH switch in tumor microenvironments modulates the peptide/antibody affinity. The
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`peptide is released in tumor tissue, and enables the antibody to bind to its target antigen.
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`[0009] Fig. 4 shows an exemplary modified antibody. In this example, the peptide is engineered to
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`contain a histidine. At physiological pH, the histidine of the peptide interacts with the antibody
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`binding site. At acidic pH, such as in a tumor microenvironment, the interaction between the peptide
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`and the antibody binding site is disfavored because the histidine is protonated. The antibody binding
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`site is available for binding to its target antigen in a tumor microenvironment.
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`[0010] Fig. 5 is an exemplary schematic of phage panning screening platform to identify pH
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`responsive peptide candidates.
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`[0011] Fig. 6 illustrates a phagemid ELISA of a collection of enriched clones resulting from three
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`rounds of biopanning against anti-mouse CTLA-4 (clone 9D9).
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`[0012] Fig. 7 is an exemplary phagemid competition ELISA from a collection of enriched clones
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`isolated after three rounds of biopanning against anti-mouse CTLA-4 (clone 9D9).
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`[0013] Fig. 8 is an exemplary pH-dependent “binding” assay of a collection of enriched clones
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`isolated after three rounds of biopanning against anti-mouse CTLA-4 (clone 9D9).
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`[0014] Fig. 9 illustrates a pH-dependent “dissociation” assay of a collection of enriched clones
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`isolated after three rounds of biopanning against anti-mouse CTLA-4 (clone 9D9).
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`[0015] Fig. 10A-Fig. 10B illustrate pH responsive anti-CTLA4 peptide candidates identified with
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`phage panning screening platform. Fig. 10A illustrates peptide candidates identified that exhibit pH
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`dependent binding to anti-CTLA4. Fig. 10B illustrates peptide candidates identified that exhibit pH
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`dependent dissociation to anti-CTLA4.
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`[0016] Fig. 11A-Fig. 11C illustrate significant pH responsiveness of unoptimized peptide candidates
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`Peptide 5 (Fig. 11A), Peptide 6 (Fig. 11B) and Peptide 10 (Fig. 11C).
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`[0017] Fig. 12 illustrates multiple pH response anti-CTLA4 peptide candidates with pH dependent
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`dissociation identified from biased library.
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`[0018] Fig. 13A-Fig. 13B illustrate pH biased library generated peptide candidates Peptide 14 (Fig.
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`13A) and Peptide 15 (Fig. 13B).
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`[0019] Fig. 14 illustrates a Peptide lO ELISA to anti-mouse antibody (clone 9D9).
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`[0020] Fig. 15 illustrates a Peptide lO competition ELISA.
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`[0021] Fig. 16A-Fig. 16C illustrate octet binding curve of Peptide 15 (Fig. 16A), Peptide 17 (Fig.
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`16C) and Peptide 18 (Fig. 16C).
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`[0022] Fig. 17 illustrates pH-dependent binding of 0t-mCTLA-4 Fab to Peptide 15 by ELISA.
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`[0023] Fig. 18 illustrates CTLA4 antibody/ligand competition ELISA.
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`[0024] Fig. 19 illustrates a phagemid ELISA of a collection of enriched clones resulting from
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`biopanning against PD-Ll antibody.
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`[0025] Fig. 20 illustrates anti-PD-Ll phage competition ELISA.
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`[0026] Fig. 21 illustrates pH-dependent phagemid PD-Ll antibody binding ELISA.
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`[0027] Fig. 22 illustrates pH-dependent phagemid PD-Ll antibody dissociation ELISA.
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`[0028] Fig. 23 illustrates Peptide 23 PD-Ll antibody binding.
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`[0029] Fig. 24 illustrates Peptide 23 PD-Ll competition ELISA.
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`[0030] Fig. 25 illustrates PD-Ll antibody/ligand competition ELISA.
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`DETAILED DESCRIPTION OF THE INVENTION
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`[0031] Protein-based therapies, including antibody therapies, are effective treatments for a variety of
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`diseases. A strategy to improve toxicity and side effects of such treatments is to engineer a peptide
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`that binds to the protein-based therapy at physiological pH, but does not bind to the protein-based
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`therapy at acidic pH. While peptides have been shown to bind to antibodies with varying affinities,
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`peptides which bind to antibodies in a pH dependent manner are not known. These pH-dependent
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`peptides enable the protein-based therapy to be activated in certain acidic microenvironments while
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`not affecting healthy tissues.
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`Certain Definitions
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`[0032] The terminology used herein is for the purpose of describing particular cases only and is not
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`intended to be limiting. As used herein, the singular forms a ,
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`an” and “the” are intended to
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`include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the
`77
`(L
`77
`(L
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`extent that the terms “including
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`7
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`includes”, “having
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`has”, “with”, or variants thereof are used in
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`7
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`either the detailed description and/or the claims, such terms are intended to be inclusive in a manner
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`similar to the term “comprising.”
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`[0033] The term “about” or “approximately” means within an acceptable error range for the
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`particular value as determined by one of ordinary skill in the art, which will depend in part on how
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`the value is measured or determined, e.g., the limitations of the measurement system. For example,
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`“about” can mean within 1 or more than 1 standard deviation, per the practice in the given value.
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`Where particular values are described in the application and claims, unless otherwise stated the term
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`“about” should be assumed to mean an acceptable error range for the particular value.
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`[0034] As used herein, “physiological pH” is used to refer to the pH of a non-diseased state cellular
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`environment.
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`In some embodiments, physiological pH is greater than pH 6.9. In some
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`embodiments, physiological pH is about pH 7.0 to about pH 8.0. In some embodiments,
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`physiological pH is about 7.4.
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`[0035] Described herein are modified antibodies, pharmaceutical compositions thereof, as well as
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`nucleic acids, and methods for discovering the same.
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`[0036] The modified antibodies described herein are connected by a linking moiety to a peptide.
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`The peptide is designed to reduce binding of the modified antibody to its target antigen when at
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`physiological pH. At acidic pH, for example at a tumor microenvironment, the peptide does not
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`reduce binding of the modified antibody to its target antigen. The peptide is designed to activate the
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`modified antibody at tumor microenvironments, thus improving the safety profile of such therapies.
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`While antibody-based therapies have proven effective for some diseases in some cases, there is a
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`need for increased targeting of antibodies to the disease site to reduce systemic-based toxicities.
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`[0037] Disclosed herein, in certain embodiments, are modified antibodies comprising a formula:
`
`A-L-P
`
`wherein A is an antibody or antibody fragment that binds to a target antigen, P is a peptide that
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`reduces binding of A to the target antigen at physiological pH, and that does not reduce binding of A
`
`to the target antigen at acidic pH, and L is a linking moiety that connects A to P at physiological pH
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`and in a tumor microenvironment and L is bound to A outside an antigen binding site.
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`Peptide [P]
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`[0038] The peptide of the modified antibodies, in some embodiments, reversibly binds to A in such
`
`a way that P sterically blocks, inhibits, or reduces the binding of affinity of A for its target antigen at
`
`physiological pH.
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`In some embodiments, P reversibly binds to A through ionic, electrostatic,
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`hydrophobic, Pi-stacking, or H-bonding interactions, or a combination thereof. In some
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`embodiments, P binds to the antigen binding site of A at physiological pH. In some embodiments, P
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`binds to A at amino acid residues which are near the antigen binding site of A. In some
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`embodiments, P binds to amino acid residues within the antigen binding site.
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`[0039] In some embodiments, at acidic pH, P is not reversibly bound to the antigen binding site of
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`A. In some embodiments, at acidic pH, P is not reversibly bound to amino acid residues near the
`
`antigen binding site of A. The peptide activates the modified antibody at acidic pH by exposing the
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`antigen binding site of A for engagement with its respective target antigen. In some cases, P has a
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`different conformation at acidic pH, compared to the conformation of P at physiological pH. In
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`some embodiments, at acidic pH, P does not form any interactions with A. In some embodiments, at
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`acidic pH, P does not form any interactions with the linking moiety (L). In some embodiments, at
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`acidic pH, P forms an interaction with L. In some embodiments, P and L reversibly bind through
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`ionic, electrostatic, hydrophobic, Pi-stacking, or H-bonding interactions, or a combination thereof.
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`[0040] In some cases, P comprises a peptide sequence. In some cases, P comprises a peptide
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`sequence disclosed in Table 1. In some cases, P comprises a peptide sequence at least 80% identical
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`to a peptide sequence disclosed in Table 1. In some cases, P comprises a peptide sequence at least
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`90% identical to a peptide sequence disclosed in Table 1. In some cases, P comprises a peptide
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`sequence at least 95% identical to a peptide sequence disclosed in Table 1. In some cases, the
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`peptides disclosed herein are linear peptides. In some cases, the peptides disclosed herein are cyclic
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`peptides.
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`Table 1. Peptide Sequences (P)
`
`Peptide ID
`
`Sequence
`
`SEQ
`
`ID NO
`
`1—:1—:
`kl‘lL»)
`
`38
`
`39
`
`40
`
`41
`
`42
`
`43
`
`44
`
`45
`
`46
`
`47
`
`48
`
`49
`
`50
`
`52
`
`53
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`54
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`55
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`Peptide 10
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`Peptide 11
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`Peptide 12
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`Peptide 13
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`Peptide 26
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`Peptide 14
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`Peptide 15
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`Peptide 16
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`Peptide 17
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`Peptide 18
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`Peptide 19
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`Peptide 20
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`Peptide 27
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`Peptide 22
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`Peptide 23
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`Peptide 28
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`Peptide 29
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`

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`Peptide 30
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`SDFSGLLFYDYQ
`
`Peptide 31
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`GGCVHFFHHQRPDC Peptide 24
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`GGCHNKSGLFYHYC
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`Peptide 25
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`GGCFYPGHHHQLLC
`
`[0041] In some embodiments, P comprises an amino acid sequence selected from the group
`
`consisting of Peptide 1, Peptide 2, Peptide 5, Peptide 6, Peptide 10, Peptide 13, Peptide 26, Peptide
`
`14, Peptide 15, Peptide 20, Peptide 27, Peptide 21, Peptide 22, Peptide 23, Peptide 28, Peptide 29,
`
`Peptide 30, Peptide 31, and Peptide 24. In some embodiments, P comprises an amino acid sequence
`
`selected from the group consisting of Peptide 10, Peptide 13, Peptide 26, Peptide 14, Peptide 15,
`
`Peptide 27, Peptide 22, Peptide 23, and Peptide 31. In some embodiments, P comprises an amino
`
`acid sequence selected from the group consisting of Peptide 15 and Peptide 23. In some
`
`embodiments, P comprises an amino acid sequence of Peptide 15. In some embodiments, P
`
`comprises an amino acid sequence of Peptide 23.
`
`[0042] In some embodiments, P is designed to incorporate amino acid residues which cause a
`
`conformational change when triggered by an environmental change. In some cases, the
`
`environmental change is the difference in pH from normal, healthy tissue to an acidic pH that is
`
`found at tumor microenVironments.
`
`[0043] Cancer cells in a solid tumor are able to form a tumor microenVironment in their
`
`surroundings to support the growth and metastasis of the cancer cells. A tumor microenVironment is
`
`often hypoxic. As the tumor mass increases, the interior of the tumor grows farther away from
`
`existing blood supply, which leads to difficulties in fully supplying oxygen to the tumor
`
`microenVironment. As a result, the tumor cells tend to rely on energy generated from lactic acid
`
`fermentation, which does not require oxygen. As a consequence of using lactic acid fermentation is
`
`that the tumor microenVironment is acidic (approximately pH 6.0-6.9) in contrast to other parts of
`
`the body which are typically either neutral or slightly basic. For example, human blood plasma has a
`
`pH of about 7.4.
`
`[0044] In some embodiments, P contains at least one histidine residue. In some embodiments, at
`
`physiological pH, at least one histidine residue of P forms a binding interaction with at least one
`
`amino acid residue of the antigen binding site of A.
`
`In some embodiments, at physiological pH, at
`
`least one histidine residue of P forms a binding interaction with at least one amino acid residue that
`
`is near the antigen binding site of A.
`
`In some embodiments, at acidic pH, at least one histidine
`
`residue of P forms a binding interaction with a glutamic acid or aspartic acid located on L.
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`[0045] In some embodiments, P contains more than one histidine residue. In some embodiments, P
`
`contains at least two histidine residues. In some embodiments, at physiological pH, at least two
`
`histidine residues of P form a binding interaction with an amino acid residue of the antigen binding
`
`site of A. In some embodiments, at physiological pH, at least two histidine residues of P form a
`
`binding interaction with an amino acid residue that is near the antigen binding site of A.
`
`In some
`
`embodiments, at physiological pH, at least two histidine residues of P form binding interactions with
`
`amino acid residues that are at or near the antigen binding site of A. In some embodiments, at acidic
`
`pH, at least two histidine residues of P form a binding interaction with a glutamic acid or aspartic
`
`acid located on L.
`
`[0046] In some embodiments, P contains at least two charged amino acid residues. In some
`
`embodiments, P contains at least three charged amino acid residues. In some embodiments, the
`
`charged amino acid residues are selected from the group consisting of lysine, arginine, histidine,
`
`aspartate, and glutamate. In some embodiments, the charged amino acid residues are selected from
`
`the group consisting of glutamate, histidine, and aspartate. In some embodiments, P contains at least
`
`glutamate and histidine. In some embodiments, P contains histidine and aspartate. In some
`
`embodiments, P contains aspartate and glutamate. In some embodiments, P contains at least one
`
`histidine and at least two aspartate residues. In some embodiments, P contains at least two histidine
`
`and at least one glutamate residues. In some embodiments, P contains at least one histidine and at
`
`least two glutamate residues. In some embodiments, P contains at least two histidine and at least one
`
`aspartate residues.
`
`[0047] In some embodiments, P contains at least two polar amino acid residues. In some
`
`embodiments, P contains at least three polar amino acids. In some embodiments, P contains at least
`
`four polar amino acids. In some embodiments, the polar amino acid residues are selected from the
`
`group consisting of serine, threonine, cysteine, asparagine, glutamine, and tyrosine.
`
`In some
`
`embodiments, P contains at least one glutamine. In some embodiments, P contains at least two
`
`glutamines. In some embodiments, P contains at least one glutamine and at least one serine. In some
`
`embodiments, P contains at least one glutamine and at least one cysteine. In some embodiments, P
`
`contains at least one glutamine and at least one asparagine. In some embodiments, P contains at least
`
`two glutamines and at least one threonine. In some embodiments, P contains at least two glutamines,
`
`and at least one asparagine. In some embodiments, P contains at least two glutamines, at least one
`
`asparagine, and at least one cysteine. In some embodiments, P contains at least two glutamines, at
`
`least one asparagine, at least one cysteine, and at least one threonine.
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`-12-
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`WSGR Docket No. 52426-701201
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`[0048] In some embodiments, P contains at least one cysteine. In some embodiments, P contains at
`
`least two cysteines. In some embodiments, P contains at least three cysteines.
`
`[0049] In some embodiments, P contains at least one glutamine and at least one methionine. In some
`
`embodiments, P contains at least two glutamines and at least one methionine. In some embodiments,
`
`P contains at least two glutamines at le