MODIFED ANTIBODIES
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`BACKGROUNDOF THE INVENTION
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`[0001] Protein-based therapies, including antibody therapies, have proven effective as treatments for
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`a variety of diseases. As with any therapeutic class, there is a need to improvetoxicity and side
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`effects of such treatments. One suchstrategy is to engineer a peptide to bind to the protein-based
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`therapy at physiological pH, but does not bind to the protein-based therapy at acidic pH. This
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`enables the protein-based therapy to be activated in certain acidic microenvironments while not
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`affecting healthy tissues.
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`REFERENCE TO A SEQUENCELISTING
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`[0002] The instant application contains a Sequence Listing which has beenfiled 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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`May26, 2017, is named 52426-701_102_SL.txt and is 40,802 bytesin size.
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`SUMMARYOF THE INVENTION
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`[0003] Disclosed herein, in certain embodiments, are modified antibodies comprising a formula A-
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`L-P wherein A is an antibody or antibody fragmentthat binds to a target antigen, P is a peptide that
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`reducesbinding ofA 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 and L is bound to A
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`outside an antigen binding site. In some embodiments, P is reversibly bound to A at physiological
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`pHthroughionic, electrostatic, hydrophobic, Pi-stacking, and H-bondinginteractions, or a
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`combination thereof. In some embodiments, P is reversibly bound to A at physiological pH at or
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`near the antigen binding site. In some embodiments, P inhibits the binding of A to the target antigen
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`at physiological pH and P doesnotinhibit the binding of A to the target antigen at acidic pH. In
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`some embodiments, in tissue other than a tumor microenvironment,P sterically blocks A from
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`binding to the target antigen. In some embodiments, at a tumor microenvironment, P is removed
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`from the antigen binding site, and the antigen binding site of A is exposed. In some embodiments,
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`the modified antibody has an increased binding affinity for the target antigen in a tumor
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`microenvironment compared to the binding affinity of the modified antibodyfor the target antigen in
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`a non-tumor microenvironment. In some embodiments, P comprises a peptide sequence with at least
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`one histidine. In some embodiments, the histidine forms a binding interaction at or near the antigen
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`binding site of A at physiological pH. In some embodiments, at acidic pH P is reversibly bound to A
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`in a region of A that is not the antigen binding site. In some embodiments, at acidic pHPis
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`reversibly bound to A in a region of A thatis not the antigen binding site through ionic, electrostatic,
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`hydrophobic, Pi-stacking and H-bonding interactions, or a combination thereof. In some
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`embodiments, P is resistant to cleavage by a protease. In some embodiments, physiological pH is
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`about pH 7.4. In some embodiments, acidic pH is about pH 6.0 to about pH 7.0. In some
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`embodiments, P comprises a peptide sequence of at least 6 amino acidsin length. In some
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`embodiments, P comprises a peptide sequence of at least 10 amino acids in length. In some
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`embodiments, P comprises a peptide sequence of at least 6 to 20 amino acids in length. In some
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`embodiments, P comprises a modified amino acid, a non-natural amino acid, or a modified non-
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`natural amino acids, or combination thereof. In some embodiments, the modified amino acid or
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`modified non-natural amino acid comprises a post-translational modification. In some embodiments,
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`at acidic pH, P is reversibly bound to L. In some embodiments, L comprises a peptide sequence with
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`at least one aspartic acid or glutamic acid, or a combination thereof. In some embodiments, the
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`histidine of P forms an interaction with the aspartic acid or glutamic acid of L. In some
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`embodiments, L is a peptide sequence havingat least 5 to no more than 50 aminoacids. In some
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`embodiments, L has a formula selected from the group consisting of: (GS),, wherein n is an integer
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`from 6 to 20 (SEQ ID NO: 1); (G2S)n, wherein n is an integer from 4 to 13 (SEQ ID NO: 2); (G3S)n,
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`wherein n is an integer from 3 to 10 (SEQ ID NO: 3); and (G4S)n, wherein n is an integer from 2 to 8
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`(SEQ ID NO: 4); and (G),, wherein n is an integer from 12 to 40 (SEQ ID NO: 5). In some
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`embodiments, L has a formula comprising (GGSGGD),, wherein n is an integer from 2 to 6 (SEQ ID
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`NO: 8). In some embodiments, L has a formula comprising (GGSGGE),, wherein n is an integer
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`from 2 to 6 (SEQ ID NO: 9). In some embodiments, L has a formula comprising
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`(GGGSGSGGGGS),, wherein n is an integer from 1 to 3 (SEQ ID NO: 6). In some embodiments, L
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`has a formula comprising (GGGGGPGGGGP),, wherein n is an integer from 1 to 3 (SEQ ID NO: 7).
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`In some embodiments, L has a formula selected from (GX),, wherein X is serine, aspartic acid,
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`glutamic acid, threonine, or proline andnis at least 20 (SEQ ID NO: 24); (GGX),, wherein X is
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`serine, aspartic acid, glutamic acid, threonine, or proline and n isat least 13 (SEQ ID NO: 25);
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`(GGGX),, wherein X is serine, aspartic acid, glutamic acid, threonine, or proline andnis at least 10
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`(SEQ ID NO: 26); (GGGGX),, wherein X is serine, aspartic acid, glutamic acid, threonine, or
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`proline and n isat least 8 (SEQ ID NO: 27); (G;X)n, wherein X is serine, aspartic acid, glutamic
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`acid, threonine, or proline and n is at least 15, and z is between 1 and 20 (SEQ ID NO: 28). In some
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`embodiments, L is resistant to cleavage by a protease. In some embodiments, L comprises a
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`modified amino acid. In some embodiments, the modified amino acid comprisesa post-translational
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`modification. In some embodiments, L comprises a non-natural amino acid or a modified non-
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`natural amino acid, or combination thereof. In some embodiments, the modified non-natural amino
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`acid comprises a post-translational modification. In some embodiments, the target antigen is selected
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`from the group consisting of: 4-1BB, CTLA4, and PD-1. In some embodiments, the target antigen is
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`4-1BB. In some embodiments, the target antigen is CTLA4. In some embodiments, the target antigen
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`is PD-1. In some embodiments, A is a full length antibody, a single-chain antibody, an Fab fragment,
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`an Fab’ fragment, an (Fab’)2 fragment, an Fv fragment, a divalent single chain antibody, bispecific
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`antibody, a trispecific antibody, a tetraspecific antibody, or an antibody drug conjugate. In some
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`embodiments, A is selected from the group consisting of utomilumab, urelumab, ipilimumab,
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`tremelimumab, pembrolizumab,and nivolumab. In some embodiments, A is utomilumab. In some
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`embodiments, A is urelumab. In some embodiments, A is ipilimumab. In some embodiments, A is
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`tremelimumab. In some embodiments, A is pembrolizumab. In some embodiments, A is nivolumab.
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`[0004] Disclosed herein, in certain embodiments, are pharmaceutical compositions, comprising a
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`modified antibody according to the compositions described herein and 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] Figure 1 exemplifies an antibody that does not comprise a peptide modification. Such
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`antibodies bind to unique antigensthat exist in abundancein tumortissue. But, the unique antigens
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`are also found in somehealthy tissues, which can trigger systemic immuneactivation in a subject,
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`and cause toxicity.
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`[0007] Figure 2 shows an exemplary modified antibody. In this example, the modified antibody is
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`linked to a peptide which bindsat or near the antigen binding site of the modified antibody at pH
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`7.4. This reduces binding of the modified antibodyto its target antigen in healthy tissue. In tumor
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`tissue, the acidic tumor microenvironmentdisrupts the interaction of the peptide with the modified
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`antibody. The antigen bindingsite of the modified antibody is exposed, and the modified antibody
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`selectively binds to its target antigen in tumortissue.
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`[0008] Figure 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 bindingto 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 tumortissue, and enables the antibodyto bindtoits target antigen.
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`[0009] Figure 4 shows an exemplary modified antibody. In this example, the peptide is engineered
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`to 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 bindingsite is disfavored becausethe histidine is protonated. The antibody binding
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`site is available for bindingto its target antigen in a tumor microenvironment.
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`DETAILED DESCRIPTION OF THE INVENTION
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`[0010] While preferred embodiments of the present invention have been shownand described
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`herein, it will be obvious to those skilled in the art that such embodiments are provided by way of
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`example only. Numerous variations, changes, and substitutions will now occurto those skilled in the
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`art without departing from the invention. It should be understood that various alternatives to the
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`embodiments of the invention described herein may be employedin practicing the invention.It is
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`intended that the following claims define the scope of the invention and that methodsandstructures
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`within the scope of these claims and their equivalents be covered thereby
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`Certain Definitions
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`[0011] The terminology used herein is for the purpose of describing particular cases only and is not
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`intendedto be limiting. As used herein, the singular forms “a’, “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
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`extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in
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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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`[0012] The term “about” or “approximately” means within an acceptable error range for the
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`particular value as determined by one ofordinary 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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`[0013] As used herein, “physiological pH” is used to refer to the pH of a non-diseasedstate cellular
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`environment. 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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`[0014] 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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`[0015] 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 antibodyto 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 antibodyto 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 somecases, 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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`[0016] Disclosed herein, in certain embodiments, are modified antibodies comprising a formula:
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`A-L-P
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`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 ofA 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 and L is bound to A
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`outside an antigen bindingsite.
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`Peptide (P)
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`[0017] The peptide of the modified antibodies, in some embodiments, reversibly binds to A in such
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`a waythat P sterically blocks, inhibits, or reduces the binding of affinity of A forits target antigen at
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`physiological pH. In some embodiments, P reversibly binds to A throughionic, 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 whichare near the antigen binding site of A. In some
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`embodiments, P binds to amino acid residues within the antigen bindingsite.
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`[0018] In some embodiments, at acidic pH, P is not reversibly boundto the antigen bindingsite of
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`A. In some embodiments, at acidic pH,P is not reversibly bound to amino acid residues near the
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`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 anyinteractions with the linking moiety (L). In some embodiments, at
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`acidic pH, P formsan 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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`[0019] In some cases, P comprises a peptide sequence. In some embodiments, P is designed to
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`incorporate amino acid residues which cause a conformational change whentriggered by an
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`environmental change. In some cases, the environmental changeis the difference in pH from
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`normal, healthy tissue to an acidic pH that is found at tumor microenvironments.
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`[0020] Cancercells in a solid tumor are able to form a tumor microenvironment in their
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`surroundings to support the growth and metastasis of the cancer cells. A tumor microenvironmentis
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`often hypoxic. As the tumor mass increases, the interior of the tumor grows farther away from
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`existing blood supply, which leads to difficulties in fully supplying oxygen to the tumor
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`microenvironment. As a result, the tumorcells tend to rely on energy generated from lactic acid
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`fermentation, which does not require oxygen. As a consequence ofusing lactic acid fermentation is
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`that the tumor microenvironmentis acidic (approximately pH 6.0-6.9) in contrast to other parts of
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`the body whichare typically either neutral or slightly basic. For example, human blood plasma has a
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`pHofabout7.4.
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`[0021] In some embodiments, P contains at least one histidine residue. In some embodiments, at
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`physiological pH,at least one histidine residue of P forms a binding interaction with at least one
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`amino acid residue of the antigen binding site of A. In some embodiments, at physiological pH, at
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`least one histidine residue of P forms a binding interaction with at least one amino acid residuethat
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`is near the antigen binding site of A. In some embodiments, at acidic pH,at least one histidine
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`residue of P forms a binding interaction with a glutamic acid or aspartic acid located on L.
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`[0022] In some embodiments, P contains more than one histidine residue. In some embodiments, at
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`physiological pH,at least two histidine residues of P form a binding interaction with an amino acid
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`residue of the antigen binding site of A. In some embodiments, at physiological pH,at least two
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`histidine residues of P form a binding interaction with an amino acid residue that is near the antigen
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`binding site of A. In some embodiments, at physiological pH,at least two histidine residues of P
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`form binding interactions with amino acid residuesthat are at or near the antigen bindingsite of A.
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`In some embodiments, at acidic pH,at least two histidine residues of P form a binding interaction
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`with a glutamic acid or aspartic acid located on L.
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`[0023] In some embodiments, P is a peptide sequenceat least 5 amino acids in length. In some
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`embodiments, P is a peptide sequenceat least 6 amino acids in length. In some embodiments, P is a
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`peptide sequenceat least 10 amino acids in length. In some embodiments, P is a peptide sequence at
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`least 20 amino acids in length. In some embodiments, P is resistant to cleavage by a protease.
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`[0024] In some embodiments, P is not a natural binding partner of A. In someinstances, P is a
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`modified binding partner for A which contains amino acid changesthat atleast slightly decrease
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`affinity and/or avidity of binding to A. In some embodiments, P contains no or substantially no
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`homologyto A’s natural binding partner. In some embodiments, P is no more than 5%, 10%, 15%,
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`20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to the natural
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`binding partnerof A.
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`[0025] In some embodiments, P comprises a modified amino acid or non-natural amino acid, or a
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`modified non-natural amino acid, or a combination thereof. In some embodiments, the modified
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`amino acid or a modified non-natural amino acid comprises a post-translational modification. In
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`some embodiments P comprises a modification including, but not limited to acetylation, acylation,
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`ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety,
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`covalent attachment of a nucleotide or nucleotide derivative, covalent attachmentofa lipid or lipid
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`derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond
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`formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of
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`pyroglutamate, formylation, gammacarboxylation, glycosylation, GPI anchor formation,
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`hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing,
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`phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition
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`of amino acidsto proteins such as arginylation, and ubiquitination. Modifications are made
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`anywhereto P including the peptide backbone, the amino acid side chains, and the terminus.
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`Linking Moiety
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`(L
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`[0026] In some embodiments, L is a peptide sequence ofat least 5 amino acid residues. In some
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`embodiments, L is a peptide sequence of no more than 50 amino acids. Regarding the amino acid
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`composition of L, peptides are selected with properties that confer flexibility and facilitate a
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`conformational change of P during a change in pH.
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`[0027] In some embodiments,L is resistant to protease cleavage. For example, glycine and serine
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`residues generally provide protease resistance. Examples of suitable linking moieties for connecting
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`A or C to P include,but are not limited to (GS),, wherein n is an integer from 6 to 20 (SEQ ID NO:
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`1); (G2S),, wherein n is an integer from 4 to 13 (SEQ ID NO: 2); (G3S),, wherein n is an integer
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`from 3 to 10 (SEQ ID NO: 3); and (G4S),, wherein n is an integer from 2 to 8 (SEQ ID NO: 4); and
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`(G),, wherein n is an integer from 12 to 40 (SEQ ID NO: 5). Additional examples include, but are
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`not limited to, (GGGSGSGGGGS) », wherein n is an integer from 1 to 3 (SEQ ID NO: 6), or
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`(GGGGGPGGGGP) », wherein n is an integer from 1 to 3 (SEQ ID NO: 7).
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`[0028] In some embodiments, L bindsto P at acidic pH, for example, at a tumor microenvironment.
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`In some embodiments, at acidic pH, L is reversibly bound to P. In some embodiments, the
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`interaction of L and P at acidic pH is mediated through a histidine residue on P and a glutamic acid
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`or aspartic acid residue on L. Examplesof such linking moieties include, but are not limited to,
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`(GGSGGD),, wherein n is an integer from 2 to 6 (SEQ ID NO: 8); or (GGSGGE),, wherein n is an
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`integer from 2 to 6 (SEQ ID NO: 9).
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`[0029] In some embodiments, L has a formula comprising, (GS),(GGSGGD), (GS),, wherein x is an
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`integer from 0 to 20,y is an integer from 2 to 6, and z is an integer from 0 to 20 (SEQ ID NO: 10).
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`In some embodiments, L has a formula comprising, (G2S),(GGSGGD), (G2S),, wherein x is an
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`integer from 0 to 13, y is an integer from 2 to 6, and z is an integer from 0 to 13 (SEQ ID NO: 11).
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`In some embodiments, L has a formula comprising, (G3S);(GGSGGD), (G3S),, wherein x is an
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`integer from 0 to 10, y is an integer from 2 to 6, and z is an integer from 0 to 10 (SEQ ID NO: 12).
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`In some embodiments, L has a formula comprising, (G4S);(GGSGGD), (G4S),, wherein x is an
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`integer from 0 to 8, y is an integer from 2 to 6, and z is an integer from 0 to 8 (SEQ ID NO: 13). In
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`some embodiments, L has a formula comprising, (G);(GGSGGD), (G),, wherein x is an integer from
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`0 to 40, y is an integer from 2 to 6, and z is an integer from 0 to 40 (SEQ ID NO: 14). In some
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`embodiments, L has a formula comprising, (GGGSGSGGGGS ),(GGSGGD), (GGGSGSGGGGS),,
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`wherein x is an integer from 0 to 3, y is an integer from 2 to 6, andzis an integer from 0 to 3 (SEQ
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`ID NO: 15). In some embodiments, L has a formula comprising, (GGGSGSGGGGP),(GGSGGD),
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`(GGGSGSGGGGP),wherein x is an integer from 0 to 3, y is an integer from 2 to 6, and z is an
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`integer from 0 to 3 (SEQ ID NO: 16).
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`[0030] In some embodiments, L has a formula comprising, (GS),(GGSGGE),(GS),, wherein x is an
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`integer from 0 to 20,y is an integer from 2 to 6, and z is an integer from 0 to 20 (SEQ ID NO: 17).
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`In some embodiments, L has a formula comprising, (G2S);(GGSGGE), (G2S),, wherein x is an
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`integer from 0 to 13, y is an integer from 2 to 6, and z is an integer from 0 to 13 (SEQ ID NO: 18).
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`In some embodiments, L has a formula comprising, (G3S),(GGSGGE), (G3S),, wherein x is an
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`integer from 0 to 10, y is an integer from 2 to 6, and z is an integer from 0 to 10 (SEQ ID NO: 19).
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`In some embodiments, L has a formula comprising, (G4S);(GGSGGE), (G4S),, wherein x is an
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`integer from 0 to 8, y is an integer from 2 to 6, and z is an integer from 0 to 8 (SEQ ID NO: 20). In
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`some embodiments, L has a formula comprising, (G);(GGSGGE),(G),, wherein x is an integer from
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`0 to 40, y is an integer from 2 to 6, and z is an integer from 0 to 40 (SEQ ID NO: 21). In some
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`embodiments, L has a formula comprising, (GGGSGSGGGGS),(GGSGGE), (GGGSGSGGGGS),,
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`wherein x is an integer from 0 to 3, y is an integer from 2 to 6, andzis an integer from 0 to 3 (SEQ
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`ID NO: 22). In some embodiments, L has a formula comprising, (GGGSGSGGGGP),(GGSGGE),
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`(GGGSGSGGGGP),wherein x is an integer from 0 to 3, y is an integer from 2 to 6, and z is an
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`integer from 0 to 3 (SEQ ID NO: 23).
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`[0031] Additional examples of linking moieties include, but are not limited to wherein L has a
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`formula selected from (GX)n, wherein X is serine, aspartic acid, glutamic acid, threonine, or proline
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`andnis at least 20 (SEQ ID NO: 24); (GGX),, wherein X is serine, aspartic acid, glutamic acid,
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`threonine, or proline and n is at least 13 (SEQ ID NO: 25); (GGGX),, wherein X is serine, aspartic
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`acid, glutamic acid, threonine, or proline and n is at least 10 (SEQ ID NO: 26); (GGGGX),, wherein
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`X is serine, aspartic acid, glutamic acid, threonine, or proline and n is at least 8 (SEQ ID NO: 27);
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`(G,X)n, wherein X is serine, aspartic acid, glutamic acid, threonine, or proline andnis at least 15,
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`and z is between 1 and 20 (SEQ ID NO: 28).
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`[0032] In some embodiments, L comprises a modified amino acid or non-natural amino acid, or a
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`modified non-natural amino acid, or a combination thereof. In some embodiments, the modified
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`amino acid or a modified non-natural amino acid comprises a post-translational modification. In
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`some embodiments L comprises a modification including, but not limited, to acetylation, acylation,
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`ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety,
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`covalent attachment of a nucleotide or nucleotide derivative, covalent attachment ofa lipid or lipid
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`derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond
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`formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of
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`pyroglutamate, formylation, gammacarboxylation, glycosylation, GPI anchor formation,
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`hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing,
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`phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition
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`of amino acidsto proteins such as arginylation, and ubiquitination. Modifications are made
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`anywhereto L including the peptide backbone,or the amino acid side chains.
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`Antibody or Antibody Fragments (A
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`[0033] In some embodiments, A is a full length antibody, a single-chain antibody, a Fab Fragment,
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`an Fab’ fragment, an (Fab’)2 fragment, an Fv fragment, a divalent single chain antibody, a bispecific
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`antibody, trispecific antibody, tetraspecific antibody, or an antibody drug conjugate.
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`[0034] In some embodiments A is an antagonist, agonist, conditionally active antibody, or a
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`sweeping body.
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`[0035] In some embodiments, A is an antibody or antibody fragment including, but not limited to,
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`utomilumab, urelumab,ipilimumab, tremelimumab, pembrolizumab, and nivolumab. In some
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`embodiments, A is utomilumab. In some embodiments, A is urelumab. In some embodiments, A is
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`ipilimumab. In some embodiments, A is tremelimumab. In some embodiments, A is
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`pembrolizumab. In some embodiments, A is nivolumab.
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`[0036] In some embodiments, A bindsto a target antigen. In some embodiments, the target antigen
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`includes, butis not limited to, 4-1BB, CTLA4, and PD-1. In some embodiments, the target antigen is
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`4-1BB. In some embodiments, the target antigen is CTLA4. In some embodiments, the target
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`antigen is PD-1.
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`[0037] In some embodiments, A contains a modification so as to increase the bioavailability,
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`improvestability, or solubility of the modified antibody. In some embodiments, A is conjugated to
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`polyethylene glycol, polysialic acid (PSA), HPMA copolymer, dextran, albumin, a glycosyl group or
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`a combination thereof.
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`[0038] In some embodiments, A comprises a modified amino acid or non-natural amino acid, or a
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`modified non-natural amino acid, or a combination thereof. In some embodiments, the modified
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`amino acid or a modified non-natural amino acid comprises a post-translational modification. In
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`some embodiments A comprises a modification including, but not limited to acetylation, acylation,
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`ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety,
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`covalent attachment of a nucleotide or nucleotide derivative, covalent attachmentofa lipid or lipid
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`derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond
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`formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of
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`pyroglutamate, formylation, gammacarboxylation, glycosylation, GPI anchor formation,
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`hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing,
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`phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition
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`of amino acidsto proteins such as arginylation, and ubiquitination. Modifications are made
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`anywhere to A including the peptide backbone, the amino acid side chains, or the termini or teminus.
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`Polynucleotides Encoding Modified Antibodies
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`[0039] Also provided, in some embodiments, are polynucleotide molecules encoding a modified
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`antibody described herein. In some embodiments, the polynucleotide molecules are provided as a
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`DNAconstruct. In other embodiments, the polynucleotide molecules are provided as a messenger
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`RNAtranscript.
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`[0040] The polynucleotide molecules are constructed by known methods such as by combining the
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`genes encoding the domainseither separated by peptidelinkers or, in other embodiments, directly
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`linked by a peptide bond,into a single genetic construct operably linked to a suitable promoter, and
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`optionally a suitable transcription terminator, and expressing it in bacteria or other appropriate
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`expression system such as, for example CHO cells. Depending on the vector system and host
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`utilized, any numberof suitable transcription and translation elements, including constitutive and
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`inducible promoters, may be used. The promoteris selected suchthat it drives the expression of the
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`polynucleotide in the respective hostcell.
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`[0041] In some embodiments, the polynucleotide is inserted into a vector, preferably an expression
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`vector, which represents a further embodiment. This recombinant vector can be constructed
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`according to known methods. Vectors of particular interest include plasmids, phagemids, phage
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`derivatives, virii (e.g., retroviruses, adenoviruses, adeno-associated viruses, herpes viruses,
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`lentiviruses, and the like), and cosmids.
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`[0042] A variety of expression vector/host systems may be utilized to contain and express the
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`polynucleotide encoding the polypeptide of the described antigen-binding protein. Examples of
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`expression vectors for expression in E.coli are pSKK (Le Gallet al., J Immunol Methods. (2004)
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`285(1):111-27) or peDNAS (Invitrogen) for expression in mammaliancells.
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`[0043] Thus, the modified antibodies as described herein, in some embodiments, are produced by
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`introducing a vector encoding the protein as described above into a host cell and culturing said host
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`cell under conditions whereby the protein domains are expressed, may be isolated and, optionally,
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`further purified.
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`Pharmaceutical Compositions
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`[0044] Also provided, in some embodiments, are pharmaceutical compositions comprising a
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`modified antibody described herein, a vector comprising the polynucleotide encoding the
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`polypeptide of the modified antibodyor a host cell transformed by this vector and at least one
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`pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" includes, butis
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`not limited to, any carrier that does not interfere with the effectiveness of the biological activity of
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`the ingredients and that is not toxic to the patient to whom it is administered. Examples of suitable
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`pharmaceutical carriers are well knownin the art and include phosphate buffered saline solutions,
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`water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutionsetc.
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`Such carriers can be formulated by conventional methods and can be administered to the subject at a
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`suitable dose. Preferably, the compositionsare sterile. These compositions mayalso contain
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`adjuvants such as preservative, emulsifying agents and dispersing agents. Prevention of the action of
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`microorganisms maybe ensured bythe inclusion of various antibacterial and antifungal agents.
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`[0045] In some embodiments of the pharmaceutical compositions, the modified antibody described
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`herein is encapsulated in nanoparticles. In some embodiments, the nanoparticles are fullerenes,
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`liquid crystals, liposome, quantum dots, superparamagnetic nanoparticles, dendrimers, or nanorods.
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`In other embodimentsof the pharmaceutical compositions, the modified antibody, is attached to
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`liposomes. In some instances, the modified antibody is conjugated to the surface of liposomes. In
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`someinstances, the modified antibody is encapsulated within the shell of a liposome. In some
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`instances, the liposomeis a cationic liposome.
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`[0046] The modified antibodies described herein are contemplated for use as a medicament.
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`Administration is effected by different ways, e.g. by intravenous, intraperitoneal, subcutaneous,
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`intramuscular, topical or intradermal administration. In some embodiments, the route of
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`administration depends on the kind of therapy and the kind of compoundcontained in the
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`pharmace