Patent Publication Number: US-9834597-B2

Title: Modified FC polypeptides, FC conjugates, and methods of use thereof

Description:
CROSS-REFERENCE 
     This application claims the benefit of U.S. Provisional Patent Application No. 61/704,166, filed Sep. 21, 2012, and U.S. Provisional Patent Application No. 61/711,589, filed Oct. 9, 2012, which applications are incorporated herein by reference in their entirety. 
    
    
     BACKGROUND 
     Generation of fusion proteins comprising segments derived from two or more different precursor proteins has represented an important advance in the field of protein engineering. Fusion proteins allow multiple biological functions, such as binding and therapeutic activity, to be combined in a single entity. 
     The crystallizable fragment (Fc) of an immunoglobulin molecule binds to Fc receptors and/or complement proteins. The Fc domain mediates various physiological effects including lysis of target cells. 
     There is a need in the field for methods of a conjugating functional group to the Fc domain of an immunoglobulin. 
     LITERATURE 
     
         
         Gilmore et al. (2006)  Angew. Chem. Int. Ed.  45:5307; Scheck et al. (2007)  ACS Chem. Biol.  2:247; Scheck et al. (2008)  J. Am. Chem. Soc.  130:11762; Witus et al. (2010)  J. Am. Chem. Soc.  132:16812. 
       
    
     SUMMARY 
     The present disclosure provides modified Fc domains having one or more attachment moieties for attaching a heterologous functional moiety; and methods of generating the modified Fc domains. The present disclosure provides Fc conjugates; and methods of making the conjugates. The Fc conjugates are useful in various methods, which are also provided. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIGS. 1A-C  depict the structure and an expression vector for Fc of human IgG1.  FIG. 1C  depicts the nucleotide (SEQ ID NO:1) and amino acid (SEQ ID NO:2) sequences of an exemplary Fc. 
         FIG. 2  presents modification schemes for Fc proteins. 
         FIGS. 3A-C  depict LCMS analysis of (a) the Fc protein collected 2 days after transfection; (b) the Fc protein collected 5 d after transfection (fresh Opti-MEM media was replaced after 2 d); and (c) the Fc protein after treatment with PNGase F. 
         FIGS. 4A-C  depict analysis of transamination efficiency for AKT-Fc domains. 
         FIGS. 5A-D  depict modification of Fc domains via oxidative coupling. 
         FIGS. 6A-D  depict construction of Fc-aptamer conjugates. 
         FIGS. 7A-D  depict cell binding specificity and C1q binding ability of Fc-aptamer conjugates. 
         FIG. 8  provides a scheme for the synthesis of hydrazide-DNA oligonucleotide 5. 
         FIG. 9  provides a scheme for synthesis of aminophenol-DNA oligonucleotide 10b. 
         FIGS. 10A-C  provide amino acid sequences of immunoglobulin Fc domains. 
         FIGS. 11A and 11B  depict functionalization of anti-HER2 human IgG1 antibodies with DNA aptamers both through N-terminal modification and through lysine modification. 
         FIGS. 12A and 12B  depict the binding specificity of antibody-DNA aptamer conjugates. 
         FIGS. 13A-C  provide amino acid sequences ( FIG. 13C ) of anti-HER2 heavy and light chains and nucleotide sequences ( FIGS. 13A and 13B ) encoding same. 
     
    
    
     DEFINITIONS 
     “ADCC” or “antibody dependent cell-mediated cytotoxicity,” as used herein, refers to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fcγ receptors (FcγRs) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. 
     “ADCP” or “antibody dependent cell-mediated phagocytosis,” as used herein, refers to a cell-mediated reaction in which nonspecific cytotoxic cells that express FcγRs recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell. 
     “CDC” or “complement-dependent cytotoxicity,” as used herein, refers to a reaction initiated by multiple Fc-domains interacting with C1q, which can ultimately result in one or more of lysis of a target cell through the formation of the membrane attack complex (MAC), phagocyte recruitment, and opsonization of a target. 
     A modified Fc polypeptide of the present disclosure, or an Fc conjugate of the present disclosure, can specifically bind to an Fc receptor. By “specifically bind” is meant that the modified Fc polypeptide or the Fc conjugate binds an Fc receptor with an affinity in the range of from about 10 −6  M to about 10 −7 M, from about 10 −7  M to about 10 −8  M, or from about 10 −8  M to about 10 −9  M, or greater than 10 −9  M. In some cases, a modified Fc polypeptide, or an Fc conjugate, binds an Fc receptor with an affinity of less than 10 −6 M. 
     Fc receptors are known in the art, and are classified based on the isotype of the Fc that they bind. Fc receptors include neonatal Fc receptor (FcRn), Fcγ receptors (FcγR), an Fcα receptor (FcαR), Fcε receptors (FcεR), and Fcα/μ, receptor (Fcα/μR). FcγRs bind IgG isotype Fc domains and include FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16a), and FcγRIIIB (CD16b). Binding of an IgG isotype Fc domain (Fcγ) to an FcγR can result in phagocytosis, ADCC, and the like. The Fcα receptor (FcαRI) binds IgA Fc domains, and is found on the surface of neutrophils, eosinophils, monocytes, some macrophages (including Kupffer cells), and some dendritic cells. Binding of an IgA Fc domain (Fcα) to an FcαR can result in phagocytosis or induction of microbe killing FcεRs bind IgE Fc domains, and include the high-affinity FcεRI, found on epidermal Langerhans cells, eosinophils, mast cells and basophils; and the low-affinity FcεRII (CD23). Binding of an IgE isotype Fc domain (Fcε) to an FcεR (e.g., FcεRI) can result in degranulation. Fcα/μR binds IgA Fc domains and IgM Fc domains, and is found on B cells, mesangial cells, and macrophages. 
     By “effector function” as used herein is meant a biochemical event that results from the interaction of an antibody Fc region (“an Fc domain”) with an Fc receptor or ligand. Effector functions include but are not limited to ADCC, ADCP, and complement-dependent cytotoxicity (CDC). By “effector cell” as used herein is meant a cell of the immune system that expresses one or more Fc receptors and mediates one or more effector functions. Effector cells include but are not limited to monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and cytotoxic T cells. 
     By “Fc”, “Fc region,” Fc polypeptide,” “Fc domain,” etc. as used herein is meant a polypeptides comprising the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM Fc may include the J chain. For IgG, the Fc domain comprises immunoglobulin domains Cy2 and Cy3 (CH2 and CH3) and the hinge between Cy1 (CH1) and Cy2 (CH2). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat. “Fc” can refer to the Fc domain in isolation, or the Fc region in the context of an antibody, antibody fragment, or Fc fusion. An Fc may be an antibody, Fc fusion, or an protein or protein domain that comprises Fc. 
     By “Fc ligand” as used herein is meant a factor (e.g., a polypeptide) that binds to the Fc region to form an Fc-ligand complex. Fc ligands include but are not limited to Fc receptors, C1q, C3, mannan binding lectin, mannose receptor, staphylococcal protein A, streptococcal protein G, and viral FcγR. 
     Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. 
     Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. 
     Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. 
     It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an Fc polypeptide” includes a plurality of such polypeptide and reference to “the attachment moiety” includes reference to one or more attachment moieties and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. 
     It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. 
     The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. 
     DETAILED DESCRIPTION 
     The present disclosure provides modified Fc domains having one or more attachment moieties for attaching a heterologous functional moiety; and methods of generating the modified Fc domains. The present disclosure provides Fc conjugates; and methods of making the conjugates. The Fc conjugates are useful in various methods, which are also provided. 
     Modified Fc Polypeptides 
     The present disclosure provides a modified Fc polypeptide comprising one or more attachment moieties for attaching a heterologous functional moiety. 
     In some cases, a modified Fc polypeptide comprises a single (no more than one) attachment moiety for attaching a heterologous functional moiety. In other cases, a modified Fc polypeptide comprises 2, 3, 4, or 5 attachment moieties. 
     As used herein, the term “attachment moiety” refers to a moiety that is capable of reacting directly either spontaneously or after activation, with an accessible functional group of a heterologous functional moiety under aqueous conditions to produce a covalent linkage to the modified Fc polypeptide. The attachment moiety is capable of reacting under aqueous conditions at which Fc polypeptides of interest are able to be maintained in a folded state (e.g., physiological conditions). 
     The attachment moiety may include a first functional group that is reactive with a second functional group of the heterologous functional moiety and conjugates the attachment moiety and the heterologous functional moiety. Any suitable first and second functional groups and conjugation chemistries may find use in the subject modified Fc polypeptides and Fc conjugates, and methods for making the same. In some cases the first functional group includes an aldehyde or a ketone group. In other cases, the first functional group includes an aryl amino group (e.g., an aniline group or a aminophenol group). Further first and second functional groups of interest and methods of conjugating the same include but are not limited to those groups and methods described in Hermanson, “Bioconjugate Techniques” 2nd Edition, Academic Press, 2008. 
     As used herein, the terms “to crosslink” or “conjugate” are used interchangeably and refer to the process of linking two moieties or atoms to each other via a covalent bond. An exemplary cross-linking process is the reaction of a ketone group with a hydroxylamino group to produce a stable oxime covalent linkage. 
     In some instances, the modified Fc polypeptide is described by the structure of formula (I): 
     
       
         
         
             
             
         
       
     
     where R 1  is an amino acid sidechain, hydrogen, an alkyl or an aryl group, L is an optional linker and Z is an Fc polypeptide. In some embodiments, in formula (I), R 1  is methyl, L is a carbonyl group (—C(═O)—), and Z is an Fc polypeptide. In certain embodiments, R 1  is the amino acid sidechain of alanine or glutamic acid. 
     In some embodiments, the modified Fc polypeptide is described by the structure of formula (II): 
     
       
         
         
             
             
         
       
     
     where R 1  is as described for formula (I), and Z 1  is an Fc polypeptide. In some embodiments, Z 1  includes N-terminal lysine-threonine residues, and R 1  is the amino acid side chain of alanine or glutamic acid. 
     In other instances, the modified Fc polypeptide is described by the structure of formula (III): 
     
       
         
         
             
             
         
       
     
     where R is one or more optional aryl substituents, R′ and R″ are each independently H, an alkyl or an aryl, L is a linker and Z is a Fc polypeptide. In some cases, each R substituent is independently selected from hydroxyl, alkyl, and aryl. In certain embodiments, R′ and R″ are each H. In some cases, the modified Fc polypeptide includes an aminophenol group. In certain cases, R is a hydroxyl substituent that is located at a position para to L, and L is located at a position meta to the amino group. In other cases, R is absent, and L is attached at the para position to the amino group. 
     In some embodiments, the modified Fc polypeptide is described by the structure: 
     
       
         
         
             
             
         
       
     
     where L 1  is a linker, X is O or NH, R′ and R″ are each independently H, an alkyl or an aryl, R is one or more groups, independently selected from hydrogen, an alkyl, an aryl, a hydroxy, an alkoxy, an aryloxy, a heterocycle, a cyano, a halogen, an amino, an acyl, an acyloxy, an amido and nitro; R 1  is the sidechain of an amino acid, H, an alkyl or an aryl; and Z 1  is an Fc polypeptide. In certain embodiments, R′ and R″ are each H. In certain embodiments, L 1  is —(CH 2 ) m —NHCO—CH 2 — and X is O or NH, where m is 0, 1, 2, 3, 4, 5 or 6. In some embodiments, L 1  is —(CH 2 ) m —NHCO— and X is NH, where m is 0, 1, 2, 3, 4, 5 or 6. In certain embodiments, L 1  is —(CH 2 ) m —CONH—CH 2 — and X is O or NH, where m is 0, 1, 2, 3, 4, 5 or 6. In certain embodiments, L 1  is —(CH 2 ) m —CONH— and X is O or NH, where m is 0, 1, 2, 3, 4, 5 or 6. 
     In some embodiments, the modified Fc polypeptide is described by the structure of formula (IV): 
     
       
         
         
             
             
         
       
     
     where L 1  is a linker, X is O or NH, R′ and R″ are each independently H, an alkyl or an aryl and R 1  and Z 1  are as defined above in formula (II). In certain embodiments, R′ and R″ are each H. In certain embodiments, L 1  is —(CH 2 ) m —NHCO—CH 2 — and X is O, where m is 0, 1, 2, 3, 4, 5 or 6. In some embodiments, L 1  is —(CH 2 ) m —NHCO— and X is NH, where m is 0, 1, 2, 3, 4, 5 or 6. In certain embodiments, m is 1 or 2. In certain embodiments, L 1  is —(CH 2 ) m —CONH—CH 2 — and X is O or NH, where m is 0, 1, 2, 3, 4, 5 or 6. In certain embodiments, L′ is —(CH 2 ) m —CONH— and X is O or NH, where m is 0, 1, 2, 3, 4, 5 or 6. 
     In certain embodiments, the modified Fc polypeptide is described by the structure of formula (V): 
     
       
         
         
             
             
         
       
     
     where m is 1 or 2, R′ and R″ are each independently H, an alkyl or an aryl, and R 1  and Z 1  are as described in formula (II). In certain embodiments, R′ and R″ are each H. In certain embodiments, R 1  is a lower alkyl (e.g., methyl). In certain embodiments, Z 1  is a Fc polypeptide that includes N-terminal lysine-threonine residues, and R 1  is the amino acid side chain of alanine. 
     As used herein, the term “linker” or “linkage” refers to a linking moiety that connects two groups and has a backbone. In some cases, the backbone of the linker is 100 atoms or less in length, such as 50 atoms or less, or 20 atoms or less in length. In other cases, the backbone of the linker is 100 atoms or greater in length. A linker or linkage may be a covalent bond that connects two groups or a chain of between 1 and 100 atoms in length, such as between 1 and 50 atoms in length or 1 and 20 atoms in length, for example of about 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18 or 20 carbon atoms in length, where the linker may be linear, branched, cyclic or a single atom. In certain cases, one, two, three, four or five or more carbon atoms of a linker backbone may be optionally substituted with a sulfur, nitrogen or oxygen heteroatom. The bonds between backbone atoms may be saturated or unsaturated, usually not more than one, two, or three unsaturated bonds will be present in a linker backbone. The linker may include one or more substituent groups, for example with an alkyl, aryl or alkenyl group. A linker may include, without limitations, oligo(ethylene glycol); ethers, thioethers, tertiary amines, alkyls, which may be straight or branched, e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), and the like. The linker backbone may include a cyclic group, for example, an aryl, a heterocycle or a cycloalkyl group, where 2 or more atoms, e.g., 2, 3 or 4 atoms, of the cyclic group are included in the backbone. A linker may be peptidic or non-peptidic. A linker may be cleavable or non-cleavable. 
     Fc Polypeptides 
     Fc polypeptides that can be modified to generate a subject modified Fc polypeptide include Fc polypeptides of any of a variety of species, including, e.g., human Fc polypeptide, mouse Fc polypeptides, rat Fc polypeptides, and the like. Fc polypeptides that can be modified to generate a subject modified Fc polypeptide include synthetic (non-naturally occurring) Fc polypeptides, e.g., Fc polypeptides comprising amino acid sequences not found in nature. Fc polypeptides that can be modified to generate a subject modified Fc polypeptide include Fcγ (including Fcγ1, Fcγ2, Fcγ3, Fcγ4), Fcδ, Fcμ, Fcα, and Fcε. In some cases, the Fc polypeptide is part of a longer polypeptide, e.g., in some embodiments, the Fc polypeptide is part of a full-length immunoglobulin polypeptide comprising an antigen-binding domain. Where the Fc polypeptide is part of a longer polypeptide, the attachment site can be the native N-terminal amino acid of the longer polypeptide, or the N-terminal amino acid of the longer polypeptide can be mutated, as described herein. 
     An Fc polypeptide that is modified to comprise one or more attachment moieties for attaching a heterologous functional moiety may be referred to herein as a “parent” Fc polypeptide. 
     Generally, an Fc polypeptide that is suitable for modification to generate a subject modified Fc polypeptide is an Fc polypeptide that can bind an Fc receptor. 
     In some cases, an Fc polypeptide that is suitable for modification to generate a subject modified Fc polypeptide comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to a contiguous stretch of at least about 200 amino acids (e.g., 212 amino acids, 227 amino acids, 234 amino acids, or 238 amino acids; or from about 200 amino acids (aa) to about 210 aa, from about 210 aa to about 220 aa, from about 220 aa to about 225 aa, from about 225 aa to about 230 aa, from about 230 aa to about 240 aa, from about 240 aa to about 250 aa, or from about 250 aa to about 276 aa) of one of the amino acid sequences depicted in  FIGS. 10A-C . 
     For example, in some cases, an Fc polypeptide that is suitable for modification to generate a subject modified Fc polypeptide comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to a contiguous stretch of at least about 200 amino acids (from about 200 aa to about 210 aa, from about 210 aa to about 220 aa, from about 220 aa to about 225 aa, or from about 225 aa to about 227 aa) of the IgG1 Fc amino acid sequence set forth in SEQ ID NO:3 and depicted in  FIG. 10A . 
     As another example, in some cases, an Fc polypeptide that is suitable for modification to generate a subject modified Fc polypeptide comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to a contiguous stretch of at least about 200 amino acids (from about 200 aa to about 210 aa, from about 210 aa to about 220 aa, from about 220 aa to about 225 aa, or from about 225 aa to about 227 aa) of amino acids 99-325 of the IgG2 Fc amino acid sequence set forth in SEQ ID NO:4 and depicted in  FIG. 10A . 
     As another example, in some cases, an Fc polypeptide that is suitable for modification to generate a subject modified Fc polypeptide comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to a contiguous stretch of at least about 200 amino acids (from about 200 aa to about 210 aa, from about 210 aa to about 220 aa, from about 220 aa to about 225 aa, from about 225 aa to about 230 aa, or from about 230 aa to about 238 aa) of amino acids 19-246 of the IgG3 Fc amino acid sequence set forth in SEQ ID NO:5 and depicted in  FIG. 10A . 
     As another example, in some cases, an Fc polypeptide that is suitable for modification to generate a subject modified Fc polypeptide comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to a contiguous stretch of at least about 200 amino acids (from about 200 aa to about 210 aa, from about 210 aa to about 220 aa, from about 220 aa to about 222 aa) of amino acids 162-383 of the IgD Fc amino acid sequence set forth in SEQ ID NO:6 and depicted in  FIG. 10B . 
     As another example, in some cases, an Fc polypeptide that is suitable for modification to generate a subject modified Fc polypeptide comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to a contiguous stretch of at least about 200 amino acids (from about 200 aa to about 210 aa, from about 210 aa to about 220 aa, from about 220 aa to about 225 aa, from about 225 aa to about 230 aa, from about 230 aa to about 240 aa, from about 240 aa to about 250 aa, or from about 250 aa to about 276 aa) of the IgM Fc amino acid sequence set forth in SEQ ID NO:7 and depicted in  FIG. 10B . 
     As another example, in some cases, an Fc polypeptide that is suitable for modification to generate a subject modified Fc polypeptide comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to a contiguous stretch of at least about 200 amino acids (from about 200 aa to about 210 aa, from about 210 aa to about 220 aa, from about 220 aa to about 225 aa, from about 225 aa to about 230 aa, or from about 230 aa to about 234 aa) of amino acids 120-353 of the IgA Fc amino acid sequence set forth in SEQ ID NO:8 and depicted in  FIG. 10C . 
     As another example, in some cases, an Fc polypeptide that is suitable for modification to generate a subject modified Fc polypeptide comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to a contiguous stretch of at least about 200 amino acids (from about 200 aa to about 210 aa, or from about 210 aa to about 212 aa) of amino acids 6-222 of the IgE Fc amino acid sequence set forth in SEQ ID NO:9 and depicted in  FIG. 10C . 
     As another example, in some cases, an Fc polypeptide that is suitable for modification to generate a subject modified Fc polypeptide comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to a contiguous stretch of at least about 200 amino acids (from about 200 aa to about 210 aa, or from about 210 aa to about 228 aa) of amino acids 100-327 of the IgG4 Fc amino acid sequence set forth in SEQ ID NO:10 and depicted in  FIG. 10C . 
     Additional Sequences 
     In some cases, the parent Fc polypeptide is part of a larger polypeptide, e.g., the parent polypeptide comprising the parent Fc polypeptide, and also comprises additional (non-Fc) amino acid sequences. 
     In some cases, the parent Fc polypeptide includes one or more of: an antigen-binding region; a secretion signal sequence; a hinge sequence; a non-immunoglobulin sequence; and the like. 
     Antigen-Binding Region 
     As noted above, in some cases, the Fc polypeptide part of an immunoglobulin polypeptide comprising an antigen-binding domain and the Fc polypeptide. In these embodiments, the attachment site is at the N-terminus of the immunoglobulin polypeptide. Thus, for example the N-terminal amino acid can be the native N-terminal amino acid, or the native N-terminal amino acid can be mutated, as described herein. 
     An antigen-binding region can include VH and/or VL regions. VH and VL sequences are known in the art. An antigen-binding region can be an antibody fragment that retain specific binding to antigen, including, but not limited to, Fab, Fab′, F(ab′) 2 , Fv, scFv, and Fd fragments. An antigen-binding region can be a single-chain Fv (scFv). An antigen-binding region can be a diabody. 
     “Fv” is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the V H -V L  dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. 
     The “Fab” fragment also contains the constant domain of the light chain and the first constant domain (CH 1 ) of the heavy chain. Fab fragments differ from Fab′ fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH 1  domain including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab) 2  antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. 
     The “light chains” of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. 
     “Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). 
     The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993). 
     As used herein, the term “CDR” or “complementarity determining region” is intended to mean the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991); by Chothia et al., J. Mol. Biol. 196:901-917 (1987); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); and in “Antibody Engineering” (Springer Lab Manuals), Roland Kontermann and Stefan Duebel (2001), Chapter 21: “Protein sequence analysis and structure analysis of antibody variable domains” by Andrew Martin, pages 422-442, where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of any of these definitions to refer to a CDR of an antibody or grafted antibodies or variants thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues which encompass the CDRs as defined by the above-cited Kabat, Chothia, and MacCallum references are set forth below in Table 1 as a comparison. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 CDR Definitions 
               
            
           
           
               
               
               
               
            
               
                   
                 Kabat 1   
                 Chothia 2   
                 MacCallum 3   
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 V H  CDR1 
                 31-35 
                 26-32 
                 30-35 
               
               
                   
                 V H  CDR2 
                 50-65 
                 53-55 
                 47-58 
               
               
                   
                 V H  CDR3 
                 95-102 
                 96-101 
                 93-101 
               
               
                   
                 V L  CDR1 
                 24-34 
                 26-32 
                 30-36 
               
               
                   
                 V L  CDR2 
                 50-56 
                 50-52 
                 46-55 
               
               
                   
                 V L  CDR3 
                 89-97 
                 91-96 
                 89-96 
               
               
                   
                   
               
               
                   
                   1 Residue numbering follows the nomenclature of Kabat et al., supra 
               
               
                   
                   2 Residue numbering follows the nomenclature of Chothia et al., supra 
               
               
                   
                   3 Residue numbering follows the nomenclature of MacCallum et al., supra 
               
            
           
         
       
     
     As used herein, the term “framework” when used in reference to an antibody variable region is intended to mean all amino acid residues outside the CDR regions within the variable region of an antibody. A variable region framework is generally a discontinuous amino acid sequence between about 100-120 amino acids in length but is intended to reference only those amino acids outside of the CDRs. As used herein, the term “framework region” is intended to mean each domain of the framework that is separated by the CDRs. 
     Hinge Region 
     In some cases, the parent Fc polypeptide includes a hinge region, e.g., an immunoglobulin heavy chain hinge region, or other suitable spacer (also referred to herein as a “linker”) Immunoglobulin hinge region amino acid sequences are known in the art; see, e.g., Tan et al. (1990)  Proc. Natl. Acad. Sci. USA  87:162; and Huck et al. (1986)  Nucl. Acids Res.  14:1779. As non-limiting examples, an immunoglobulin hinge region can include one of the following amino acid sequences: ISAM (as depicted in  FIG. 1C ); CPEPKSCDTPPPCPR (SEQ ID NO:11) (see, e.g., Glaser et al. (2005)  J. Biol. Chem.  280:41494); ELKTPLGDTTHT (SEQ ID NO:12); KSCDKTHTCP (SEQ ID NO:13); KCCVDCP (SEQ ID NO:14); KYGPPCP (SEQ ID NO:15); and the like. 
     A non-immunoglobulin linker peptide can be used. The linker peptide may have any of a variety of amino acid sequences. Proteins can be joined by a spacer peptide, generally of a flexible nature, although other chemical linkages are not excluded. A linker can be a peptide of between about 6 and about 40 amino acids in length, or between about 6 and about 25 amino acids in length. These linkers are generally produced by using synthetic, linker-encoding oligonucleotides to couple the proteins. Peptide linkers with a degree of flexibility will generally be preferred. The linking peptides may have virtually any amino acid sequence, bearing in mind that suitable linkers will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art. 
     Secretion Signal Peptide 
     In some cases, the parent Fc polypeptide includes a secretion signal peptide, a variety of which are known in the art, which secretion signal peptide provides for secretion from a eukaryotic cell or a prokaryotic cell used to synthesize the parent Fc polypeptide. 
     Secretion signals that are suitable for use in bacteria include, but are not limited to, the secretion signal of Braun&#39;s lipoprotein of  E. coli, S. marcescens, E. amylosora, M. morganii,  and  P. mirabilis , the TraT protein of  E. coli  and  Salmonella ; the penicillinase (PenP) protein of  B. licheniformis  and  B. cereus  and  S. aureus ; pullulanase proteins of  Klebsiella pneumoniae  and  Klebsiella aerogenese; E. coli  lipoproteins 1pp-28, Pal, Rp1A, Rp1B, OsmB, NIpB, and Orl17; chitobiase protein of  V. harseyi ; the β-1,4-endoglucanase protein of  Pseudomonas solanacearum , the Pal and Pcp proteins of  H. influenzae ; the OprI protein of  P. aeruginosa ; the MalX and AmiA proteins of  S. pneumoniae ; the 34 kda antigen and TpmA protein of  Treponema pallidum ; the P37 protein of  Mycoplasma hyorhinis ; the neutral protease of  Bacillus amyloliquefaciens ; and the 17 kda antigen of  Rickettsia rickettsii . Secretion signal sequences suitable for use in yeast are known in the art, and can be used. See, e.g., U.S. Pat. No. 5,712,113. 
     Secretion signals that are suitable for use in eukaryotic cells, e.g., mammalian cells, are known in the art. See, e.g., Notwehr and Gordon (1990)  Bioessays  12:479; Choo et al. (2005)  BMC Bioinformatics  6:249; Nielsen et al. (1997)  Protein Engineering  10:1. A non-limiting example of a secretion signal sequence suitable for use in a mammalian cell includes, e.g., an IL2 signal sequence (e.g., MYRMQLLSCIALSLALVTNS (SEQ ID NO:16); as depicted in  FIG. 1C ). Other examples include, e.g., MGVKVLFALICIAVAEA (SEQ ID NO:17); MKWVTFISLLFLFSSAYS (SEQ ID NO:18); MAFLWLLSCWALLGTTFG (SEQ ID NO:19); and MNLLLILTFVAAAVA (SEQ ID NO:20). 
     Attachment Site 
     As noted above, a subject modified Fc polypeptide comprises one or more attachment moieties for attaching a heterologous functional moiety. In some embodiments, the attachment moiety is attached via the N-terminus of the Fc polypeptide. In other embodiments, the attachment moiety is attached via the side chain of a Lys residue at or near (e.g., within 1-5 amino acids of) the N-terminus of the Fc polypeptide. In other embodiments, the attachment moiety is attached via the side chain of a Lys residue at or near (e.g., within 1-5 amino acids of) the N-terminus of the Fc polypeptide, where the attachment is substantially selective for the Lys residue at or near the N-terminus of the Fc polypeptide (e.g., only one Lys residue, i.e., the Lys residue at or near the N-terminus of the Fc polypeptide, is modified with the attachment moiety). In other embodiments, the attachment moiety is attached via the side chain of a Lys residue at any position within the Fc polypeptide. Where the Fc polypeptide is part of a longer polypeptide, the attachment moiety is attached via the N-terminus of the longer polypeptide. For simplicity, the discussion below refers to the “N-terminal amino acid of the Fc polypeptide”; where the Fc polypeptide is part of a longer polypeptide, the “N-terminal amino acid of the Fc polypeptide” should be understood to refer to the N-terminal amino acid of the longer polypeptide. The N-terminal amino acid of the Fc polypeptide may be modified to install any suitable attachment moiety. In some cases, the amino acid sequence of an Fc polypeptide can be altered to include, at or near the N-terminus of the Fc polypeptide (e.g., at the first, second, or third N-terminal position), a positively charged and/or basic amino acid residue, including but not limited to, arginine or lysine. In other cases, the native N-terminal sequence of the Fc polypeptide is retained and attached to the attachment moiety. In certain cases, the amino acid sequence of an Fc polypeptide can be altered (e.g., by mutation) to include, at or near the N-terminus of the Fc polypeptide (e.g., at either the first or second position) an amino acid sequence that is selected to undergo an N-terminal modification reaction to install an attachment moiety. In some cases, one or more of the three N-terminal amino acid residues of an Fc polypeptide of interest are mutated to include one or more mutated amino acid residues that are desirable for modulating the installation of an attachment moiety. Any suitable mutated residues may be included at or near the N-terminal of an Fc polypeptide of interest. In other cases, the native Fc polypeptide N-terminal sequence includes desirable amino acid residues (e.g., any one of the mutated N-terminal sequences described herein) for installation of an attachment moiety. 
     In some embodiments, the N-terminus of the Fc polypeptide includes a three-residue sequence: Xaa 1 -Xaa 2 -Xaa 3 , where Xaa 1  is alanine, glycine, aspartic acid, glutamic acid, asparagine, cysteine, arginine, threonine, tyrosine, leucine, or serine; wherein Xaa 2  and Xaa 3  are independently amino acid residues and at least one of Xaa 2  and Xaa 3  is a basic amino acid residue (e.g., lysine, arginine, or histidine). In certain embodiments, Xaa 1  is alanine or glutamic acid. In some cases, each of Xaa 2  and Xaa 3  is independently a basic amino acid (e.g., Lys, His, or Arg). In some cases, only one of Xaa 2  and Xaa 3  is a basic amino acid (e.g., Lys, His, or Arg). In some cases, both Xaa 2  and Xaa 3  are a basic amino acid (e.g., Lys, His, or Arg). In some cases, both of Xaa 2  and Xaa 3  are independently basic amino acid residues (e.g., Lys-Lys, Arg-Arg, Lys-Arg, Arg-Lys, Arg-His, Lys-His, His-His, His-Lys or His-Arg). Where both of Xaa 2  and Xaa 3  are independently basic amino acid residues (e.g., Lys-Lys, Arg-Arg, Lys-Arg, Arg-Lys, Arg-His, Lys-His, His-His, His-Lys or His-Arg), Xaa 1  is Ala. In some embodiments, one, two, or all three of Xaa 1 , Xaa 2 , and Xaa 3  is/are an acidic amino acid (e.g., Glu or Asp). For example, in some cases one of Xaa 1 , Xaa 2 , and Xaa 3  is an acidic amino acid (e.g., Glu or Asp); in other cases, two of Xaa 1 , Xaa 2 , and Xaa 3  are acidic amino acids (e.g., Glu or Asp); and in other instances, each of Xaa 1 , Xaa 2 , and Xaa 3  is an acidic amino acid (e.g., Glu or Asp), In some instances, the Fc polypeptide includes an N-terminal sequence selected from Xaa 1 -Lys-Xaa 3 , Xaa 1 -Arg-Xaa 3 , Xaa 1 -Xaa 2 -Lys and Xaa 1 -Xaa 2 -Arg, wherein Xaa 1  is Ala, and Xaa 2  and Xaa 3  are independently an amino acid residue. In some embodiments, the N-terminus of the Fc polypeptide includes an alanine-lysine-threonine (AKT) amino acid sequence, where the amino group of the terminus of the AKT sequence can be modified to install the attachment moiety. In some embodiments, the N-terminus of the Fc polypeptide includes an AKT amino acid sequence, where the epsilon amino group of the Lys can be modified to install the attachment moiety. In some embodiments, the N-terminus of the Fc polypeptide is mutated to include at least an alanine-lysine (AK) amino acid sequence. In certain embodiments, the N-terminus of the Fc polypeptide is mutated at least at the first position to include an alanine, glycine, aspartic acid, glutamic acid, asparagine, cysteine, arginine, threonine, tyrosine, leucine, or serine residue. In certain embodiments, the amino group of the N-terminus is modified to include a ketone or an aldehyde group via a transamination reaction. 
     As noted above, in some cases, one or both of Xaa 2  and Xaa 3  are independently basic amino acid residues (e.g., Lys-Lys, Arg-Arg, Lys-Arg, Arg-Lys, Arg-His, Lys-His, His-His, His-Lys or His-Arg), Xaa 1  is Ala. Where one or both of Xaa 2  and Xaa 3  are independently basic amino acid residues (e.g., Lys-Lys, Arg-Arg, Lys-Arg, Arg-Lys, Arg-His, Lys-His, His-His, His-Lys or His-Arg), Xaa 1  is Ala; and the transamination reaction to convert the N-terminal amino group to a ketone or aldehyde group is carried out with pyridoxal 5′-phosphate (PLP). As noted above, in some cases, one, two, or three of Xaa 1 , Xaa 2 , and Xaa 3  is an acidic amino acid (e.g., Glu or Asp). Where one, two, or three of Xaa 1 , Xaa 2 , and Xaa 3  is an acidic amino acid (e.g., Glu or Asp), the transamination reaction to convert the N-terminal amino group to a ketone or aldehyde group is carried out with Rapoport&#39;s Salt (e.g., N-methylpyridinium-4-carboxaldehyde or 4-Formyl-1-methylpyridinium salt). 
     Methods of Generating Modified Fc Polypeptides 
     The subject modified Fc domains may be prepared by using a variety of methods, the chemistry and steps of which are known to one of ordinary skill in the art. One exemplary method is illustrated in  FIG. 2 ; however there are many other routes that would lead to the same molecule. In general terms, in the first step of the synthesis the N-terminal of a Fc domain of interest is mutated to include a sequence that is highly reactive to transamination reaction with an aldehyde. The N-terminal sequence of the Fc polypeptide may be selected to provide for a desired efficiency of transamination. For example, the N-terminal of the Fc polypeptide may be mutated to include alanine-lysine-threonine (AKT-Fc). 
     In the second step of the synthesis, the amino terminal of the Fc polypeptide undergoes a transamination reaction with an aldehyde reagent to convert the N-terminal amino group to a ketone or aldehyde group. Any convenient aldehyde reagent may be utilized in the subject methods. Reagents of interest include, but are not limited to, pyridoxal 5′-phosphate (PLP), Rapoport&#39;s Salt (e.g., N-methylpyridinium-4-carboxaldehyde or 4-Formyl-1-methylpyridinium salt), and those reagents described by Francis et al.,  J. Am. Chem. Soc.  2010, 132, 16812-16817 and Francis et al.  Angew. Chem. Int. Ed.  2006, 45, 5307-5311, the disclosures of which are herein incorporated by reference in their entirety. 
     In an optional third step of the synthesis, the aldehyde or ketone group of the N-terminal residue of the modified Fc polypeptide may be covalently crosslinked with any convenient functional group and utilizing any convenient chemistry to install a further attachment moiety on the modified Fc polypeptide. In some embodiments, the aldehyde or ketone group is further derivatized to install a further attachment moiety, via an aldol reaction with a second aldehyde or ketone group, or via a coupling reaction with a hydroxylamino, a hydrazine or a hydrazido group. In such cases, the further attachment moiety may include an aryl amino group, such as an aniline group or an aminophenol group. 
     In certain embodiments, the aldehyde or ketone containing modified Fc polypeptide may be contacted with a bifunctional moiety including an aniline group linked to an aldehyde or ketone reactive group. In certain cases, the bifunctional moiety is described by the formula: 
                         
where R is one or more optional aryl substituents, R′ and R″ are each independently H, an alkyl or an aryl, L 6  is an optional linker and Y 1  is an aldehyde or ketone-reactive functional group. In certain embodiments, R′ and R″ are each H.
 
     In certain embodiments, Y 1  is —CONHNH 2 , —O—NH 2  or —NHNH 2 . In certain embodiments, the bifunctional moiety is described by the formula: 
                         
where R′ and R″ are each independently H, an alkyl or an aryl, L 6  is an optional linker and Y 1  is —CONHNH 2 , —O—NH 2  or —NHNH 2 . In certain embodiments, R′ and R″ are each H.
 
Fc Conjugates
 
     The present disclosure provides Fc conjugates; compositions comprising the conjugates; and methods of making the conjugates. An Fc conjugate of the present disclosure comprises a subject modified Fc polypeptide and a covalently linked heterologous functional moiety. 
     A subject Fc conjugate retains the ability to bind one or more Fc ligands. For example, a subject Fc conjugate retains at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 99%, or 100%, of the ability of the parent Fc polypeptide to bind an Fc ligand. For example, in some embodiments, a subject Fc conjugate retains the ability to bind an Fc receptor. In other embodiments, a subject Fc conjugate retains the ability to bind C1q. 
     A subject Fc conjugate retains at least one function of a parent Fc polypeptide. For example, a subject Fc conjugate retains at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, about 99%, or 100%, of the function of a parent Fc polypeptide. For example, in some cases, a subject Fc conjugate retains activity in mediating or inducing an immune response to a target cell. For example, in some cases, a subject Fc conjugate retains activity in inducing cytolytic activity against a target cell. In other cases, a subject Fc conjugate retains activity in inducing phagocytosis of a target. In other instances, a subject Fc conjugate retains the ability to bind to FcRn. 
     In some embodiments, a subject Fc conjugate exhibits enhanced pharmacokinetic properties, relative to a parent Fc polypeptide used to generate the Fc conjugate. 
     In some embodiments, a subject Fc conjugate exhibits an increased serum half-life, compared to the serum half-life of the parent Fc polypeptide used to generate the Fc conjugate. For example, in some embodiments, a subject Fc conjugate has a serum half-life that is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 75%, at least about 100% (or 2-fold), at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold, higher than the serum half-life of the parent Fc polypeptide used to generate the Fc conjugate. In some of these embodiments, a subject Fc conjugate exhibits an increased serum half-life by virtue of its ability to bind FcRn. 
     In some embodiments, a subject Fc conjugate exhibits an increased serum half-life, compared to the serum half-life (also referred to herein as “circulating half-life”) of the heterologous functional moiety when not present in the Fc conjugate (e.g., compared to the free heterologous functional moiety not conjugated to an Fc polypeptide). For example, in some embodiments, a subject Fc conjugate has a serum half-life that is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 75%, at least about 100% (or 2-fold), at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold, higher than the serum half-life of the unconjugated heterologous functional moiety. In some of these embodiments, a subject Fc conjugate exhibits an increased serum half-life by virtue of its ability to bind FcRn. 
     The present disclosure provides a method of increasing the serum half-life of a molecule (e.g., a functional moiety, as described herein). The method comprises conjugating the molecule to a modified Fc polypeptide of the present disclosure, thereby generating an Fc conjugate. The serum half-life of the Fc conjugate is greater than the serum half-life of the unconjugated molecule. 
     In some embodiments, the Fc conjugate is described by the structure of formula (VI): 
                         
where R 1  and Z 1  are as described above in formula (II), X is O or NH, and R 2  is L 2 -Z 2 , where L 2  is an optional linker and Z 2  is a heterologous functional moiety (e.g., as described below).
 
     In some embodiments, in formula (VI), X is O such that the Fc polypeptide is crosslinked to the heterologous functional moiety via an oxime. In some embodiments, in formula (VI), X is NH such that the Fc polypeptide is crosslinked to the heterologous functional moiety via a hydrazone. 
     In some embodiments, the Fc conjugate is described by the structure of one of formulas (VII) and (VIII): 
                         
where R 1  and Z 1  are as described above in formula (II), X is O or NH, R 3  is L 3 -Z 2 , R 4  is L 4 -Z 2 , where L 3  and L 4  are optional linkers and Z 2  is a heterologous functional moiety (e.g., as described below).
 
     In some embodiments, the Fc conjugate includes a linker having a structure derived from oxidative coupling of an aniline group and an aminophenol group (or alternative convenient oxidative coupling group). A number of linker structures are envisaged as products of the oxidative coupling of a modified Fc polypeptide (e.g., an aniline-containing modified Fc polypeptide) and a suitable heterologous functional moiety (e.g., aminophenol containing heterologous functional moiety). In some cases, the heterologous functional moiety includes a group suitable for oxidative coupling to the modified Fc polypeptide, including but limited to, an aminophenol, a 2-methoxyphenol, an aniline, an azidophenol, and a phenylene diamine. Without wishing to be bound by theory, in certain embodiments, in formulas (VI), (VII) or (VIII), linker L 2 , L 3  or L 4  includes a group described by one of the following structures: 
     
       
         
         
             
             
         
       
     
     In some embodiments, in formulas (VII) or (VIII), R 3  and R 4  may include one of the following structures: 
                         
where L 5  is an optional linker and Z 2  is a heterologous functional moiety (e.g., as described below). R 3  and R 4  may further include a linker that connects the aniline group to the N-terminal of the Fc polypeptide (e.g., as described herein).
 
     In some embodiments, the Fc conjugate includes one of the following structures: 
                         
where L 5  is an optional linker and Z 2  is a heterologous functional moiety (e.g., as described below). The Fc conjugate may further include a linker that connects the aniline group to the N-terminal of the Fc polypeptide. Any convenient linker may be utilized to connect the aniline group and the Fc polypeptide.
 
     In some embodiments, the Fc conjugate is described by one of the following structures: 
     
       
         
         
             
             
         
       
     
     where L 1  and L 5  are optional linkers, Z 2  is a heterologous functional moiety, X is O or NH, R 1  is as described above for formula (II), and Z 1  is an Fc polypeptide. In certain embodiments, Z 1  includes N-terminal lysine-threonine residues, and R′ is the amino acid sidechain of alanine. 
     Methods of Generating Fc Conjugates 
     The subject Fc conjugates may be prepared by conjugating the subject modified Fc polypeptides with a heterologous functional moiety by using a variety of methods, the chemistry and steps of which are known to one of ordinary skill in the art. Methods and chemistries of interest that may be adapted for use in preparing the subject Fc conjugates include those described in Hermanson, “Bioconjugate Techniques” 2nd Edition, Academic Press, 2008. 
     In some embodiments, the method includes conjugating a modified Fc polypeptide that includes an aldehyde or a ketone group with a heterologous functional moiety that includes a hydroxylamino group, or a hydrazine or hydrazide group to produce oxime or hydrazone cross-links, respectively. Any convenient methods of conjugating to aldehyde or ketone groups to produce an oxime or hydrazone linkage may be utilized. 
     In other embodiments, the method includes chemoselectively conjugating a modified Fc polypeptide that includes an aniline group with a heterologous functional moiety that includes an aminophenol group (or alternative convenient oxidative coupling group) to oxidatively couple the aniline and aminophenol groups. Any convenient methods of oxidatively coupling, and coupling reagents or functional groups for achieving the same, may be adapted for use in the subject methods, including but not limited to, those methods and reagents described by Geoghegan et al.,  Bioconj. Chem.  1992, 3, 138-146; and Liu et al.,  J. Am. Chem. Soc.  2006, 128, 15228-15235. In certain embodiments, the conjugation is mediated by sodium periodate or ferricyanide (K 3 Fe(CN) 6 ). 
     Heterologous Functional Moieties 
     Suitable heterologous functional moieties can be of any of a variety of classes of molecules including, e.g., polypeptides, nucleic acids, DNA aptamers, peptide aptamers, small molecules, nanoparticles, drug delivery vehicles, ligands, polymers, liposomes, and oligosaccharides. 
     A heterologous functional moiety can provide for a variety of functionalities, including, but not limited to, pro-angiogenic activity; anti-angiogenic activity; cytotoxicity; reduction of IgE production; specific modulation of gene expression; cell binding activity; growth factor activity; and the like. 
     Polypeptides 
     In some cases, the heterologous functional moiety is a polypeptide, where suitable polypeptides can have a length of from about 3 amino acids to 1000 amino acids (or longer than 1000 amino acids), e.g., from about 3 amino acids (aa) to about 5 aa, from about 5 aa to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 25 aa, from about 25 aa to about 50 aa, from about 50 aa to about 75 aa, from about 75 aa to about 100 aa, from about 100 aa to about 250 aa, from about 250 aa to about 500 aa, from about 500 aa to about 750 aa, or from about 750 aa to about 1000 aa (or more than 1000 aa). 
     The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term “polypeptide” includes polypeptides comprising one or more of a fatty acid moiety, a lipid moiety, a sugar moiety, and a carbohydrate moiety. The term “polypeptides” includes post-translationally modified polypeptides. 
     Suitable polypeptides include, e.g., an enzyme, a hormone, a cytokine, a binding protein, a toxin, and the like. Exemplary therapeutic peptides include, but are not limited to, hormones, growth factors, cytokines, chemokines, binding peptides, blocking peptides, toxins, angiogenic factors, anti-angiogenic factors, antibiotics, anti-cancer peptides, anti-viral peptides, pharmaceutical peptides, enzymes, agonists, antagonists, hematopoietic agents such as erythropoietin, and the like. 
     In some cases, the polypeptide heterologous functional moiety comprises one or more naturally occurring non-genetically encoded L-amino acids, synthetic L-amino acids or D-enantiomers of an amino acid. For example, a Peptide can comprise only D-amino acids. For example, a Peptide can comprise one or more of the following residues: hydroxyproline, β-alanine, o-aminobenzoic acid, m-aminobenzoic acid, p-aminobenzoic acid, m-aminomethylbenzoic acid, 2,3-diaminopropionic acid, α-aminoisobutyric acid, N-methylglycine (sarcosine), ornithine, citrulline, t-butylalanine, t-butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine, norleucine, naphthylalanine, pyridylalanine 3-benzothienyl alanine, 4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, β-2-thienylalanine, methionine sulfoxide, homoarginine, N-acetyl lysine, 2,4-diamino butyric acid, rho-aminophenylalanine, N-methylvaline, homocysteine, homoserine, ε-amino hexanoic acid, ω-aminohexanoic acid, ω-aminoheptanoic acid, ω-aminooctanoic acid, ω-aminodecanoic acid, ω-aminotetradecanoic acid, cyclohexylalanine, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, δ-amino valeric acid, and 2,3-diaminobutyric acid. 
     In some cases, the heterologous functional moiety is a mimetic. Mimetics are peptide-containing molecules that mimic elements of protein secondary structure. See, for example, Johnson et al., “Peptide Turn Mimetics” in “Biotechnology and Pharmacy”, Pezzuto et al., Eds., Chapman and Hall, New York (1993). The rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains so as to facilitate molecular interactions, such as those of antibody and antigen. A peptide mimetic is expected to permit molecular interactions similar to the natural molecule. These principles may be used to engineer second generation molecules having many of the natural properties of a naturally-occurring peptide, but with altered or improved characteristics, such as increased absorption across the stomach or intestine and/or improved stability or activity in vivo. 
     Drugs 
     Drugs that can be conjugated to a parent Fc to generate a subject drug-Fc conjugate include, but are not limited to, anthracycline, an epipodophyllotoxin, a taxane, an antimetabolite, an alkylating agent, an antibiotic, a COX-2 inhibitor, an antimitotic, an antiangiogenic agent, a proapoptotic agent, doxorubicin, methotrexate, CPT-11, 5-fluorouracil, bleomycin, busulfan, carboplatin, chlorambucil, cisplatin (CDDP), cyclophosphamide, dactinomycin, daunorubicin, an estrogen receptor binding agent, etoposide (VP16), farnesyl-protein transferase inhibitor, gemcitabine, ifosfamide, mechlorethamine, melphalan, mitomycin, navelbine, nitrosourea, plicamycin, procarbazine, raloxifene, tamoxifen, paclitaxel, temazolomide, transplatinum, vinblastine, vincristine, a camptothecan, a nitrogen mustard, an alkyl sulfonate, a triazene, a folic acid analog, a pyrimidine analog, a purine analog, a platinum coordination complex, and a hormone. 
     Drugs that can be conjugated to a parent Fc to generate a subject drug-Fc conjugate include, but are not limited to, aplidin, azaribine, anastrozole, azacytidine, bleomycin, bortezomib, bryostatin-1, busulfan, calicheamycin, camptothecin, 10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin, irinotecan (CPT-11), SN-38, carboplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, daunomycin glucuronide, daunorubicin, dexamethasone, diethylstilbestrol, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, ethinyl estradiol, estramustine, etoposide, etoposide glucuronide, etoposide phosphate, floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, fluorouracil, fluoxymesterone, gemcitabine, hydroxyprogesterone caproate, hydroxyurea, idarubicin, ifosfamide, L-asparaginase, leucovorin, lomustine, mechlorethamine, medroprogesterone acetate, megestrol acetate, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, phenyl butyrate, prednisone, procarbazine, paclitaxel, pentostatin, PSI-341, semustine streptozocin, tamoxifen, taxanes, taxol, testosterone propionate, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, velcade, vinblastine, vinorelbine, vincristine, ricin, abrin, ribonuclease, onconase, rapLR1, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin,  Pseudomonas  exotoxin, and  Pseudomonas  endotoxin. 
     Anti-Angiogenic Agents 
     Exemplary anti-angiogenic agents suitable for conjugation to a parent Fc to generate a subject Fc conjugate include, but are not limited to, angiostatin, baculostatin, canstatin, maspin, anti-VEGF antibodies or peptides, anti-placental growth factor antibodies or peptides, anti-Flk-1 antibodies, anti-Flt-1 antibodies or peptides, laminin peptides, fibronectin peptides, plasminogen activator inhibitors, tissue metalloproteinase inhibitors, interferons, interleukin 12, IP-10, Gro-β, thrombospondin, 2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole, CM101, Marimastat, pentosan polysulphate, angiopoietin 2, interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment, Linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4, and minocycline. 
     Nucleic Acids 
     In some cases, the heterologous functional moiety is a nucleic acid. The terms “DNA”, “nucleic acid”, “nucleic acid molecule”, “polynucleotide” and the like are used interchangeably herein to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include linear and circular nucleic acids, messenger RNA (mRNA), complementary DNA (cDNA), recombinant polynucleotides, and the like. 
     Suitable nucleic acids include a nucleic acid comprising a nucleotide sequence encoding a polypeptide; a nucleic acid comprising a nucleotide sequence encoding an RNA; an antisense RNA; a siRNA; a microRNA; an RNA or a DNA aptamer; a decoy RNA; and the like. 
     Aptamers 
     In some cases, the heterologous functional moiety is an aptamer. See, e.g., Keefe et al. (2010)  Nat. Rev. Drug Discov.  9:537. An aptamer binds to a non-nucleic acid ligand, such as a small organic molecule or protein, e.g., a transcription or translation factor, and subsequently modifies (e.g., inhibits) activity. An aptamer can fold into a specific structure that directs the recognition of the targeted binding site on the non-nucleic acid ligand. 
     Aptamers may be prepared by any known method, including synthetic, recombinant, and purification methods, and may be used alone or in combination with other ligands specific for the same target. In general, a minimum of approximately 3 nucleotides, e.g., at least 5 nucleotides, are necessary to effect specific binding. Aptamers of sequences shorter than 10 bases may be used; alternatively, aptamers of 10, 20, 30 or 40 nucleotides, or longer than 40 nucleotides may be used. For example, an aptamer can have a length of from about 10 nucleotides (nt) to about 20 nt, from about 20 nt to about 30 nt, from about 30 nt to about 40 nt, to about 50 nt, from about 50 nt to about 60 nt, from about 60 nt to about 70 nt, from about 70 nt to about 80 nt, from about 80 nt to about 90 nt, or from about 90 to about 100 nt. 
     Aptamers need to contain the sequence that confers binding specificity, but may be extended with flanking regions and otherwise derivatized. In preferred embodiments, the binding sequences of aptamers may be flanked by primer-binding sequences, facilitating the amplification of the aptamers by PCR or other amplification techniques. In a further embodiment, the flanking sequence may comprise a specific sequence that preferentially recognizes or binds a moiety to enhance the immobilization of the aptamer to a substrate. 
     Aptamers may be isolated, sequenced, and/or amplified or synthesized as conventional DNA or RNA molecules. Alternatively, aptamers of interest may comprise modified oligomers. Any of the hydroxyl groups ordinarily present in aptamers may be replaced by phosphonate groups, phosphate groups, protected by a standard protecting group, or activated to prepare additional linkages to other nucleotides, or may be conjugated to solid supports. One or more phosphodiester linkages may be replaced by alternative linking groups, such as P(O)O replaced by P(O)S, P(O)NR 2 , P(O)R, P(O)OR′, CO, or CNR 2 , wherein R is H or alkyl (1-20C) and R′ is alkyl (1-20C); in addition, this group may be attached to adjacent nucleotides through O or S, Not all linkages in an oligomer need to be identical. 
     Methods for preparation and screening of aptamers that bind to particular targets of interest are well known, for example U.S. Pat. No. 5,475,096 and U.S. Pat. No. 5,270,163. The technique generally involves selection from a mixture of candidate aptamers and step-wise iterations of binding, separation of bound from unbound aptamers and amplification. Because only a small number of sequences (possibly only one molecule of aptamer) corresponding to the highest affinity aptamers exist in the mixture, it is generally desirable to set the partitioning criteria so that a significant amount of aptamers in the mixture (approximately 5-50%) is retained during separation. Each cycle results in an enrichment of aptamers with high affinity for the target. Repetition for between three to six selection and amplification cycles may be used to generate aptamers that bind with high affinity and specificity to the target. 
     In some embodiments, the aptamer is an anti-VEGF aptamer. For anti-VEGF aptamers, see, e.g., Ng et al. (2006)  Nature Reviews Drug Discovery  5:123; and U.S. Pat. Nos. 6,426,335; 6,168,778; 6,147,204; 6,051,698; and 6,011,020. See also, U.S. Patent Publication Nos. 2012/0207671 and 2012/0148584. 
     Nanoparticles 
     In some cases, the heterologous functional moiety is a nanoparticle. 
     Drug Delivery Vehicles 
     In some cases, the heterologous functional moiety is a drug delivery vehicle, where suitable drug delivery vehicles include, but are not limited to, a viral capsid, a liposome (e.g., a lipid vesicle), a dendrimer, a biodegradable particle, and the like. 
     Ligands 
     In some cases, the heterologous functional moiety is a small molecule targeting group, e.g., a ligand for a cell-surface receptor. Suitable ligands include, e.g., neurotransmitters, hormones, ligands that bind cancer biomarkers, kinase inhibitors, integrin-binding ligands, and the like. A ligand can be a naturally-occurring ligand, or a synthetic ligand. 
     Polymers 
     In some cases, the heterologous functional moiety is a polymer (e.g., a polymer other than a polypeptide or a nucleic acid). Suitable polymers include natural polymers, semisynthetic polymers, and synthetic polymers. Suitable synthetic polymers include, but are not limited to, polymers or copolymers derived from polydioxane, polyphosphazene, polysulphone resins, poly(acrylic acid), poly(acrylic acid) butyl ester, poly(ethylene glycol), poly(propylene), polyurethane resins, poly(methacrylic acid), poly(methacrylic acid)-methyl ester, poly(methacrylic acid)-n butyl ester, poly(methacrylic acid)-t butyl ester, polytetrafluoroethylene, polyperfluoropropylene, poly N-vinyl carbazole, poly(methyl isopropenyl ketone), poly alphamethyl styrene, polyvinylacetate, poly(oxymethylene), poly(ethylene-co-vinyl acetate), a polyurethane, a poly(vinyl alcohol), and polyethylene terephthalate; ethylene vinyl alcohol copolymer (commonly known by the generic name EVOH or by the trade name EVAL); polybutylmethacrylate; poly(hydroxyvalerate); poly(L-lactic acid) or poly(L-lactide); poly(e-caprolactone); poly(lactide-co-glycolide); poly(hydroxybutyrate); poly(hydroxybutyrate-co-valerate); polydioxanone; polyorthoester; polyanhydride; poly(glycolic acid) (PGA); poly(D,L-lactide) (PDLL); poly(L-Lactide)(PLL); copolymers of PGA, poly(D,L-lactic acid) (PDLA), and/or poly(lactic acid) (PLA); poly(glycolic acid-co-trimethylene carbonate); polyphosphoester; polyphosphoester urethane; poly(amino acids); cyanoacrylates; poly(trimethylene carbonate); poly(iminocarbonate); copoly(ether-esters) (e.g., poly(ethylene oxide) (PEO)/PLA); polyalkylene oxalates; polyphosphazenes; polyurethanes; silicones; polyesters; polyolefins; polyisobutylene and ethylene-alphaolefin copolymers; acrylic polymers and copolymers; vinyl halide polymers and copolymers, such as polyvinyl chloride; polyvinyl ethers, such as polyvinyl methyl ether; polyvinylidene halides, such as polyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile; polyvinyl ketones; polyvinyl aromatics, such as polystyrene; polyvinyl esters, such as polyvinyl acetate; copolymers of vinyl monomers with each other and olefins, such as ethylene-methyl methacrylate copolymers, acrylonitrile-styrene copolymers, ABS resins, and ethylene-vinyl acetate copolymers; polyamides, such as Nylon 66 and polycaprolactam; alkyd resins; polycarbonates; polyoxymethylenes; polyimides; polyethers; epoxy resins; polyurethanes; rayon; rayon-triacetate; cellulose; cellulose acetate; cellulose butyrate; cellulose acetate butyrate; cellophane; cellulose nitrate; cellulose propionate; cellulose ethers; amorphous Teflon; and carboxymethyl cellulose. 
     Suitable hydrophobic polymers include poly(L-lactide), poly(glycolide), poly(e-caprolactone), copolymers of lactide and/or glycolide or/and poly(e-caprolactone), hydrophobic peptides or a combination of hydrophobic peptides, polyurethanes. Any hydrophobic polymer that can form a micelle in water is suitable for use as a hydrophobic polymer. Suitable hydrophobic polymers include, e.g., poly(glycolide) or poly(glycolic acid); poly(e-caprolactone); poly(D,L-lactide); poly(L-Lactide); copolymers of these and other polyesters; polyamides; polyanhydrides; polyurethanes; poly(ortho esters); poly(iminocarbonates). In some embodiments, the hydrophobic polymer of the nanoparticle (or microparticle) is poly-L-lactide. 
     Suitable hydrophilic polymers include, but are not limited to, poly(ethylene glycol); poly(vinyl alcohol); polyethers; poly(methacrylic acid); poly(acrylic acid); poly(hydroxyethylmethacrylate) (pHEMA); hyaluronic acid; and hyaluronate. 
     Compositions 
     The present disclosure provides compositions, including pharmaceutical compositions, comprising a subject Fc conjugate. Compositions comprising a subject compound can include one or more of: a salt, e.g., NaCl, MgCl 2 , KCl, MgSO 4 , etc.; a buffering agent, e.g., a Tris buffer, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N-Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; a solubilizing agent; a detergent, e.g., a non-ionic detergent such as Tween-20, etc.; a protease inhibitor; a membrane penetration facilitator; and the like. 
     The present disclosure provides pharmaceutical compositions comprising a subject Fc conjugate. A subject compound can be formulated with one or more pharmaceutically acceptable excipients. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy,” 20th edition, Lippincott, Williams, &amp; Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds., 7 th  ed., Lippincott, Williams, &amp; Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3 rd  ed. Amer. Pharmaceutical Assoc. 
     Utility 
     A modified Fc, or an Fc conjugate, of the present disclosure finds use in a variety of applications. For example, a subject Fc conjugate can be used in various research, diagnostic, and therapeutic applications. 
     Cytotoxicity 
     A subject Fc conjugate is in some embodiments useful for mediating cytotoxicity toward a target cell. A subject Fc conjugate binds to an Fc receptor on a cytotoxic immune cell (e.g., a cytotoxic T cell, or an NK cell), thereby mediating killing of a target cell. 
     Target cells include, but are not limited to, cancer cells. Thus, the present disclosure provides methods of killing, or inhibiting the growth of, a target cancer cell, the method involving contacting a cytotoxic immune effector cell (e.g., a cytotoxic T cell, or an NK cell) with a subject Fc conjugate, such that the Fc conjugate binds to the cytotoxic immune effector cell and, in the presence of a target cancer cell, the cytotoxic immune effector cell mediates killing of the target cell. The heterologous functional moiety on the Fc conjugate can provide additional specificity for a particular target cell and/or can provide for an additional cytotoxic effect. 
     The present disclosure provides a method of treating cancer in an individual having a cancer, the method involving administering to the individual an effective amount of a subject Fc conjugate. The Fc conjugate can be provided in a pharmaceutical composition, together with one or more pharmaceutically acceptable excipients. The Fc conjugate (or pharmaceutical composition comprising the Fc conjugate) can be administered systemically (e.g., intravenously, etc.) or locally (e.g., at or near the site of a cancer cell or tumor). 
     Carcinomas that can be amenable to therapy by a method disclosed herein include, but are not limited to, esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder carcinoma, including transitional cell carcinoma (a malignant neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm&#39;s tumor, cervical carcinoma, uterine carcinoma, testicular carcinoma, osteogenic carcinoma, epithelieal carcinoma, and nasopharyngeal carcinoma. 
     Sarcomas that can be amenable to therapy by a method disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing&#39;s sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas. 
     Other solid tumors that can be amenable to therapy by a method disclosed herein include, but are not limited to, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma. 
     Leukemias that can be amenable to therapy by a method disclosed herein include, but are not limited to, a) chronic myeloproliferative syndromes (neoplastic disorders of multipotential hematopoietic stem cells); b) acute myelogenous leukemias (neoplastic transformation of a multipotential hematopoietic stem cell or a hematopoietic cell of restricted lineage potential; c) chronic lymphocytic leukemias (CLL; clonal proliferation of immunologically immature and functionally incompetent small lymphocytes), including B-cell CLL, T-cell CLL prolymphocytic leukemia, and hairy cell leukemia; and d) acute lymphoblastic leukemias (characterized by accumulation of lymphoblasts). Lymphomas that can be treated using a subject method include, but are not limited to, B-cell lymphomas (e.g., Burkitt&#39;s lymphoma); Hodgkin&#39;s lymphoma; non-Hodgkin&#39;s lymphoma, and the like. 
     Other cancers that can be amenable to treatment according to the methods disclosed herein include atypical meningioma (brain), islet cell carcinoma (pancreas), medullary carcinoma (thyroid), mesenchymoma (intestine), hepatocellular carcinoma (liver), hepatoblastoma (liver), clear cell carcinoma (kidney), and neurofibroma mediastinum. 
     EXAMPLES 
     The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like. 
     Example 1: Preparation and Characterization of Fc Conjugates 
     Fc molecules with added functionality were created by installing synthetic molecules at the N-termini of crystallizable fragment domains (Fc&#39;s) via a chemical modification approach. First, a pyridoxal 5′-phosphate (PLP) mediated N-terminal transamination reaction provided a compatible method for site-selectively installing ketones as reactive handles on Fc domains. High levels of conversion were achieved. For elaboration of the newly installed chemical handles, two different approaches for the ligation of heterologous groups to the Fc domain were used. In the first approach, alpha-effect amines were used to create oxime or hydrazone linkages. In the second approach, the ketone was used as a site to introduce a second reaction handle: an aniline group that can participate in a recently reported oxidative coupling reaction. The oxidative coupling provides a highly efficient ligation strategy requiring very short reaction times (two min or less) at room temperature. 
     Fc domains were functionalized with DNA aptamers. The specificity of the aptamers for binding their cellular targets was demonstrated, as was their ability of the modified Fc domains to bind to complement proteins. 
     Materials and Methods 
     General Experimental Procedures and Materials 
     Unless otherwise noted, all chemicals and solvents were of analytical grade and used as received from commercial sources. Water (dd-H 2 O) used in biological procedures or as the reaction solvent was deionized using a NANOpure purification system (Barnstead, USA). All oligonucleotides were obtained from Integrated DNA Technologies (Coralville, Iowa). All cell culture reagents were obtained from Gibco/Invitrogen Corp. (Carlsbad, Calif.) unless otherwise noted. Cell culture was conducted using standard techniques. Jurkat and Ramos cells were grown in T-25 culture flasks (Corning) in RPMI Medium 1640 supplemented with 10% (v/v) fetal bovine serum (FBS, Omega Scientific) and 1% penicillin/streptomycin (P/S, Sigma). U266 cells were grown in T-25 culture flasks (Corning) in RPMI Medium 1640 supplemented with 15% (v/v) fetal bovine serum (FBS, Omega Scientific) and 1% penicillin/streptomycin (P/S, Sigma). 
     Instrumentation and Sample Analysis 
     Liquid Chromatography-Mass Spectrometry (LC-MS). 
     Fc protein bioconjugates were analyzed using an Agilent 1200 liquid chromatograph (LC; Santa Clara, Calif.) that was connected in-line with an LTQ Orbitrap XL hybrid mass spectrometer equipped with an electrospray ionization source (ESI; Thermo Fisher Scientific, Waltham, Mass.), located in the QB3/Chemistry Mass Spectrometry Facility at UC Berkeley. The LC was equipped with a reversed-phase C 8  column (100 mm×1.0 mm, 5 μm particles, Restek, Bellefonte, Pa.). Solvent A was 0.1% formic acid/99.9% water and solvent B was 0.1% formic acid/99.9% acetonitrile (v/v). For each sample, approximately 200 picomoles of protein analyte was injected onto the column. Following sample injection, analyte trapping was performed for 5 min with 99.5% A. The elution program consisted of a linear gradient from 30% to 95% B over 19.5 min, isocratic conditions at 95% B for 5 min, a linear gradient to 0.5% B over 0.5 min, and then isocratic conditions at 0.5% B for 9.5 min. Solvent (Milli-Q water) blanks were run between samples, and the autosampler injection needle was rinsed with Milli-Q water after each sample injection, to avoid cross-contamination between samples. Mass spectra were recorded in the positive ion mode over the range m/z=450-2000. Raw mass spectra were processed using Xcalibur software (version 2.0.7 SP1, Thermo) and protein charge state distributions were deconvoluted using ProMass software (version 2.5 SR-1, Novatia, Monmouth Junction, N.J.). 
     High Performance Liquid Chromatography (HPLC). 
     HPLC was performed on an Agilent 1100 Series HPLC System (Agilent Technologies, USA). Sample analysis for all HPLC experiments was achieved with an inline diode array detector (DAD). Anion exchange HPLC of Fc-DNA conjugates was accomplished using Biosep-DEAE-PEI column (Phenomenex). 
     Gel Analyses. 
     For protein analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on a Mini-Protean apparatus from Bio-Rad (Hercules, Calif.), following the protocol of Laemmli. 1  The reducing protein electrophoresis samples were heated for 10 minutes at 95° C. in the presence of β-mercaptoethanol to ensure reduction of any disulfide bonds. Gels were run for 40 minutes at 200 V to allow good separation of bands. Commercially available markers (Fisher) were applied to at least one lane of each gel for assignment of apparent molecular masses. Visualization of protein bands was accomplished by staining with Coomassie Brilliant Blue R-250 (Bio-Rad). For fluorescent protein conjugates, visualization was accomplished on a Typhoon 9410 (Amersham Biosciences). 
     Construction of the Plasmids for the Expression of Fc Domains 
     To express the Fc substrates, the pInFuse-hIgG1-Fc2 (InvivoGen, San Diego, Calif.) plasmid, which contained the human IgG1-Fc gene with its introns and an IL2 signal sequence, was used. A short intron was present between each region: one intron was located between the hinge and the CH2 domain and one intron was located between CH2 and CH3 ( FIG. 1B ). The Fc protein expressed from this plasmid was comprised of the CH2 and CH3 domains of the IgG1 heavy chain. Intracellular cleavage of this sequence occurs after Ser20 and leads to the secretion of the protein to extracellular medium ( FIG. 1B ). As in the sequence shown in  FIG. 1C , Ile-Ser-Ala remains at the N-terminus of the secreted Fc protein after the IL2 signal sequence is cleaved. The DNA sequence ATATCGGCC encoding Ile-Ser-Ala at the N-terminus was replaced with GCAAAGACC, encoding Ala-Lys-Thr (the optimized sequence for PLP-mediated transamination) using Quikchange site-directed mutagenesis kit (Stratagene, La Jolla, Calif.). Moreover, the ATG following these 9 bases was also changed to ACG to eliminate the potential of having another translation initiation site. This was accomplished via two rounds of Quikchange site-directed mutagenesis: the first round mutated ISA to AKT and the second mutated M to T. 
       FIGS. 1A-C . Structure and expression vector for Fc of human IgG1. (a) Structure of Fc region of human immunoglobulin G1 (IgG1) is comprised of two monomers, each containing two domains (CH2 and CH3), with glycosylation at Asn297. The hinge region, which contains two disulfide bonds, serves as a flexible spacer between Fc and Fab. (b) A plasmid expressing human IgG1 Fc protein with introns and IL2 signal sequence (IL2ss) was constructed and used for these experiments. The introns helped to increase the expression level of the IgG1 Fc, and IL2ss signaled the secretion of the Fc protein to the extracellular medium. (c) The sequence of the IL2ss (grey) and human IgG1 Fc (upper case letters) is shown with introns (lower case letters) inserted between hinge and CH2 and between CH2 and CH3 (red). The mutated region is in bold. 
     The sequences of mutagenic and sequencing primers used in this study are listed in Table 1. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Primer sequences used for construction of plasmid  
               
               
                 expressing AKT-Fc and sequencing primers. 
               
            
           
           
               
               
            
               
                 Purpose 
                 Primers* 
               
               
                   
               
               
                 Mutating  
                 5′-GCACTAAGTCTTGCACTTGTCACGAATTCG GCAA   
               
               
                 ISA to AKT 
                   AGACC ATGGTTAGATCTGACAAAACT-3′ (SEQ ID  
               
               
                   
                 NO: 21) 
               
               
                   
                 5′-ATGTGTGAGTTTTGTCAGATCTAACCAT GGTCTT   
               
               
                   
                   TGC CGAATTCGTGACAAGTGCAAGAC-3′ (SEQ ID 
               
               
                   
                 NO: 22) 
               
               
                   
               
               
                 Mutating 
                 5′-TGTCACGAATTCGGCAAAGACC ACG GTTAGATCT 
               
               
                 M to T 
                 GA-3′ (SEQ ID NO: 23) 
               
               
                   
                 5′-AGTTTTGTCAGATCTAAC CGT GGTCTTTGCCGAA 
               
               
                   
                 TTC-3′ (SEQ ID NO: 24) 
               
               
                   
               
               
                 Sequencing 
                 5′-TGCTTGCTCAACTCTACGTC-3′ (SEQ ID 
               
               
                   
                 NO: 25) 
               
               
                   
                 5′-TTGCAGCTTATAATGGTTACAAA-3′ (SEQ ID 
               
               
                   
                 NO: 26) 
               
               
                   
               
               
                 *The mutated sites are in bold. 
               
            
           
         
       
     
     The polymerase chain reaction (PCR) mixture was composed of 1 μL of 50 ng/μL plasmid template, 5 μL of each primer (25 ng/μL), 1 μL of dNTP mixture, 5 μL of 10× buffer, 1 μL of PfuTurbo DNA polymerase (2.5 units/μL) and 33 of μL of dH 2 O. The reaction was started with 2 min at 95° C. to predenature the template, followed by 18 cycles of 30 sec at 95° C., 1 min at 55° C. and 6 min at 70° C. The PCR ended with final polymerization at 70° C. for 10 min and the reaction mixture was left at 10° C. until the next step. After the PCR reaction, 1 μL of DpnI (10 units/μL) was added and the mixture was incubated at 37° C. for 2 h to degrade the original unmodified plasmid templates. After DpnI digestion, 2 μL of the mixture was used to transform  E. coli  XL1-Blue competent cells by heat-shock following the manufacturer&#39;s protocol. The transformed  E. coli  XL1-Blue was spread on Luria Broth (LB) plates containing Zeocin (Fast-Media Zeo Agar, InvivoGen) and incubated at 37° C. overnight (˜16 h). Colonies were selected and grown in 5 mL terrific broth (TB) media containing Zeocin (Fast-Media Zeo TB, InvivoGen) at 37° C. overnight (12-16 h). Plasmid DNA was isolated using a QIAprep Spin Miniprep Kit (Qiagen). The DNA sequences were confirmed by gene sequencing. The sequences of the primers used for sequencing are listed in Table 1. 
                     TABLE 2                  Sequence of DNA oligonucleotides used in the       protein modification.                     DNA oligo-           nucleotides   Sequence               Sgc8c   5′-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACG       (41-mer)   GTTAGA-3′ (SEQ ID NO: 27)               TD05.1   5′-AGGAGGATAGTTCGGTGGCTGTTCAGGGTCTCCTC       (37-mer)   CT-3′ (SEQ ID NO: 28)               M2M2   5&#39;-CCCTAGAGTGAGTCGTATGACCCTAGAGTGAGTCG       (41-mer)   TATGAA-3′ (SEQ ID NO: 29)                    
General Procedure for Expression of AKT-Fc
 
     The plasmids expressing AKT-Fc constructed above were transiently transfected into human embryonic kidney (HEK) 293T cells using Lipofectamine 2000 (Invitrogen) in Opti-MEM medium following the protocol from Invitrogen. The cells were incubated at 37° C. in 5% CO 2 . After 2 days, media were collected and secreted antibodies were purified using protein G affinity chromatography, according to the procedure from the manufacturer (Pierce). Fresh media were added and cultures were grown for additional 3 days, after which additional antibodies were harvested and purified as above. Purified protein was buffer exchanged into phosphate buffered saline (PBS) using Amicon Ultra 4 mL 10,000 MWCO (Millipore) centrifugal ultrafiltration membranes. Purity was evaluated by SDS-PAGE with Coomassie staining. 
     General Procedure for PLP Transamination 
     The 2× protein stock solutions were prepared at 10-40 μM using 25 mM phosphate buffer at pH 6.5. The 2× (200 mM) PLP stock solutions were prepared in 25 mM phosphate buffer and the pH of the solution was adjusted to 6.5 using NaOH solution. Protein and PLP stock solutions were mixed in equal volumes. The reaction mixture was briefly agitated to ensure mixing and then incubated without further agitation at 37° C. for 1 h. After incubation, the PLP was removed using NAP Sephadex size exclusion columns (GE Healthcare). The resulting keto-Fc solution was then concentrated and the buffer was exchanged with 25 mM phosphate buffer (pH 6.5), using Millipore 0.5 or 4 mL spin concentrators (MWCO 10 kDa), following the protocol from the manufacturer. 
     General Procedure for Hydrazone and Oxime Formation 
     The reaction was performed with 10-40 μM keto-Fc and RONH 2  or R(CO)NHNH 2  at varied concentrations. For the analysis of PLP transamination efficiency, BnONH 2  and Alexfluor 488-ONH 2  were added to keto-Fc to a final concentration of 100 mM and 80 μM, respectively. For the attachment of an oxidative coupling partner, aniline-ONH 2  was added to a final concentration of 25 mM. To make Fc-aptamer constructs, keto-Fc was mixed with the hydrazide-aptamer at a final concentration of 100 μM in the presence of 100 mM aniline, which is known to enhance the rate of hydrazone formation. Dirksen, A.; Hackeng, T. M.; Dawson, P. E.  Angew. Chem. Int. Ed.  2006, 45, 7581-7584. The reaction mixture was incubated at RT for 18-50 h. All the reactions were carried out in 25 mM phosphate buffer (pH 6.5), except for oxime formation with aniline-ONH 2 , which was done in 25 mM phosphate buffer (pH 5). Following the reaction, the small molecules were removed using NAP Sephadex size exclusion columns (GE Healthcare) and the resulting product mixtures were concentrated using Millipore 0.5 or 4 mL spin concentrators (Molecular weight cutoff (MWCO)=10 kDa), following the protocol from the manufacturer. The percent reaction conversion for the Fc samples with small molecules was analyzed using liquid chromatography-mass spectroscopy (LCMS) and the modification with large molecules was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie staining. 
     The General Procedure for Oxidative Coupling 
     To a solution of 10-40 μM Fc-aniline (9) in 25 mM phosphate buffer (pH 6.5) was added a solution of aminophenol-aptamer (10b) or aminophenol-2k PEG (10a) to a final concentration of 100 μM. Sodium periodate (Sigma-Aldrich) was dissolved to a concentration of 10 mM in 25 mM phosphate buffer (pH 6.5). The sodium periodate was then added to the reaction mixture to reach a final concentration of 1 mM, and the reaction was allowed to proceed for 2 min at RT. In some cases, a solution of mannose was also added to a final concentration of 10 mM or 100 mM before addition of the periodate solution. The resulting protein samples were purified on NAP Sephadex size exclusion columns (GE Healthcare) and concentrated using Millipore 0.5 or 4 mL spin concentrators (MWCO 10 kDa), following the protocol from the manufacturer. 
     Purification of Fc-Aptamer Constructs 
     The resulting Fc-DNA conjugates from both hydrazone formation and oxidative coupling were purified from unreacted Fc and DNA using anion exchange high performance liquid chromatography (HPLC) with a 20-min gradient of 100% buffer A to 5% buffer A 95% buffer B, where buffer A is 25 mM sodium phosphate buffer pH 6.5 and buffer B is 25 mM sodium phosphate buffer pH 6.5 with 1 M NaCl. The fractions collected were analyzed using SDS-PAGE and those containing Fc-DNA constructs were combined and concentrated using Millipore 0.5 mL spin concentrators (MWCO 10 kDa). 
     Flow Cytometry Experiments 
     Flow cytometry was used to determine binding ability of all the Fc-aptamer constructs. All experiments were carried out in triplicate. For all samples, 100 μL of 3×10 6  cells/mL of Jurkat, Ramos, and U266 was used, suspended in binding buffer (4.5 g/L glucose, 5 mM MgCl 2 , 0.1 mg/mL yeast tRNA (Sigma) and 1 mg/mL bovine serum albumin (BSA) (Fisher) in Dulbecco&#39;s PBS with calcium chloride and magnesium chloride (Invitrogen)). To these cells were added 10 μL of a series of 400 nM Fc-aptamer construct solutions. The samples were then incubated on ice for 1 h. The resulting cells were washed with 500 μL of binding buffer and resuspended in an additional 100 μL of binding buffer. Anti-human IgG1 antibody (specific for the Fc domain) with fluorescein isothiocyanate (FITC)-conjugated (Sigma) was then added to a final concentration of ˜0.30 μM. Cells were incubated for 1 h on ice in the dark, then washed with 500 μL of binding buffer, and resuspended in 200 μL binding buffer. The cells were analyzed by flow cytometry to determine the amount of FITC fluorescence. For each sample, 10,000 cells were counted. 
     ELISA for C1q Binding 
     The binding of human C1q to AKT-Fc, chemically-modified Fc, and Fc-DNA conjugates were assessed by an enzyme-linked immunosorbent assay (ELISA) binding assay, adjusted from a published procedure (Idusogie, E. E.; Presta, L. G.; Totpal, K.; Wong, P. Y.; Meng, Y. G.; Mulkerrin, M. G.; Alerts, E.  J. Immunol  2000, 164, 4178-4184). High binding Costar 96-well plates (Corning, N.Y.) were coated with varying concentrations of Fc samples in coating buffer (0.05 M sodium carbonate buffer, pH 9) overnight at 4° C. All samples were run in duplicate. The plates were washed three times after each incubation step with 300 μL of PBS+0.05% Tween 20, pH 7.4. After coating, the plates were blocked with 200 μL of ELISA diluent (0.1 M NaPO 4 /0.1 M NaCl/0.1% gelatin/0.05% Tween 20/0.05% ProClin300) for 1 h at RT, followed by incubation with 100 μL of 2 μg/mL human C1q (Quidel, San Diego, Calif.) in ELISA diluent for 2 h. Then, 100 μL of a 1:400 dilution of sheep anti-human C1q peroxidase-conjugated antibody (Abcam) in ELISA diluent was added and incubated at RT for 1 h. The plates were developed with 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate buffer (Sigma) at RT for 15 min. The reaction was stopped upon the addition of 100 μL of stop reagent for TMB substrate (Sigma), and the OD was measured at 450 nm using a Spectramax M3 microplate reader (Molecular Devices, Sunnyvale, Calif.). The obtained Hill plots displayed different amplitudes for the binding curves, which were taken to arise from different amounts of Fc samples binding to the wells. The plots were therefore normalized based on their maximum overall absorbance. 
     Synthesis of Hydrazide-DNA (6) 
     The 5′ thiol DNA oligonucleotide supplied by IDT was reduced in 40 mM TCEP in PBS, pH 7.4 for 2 h at RT. The TCEP was removed using NAP Sephadex size exclusion columns (GE Healthcare). Ten equivalents of 3,3′-N-[ε-maleimidocaproic acid]hydrazide, trifluoroacetic acid salt (EMCH, Pierce), were added to a sample of reduced 5′ thiol oligonucleotide in PBS, pH 7.4. The reaction mixture was incubated for 2.5 h at RT. The resulting hydrazide-DNA (6) was purified using NAP Sephadex size exclusion columns (GE Healthcare), followed by spin concentration with Millipore 0.5 mL spin concentrators (MWCO 10 kDa). 
     The scheme for the synthesis of hydrazide-DNA oligonucleotide 5 is depicted in  FIG. 8 . 
     Synthesis of Aminophenol-DNA (11b) 
     The 5′ thiol DNA oligonucleotide supplied by IDT was reduced in 40 mM TCEP in PBS, pH 7.4 for 2 h at RT. The TCEP was removed using NAP Sephadex size exclusion columns (GE Healthcare). The resulting product was buffered exchanged into 25 mM phosphate buffer at pH 8 by spin concentration with Millipore 0.5 mL spin concentrators (MWCO 10 kDa). Approximately one equivalent of a nitrophenol-maleimide linker was added to the reduced 5′ thiol DNA oligonucleotide and the reaction was carried out in 25 mM phosphate buffer pH 8 at room temperature (RT) for 1 h. The resulting nitrophenol-DNA was then purified using NAP Sephadex size exclusion columns (GE Healthcare), followed by spin concentration with Millipore 0.5 mL spin concentrators (molecular weight cut-off (MWCO) 10 kDa) into PBS. The nitro group was reduced to amine in the presence of 10 mM sodium dithionite at RT for 10 min. The final product aminophenol-DNA was purified using NAP Sephadex size exclusion columns (GE Healthcare), followed by spin concentration with Millipore 0.5 mL spin concentrators (MWCO 10 kDa) into 25 mM phosphate buffer pH 6.5 for subsequent conjugation with Fc proteins. 
     The scheme for synthesis of aminophenol-DNA oligonucleotide 10b is depicted in  FIG. 9 . 
     Synthesis of Aniline-ONH 2  (8) 
     To (boc-aminooxy)acetic acid (280 mg, 1.5 mmol) dissolved in methylene chloride was added dicyclohexylcarbodiimide (362 mg, 1.7 mmol) and N-hydroxysuccinimide (168 mg, 1.5 mmol). After 15 min with stirring, the precipitate was filtered through Celite, followed by a 0.22 μm PVDF syringe filter. To the remaining solution was added 2-(4-aminophenyl)ethylamine (200 mg, 1.5 mmol) and triethylamine (400 mg, 4 mmol). After 1 h of stirring the solution was concentrated under reduced pressure and applied to a silica gel column. Purification using ethyl acetate as the mobile phase afforded approximately 200 mg of the product (45% yield).  1 H NMR (400 MHz, CDCl 3 ) δ 1.4 (s, 9H), 2.7 (t, 2H), 3.4 (q, 2H), 3.5 (br s, 2H), 4.2 (s, 2H), 6.6 (d, 2H), 7.0 (d, 2H), 8.1 (br s, 1H), 8.5 (s, 1H). The Boc group was removed via exposure to 1:1 trifluoroacetic acid:methylene chloride for 10 min, after which the solvent was removed under a stream of nitrogen. The resulting oil was placed under vacuum overnight. It was then dissolved to 100 mM in water and stored frozen until used. To avoid precipitation, it was necessary to neutralize the residual trifluoroacetic acid by adding phosphate buffer before addition to protein-containing solutions. 
     Synthesis of 2k PEG-Aminophenol (10a) 
     2k poly(ethylene glycol) (PEG)-aminophenol (10a) was synthesized according to published protocol (Behrens, C. R.; Hooker, J. M.; Obermeyer, A. C.; Romanini, D. W.; Katz, E. M.; Francis, M. B.  J. Am. Chem. Soc.  2011, 133, 16398-16401). 
     Synthesis of Nitrophenol-Maleimide Linker 
     To tyramine was added dropwise one equivalent of fuming nitric acid at 4° C. using trifluoroacetic acid as the solvent and this resulted in quantitative conversion to o-nitrotyramine. O-nitrotyramine (50 mg, 0.27 mmol) was dissolved in 10 mL DMF and treated with one equivalent of succinimidyl-6-N-maleimidohexanoate (as described in Nielsen, O.; Buchardt, O.  Synthesis  1991, 10, 819-821) along with sufficient triethylamine to reach pH 8. Multiple equivalents of triethylamine were required due to residual trifluoroacetic acid from the nitration step. After 45 min, 20 mL of 0.1 M NaHSO 4  was added to the reaction. The product was extracted with methylene chloride, dried over Na 2 SO 4 , and purified on a silica column using ethyl acetate as the mobile phase. Isolated yield was 33%.  1 H NMR (300 MHz, CDCl 3 ) δ 1.2 (m, 2H), 1.6 (m, 4H), 2.1 (t, 2H), 2.8 (t, 2H), 3.5 (t, 4H), 5.6 (br s, 1H), 6.6 (s, 2H), 7.0 (d, 1H), 7.5 (d, 1H), 8.0 (s, 1H), 10.5 (br s, 1H). 
     Results 
     Recently, the potential of using pyridoxal 5′-phosphate (PLP)-mediated transamination has been demonstrated as a convenient way to install one new functional group in a single location without modifying native free cysteine or lysine residues. 15  Taking advantage of this selectivity, the synthesis of Fc-synthetic molecule hybrids was designed around using PLP-mediated N-terminal transamination to provide a unique ketone group at the N-terminus of the Fc-domain for further functionalization ( FIG. 2 ). The synthetic moiety can then be conjugated to the N-terminus using traditional oxime or hydrazone formation. 16  The use of an oxidative coupling reaction was explored as a new approach to ligate synthetic groups to the protein with significantly enhanced efficiency 17 —a useful feature for the installation of high-value cargo, such as complex drug molecules or the nucleic acid aptamers described herein. 
       FIG. 2 . Modification scheme for Fc proteins. First, ketone functional groups are installed at the N-termini through PLP-mediated transamination. These groups can then be functionalized using two different approaches. The first involves the direct attachment of molecules of interest via oxime formation (with 4) or hydrazone formation (with 5). The second strategy uses a highly efficient oxidative coupling reaction. This approach involves the chemoselective coupling of aniline groups on the Fc proteins (9) with aminophenol-containing reagents (10). 
     Since the efficiency of PLP-mediated transamination depends on the sequence of the N-terminus, 15c  the Fc protein was first mutated to contain a highly reactive alanine-lysine-threonine (AKT) sequence 15d  immediately following the IL2 signaling peptide (see sequence in  FIG. 1C ). This leader peptide was cleaved during the secretion of the AKT-Fc proteins from the host cells. The protein was expressed in glycosylated form in transfected HEK 293T cells (see mass spectrometry characterization in  FIG. 3A-C ) and purified using a protein G column. 
       FIGS. 3A-C . LCMS analysis of (a) the Fc protein collected 2 d after transfection; (b) the Fc protein collected 5 d after transfection (fresh Opti-MEM media was replaced after 2 d); and (c) the Fc protein after treatment with PNGase F (both 2 d and 5 d samples were identical). The heterogeneity was a result of differences in the number of galactose (G, 162 Da) and N-acetylglucosamine (N, 203 Da) residues that were incorporated. Because each monomer of the Fc protein could contain up to two galactose residues, there are five possibilities of glycosylation patterns for the Fc dimer, corresponding to a normal distribution of M+0G (55206 Da), M+1G (55368 Da), M+2G (55530 Da), M+3G (55692 Da), and M+4G (55854 Da). Additionally, peaks at 55005, 55166, 55329, and 55492 Da appear to match M+0G-N, M+1G-N, M+2G-N, and M+3G-N, and M+3G-N, respectively. 
     The AKT-Fc proteins were then exposed to a freshly prepared 100 mM solution of PLP in pH 6.5 phosphate buffer at 37° C. for 1 h. Due to the small mass difference (1 Da) corresponding to the transamination of each terminus, the resulting protein was exposed to benzyl hydroxylamine (BnONH 2 , 4a) for 2 d at RT before characterization using mass spectrometry. To simplify the analysis, the carbohydrate domain was removed from the samples using PNGase F. As shown for the non-reduced Fc products in  FIGS. 4A and 4B , very high conversion was observed. Two major products were obtained (6a and 6a+PLP), both resulting from the desired oxime formation reaction at the two Fc termini. The higher mass product resulted from an aldol reaction between the aldehyde of PLP and the transaminated terminus during the first reaction step (shown as 3+PLP in  FIG. 2 ). The only other product that was evident was a small amount of Fc with a single oxime modification (species 12). In samples lacking PLP treatment, exposure to BnONH 2  led to no observable modification. Oxime formation using AlexaFluor 488 alkoxyamine was also used to detect reaction progress via SDS-PAGE ( FIG. 4C ). Fluorescence was detected in the +PLP lanes using both reducing and non-reducing gel loading buffers, suggesting that AKT-Fc underwent transamination by PLP and that the Fc remained in dimeric state under the reaction conditions. In addition to the expected products, a small amount of residual Fc dimer (˜4-7% by densitometry) was observed in the reduced lane. It was presumed that this resulted from an aldol reaction between the two terminal ketones, which are directly adjacent to one another in the dimeric Fc structure. Such a species would still possess a single remaining ketone group, thus allowing its labeling with the dye molecule. The presence of this minor species could also explain species 12 in  FIG. 4B , but the identities of these two byproducts have not been confirmed further due to their very low abundance. 
       FIGS. 4A-C . Analysis of transamination efficiency for AKT-Fc domains. AKT-Fc (1) was exposed to 50 mM 4a for 40 h at pH6.5, followed by treatment with PNGaseF (a) without prior transamination with PLP (as a negative control), or (b) following transamination with 100 mM PLP at 37° C. for 1 h. The peak at 52315 Da corresponded to unmodified AKT-Fc (expected: 52315 Da). The double-oxime product (6a) appeared at 52525 Da (expected: 52523 Da), and the peak at 52772 Da corresponded to product 6a plus a single PLP addition (expected: 52770 Da). The peak at 52419 Da corresponded to the addition of one molecule of 4a to the AKT-Fc fragment (12, expected: 52419 Da). (c) Samples of AKT-Fc with and without transamination using PLP (100 mM PLP at 37° C. for 1 h) were exposed to 80 μM Alexa-Fluor 488 alkoxyamine (4b) for 43 h in the presence of 100 m Maniline as a catalyst. They were then analyzed by SDS-PAGE under reducing (Lanes 1 and 2) or nonreducing (Lanes 3 and 4) conditions. The fluorescent images of the Fc-AlexaFluor488 oxime products (top) were taken using a Typhoon imaging system. The bottom gel was stained using Coomassie blue. 
     Due to slow kinetics of oxime and hydrazone formation, long reaction times and large excesses of reagent are typically used to reach full conversion. While this is fine for inexpensive small molecules, it poses a problem when the group to be attached is a large or expensive reagent (such as a drug). With these cases in mind, oxime formation was used to attach an aniline-containing small molecule to allow further ligation using oxidative coupling reactions. As reported recently, the periodate-mediated oxidative coupling 18  between anilines and aminophenols can achieve chemoselective protein modification with very high levels of product conversion in less than 2 min. Using the transamination/oxime formation procedure described above with compound 8 produced Fc proteins bearing an aniline group at each of the two termini (species 9 and 9+PLP in  FIG. 5B ). 
     Following successful installation of aniline groups on the Fc proteins, the oxidative coupling reaction was then investigated using aminophenol containing polyethyleneglycol 10a (MW ˜2000 Da). 12a  Aniline Fc 9 (10-40 μM) was combined with 100 μM 10a in pH 6.5 phosphate buffer. A stock solution of sodium periodate was added to a final concentration of 1 mM, and the reaction was incubated for 2 min. The resulting solution was then passed through a NAPS gel filtration column to quench and remove the periodate. SDS-PAGE analysis under reducing conditions (and without the use of PNGase F) indicated that 50% of the individual Fc chains were converted to the singly PEGylated product ( FIG. 5C , lane 6), as indicated by optical densitometry. This yield likely results from the close proximity of the two N-termini in the intact Fc domains, resulting in the attachment of only one PEG chain to each protein dimer. Nonetheless, a high degree of modification was observed using very short coupling times. 
     As one potential concern with this strategy, immunoglobulin proteins contain oligosaccharides, which could be cleaved to form aldehydes in the presence of sodium periodate. To determine to what degree this occurs, unmodified Fc domains were exposed to NaIO 4  under the oxidative coupling conditions for 2 min and 1 h. The resulting aldehyde groups were then visualized by subsequent reaction with AlexaFluor 488 alkoxyamine (4b). As seen in  FIG. 5D , the oligosaccharides on the Fc protein were only minimally oxidized by NaIO 4  at the 2 min time point (compare lanes 2 and 5 to the background labeling for unmodified Fc in lane 1). Furthermore, it was found that the addition of 10 mM to 100 mM mannose could suppress this oxidation of the carbohydrate groups completely. Interestingly, the oxidative coupling reaction still proceeded with similar conversion in the presence of 10 mM mannose ( FIG. 5C , lane 7), and with somewhat reduced conversion in the presence of 100 mM mannose (lane 8). Thus, this strategy provides a method to protect glycoproteins from undesired oxidation with this procedure. Even though it should be noted that the Fc protein examined here does not contain sialic acids, which are the most susceptible to oxidation, it is anticipated that oxidative coupling will still occur in a much shorter timescale in comparison to oxidation of oligosaccharides. 
       FIGS. 5A-D . Modification of Fc domains via oxidative coupling (O.C.). AKT-Fc (1) was exposed to 25 mM 8 for 40 h at pH 4.5, followed by PNGaseF treatment (a) without prior transamination with PLP (as a negative control), or (b) following transamination with 100 mM PLP at 37° C. for 1 h. The peak at 52315 Da corresponded to unmodified AKT-Fc (expected: 52315 Da). The double-oxime product (9) appeared at 52697 Da (expected: 52695 Da), and the peak at 52944 Da corresponded to product 9 plus a single PLP addition (expected: 52942 Da). The peak at 52506 Da corresponded to the addition of one molecule of 8 to the AKT-Fc fragment (13, expected: 52506 Da). (c) Samples of unmodified Fc (1) and Fc-aniline (9) were exposed to 100 μM 2k PEG-aminophenol (10a) and 1 mM NaIO 4  for 2 min at RT. Lanes 1-5 display negative controls. Only in the presence of both aniline on the Fc and NaIO 4  did the attachment of 2 k PEG-aminophenol occur (lane 6). In the presence of 10 mM mannose (lane 7), the O.C. still proceeded; however, the yield suffered when the mannose concentration was increased to 100 mM (lane 8). (d) The extent of oxidation of the oligosaccharides on the Fc protein within the reaction time of the O.C. was analyzed. The Fc proteins were exposed to 1 mM NaIO 4  for 2 and 60 min. The reaction was stopped upon addition of TCEP and any aldehydes formed from the oxidation were then detected by Alexafluor 488-ONH 2 . The fluorescent images of Fc-AlexaFluor488 oxime products (top) were taken using a Typhoon imaging system. The oligosaccharides on the Fc were minimally oxidized under the O.C. reaction time of 2 min (lane 2) and this oxidation was lowered to background level upon addition of 10 mM mannose or higher concentration (lanes 3 and 4). The oxidation of oligosaccharides on Fc with NaIO 4  for 1 h was shown as a positive control (lane 5). Lane 1 and 6 display the background level of oxidized sugar in the absence of NaIO 4 . 
     Since the 1990s, small RNA and DNA aptamers have emerged as a new class of molecules for therapeutic and diagnostic purposes, owing to the successful development of the systematic evolution of ligands by an exponential enrichment process, known as SELEX. 7,19  Using SELEX, new aptamers can be evolved to bind to cells with high specificity and affinity, often without prior knowledge of the specific molecular targets. These readily evolved binding groups could endow Fc domains with specific cell binding abilities, and, conversely, the Fc domains could improve the in vivo circulation times of the oligonucleotides, as shown by Barbas et al. 6a  Two aptamers were selected for attachment to the Fc proteins as a proof of principle for the production of Fc-synthetic molecule hybrids: (1) sgc8c, which targets protein tyrosine kinase 7 (PTK7) 20  and (2) TD05.1, which targets membrane-bound IgM (mIgM, also known as the B-cell receptor). 21  The sgc8c aptamer has been used in many applications 22,17c  and shown high specificity to its target. The use of TD05.1 could be advantageous because there is currently no antibody that is specific to binding mIgM without also binding to circulating IgM in the blood. 21  For use as a negative control in cell binding experiments, a scrambled 41-nucleotide DNA sequence (M2M2) was also attached to Fc. 
     Using the hydrazone formation strategy with 100 μM hydrazide-oligonucleotides (5) and 100 mM aniline as a coupling catalyst 23  over a 48 h period, the Fc-aptamer conjugates could be obtained as identified by SDS-PAGE analysis ( FIG. 6B ). Due to the high negative charge of the DNA portion, the Fc conjugates could also be separated using anion exchange chromatography ( FIG. 6C ), allowing more accurate determination of the product ratios. The major product (64%) possessed a single attachment of the oligonucleotide to one of the Fc N-termini, likely due to steric hindrance and electronic repulsion between two DNA molecules. The doubly modified product corresponded to 20% yield, with only 16% of unconjugated Fc protein remaining. The alternative method, the attachment of aminophenol-DNA oligonucleotides (10b, 100 μM) to Fc-aniline (9) via oxidative coupling reaction, was achieved at RT in 2 min. In this case, 58% of the protein product corresponded to the single aptamer conjugate and 39% corresponded to the double aptamer conjugate, representing a total of &gt;95% of the protein species. The overall yields were therefore slightly higher than those achieved by direct hydrazone formation ( FIG. 6A , lanes 4-6), but they were obtained with drastically reduced coupling times. Anion exchange chromatography was again used to obtain pure conjugates for use in subsequent binding studies,  FIG. 6D . 
       FIGS. 6A-D . Construction of Fc-aptamer conjugates. Two different aptamers, sgc8c targeting PTK7 and TD05.1 targeting membrane-bound IgM, were attached to keto-Fc using the two approaches shown in  FIG. 1 . (a) Structure of hydrazide- and aminophenol-DNA oligonucleotides used for hydrazone formation and the oxidative coupling reaction, respectively. (b) SDS-PAGE analysis under non-reducing conditions showed the formation of Fc hybrids using either hydrazone formation (labeled as ‘h’, lanes 1-3) or oxidative coupling (′o′, lanes 4-6). Single aptamer conjugates were the major products, along with lesser amounts of doubly-labeled conjugated species. (c) Anion exchange-HPLC analysis of the crude Fc-TD05.1 adduct following the oxidative coupling reaction, indicating the relative quantities of Fc, Fc-DNA, and Fc-(DNA). The shaded fraction was 2 collected for further use. (d) SDS-PAGE analysis (non-reducing) of the purified Fc-aptamer conjugates used for cell binding analysis. 
     The specificities of Fc-sgc8c, Fc-TD05.1, and Fc-M2M2 hybrid constructs generated from both the hydrazone and the oxidative coupling strategies were next evaluated for selective cell binding using flow cytometry. Jurkat cells, a T-cell leukemia cell line overexpressing PTK7, were used as the targeted cells for the Fc-sgc8c constructs. Ramos cells, a Burkitt&#39;s lymphoma cell line overexpressing mIgM, were used as target cells for Fc-TD05.1. U266 cells, a B-cell line overexpressing neither membrane protein, were chosen as a negative control sample. The binding assay was conducted as outlined in  FIG. 7A , with detection of the cell-bound Fc conjugates using fluorescently labeled anti-human IgG1. Only Jurkat cells were bound by Fc-sgc8c, and only Ramos cells were recognized by Fc-TD05.1 ( FIG. 7B ). Neither cell line was recognized by Fc-M2M2. Moreover, the U266 negative control cell line did not bind to any of the Fc-aptamer constructs. In addition to confirming that the aptamers retained their specificity after attachment to the Fc domains, these results also indicated that the Fc region retained its proper folding and thus could still be recognized by the fluorescent secondary antibodies. 
       FIGS. 7A-D . Cell binding specificity and C1q binding ability of Fc-aptamer conjugates. (a) The general cell binding analysis scheme is shown. The cells were incubated with Fc-aptamer samples, which were subsequently detected using FITC-labeled secondary antibodies specific for Fc of human IgG1. (b) Flow cytometry data are shown for the binding of Fc-aptamer conjugates to Jurkat cells (overexpressing PTK7, the target of the sgc8c aptamer, ref 20), Ramos cells (overexpressing membrane-bound IgM, the target for the TD05.1 aptamer, ref 21), and U266 cells as a negative control. All Fc-sgc8c and Fc-TD05.1 conjugates retained their targeting specificity, whether they were generated using hydrazone formation (labeled ‘h’) or oxidative coupling (labeled ‘o’). Unmodified Fc proteins and an Fc-M2M2 conjugate (bearing a scrambled 41-base oligonucleotide) did not bind to any cell lines. (c) ELISA data are shown for C1q binding to unmodified Fc, PLP-treated Fc, and Fc after oxime formation with BnONH. (d) ELISA data are shown for C1q binding to the 2 Fc-aptamer conjugates used in (b). 
     A key feature associated with the toxicity of many antibodies is the recruitment of complement proteins found in blood serum. The ability of the modified Fc proteins to bind to the C1q complement protein was evaluated using ELISA. 24  Briefly, varying concentrations of the Fc conjugates were bound to a polystyrene 96-well plate, after which a 2 μg/mL solution of human C1q was added. Binding ability was confirmed using an anti-C1q antibody conjugated to horseradish peroxidase (HRP). These results showed that the PLP-treated Fc and Fc-aptamer constructs still retained their ability to bind to C1q with similar affinities. This suggests that the bulk of the immunological activating properties of the Fc domains remained intact following the modification process ( FIGS. 7A and 7D ). 
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         14. (a) Hutchins, B. M.; Kazane, S. A.; Staflin, K.; Forsyth, J. S.; Felding-Habermann, B.; Schultz, P. G.; Smider, V. V. J. Mol. Biol. 2011, 406, 595-603. (b) Hutchins, B. M.; Kazane, S. A.; Staflin, K.; Forsyth, J. S.; Felding-Habermann, B.; Smider, V. V.; Schultz, P. G. Chem. Biol. 2011, 18, 299-303. 
         15. (a) Gilmore, J. M.; Scheck, R. A.; Esser-Kahn, A. P.; Joshi, N. S.; Francis, M. B. Angew. Chem. Int. Ed. 2006, 45, 5307-5311. (b) Scheck, R. A.; Francis, M. B. ACS Chem. Biol. 2007, 2, 247-251. (c) Scheck, R. A.; Dedeo, M. T.; Iavarone, A. T.; Francis, M. B. J. Am. Chem. Soc. 2008, 130, 11762-11770. (d) Witus, L. S.; Moore, T.; Thuronyi, B. W.; Esser-Kahn, A. P.; Scheck, R. A.; Iavarone, A. T.; Francis, M. B. J. Am. Chem. Soc. 2010, 132, 16812-16817. For previous studies of transamination with PLP, see (e) Snell, E. E. J. Am. Chem. Soc. 1945, 67, 194-197. (f) Dixon, H. B. F. J. Protein Chem. 1984, 3, 99-108. (g) Dixon, H. B. F.; Fields, R. Methods Enzymol. 1972, 25, 409-419. 
         16. (a) Jencks, W. P. J. Am. Chem. Soc. 1959, 81, 475-481. (b) Cornish, V. W.; Hahn, K. M.; Schultz, P. G. J. Am. Chem. Soc. 1996, 118, 8150-8151. (c) Mahal, L. K.; Yarema, K. J.; Bertozzi, C. R. Science 1997, 276, 1125-1128. (d) Kalia, J.; Raines, R. Angew. Chem. Int. Ed. 2008, 47, 7523-7526. 
         17. (a) Hooker, J. M.; Esser-Kahn, A. P.; Francis, M. B. J. Am. Chem. Soc. 2006, 128, 15558-15559. (b) Behrens, C. R.; Hooker, J. M.; Obermeyer, A. C.; Romanini, D. W.; Katz, E. M.; Francis, M. B. J. Am. Chem. Soc. 2011, 133, 16398-16401. (c) Tong, G. J.; Hsiao, S. C.; Carrico, Z. M.; Francis, M. B. J. Am. Chem. Soc. 2009, 131, 11174-11178. 
         18. For examples of other bioconjugation reactions involving sodium periodate, see: (a) Geoghegan, K. F.; Stroh, J. G. Bioconj. Chem. 1992, 3, 138-146. (b) Liu, B.; Burdine, L.; Kodadek, T. J. Am. Chem. Soc. 2006, 128, 15228-15235. 
         19. (a) Fang, X.; Tan, W.  Acc. Chem. Res.  2010, 43, 48-57. (b) Keefe, A. D.; Pai, S.; Ellington, A.  Nat. Rev. Drug Discov.  2010, 9, 537-550. 
         (c) Jayasena, S. D.  Clin. Chem.  1999, 45, 1628-1650. (d) Nimjee, S. M.; Rusconi, C. P.; Sullenger, B. A.  Ann. Rev. Med.  2005, 56, 555-583. (e) Bouchard, P. R.; Hutabarat, R. M.; Thompson, K. M.  Ann. Rev. Pharm. Tox.  2010, 50, 237-257. 
         20. (a) Shangguan, D.; Li, Y.; Tang, Z.; Cao, Z. C.; Chen, H. W.; Mallikaratchy, P.; Sefah, K.; Yang, C. J.; Tan, W.  Proc. Natl. Acad. Sci.  2006, 103, 11838-11843. (b) Shangguan, D.; Tang, Z.; Mallikaratchy, P.; Xiao, Z.; Tan, W.  Chembiochem  2007, 8, 603-606. (c) Shangguan, D.; Cao, Z.; Meng, L.; Mallikaratchy, P.; Sefah, K.; Wang, H.; Li, Y.; Tan, W.  J. Proteome Res.  2008, 7, 2133-2139. 
         21. (a) Mallikaratchy, P.; Tang, Z.; Kwame, S.; Meng, L.; Shangguan, D.; Tan, W.  Mol. Cell. Proteomics  2007, 6, 2230-2238. (b) Mallikaratchy, P. R.; Ruggiero, A.; Gardner, J. R.; Kuryavyi, V.; Maguire, W. F.; Heaney, M. L.; McDevitt, M. R.; Patel, D. J.; Scheinberg, D. A.  Nucl. Acids Res.  2011, 39, 2458-2469. 
         22. (a) Shi, H.; He, X.; Wang, K.; Wu, X.; Ye, X.; Guo, Q.; Tan, W.; Qing, Z.; Yang, X.; Zhou, B.  Proc. Natl. Acad. Sci. U.S.A.  2011, 108, 3900-3905. (b) Douglas, S. M.; Bachelet, I.; Church, G. M.  Science  2012, 335, 831-834. (c) Luo, Y.-L.; Shiao, Y.-S.; Huang, Y.-F.  ACS Nano  2011, 5, 7796-7804. (d) Stephanopoulos, N.; Tong, G. J.; Hsiao, S. C.; Francis, M. B.  ACS Nano  2010, 4, 6014-6020. 
         23. (a) Dirksen, A.; Hackeng, T. M.; Dawson, P. E.  Angew. Chem. Int. Ed.  2006, 45, 7581-7584. (b) Dirksen, A.; Dirksen, S.; Hackeng, T. M.; Dawson, P. E.  J. Am. Chem. Soc.  2006, 128, 15602-15603. 
         24. Idusogie, E. E.; Presta, L. G.; Totpal, K.; Wong, P. Y.; Meng, Y. G.; Mulkerrin, M. G.; Alerts, E.  J. Immunol.  2000, 164, 4178-4184. 
       
    
     Example 2: Preparation and Characterization of Antibody Conjugates 
     Anti-HER2 human IgG1 antibodies were functionalized with DNA aptamers both through N-terminal modification and through lysine modification. The binding specificity of antibody-DNA aptamer conjugates was determined by flow cytometry. 
     Construction of Light Chain Anti-HER2 Human IgG1 Expression Plasmids. 
     To clone a plasmid for the expression of the anti-HER2 human IgG1 light chain, the sequence for the variable domain of the light chain (V L ) was obtained from the literature, assembled into a gene, then cloned into a plasmid containing the light chain constant region. Gene2Oligo was used to generate the following set of oligonucleotides for gene assembly from the V L  sequence. An IL2 signaling sequence was also included in the N-terminal region. The bases in lower case were added by the Gene2Oligo program and did not belong to the input sequence: 
     
       
         
           
               
               
            
               
                   
                 R0: 
               
               
                   
                 (SEQ ID NO: 30) 
               
               
                   
                 ACCTTTTTTTacattgaagtgcag; 
               
               
                   
                   
               
               
                   
                 F0: 
               
               
                   
                 (SEQ ID NO: 31) 
               
               
                   
                 ctgcacttcaatgtAAAAAAAGGTCACCATGTACAGGATGCA; 
               
               
                   
                   
               
               
                   
                 R24: 
               
               
                   
                 (SEQ ID NO: 32) 
               
               
                   
                 GCAATGCAAGACAGGAGTTGCATCCTGTACATGGTG; 
               
               
                   
                   
               
               
                   
                 F42: 
               
               
                   
                 (SEQ ID NO: 33) 
               
               
                   
                 ACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCA; 
               
               
                   
                   
               
               
                   
                 R60: 
               
               
                   
                 (SEQ ID NO: 34) 
               
               
                   
                 TCAGTCTTAGCCGAATTCGTGACAAGTGCAAGACTTAGT; 
               
               
                   
                   
               
               
                   
                 F80: 
               
               
                   
                 (SEQ ID NO: 35) 
               
               
                   
                   
               
               
                   
                 CGAATTCGGCTAAGACTGACATCCAAATGACTCAGAGCC; 
               
               
                   
                   
               
               
                   
                 R99: 
               
               
                   
                 (SEQ ID NO: 36) 
               
               
                   
                 GCGCTCAGGGAACTGGGGCTCTGAGTCATTTGGATG; 
               
               
                   
                   
               
               
                   
                 F119: 
               
               
                   
                 (SEQ ID NO: 37) 
               
               
                   
                 CCAGTTCCCTGAGCGCTTCCGTAGGGGACAGG; 
               
               
                   
                   
               
               
                   
                 R135: 
               
               
                   
                 (SEQ ID NO: 38) 
               
               
                   
                 GCCCGACATGTTATTGTCACCCTGTCCCCTACGGAA; 
               
               
                   
                   
               
               
                   
                 F151: 
               
               
                   
                 (SEQ ID NO: 39) 
               
               
                   
                 GTGACAATAACATGTCGGGCTAGCCAGGATGTCAATACAG; 
               
               
                   
                   
               
               
                   
                 R171: 
               
               
                   
                 (SEQ ID NO: 40) 
               
               
                   
                 CTGGTACCAAGCGACAGCTGTATTGACATCCTGGCTA; 
               
               
                   
                   
               
               
                   
                 F191: 
               
               
                   
                 (SEQ ID NO: 41) 
               
               
                   
                 CTGTCGCTTGGTACCAGCAAAAGCCCGGAAAGGC; 
               
               
                   
                   
               
               
                   
                 R208: 
               
               
                   
                 (SEQ ID NO: 42) 
               
               
                   
                 GCTGTATATAAGAAGCTTTGGCGCCTTTCCGGGCTTTTG; 
               
               
                   
                   
               
               
                   
                 F225: 
               
               
                   
                 (SEQ ID NO: 43) 
               
               
                   
                 GCCAAAGCTTCTTATATACAGCGCCAGTTTCCTCTATTCTGG; 
               
               
                   
                   
               
               
                   
                 R247: 
               
               
                   
                 (SEQ ID NO: 44) 
               
               
                   
                 GAACCTGCTCGGCACGCCAGAATAGAGGAAACTGGC; 
               
               
                   
                   
               
               
                   
                 F267: 
               
               
                   
                 (SEQ ID NO: 45) 
               
               
                   
                 CGTGCCGAGCAGGTTCTCTGGATCTCGGTCCG; 
               
               
                   
                   
               
               
                   
                 R283: 
               
               
                   
                 (SEQ ID NO: 46) 
               
               
                   
                 TCAGTGTGAAATCGGTCCCGGACCGAGATCCAGA; 
               
               
                   
                   
               
               
                   
                 F299: 
               
               
                   
                 (SEQ ID NO: 47) 
               
               
                   
                 GGACCGATTTCACACTGACCATTAGTTCTCTGCAGCC; 
               
               
                   
                   
               
               
                   
                 R317: 
               
               
                   
                 (SEQ ID NO: 48) 
               
               
                   
                 TAGTATGTTGCAAAGTCCTCTGGCTGCAGAGAACTAATGG; 
               
               
                   
                   
               
               
                   
                 F336: 
               
               
                   
                 (SEQ ID NO: 49) 
               
               
                   
                 AGAGGACTTTGCAACATACTACTGCCAGCAGCACTAT; 
               
               
                   
                   
               
               
                   
                 R357: 
               
               
                   
                 (SEQ ID NO: 50) 
               
               
                   
                 AGGTTGGGGGTGTGGTATAGTGCTGCTGGCAG; 
               
               
                   
                   
               
               
                   
                 F373: 
               
               
                   
                 (SEQ ID NO: 51) 
               
               
                   
                 ACCACACCCCCAACCTTTGGTCAGGGCACGAA; 
               
               
                   
                   
               
               
                   
                 R389: 
               
               
                   
                 (SEQ ID NO: 52) 
               
               
                   
                 CGTACGCTTGATTTCCACCTTCGTGCCCTGACCAA; 
               
               
                   
                   
               
               
                   
                 F405: 
               
               
                   
                 (SEQ ID NO: 53) 
               
               
                   
                 GGTGGAAATCAAGCGTACGAAAAAAAcccccaactttgt; 
               
               
                   
                   
               
               
                   
                 F424: 
               
               
                   
                 (SEQ ID NO: 54) 
               
               
                   
                 acaaagttgggggTTTTTTT. 
               
            
           
         
       
     
     An additional GCTAAAACT was added to the 5′ end in order to create a three residue N-terminal extension (AKT). The resulting V L  gene was inserted into a vector at BsiWI and BstEII restriction sites using standard cloning techniques. The vector used, pFUSE2-CLIg-hk from Invivogen (San Diego, Calif.), already contained the constant region of the kappa light chain. 
     Construction of Heavy Chain Anti-HER2 Human IgG1 Expression Plasmids. 
     A plasmid for the expression of the anti-HER2 heavy chain was cloned in a similar fashion to that of light chain. In brief, the variable and constant region 1 of heavy chain (V H  and C H 1) was constructed from the following set of oligonucleotides with additional bases (CTCCAAACA) at the 5′ end (corresponding to three N-terminal residues, LQT, for use in other work and later mutated to AKT for this work). 
     
       
         
           
               
               
            
               
                   
                 R0: 
               
               
                   
                 (SEQ ID NO: 55) 
               
               
                   
                 TTTTTTTcttagctgctttga; 
               
               
                   
                   
               
               
                   
                 F0: 
               
               
                   
                 (SEQ ID NO: 56) 
               
               
                   
                 tcaaagcagctaagAAAAAAAGAATTCGCTCCAAACAG; 
               
               
                   
                   
               
               
                   
                 R21: 
               
               
                   
                 (SEQ ID NO: 57) 
               
               
                   
                 CGACGAGTTGGACTTCTGTTTGGAGCGAATTC; 
               
               
                   
                   
               
               
                   
                 F38: 
               
               
                   
                 (SEQ ID NO: 58) 
               
               
                   
                 AAGTCCAACTCGTCGAAAGCGGAGGTGGC; 
               
               
                   
                   
               
               
                   
                 R53: 
               
               
                   
                 (SEQ ID NO: 59) 
               
               
                   
                 CCAGGCTGAACCAGGCCACCTCCGCTTT; 
               
               
                   
                   
               
               
                   
                 F67: 
               
               
                   
                 (SEQ ID NO: 60) 
               
               
                   
                 CTGGTTCAGCCTGGCGGAAGCCTGCGC; 
               
               
                   
                   
               
               
                   
                 R81: 
               
               
                   
                 (SEQ ID NO: 61) 
               
               
                   
                 GCAGCACAGCTCAAGCGCAGGCTTCCG; 
               
               
                   
                   
               
               
                   
                 F94: 
               
               
                   
                 (SEQ ID NO: 62) 
               
               
                   
                 TTGAGCTGTGCTGCCTCCGGATTTAATATCAAAGA; 
               
               
                   
                   
               
               
                   
                 R108: 
               
               
                   
                 (SEQ ID NO: 63) 
               
               
                   
                 CGAACCCAGTGTATATAAGTATCTTTGATATTAAATCCGGAG; 
               
               
                   
                   
               
               
                   
                 F129: 
               
               
                   
                 (SEQ ID NO: 64) 
               
               
                   
                 TACTTATATACACTGGGTTCGCCAGGCTCCTGGA; 
               
               
                   
                   
               
               
                   
                 R150: 
               
               
                   
                 (SEQ ID NO: 65) 
               
               
                   
                 CCACTCCAGACCCTTTCCAGGAGCCTGG; 
               
               
                   
                   
               
               
                   
                 F163: 
               
               
                   
                 (SEQ ID NO: 66) 
               
               
                   
                 AAGGGTCTGGAGTGGGTGGCGAGAATCTACC; 
               
               
                   
                   
               
               
                   
                 R178: 
               
               
                   
                 (SEQ ID NO: 67) 
               
               
                   
                 GGGTATAACCATTGGTTGGGTAGATTCTCGCCAC; 
               
               
                   
                   
               
               
                   
                 F194: 
               
               
                   
                 (SEQ ID NO: 68) 
               
               
                   
                 CAACCAATGGTTATACCCGCTATGCAGACAGCG; 
               
               
                   
                   
               
               
                   
                 R212: 
               
               
                   
                 (SEQ ID NO: 69) 
               
               
                   
                 GTAAACCGCCCTTTCACGCTGTCTGCATAGC; 
               
               
                   
                   
               
               
                   
                 F227: 
               
               
                   
                 (SEQ ID NO: 70) 
               
               
                   
                 TGAAAGGGCGGTTTACAATTAGTGCCGACACA; 
               
               
                   
                   
               
               
                   
                 R243: 
               
               
                   
                 (SEQ ID NO: 71) 
               
               
                   
                 GGTAAGCGGTATTTTTAGATGTGTCGGCACTAATT; 
               
               
                   
                   
               
               
                   
                 F259: 
               
               
                   
                 (SEQ ID NO: 72) 
               
               
                   
                 TCTAAAAATACCGCTTACCTCCAGATGAACTCTCTG; 
               
               
                   
                   
               
               
                   
                 R278: 
               
               
                   
                 (SEQ ID NO: 73) 
               
               
                   
                 TGTCCTCGGCCCTCAGAGAGTTCATCTGGA; 
               
               
                   
                   
               
               
                   
                 F295: 
               
               
                   
                 (SEQ ID NO: 74) 
               
               
                   
                 AGGGCCGAGGACACGGCTGTGTATTATTGC; 
               
               
                   
                   
               
               
                   
                 R308: 
               
               
                   
                 (SEQ ID NO: 75) 
               
               
                   
                 CACCCCACCGGCTGCAATAATACACAGCCG; 
               
               
                   
                   
               
               
                   
                 F325: 
               
               
                   
                 (SEQ ID NO: 76) 
               
               
                   
                 AGCCGGTGGGGTGGAGACGGATTCTATGCT; 
               
               
                   
                   
               
               
                   
                 R338: 
               
               
                   
                 (SEQ ID NO: 77) 
               
               
                   
                 TGACCCCAATAGTCCATAGCATAGAATCCGTCTC; 
               
               
                   
                   
               
               
                   
                 F355: 
               
               
                   
                 (SEQ ID NO: 78) 
               
               
                   
                 ATGGACTATTGGGGTCAGGGCACTCTCGTCA; 
               
               
                   
                   
               
               
                   
                 R372: 
               
               
                   
                 (SEQ ID NO: 79) 
               
               
                   
                 TGGCACTGCTTACAGTGACGAGAGTGCCC; 
               
               
                   
                   
               
               
                   
                 F386: 
               
               
                   
                 (SEQ ID NO: 80) 
               
               
                   
                 CTGTAAGCAGTGCCAGCACAAAGGGGCC; 
               
               
                   
                   
               
               
                   
                 R401: 
               
               
                   
                 (SEQ ID NO: 81) 
               
               
                   
                 CAAGGGGAAAGACACTAGGCCCCTTTGTGC; 
               
               
                   
                   
               
               
                   
                 F414: 
               
               
                   
                 (SEQ ID NO: 82) 
               
               
                   
                 TAGTGTCTTTCCCCTTGCTCCATCTAGCAAATCTAC; 
               
               
                   
                   
               
               
                   
                 R431: 
               
               
                   
                 (SEQ ID NO: 83) 
               
               
                   
                 GGTGCCCCCGCTGGTAGATTTGCTAGATGGAG; 
               
               
                   
                   
               
               
                   
                 F450: 
               
               
                   
                 (SEQ ID NO: 84) 
               
               
                   
                 CAGCGGGGGCACCGCCGCCCTGGGAT; 
               
               
                   
                   
               
               
                   
                 R463: 
               
               
                   
                 (SEQ ID NO: 85) 
               
               
                   
                 GTCCTTGACCAGGCATCCCAGGGCGGC; 
               
               
                   
                   
               
               
                   
                 F476: 
               
               
                   
                 (SEQ ID NO: 86) 
               
               
                   
                 GCCTGGTCAAGGACTATTTTCCTGAGCCAGT; 
               
               
                   
                   
               
               
                   
                 R490: 
               
               
                   
                 (SEQ ID NO: 87) 
               
               
                   
                 TCCAGGACACGGTGACTGGCTCAGGAAAATA; 
               
               
                   
                   
               
               
                   
                 F507: 
               
               
                   
                 (SEQ ID NO: 88) 
               
               
                   
                 CACCGTGTCCTGGAATAGTGGCGCCTTGA; 
               
               
                   
                   
               
               
                   
                 R521: 
               
               
                   
                 (SEQ ID NO: 89) 
               
               
                   
                 TGTGTGAACACCAGAAGTCAAGGCGCCACTAT; 
               
               
                   
                   
               
               
                   
                 F536: 
               
               
                   
                 (SEQ ID NO: 90) 
               
               
                   
                 CTTCTGGTGTTCACACATTTCCCGCCGTCC; 
               
               
                   
                   
               
               
                   
                 R553: 
               
               
                   
                 (SEQ ID NO: 91) 
               
               
                   
                 CAGCCCACTAGATTGAAGGACGGCGGGAAA; 
               
               
                   
                   
               
               
                   
                 F566: 
               
               
                   
                 (SEQ ID NO: 92) 
               
               
                   
                 TTCAATCTAGTGGGCTGTACTCTCTCTCCAGTGT; 
               
               
                   
                   
               
               
                   
                 R583: 
               
               
                   
                 (SEQ ID NO: 93) 
               
               
                   
                 TGGGTACCGTCACCACACTGGAGAGAGAGTA; 
               
               
                   
                   
               
               
                   
                 F600: 
               
               
                   
                 (SEQ ID NO: 94) 
               
               
                   
                 GGTGACGGTACCCAGTTCAAGCTTGGGCA; 
               
               
                   
                   
               
               
                   
                 R614: 
               
               
                   
                 (SEQ ID NO: 95) 
               
               
                   
                 TGCAGATATAGGTCTGTGTGCCCAAGCTTGAAC; 
               
               
                   
                   
               
               
                   
                 F629: 
               
               
                   
                 (SEQ ID NO: 96) 
               
               
                   
                 CACAGACCTATATCTGCAATGTGAACCACAAGCC; 
               
               
                   
                   
               
               
                   
                 R647: 
               
               
                   
                 (SEQ ID NO: 97) 
               
               
                   
                 CCACCTTTGTATTGCTGGGCTTGTGGTTCACAT; 
               
               
                   
                   
               
               
                   
                 F663: 
               
               
                   
                 (SEQ ID NO: 98) 
               
               
                   
                 CAGCAATACAAAGGTGGACAAAAAAGTCGAGCCT; 
               
               
                   
                   
               
               
                   
                 R680: 
               
               
                   
                 (SEQ ID NO: 99) 
               
               
                   
                 TGTCACAGCTCTTTGGAGGCTCGACTTTTTTGT; 
               
               
                   
                   
               
               
                   
                 F697: 
               
               
                   
                 (SEQ ID NO: 100) 
               
               
                   
                 CCAAAGAGCTGTGACAAAACTCACACATGCCC; 
               
               
                   
                   
               
               
                   
                 R713: 
               
               
                   
                 (SEQ ID NO: 101) 
               
               
                   
                 TACCTGGGCACGGTGGGCATGTGTGAGTTT; 
               
               
                   
                   
               
               
                   
                 F729: 
               
               
                   
                 (SEQ ID NO: 102) 
               
               
                   
                 ACCGTGCCCAGGTAAGCCAGCCCAGGC; 
               
               
                   
                   
               
               
                   
                 R743: 
               
               
                   
                 (SEQ ID NO: 103) 
               
               
                   
                 ccccattgactTTTTTTTAGGCCTGGGCTGGCT; 
               
               
                   
                   
               
               
                   
                 R756: 
               
               
                   
                 (SEQ ID NO: 104) 
               
               
                   
                 CTAAAAAAAagtcaatgggg. 
               
            
           
         
       
     
     The BglII site was introduced using PCR with forward primer F0 and a reverse primer containing a BglII restriction site (sequences shown below). Forward: tcaaagcagctaagAAAAAAAGAATTCGCTCCAAACAG (SEQ ID NO:56); Reverse: tttttttAGATCTCTTTGGAGGCTCGACTTTTTTGT (SEQ ID NO:105). 
     The gene encoding V H  and C H 1 was inserted into a vector comprising the crystallizable fragment (Fc) domain (i.e. C H 2 and C H 3 domains) of human IgG1 heavy chain (pinfuse-higG1-fc2 from Invivogen) at the EcoRI and BglII restriction sites. A Quikchange site-directed mutagenesis kit was used to generate the desired AKT N-terminal mutant. Incorporation of these mutations was verified by sequencing. 
     Procedure for Expression and Purification of Mutant Antibodies 
     The plasmids for the light and heavy chains of the anti-HER2 antibody were transiently co-transfected into human embryonic kidney 293T cells in a 3:2 ratio using lipofectamine 2000 (Invitrogen, Grand Island, N.Y.) in Opti-MEM medium following the protocol from Invitrogen. The cells were incubated at 37° C. in 5% CO 2 . After two days, the media was collected and the secreted antibodies were purified using protein G affinity chromatography, according to the procedure from the manufacturer (Pierce, Rockford, Ill.). The media was replaced and cultures were grown for an additional 3 days, after which the additional antibodies were harvested and purified as above. Purified protein was buffer exchanged into PBS using Amicon Ultra 4 mL 10,000 MWCO (Millipore) centrifugal ultrafiltration membranes. Purity was evaluated by SDS-PAGE with Coomassie staining. 
     Procedure for PLP N-Terminal Transamination 
     The 2× protein stock solutions were prepared at 1-40 μM using 25 mM phosphate buffer at pH 6.5. The 2× (200 mM) PLP stock solutions were prepared in 25 mM phosphate buffer and the pH of the solution was adjusted to 6.5 using NaOH solution. Protein and PLP stock solutions were mixed in equal volumes. The reaction mixture was briefly agitated to ensure mixing and then incubated without further agitation at 37° C. for 1 h. After incubation, the PLP was removed using NAP Sephadex size exclusion columns (GE Healthcare). The resulting keto-antibody solution was then concentrated and the buffer was exchanged with 25 mM phosphate buffer (pH 6.5), using Millipore 0.5 or 4 mL spin concentrators (MWCO 10 kDa), following the protocol from the manufacturer. 
     Procedure for Hydrazone and Oxime Formation 
     The reaction was performed with 1-40 μM keto-antibody and RONH 2  or R(CO)NHNH 2  at varied concentrations. For the analysis of PLP transamination efficiency, BnONH 2  were added to keto-antibody to a final concentration of 100 mM. For the attachment of an oxidative coupling partner, aniline-ONH 2  was added to a final concentration of 25 mM. The reaction mixture was incubated at RT for 18-50 h. All the reactions were carried out in 25 mM phosphate buffer (pH 6.5), except for oxime formation with aniline-ONH 2 , which was done in 25 mM phosphate buffer (pH 5). Following the reaction, the small molecules were removed using NAP Sephadex size exclusion columns (GE Healthcare) and the resulting product mixtures were concentrated using Millipore 0.5 or 4 mL spin concentrators (MWCO 10 kDa), following the protocol from the manufacturer. The percent reaction conversion for the antibody samples with small molecules was analyzed using LCMS. 
     Procedure for Lysine Modification 
     The reaction was performed with 1-40 μM antibody in phosphate buffer pH 8 and R—NHS ester at varied concentrations. To make antibody-aniline conjugates, a mixture of 3-(4-aminophenyl) propionic acid, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was added to the wild-type antibody to a final concentration of 2.5 mM, and the solution was incubated at RT for 1 h. The reaction was quenched upon adding excess hydroxylamine and the small molecules were removed using NAP Sephadex size exclusion columns (GE Healthcare). The resulting antibody-aniline conjugates were concentrated using Millipore 0.5 or 4 mL spin concentrators (MWCO 10 kDa). 
     Procedure for Oxidative Coupling 
     To a solution of 1-40 μM antibody-aniline in 25 mM phosphate buffer (pH 6.5) was added a solution of 5-100 equivalents of aminophenol-aptamer or aminophenol-2k PEG. Sodium periodate (Sigma-Aldrich) was dissolved to a concentration of 10 mM in 25 mM phosphate buffer (pH 6.5). The sodium periodate was then added to the reaction mixture to reach a final concentration of 1 mM, and the reaction was allowed to proceed for 2-5 min at RT. In some cases, a solution of mannose was also added to a final concentration of 10 mM or 100 mM before addition of the periodate solution. The resulting protein samples were purified on NAP Sephadex size exclusion columns (GE Healthcare) and concentrated using Millipore 0.5 or 4 mL spin concentrators (MWCO 10 kDa), following the protocol from the manufacturer. 
     Purification of Antibody-Aptamer Constructs 
     The resulting antibody-DNA conjugates from both N-terminal and lysine modification were purified from unreacted DNA using sized exclusion FPLC and the fractions containing antibody-DNA constructs were combined and concentrated using Millipore 0.5 or 4 mL spin concentrators (MWCO 10 kDa). 
     Flow Cytometry Experiments 
     Flow cytometry was used to determine binding ability of all the antibody-aptamer constructs. For all samples, 100 μL of 1-3×10 6  cells/mL of Jurkat, Ramos, and MCF7 clone 18 were used, suspended in binding buffer (4.5 g/L glucose, 5 mM MgCl 2 , 0.1 mg/mL yeast tRNA (Sigma) and 1 mg/mL BSA (Fisher) in Dulbecco&#39;s PBS with calcium chloride and magnesium chloride (Invitrogen)). To these cells were added 10 μL of a series of 2-200 nM antibody-aptamer construct solutions. The samples were then incubated on ice for 45 min. The resulting cells were washed with 500 μL of binding buffer and resuspended in an additional 100 μL of binding buffer. Anti-human IgG1 antibody (specific for the Fc domain) with FITC conjugated (Sigma) was then added to a final concentration of ˜0.30 μM. Cells were incubated for 30 min on ice in the dark, then washed with 500 μL of binding buffer, and resuspended in 200 μL binding buffer. The cells were analyzed by flow cytometry to determine the amount of FITC fluorescence. For each sample, 10,000 cells were counted. 
     Results 
     The results are depicted in  FIGS. 11A and 11B , and in  FIGS. 12A and 12B . 
     Anti-HER2 human IgG1 antibodies were functionalized with DNA aptamers both through N-terminal modification and through lysine modification ( FIG. 11A-B ). The production of constructs was confirmed by SDS-PAGE analysis ( FIG. 12A ). The binding specificity of antibody-DNA aptamer conjugates was determined by flow cytometry. Both the antibody and the DNA aptamer components of the conjugates retained their binding specificities to the target cells ( FIG. 12B ). Jurkat (a target cell line of sgc8c aptamer) and Ramos (a target cell line of TD05.1 aptamer) cells were used to confirm the binding specificities of the aptamer moieties of the antibody-aptamer conjugates. Various anti-HER2 antibody-aptamer conjugates were also shown to bind to the MCF7 clone 18 cell line that overexpresses HER2 receptors. 
     Examples of amino acid sequences of anti-HER2 heavy and light chains, and nucleotide sequence encoding same, are presented in  FIGS. 13A-C . 
     While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.