Patent Application: US-201313739793-A

Abstract:
the invention generally relates to the field of immunochemistry including antibody therapy , diagnostics , and basic research and specifically relates to the area of alternatives to natural antibodies including artificial antibodies or antibody mimics . the invention relates particularly to the cooperative assembly of stable affinity complexes .

Description:
in some embodiments , the present invention provides compositions , systems , and methods related to affinity complexes . in some embodiments , the present invention provides compositions , systems , and methods related to affinity complexes configured to form in a cooperative and dynamic process in the presence of a target . in some embodiments , a target contains two or more discontinuous epitopes to which the affinity complex interacts . in some embodiments , the affinity complex comprises or consists of three or more components : ( 1 ) a primary affinity molecule that interacts with one epitope of the target , ( 2 ) a second primary affinity molecule that interacts with a second epitope of the same target , and ( 3 ) a linker that interacts with the 2 primary affinity molecules . in some embodiments , two or more primary affinity molecules bind to epitopes on a target molecule . in some embodiments , the epitopes are discontinuous . in some embodiments , the primary affinity molecules recognize the same epitopes . in some embodiments , the primary affinity molecules recognize different epitopes . in some embodiments , a linker affinity molecule comprises two or more secondary affinity domains . in some embodiments , a linker affinity molecule comprises two or more secondary affinity domains connected by one or more flexible linkers . in some embodiments , each secondary affinity domain of the linker affinity molecule recognizes and binds to a primary affinity molecule of the present invention . in some embodiments , secondary affinity domains on the linker affinity molecule recognize the linker interaction domain of the primary affinity molecules . in some embodiments , two or more primary affinity molecules of the invention have the same linker interaction domains . in some embodiments , two or more primary affinity molecules of the invention have different linker interaction domains . in some embodiments , linker affinity molecules of the present invention bind to two or more primary affinity molecules through interactions between secondary affinity domains and linker interaction domains . in some embodiments , the cooperativity of the complex of interactions of the present invention increases the specificity of target recognition and binding . in some embodiments , the cooperativity of the complex of interactions of the present invention increases the affinity and strength of target recognition and binding . in some embodiments , the present invention comprises a more complex set of interactions , as described below . in some embodiments , an affinity complex of the present invention provides recognition of multiple targets . in some embodiments , an affinity complex of the present invention provides multiple different linker affinity molecules . in some embodiments , an affinity complex of the present invention provides linker affinity molecules that recognize and bind multiple different primary affinity molecules . in some embodiments , an affinity complex of the present invention provides linker affinity molecules which recognize and bind multiple different linker interaction domains . in some embodiments , primary affinity molecule comprises or consists of a scaffold that has a target epitope interaction domain or region known as a paratope , and a linker interaction domain or region . in some embodiments , the paratope and linker interaction domain are situated to allow interaction with a target epitope and a linker molecule . in some embodiments , each primary affinity molecule can comprise or consist of the same scaffold . in some embodiments , each primary affinity molecule can comprise or consist of different scaffolds . in some embodiments , primary affinity molecules can be any antibody , antibody fragment , scaffold or molecular construct that has a paratope domain or region and a linker interaction domain or region . for example , igg antibodies known to interact with a single target at two discontinuous epitopes can used with a linker molecule that interacts with each fc domain of the igg ( such as protein a or g ). the thermodynamic kinetics for this complex should take into account that each igg antibody contains 2 binding domains . in some embodiments , fab fragments of an igg antibody are employed as the primary affinity molecule . in embodiments where , fab fragments of an igg antibody are employed as the primary affinity molecule , the preferred linker molecule would interact preferentially with the constant domains ( cl and ch1 ) of the molecule . in some embodiments , single chain fragments of the variable domains ( scfv ) are employed due to their increased stability . in some embodiments , the smaller size of the vhh domain of camelids ( nanobodies ) is a preferred affinity molecule . in some embodiments , the primary affinity molecule is a monobody ( fibronectin type iii domain ) derived from a human cell surface protein . this scaffold is structurally similar to antibody variable domains , but does not contain disulfide bonds that can hinder expression in prokaryotic systems . in some embodiments , monobodies have a molecular weight of ˜ 10 , 000 daltons , they are very soluble , and thermally and proteolytically stable . in some embodiments , the monobody scaffold contains three loops ( bc , de , and fg loops ) that can be collectively employed as a paratope , similar to the cdr regions of an immunoglobulin . the polar opposite end of the paratope region contains three additional loops ( ab , cd , and ef loops ). in some embodiments , the ab , cd , and ef loops can be employed as interaction domains of the secondary affinity reagent . in some embodiments , monobodies are the secondary affinity molecules of the present invention . in some embodiments , monobodies serving as secondary affinity molecules can be linked as described above , or expressed as a single polypeptide with the c - terminus of one monobody linked to the n - terminus of the other monobody using a glycine / serine linker . in some embodiments , other linkers can be used , such as an abbreviated rpeg . in some embodiments , the primary affinity molecule is a darpin ( designed ankyrin repeat protein ) that is derived from a large class of repeat proteins found in various cellular sections in a variety of species . each repeat consists of 33 amino acid residues that form a beta - turn followed by two anti - parallel helices and a randomized loop that is joined to the beta - turn of the next repeat and functions to “ stack ” the repeats generating a very stable hydrophobic core . in some embodiments , the loop and beta - turn sequences are involved in the paratope of the molecule . in some embodiments , residues of the helices can contribute to the paratope . in some embodiments , the combination of the loop and beta - turn sequences and the residues of the helicies generate a broad paratope interface . in some embodiments , three or more of these repeats are created to generate a molecule with very high affinity . in some embodiments , the ends of the repeats are “ capped ” to preserve the hydrophobic core , increase its solubility and stability , and can be used for labeling or immobilization . in some embodiments , n - terminal and c - terminal caps are employed as interaction domains of the secondary affinity reagent . in some embodiments , the primary affinity molecule is an affibody ( the z domain of staphylococcal protein a ) that comprises or consists of 58 amino acids arranged as a bundle of 3 anti - parallel alpha helices . in some embodiments , the small size of the affibody molecule provides easier expression and solubility in prokaryotic systems . in some embodiments , the affibody polypeptide is chemically synthesized then folded , which allows the introduction of non - canonical amino acids in the interaction domain or the addition of labels or reactive groups . in some embodiments , the interaction domain comprises or consists of 13 amino acid residues that are randomized to generate a library from which an affinity molecule is panned . in some embodiments , binding affinities for affibodies and their substrates are in the nanomolar range . in some embodiments , the primary affinity molecule is a microbody ( nascacell technologies ). in some embodiments , a microbody is based on natural cysteine - knot microproteins and cyclical knottins . in some embodiments , microbodies are small ( 28 to 45 amino acids ), yet very stable due to three disulfide bonds within the structure , which allows the display of a single peptide loop up to 20 amino acids . in some embodiments , microbodies are very soluble and are expressed from bacteria or synthesized by chemical means then properly folded . in some embodiments , the stability and solubility of these proteins provides alternative therapeutic delivery modes to the standard injection of most biologicals . in some embodiments , a very similar molecule called a versabody ( amunix ), acts as a primary affinity molecule . in some embodiments , a versabody is a very small high disulfide density scaffold based on natural biopharmaceuticals , such as scorpion toxin . versabodies are extremely stable , soluble , and non - immunogenic . in some embodiments , the primary affinity molecule is an anticalin , an avimer or the domain a of an avimer , a thioredoxin , an ubiquitin , a gamma - crystallin , ctla - 4 ( evibody ), or other recombinant artificial antibodies . in some embodiments , a primary affinity molecule is any molecule capable of binding a target with a suitable affinity . in some embodiments , primary affinity molecules have an auxiliary interaction domain or region added to their structure that allows the binding of an auxiliary functional group ( e . g . detectable label , radioisotope , drug , toxin , enzyme or other moiety ). in some embodiments , for example , the c - terminus of a fibronectin type iii scaffold is extended with the nano - tag peptide sequence that binds streptavidin with nanomolar affinity and labeled streptavidin conjugates added to bind the affinity molecules . in some embodiments , an auxiliary interaction domain is added to a linker molecule of the present invention . in some embodiments , the linker molecule comprises or consists of two or more ( e . g . 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , . . . , 30 , . . . , etc .) linked secondary affinity molecules that interact with the interaction domains of a primary affinity molecule . in some embodiments , the configurational cooperativity or avidity of the complex assembly is dependent on the affinity of the secondary affinity molecules for the primary affinity molecules . in some embodiments , the configurational cooperativity or avidity of the complex assembly is dependent on the degree of freedom of each affinity molecule . in some embodiments , the secondary affinity molecules of the linker molecule are covalently linked via a flexible polymer such as a polypeptide ( e . g . glycine / serine polypeptides ), a nucleic acid strand , polyethylene glycol , and peptide nucleic acid ( pna ) that has sufficient degree of freedom to allow the interaction of the secondary affinity molecules with the primary affinity molecules . in some embodiments , the secondary affinity molecules of the linker molecule are linked , either directly or linked via a suitable linker . the present invention is not limited to any particular linker group . indeed , a variety of linker groups are contemplated , suitable linkers could comprise , but are not limited to , alkyl groups , ether , polyether , alkyl amide linker , a peptide linker , a modified peptide linker , a poly ( ethylene glycol ) ( peg ) linker , a streptavidin - biotin or avidin - biotin linker , polyaminoacids ( eg . polylysine ), functionalised peg , polysaccharides , glycosaminoglycans , dendritic polymers such as described in wo93 / 06868 and by tomalia et al . in angew . chem . int . ed . engl . 29 : 138 - 175 ( 1990 ), peg - chelant polymers such as described in w94 / 08629 , wo94 / 09056 and wo96 / 26754 , oligonucleotide linker , phospholipid derivatives , alkenyl chains , alkynyl chains , disulfide , or a combination thereof . in some embodiments the linker comprises a single chain connecting secondary affinity molecule to a second secondary affinity molecule . in some embodiments , there are multiple linkers connecting secondary affinity molecules to a single secondary affinity molecule . in some embodiments , a linker may connect multiple secondary affinity molecules to each other . in some embodiments , a linker attaches an additional functional portion to secondary affinity molecule . in some embodiments , a linker may be branched , connecting more than two secondary affinity molecules . in some embodiments , the linker may be flexible , or rigid . in some embodiments , the linker of the present invention is cleavable or selectively cleavable . in some embodiments , the linker is cleavable under at least one set of conditions , while not being substantially cleaved ( e . g . approximately 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 99 %, or greater remains uncleaved ) under another set ( or other sets ) of conditions . in some embodiments , the linker is susceptible to enzymatic cleavage ( e . g . proteolysis ). in some embodiments , the enzymatic cleavage is site specific ( e . g . sequence specific ). in some embodiments , the enzymatic cleavage is at a random site along the linker . in some embodiments , the enzymatic cleavage may occur at multiple random sites along the linker . in some embodiments , the linker is susceptible to cleavage under specific conditions relating to ph , temperature , oxidation , reduction , uv exposure , exposure to radical oxygen species , chemical exposure , light exposure ( e . g . photo - cleavable ), etc . in some embodiments , an affinity complex of the present invention provides high avidity binding and greater specificity than do the interactions of the two discontinuous target epitopes . as a result , primary affinity molecules with moderate affinities (& gt ; 10 nm ) are preferred . in some embodiments , linker molecules that can form a complex with primary affinity molecules are be prepared by any suitable method . in some embodiments , igg antibodies are used as affinity molecules , and anti - fc antibodies or fab fragments can be linked via their reduced thiol groups , preferably using a crosslinking system from solulink . in some embodiments , one part of the anti - fc igg / fab is labeled with mhph ( 3 - n - maleimido - 6 - hydraziniumpyridine hydrochloride ) and the other part with mtfb ( maleimido trioxa - 6 - formyl benzamide ). in some embodiments , the hydrazine moiety of the mhph - modified molecules react with 4 - formylbenzamide of the mtfb - modified molecules to form stable bis - arylhydrazone - mediated conjugates . in some embodiments , alternative methods for crosslinking proteins , known to those skilled in the art , are utilized . in some embodiments , an oligonucleotide can be synthesized with chemically reactive moieties ( for example , a maleimide ) on each end that would react with the secondary affinity molecule . in some embodiments , each molecule could be conjugated to an oligonucleotide , one with a 3 ′ is free and the other with a 5 ′ free so that the two strands can be ligated . in some embodiments , any suitable methods to link the secondary affinity molecules would also be appropriate so long as the linker has flexibility to allow interaction of the secondary affinity molecules . in some embodiments , an affinity complex comprises a quaternary system with four components : a target , two primary affinity molecules and a secondary affinity molecule . in some embodiments , more intricate affinity complex designs , involving more than 4 components are used ( e . g . 5 components , 6 components , 7 components , 8 components , 9 components , 10 components , 11 components , 12 components , 13 components , 14 components , 15 components , 16 components , 17 components , 18 components , 19 components , 20 components , & gt ; 20 components , etc .). in some embodiments , a quinary system ( i . e . 5 components ) could consist of a target , 2 primary affinity molecules , and 2 secondary affinity molecules ( linker affinity molecules ) that have domains that can freely interact . an additional affinity molecule that labels the complex , binding to either primary affinity molecule or the linker with its secondary affinity molecules , provides another example of a quinary system . in some embodiments , 3 primary affinity molecules that interact with 3 discontinuous epitopes of the target and are assembled with a secondary linker that interacts with each primary affinity molecule . the complexity and component possibilities are significantly amplified with senary ( 6 components ), septenary ( 7 components ), octonary ( 8 components ), nonary ( 9 components ), and denary 10 components ) systems . in some embodiments , a linker complex in a quinary system is heterodimeric helical coiled - coil domains , where one of the affinity molecules has a k - coil , preferable to the n - or c - terminus , while the other affinity molecule has an e - coil attached or fused . the multimer heptad k - coil naturally forms an alpha helix and aligns the positive charges of the lysine ( k ) amino acids on one side of the coil . the multimer heptad e - coil does the same , except it aligns the negative charges of the glutamic acid ( e ) amino acids . the positive charges of the k - coil form salt bridges with the negative charges of the e - coil and the binding interaction strength is determined by the number of heptads ( number of salt bridges ). in some embodiments , a linker complex is a single stranded dna covalently attached to each secondary affinity molecule . in some embodiments , the free end of the dna strand of one affinity molecule would be complementary to the free end of the other affinity molecule . in some embodiments , hybridization of the complementary dna strands would provide formation of the linker . in some embodiments , protein : oligonucleotide conjugate linkers can be prepared by many different methods ( e . g . by labeling the protein with a moiety that reacts with a moiety placed on the end of the synthetic oligonucleotide ). in some embodiments , the thermodynamics of the affinity complex assembly are an important aspect of the present invention . fig2 represents a thermodynamic cycle of a quaternary system where the primary affinity molecules interact with the epitopes of a target and also with the linker . association and dissociation constants of each interaction are represented by k a and k d , respectively , and together determine the affinity ( k d ) of the interaction . the free energy of formation is symbolized by ag ° and is equal to − rtlnk d . each interaction contributes to the formation of the complex in a cooperative manner , which results in an interaction called avidity that corresponds to coupling of the free energies , or δδg . in some embodiments , the affinity constants within a complex are also dependent on molecular “ stress ” of the system , including the intramolecular flexibility of the components , intermolecular alignment within the complex , and steric hindrance in binding . all of these parameters contribute to the δδg . in some embodiments , the binding interaction of a paratope to its epitope is based upon a combination of molecular contacts that together account for the affinity strength ( e . g . van der waals interactions , hydrogen bonding , and hydrophobic interactions ), specific amino acid side groups of the paratope polypeptide form bonds with amino acid side groups of the epitope polypeptide . in some embodiments , a portion of the amino acids in the paratope function as structural support . in some embodiments , antibody mimics have a single polypeptide paratope , such as affibodies and versabodies . in these embodiments , the sum of those interactions determines the affinity . in some embodiments , affinity molecules comprise multiple polypeptide loops or cdrs ( complementarity determining regions ), such as fibronectin type iii domains , ankyrin repeats , and igg molecules . these embodiments demonstrate additional number and spacing of those interactions . in some embodiments , the structure of the paratope should be adaptable to fit the epitope . in some embodiments , the paratope has enough flexibility to form bonds with the epitope without introducing intramolecular strain . in some embodiments , a large number of affinity molecules are be screened ( e . g ., in a binding assay ) to achieve a suitable structure . in some embodiments , preparing and panning affinity molecules from translated dna libraries to identify those with high affinity and specificity carried out as part of the creation of an affinity mimic . there are numerous methods for performing this function . in some embodiments , preparing and panning the affinity mimic is linked to its genetic code in by some means . some embodiments use in vivo display techniques , such as phage , yeast , and bacterial display , where the affinity molecule is “ displayed ” on the surface of a cell or virus based on the genetic code inside . in some embodiments , in vitro display methods that use translation cocktails , such as s30 , wheat germ , or rabbit reticulocyte lysates , to express the affinity polypeptide , while retaining its association to the nucleic acid , are used . in some embodiments , ribosome display is used . in ribosome display , the ribosome structure is the connection between the mrna and polypeptide while with rna display it is a linker attached to the 3 ′ end of the mrna that attaches to the polypeptide . in some embodiments dna display is used . for dna display , a polypeptide is co - expressed with the affinity molecule that specifically binds to the dna coding region that transcribed its mrna . in some embodiments , one of several in vivo techniques that express both the antigen ( target ) and the affinity binder within the cell and the binding events are detected by activation of a reporter gene ( two - hybrid techniques ) or by protein - fragment complementation assay . in some embodiments , the size and diversity of the coding library that expresses the affinity mimics are a key to the method used . in some embodiments , numerous rounds of affinity molecule “ enhancement ” using sophisticated gene sequence shuffling is required to achieve high affinity binding interactions . in some embodiments , only moderate affinity interactions are required . in some embodiments , only moderate affinity interactions are preferred . in embodiments , increased effectiveness of screening libraries is achieved when moderate affinity is sought . in some embodiments , binary - or tertiary - code library systems reduce the size of the libraries , increase their effectiveness , and further simplify the process . in some embodiments , the basis of the binary - code interface within affinity molecules is that effective affinity binders can be generated by using only 2 amino acids , tyrosine and serine ( e . g . fibronectin type iii domains that were developed using the tyr / ser binary - code interface demonstrated affinities to 3 different proteins of 5 to 90 nm ( koide , a ., et al ., proc . nat . acad . sci . 104 , 6632 - 6637 , herein incorporated by reference in its entirety )). in some embodiments , a nanomolar affinity level , which can be achieved in binary - code interface , is very effective in an affinity complex where the binding affinities are multiplied by the linkage of the affinity molecules . in some embodiments , the combination of a simplified binary - code interface library system and a cooperative affinity complex system greatly reduces the time and resources necessary to development high affinity and specific affinity complexes . in some embodiments , binding interactions are observed by labeling the components ( e . g . with small molecule fluorescent probes ). in some embodiments , fluorescent molecules with distinct fluorescence characteristics ( excitation and emission ) and reactive moieties for covalent linkage to proteins can be utilized to label proteins ( e . g . alexa fluors ( invitrogen ), cydye fluors ( ge healthcare life sciences ), dylight fluors ( dyomics gmbh ), hilyte fluors ( anaspec ) and the irdye near infrared fluors ( li - cor )). in some embodiments , fluorophores are linked to either a nhs ester reactive group ( reacts with ε - amine of lysine and the α - amine of the polypeptide n - terminal ) or a maleimide reactive group ( reacts with reduced sulfhydryl of cysteine ). in some embodiments , labeling proteins non - specifically , especially small polypeptides can potentially interfere with their function . in some embodiments , it is important to demonstrate no loss of utility of the affinity molecule . in some embodiments , if the affinity molecule does not have a cysteine in the polypeptide sequence ( such as an affibody or fibronectin scaffold ), a cysteine can be introduced at the c - terminal and specifically labeled with any maleimide fluorophore . in some embodiments , the affinity molecules and linker are each covalently labeled with a distinct fluorescent molecule , each with different fluorescent properties , so that each could be detected simultaneously , to determine binding of each component . in some embodiments , the target is immobilized on a surface ( e . g . plastic , glass , nitrocellulose , etc . ), the surface blocked to reduce non - specific binding , and the components of the affinity complex allowed to bind . in some embodiments , the binding surface is scanned by a fluorometer ( plate or slide reader , or digital camera imager ) to detect the fluorescence . in some embodiments , a fluorescent bead format , where the target is immobilized onto the surface of a bar - coded bead and the binding events detected by flow cytometry , is utilized . in some embodiments , the formation of the affinity complex is detected using fret ( fluorescence resonance energy transfer ) or bret ( bioluminescence resonance energy transfer ). fret and bret technologies are homogeneous assays , in which the target does not need to be immobilized . in some embodiments , when using fret , one of the primary affinity molecules is labeled with a donor fluorophore and either the other primary affinity molecule or the linker is labeled with an acceptor fluorophore . the fluorophores could be either fluorescent dyes or proteins . excitation of the donor fluorophore with a specific wavelength of light that does not excite the acceptor fluorophore can allow the donor to transfer its potential energy ( called forester resonance energy ) to the acceptor molecule if it is within 10 nm of the donor . in some embodiments , when using bret , the enzymatic activation of renilla luciferase ( using a coelenterazine luciferin ) is used as the energy source that is transferred to the acceptor , a fluorescent protein . in some embodiments , steric hindrance of large biomolecules could interfere with affinity interactions when using fret or bret . in some embodiments , detection of affinity complexes does not require affinity labels ( e . g . surface plasmon resonance spectroscopy ( spr ), atomic force microscopy ( afm ) or quartz crystal microbalance ( qcm )). in some embodiments , the target is immobilized onto a surface , affinity reagents are added to the surface , and the affinity complex forms on the surface at the target . in some embodiments , binding of component molecules is observed and differentiated from binding of the entire complex . in embodiments using spr , the addition of mass to the surface is detected by a change in the resonance angle , which is a function of the refractive index of the area immediately above the surface . in embodiments using afm , a high resolution scanning probe technique physically measures minute structural changes of a surface using a cantilever . in embodiments using qcm , inorganic films are measured on surfaces . in some embodiments , a continuous - flow qcm system is used for the detection of biological interactions . in some embodiments , a microbalance is a piezoelectric quartz crystal that oscillates at a resonance frequency and this frequency changes with the addition of molecules to the surface of the crystal . in some embodiments , binding can be detected in real time as a solution containing the affinity reagents streams over the surface containing the target molecule . in some embodiments , affinity binders are identified from very large dna libraries using established panning methods . in some embodiments , affinity molecules are very stable , are easily modified or conjugated , and have low immunogenicity . in some embodiments , the compositions and method of embodiments of the invention are ideally suited for scaffold - based affinity therapeutics . all of the attributes afforded single domain affinity reagents are applicable to affinity complexes , but with improved binding affinity and specificity . in some embodiments , the small size of the components allows more rapid tissue penetration from the circulatory system and the lower affinities of the primary affinity molecules allows deeper penetration into , for example , a tumor ( thurber , et al . adv drug deliv . rev . 60 , 1421 - 34 ). in some embodiments , the parameters of molecular size , binding affinity , clearance ( both systematic and endocytic ), and dosing effect tumor penetration . in some embodiments , small affinity molecules are highly soluble and allow high dosing of the therapeutic . in some embodiments , an optimum linker dose balances the thermodynamics of forming the affinity complex with penetration into a tissue . in some embodiments , systematic clearing of the small molecules is attenuated by including albumin or fc binding domains to the molecules . in some embodiments , aspects of uniform therapeutic coverage are important for drug delivery , and for improved tumor or tissue imaging . the versatility of embodiments of the invention as a therapeutic is demonstrated in the countless number of available affinity complexes . in some embodiments , at least two primary affinity molecules are used for complex formation . in some embodiments , as many primary affinity molecules as there are epitopes are used in a single reagent . in some embodiments , primary affinity molecules are interchangeable ( e . g . affibodies can be used with monobodies or microbodies or nanobodies or darpins , etc .). in some embodiments , linker molecules are interchangeable . in some embodiments , it is advantageous to rely on one or two affinity binders . in some embodiments , the linker molecule is two secondary affinity molecules tethered with a flexible polypeptide . in some embodiments , the linker molecule introduces a myriad of tags . in some embodiments , fluorescent dyes or proteins , radionucleotide or heavy metal chelates , and paramagnetic nanoparticles are used to image specific structures , tissues , and cells within the body . in some embodiments , drugs or toxins are used on the linkers to deliver their payloads directly to specific cells . in some embodiments , the therapeutic treatment adept ( antibody - directed enzyme prodrug therapy ) is utilized with the present invention ( e . g . an enzyme is added to the linker that catalyzes an inactive prodrug into its active form ). in some embodiments , the fc fragment of an igg is fused to the linker molecule in order to activate natural cytotoxic cells . in some embodiments , the present invention provides affinity molecules specific to cellular antigens that are expressed within a cell , termed intrabodies . in some embodiments , intrabody techniques have the capability to generate protein knockdown conditions similar to interference rna ( rnai ), phenotypic knockouts , and controlling specific biological processes in order to investigate genomic and proteomic functionality . in some embodiments , intracellular localization peptide sequences provide affinity molecules with the functionality to be directed to specific cell organelles ( e . g . mitrochondria , nucleus , etc .) to improve their effect . in some embodiments , this technology requires that the gene coding sequences for the affinity molecules are introduced into the cells . in some embodiments , cell - permeable peptides ( cpp ) or protein transduction domains ( ptd ) connected to affinity molecules obviate the need to introduce genes encoding sequences for the affinity molecules into cells . in some embodiments , transducible affinity reagents , sometimes called transbodies , bind to the surface of a cell and are internalized by endocytosis . in some embodiments , affinity complexes , in which each component is tagged with a cpp or ptd are internalized , and allowed to form the affinity complex at the site of the target . in some embodiments , this approach significantly increases the effectiveness of this tool as a therapeutic , as well as a research tool . immunochemistry is a major segment of in vitro diagnostics ( ivd ) and is dominated by immune response derived antibodies ( polyclonal and monoclonal ). the cost of developing antibody mimics has for the most part precluded their use in this cost - conscious area , but could be advantageous where they can improve methodology , sensitivity , and practicality . most ivd immunoassays are heterogeneous , requiring a surface ( a plastic well , glass slide , bead ) to which a reagent is attached that allows extensive washing to remove background contaminants and improve detection . single - step homogeneous immunoassays have existed for over 20 years , but typically sacrifice sensitivity for convenience . examples of older homogeneous immunoassays are fluorescence quenching or polarization ( abbott &# 39 ; s tds assay ) and enzymatic steric hindrance assays ( miles laboratories ). fret and bret are more recent technologies to enter this field as well as a plethora of protein - fragment complementation assays ( pca ). more recently , a highly sensitive one - step homogeneous immunoassay called nanodlsa that measures the degree of nanoparticle aggregation by dynamic light scattering has been developed , but not commercialized ( liu et al . j . am . chem . soc . 2008 , 130 , 2780 - 2782 ). in some embodiments , the present invention finds utility in highly sensitive homogeneous immunochemistry assays . in some embodiments , affinity complexes for bret and fret have been described above and improve the versatility of these assays by labeling the secondary affinity molecules in a quinary affinity complex using coiled - coil or similar association mechanism . in some embodiments , pca assays are suited for affinity complex dynamics , in which the affinity complex is a quinary system in which the complementary protein fragments have a specific affinity for each other . in some embodiments , enzymes that have been developed for this assay include β - galactosidase , β - lactamase , and renilla or firefly luciferase as well as green fluorescent protein ( gfp ) as a non - enzymatic example . in some embodiments , one fragment of the enzyme , or fluorescent protein , is expressed as a fusion or covalently linked to a secondary affinity molecule that binds to the primary affinity molecule and the other to another secondary affinity molecule . in some embodiments , the secondary affinity molecules of the two conjugates interact with different domains of the primary affinity molecules , otherwise the same fragment can be present on the same target ( 50 %) resulting in a lack of signal . the present invention further provides systems and kits ( e . g ., commercial therapeutic , diagnostic , or research products , reaction mixtures , etc .) that contain one or more or all components sufficient , necessary , or useful to practice any of the methods described herein . these systems and kits may include buffers , detection / imaging components , positive / negative control reagents , instructions , software , hardware , packaging , or other desired components . a specific example of a therapeutic application of the invention involves a reagent that interacts with a tumor - specific cell surface protein . trastuzumab , more commonly known as herceptin ( genentech ), is a humanized monoclonal antibody that recognizes human epidermal growth factor receptor - 2 ( her2 / neu or erbb - 2 ) when it is over - expressed in aggressive tumors that occur in 20 to 25 % of the estimated 200 , 000 breast cancer cases per year . her2 / neu is a large ( p185 ) orphan receptor - based tyrosine kinase that forms heterodimers with other erbb receptors on the cell surface to effect cell differentiation and growth . a therapeutic product is created that contains at least two , and potentially more , monobodies with binary code interfaces that interact with discontinuous epitopes of the her2 / neu protein , each with a kd of ˜ 10 nm . each monobody has a 46 amino acid serum albumin binding domain ( abd ) linked to its c - terminus that would extend serum half - life of the molecules . the therapeutic reagent contains a linker molecule consisting of a single chain , bimolecular affibody with a 20 amino acid gly / ser linker that interacts with the ef loop of the monobody at a kd of ˜ 10 nm . the designed linker interacts with only 2 monobodies at a time , but can form a complex with several different monobodies , generating a “ web ” that blocks the receptor &# 39 ; s dimerization and , therefore , signal transduction pathway . the linker molecule contains a high - contrast radionuclide imaging agent , such as 111 indium chelated to benzyl - diethylenetriaminepentaacetic acid ( dtpa ), to track the affinity complex formation by positron emission tomography . a specific example of a diagnostic application of the invention involves a reagent that contains at least 2 , and potentially more , monobodies with binary code interfaces that interact with discontinuous epitopes of the protein interleukin - 6 ( il - 6 , a modulator of inflammation ), each with a kd of ˜ 10 nm . the reagent contains 2 more secondary monobodies with binary code interfaces that specifically interact with the ef loop of the primary monobodies ( but not the secondary monobodies ) at a kd of ˜ 10 nm . the secondary monobodies are linked via a gly / ser linker to a fragment of firefly luciferase , either n - luc or c - luc . the reagent contains components to reduce non - specific interactions ( such as serum albumin ), a buffer optimal for both binding interactions and luciferase activity , and the substrates luciferin and atp . a sample of blood or serum is diluted into the reagent , the complexes are allowed to form on the il - 6 , the proximity luciferase fragments allowed to complement / fold together , and complete enzymes generate bioluminescent light from the reaction with atp and luciferin . the amount of sustained light output from this homogeneous assay is directly relative to the target protein concentration in the reaction . the volume of the reaction can be as large as 100 μl for use in a 96 - well microtiter plate luminometer or less than a microliter for use in microfluidic devices . in addition , other targets can be assayed by simply changing the primary monobodies in the reagent . this can also be used to assay membrane proteins on the surface of intact cells , such as gpcr or other receptors , and detect protein : protein interactions . the following references are herein incorporated by reference in their 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