Quantum defect sensitization via phase-changing supercharged antibody fragments

Abstract

The present technology relates generally to carbon nanotube antibody fragment conjugates useful for the detection of a bioanalyte, and methods of making and using the same. In particular, a conjugate for detection of a bioanalyte includes a single-walled carbon nanotube with a sp3 quantum-well defect site (QWNT) conjugated to an antibody fragment via an amide bond at the sp3 quantum-well defect site; wherein the antibody fragment is a single-chain variable fragment with an overall net charge of about -10 to about -30 at a pH of about 6.5 to about 7.5, the antibody fragment changes phase in response to interaction with the bioanalyte, and the antibody fragment comprises Vu-linker-Vr, wherein VH is a variable heavy chain domain, Vi. is a variable light chain domain; and wherein the antibody fragment comprises a number of D residues to provide the overall net charge.

Description

Atty Dkt No: 115872-1955 (SK2024-041-02)QUANTUM DEFECT SENSITIZATION VIA PHASE-CHANGING SUPERCHARGED ANTIBODY FRAGMENTSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 635,191, filed April 17, 2024, the contents of which are incorporated herein by reference in their entirety for any and all purposes.U.S. GOVERNMENT SUPPORT

[0002] This invention was made with government support under W81XWH-22-1-0563 awarded by the United States Army Medical Research and Development Command, EB033580, EB033651, CA132378, and CA008748 awarded by the National Institutes of Health, and 1752506 and 2323759 awarded by the National Science Foundation.. The government has certain rights in the invention.FIELD

[0003] The present technology relates generally to carbon nanotube antibody fragment conjugates useful for the detection of a bioanalyte, and methods of making and using the same.SUMMARY

[0004] In an aspect, the present disclosure provides a conjugate for detection of a bioanalyte comprising a single-walled carbon nanotube with a sp3quantum-well defect site (QWNT) conjugated to an antibody fragment via an amide bond at the sp3quantum-well defect site; The antibody fragment is a single-chain variable fragment with an overall net charge of about -10 to about -30. The antibody fragment changes phase in response to interaction with the bioanalyte, and the antibody fragment comprises Vu-linker-Vr, wherein VH is a variable heavy chain domain, VL is a variable light chain domain, and the linker comprises (SGG)X, GG(SGG)X, (GGGGSJx, (SGG)x, SS(GGGS)X, or a combination of any two or more thereof, where x is 1 to 12. The antibody fragment comprises a number of D residues to provide the overall net charge of about -10 to about -30. The bioanalyte comprises a cytokine, an epidermal growth factor, an interferon, or a hormone.-1-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)

[0005] In a related aspect, a pharmaceutical composition for detecting the bioanalyte is provided that includes about 10'2nM to about 103nM of a conjugate disclosed herein; and a pharmaceutically acceptable solvent.

[0006] In a related aspect, a method for selecting a subject for treatment with anti-IL-6 therapy is provided that includes (a) detecting IL-6 above a predetermined threshold in a biological sample obtained from the subject using the pharmaceutical composition of a conjugate disclosed herein; and (b) administering the anti-IL-6 therapy to the subject.

[0007] In a related aspect, a method for treating or preventing cytokine release syndrome in a subject is provided that includes (a) detecting IL-6 above a predetermined threshold in a biological sample obtained from the subject using the pharmaceutical composition of a conjugate disclosed herein; and (b) administering an anti -IL-6 therapy to the subject.

[0008] In a related aspect, a method for prolonging survival of a cancer patient is provided that includes administering to the cancer patient an effective amount of an anti-IL-6 therapy, wherein an IL-6 level in a biological sample obtained from the cancer patient is at or above a predetermined threshold measured using the composition of a conjugate disclosed herein.

[0009] Further aspects and embodiments of the present technology are described herein.BRIEF DESCRIPTION OF THE DRAWING

[0010] FIG. 1 is an illustration of supercharged single-chain antibody fragment (scFv) coupled quantum defect-modified single walled carbon nanotubes (QWNT) synthesis and signal transduction mechanism. A 4-carboxylaryl functional group is covalently incorporated into a (6,5)-SWCNT via diazonium reaction to create QWNT. Covalent functionalization of quantum defects results in a new fluorescence emission that is localized at the defect site. Supercharged scFv can be bioconjugated to the 4-carboxylaryl defects via EDC-NHS chemistry. In the presence of target analyte, human interleukin-6 (hIL-6), the scFv of the scFv- QWNT complex can undergo ligand-induced folding in proximity to the defect center, resulting in solvatochromic shifts in its NIR fluorescence.

[0011] FIGS. 2A-2G: Supercharged a-IL6 scFv design and characterization. FIG. 2A provides spatial distribution of acidic, basic, and disulfide linker side chains in each step of the design of scFVs. Spheres are the beta carbons of each ionizable residue. FIG. 2A also provides-2-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02) sequences of a-IL6wt and a-IL6sc. Basic and acidic residues and disulfide linkers are highlighted, CDR regions over- or underlined. FIG. 2B provides microscale thermophoresis (MST) quantification of a-IL6sc:hIL-6 binding affinity. Line drawn is the best fit of the data with a Kd of 400 nM. FIG. 2C provides salt-induced folding of a-IL6sc. Line drawn is the best fit of the data with Equation 1 with AGfoiding, o of +4.5 kcal / mol and the salt-dependent folding stabilization of -3.4 kcal / mol M, Ip = 0.066, 1 = 0.049, mr = 0.013 M’1, and mu = - 9.76»10'3M'1. FIG. 2D provides selected a-IL6sc:hIL-6 binding experiments performed using MST at NaCl activities ranging from 0.5 M to 2 M. FIG. 2E provides dissociation constants of IL6sc and hIL-6 as a function of NaCl activity. Error bars are errors of the individual fits at each NaCl concentration. Filled circles with different color codes correspond to the data obtained from the MST curve in FIG. 2D. FIG. 2F provides folding and binding equilibrium thermodynamics of IL6sc as a function of NaCl activity. Filled circles are the NaCl dependence of AGfoiding derived using the data from FIG. 2C and Equation 1. Black line is the best linear fit of the AGfoiding data. Open circles are AGbinding calculated from the dissociation constants in FIG. 2E. The red and purple lines are the best linear fit of the AGbinding data below and above NaCl activities of 0.8 M, respectively. The black and red lines have identical slopes within error, and the distance between them, 10.46 kcal / mol, is the interaction energy between the folded protein and the ligand, AAGdock. FIG. 2G provides pH invariance of a-IL6sc:hIL-6 binding over the physiologically relevant pH range.

[0012] FIGS. 3A-3D: Characterization of scFv-QWNT nanosensors. FIG. 3A provides fluorescence spectra of unfunctionalized (6,5)-SWCNT, QWNT, and supercharged scFv- coupled QWNT. The excitation wavelength was 808 nm. FIG. 3B provides Zeta potential of QWNT, wild-type scFv-coupled QWNT (WT), and supercharged scFv-coupled QWNT. FIG. 3C provides a correlogram of QWNT and supercharged scFv-coupled QWNT. FIG. 3D provides an atomic force microscopy image of scFv-conjugated QWNT. Scale bar is 100 nm. Color bar is the height in nanometers.

[0013] FIGS. 4A-4F: scFv-QWNT response to human IL-6 and effects of supercharging. FIG. 4A provides fluorescence spectra of before and after adding 2.5 pM hIL-6 in 20% FBS. FIG. 4B provides wavelength shift of En and Eif peaks as a function of hIL-6 concentration in 20% FBS. FIG. 4C provides time dependent wavelength shift of En and Eif peaks after the addition of 1 pM hIL-6 in 20% FBS. FIG. 4D provides Eif wavelength shift in the presence of non-specific proteins in 20% FBS. FIG. 4E provides Eif wavelength shift for-3-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02) freshly prepared and 7-day old scFv-QWNT complexes. Error bars are standard deviation of triplicate. FIG. 4F provides Eif wavelength shift of wild-type (open circles) and supercharged scFv-coupled QWNTs (filled circles) as a function of hIL-6 concentration in 20% FBS.

[0014] FIGS. 5A-5E: Quantum chemical modeling of scFv-QWNT. FIG. 5A provides scale comparison between 4 nm long (6,5)-SWCNT and folded supercharged scFv protein. FIG. 5B provides point charge model that approximates the effect of protein supercharging in scFv-QWNT as two point charges that are symmetrically positioned at a distance of 1.41 nm (1 nm away along the CNT axis and 1 nm away from the CNT surface, as indicated by dashed lines) from a quantum defect pair. The defect pair consists of an N-methylbenzamide and a hydroxyl group pair, arranged in the ortho L90 configuration, at the center of the (6,5)-SWCNT. FIG. 5C provides absorption spectra calculated for unfunctionalized SWCNT (top) and QWNT (bottom) with varying charges from 0 to -le'. The E11 and Eif peaks are labeled. FIG. 5D provides E11 wavelength shifts of unfunctionalized SWCNT as a function of point charge. FIG. 5E provides E11 and Eif wavelength shifts of QWNT as a function of point charge.

[0015] FIGS. 6A-6B: Isothermal titration calorimetry of scFv binding IL-6. FIG. 6A is pWatts vs. time (minutes) for scFv binding IL-6. FIG. 6B is kcal mol'1of inj ectant vs. molar ratio for scFv binding IL-6.

[0016] FIGS. 7A-7B: Binding experiments for scFV having different numbers of disulfide bridges with IL-6. FIG. 7A provides binding experiment data for binding of IL-6 by scFV comprising a low concentration of disulfide bridges, with characterization performed using MST. FIG. 7B provides binding experiment data for binding of IL-6 by scFV comprising a high concentration of disulfide bridges, with characterization performed using MST.

[0017] FIGS. 8A-8B: Binding experiments for different scFVs with luteinizing hormone (LH). FIG. 8A provides binding experiment data for binding LH by scFV having a net charge of -17. FIG. 8B provides binding experiment data for binding LH by scFV having a net charge of -22.DETAILED DESCRIPTION

[0018] It is to be appreciated that certain aspects, modes, embodiments, variations, and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.-4-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)Definitions

[0019] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

[0020] As used herein and in the appended claims, singular articles such as “a” and “an” and “the” and similar referents in the context of describing the elements (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the embodiments, and does not pose a limitation on the scope of the claims unless otherwise stated. No language in the specification should be construed as indicating any non-claimed element as essential. For example, reference to “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.

[0021] As used herein, “about” will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art, given the context in which it is used, “about” will mean up to plus or minus 10% of the particular term - for example, “about 10 wt.%” would be understood to mean “9 wt.% to 11 wt.%.” It is to be understood that when “about” precedes a term, the term is to be construed as disclosing “about” the term as well as the term without modification by “about” — for example, “about 10 wt.%” discloses “9 wt.% to 11 wt.%” as well as disclosing “10 wt.%.”

[0022] As used herein, the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including orally, intranasally,-5-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02) parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), or topically. Administration includes self-administration and the administration by another.

[0023] The phrase “and / or” as used in the present disclosure will be understood to mean any one of the recited members individually or a combination of any two or more thereof - for example, “A, B, and / or C” would mean “A, B, C, A and B, A and C, B and C, or the combination of A, B, and C ”

[0024] As used herein, a “control” is an alternative sample used in an experiment for comparison purpose. A control can be “positive” or “negative.” For example, where the purpose of the experiment is to determine a correlation of the efficacy of a therapeutic agent for the treatment for a particular type of disease or condition, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or a sample that does not receive the therapy or receives a placebo) are typically employed.

[0025] As used herein, “prevention,” “prevent,” or “preventing” of a disease or condition refers to one or more compounds that, in a statistical sample, reduces the occurrence of the disease or condition in the treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of the disease or condition relative to the untreated control sample. As used herein, prevention includes preventing or delaying the initiation of symptoms of the disease or condition. As used herein, prevention also includes preventing a recurrence of one or more signs or symptoms of a disease or condition.

[0026] As used herein, the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.

[0027] As used herein, the term “sequential” therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.-6-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)

[0028] As used herein, the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.

[0029] Generally, reference to a certain element such as hydrogen or H is meant to include all isotopes of that element. For example, if an R group is defined to include hydrogen or H, it also includes deuterium and tritium. Compounds comprising radioisotopes such as tritium,14C,32P, and35S are thus within the scope of the present technology. Procedures for inserting such labels into the compounds of the present technology will be readily apparent to those skilled in the art based on the disclosure herein.

[0030] In general, “substituted” refers to an organic group as defined below (e.g., an alkyl group) in which one or more bonds to a hydrogen atom contained therein are replaced by a bond to non-hydrogen or non-carbon atoms. Substituted groups also include groups in which one or more bonds to a carbon(s) or hydrogen(s) atom are replaced by one or more bonds, including double or triple bonds, to a heteroatom. Thus, a substituted group is substituted with one or more substituents, unless otherwise specified. In some embodiments, a substituted group is substituted with 1, 2, 3, 4, 5, or 6 substituents. Examples of substituent groups include: halogens (z.e., F, Cl, Br, and I); hydroxyls; alkoxy, alkenoxy, aryloxy, aralkyloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, and heterocyclyl alkoxy groups; carbonyls (oxo); carboxylates; esters; urethanes; oximes; hydroxylamines; alkoxyamines; aralkoxyamines; thiols; sulfides; sulfoxides; sulfones; sulfonyls; pentafluorosulfanyl (z.e., SFs), sulfonamides; amines; N-oxides; hydrazines; hydrazides; hydrazones; azides; amides; ureas; amidines; guanidines; enamines; imides; isocyanates; isothiocyanates; cyanates; thiocyanates; imines; nitro groups; nitriles (z.e., CN); and the like.

[0031] Substituted ring groups such as substituted cycloalkyl, aryl, heterocyclyl and heteroaryl groups also include rings and ring systems in which a bond to a hydrogen atom is replaced with a bond to a carbon atom. Therefore, substituted cycloalkyl, aryl, heterocyclyl and heteroaryl groups may also be substituted with substituted or unsubstituted alkyl, alkenyl, and alkynyl groups as defined below.

[0032] As used herein, Cx-Cy, such as C1-C12, Ci-Cs, or Ci-Ce when used before a group refers to that group containing x to y carbon atoms-7-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)

[0033] Alkyl groups include straight chain and branched chain alkyl groups having from 1 to 12 carbon atoms, and typically from 1 to 10 carbons or, in some embodiments, from 1 to 8, 1 to 6, or 1 to 4 carbon atoms. Alkyl groups may be substituted or unsubstituted. Examples of straight chain alkyl groups include groups such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, tert-butyl, neopentyl, isopentyl, and 2,2- dimethylpropyl groups. Representative substituted alkyl groups may be substituted one or more times with substituents such as those listed above, and include without limitation haloalkyl (e.g., trifluoromethyl), hydroxyalkyl, thioalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkoxyalkyl, carboxyalkyl, and the like.

[0034] Cycloalkyl groups include mono-, bi- or tricyclic alkyl groups having from 3 to 12 carbon atoms in the ring(s), or, in some embodiments, 3 to 10, 3 to 8, or 3 to 4, 5, or 6 carbon atoms. Cycloalkyl groups may be substituted or unsubstituted. Exemplary monocyclic cycloalkyl groups include, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group has 3 to 8 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 5, 3 to 6, or 3 to 7. Bi- and tricyclic ring systems include both bridged cycloalkyl groups and fused rings, such as, but not limited to, bicyclo[2.1.1]hexane, adamantyl, decalinyl, and the like. Substituted cycloalkyl groups may be substituted one or more times with nonhydrogen and non-carbon groups as defined above. However, substituted cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined above. Representative substituted cycloalkyl groups may be mono-substituted or substituted more than once, such as, but not limited to, 2,2-, 2,3-, 2,4- 2,5- or 2, 6-di substituted cyclohexyl groups, which may be substituted with substituents such as those listed above.

[0035] Cycloalkylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a cycloalkyl group as defined above. Cycloalkylalkyl groups may be substituted or unsubstituted. In some embodiments, cycloalkylalkyl groups have from 4 to 16 carbon atoms, 4 to 12 carbon atoms, and typically 4 to 10 carbon atoms. Substituted cycloalkylalkyl groups may be substituted at the alkyl, the cycloalkyl or both the alkyl and cycloalkyl portions of the group. Representative substituted cycloalkylalkyl groups may be mono-substituted or substituted more than once, such as, but not limited to, mono-, di- or tri -substituted with substituents such as those listed above.-8-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)

[0036] Alkenyl groups include straight and branched chain alkyl groups as defined above, except that at least one double bond exists between two carbon atoms. Alkenyl groups may be substituted or unsubstituted. Alkenyl groups have from 2 to 12 carbon atoms, and typically from 2 to 10 carbons or, in some embodiments, from 2 to 8, 2 to 6, or 2 to 4 carbon atoms. In some embodiments, the alkenyl group has one, two, or three carbon-carbon double bonds. Examples include, but are not limited to vinyl, allyl, -CH=CH(CH3), -CH=C(CH3)2, -C(CH3)=CH2, -C(CH3)=CH(CH3), -C(CH2CH3)=CH2, among others. Representative substituted alkenyl groups may be mono-substituted or substituted more than once, such as, but not limited to, mono-, di- or tri-substituted with substituents such as those listed above.

[0037] Cycloalkenyl groups include cycloalkyl groups as defined above, having at least one double bond between two carbon atoms. Cycloalkenyl groups may be substituted or unsubstituted. In some embodiments the cycloalkenyl group may have one, two or three double bonds but does not include aromatic compounds. Cycloalkenyl groups have from 4 to 14 carbon atoms, or, in some embodiments, 5 to 14 carbon atoms, 5 to 10 carbon atoms, or even 5, 6, 7, or 8 carbon atoms. Examples of cycloalkenyl groups include cyclohexenyl, cyclopentenyl, cyclohexadienyl, cyclobutadienyl, and cyclopentadienyl.

[0038] Cycloalkenylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of the alkyl group is replaced with a bond to a cycloalkenyl group as defined above. Cycloalkenylalkyl groups may be substituted or unsubstituted. Substituted cycloalkenylalkyl groups may be substituted at the alkyl, the cycloalkenyl or both the alkyl and cycloalkenyl portions of the group. Representative substituted cycloalkenylalkyl groups may be substituted one or more times with substituents such as those listed above.

[0039] Alkynyl groups include straight and branched chain alkyl groups as defined above, except that at least one triple bond exists between two carbon atoms. Alkynyl groups may be substituted or unsubstituted. Alkynyl groups have from 2 to 12 carbon atoms, and typically from 2 to 10 carbons or, in some embodiments, from 2 to 8, 2 to 6, or 2 to 4 carbon atoms. In some embodiments, the alkynyl group has one, two, or three carbon-carbon triple bonds. Examples include, but are not limited to -C=CH, -C=CCH3, -CH2C=CCH3, -C=CCH2CH(CH2CH3)2, among others. Representative substituted alkynyl groups may be mono-substituted or substituted more than once, such as, but not limited to, mono-, di- or tri-substituted with substituents such as those listed above.-9-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)

[0040] Aryl groups are cyclic aromatic hydrocarbons that do not contain heteroatoms. Aryl groups herein include monocyclic, bicyclic, and tricyclic ring systems. Aryl groups may be substituted or unsubstituted. Thus, aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, fluorenyl, phenanthrenyl, anthracenyl, indenyl, indanyl, pentalenyl, and naphthyl groups. In some embodiments, aryl groups contain 6-14 carbons, and in others from 6 to 12 or even 6-10 carbon atoms in the ring portions of the groups. In some embodiments, the aryl groups are phenyl or naphthyl. The phrase “aryl groups” includes groups containing fused rings, such as fused aromatic-aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, and the like). Representative substituted aryl groups may be mono-substituted (e.g., tolyl) or substituted more than once. For example, monosubstituted aryl groups include, but are not limited to, 2-, 3-, 4-, 5-, or 6-substituted phenyl or naphthyl groups, which may be substituted with substituents such as those listed above.

[0041] Aralkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined above. Aralkyl groups may be substituted or unsubstituted. In some embodiments, aralkyl groups contain 7 to 16 carbon atoms, 7 to 14 carbon atoms, or 7 to 10 carbon atoms. Substituted aralkyl groups may be substituted at the alkyl, the aryl or both the alkyl and aryl portions of the group. Representative aralkyl groups include but are not limited to benzyl and phenethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-indanylethyl. Representative substituted aralkyl groups may be substituted one or more times with substituents such as those listed above.

[0042] Heterocyclyl groups include aromatic (also referred to as heteroaryl) and nonaromatic ring compounds containing 3 or more ring members, of which one or more is a heteroatom such as, but not limited to, N, O, and S. Heterocyclyl groups may be substituted or unsubstituted. In some embodiments, the heterocyclyl group contains 1, 2, 3 or 4 heteroatoms. In some embodiments, heterocyclyl groups include mono-, bi- and tricyclic rings having 3 to 16 ring members, whereas other such groups have 3 to 6, 3 to 10, 3 to 12, or 3 to 14 ring members. Heterocyclyl groups encompass aromatic, partially unsaturated and saturated ring systems, such as, for example, imidazolyl, imidazolinyl and imidazolidinyl groups. The phrase “heterocyclyl group” includes fused ring species including those comprising fused aromatic and non-aromatic groups, such as, for example, benzotriazolyl, 2,3- dihydrobenzo[l,4]dioxinyl, and benzo[l,3]dioxolyl. The phrase also includes bridged polycyclic ring systems containing a heteroatom such as, but not limited to, quinuclidyl. The-10-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02) phrase includes heterocyclyl groups that have other groups, such as alkyl, oxo or halo groups, bonded to one of the ring members, referred to as “substituted heterocyclyl groups.” Heterocyclyl groups include, but are not limited to, aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydrothiophenyl, tetrahydrofuranyl, dioxolyl, furanyl, thiophenyl, pyrrolyl, pyrrolinyl, imidazolyl, imidazolinyl, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, thiazolinyl, isothiazolyl, thiadiazolyl, oxadiazolyl, piperidyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydropyranyl, tetrahydrothiopyranyl, oxathiane, dioxyl, dithianyl, pyranyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, dihydropyridyl, dihydrodithiinyl, dihydrodithionyl, homopiperazinyl, quinuclidyl, indolyl, indolinyl, isoindolyl,azaindolyl (pyrrolopyridyl), indazolyl, indolizinyl, benzotri azolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, benzthiazolyl, benzoxadiazolyl, benzoxazinyl, benzodithiinyl, benzoxathiinyl, benzothiazinyl, benzoxazolyl, benzothiazolyl, benzothiadi azolyl, benzo [1,3] dioxolyl, pyrazolopyridyl, imidazopyridyl (azabenzimidazolyl), tri azol opyridyl, isoxazolopyridyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, quinolizinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl, pteridinyl, thianaphthyl, dihydrobenzothiazinyl, dihydrobenzofuranyl, dihydroindolyl, dihydrobenzodioxinyl, tetrahydroindolyl, tetrahydroindazolyl, tetrahydrobenzimidazolyl, tetrahydrobenzotriazolyl, tetrahydropyrrolopyridyl, tetrahydropyrazolopyridyl, tetrahydroimidazopyridyl, tetrahydrotriazolopyridyl, and tetrahydroquinolinyl groups. Representative substituted heterocyclyl groups may be monosubstituted or substituted more than once, such as, but not limited to, pyridyl or morpholinyl groups, which are 2-, 3-, 4-, 5-, or 6-substituted, or disubstituted with various substituents such as those listed above.

[0043] Heteroaryl groups are aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S. Heteroaryl groups may be substituted or unsubstituted. Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiophenyl, benzothiophenyl, furanyl, benzofuranyl, indolyl, azaindolyl (pyrrolopyridinyl), indazolyl, benzimidazolyl, imidazopyridinyl (azabenzimidazolyl), pyrazolopyridinyl, triazolopyridinyl, benzotri azolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Heteroaryl groups include fused-11-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02) ring compounds in which all rings are aromatic such as indolyl groups and include fused ring compounds in which only one of the rings is aromatic, such as 2,3-dihydro indolyl groups. Representative substituted heteroaryl groups may be substituted one or more times with various substituents such as those listed above.

[0044] Heterocyclylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heterocyclyl group as defined above. Heterocyclylalkyl groups may be substituted or unsubstituted. Substituted heterocyclylalkyl groups may be substituted at the alkyl, the heterocyclyl or both the alkyl and heterocyclyl portions of the group. Representative heterocyclyl alkyl groups include, but are not limited to, morpholin-4-yl-ethyl, furan-2-yl-m ethyl, imidazol-4-yl-m ethyl, pyri din-3 -yl-methyl, tetrahydrofuran-2-yl-ethyl, and indol-2-yl-propyl. Representative substituted heterocyclylalkyl groups may be substituted one or more times with substituents such as those listed above.

[0045] Heteroaralkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heteroaryl group as defined above. Heteroaralkyl groups may be substituted or unsubstituted. Substituted heteroaralkyl groups may be substituted at the alkyl, the heteroaryl or both the alkyl and heteroaryl portions of the group. Representative substituted heteroaralkyl groups may be substituted one or more times with substituents such as those listed above.

[0046] Groups described herein having two or more points of attachment (i.e., divalent, trivalent, or polyvalent) within the compound of the present technology are designated by use of the suffix, “ene.” For example, divalent alkyl groups are alkylene groups, divalent aryl groups are arylene groups, divalent heteroaryl groups are divalent heteroarylene groups, and so forth. Substituted groups having a single point of attachment to the compound of the present technology are not referred to using the “ene” designation. Thus, e.g., chloroethyl is not referred to herein as chloroethylene.

[0047] Alkoxy groups are hydroxyl groups (-OH) in which the bond to the hydrogen atom is replaced by a bond to a carbon atom of a substituted or unsubstituted alkyl group as defined above. Alkoxy groups may be substituted or unsubstituted. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, and the like. Examples of branched alkoxy groups include but are not limited to isopropoxy, sec-butoxy,-12-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02) tert-butoxy, isopentoxy, isohexoxy, and the like. Examples of cycloalkoxy groups include but are not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. Representative substituted alkoxy groups may be substituted one or more times with substituents such as those listed above.

[0048] The terms “alkanoyl” and “alkanoyloxy” as used herein can refer, respectively, to -C(O)-alkyl groups and -O-C(O)-alkyl groups, each containing 2-5 carbon atoms. Similarly, “aryloyl” and “aryloyloxy” refer to -C(O)-aryl groups and -O-C(O)-aryl groups.

[0049] The terms "aryloxy" and “arylalkoxy” refer to, respectively, a substituted or unsubstituted aryl group bonded to an oxygen atom and a substituted or unsubstituted aralkyl group bonded to the oxygen atom at the alkyl. Examples include but are not limited to phenoxy, naphthyloxy, and benzyloxy. Representative substituted aryloxy and arylalkoxy groups may be substituted one or more times with substituents such as those listed above.

[0050] The term “carboxylate” as used herein refers to a -COOH group.

[0051] The term “ester” as used herein refers to -COOR70and -C(O)O-G groups. R70is a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heterocyclylalkyl or heterocyclyl group as defined herein. G is a carboxylate protecting group. Carboxylate protecting groups are well known to one of ordinary skill in the art. An extensive list of protecting groups for the carboxylate group functionality may be found in Protective Groups in Organic Synthesis, Greene, T.W.; Wuts, P. G. M., John Wiley & Sons, New York, NY, (3rd Edition, 1999) which can be added or removed using the procedures set forth therein and which is hereby incorporated by reference in its entirety and for any and all purposes as if fully set forth herein.

[0052] The term “amide” (or “amido”) includes C- and N-amide groups, i.e., -C(O)NR71R72, and -NR71C(O)R72groups, respectively. R71and R72are independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl or heterocyclyl group as defined herein. Amido groups therefore include but are not limited to carbamoyl groups (-C(O)NH2) and formamide groups (-NHC(O)H). In some embodiments, the amide is -NR71C(O)-(CI-5 alkyl) and the group is termed "carbonylamino," and in others the amide is -NHC(O)-alkyl and the group is termed "alkanoylamino."

[0053] The term “nitrile” or “cyano” as used herein refers to the -CN group.-13-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)

[0054] Urethane groups include N- and O-urethane groups, i.e., -NR73C(O)OR74and -OC(O)NR73R74groups, respectively. R73and R74are independently a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein. R73may also be H.

[0055] The term “amine” (or “amino”) as used herein refers to -NR75R76groups, wherein R75and R76are independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl or heterocyclyl group as defined herein. In some embodiments, the amine is alkylamino, dialkylamino, arylamino, or alkylarylamino. In other embodiments, the amine is NH2, methylamino, dimethylamino, ethylamino, diethylamino, propylamino, isopropylamino, phenylamino, or benzylamino.

[0056] The term “sulfonamido” includes S- and N-sulfonamide groups, i.e., -SO2NR78R79and -NR78SO2R79groups, respectively. R78and R79are independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein. Sulfonamido groups therefore include but are not limited to sulfamoyl groups (-SO2NH2). In some embodiments herein, the sulfonamido is - NHSCh-alkyl and is referred to as the "alkylsulfonylamino" group.

[0057] The term “thiol” refers to -SH groups, while “sulfides” include -SR80groups, “sulfoxides” include -S(O)R81groups, “sulfones” include -SO2R82groups, and “sulfonyls” include -SO2OR83. R80, R81, R82, and R83are each independently a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein. In some embodiments the sulfide is an alkylthio group, -S-alkyl.

[0058] The term “urea” refers to -NR84-C(O)-NR85R86groups. R84, R85, and R86groups are independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclyl, or heterocyclylalkyl group as defined herein.

[0059] The term “amidine” refers to -C(NR87)NR88R89and -NR87C(NR88)R89, wherein R87, R88, and R89are each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.-14-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)

[0060] The term “guanidine” refers to -NR90C(NR91)NR92R93, wherein R90, R91, R92and R93are each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.

[0061] The term “enamine” refers to -C(R94)=C(R95)NR96R97and -NR94C(R95)=C(R96)R97, wherein R94, R95, R96and R97are each independently hydrogen, a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.

[0062] The term “halogen” or “halo” as used herein refers to bromine, chlorine, fluorine, or iodine. In some embodiments, the halogen is fluorine. In other embodiments, the halogen is chlorine or bromine.

[0063] The term “hydroxyl” as used herein can refer to -OH or its ionized form, -O . A “hydroxyalkyl” group is a hydroxyl-substituted alkyl group, such as HO-CH2-.

[0064] The term “imide” refers to -C(O)NR98C(O)R", wherein R98and R" are each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.

[0065] The term “imine” refers to -CR100(NR101) and -N(CR100R101) groups, wherein R100and R101are each independently hydrogen or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein, with the proviso that R100and R101are not both simultaneously hydrogen.

[0066] The term “nitro” as used herein refers to an -NO2 group.

[0067] The term “trifluorom ethyl” as used herein refers to -CF3.

[0068] The term “trifluorom ethoxy” as used herein refers to -OCF3.

[0069] The term “azido” refers to -N3.

[0070] The term “trialkyl ammonium” refers to a -N(alkyl)s group. A trialkylammonium group is positively charged and thus typically has an associated anion, such as halogen anion.

[0071] The term “isocyano” refers to -NC.

[0072] The term “isothiocyano” refers to -NCS.-15-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)

[0073] The term “pentafluorosulfanyl” refers to -SFs.

[0074] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 atoms refers to groups having 1, 2, or 3 atoms. Similarly, a group having 1-5 atoms refers to groups having 1, 2, 3, 4, or 5 atoms, and so forth.

[0075] Pharmaceutically acceptable salts of compounds described herein are within the scope of the present technology and include acid or base addition salts which retain the desired pharmacological activity and is not biologically undesirable (e.g., the salt is not unduly toxic, allergenic, or irritating, and is bioavailable). When the compound of the present technology has a basic group, such as, for example, an amino group, pharmaceutically acceptable salts can be formed with inorganic acids (such as hydrochloric acid, hydroboric acid, nitric acid, sulfuric acid, and phosphoric acid), organic acids (e.g., alginate, formic acid, acetic acid, benzoic acid, gluconic acid, fumaric acid, oxalic acid, tartaric acid, lactic acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, naphthalene sulfonic acid, and p-toluenesulfonic acid) or acidic amino acids (such as aspartic acid and glutamic acid). When the compound of the present technology has an acidic group, such as for example, a carboxylic acid group, it can form salts with metals, such as alkali and earth alkali metals (e.g., Na+, Li+, K+, Ca2+, Mg2+, Zn2+), ammonia or organic amines (e.g., dicyclohexylamine, trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine) or basic amino acids (e.g., arginine, lysine and ornithine). Such salts can be prepared in situ during isolation and purification of the compounds or by separately reacting the purified compound in its free base or free acid form with a suitable acid or base, respectively, and isolating the salt thus formed.-16-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)

[0076] Those of skill in the art will appreciate that compounds of the present technology may exhibit the phenomena of tautomerism, conformational isomerism, geometric isomerism and / or stereoisomerism. As the formula drawings within the specification and claims can represent only one of the possible tautomeric, conformational isomeric, stereochemical or geometric isomeric forms, it should be understood that the present technology encompasses any tautomeric, conformational isomeric, stereochemical and / or geometric isomeric forms of the compounds having one or more of the utilities described herein, as well as mixtures of these various different forms.

[0077] Tautomers” refers to isomeric forms of a compound that are in equilibrium with each other. The presence and concentrations of the isomeric forms will depend on the environment the compound is found in and may be different depending upon, for example, whether the compound is a solid or is in an organic or aqueous solution. For example, in aqueous solution, quinazolinones may exhibit the following isomeric forms, which are referred to as tautomers of each other:As another example, guanidines may exhibit the following isomeric forms in protic organic solution, also referred to as tautomers of each other:Because of the limits of representing compounds by structural formulas, it is to be understood that all chemical formulas of the compounds described herein represent all tautomeric forms of compounds and are within the scope of the present technology.

[0078] Stereoisomers of compounds (also known as optical isomers) include all chiral, diastereomeric, and racemic forms of a structure, unless the specific stereochemistry is-17-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02) expressly indicated. Thus, compounds used in the present technology include enriched or resolved optical isomers at any or all asymmetric atoms as are apparent from the depictions. Both racemic and diastereomeric mixtures, as well as the individual optical isomers can be isolated or synthesized so as to be substantially free of their enantiomeric or diastereomeric partners, and these stereoisomers are all within the scope of the present technology.

[0079] The compounds of the present technology may exist as solvates, especially hydrates. Hydrates may form during manufacture of the compounds or compositions comprising the compounds, or hydrates may form over time due to the hygroscopic nature of the compounds. Compounds of the present technology may exist as organic solvates as well, including DMF, ether, and alcohol solvates, among others. The identification and preparation of any particular solvate is within the skill of the ordinary artisan of synthetic organic or medicinal chemistry.

[0080] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel el al. eds. (2007) Current Protocols in Molecular Biology, the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach,' Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual,' Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis,' U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization,' Anderson (1999) Nucleic Acid Hybridization,' Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir ’s Handbook of Experimental Immunology. Methods to detect and measure levels of polypeptide gene expression products (i.e., gene translation level) are well-known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also,-18-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).

[0081] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in the present disclosure. Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.

[0082] The term “amino acid” refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrolysine and selenocysteine. Amino acid analogs refer to agents that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (such as, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. In some embodiments, amino acids forming a polypeptide are in the D form. In some embodiments, the amino acids forming a polypeptide are in the L form. In some embodiments, a first plurality of amino acids forming a polypeptide is in the D form and a second plurality is in the L form.

[0083] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter code.-19-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)

[0084] As used herein, the term “analog” refers to a structurally related polypeptide or nucleic acid molecule having the function of a reference polypeptide or nucleic acid molecule.

[0085] As used herein, the term “antibody” means not only intact antibody molecules, but also fragments of antibody molecules that retain immunogen-binding ability. Such fragments are also well known in the art and are regularly employed both in vitro and in vivo. Accordingly, as used herein, the term “antibody” means not only intact immunoglobulin molecules but also the well-known active fragments F(ab')2, and Fab. F(ab')2, and Fab fragments that lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl etal., J. NucL Med. 24:316-325 (1983)). Antibodies may comprise whole native antibodies, monoclonal antibodies, human antibodies, humanized antibodies, camelised antibodies, multispecific antibodies, bispecific antibodies, chimeric antibodies, Fab, Fab', single chain V region fragments (scFv), single domain antibodies (e.g., nanobodies and single domain cam elid antibodies), VNAR fragments, Bi-specific T-cell engager (BiTE) antibodies, minibodies, disulfide-linked Fvs (sdFv), and anti -idiotypic (anti-Id) antibodies, intrabodies, fusion polypeptides, unconventional antibodies and antigen binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, z.e., molecules that contain an antigen binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass.

[0086] In certain embodiments, an antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant (CH) region. The heavy chain constant region is comprised of three domains, CHI, CH2, and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant CL region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant-20-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02) regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Cl q) of the classical complement system. As used herein interchangeably, the terms “antigen binding portion”, “antigen binding fragment”, or “antigen binding region” of an antibody, refer to the region or portion of an antibody that binds to the antigen and which confers antigen specificity to the antibody; fragments of antigen binding proteins, for example antibodies, include one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antigen binding portions encompassed within the term “antibody fragments” of an antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., Nature 341 : 544-546 (1989)), which consists of a VH domain; and an isolated complementarity determining region (CDR). An “isolated antibody” or “isolated antigen binding protein” is one which has been identified and separated and / or recovered from a component of its natural environment. “Synthetic antibodies” or “recombinant antibodies” are generally generated using recombinant technology or using peptide synthetic techniques known to those of skill in the art.

[0087] Antibodies and antibody fragments can be wholly or partially derived from mammals (e.g., humans, non-human primates, goats, guinea pigs, hamsters, horses, mice, rats, rabbits and sheep) or non-mammalian antibody producing animals (e.g., chickens, ducks, geese, snakes, and urodele amphibians). The antibodies and antibody fragments can be produced in animals or produced outside of animals, such as from yeast or phage (e.g., as a single antibody or antibody fragment or as part of an antibody library).

[0088] Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules. These are known as single chain Fv (scFv); see e.g. , Bird et al. , Science 242:423-426 (1988); and Huston et al.. Proc. Natl. Acad. Sci. 85 : 5879-5883 (1988). These antibody fragments are obtained using conventional techniques known to those of ordinary-21-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02) skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0089] As used herein, the term “single-chain variable fragment” or “scFv” is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin (e.g, mouse or human) covalently linked to form a VH: :VL heterodimer. The heavy (VH) and light chains (VL) are either joined directly or joined by a peptide-encoding linker (e.g, about 10, 15, 20, 25 amino acids), which connects the N-terminus of the VH with the C-terminus of the VL, or the C-terminus of the VH with the N-terminus of the VL. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility. The linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen binding domain.

[0090] Despite removal of the constant regions and the introduction of a linker, scFv proteins retain the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies can be expressed from a nucleic acid comprising VH- and VL-encoding sequences as described by Huston, et al. (Proc. Nat. Acad. Set. USA, 85:5879-5883 (1988)). See, also, U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754. Antagonistic scFvs having inhibitory activity have been described (see, e.g., Zhao et al., Hybridoma (Larchmt) 27(6):455-51 (2008); Peter et al., J Cachexia Sarcopenia Muscle (2012); Shieh et al. , J Imunol 183(4):2277-85 (2009); Giomarelli et al., Thromb Haemost 97(6):955-63 (2007); Fife eta., J Clin Invs 116(8):2252-61 (2006); Brocks et al., Immunotechnology 3(3): 173-84 (1997); Moosmayer et al., Ther Immunol 2(10):31- 40 (1995). Agonistic scFvs having stimulatory activity have been described (see, e.g., Peter et al., J Biol Chem 25278(38):36740-7 (2003); Xie et al., Nat Biotech 15(8):768-71 (1997); Ledbetter etal., Crit Rev Immunol 17(5-6):427-55 (1997); Ho etal., Bio Chim Biophys Acta 1638(3):257-66 (2003)).

[0091] As used herein, an “antigen” refers to a molecule to which an antibody can selectively bind. The target antigen may be a protein e.g., an antigenic peptide), carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. An antigen may also be administered to an animal subject to generate an immune response in the subject.

[0092] As used herein, a “cancer” is a disease state characterized by the presence in a subject of cells demonstrating abnormal uncontrolled replication and in some aspects, the term-22-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02) may be used interchangeably with the term “tumor.” The term “cancer or tumor antigen” refers to an antigen known to be associated and expressed in a cancer cell or tumor cell (such as on the cell surface) or tissue, and the term “cancer or tumor targeting antibody” refers to an antibody that targets such an antigen. In some embodiments, the cancer or tumor antigen is not expressed in a non-cancer cell or tissue. In some embodiments, the cancer or tumor antigen is expressed in a non-cancer cell or tissue at a level significantly lower compared to a cancer cell or tissue. In some embodiments, the cancer is a solid tumor. In other embodiments, the cancer is not a solid tumor. In some embodiments, the cancer is from a carcinoma, a sarcoma, a myeloma, a leukemia, or a lymphoma. In some embodiments, the cancer is a primary cancer or a metastatic cancer. In some embodiments, the cancer is a relapsed cancer. In some embodiments, the cancer reaches a remission, but can relapse. In some embodiments, the cancer is unresectable.

[0093] A “composition” is intended to mean a combination of active agent and another compound or composition, inert (for example, a nanoparticle, detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include carriers, such as pharmaceutically acceptable carriers. In some embodiments, the carrier (such as the pharmaceutically acceptable carrier) comprises, or consists essentially of, or yet further consists of a nanoparticle, such as a polymeric nanoparticle carrier or a lipid nanoparticle that can be used alone or in combination with another carrier, such as an adjuvant or solvent. Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri, tetra-oligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume. Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid components, which can also function in a buffering capacity, include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. Carbohydrate excipients are also intended within the scope of this technology, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose,-23-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02) maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol. A composition as disclosed herein can be a pharmaceutical composition. A “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0094] As used herein, the term “linker” refers to any amino acid sequence comprising from a total of 1 to 200 amino acid residues; or about 1 to 10 amino acid residues, or alternatively 8 amino acids, or alternatively 6 amino acids, or alternatively 5 amino acids that may be repeated from 1 to 10, or alternatively to about 8, or alternatively to about 6, or alternatively to about 5, or alternatively, to about 4, or alternatively to about 3, or alternatively to about 2 times.

[0095] “Pharmaceutically acceptable carriers” refers to any diluents, excipients, or carriers that may be used in the compositions disclosed herein. In some embodiments, a pharmaceutically acceptable carrier comprises, or consists essentially of, or yet further consists of a nanoparticle, such as a polymeric nanoparticle carrier or a lipid nanoparticle (LNP). Additionally or alternatively, pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, polyethylene glycol and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They can be selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

[0096] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally occurring amino acid, e.g., an amino acid analog. The terms encompass amino-24-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02) acid chains of any length, including full length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0097] As used herein, the terms “subject,” “individual,” or “patient” are used interchangeably and refer to an individual organism, a vertebrate, or a mammal and may include humans, non-human primates, rodents, and the like (e.g., which is to be the recipient of a particular treatment, or from whom cells are harvested). In certain embodiments, the individual, patient or subject is a human.

[0098] As used herein, "synthetic," with reference to, for example, a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide refers to a nucleic acid molecule or polypeptide molecule that is produced by recombinant methods and / or by chemical synthesis methods. As used herein, production by recombinant means by using recombinant DNA methods means the use of the well-known methods of molecular biology for expressing proteins encoded by cloned DNA.

[0099] “Treating” or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, z.e., arresting its development; (ii) relieving a disease or disorder, z.e., causing regression of the disorder; (iii) slowing progression of the disorder; and / or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission.

[0100] It is also to be appreciated that the various modes of treatment of disorders as described herein are intended to mean “substantial,” which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved. The treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.

[0101] Throughout this disclosure, various publications, patents, and published patent specifications are referenced by an identifying citation. Also within this disclosure are Arabic numerals referring to referenced citations, the full bibliographic details of which are provided immediately preceding the claims. The disclosures of these publications, patents, and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.-25-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)Quantum Defect Carbon Nanotubes of the Present Technology

[0102] Carbon nanotubes (CNTs) are allotropes of carbon with a cylindrical nanostructure. The carbon atoms are all surface atoms formed in regular structures with defined periodicity. The CNTs may be fibrillar (z.e., have an aspect (length -to-diameter) ratio greater than 1), and in any embodiment herein the CNTs may have an aspect ratio from 1.1 to about 105(such as from about 11 to about 105). The CNTs may be single-walled CNTs (SWCNTs). Semiconducting SWCNTs are cylindrical nanostructures composed of a graphitic lattice of sp2carbons. SWCNTs fluoresce in the near-infrared region (NIR, 800-1700 nm).

[0103] Quantum defects are covalent functional modifications on SWCNTs that modify the otherwise crystalline semiconductors. A quantum defect may be an imperfection in the carbon lattice structure. The imperfection in the carbon lattice structure may include a sp3quantum-well defect site. As a nonlimiting example, a sp3quantum-well defect site may include a carboxyl or a carboxyaryl, where the carboxyl may be used to conjugate proteinbinding peptides via reaction with an amine of the IDP.

[0104] Quantum defects may create new fluorescence peaks (Eif) at about 100 meV to about 300 meV redshifted from the nanotube host emission (En). The defect fluorescence may introduce new chemical sensitivities on quantum defect-modified SWCNTs (QWNTs) that are determined by the chemical nature of the defect, making quantum defects the molecular focal point for chemical detection. The quantum defect-induced fluorescence peak may be sensitive to the local microenvironment around the added functional group and may be systematically controlled through tailoring the functional groups. Conjugation of protein-binding peptides with QWNTs may modulate the dielectric properties of the surrounding environment, quantified by induction-polarity parameters, which can influence defect emission.

[0105] QWNTs may be formed by treating SWCNTs with a chemical treatment that introduces quantum defects into the SWCNTs. For example, SWCNTs may be treated with diazonium salts to form QWNTs. QWNTs of the present application may include about 1 to about 50 defects per 100 nm (e.g., about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 10, about 1 to about 5 defects per 100 nm, or any value or subrange therebetween).

[0106] In any embodiment herein, the QWNTs may have an average length of about 10- 100,000 nm, about 30-1000 nm, about 30-300 nm, about 30-100 nm, about 100-3000 nm, about-26-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)100-1000 nm, about 100-300 nm, about 300-3000 nm, or about 300-1000 nm. In any embodiment herein, the QWNTs may have an average length of about 100-600 nm, about 100- 500 nm, about 100-400 nm, about 200-600 nm, about 200-500 nm, about 200-400 nm or about 250-350 nm. In any embodiment herein, the QWNTs may have an average length of about 300 nm. Length can be determined by transmission electron or atomic force microscopy, dynamic light scatter or any appropriate physicochemical method including chromatography.

[0107] In any embodiment herein, the QWNTs may have an average diameter of about 0.1- 100 nm, about 0.1-10 nm, about 0.1-3 nm, about 0.1-1 nm, about 0.1-0.3 nm, about 0.3-30 nm, about 0.3-10 nm, about 0.3-3 nm, about 0.3-1 nm, about 1-30 nm, about 1-10 nm, about 1-3 nm, about 3-30 nm, about 3-10 nm, or about 10-30 nm. In any embodiment herein, the QWNTs may an average diameter of about 0.5-1.5 nm, about 0.6-1.4 nm, about 0.7-1.3 nm, about 0.8- 1.2 nm or about 0.9-1.1 nm. In any embodiment herein, the QWNTs may have an average diameter of about 1 nm. Diameter can be determined by transmission electron or atomic force microscopy, dynamic light scatter or any appropriate physicochemical method including Raman spectroscopy or chromatography.Supercharged Antibody Fragments of the Present Technology

[0108] Intrinsically disordered proteins (IDPs) are proteins or protein domains that undergo a phase transition between disordered and folded states as part of their function. A subclass of IDPs is unstructured until they bind to another protein or small molecule ligand - a process termed ligand-induced folding. Dramatically increasing the surface net charge of a well-folded protein, known as supercharging, can turn proteins into ligand-induced-folders by destabilizing the folded state of the protein via charge-charge repulsion, and the additional folding energy that results from ligand binding may be sufficient to drive the phase transition from an extended unliganded state to a compact folded bound state. Such modifications may improve protein solubility, stability, and reversibility, while greatly increasing the electric field changes associated with ligand binding. For these reasons, supercharged IDPs represent an attractive target for biodevices

[0109] However, IDPs are not conventionally included as part of sensing devices. One reason for this may be that conventional IDPs have simple folding topologies that are highly designable, whereas IDPs useful in sensing devices, such as antibodies and antibody fragments have complex folding topologies where design rules to engineer this functionality have yet to be determined.-27-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)

[0110] Disclosed herein are IDPs useful as molecular sensors for bioanalyte detection and biophysical studies. The IDPs may change phase in response to interaction with a bioanalyte. The phase change may be specific to the bioanalyte. The phase change may be due to specific binding of the IDP to the bioanalyte. The bioanalyte may be a cytokine, an epidermal growth factor, an interferon, or a hormone. For example, the cytokine may be selected from a lymphokine, an interleukin, a chemokine, or an interferon. For example, the interleukin may be interleukin-1 (IL-1) or interleukin-6 (IL-6). For example, the interferon may be interferon gamma (IFNG). For example, the epidermal growth factor may be transforming growth factor beta (TGFP). For example, the hormone may be luteinizing hormone (LH).[OHl] IL-6 is a proinflammatory cytokine that is closely associated with multiple medical conditions. Serum IL-6 level is elevated in patients with cytokine release syndrome (CRS). CRS is an acute systemic inflammatory syndrome caused by infections, autoimmune diseases, and hematologic conditions, including sepsis. For instance, cytokine release syndrome is common in cancer patients receiving chimeric antigen receptor T-cell (CAR-T) therapy, occurring in up to 50% of patients. Close monitoring and aggressive use of anti-IL6 therapy have been integral in managing patients receiving CAR-T cell therapy. Therefore, the development of robust biosensors may facilitate the early assessment of risk and timely measurement of disease progression.

[0112] The IDP may be derived from a monoclonal antibody (mAb) that binds to the bioanalyte (e.g., IL-6, IL-1, TGFP, IFNG, or LH). The IDP may comprise, or consist essentially of, or yet further consist of an scFv. The IDP may comprise, or consist essentially of, or yet further consist of a human scFv that binds specifically to a bioanalyte.

[0113] The IDP may be a wild-type (WT) version of a protein or a mutant version. The IDP may be a wild-type (WT) version of a scFv or a mutant version of scFv. For example, the IDP may be a mutant scFv with a sequence modified from the WT scFv to provide a supercharged scFv. The mutant scFv may include a number of residues changed in its sequence as compared to the WT scFV, while still maintaining all or a portion of the binding affinity for the bioanalyte.

[0114] The IDP may be a supercharged IDP having a negative net charge. The IDP may be a supercharged mutant scFv. The negative net charge may be about -1 to about -35 (e.g., greater than -5, greater than -10, greater than -15, greater than -20, -5 to -30, -10 to -30, -10 to-28-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)-25, -18 to -25, -20 to -25, or any value or subrange therebetween). The negative net charge may be provided, at least in part, by a number of D residues present in the sequence of the IDP. For example, the IDP may include 1 to 50 D residues (e.g., 5 to 40, 10 to 40, 15 to 40, 20 to 40, 25 to 35, 27 to 32, or any value or subrange therebetween).

[0115] The IDP may include a number of acidic residues. For example, the percentage of acidic residues may be about 1% to about 50% (e.g., about 1% to about 40%, about 1 % to about 30%, about 5% to about 30%, about 10% to about 30%, about 10% to about 20%, about 15% to about 20%, or any value or subrange therebetween).

[0116] The IDP may have a high binding specificity and high binding affinity to a bioanalyte. For example, the IDP (embodied, for example as an antibody fragment) binds to a particular bioanalyte with a dissociation constant (Ka) of about 1 x 10'5M or less. In certain embodiments, the Ka is about 5 x 10'6M or less, about 1 x 10'6M or less, about 5 x 10'7M or less, about 1 x 10'7M or less, about 5 x 10'8M or less, about 1 x 10'8M or less, about 5 x 10'9or less, about 4 x 10'9M or less, about 3 x 10'9M or less, about 2 x 10'9M or less, or about 1 x 10'9M or less. In certain non-limiting embodiments, the Ka is from about 3 x 10'9M or less. In certain non-limiting embodiments, the Ka is from about 1 x 10'7M to about 1 x 10'6M.

[0117] Binding of the IDP to the bioanalyte may be confirmed by, for example, enzyme- linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western Blot assay. Each of these assays generally detect the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody, or an scFv) specific for the complex of interest. For example, the scFv can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March 1986, which is incorporated by reference herein). The radioactive isotope can be detected by such means as the use of a y counter or a scintillation counter or by autoradiography.

[0118] The IDP (e.g., human scFv) may comprise a heavy chain variable (VH) region and a light chain variable (VL) region, optionally linked with a linker sequence, for example a linker peptide (e.g., comprising S and G residues), between the heavy chain variable (VH) region and the light chain variable (VL) region. The IDP may be in the sequence Vn-linker-Vr. The linker sequence may include one or more repeating sequences comprising or consisting of S and G-29-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02) residues. For example, the linker may be (SGG)X, GG(SGG)X, (GGGGS)X, (SGG)X, SS(GGGS)X, or a combination of any two or more thereof, where x is 1 to 12, 1 to 10, 1 to 8, 1 to 5, 2 to 5, or any value or subrange therebetween, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. For example, the linker may be any sequence listed in Table 1.Table 1: Linker sequences

[0119] The IDP may comprise a linker connecting the heavy chain variable (VH) region and light chain variable (VL) region of the extracellular antigen-binding domain. As used herein, the term “linker” refers to a functional group (e.g, chemical or polypeptide) that covalently attaches two or more polypeptides or nucleic acids so that they are connected to one another. As used herein, a “peptide linker” refers to one or more amino acids used to couple two proteins together (e.g, to couple VH and VL domains). The linker may be a peptide linker including G and S residues. For example, the linker may include amino acids having the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or a combination of any two or more thereof.

[0120] The IDP may be a mutant scFv with a sequence modified from the WT scFv to provide a supercharged scFv. For example, the number of mutant residues in the mutant scFv sequence may be 1 to 50 (e.g., greater than 5, greater than 10, greater than 15, greater than 20, greater than 30, 5 to 50, 10 to 40, 10 to 40, 15 to 40, 20 to 40, 25 to 35, or any value or subrange therebetween). For example, the number of mutant residues may be 25 to 30. The mutant residues may be on solvent-exposed regions of the IDP. For example, the mutant residues may be on solvent-exposed side chains on beta strands of the IDP.

[0121] At least a portion of the mutant residues may be changed to D residues. The positions of the mutant D residues may be chosen to increase the distance between solvent- exposed acidic side chains located of the IDP. The number of mutant residues may provide the supercharged IDP having the negative net charge, where the mutant D residues contribute to the negative net charge. For example, the number of different residues changed to D residues may be 1 to 50 (e.g., greater than 5, greater than 10, greater than 15, greater than 20, greater-30-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02) than 30, 5 to 50, 10 to 40, 10 to 40, 15 to 40, 20 to 40, 25 to 35, or any value or subrange therebetween). For example, the number of different residues may be 25 to 30.

[0122] The total number of D residues in the IDP may be 1 to 50 (e.g., greater than 5, greater than 10, greater than 15, greater than 20, greater than 30, 5 to 50, 10 to 40, 10 to 40, 15 to 40, 20 to 40, 25 to 35, or any value or subrange therebetween). D residues may be present in the solvent-exposed side chain or chains located on beta strands of the IDP. For example, the number of D residues may be 25 to 35 solvent-exposed side chain residues located on beta strands of the IDP.

[0123] At least a portion of the mutant residues may be changed to L. Residues may be changed from R to L residues to reduce or exclude instances in the sequence of the IDP of two residues with negative charges placed in close contact (e.g., within 10 residues, 5 residues, 3 residues, or immediately next to). For example, the number of mutant residues changed to L residues may be 0 to 10 (e.g., 0 to 8, 0 to 6, 0 to 5, 0 to 4, 0 to 2, or any value or subrange therebetween). For example, the number of mutant residues changed to L may be 2. For example, the IDP may include 0 to about 5 R residues replaced by L residues as compared to the WT.

[0124] The IDP may be free of or have fewer disulfide bridges and cysteine pairs. This may aid the extended-to-folded phase transition of the IDP. For example, the IDP may be a mutant IDP with a sequence modified from the WT to replace cysteine pairs with other residues (e.g., V and A residues). For example, the number of mutant V and A pairs changed from C pairs in the WT version may be 0 to 10 (e.g., 0 to 8, 0 to 6, 0 to 5, 0 to 4, 0 to 2, or any value or subrange therebetween). For example, the number of mutant residues changed to L may be 2.

[0125] Exemplary amino acid sequences of the heavy chain variable (VH) domain and light chain variable (VL) domain present in the extracellular antigen-binding domain of the IDP described herein include, but are not limited to:

[0126] a-IL6sc VH domain (SEQ ID NO: 5)EVQLDESGPGLVKPSQTLSLDVDVSGGSITTRYYAWSWILQPPGKGLEWMGVIDYDGDTY YSPSLKSRTDIDWDTSKNVFDLDLSSVTPEDTAVYYAADDPDWTGFHYDYWGQGTDVTV S-31-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)

[0127] a-IL6sc VL domain (SEQ ID NO: 6)QAVLTQPPLVDGTPGQTVTIDVAGANNDIGTYAYVSWYQQLPGTAPDLLIYKVTTRASGIPSRFDGDKSGNDADLDISGLQSEDEADYYAASYLNFNNAVFGRGTDLTV

[0128] a-IL6-a VH domain (SEQ ID NO: 7)EVQLQESGPGLVKPSQTLSLTVTVSGGSITTRYYAWSWILQPPGKGLEWMGVIDYDGDTYY SPSLKSRTDISWDTSKNVFSLDLSSVTPEDTAVYYAADDPDWTGFHYDYWGQGTDVTVS

[0129] a-IL6-b VH domain (SEQ ID NO: 8)EVQLQESGPGLVKPSQTLSLDVTVSGGSITTRYYAWSWIRQPPGKGLEWMGVIDYDGDTY YSPSLKSRTSISWDTSKNVFDLDLSSVTPEDTAVYYAARDPDWTGFHYDYWGQGTQVTVS

[0130] a-IL6-b VL domain (SEQ ID NO: 9)QAVLTQPPLVSGTPGQTVTIDVAGANNDIGTYAYVSWYQQLPGTAPKLLIYKVTTRASGIPS RFDGDKSGNDADLDISGLQSEDEADYYAASYRNFNNAVFGRGTDLTV

[0131] a-IL6-c VH domain (SEQ ID NO: 10)EVQLQESGPGLVKPSQTLSLTVDVSGGSITTRYYAWSWILQPPGKGLEWMGVIDYDGDTY YSPSLKSRTDISWDTSKNQFSLQLSSVTPEDTAVYYAARDPDWTGFHYDYWGQGTDVTVS

[0132] a-IL6-c VL domain (SEQ ID NO: 11)QAVLTQPPLVDGTPGQTVTIDVAGANNDIGTYAYVSWYQQLPGTAPKLLIYKVTTRASGIPS RFSGDKSGNDASLDISGLQSEDEADYYAASYLNFNNAVFGRGTDLTV

[0133] a-IL6-d VH domain (SEQ ID NO: 12)EVQLDESGPGL VKPSQTLSLDCD VSGGSITTRYYA WSWILQPPGKGLEWMGVIDYDGDTY YSPSLKSRTDIDWDTSKNVFDLDLSSVTPEDTAVYYCADDPDWTGFHYDYWGQGTDVTV S

[0134] a-IL6-e VL domain (SEQ ID NO: 13)QAVLTQPPLVDGTPGQTVTIDCAGANNDIGTYAYVSWYQQLPGTAPDLLIYKVTTRASGIP SRFDGDKSGNDADLDISGLQSEDEADYYACSYLNFNNAVFGRGTDLTV-32-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)

[0135] a-ILip VH domain (SEQ ID NO: 14)ENLYFQGDIQMDQSPSSLDASVGDAVTIAADTSGNIHNYLTWYQQKPGAAPQLLIYNADTLADGVPSAFDGSGSGTQFDLDISSLQPEDFANYYVQHFWSLPFTFGQGTDVEIAAT

[0136] a-ILip VL domain (SEQ ID NO: 15)EVDL VESGGGL VQPGGSLDLDADASGFDFSA YDMSWVRQAPGAALEWVA YISSGGGSTYFPDTVKGAFDISRDNAANTLDLQMNSLRAEDTAVYYAARQNDKLTWFDYWGQGTLVTV

[0137] a-TGFp VH domain (SEQ ID NO: 16)QVQLDQSGAEVDKPGSSVWDWASGYTFSSNVISWVRQAPGQGLEWMGGVIPIVDIANYAQRFKGRVDIDADESTSTTDMELSSLRSEDTADYYAASTLGLVLDAMDYWGQGTDVDV

[0138] a-TGFp VL domain (SEQ ID NO: 17)LETVLDQSPGTLDLSPGERA TLDWASQSLGSSYLA WYQQKPGQAPVLLIYGASSDAPGIPDRFDGDGSGTDFDLDISRLEPEDFAVYYAQQYADSPITFGQGTDLEIK

[0139] a-Interferon gamma VH domain (SEQ ID NO: 18)QVQLQQSGPELEEPGETVEISCEASGYTFTDYGMNWVKQAPGQGLKWMGWINTYTGESTYVDDFEGEFVFSLETSASAAYLQINNLENEDTATYFCARRGFYAMDYWGQGTSVTV

[0140] a-Interferon gamma VL domain (SEQ ID NO: 19)NVLTQSPAIMSASPGEEVTLTCSASSSISYMFWYHQEPGSSPRLLIYDTSNLASGVPVEFSGSGSGTSYSLTISEMEAEDAATYFCHQSSSYPFTFGSGTKLEIE

[0141] a-LH-a VH domain (SEQ ID NO: 20)DIKMTQSPSSMYASLGERVDITVAASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFDGDGSGQDYDLTISSLEYEDMGIYYALQYDEFPYTFGGGTKLEIKA

[0142] a-LH-a VL domain (SEQ ID NO: 21)EVDLQQSDAEWKPGASVALSVDASGFNIKDTYMHWVNLRPDQGLEWIGRJDPANADTRYDSKFQGKADIDADTSSNDAYLAFSSLTSEDTAVYYAARSTYYHGSGYGWDWYFDVWGAGTDVTVSSGGC-33-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)

[0143] a-LH-b VH domain (SEQ ID NO: 22)DIKMTQSPSSMYASLGERVDITVDASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRF DGDGSGQDYDLTISSLEYEDMGIYYALQYDEFPYTFGGGTKLEIKD

[0144] a-LH-b VL domain (SEQ ID NO: 23)EVDLQQSDAEWKPGASVDLSVDASGFNIKDTYMHWVNLRPDQGLEWIGRIDPANADTR YDSKFQGKADIDADTSSNDAYLDFSSLTSEDTAVYYAARSTYYHGSGYGWDWYFDVWGAG TDVDVSSGGC

[0145] a-LH-c VH domain (SEQ ID NO: 24)DIKMTQSPSSMDASLGEAVDIDVAASQDINSYLSWFQQKPGKSPKTLIYAANRLVDGVPSR FDGDGSGQDYDLDISSLDYADMGIYYALQYDEFPYTFGGGTDLEIDDT

[0146] a-LH-c VL domain (SEQ ID NO: 25)VQLQQSGAEVMKPGASVAISVAGTGYTFSSYWIEWVKQRPGHGLERJGEILPGSGSTNYNE KFRGKADFDADKSSKTADMDLSSLTSEDSAVYYAARYLPYYYAMDYWGQGTSVTVSSGGC

[0147] a-LH-d VH domain (SEQ ID NO: 26)DIKMTQSPSSMDASLGEDVDIDVDASQDINSYLSWFQQKPGKSPKTLIYDANRLVDGVPSR FDGDGSGQDYDLDISSLDYADMGIYYALQYDEFPYTFGGGTDLEIDDT

[0148] a-LH-d VL domain (SEQ ID NO: 27)SVQLQQSGAEVMKPGASVDISVDGTGYTFSSYWIEWVKQRPGHGLERIGEILPGSGSTNYN EKFRGKADFDADKSSKTADMDLSSLTSEDSAVYYAARYLPYYYAMDYWGQGTSVTVSSGG C

[0149] Examples of other heavy chain variable (VH) region and light chain variable (VL) region amino acid sequences that bind bioanalytes are known in the art, and are described in US Patent Nos. US 7,919,095 and US 8,198,414 (binding IL-6), US Patent No. 7,446,175 (binding IL-1), US Patent No. US 7,723,486 (binding TGFP), US Patent No. US 7,700,098 (binding a-Interferon gamma), the contents of which are incorporated by reference herein in their entireties.-34-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)

[0150] Exemplary amino acid sequences of the supercharged scFv described herein include, but are not limited to:

[0151] a-IL6sc (SEQ ID NO: 28), having a net charge of -24EVQLDESGPGLVKPSQTLSLDVDVSGGSITTRYYAWSWILQPPGKGLEWMGVIDYDGDTYYSPSLKSRTDIDWDTSKNVFDLDLSSVTPEDTAVYYAADDPDWTGFHYDYWGQGTDVTVSGGSGGSGGSGGSGGSGGSGGSGGQAVLTQPPLVDGTPGQTVTIDVAGANNDIGTYAYVSWYQQLPGTAPDLLIYKVTTRASGIPSRFDGDKSGNDADLDISGLQSEDEADYYAASYLNF NNAVFGRGTDLTV

[0152] a-IL6-a (SEQ ID NO: 29), having a net charge of -19EVQLQESGPGLVKPSQTLSLTVTVSGGSITTRYYAWSWILQPPGKGLEWMGVIDYDGDTYYSPSLKSRTDISWDTSKNVFSLDLSSVTPEDTAVYYAADDPDWTGFHYDYWGQGTDVTVSGGSGGSGGSGGSGGSGGSGGSGGQAVLTQPPLVDGTPGQTVTIDVAGANNDIGTYAYVSWYQQLPGTAPDLLIYKVTTRASGIPSRFDGDKSGNDADLDISGLQSEDEADYYAASYLNFNN AVFGRGTDLTV

[0153] a-IL6-b (SEQ ID NO: 30), having a net charge of -13EVQLQESGPGLVKPSQTLSLDVTVSGGSITTRYYAWSWIRQPPGKGLEWMGVIDYDGDTYYSPSLKSRTSISWDTSKNVFDLDLSSVTPEDTAVYYAARDPDWTGFHYDYWGQGTQVTVSGGSGGSGGSGGSGGSGGSGGSGGQAVLTQPPLVSGTPGQTVTIDVAGANNDIGTYAYVSWYQQLPGTAPKLLIYKVTTRASGIPSRFDGDKSGNDADLDISGLQSEDEADYYAASYRNFN NAVFGRGTDLTV

[0154] a-IL6-c (SEQ ID NO: 31), having a net charge of -13EVQLQESGPGLVKPSQTLSLTVDVSGGSITTRYYAWSWILQPPGKGLEWMGVIDYDGDTYYSPSLKSRTDISWDTSKNQFSLQLSSVTPEDTAVYYAARDPDWTGFHYDYWGQGTDVTVSGGSGGSGGSGGSGGSGGSGGSGGQAVLTQPPLVDGTPGQTVTIDVAGANNDIGTYAYVSWYQQLPGTAPKLLIYKVTTRASGIPSRFSGDKSGNDASLDISGLQSEDEADYYAASYLNFNN AVFGRGTDLTV

[0155] a-IL6-d (SEQ ID NO: 32), having a net charge of -24-35-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)EVQLDESGPGL VKPSQTLSLDCD VSGGSITTRYYA WSWILQPPGKGLEWMGVIDYDGDTY YSPSLKSRTDIDWDTSKNVFDLDLSSVTPEDTAVYYCADDPDWTGFHYDYWGQGTDVTV SGGSGGSGGSGGSGGSGGSGGSGGQAVLTQPPLVDGTPGQTVTIDVAGANNDIGTYAYV SWYQQLPGTAPDLLIYKVTTRASGIPSRFDGDKSGNDADLDISGLQSEDEADYYAASYLNF NNAVFGRGTDLTV

[0156] a-IL6-e (SEQ ID NO: 33), having a net charge of -24EVQLDESGPGLVKPSQTLSLDVDVSGGSITTRYYAWSWILQPPGKGLEWMGVIDYDGDTY YSPSLKSRTDIDWDTSKNVFDLDLSSVTPEDTAVYYAADDPDWTGFHYDYWGQGTDVTV SGGSGGSGGSGGSGGSGGSGGSGGQAVLTQPPLVDGTPGQTVTIDCAGANNDIGTYAYV SWYQQLPGTAPDLLIYKVTTRASGIPSRFDGDKSGNDADLDISGLQSEDEADYYACSYLNF NNAVFGRGTDLTV

[0157] a-ILip (SEQ ID NO: 34), having a net charge of -25ENLYFQGDIQMDQSPSSLDASVGDAVTIAADTSGNIHNYLTWYQQKPGAAPQLLIYNADTL ADGVPSAFDGSGSGTQFDLDISSLQPEDFANYYVQHFWSLPFTFGQGTDVEIAATGGGGS GGGGSGGGGSGGGGSEVDLVESGGGLVQPGGSLDLDADASGFDFSAYDMSWVRQAPG AALEWVA YISSGGGSTYFPDTVKGAFDISRDNAANTLDLQMNSLRAEDTA VYYAARQNDKL TWFDYWGQGTL VTV

[0158] a-TGFp (SEQ ID NO: 35), having a net charge of -26QVQLDQSGAEVDKPGSSVWDWASGYTFSSNVISWVRQAPGQGLEWMGGVIPIVDIANYAQRFKGRVDIDADESTSTTDMELSSLRSEDTADYYAASTLGLVLDAMDYWGQGTDVDVSG GSGGSGGSGGSGGSGGSGGSGGLETVLDQSPGTLDLSPGERA TLDWASQSLGSSYLA WY QQKPGQAPVLLIYGASSDAPGIPDRFDGDGSGTDFDLDISRLEPEDFAVYYAQQYADSPIT FGQGTDLEIK

[0159] a-Interferon gamma (SEQ ID NO: 36), having a net charge of -22QVQLQQSGPELEEPGETVEISCEASGYTFTDYGMNWVKQAPGQGLKWMGWINTYTGESTYVDDFEGEFVFSLETSASAAYLQINNLENEDTATYFCARRGFYAMDYWGQGTSVTVSSGG GSGGGSGGGSNVLTQSPAIMSASPGEEVTLTCSASSSISYMFWYHQEPGSSPRLLIYDTSNL ASGVPVEFSGSGSGTSYSLTISEMEAEDAATYFCHQSSSYPFTFGSGTKLEIE-36-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)

[0160] a-LH-a (SEQ ID NO: 37), having a net charge of -17DIKMTQSPSSMYASLGERVDITVAASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFDGDGSGQDYDLTISSLEYEDMGIYYALQYDEFPYTFGGGTKLEIKAGGGGSGGGGSGGGGSGGGGSEVDLQQSDAEWKPGASVALSVDASGFNIKDTYMHWVNLRPDQGLEWIGRIDPANADTRYDSKFQGKADIDADTSSNDAYLAFSSLTSEDTAVYYAARSTYYHGSGYGWDWYF DVWGAGTDVTVSSGGC

[0161] a-LH-b (SEQ ID NO: 38), having a net charge of -22DIKMTQSPSSMYASLGERVDITVDASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFDGDGSGQDYDLTISSLEYEDMGIYYALQYDEFPYTFGGGTKLEIKDGGGGSGGGGSGGGGSGGGGSEVDLQQSDAEWKPGASVDLSVDASGFNIKDTYMHWVNLRPDQGLEWIGRIDPANADTRYDSKFQGKADIDADTSSNDAYLDFSSLTSEDTAVYYAARSTYYHGSGYGWDWY FDVWGAGTDVDVSSGGC

[0162] a-LH-c (SEQ ID NO: 39), having a net charge of -17DIKMTQSPSSMDASLGEAVDIDVAASQDINSYLSWFQQKPGKSPKTLTYAANRLVDGVPSRFDGDGSGQDYDLDISSLDYADMGIYYALQYDEFPYTFGGGTDLEIDDTGGGGSGGGGSGGGGSGGGGSSVQLQQSGAEVMKPGASVAISVAGTGYTFSSYWIEWVKQRPGHGLERIGEILPGSGSTNYNEKFRGKADFDADKSSKTADMDLSSLTSEDSAVYYAARYLPYYYAMDYWGQ GTSVTVSSGGC

[0163] a-LH-d (SEQ ID NO: 40), having a net charge of -22DIKMTQSPSSMDASLGEDVDIDVDASQDINSYLSWFQQKPGKSPKTLIYDANRLVDGVPSRFDGDGSGQDYDLDISSLDYADMGIYYALQYDEFPYTFGGGTDLEIDDTGGGGSGGGGSGGGGSGGGGSSVQLQQSGAEVMKPGASVDISVDGTGYTFSSYWIEWVKQRPGHGLERIGEILPGSGSTNYNEKFRGKADFDADKSSKTADMDLSSLTSEDSAVYYAARYLPYYYAMDYWGQGTSVTVSSGGCConjugates of Quantum Defect Carbon Nanotubes and Supercharged AntibodyFragments of the Present Technology

[0164] Disclosed herein are conjugates useful as molecular sensors for bioanalyte detection and biophysical studies. The conjugates provide quantum defect responses to localized-37-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02) environmental changes using proteins designed to amplify the transduction of bioanalyte binding (FIG. 1). The conjugates may include phase-changing IDPs (e.g., scFvs) that recognize a bioanalyte (e.g., a cytokine, an epidermal growth factor, interferon, or a hormone) . The IDP may be capable of performing the full phase transition from an intrinsically disordered state to a compact folded state upon bioanalyte binding. The IDP may be a supercharged scFv without internal disulfide bonds. The IDPs may be conjugated to QWNTs via quantum-well defect sites. For example, the QWNT may include at a sp3quantum-well defect site a carboxyl or a carboxyaryl, which may be used to conjugate to the IDP. Conjugation may be direct covalent bond conjugation between the QWNT and the IDP, with the carboxyl reacting with an amine of the IDP, forming an amide bond (e.g., via carbodiimide crosslinker chemistry). These conjugates may be used to observe quantitative and specific wavelength shifts of quantum defect fluorescence in response to the presence of a bioanalyte (e.g., a cytokine, an epidermal growth factor, interferon, or a hormone) in solution, including biologically relevant solutions (e.g., serum). Conjugation of the IDP to the QWNT may provide sensitization of the quantum defects to changes in local electric fields at the defect site, in this case induced by a protein phase transition.

[0165] The density of IDPs conjugated to a QWNT may be about 1 to about 20 defects per 100 nm (e.g., about 1 to about 15, about 1 to about 10, about 1 to about 80, about 1 to about 6, about 1 to about 4, about 1 to about 2 IDPs per 100 nm, or any value or subrange therebetween). For example, the conjugates may include about 1-4 IDPs conjugated to the QWNT per 100 nm. Density of IDPs conjugated to QWNTs may be quantified with atomic force microscopy (AFM).Methods for Detecting a Bioanalyte

[0166] The diagnostic methods of the present technology involve detecting a bioanalyte or determining a bioanalyte concentration in a biological sample obtained from a subject using the conjugates of the present technology. The methods of detecting the bioanalyte may include contacting the conjugate with the bioanalyte. Contacting may be conducted in solution. For example, the methods of detecting may include preparing a pharmaceutical composition including the conjugate, as described herein, and contacting the bioanalyte with the pharmaceutical composition. Contacting may include immersing, mixing, and the like.-38-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)

[0167] The methods of detecting the cytokine IL-6 may involve using a conjugate with a supercharged antibody fragment that recognizes the cytokine IL-6. For example, methods of detecting IL-6 may include using one or more conjugates comprising SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or a combination of any two or more thereof.

[0168] The methods of detecting the cytokine IL-1 may involve using a conjugate with a supercharged antibody fragment that recognizes the cytokine IL-1. For example, methods of detecting IL-1 may include using one or more conjugates comprising SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 34, or a combination of any two or more thereof.

[0169] The methods of detecting epidermal growth factor TGFP may involve using a conjugate with a supercharged antibody fragment that recognizes TGFp. For example, methods of detecting TGFP may include using one or more conjugates comprising SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 35, or a combination of any two or more thereof.

[0170] The methods of detecting IFNG may involve using a conjugate with a supercharged antibody fragment that recognizes IFNG. For example, methods of detecting IFNG may include using one or more conjugates comprising SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 36, or a combination of any two or more thereof.

[0171] The methods of detecting LH may involve using a conjugate with a supercharged antibody fragment that recognizes LH. For example, methods of detecting LH may include using one or more conjugates comprising SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO :27, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or a combination of any two or more thereof.Methods for Selecting Treatments for Patients Based on Bioanalyte Detection

[0172] In an aspect, a method for selecting a subject for treatment with anti-IL-6 therapy includes (a) detecting IL-6 above a predetermined threshold in a biological sample obtained from the subject using a pharmaceutical composition disclosed herein; and (b) administering the anti-IL-6 therapy to the subject. The pharmaceutical composition may be a pharmaceutical composition for the detection of IL-6, as described herein.-39-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)

[0173] The method of selecting treatments may be useful for treating or preventing cytokine release syndrome. Treatment may include administering an anti-IL-6 therapy to the subject, according to treatments known in the art.

[0174] In another aspect, a method for treating or preventing cytokine release syndrome in a subject includes (a) detecting IL-6 above a predetermined threshold in a biological sample obtained from the subject using a pharmaceutical composition disclosed herein; and (b) administering an anti-IL-6 therapy to the subject. The pharmaceutical composition may be a pharmaceutical composition for the detection of IL-6, as described herein. The anti-IL-6 therapy may be administered according to methods known in the art.

[0175] In another aspect, a method for prolonging survival of a cancer patient includes administering to the cancer patient an effective amount of an anti-IL-6 therapy, wherein an IL- 6 level in a biological sample obtained from the cancer patient is at or above a predetermined threshold measured using a pharmaceutical composition disclosed herein. The pharmaceutical composition may be a pharmaceutical composition for the detection of IL-6, as described herein. The anti-IL-6 therapy may be administered according to methods known in the art.

[0176] In any embodiment, the subject may be diagnosed with and / or may have previously been diagnosed with cytokine release syndrome. The subject may be diagnosed with and / or may have previously been diagnosed with cancer. The subject may be receiving immunotherapy treatment, and / or have previously been treated with an immunotherapy. Nonlimiting examples of immunotherapy include monoclonal antibody therapy, CAR T-cell therapy, or a combination thereof. Nonlimiting examples of CAR T-cell therapy include tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, lisocabtagene maraleucel, idecabtagene vicleucel, ciltacabtagene autoleucel, obecabtagene autoleucel, or a combination of any two or more thereof.

[0177] In any embodiment, the biological sample used for detection of IL-6 may include plasma, blood, serum, or biopsied tissue. The subject may be a mammal. For example, the subject may be a human, a rat, or another mammal.Pharmaceutical Compositions and Preparations of the Present Technology

[0178] In any and all embodiments of the conjugate composition disclosed herein, the composition may further comprise a pharmaceutically acceptable carrier selected from the-40-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02) group consisting of a solution, cream, emulsion, gel, liposome, nanoparticle, or ointment. Disclosed herein are pharmaceutical compositions comprising conjugates of the present disclosure that may contain a carrier or diluent, which can be a solvent or dispersion medium containing, for example, water, saline, Tris buffer, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be affected by various antibacterial and antifungal agents and preservatives, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In some embodiments, isotonic agents, for example, sugars (e.g., sucrose) or sodium chloride, and buffering agents are included. Isotonic agents may be present in an amount of about 1% (w / v) to about 40% (w / v), about 5 % (w / v) to about 30% (w / v), about 5% (w / v) to about 20% w / v), or about 10% (w / v).

[0179] Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin or carrier molecules. Other excipients may include wetting or emulsifying agents. In general, excipients suitable for injectable preparations can be included as apparent to those skilled in the art.

[0180] Pharmaceutical compositions and preparations comprising conjugates of the present technology may be manufactured by means of conventional mixing, dissolving, granulating, emulsifying, encapsulating, entrapping, or lyophilizing processes. Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients, or auxiliaries that facilitate formulating preparations suitable for in vitro, in vivo, or ex vivo use. The compositions can be combined with one or more additional biologically active agents and may be formulated with a pharmaceutically acceptable carrier, diluent, or excipient to generate pharmaceutical (including biologic) or veterinary compositions of the instant disclosure suitable for parenteral or intravenous administration.

[0181] Many types of formulation are possible as is appreciated by those skilled in the art. The particular type chosen is dependent upon the route of administration chosen, as is well- recognized in the art. For example, systemic formulations will generally be designed for-41-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02) administration by injection, e.g., intravenous, as well as those designed for intratumoral delivery. In some embodiments, the systemic or intratumoral formulation is sterile.

[0182] Sterile injectable solutions are prepared by incorporating conjugates of the present disclosure in the required amount of the appropriate solvent with various other ingredients enumerated herein, as required, followed by suitable sterilization means. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle that contains the basic dispersion medium and the required other ingredients from those enumerated above.

[0183] In some embodiments, the conjugate compositions of the present disclosure may be formulated in aqueous solutions, or in physiologically compatible solutions or buffers such as Hanks’s solution, Ringer’s solution, mannitol solutions or physiological saline buffer. In certain embodiments, any of the conjugate compositions may contain formulator agents, such as suspending, stabilizing, penetrating or dispersing agents, buffers, lyoprotectants or preservatives such as polyethylene glycol, polysorbate 80, l-dodecylhexahydro-2H-azepin-2- one (laurocapran), oleic acid, sodium citrate, Tris HC1, dextrose, propylene glycol, mannitol, polysorbate polyethylene sorbitan monolaurate (Tween®-20), isopropyl myristate, benzyl alcohol, isopropyl alcohol, ethanol sucrose, trehalose and other such generally known in the art may be used in any of the compositions of the instant disclosure. (Pramanick et al., Pharma Times 45(3) 65-16 (2013)).

[0184] Pharmaceutical compositions for detecting the cytokine IL-6 may include a conjugate with a supercharged antibody fragment that recognizes the cytokine IL-6. For example, pharmaceutical compositions for detecting IL-6 may include one or more conjugates comprising SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or a combination of any two or more thereof.

[0185] Pharmaceutical compositions for detecting the cytokine IL-1 may include a conjugate with a supercharged antibody fragment that recognizes the cytokine IL-1. For example, pharmaceutical compositions for detecting IL-1 may include one or more conjugates comprising SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 34, or a combination of any two or more thereof.-42-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)

[0186] Pharmaceutical compositions for detecting epidermal growth factor TGFP may include a conjugate with a supercharged antibody fragment that recognizes TGFp. For example, pharmaceutical compositions for detecting TGFP may include one or more conjugates comprising SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 35, or a combination of any two or more thereof.

[0187] Pharmaceutical compositions for detecting IFNG may include a conjugate with a supercharged antibody fragment that recognizes IFNG. For example, pharmaceutical compositions for detecting IFNG may include one or more conjugates comprising SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 36, or a combination of any two or more thereof.

[0188] Pharmaceutical compositions for detecting LH may include a conjugate with a supercharged antibody fragment that recognizes LH. For example, pharmaceutical compositions for detecting LH may include one or more conjugates comprising SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO :27, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or a combination of any two or more thereof.Effective Amount of Compositions of the Present Technology

[0189] An effective amount of conjugate compositions of the present technology can be utilized for bioanalyte detection, with the effective amount in one or more divided doses for a prescribed period of time and at a prescribed frequency of administration.

[0190] For example, as is apparent to those skilled in the art, an effective amount of conjugate compositions of the present technology in accordance with the present disclosure may vary according to factors such as desired detection limit, composition, concentration of the bioanalyte, and the ability of the conjugate compositions of the present technology to detect a desired bioanalyte.EXAMPLES

[0191] The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way. The examples herein are provided to illustrate advantages of the present technology and to further assist a person of ordinary skill in the art with preparing or using the compositions and systems of the present technology. The examples-43-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02) should in no way be construed as limiting the scope of the present technology, as defined by the appended claims. The examples can include or incorporate any of the variations, aspects, or embodiments of the present technology described above. The variations, aspects, or embodiments described above may also further each include or incorporate the variations of any or all other variations, aspects, or embodiments of the present technology. The following Examples demonstrate the preparation, characterization, and use of illustrative compositions of the present technology to detect bioanalytes using the conjugates disclosed herein.EXAMPLE 1: IL-6 Detection with scFv / QWNT Conjugates

[0192] Quantum defects in single-walled carbon nanotubes promoted exciton localization that provide potential applications in biodevices. Quantum defect sensitization was explored by forming an intrinsically disordered antibody fragment protein to undergo a phase change at a quantum defect site. A supercharged single-chain antibody fragment (scFv) was provided that had a full ligand-induced folding transition from an intrinsically disordered state to a compact folded state in the presence of the cytokine IL-6. The supercharged scFv was conjugated to a quantum defect in the QWNT to induce a substantial local electric field change upon ligand binding. Employing the detection of a proinflammatory biomarker, interleukin-6, as a representative model system, supercharged scFv-coupled quantum defects exhibited robust fluorescence wavelength shifts concomitant with the protein folding transition. Quantum chemical simulations suggested that the quantum defects amplified the optical response to the localization of charges produced upon antigen-induced folding of the proteins, which is difficult to achieve in unmodified nanotubes. These findings indicate these conjugates may be used for biomarker sensing and studying protein biophysics. Unless otherwise specified, the scFv in Example 1 was SEQ ID. No. 28.Experimental Materials and Methods

[0193] Supercharged scFv design. The structure of IL-6 in complex with a neutralizing antibody (PDB ID: 4ZS7) was used as the basis for scFv design. A GG(SGG)? sequence was used as linker to link together the antibody variable regions in a heavy / light chain orientation. Disulfide bridges were replaced with alanine-leucine pairs to provide a full folding transition. This sequence was referred to as the wildtype anti-hIL-6 scFv, a-IL6wt. Using the solved crystal structure as a reference, solvent-exposed residues on beta strands that were not involved in antigen binding or at the interface between variable regions were chosen for mutation to-44-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02) aspartic acid. The introduction of these mutations changed the overall net charge of the scFv from -2 to -25. This sequence is referred to as the supercharged anti-hIL-6 scFv, a-IL6sc (SEQ ID. No. 28).

[0194] Protein purification. scFvs were expressed as insoluble inclusion bodies in NiCo(DE3) (New England Biolabs, Ipswich, MA) e. coli cells in the pET28a vector (EMD Biosciences Inc., Darmstadt, Germany). Cells were grown at 37 °C until an optical density at 600 nm of 1 was reached, after which the temperature was dropped to 18 °C, 0.5 mM IPTG (GoldBio Inc., St. Louis, MO) was added, and the cells were incubated overnight. Cell pellets were harvested and resuspended in 50 mM sodium phosphate, 300 mM sodium chloride, 10 mM imidazole pH 8 (Buffer A). After adding 50 pg / mL DNAse and AEBSF, cells were disrupted via press and centrifuged at 17,000 g for 30 minutes. The supernatant was discarded, and the remaining fraction was resuspended in 50 mM sodium phosphate, 8 M guanidinium hydrochloride pH 8 (Buffer B). Cells underwent three rounds of freeze-thaw cycles and were centrifuged at 17,000 g for 30 minutes. The supernatant was added to Ni-NTA beads, washed with Buffer B, and eluted with 50 mM sodium phosphate, 8 M guanidinium hydrochloride at pH 4.5. Proteins were then dialyzed overnight into 20 mM Tris, 100 mM sodium chloride, 1 mM EDTA, 1 mM DTT at pH 8. TEV protease was added to the proteins, which had undergone partial precipitation, and the reaction was allowed to occur at 20 °C for 5 hours. The sample was centrifuged at 5,000 g for 10 minutes, the supernatant was discarded, and the precipitated protein was resuspended in Buffer B. Samples were again incubated with Ni-NTA beads and the flow-through was collected. Proteins were further purified and refolded in a single step on a superose 12 gel filtration column (50 mM phosphate, 500 mM NaCl pH 8. The column was preloaded with a bolus of 50 mM phosphate, 500 mM NaCl, 1 M arginine at pH 8. hIL-6 was expressed as an MBP-fusion protein in an identical manner as the scFvs. Cell pellets were resuspended in Buffer A, lysed via press, centrifuged at 17,000 g for 30 minutes and the supernatant was loaded onto Ni-NTA beads. Proteins were eluted using 50 mM sodium phosphate, 300 mM sodium chloride, and 250 mM imidazole at pH 8. TEV protease and 1 mM DTT were added and incubated overnight at 4 °C. Proteins were dialyzed into Buffer A and reloaded onto Ni-NTA beads. The flowthrough was collected and purified using a superose 12 gel filtration column.

[0195] Circular dichroism. Circular dichroism spectra were recorded with an Aviv Biomedical Circular Dichroism Spectrometer Model 400 in a 0.2 cm light path quartz cell,-45-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02) using a bandwidth of 1-2 nm, an averaging time of 2 seconds, and averaged over two scans. Because of the high sodium chloride concentrations, spectra were recorded between 215 nm and 250 nm, with background correction.

[0196] Microscale thermophoresis. Human IL-6 was fluorescently labeled using fluorescein NHS according to manufacturer instructions: The labeling reaction was carried out in 50 mM borate, 100 mM sodium chloride, pH 8.5. Unreacted dye was removed by dialysis and a G-25 desalting column. MST experiments were performed in 50 mM sodium phosphate pH 8 using a NanoTemper Monolith NT.l 15 and standard capillaries. The concentrations of scFv were determined by UV absorbance. Data was analyzed with PALMIST.

[0197] Intrinsic tryptophan fluorescence measurements. Salt-induced folding was measured using a DM45 spectrofluorometer (Olis Inc. Athens, GA). Samples were excited at 280 nm and emission measured at 348 nm. Slit widths were 3.06 nm. 100 data points were collected for each scan with a 0.5 sec integration time. Five scans were performed for each NaCl concentration and averaged to the final value. Measurements were performed in 50 mM sodium phosphate, pH 8 with varying concentrations of NaCl. Fluorescence intensity as a function of NaCl was fit with Equation 1, modified from that of Santoro and Bolen: (Equation 1)where Ip and ly refer to the fluorescence intensity of the folded and unfolded states at zero [NaCl], mu and mr account for the slopes of the pre- and post-transition regions. AGfOiding 0is the free energy of folding at 0 [NaCl] and m is the dependence of the free energy of folding on NaCl activity. This equation assumes a linear dependence of the folding free energy on otNaci over the experimental range of 0.1 - 4.0 M. This has been shown to be true for several cations in the case of the designed heme binding protein H4(-24) over the cation activity range at which the average acidic side chain-side chain distance is significantly shorter than the Debye length.

[0198] Purification of (6,5)-SWCNT. Raw SWCNT material, CoMoCAT SG65i (Sigma- Aldrich) was dispersed in 1 wt. / v% sodium deoxycholate (DOC, 99.9%, Sigma-Aldrich) aqueous solution at a nanotube concentration of 1 mg / mL using tip-sonication at 6W (Sonics & Materials, Inc) and 4 °C for 1 hour, followed by ultracentrifugation at 100,000 g for 30 min.-46-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)The 85% supernatant was used to obtain (6,5) enriched SWCNT solution based on the previously reported protocol. The final purified (6,5) SWCNTs were stabilized in 1.04% DOC solution to maintain long-term colloidal stability.

[0199] Covalent functionalization of purified (6,5)-SWCNT. 4-carboxylbenzene tetrafluoroborate was freshly synthesized from 4-aminobenzoic acid (>99%, Sigma Aldrich) and nitrous acid. The synthesized diazonium salts were added to the aqueous solution of purified (6,5) enriched SWCNTs in 1% sodium dodecyl sulfate (SDS, >99.0%, Sigma Aldrich) at the diazonium salt to carbon of (6,5)-SWCNT molar ratio of 1 to 275. The SWCNT and diazonium mixture was illuminated with a mercury arc lamp (X-Cite 120Q, Excelitas) at about 20 °C. After 20 minutes of illumination, the QWNT solution was filtrated through Cytiva Vivaspin® 20 filters (lOOkDa MWCO) to remove unreacted diazonium salts and concentrate the QWNT solution for bioconjugation.

[0200] Protein conjugation to 4-carboxylaryl QWNT. The QWNTs in 1% SDS were reacted with N-Ethyl-N’-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, >99%, Sigma Aldrich) and N-hydroxysuccinimide (NHS, >98%, Sigma Aldrich) in a [quantum defects]: [EDC] :[NHS] ratio of 1 : 10:25 for 30 minutes at 20 °C. 1.2 pL of P-mercaptoethanol was added to the solution to quench unreacted EDC. After 15 minutes, the solution was diluted 10 times using 1 wt. / v % DOC, ultrafiltered by AMICON filters (100 kDaMWCO), and diluted with 1XPBS to adjust the final concentration of DOC to 0.1%. Purified scFv solution in 1XPBS was added in a ratio [sulfo-NHS ester-aryl defect]:[scFv]=l : 10. Solution was reacted for 3 hours on a rotator at 4 °C. The resulting solution was dialyzed in a dialysis device (Spectrum™ Spectra / Por™ Float-A-Lyzer™ G2, lOOOkDa MWCO) using 0.1% DOC in 1XPBS at 4 °C to reduce free scFvs in the solution by a factor of 10.

[0201] Near-infrared fluorescence spectroscopy of scFv-QWNTs. Fluorescence emission spectra of (6,5)-SWCNTs with scFv-coupled quantum defects (scFv-QWNTs) were acquired using a fiber-coupled laser system and a ID InGaAs NIR detector. The samples were excited with an 808 nm laser power supply (1 W; Optoelectronics Tech. Co., Ltd). The light path was shaped and fed into the back of an inverted IX-71 microscope (Olympus), where it passed through a 20* NIR objective (Olympus) and illuminated the samples in a 96-well clear flat bottom UV-transparent microplate (Corning). Emission from the SWCNTs was collected through the objective and passed through a dichroic mirror (875 nm cutoff, Semrock). The light was f / # matched to the spectrometer using several lenses and injected into a Shamrock-47-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)303i spectrograph (Andor, Oxford Instruments) with a slit width of 100 pm, which dispersed the emission using an 86 g / mm grating with 1.35 pm blaze wavelength. The spectral range was 723 nm to 1694 nm with a resolution of 1.89 nm. The light was collected by an iDus 1.7 pm InGaAs (Andor, Oxford Instruments) with an exposure time of 0.1 seconds to 0.25 seconds. An HL-3-CAL-EXT halogen calibration light source (Ocean Optics) was used to correct for wavelength-dependent features in the emission intensity arising from the spectrometer, detector, and other optics. A Hg / Ne pencil-style calibration lamp (Newport) was used to calibrate the spectrometer wavelength. Background subtraction was conducted using a well in a 96-well plate filled with 20% FBS. Following the acquisition, the data were processed with custom codes written in MATLAB that applied the aforementioned spectral corrections and background subtraction and fitted the fluorescence emission peaks with Lorentzian functions.

[0202] Atomic force microscopy (AFM). A stock solution of scFv conjugated (6,5)- QWNTs at 1 mg / mL in IX PBS was diluted 20* in dELO and plated on a freshly cleaved mica substrate (SPI) for 4 min before washing with 10 mL of dELO and blowing dry with argon gas. An Olympus AC240TS AFM probe (Asylum Research) in an Asylum Research MFP-3D-Bio instrument was used to image in AC mode. Data was captured at 2.93 nm / pixel XY resolution and 15.63 pm Z resolution.

[0203] Quantum chemical modelins. The charge effects were modeled using a 4 nm (1 unit cell) long (6,5)-SWCNT that contained an N-methylbenzamide and hydroxyl quantum defect pair in the ortho L90 configuration (using the notation of Gifford, B. J., Kilina, S., Htoon,H., Doom, S.K. & Tretiak, S. Exciton localization and optical emission in aryl-functionalized carbon nanotubes. J Phys Chem C 122, 1828-1838 (2018), incorporated by reference herein in its entirety for all purposes) located at the center of the nanotube. The pairing group was required to maintain a closed shell electronic structure and the choice of a hydroxyl group was based on its likelihood of formation in aqueous environments. To reduce end effects and prevent edge states from altering the band gap of the nanotube, the nanotube ends were terminated with hydrogen atoms. The structure was optimized using the B3LYP functional and the STO3G basis set, as implemented in the Gaussian 16 software package. For timedependent density functional theory (TD-DFT) calculations, two point charges were positionedI.41 nm away from the defect center. Specifically, the charges were placed 1 nm away along the QWNT axis and 1 nm perpendicular to the QWNT surface. Note that the geometry changes-48-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02) due to the presence of charges were negligible (<0.05 A). These charges were varied from 0 to 1 e’, providing the study of effects of different charge states on the nanotube.

[0204] Isothermal titration calorimetry (ITC). scFvs and IL-6 ligand were purified as described above. Samples were dialyzed overnight into 50 mM sodium phosphate, pH 8 with varying amounts of sodium chloride. Samples were dialyzed into the same buffer to prevent any background noise from differences in buffer concentrations. After dialysis, samples were filtered and the concentrations were measured by UV-VIS. scFV concentrations ranged from 1 pM to 10 pM, and IL-6 ligand concentrations were adjusted to be between 10x and 100* the scFv concentration. Measurements were made using a Microcal ITC200 instrument at 30 °C. Data was analyzed using the Origin 7 Software Package.Design of signal-amplifying scFvs

[0205] The design of the supercharged IDP was based on the original sequence of an antibody against human IL-6 (hIL-6) with the goal of producing a large conformational and electric field change upon antigen binding. First, a WT scFv was produced using an X-ray structure of an hIL-6 neutralizing antibody fragment (PDB ID: 4ZS7). The sequences of the two variable regions (VH and VL) were linked together by a flexible glycine-serine linker with the orientation Vn-linker-Vi. to generate the scFv sequence termed a-IL6wt (FIG. 2A). To provide a complete extended-to-folded phase transition, each of the two cysteine pairs was mutated to valine and alanine. Structure modeling using Alphafold2 and RoseTTAFold2 predicted that the a-IL6wt sequence had an identical fold and binding interface compared to the modeled complex of a-IL6wt and hIL-6. This protein, termed a-IL6wt, was expressed and purified and its hIL-6 binding affinity quantified using microscale thermophoresis with fluorescently-labeled hIL-6 (FIG. 2B). a-IL6wt recognized its target with a dissociation constant (KD) of 400 ± 100 nM. The reverse construct, Vi -linker-Vn, did not bind hIL-6.

[0206] The a-hIL-6 scFv was supercharged by changing 28 solvent-exposed side chains located on beta strands of a-IL6wt into aspartate residues, as well as two arginine-to-leucine mutations which would have placed two negative charges in close contact, resulting in a protein in which 13.7% of the total protein sequence was acidic (FIG. 2A). Attempts to incorporate glutamate residues resulted in insoluble proteins. Aspartate positions were chosen to increase the distance between surface-exposed acidic side chains. The resultant protein, a-IL6sc, increased the net charge from -2 to -24 at a pH of about 6.5 to about 7.5. Circular dichroism-49-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02) and fluorescence measurements (FIG. 2C) demonstrated that a-IL6sc underwent a disordered- to-ordered transition with increasing sodium chloride activities: The free energy of folding at no NaCl was +4.5 kcal / mol and NaCl linearly stabilized folding at a rate of -3.4 kcal / mol M. Microscale thermophoresis measurements were conducted that demonstrated a-IL6sc binds hIL-6 with a sodium chloride-dependent affinity - increasingly higher sodium chloride activities screen sidechain charge-charge repulsion, reducing the electrostatic folding energy penalty for binding and increasing net affinity (FIGS. 2D and 2E). At salt concentrations greater than 2 M, when the scFv was fully folded prior to binding, the KD value was 800 ± 200 nM, similar to that of a-IL6wt. At lower salt concentrations (0.5 M), where the energy barrier to a-IL6sc folding was larger, the KD value increased up to 8 ± 1 pM. Plotting AG of both folding and binding showed that both values scaled linearly with NaCl activity above the folding transition, with a AG difference between folding and binding (AAGdock, the interaction energy between the folded protein and the ligand) of -10.48 kcal / mol (FIG. 2F). Apparent binding affinity stopped increasing at the sodium chloride activities than those at the protein folds, suggesting that a-IL6sc has at least one partially folded intermediate. The ionic strengthdependent folding and ligand affinity did not significantly change within the approximate pH range of serum (pH values between 7 and 8, FIG. 2G), likely due to the fact that serum pH values were significantly higher than aspartate side chain pKa’s. The binding affinity and the salt-induced folding behavior of the scFv were not significantly changed up to sodium deoxycholate concentration of 0.1% while the dynamic range started to decrease at higher concentrations.Synthesis of scFv-coupled QWNTs.

[0207] QWNTs were developed to couple the scFv covalently to the SWCNT sidewall via a sp3quantum-well defect site. The (6,5) chirality SWCNT was covalently functionalized with 4-carboxyl aryl defects via diazonium chemistry. Functionalization was confirmed by the decreased host nanotube emission (En) at about 993 nm and the evolution of bright Eif emission at 1,149 nm (FIG. 3A). The intensity ratio between En and Eif of QWNTs was adjusted to 1 :2 to 1 :5 via functionalization to ensure that both emission bands were visible to provide observation of subtle changes in emission wavelength. The scFv was then conjugated to the QWNT via carbodiimide crosslinker chemistry. Note that the conjugation was not sitespecific, and multiple primary amine groups of scFvs (15 and 11 in a-IL6wt and a-IL6sc, respectively) can be attached to a carboxyl aryl defect. Free proteins were then removed by-50-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02) dialysis in phosphate-buffered saline with 0.1% sodium deoxycholate. The absorption spectrum and fluorescence emission of the QWNT remained the same after scFv conjugation (FIG. 3A), suggesting no aggregation occurred throughout the conjugation and dialysis processes. The surface charge (zeta potential) of a-IL6sc-coupled QWNTs (a-IL6sc-QWNTs) was more negative than the QWNTs and a-IL6wt-QWNTs. Without being bound by any theory, this may be due to the highly negative a-IL6sc on the QWNT surface (FIG. 3B). Correlograms of dynamic light scattering measurements shifted to a longer lag time after the bioconjugation, indicating an increase in the average hydrodynamic size of the scFv-QWNT complexes (FIG. 3C). Atomic force microscopy (AFM) imaging (FIG. 3D) also suggested the covalent conjugation of scFvs to QWNTs. In contrast to the smooth surface along the QWNT, the scFv-coupled QWNTs showed surface patterns due to the surface-bound scFvs. The density of scFvs on QWNTs ranged from 1-4 per 100 nm.Fluorescence response of scFv-coupled QWNTs.

[0208] The fluorescence response of the scFv-coupled QWNTs in response to the ligand- induced scFv folding transition was investigated. scFv-QWNT complexes were titrated with hIL-6 in 20% serum. 2.5 pM hIL-6 resulted in a 2 nm shift in the wavelength of the Eif band of a-IL6sc-coupled QWNTs (FIG. 4A). The Eif band exhibited quantitative redshifting at increasing concentrations of hIL-6, while the En band remained at the same wavelength (FIG. 4B) The dynamic range of the spectral response was 0.08 pM to 10 pM of hIL-6, consistent with the binding isotherm curve of free a-IL6sc observed by microscale thermophoresis (FIG. 2D) The Eif fluorescence red-shifted up to 4 nanometers at increasing concentrations of hlL- 6, with a detection limit of 80 nM and sensitivity up to 189 nM. Spectral responses saturated within 4 hours of incubation (FIG. 4C). The slow response of the Eif may be attributed to the screening by other proteins in 20% serum (6.4 mg / mL - 14 mg / mL total proteins in the solution) and the interactions between deoxy cholate (bile surfactant stabilizing QWNTs in aqueous media, 0.1 % in solution), scFvs, and ligand that may modulate the binding kinetics of scFvs. Non-target proteins, including bovine serum albumin (BSA), tumor necrosis factor a (TNF-a), and tumor growth factor pi (TGFpi) did not induce wavelength shifts in the scFv- coupled QWNTs (FIG. 4D). A high salt concentration (2 M of sodium chloride) was explored for its effects on the spectral response. No hIL-6-dependent spectral response was observed, presumably due to smaller changes in ligand binding-induced local electric field near the quantum defects because the protein was already folded.-51-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)

[0209] The long-term functionality of the scFv-coupled QWNTs was assessed (FIG. 4E). Sensor complexes stored at 4 °C for a week remained physically stable, detecting hIL-6 in serum with the same sensitivity as when freshly prepared. Without being bound by any theory, this high physical stability and long-term functionality may be due to the fact that the stronger electrostatic charge of supercharged proteins and the relatively low density on the QWNT sidewall reduce the chance of protein aggregation, improving the stability of a-IL6sc on QWNTs.

[0210] To assess the effects of protein supercharging on sensor functionality, identical experiments were performed using a-IL6wt as with a-IL6sc-coupled QWNTs. In hIL-6 titration experiments, a much smaller shift in Eif peak was observed using a-IL6wt (1 nm at the highest concentration, 10 pM), as compared to the supercharged a-IL6sc (FIG. 4F). There was no significant difference in the Eif peak wavelengths between a-IL6wt and a-IL6sc coupled QWNTs in the presence of hIL-6. These comparative studies suggested that protein supercharging engendered significant changes in the local electric field upon ligand-induced folding, inducing significantly-enhanced spectral shifts on the quantum defects to which they are attached.Quantum Chemical Modeling.

[0211] TD-DFT modeling was used to elucidate how protein charges may induce spectral shifts of the Eif band (FIGS. 5A-5E). The computational model included a (6,5)-SWCNT, 4 nm in length with hydrogen end capping, an N-methylbenzamide defect, and a hydroxyl pairing group located near the midsection of the tube. Due to the substantial size (about 5 nanometers) and complex geometry of the folded a-IL6sc, it was impractical to explicitly include it in the DFT calculation (FIG. 5A). Instead, a methyl amide bond was simulated to represent the peptide bond between the aryl defect and a-IL6sc. Given the highly charged surface and size of the scFv relative to the QWNT, it was reasonable to assume that several charges from the scFv may be brought close to the defect in various directions. Although the exact distances between any charged groups on the scFv and the N-methylbenzamide defect were not experimentally determined, the primary effect anticipated from varying these distances was a change in the effective charge near the defect. To emulate the collective charge effects of the scFv to the QWNT emission, two point-charges were positioned at a distance of 1.41 nm from the defect center and varied these charges from 0 to 1 e' (FIG. 5B).-52-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)

[0212] These TD-DFT simulations showed that, with increasing local negative charge, both the En and Eif peaks steadily redshifted (FIG. 5C). In the unfunctionalized SWCNT model, there were minimal shifts in the En peak (FIG. 5D). For the QWNT, the simulation indicated that the En exhibited a relatively small red shift, similar to the unfunctionalized SWCNT, but Eif showed a substantially larger shift, consistent with the experimental observation. When both charges had a magnitude of -le each, the Eif peak shifted by about 5.5 nm, while the En peak only shifted by about 1.3 nm. Without being bound by any theory, these TD-DFT results indicated the strong negative charges of a-IL6sc draw closer to the defect center when a-IL6sc couples to hIL-6 and folds, and that this increased local electric field leads to larger spectral shifts on the Eif peak as compared to the SWCNT En peak.Isothermal Titration Calorimetry.

[0213] FIGS. 6A-6B provide isothermal titration calorimetry of scFv binding IL-6. FIG. 6A is pWatts vs. time (minutes) for scFv binding IL-6. FIG. 6B is kcal mol'1of inj ectant vs. molar ratio for scFv binding IL-6.EXAMPLE 2: Comparison of IL-6 Binding Affinity for Different scFVs

[0214] scFVs were prepared and characterized according to the methods in Example 1. FIGS. 7A-7B provide binding experiments for scFV with different numbers of disulfide bridges. FIG. 7A provides binding experiment data for binding of IL-6 by scFV comprising a low concentration of disulfide bridges, with characterization performed using MST. FIG. 7B provides binding experiment data for binding of IL-6 by scFV comprising a high concentration of disulfide bridges, with characterization performed using MST. The scFV in FIG. 7A was SEQ ID NO: 32. The scFV in FIG. 7B was SEQ ID NO: 33. Results indicated a KD of 170 nM for SEQ ID NO: 32 and a KD of 130 nM for SEQ ID NO: 33.EXAMPLE 3: Comparison of scFV Binding Affinity for IH

[0215] scFVs were prepared and characterized according to the methods in Example 1. FIGS. 8A-8B: Binding experiments for different scFVs with luteinizing hormone (LH). FIG. 8A provides binding experiment data for binding LH by scFV having a net charge of -17. FIG. 8B provides binding experiment data for binding LH by scFV having a net charge of -22. Characterization was performed using MST. The scFV in FIG. 8A was SEQ ID NO: 37. The-53-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02) scFV in FIG. 8B was SEQ ID NO: 38. Results indicated a KD of 2200 nM for SEQ ID NO: 37 and a KD of 60000 nM for SEQ ID NO: 38.REFERENCES

[0216] Bachilo, S.M. et al. Structure-assigned optical spectra of single-walled carbon nanotubes. Science 298, 2361-2366 (2002).

[0217] Brozena, A.H., Kim, M., Powell, L.R. & Wang, Y. Controlling the optical properties of carbon nanotubes with organic colour-centre quantum defects. Nat. Rev. Chem. 3, 375-392 (2019).

[0218] Piao, Y. et al. Brightening of carbon nanotube photoluminescence through the incorporation of sp3 defects. Nat. Chem. 5, 840-845 (2013).

[0219] Kwon, H. et al. Optical probing of local pH and temperature in complex fluids with covalently functionalized, semiconducting carbon nanotubes. J. Phys. Chem. C 119, 3733-3739 (2015).

[0220] Kim, M. et al. Mapping structure-property relationships of organic color centers. Chem 4, 2180-2191 (2018).

[0221] Hartmann, N.F. et al. Photoluminescence imaging of solitary dopant sites in covalently doped single-wall carbon nanotubes. Nanoscale 7, 20521-20530 (2015).

[0222] He, X. et al. Carbon nanotubes as emerging quantum -light sources. Nat Mater 17, 663-670 (2018).

[0223] Kim, M. et al. Nanosensor-based monitoring of autophagy-associated lysosomal acidification in vivo. Nat. Chem. Biol. 19, 1448-1457 (2023).

[0224] Niidome, Y., Wakabayashi, R., Goto, M., Fujigaya, T. & Shiraki, T. Proteinstructure-dependent spectral shifts of near-infrared photoluminescence from locally functionalized single-walled carbon nanotubes based on avidin-biotin interactions. Nanoscale 14, 13090-13097 (2022).-54-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)

[0225] Niidome, Y. et al. Near-infrared photoluminescent detection of serum albumin using single-walled carbon nanotubes locally functionalized with a long-chain fatty acid. Carbon 216, 118533 (2024).

[0226] Metternich, J.T. et al. Near-infrared fluorescent biosensors based on covalent DNA anchors. J Am Chem Soc 145, 14776-14783 (2023).

[0227] Uversky, V.N. Unusual biophysics of intrinsically disordered proteins. Biochim. Biophys. Acta, Proteins Proteomics 1834, 932-951 (2013).

[0228] Toto, A. et al. Templated folding of intrinsically disordered proteins. J. Biol. Chem. 295, 6586-6593 (2020).

[0229] Mao, A.H., Crick, S.L., Vitalis, A., Chicoine, C.L. & Pappu, R.V. Net charge per residue modulates conformational ensembles of intrinsically disordered proteins. Proc. Natl. Acad. Sci. U.S.A. 107, 8183-8188 (2010).

[0230] Lawrence, M.S., Phillips, K.J. & Liu, D.R. Supercharging proteins can impart unusual resilience. J. Am. Chem. Soc. 129, 10110-10112 (2007).

[0231] Lepak, L. A. et al. Handheld chem / biosensor using extreme conformational changes in designed binding proteins to enhance surface plasmon resonance (SPR). Proc. SPIE Adv. Environ. Chem. Biol. Sensing Technol. XII 9862 (2016).

[0232] Emberly, E.G., Wingreen, N.S. & Tang, C. Designability of alpha-helical proteins. Proc. Natl. Acad. Sci. U. S. A. 99, 11163-11168 (2002).

[0233] Murthy, H., Iqbal, M., Chavez, J.C. & Kharfan-Dabaja, M.A. Cytokine release syndrome: Current perspectives. ImmunoTargets Ther. 8, 43-52 (2019).

[0234] Chen, H. et al. Management of cytokine release syndrome related to CAR-T cell therapy. Front. Med. 13, 610-617 (2019).

[0235] Norelli, M. et al. Monocyte-derived IL-1 and IL-6 are differentially required for cytokine-release syndrome and neurotoxicity due to CAR T cells. Nat. Med. 24, 739-748 (2018).-55-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)

[0236] Worn, A. & Pluckthun, A. An intrinsically stable antibody scFv fragment can tolerate the loss of both disulfide beads and fold correctly. FEBS Lett. 427, 357-361 (1998).

[0237] Scheuermann, T.H., Padrick, S.B., Gardner, K.H. & Brautigam, C.A. On the acquisition and analysis of microscale thermophoresis data. Anal Biochem 496, 79-93 (2016).

[0238] Santoro, M.M. & Bolen, D.W. Unfolding free-energy changes determined by the linear extrapolation method .1. Unfolding of phenylmethanesulfonyl alpha-chymotrypsin using different denaturants. Biochemistry 27, 8063-8068 (1988).

[0239] Schnatz, P.J. et al. Designing heterotropically activated allosteric conformational switches using supercharging. Proc. Natl. Acad. Sci. U. S. A. 117, 5291-5297 (2020).

[0240] Subbaiyan, N.K. et al. Role of surfactants and salt in aqueous two-phase separation of carbon nanotubes toward simple chirality isolation. ACS Nano 8, 1619-1628 (2014).

[0241] Gifford, B.J., Kilina, S., Htoon, H., Doorn, S.K. & Tretiak, S. Exciton localization and optical emission in aryl-functionalized carbon nanotubes. J Phys Chem C 122, 1828-1838 (2018).

[0242] Wang, P. et al. Quantum defects: What pairs with the aryl group when bonding to the sp2 carbon lattice of single-wall carbon nanotubes? J Am Chem Soc 144, 13234-13241 (2022).

[0243] Kilina, S. & Tretiak, S. Excitonic and vibrational properties of single-walled semiconducting carbon nanotubes. Adv Func Mater 17, 3405-3420 (2007).

[0244] Frisch, M.J. et al. Gaussian 16, Revision B.01. Gaussian, Inc., Wallingford CT (2016).

[0245] Blanchetot, C. et al. Structural mimicry of receptor interaction by antagonistic interleukin-6 (IL-6) antibodies. J. Biol. Chem. 291, 13846-13854 (2016).

[0246] Ramm, K., Gehrig, P. & Pluckthun, A. Removal of the conserved disulfide bridges from the scFv fragment of an antibody: Effects on folding kinetics and aggregation. J Mol Biol 290, 535-546 (1999).-56-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)

[0247] Worn, A. & Pluckthun, A. An intrinsically stable antibody scFv fragment can tolerate the loss of both disulfide bonds and fold correctly. FEBS Lett 427, 357-361 (1998).

[0248] Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583-589 (2021).

[0249] Mirdita, M. et al. ColabFold: Making protein folding accessible to all. Nat. Methods 19, 679-682 (2022).

[0250] Baek, M. et al. Efficient and accurate prediction of protein structure using RoseTTAFold2. bioRxiv (2023) doi: 10.1101 / 2023.05.24.542179 (accessed 2024-03-01).

[0251] McCann, J. J. et al. Computational design of a sensitive, selective phase-changing sensor protein for the VX nerve agent. Science Advances 8, 6 (2022).

[0252] Fercher, C., Jones, M.L., Mahler, S.M. & Corrie, S.R. Recombinant antibody engineering enables reversible binding for continuous protein biosensing. ACS Sensors 6, 764- 776 (2021).

[0253] Pabst, T. et al. Analysis of IL-6 serum levels and CAR T cell-specific digital PCR in the context of cytokine release syndrome. Exp. Hematol. 88, 7-14. el3 (2020).

[0254] Remy, K.E. et al. Immunotherapies for COVID-19: Lessons learned from sepsis. Lancet Respir. Med. 8, 946-949 (2020).

[0255] Blanchetot, C. et al. Structural mimicry of receptor interaction by antagonistic interleukin-6 (IL-6) antibodies. J. Biol. Chem. 291, 13846-13854 (2016).

[0256] Quintero-Hernandez, V. et al. The change of the scFv into the Fab format improves the stability and in vivo toxin neutralization capacity of recombinant antibodies. Mol Immunol 44, 1307-1315 (2007).

[0257] Pooja, G. et al. Optical nanosensor passivation enables highly sensitive detection of the inflammatory cytokine IL-6. bioRxiv (2023) doi: 10.1101 / 2023.05.10.540217 (accessed 2024-03-01).-57-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)EQUIVALENTS

[0258] While certain embodiments have been illustrated and described a person with ordinary skill in the art, after reading the foregoing specification, can effect changes, substitutions of equivalents and other types of alterations to the compounds of the present technology or salts, pharmaceutical compositions, derivatives, prodrugs, metabolites, tautomers, or racemic mixtures thereof as set forth herein. Each aspect and embodiment described above can also have included or incorporated therewith such variations or aspects as disclosed in regard to any or all of the other aspects and embodiments.

[0259] The present technology is also not to be limited in terms of the particular aspects described herein, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. It is to be understood that this present technology is not limited to particular methods, reagents, compounds, compositions, labeled compounds or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting. Thus, it is intended that the specification be considered as exemplary only with the breadth, scope and spirit of the present technology indicated only by the appended claims, definitions therein and any equivalents thereof.

[0260] The embodiments, illustratively described herein may suitably be practiced in the absence of any element or elements, limitation, or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed technology. Additionally, the phrase “consisting essentially of’ will be understood to include those elements specifically recited and those additional elements that do-58-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02) not materially affect the basic and novel characteristics of the claimed technology. The phrase “consisting of’ excludes any element not specified.

[0261] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0262] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member.

[0263] All publications, patent applications, issued patents, and other documents (for example, journals, articles and / or textbooks) referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

[0264] The present technology may include, but is not limited to, the features and combinations of features recited in the following lettered paragraphs, it being understood that the following paragraphs should not be interpreted as limiting the scope of the claims as appended hereto or mandating that all such features must necessarily be included in such claims:A. A conjugate for detection of a bioanalyte comprising:-59-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02) a single-walled carbon nanotube with a sp3quantum-well defect site (QWNT) conjugated to an antibody fragment via an amide bond at the sp3quantum-well defect site; wherein the antibody fragment is a single-chain variable fragment with an overall net charge of about -10 to about -30 at a pH of about 6.5 to about 7.5, the antibody fragment changes phase in response to interaction with the bioanalyte, and the antibody fragment comprises Vu-linker-Vr, wherein VH is a variable heavy chain domain, Vris a variable light chain domain, and the linker comprises (SGG)X, GG(SGG)X, (GGGGS)X, (SGG)X, SS(GGGS)X, or a combination of any two or more thereof, where x is 1 to 12; and wherein the antibody fragment comprises a number of D residues to provide the overall net charge.B. The conjugate of paragraph A, wherein the antibody fragment comprises about 5 to about50 D residues.C. The conjugate of paragraph A or paragraph B, wherein the antibody fragment is about10% to about 20% of acidic residues.D. The conjugate of any one of paragraphs A-C, wherein the antibody fragment comprises antibody mutations, in comparison to a wildtype antibody fragment, with about 20 to about 35 solvent-exposed side chain residues located on beta strands of the antibody fragment replaced with D residues, and 0 to about 5 R residues replaced by L residues.E. The conjugate of any one of paragraphs A-D, wherein the bioanalyte comprises a cytokine, an epidermal growth factor, an interferon, or a hormone.F. The conjugate of paragraph E, wherein the bioanalyte is the cytokine IL-6.G. The conjugate of paragraph F, wherein the antibody fragment comprises SEQ ID NO: 28,SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33.H. A pharmaceutical composition for detecting a bioanalyte, the pharmaceutical composition comprising:-60-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)10'2nM to about 103nM of the conjugate of any one of paragraphs A-G; and a pharmaceutically acceptable solvent.I. A method for selecting a subject for treatment with anti-IL-6 therapy comprising:(a) detecting IL-6 above a predetermined threshold in a biological sample obtained from the subject using the pharmaceutical composition of paragraph H; and(b) administering the anti-IL-6 therapy to the subject.J. The method of paragraph I, wherein the subject has been diagnosed with cytokine release syndrome.K. The method of paragraph I or paragraph J, wherein the subject has been diagnosed with cancer.L. The method of any one of paragraphs K, wherein the subject has been treated with an immunotherapy.M. The method of paragraph L, wherein the immunotherapy comprises monoclonal antibody therapy, CAR T-cell therapy, or a combination thereof.N. The method of paragraph M, wherein the CAR T-cell therapy comprises tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, lisocabtagene maraleucel, idecabtagene vicleucel, ciltacabtagene autoleucel, obecabtagene autoleucel, or a combination of any two or more thereof.O. The method of any one of paragraphs LN, wherein the biological sample comprises plasma, blood, serum, or biopsied tissue.P. A method for treating or preventing cytokine release syndrome in a subject comprising:(a) detecting IL-6 above a predetermined threshold in a biological sample obtained from the subject using the pharmaceutical composition of paragraph H; and(b) administering an anti-IL-6 therapy to the subject.Q. The method of paragraph P, wherein the subject has been diagnosed with cancer.R. The method of paragraph P or Q, wherein the subject has been treated with an immunotherapy.-61-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)S. The method of paragraph R, wherein the immunotherapy comprises monoclonal antibody therapy, CAR T-cell therapy, or a combination thereof.T. The method of paragraph S, wherein the CAR T-cell therapy comprises tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, lisocabtagene maraleucel, idecabtagene vicleucel, ciltacabtagene autoleucel, obecabtagene autoleucel, or a combination of any two or more thereof.U. The method of any one of paragraphs P-T, wherein the biological sample comprises plasma, blood, serum, or biopsied tissue.V. A method for prolonging survival of a cancer patient comprising: administering to the cancer patient an effective amount of an anti-IL-6 therapy, wherein an IL-6 level in a biological sample obtained from the cancer patient is at or above a predetermined threshold measured using the composition of paragraph H.W. The method of paragraph V, wherein the cancer patient has been treated with an immunotherapy.X. The method of paragraph W, wherein the immunotherapy comprises monoclonal antibody therapy, CAR T-cell therapy, or a combination thereof.Y. The method of paragraph X, wherein the CAR T-cell therapy comprises tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, lisocabtagene maraleucel, idecabtagene vicleucel, ciltacabtagene autoleucel, obecabtagene autoleucel, or a combination of any two or more thereof.Z. The method of any one of paragraphs V-Y, wherein the biological sample comprises plasma, blood, serum, or biopsied tissue.

[0265] Other embodiments are set forth in the following claims, along with the full scope of equivalents to which such claims are entitled.-62-4925-4952-6069.1

Claims

Atty Dkt No: 115872-1955 (SK2024-041-02)CLAIMS1. A conjugate for detection of a bioanalyte comprising: a single-walled carbon nanotube with a sp3quantum-well defect site (QWNT) conjugated to an antibody fragment via an amide bond at the sp3quantum-well defect site; and wherein the antibody fragment is a single-chain variable fragment with an overall net charge of about -10 to about -30 at a pH of about 6.5 to about 7.5, the antibody fragment changes phase in response to interaction with the bioanalyte, and the antibody fragment comprises Vu-linker-Vr, wherein VH is a variable heavy chain domain, Vris a variable light chain domain, and the linker comprises (SGG)X, GG(SGG)X, (GGGGS)X, (SGG)X, SS(GGGS)X, or a combination of any two or more thereof, where x is 1 to 12; wherein the antibody fragment comprises a number of D residues to provide the overall net charge; wherein the bioanalyte comprises a cytokine, an epidermal growth factor, an interferon, or a hormone.

2. The conjugate of claim 1, wherein the antibody fragment comprises about 5 to about 50 D residues.

3. The conjugate of claim 1, wherein the antibody fragment is about 10% to about 20% of acidic residues.

4. The conjugate of claim 1, wherein the antibody fragment comprises antibody mutations, in comparison to a wildtype antibody fragment, with about 20 to about 35 solvent- exposed side chain residues located on beta strands of the antibody fragment replaced with D residues, and 0 to about 5 R residues replaced by L residues.

5. The conjugate of claim 1, wherein the cytokine comprises IL-6 or IL-1, the epidermal growth factor comprises TGFP, the interferon comprises interferon gamma, and the hormone comprises luteinizing hormone.

6. The conjugate of claim 5, wherein the bioanalyte comprises the cytokine and wherein the cytokine is IL-6.-63-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)7. The conjugate of claim 6, wherein the antibody fragment comprises SEQ ID NO: 28, SEQID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33.

8. A pharmaceutical composition for detecting a bioanalyte, the pharmaceutical composition comprising:10'2nM to about 103nM of the conjugate of any one of claims 1-7; and a pharmaceutically acceptable solvent.

9. A method for selecting a subject for treatment with anti -IL-6 therapy comprising:(a) detecting IL-6 above a predetermined threshold in a biological sample obtained from the subject using the pharmaceutical composition of claim 8; and(b) administering the anti-IL-6 therapy to the subject.

10. The method of claim 9, wherein the subject has been diagnosed with cytokine release syndrome.

11. The method of claim 9, wherein the subject has been diagnosed with cancer.

12. The method of claim 9, wherein the subject has been treated with an immunotherapy.

13. The method of claim 12, wherein the immunotherapy comprises monoclonal antibody therapy, CAR T-cell therapy, or a combination thereof.

14. The method of claim 12, wherein the CAR T-cell therapy comprises tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, lisocabtagene maraleucel, idecabtagene vicleucel, ciltacabtagene autoleucel, obecabtagene autoleucel, or a combination of any two or more thereof.

15. The method of claim 9, wherein the biological sample comprises plasma, blood, serum, or biopsied tissue.

16. A method for treating or preventing cytokine release syndrome in a subject comprising:(a) detecting IL-6 above a predetermined threshold in a biological sample obtained from the subject using the pharmaceutical composition of claim 8; and(b) administering an anti-IL-6 therapy to the subject.

17. The method of claim 16, wherein the subject has been diagnosed with cancer.-64-4925-4952-6069.1Atty Dkt No: 115872-1955 (SK2024-041-02)18. The method of claim 16, wherein the subject has been treated with an immunotherapy.

19. The method of claim 18, wherein the immunotherapy comprises monoclonal antibody therapy, CAR T-cell therapy, or a combination thereof.

20. The method of claim 19, wherein the CAR T-cell therapy comprises tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, lisocabtagene maraleucel, idecabtagene vicleucel, ciltacabtagene autoleucel, obecabtagene autoleucel, or a combination of any two or more thereof.

21. The method of claim 16, wherein the biological sample comprises plasma, blood, serum, or biopsied tissue.

22. A method for prolonging survival of a cancer patient comprising: administering to the cancer patient an effective amount of an anti-IL-6 therapy, wherein an IL-6 level in a biological sample obtained from the cancer patient is at or above a predetermined threshold measured using the composition of claim 8.

23. The method of claim 22, wherein the cancer patient has been treated with an immunotherapy.

24. The method of claim 23, wherein the immunotherapy comprises monoclonal antibody therapy, CAR T-cell therapy, or a combination thereof.

25. The method of claim 19, wherein the CAR T-cell therapy comprises tisagenlecleucel, axicabtagene ciloleucel, brexucabtagene autoleucel, lisocabtagene maraleucel, idecabtagene vicleucel, ciltacabtagene autoleucel, obecabtagene autoleucel, or a combination of any two or more thereof.

26. The method of claim 22, wherein the biological sample comprises plasma, blood, serum, or biopsied tissue.-65-4925-4952-6069.1

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