Patent Application
20230408497
This application claims priority from prior Japanese Patent Application No. 2022-099159, filed on Jun. 20, 2022, and Japanese Patent Application No. 2023-012581, filed on Jan. 31, 2023, entitled “Labeled Polypeptide, Modified Polypeptide, Production Method for these Polypeptides, Reagent Containing these Polypeptides, and Measurement Method for Target Substance”, the entire contents of which are incorporated herein by reference.
The instant application contains a Sequence Listing which has been filed electronically in xml format and is hereby incorporated by reference in its entirety. Said xml copy, created on Jun. 8, 2023, is named Sequence Listing as filed.xml and is 21.969 KB in size.
The present invention relates to a labeled polypeptide and a production method therefor. The present invention relates to a modified polypeptide and a production method therefor. The present invention relates to a reagent containing a labeled polypeptide or a modified polypeptide. The present invention relates to a measurement method for a target substance.
Transglutaminase (TG) is an enzyme that recognizes a glutamine residue in a polypeptide as a substrate, and catalyzes a reaction between a carboxamide side chain of the glutamine residue and an amino group of a primary amine, to form an amide bond. More specifically, in the TG-catalyzed reaction, the amino group in the carboxamide side chain of the glutamine residue in the polypeptide condenses with the primary amine, and a substituent on the primary amine is transferred to the glutamine residue, to produce ammonia. In recent years, techniques with use of TG, for binding a functional substance, such as drug or label, to a glutamine residue-containing polypeptide have been known.
For example, U.S. Patent Application Publication No. 2018/0037921 describes that an anticancer drug-bound antibody was obtained, by reacting an antibody having a glutamine residue-containing peptide tag bound to the C-terminal of the heavy chain, with an anticancer drug bound to an amino group-containing linker, in the presence of TG. TG-catalyzed binding of a functional substance to the polypeptide usually employs a bifunctional linker that has functional groups at both ends. The functional groups of the bifunctional linker are selectable from an amino group (—NH2) as an amine donor in the TG-catalyzed reaction, and any functional group that covalently binds the functional substance to the linker. The glutamine residue in the polypeptide and the functional substance are thus bound through such linker.
The scope of the present invention is defined solely by the appended claims, and is not affected to any degree by the statements within this summary.
Efforts of the present inventors on binding the functional substance to the glutamine residue in the polypeptide, with use of the linker described in Patent Literature 1 have, however, not always succeeded in binding them with high efficiency. It is, therefore, an object of the present invention to provide a way to bind a label to a polypeptide with high efficiency.
The present inventors examined, as the bifunctional linker, a polyethylene glycol (PEG) chain having a thiol group (—SH) and an amino group, and found that such linker whose PEG chain moiety has a molecular weight of 1100 or larger could efficiently bind the linker to the glutamine residue in the polypeptide, thereby completing the present invention.
According to the present invention, there is provided a labeled polypeptide that includes a glutamine residue comprising a side chain represented by formula (I) below:
According to the present invention, there is also provided a modified polypeptide that includes a side chain-containing glutamine residue represented by formula (II) below:
According to the present invention, there is also provided a reagent that contains the aforementioned labeled polypeptide or modified polypeptide. According to the present invention, there is also provided a measurement method for a target substance, the method includes: forming an immune complex of the aforementioned labeled polypeptide, with a target substance; and,
According to the present invention, there is also provided a production method for a modified polypeptide, the method includes:
NH2—X—Y—SH (VI)
According to the present invention, there is also provided a production method for a labeled polypeptide, the method includes:
The present invention enables highly efficient binding of a label to a polypeptide, with use of a linker having an amino group and a thiol group.
FIG. TA is a schematic drawing illustrating an exemplary reagent of this embodiment;
A labeled polypeptide of this embodiment has a glutamine residue that includes a side chain. The side chain is represented by formula (I) above. In the labeled polypeptide, a glutamine residue in the polypeptide, before the label is added, is bound to the label while interposed by a bifunctional linker having a PEG chain. More specifically, the labeled polypeptide is obtainable by bonding a PEG chain, as a bifunctional linker, having a thiol group and an amino group, to a glutamine residue of the polypeptide, making use of a TG-catalyzed reaction, and then reacting the thiol group of the linker with a label having a maleimide group (for details, see the production method for a labeled polypeptide, described later).
In formula (I), (C) represents the α-carbon of the glutamine residue in the labeled polypeptide. The side chain represented by formula (I) is structured to contain a linker and a label, which are bound to the side chain of the glutamine residue. The labeled polypeptide of this embodiment is a polypeptide in which a glutamine residue, which can be a substrate for TG, in the polypeptide before the label is added, is modified to the glutamine residue having the side chain represented by the above formula (I). That is, the amino acid sequence of the labeled polypeptide is the same as that of the polypeptide before the label is added, except for containing the side chain-containing glutamine residue represented by formula (I) above. Type of the polypeptide in the labeled polypeptide is not particularly limited. For example, the labeled polypeptide may be any protein such as an antibody, antigen, ligand, or receptor. Among them, antibody is preferred.
The term “antibody” in this specification encompasses antibody fragment. The antibody fragment is exemplified by Fab, Fab′, F(ab′)2, Fd, Fd′, Fv, scFv, domain antibody (dAb), reduced IgG (rIgG), light chain, heavy chain antibody, variable domain of heavy chain antibody (VHH), diabody, and triabody. The antibody may be either monoclonal antibody or polyclonal antibody. The antibody may be derived from any origin not specifically limited, typically from animals such as mouse, rat, hamster, rabbit, goat, horse, camel, alpaca, chicken, ostrich, and shark. Isotype of antibody may be any of IgG, IgA, IgM, IgD and IgE, among which IgG is preferred. The antibody that specifically binds to the tag may be a commercially available antibody, or an antibody produced by a method known in the art.
In the polypeptide before the label is added, the glutamine residue capable of serving as a substrate for TG may be, for example, a glutamine residue that resides on the surface of the polypeptide, with the carboxamide side chain directed outwards from the polypeptide. Alternatively, a peptide tag that contains a glutamine residue (also referred to as “Q-tag”, hereinafter) may be added to the polypeptide before the label is added. The Q-tag contains a glutamine residue in a predetermined amino acid sequence, so that the glutamine residue can serve as a substrate for TG. Since not all glutamine residues in the polypeptide can serve as the substrate for TG, so that addition of the Q-tag is preferred. The Q-tag per se has been known, as typically described in U.S. Patent Application Publication No. 2018/0037921. The number of amino acid residues contained in the Q-tag is preferably 3 or larger, and more preferably 4 or larger. The number of amino acid residues contained in the Q-tag is preferably 20 or smaller, more preferably 15 or smaller, and most preferably 10 or smaller. One or two residues in the amino acid sequences of the Q-tag are glutamine residues. The C-terminal amino acid residue of the Q-tag is preferably a proline residue, from the viewpoint of protecting the Q-tag from being cleaved by peptidase. The Q-tag is exemplified by peptide tags having amino acid sequences such as GVLNLAQSP (SEQ ID NO: 1), GLLQGP (SEQ ID NO: 2), or LLQGP (SEQ ID NO: 3).
A position, to which the Q-tag is added in the polypeptide before the label is added, is preferably, but not restrictively, the N-terminal or C-terminal of the polypeptide, and more preferably the C-terminal of the polypeptide. For the polypeptide having the form of full-length antibody such as Fab, Fab′, or F(ab′)2, the Q-tag may be added to the C-terminal of the heavy chain or the light chain. A method for adding the Q-tag to the polypeptide is not particularly limited. For example, a cross-linker or a linker may be used to covalently attach the Q-tag to the polypeptide. Alternatively, a fusion polypeptide of the polypeptide and the Q-tag may be produced by a known genetic recombination method.
The labeled polypeptide is preferably a fusion polypeptide formed between the polypeptide, and the peptide tag that contains the glutamine residue having the side chain represented by formula (I) above. That is, the labeled polypeptide is a Q-tag-added polypeptide, in which a glutamine residue, which can be a substrate for TG, in the Q-tag is modified to the glutamine residue having the side chain represented by formula (I) above. More preferably, the labeled polypeptide is a fusion polypeptide formed between an antibody, and the peptide tag that contains the glutamine residue having the side chain represented by formula (I) above.
In formula (I) above, “(C)—(CH2)2—(C═O)—” is attributable to a carboxamide side chain of glutamine residue in the polypeptide, and “—NH—X—Y—S—” is attributable to a bifunctional linker, that is a PEG chain having a thiol group and an amino group. Here, X represents a straight chain alkylene group, whose number of carbon atoms is preferably 2 or larger and 10 or smaller, more preferably 2 or larger and 8 or smaller, and most preferably 2 or larger and 6 or smaller. The straight chain alkylene group, represented by X, preferably has no substituent. More preferably, X represents an unsubstituted straight chain alkylene group having 2 or more and 6 or less carbon atoms.
In formula (I) above, Y represents a PEG chain having a molecular weight of 1100 or larger. Since being a polymer, the PEG having a thiol group and an amino group, which is used for preparing the labeled polypeptide, may be an assemblage of multiple molecules whose molecular weight distributes over a certain range. Hence, the labeled polypeptides prepared from such PEG will have the molecular weight which distributes over a certain range. In this specification, the phrase “the PEG chain having a molecular weight of 1100 or larger” means that the minimum value of the molecular weight of the PEG chain moiety in the labeled polypeptide is 1100. The PEG chain represented by Y has a molecular weight whose lower limit is preferably 1200, 1300, 1400 or 1500. The PEG chain represented by Y has a molecular weight whose upper limit is typically 20000, 10000, 7500, 7000, 6500, 6000 or 5500. The molecular weight of the PEG chain may be determined typically by analyzing, by mass spectrometry, the PEG chain as a bifunctional linker having a thiol group and an amino group, before being bound to the polypeptide. The mass spectrometry is exemplified by infusion MS, LC-MS, time-of-flight mass spectrometry (TOF-MS), and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Conditions for mass spectrometry may be appropriately determined. For example, conditions for infusion MS, when applied, may be those described later in EXAMPLE.
Among the molecular weights of the PEG chain measured by mass spectrometry, the molecular weight that appears in the largest abundance is referred to as “mode molecular weight”. The PEG chain represented by Y has a molecular weight, typically with the lower limit of 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000 or 2050. The PEG chain represented by Y has a molecular weight typically with the upper limit of 18000, 15000, 12000, 9000, 8000, 7000, 6000, 5000, 4000, 3500 or 3000. Also the weight average molecular weight of the PEG chain may be estimated on the basis of the result of mass spectrometry. For example, in a case where the PEG chain as a bifunctional linker having a thiol group and an amino group, and before being bound to the polypeptide, is analyzed by Infusion MS, the weight average molecular weight of the PEG chain may be estimated from the molecular weight value and intensity of an isotopic molecular peak that appears highest in intensity. Conditions for analysis by infusion MS will be described later in EXAMPLES. The PEG chain represented by Y preferably has a weight average molecular weight of 1300 or larger, and more preferably 1700 or larger. The PEG chain represented by Y has a molecular weight, typically with the lower limit of 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000 or 2050. The PEG chain represented by Y preferably has an upper limit value of the molecular weight of smaller than 20000, which is typically 15000, 12000, 10000, 8000, 7000, 6000, 5000, 4500, 4000, 3500, 3000 or 2500.
The structure of Y is represented by formula (III) below. In formula (III), n represents an integer of 25 or larger, which is preferably 39 or larger.
—(OCH2CH2)n— or —(CH2CH2O)n— (III)
In formula (III), the lower limit of n is preferably 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 41. More preferably, the lower limit of n is 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40. The upper limit of n is typically 455, 228, 171, 159, 147, 136, 125, 113, 102, 90, 79, 68, or 56.
In the side chain represented by formula (I) above, a structural moiety represented by formula (IV) below is derived from a label having a maleimide group. Z represents a label, and L represents a spacer that binds the nitrogen atom of the imide ring to Z, or an atomic bond. The atomic bond represented by L means a bond through which the nitrogen atom of the imide ring is directly bound to Z, without any other atom interposed in between.
The label is a substance that can be added to a polypeptide, and is synonymous to “labeling substance”. The label may be a substance that is specifically detectable, a substance capable of generating some signal by itself (also referred to as “signal generating substance”, hereinafter), or a substance that catalyzes reaction of some other substance to induce signal generation. The substance that is specifically detectable is not particularly limited, as long as any target that can be specifically bound by such substance is present or obtainable. The substance that is specifically detectable is exemplified by biotins, hapten, and oligonucleotide. The biotins specifically bind to avidins. The hapten is exemplified by 2,4-dinitrophenyl (DNP) group. DNP specifically binds to anti-DNP antibody. The oligonucleotide specifically binds to an oligonucleotide having a sequence complementary to the base sequence of the former.
In this specification, the term “biotins” encompasses biotin and biotin analog. The biotin analog is exemplified by desthiobiotin and biocytin. In this specification, the term “avidins” encompasses and avidin and avidin analog. The avidin analog is exemplified by streptavidin, avidin-like protein derived from Paecilomyces tenuipes (Tamavidin (registered trademark)), bradavidin, and rhizavidin.
The signal generating substance is exemplified by fluorescent substance, radioisotope, and chemiluminescent substance. The substance that catalyzes reaction of some other substance to induce signal generation is exemplified by enzyme. The enzyme reacts with an appropriate substrate to generate a signal such as light or color. The enzyme is exemplified by alkaline phosphatase (ALP), peroxidase, β-galactosidase, and luciferase. The fluorescent substance is exemplified by fluorescent dyes such as Alexa Fluor (registered trademark), fluorescein isothiocyanate (FITC), and rhodamine; and fluorescent protein such as GFP. The radioisotope is exemplified by 125I, 14C, 32P, 99mTc, and 225Ac. The chemiluminescent substance is exemplified by ruthenium pyridine complex and acridinium ester. Since it is difficult to bind the radioisotope per se as a label, a possible way is to use a compound, which contains the radioisotope and a maleimide group, as the label. Such compound is exemplified by nucleic acid, saccharide, and oligopeptide that contain 125I, 14C, or 32P, and have a maleimide group added thereto. Alternatively usable is a maleimide derivative, which has a maleimide group bound to a chelating agent capable of coordinating to a metal element such as 99mTc or 225Ac. With the chelating agent moiety coordinated typically to 99mTc or 225Ac, the maleimide derivative is usable as the signal generating substance. The chelating agent is exemplified by deferoxamine.
Preferred labels include biotins, enzyme, fluorescent dye, fluorescent protein and hapten. ALP is particularly preferred as the enzyme.
In formula (I) above with L representing a spacer that binds the nitrogen atom of the imide ring to Z, L is typically exemplified by —(CH2)n—R—(C═O)—NH—, —(CH2)n—R—NH—(C═O)—, —(CH2)n—R—(C═O)—, —(CH2)n—R—(C═O)—O—, —(CH2)n—R—O—(C═O)—, —(CH2)n—R—(C═S)—NH—, —(CH2)n—R—NH—(C═S)—, —(CH2)n—R—O—, —(CH2)n—O—R—, —(CH2)n—R—S— or —(CH2)n—S—R—. n Represents an integer of 1 or larger and 10 or smaller.
Each R independently represents an atomic bond, an optionally substituted alkylene group having 1 to 10 carbon atoms, an optionally substituted arylene group or heteroarylene group having 6 to 12 carbon atoms, an optionally substituted cycloalkylene group or heterocycloalkylene group having 3 to 8 carbon atoms, and any combination of these components. The atomic bond represented by R means a bond enabling direct bonding without any other atom interposed in between.
R, when representing an alkylene group having 1 or more and 10 or less carbon atoms, is exemplified by alkylene groups such as methylene, ethylene, propylene, isopropylene, butylene, isobutylene, pentylene, neopentylene, hexylene, heptylene, octylene, 2-ethylhexylene, nonylene, and decylene groups. Among them, alkylene group having 1 or more and 4 or less carbon atoms is preferred. In a case where R represents an alkylene group having a substituent, the aforementioned number of carbon atoms does not include the number of carbon atoms of the substituent.
In a case where R represents an arylene group or a heteroarylene group, such group may only be an aromatic ring having 6 or more and 12 or less carbon atoms, which may contain one or more heteroatoms selected from N, S, O and P. Such group is exemplified by phenylene, naphthylene, biphenylylene, furanylene, pyrolene, thiophenylene, triazolene, oxadiazolene, pyridylene, and pyrimidylene groups. In a case where R represents an arylene group or a heteroarylene group having a substituent, the aforementioned number of carbon atoms does not include the number of carbon atoms of the substituent.
In a case where R represents a cycloalkylene group or heterocycloalkylene group, such group may only be a non-aromatic ring having 3 or more and 8 or less carbon atoms, which may contain one or more heteroatoms selected from N, S, O and P. Such group is exemplified by cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, pyrrolidinylene, piperidinylene, piperazinylene, and morpholinylene. In a case where R represents a cycloalkylene group or a heterocycloalkylene group having a substituent, the aforementioned number of carbon atoms does not include the number of carbon atoms of the substituent.
The substituent in R is exemplified by hydroxy, cyano, alkoxy, nitro, ═O, ═S, halogen, haloalkyl, heteroalkyl, carboxyalkyl, amine, amide, and thioether groups. R may have multiple substituents. The alkoxy group is represented by “—O-alkyl group”, wherein the alkyl group is a straight chain or branched saturated aliphatic hydrocarbon group having 1 or more and 5 or less carbon atoms, and more preferably having 1 or 2 carbon atoms.
One labeled polypeptide molecule may have one, or two or more side chain-containing glutamine residues represented by formula (I) above. Alternatively, two or more polypeptide molecules may share one label molecule, with each polypeptide structured to form the side chain represented by formula (I) above. In this case, the labeled polypeptide of formula (I) includes multiple structures, each having “(C)—(CH2)2—(C═O)—NH—X—Y—S—” and a moiety of formula (IV) coupled thereto, is bound to one Z. An exemplary labeled polypeptide is represented by formula (V) below. In formula (V), the definitions of (C), X, Y, Z, and L are same as those in formula (I).
The complex represented by formula (V) is formed of one label molecule having two maleimide groups, to which two modified polypeptide molecules described later are bound. The complex represented by formula (V) is an example of the labeled polypeptide represented by formula (I). The labeled polypeptide may alternatively be a complex formed of one label molecule having three or more maleimide groups, to which three or more modified polypeptide molecules are bound. This sort of complex is prepared by binding one label molecule having multiple maleimide groups, to multiple polypeptide molecules, while individually interposed by a PEG chain having a thiol group and an amino group.
In the labeled polypeptide of this embodiment, the polypeptide and the label are spaced by the PEG chain having a molecular weight of 1100 or larger. In an exemplary case where the polypeptide is an antibody, an advantage is that the label is prevented from non-specifically binding to the antibody. In an exemplary case where the polypeptide is an antibody, the antibody can swing owing to flexibility of the PEG chain having a molecular weight of 1100 or larger, yielding an advantage that the antibody will be more likely to capture an antigen, that is, a target substance. The labeled polypeptide of this embodiment having an antibody as the polypeptide, when used for detecting antigen in serum, is also advantageous in terms of suppressing the background, as described later in Example 8 and the results thereof. This is supposedly because the PEG chain having a molecular weight of 1100 or larger can hydrate in a water-containing sample, so as to suppress any serum-derived contaminant from nonspecifically binding to the labeled polypeptide.
The modified polypeptide of this embodiment has the side chain-containing glutamine residue represented by formula (II) above. In the modified polypeptide, the glutamine residue of the polypeptide is bound to the PEG chain having a thiol group. More specifically, the modified polypeptide is obtainable by bonding the PEG chain, as a bifunctional linker, having a thiol group and an amino group, to the glutamine residue of the polypeptide, making use of a TG-catalyzed reaction (for details, see the production method for the modified polypeptide, described later).
Upon contacting the modified polypeptide with the label having a maleimide group, the thiol group of the modified polypeptide reacts with the maleimide group of the label, to produce the labeled polypeptide. That is, the modified polypeptide is an intermediate for obtaining the labeled polypeptide of the present embodiment as the final product.
In formula (II), (C) represents the α-carbon of the glutamine residue in the modified polypeptide. The side chain represented by formula (II) is structured to contain the bifunctional linker, having a structure of PEG chain with a thiol group and an amino group, bound to the side chain of the glutamine residue. The modified polypeptide of this embodiment is a polypeptide in which a glutamine residue, which can be a substrate for TG, in the polypeptide before the linker is bound, is modified to the side chain-containing glutamine residue represented by formula (II) above. That is, the amino acid sequence of the modified polypeptide is the same as that of the polypeptide before the linker is bound, except for having the side chain-containing glutamine residue represented by formula (II) above. Types of polypeptide in the modified polypeptide is not particularly limited, and are the same as those described for the labeled polypeptide. That is, the modified polypeptide may be any protein such as an antibody, antigen, ligand, or receptor. Among them, antibody is preferred.
Details of X and Y in formula (II) are the same as those described for the labeled polypeptide. One modified polypeptide molecule may have one, or two or more side chain-containing glutamine residues represented by formula (II) above.
As has been widely known, the thiol group is susceptible to oxidation, and easily forms a disulfide bond between molecules having the thiol groups. Such formation of the disulfide bond is avoidable by protecting the thiol group of the modified polypeptide, with a protective group such as acetyl group, when the labeled polypeptide is prepared. On the other hand, the terminal thiol group in the modified polypeptide is less susceptible to oxidation at all, so that an advantage is that such thiol group is almost unlikely to form the disulfide bond between the modified polypeptides. This is supposedly because the modified polypeptide has enhanced dispersibility due to hydration of the PEG chain having a molecular weight of 1100 or larger, thus inhibiting contact between the thiol groups. Referring to such advantage, it is not always necessary to preliminarily protect the thiol group in the modified polypeptide. That is, use of the modified polypeptide will make operations, such as blocking and deblocking of the thiol group, no longer necessary, and will simplify preparation process of the labeled polypeptide.
Another embodiment of the present invention relates to a production method for the modified polypeptide. In this production method, first, the polypeptide that contains the glutamine residue is contacted with the linker represented by formula (VI) above, in the presence of TG, thereby binding the linker to the carboxamide side chain of the glutamine residue.
TG per se is a known enzyme, and is commercially available. TG may be a natural enzyme extracted and purified from a living body or a biological sample, or may be an enzyme obtained by genetic recombination. The origin of TG may be any organism, without special limitation. TG derived from microorganism (Streptomyces mobaraensis, for example) is preferably used.
The polypeptide is not particularly limited as long as it contains a glutamine residue that can serve as a substrate for TG. The polypeptide is selectable typically from any protein such as antibody, antigen, ligand, or receptor. Among them, antibody is preferred. The polypeptide, having a glutamine residue that can serve as a substrate for TG, may be a polypeptide having the Q-tag attached thereto. Preferred polypeptide is a fusion polypeptide of antibody and the Q-tag.
The linker represented by formula (VI) above is a bifunctional linker which is structured as a PEG chain having a thiol group and an amino group. Details of X and Y in formula (VI) are the same as those described for X and Y in formula (I), in relation to the labeled polypeptide of this embodiment. The linker represented by the formula (VI) per se has been known and commercially available. The molecular weight of the PEG chain may be determined by mass spectrometry. Details of the mass spectrometry and the conditions thereof are as described above. In a case where a commercially available linker is used, the molecular weight of the PEG chain may be a value disclosed by the manufacturer or supplier. The molecular weight of the linker, disclosed is this case, is preferably 2000 or larger.
The linker represented by formula (VI) above has improved dispersibility due to hydration of the PEG chain having a molecular weight of 1100 or larger, thus supposedly making the thiol groups less likely to contact to each other. Hence, the terminal thiol group of the linker is less susceptible to oxidation, so that the modified polypeptides are almost unlikely to form a disulfide bond in between. It is therefore unnecessary to preliminarily block the thiol group of the linker, in the step of binding the polypeptide to the linker represented by formula (VI). The reaction between the modified polypeptide and the label having a maleimide group can proceed quantitatively and efficiently, since the thiol group is hardly lost by formation of disulfide bond.
The linker represented by formula (VI) may have a free form, or a salt form with an inorganic acid (hydrochloric acid, for example). The linker represented by formula (VI) is preferably a salt of an inorganic acid (hydrochloric acid, for example). As described above, the linker even in the free from is less likely to form a disulfide bond in between, due to the PEG chain having a molecular weight is 1100 or larger. If given as a salt with an inorganic acid, the linker will have improved dispersibility not only due to hydration of the PEG chain, but also due to electrostatic interaction.
In the presence of TG, the glutamine residue-containing polypeptide is preferably contacted with the linker represented by formula (VI) in a suitable aqueous medium. For example, mixing of a TG solution, a polypeptide solution, and a linker solution can contact these components. Sequential order of the mixing is not particularly limited. The aqueous medium is exemplified by water, physiological saline, and buffer. The buffer is exemplified by phosphate-buffered saline, Tris-HCl, and Good's buffer. In a case where microorganism-derived TG is used, the aqueous medium preferably has the pH around neutrality (pH 6.5 or higher and pH 8.5 or lower), which is more preferably pH 8 or higher and 8.5 or lower. An aqueous solvent that contains dimethyl sulfoxide (DMSO) is also acceptable. TG-containing aqueous solvent preferably has a DMSO concentration of 20 w/v % or lower, which is more preferably 10 w/v % or lower.
In such contact, the solution that contains TG, polypeptide and linker as a result of mixing is preferably incubated under conditions that the TG-catalyzed reaction can proceed. Such conditions per se have been known. For example, the temperature is 4° C. or higher and 37° C. or lower, and preferably 20° C. or higher and 37° C. or lower. The incubation time is typically one hour or longer and 24 hours or shorter, although suitably selectable according to the temperature. The contact if conducted under low temperatures, such as 10° C. or lower, is preferably kept for a longer time (8 hours or longer, for example). During the contact, the amino group of the carboxamide side chain of the glutamine residue in the polypeptide is exchanged with the amino group in the linker, while catalyzed by TG, to form an amide bond, whereby the linker is bound to the side chain. The modified polypeptide of this embodiment is thus produced.
Next, the modified polypeptide thus produced by binding the carboxamide side chain and the linker is acquired. For example, the solution that contains TG, the polypeptide, and the linker after the enzymatic reaction may be purified by SEC or affinity chromatography with use of an appropriate column, to remove the enzyme and any unreacted component, thus acquiring the modified polypeptide. The unreacted component is typically the polypeptide remained unbound to the linker, and the linker remained unbound to the polypeptide. The solution that contains the thus preparative modified polypeptide may be desalted and concentrated as necessary.
Another embodiment of the present invention relates to a production method for the labeled polypeptide. In this production method, first, the polypeptide that contains the glutamine residue is contacted with the linker represented by formula (VI) above, in the presence of TG, thereby binding the linker to the carboxamide side chain of the glutamine residue. Next, the modified polypeptide thus produced by binding the carboxamide side chain and the linker is acquired. Details of these processes are the same as those described for the preparation method for the modified polypeptide. Also details of the label are the same as those described for the labeled polypeptide.
The thus preparative modified polypeptide is contacted with the label having a maleimide group, thereby bonding the label to the linker bound to the modified polypeptide. Since the modified polypeptide has the side chain represented by formula (II) above, so that the maleimide group of the label is bound to the thiol group of the side chain. This forms a thioether bond, thus binding the label to the terminal of the linker bound to the modified polypeptide.
The maleimide group-containing label preferably has a structure represented by formula (VII) below. In formula (VII), Z represents a label, L represents a spacer or an atomic bond, and R1 and R2 identically or differently represent a hydrogen atom or a bromine atom. Preferably, both of R1 and R2 individually represent a hydrogen atom, or a bromine atom. Details of L and Z are the same as those described for formula (I) that represents the labeled polypeptide of this embodiment. Given R1 and/or R2 represent a bromine atom, the label whose structure is represented by formula (VII) below is a label having a maleimide derivative, that is, a 3,4-dibromomaleimide group or a 3-bromomaleimide group. In this specification, the term “maleimide group” encompasses 3,4-dibromomaleimide group and 3-bromomaleimide group.
The label having the structure represented by formula (VII) above may be prepared by binding the label with the bifunctional linker that has a maleimide group and a predetermined reactive group. Such bifunctional linker per se has been known, and is commercially available. The predetermined reactive group may be appropriately determined according to the functional group owned by the label. In an exemplary case where the label has an amino group, employable is a bifunctional linker that has an N-hydroxysuccinimide (NHS) ester and a maleimide group. In this case, the bifunctional linker binds to the label as a result of the reaction between the amino group of the label, with the NHS ester of the linker, whereby the label having a structure represented by formula (VII) is obtainable. The label having the structure represented by formula (VII) may alternatively be a commercially available label.
The label having a 3,4-dibromomaleimide group as the maleimide group may be prepared typically according to the reaction scheme below (see Morais M., et al., Bioconjugation, Methods and Protocols (Methods Mol. Biol., 2019, 2033, SpringerLink), Chapter 2, pp. 15-24). In the reaction scheme below, a label having a structure represented by formula (VIII) is obtainable as the label having a maleimide group. In the reaction scheme and formula (VIII) below, AcOH represents acetic acid, DBM-C2-acid represents an intermediate, EEDQ represents 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline as a condensing agent, MeCN represents acetonitrile, and Z represents the label.
One 3,4-dibromomaleimide group can react with each of two thiol groups to form a thioether bond. Hence, use of the label having a 3,4-dibromomaleimide group typically enables one label molecule to bind two modified polypeptide molecules. That is, the modified polypeptide is dimerized while interposed by the 3,4-dibromomaleimide group, thereby obtaining a complex of two labeled polypeptide molecules. In an exemplary case where two modified polypeptide molecules are bound to one label molecule having the structure represented by formula (VIII), obtainable is a complex of the labeled polypeptide represented by formula (IX) below. In formula (IX), definitions of (C), X, Y, Z, and L are synonymous to those in formula (I).
The modified polypeptide is preferably contacted with the label having a maleimide group, in a suitable aqueous medium. For example, mixing of a modified polypeptide solution, and a maleimide group-containing label solution can contact these components. The aqueous medium is preferably buffer, which is exemplified by triethanolamine buffer, PBS, Tris-HCl, and Good's buffer. pH of the aqueous medium is preferably 6.5 or higher and 7.5 or lower.
In such contact, the solution that contains the modified polypeptide and the label as a result of mixing is preferably incubated under conditions that the reaction between the thiol group and the maleimide group can proceed. Such conditions per se have been known. For example, the temperature is 5° C. or higher and 37° C. or lower, and preferably 20° C. or higher and 30° C. or lower. The incubation time is typically 2 hours or longer and 6 hours or shorter, although suitably selectable according to the temperature. The contact if conducted under low temperatures, such as 10° C. or lower, is preferably kept for a longer time (16 hours or longer, for example). As a result of such contact, the thiol group of the modified polypeptide and the maleimide group of the label form a thioether bond, to produce the labeled polypeptide.
Then, the labeled polypeptide thus produced by binding the modified polypeptide and the label is acquired. For example, the solution that contains the modified polypeptide and the label after the enzymatic reaction may be purified by SEC with use of an appropriate column, to remove any unreacted component, thus acquiring the labeled polypeptide. The unreacted component is typically the label remained unbound to the modified polypeptide, or the modified polypeptide remained unbound to the label. The solution that contains the thus preparative labeled polypeptide may be desalted and concentrated as necessary.
In another embodiment, a label having a haloacetyl group may be used in place of the label having a maleimide group, to label the modified polypeptide (see Greg T. Hermanson, Bioconjugate Techniques Third Edition, Academic Press, Chapter 3 The reaction of Bioconjugation, pp. 240-241, 2. Thiol reactions, 2.1 Haloacetyl and alkyl halide derivatives). For example, in a case where the modified polypeptide and a label having a haloacetyl group represented by formula (X) (where, R3 represents a bromine atom or an iodine atom) are bound as represented by the reaction scheme below, obtainable is a labeled polypeptide represented by formula (XI) below. In the reaction scheme below, definitions of (C), X, Y, Z, and L are synonymous to those in formula (I).
Yet another embodiment relates to a reagent that contains the labeled polypeptide or the modified polypeptide (also referred to as “reagent of this embodiment”, hereinafter). In a case where the polypeptide is an antibody, the reagent of this embodiment that contains the labeled polypeptide, is used for the measurement method of this embodiment described later. The reagent of this embodiment that contains the modified polypeptide is used for a reaction for binding the modified polypeptide to any maleimide group-containing label, to obtain the labeled polypeptide. Details of the labeled polypeptide and the modified polypeptide are the same as those described above.
The reagent of this embodiment may be provided to the user, while enclosing the labeled polypeptide or the modified polypeptide in a vial. An exemplary reagent of this embodiment is shown in
Yet another embodiment relates to a reagent kit having the reagent that contains the labeled polypeptide or the modified polypeptide (also referred to as “reagent kit of this embodiment”, hereinafter). In a case where the polypeptide is an antibody, the reagent kit of this embodiment, having the reagent that contains the labeled polypeptide, is used for the measurement method of this embodiment described later. The reagent kit of this embodiment, having the reagent that contains the modified polypeptide, is used for a reaction of binding the modified polypeptide to any maleimide group-containing label, to obtain the labeled polypeptide. Details of the labeled polypeptide and the modified polypeptide are the same as those described above.
The reagent kit of this embodiment may be provided to the user, while packaging the vial that contains the labeled polypeptide or the modified polypeptide, in a box. The box may include a package insert. The package insert may contain descriptions on a reagent composition, a structure of the labeled polypeptide or modified polypeptide, use instructions of the reagent, and storage instructions of the reagent. An exemplary reagent kit of this embodiment is shown in
Yet another embodiment relates to a measurement method for a target substance with use of the labeled polypeptide of this embodiment (also referred to as “measurement method of this embodiment”). The labeled polypeptide used in the measurement method of this embodiment is a fusion polypeptide that is composed of an antibody, and a peptide tag that contains the side chain-containing glutamine residue represented by formula (I) above (also referred to as “labeled antibody of this embodiment”). The labeled antibody of this embodiment is an antibody having a label, capable of specifically binding to a target substance, and is used as a detection antibody in the measurement method of this embodiment. The detection antibody refers to an antibody that binds to a target substance, and provides a signal detectable with the aid of the label. The label is preferably a substance that catalyzes reaction of a signal generating substance or other substance, to induce signal generation. The label is more preferably enzyme, fluorescent dye, or fluorescent protein. Details of the labeled polypeptide of this embodiment are the same as described above.
In the measurement method of this embodiment, first, an immune complex of the labeled antibody and the target substance is formed. The target substance may be a substance recognizable by the labeled antibody, or may be a formed element that contains such substance. The substance recognizable by the labeled antibody is exemplified by protein, oligopeptide, nucleic acid, lipid, glycan, and hapten. The formed element that contains the substance recognizable by the labeled antibody is exemplified by cell, extracellular vesicle, microorganism, virus, and fragment thereof. The extracellular vesicle is exemplified by exosome, ectosome, microvesicle, microparticle, and apoptotic body. The microorganism is exemplified by bacteria and fungi.
The immune complex may be formed by mixing a sample that presumably contains a target substance, with the labeled antibody. Type of the sample is exemplified by, but not particularly limited to, biological samples such as blood, plasma, serum, lymph, and saliva; excreta such as urine and feces; and environmental samples such as river water, seawater, and soil. The immune complex is preferably formed in a solution. The sample that presumably contains the target substance is therefore preferably in a liquid form. The liquid sample is not limited to solution, and encompass suspension and sol. The sample, not in liquid form, may be converted to liquid form, typically by adding an appropriate aqueous medium to the sample. Any insoluble contaminant in the liquid sample may optionally be removed, typically by a known technique such as centrifugation or filtration. The liquid sample may optionally be diluted with the aforementioned aqueous medium. The labeled antibody is preferably in liquid form prepared in a suitable aqueous medium. The aqueous medium is as described above.
When forming the immune complex, the immune complex of the labeled antibody and the target substance is preferably formed on a solid phase. For example, the sample that presumably contains the target substance is mixed with the labeled antibody to form the immune complex, and the solution that contains the immune complex is then contacted with the solid phase capable of immobilizing thereon the labeled antibody or the target substance. The immune complex may be thus formed on the solid phase. Alternatively, the target substance may be preliminarily immobilized on the solid phase, and the solid phase may be contacted with the labeled antibody to form the immune complex on the solid phase.
Conditions for mixing, such as temperature and incubation time, are not particularly limited as long as they are suitable for the antigen-antibody interaction. Such conditions per se have been known. For example, the temperature is 4° C. or higher and 40° C. or lower, and preferably 20° C. or higher and 37° C. or lower. The incubation time is typically one minute or longer and 24 hours or shorter, although suitably selectable according to the temperature. The contact if conducted under low temperatures, such as 10° C. or lower, is preferably kept for a longer time (1 hour or longer, for example).
In the measurement method of this embodiment, a capture antibody that specifically binds to the target substance may be used in addition to the labeled antibody. The capture antibody refers to an antibody that is immobilized per se on the solid phase, and eventually captures the target substance on the solid phase. The capture antibody preferably recognizes an epitope which is different from that recognizable by the labeled antibody. In a case where the target substance has multiple same epitopes, the capture antibody and the labeled antibody may recognize the same epitope. With such additional use of the capture antibody, there is formed a sandwich immune complex of the labeled antibody, the target substance, and the capture antibody. The sandwich immune complex means a complex that contains the capture antibody, the target substance, and the labeled antibody, in which the capture antibody and the labeled antibody are bound to different sites on the target substance.
The sandwich immune complex may be formed by mixing the capture antibody, the sample that presumably contains the target substance, and the labeled antibody. In a preferred embodiment, the sandwich immune complex, formed of the capture antibody, the target substance, and the labeled antibody, is formed on the solid phase. For example, the sample that presumably contains the target substance, the capture antibody, and the labeled antibody are mixed to form the immune complex, and the solution that contains the immune complex is then contacted with the solid phase capable of immobilizing thereon the capture antibody. The sandwich immune complex may be thus formed on the solid phase. Alternatively employable is the capture antibody preliminarily immobilized on the solid phase. That is, the sandwich immune complex may be formed on the solid phase, by mixing the capture antibody preliminarily immobilized on the solid phase, the sample that presumably contains the target substance, and the labeled antibody. Conditions for mixing, such as temperature and incubation time, are as described above.
The solid phase may only be an insoluble carrier on which the target substance or the capture antibody may be immobilized. The solid phase is preferably an insoluble carrier on which the capture antibody may be immobilized. For example, the capture antibody may be immobilized on the solid phase, as a result of direct or indirect binding between the solid phase and the capture antibody. The direct binding between the solid phase and the capture antibody is exemplified by adsorption to the surface of the solid phase attributable to hydrophobic interaction, or formation of a covalent bond. In an exemplary case where the solid phase is an ELISA microplate, the capture antibody may be immobilized by adsorption, inside a well of the plate. In a case where the solid phase has a functional group on the surface, the capture antibody may be immobilized on the surface of the solid phase through a covalent bond with use of the functional group. In an exemplary case where the solid phase is a particle having a carboxy group, the carboxy group on the particle surface is activated with 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (WSC), and then reacted with NHS to form an NHS ester. Upon contact of the particle having the NHS ester with the capture antibody, the NHS ester reacts with the amino group of the capture antibody, whereby the capture antibody is covalently immobilized on the particle surface.
The indirect binding between the solid phase and the capture antibody is exemplified by binding while interposed by a molecule that is specifically binds to the capture antibody. By preliminarily immobilizing such molecule on the surface of the solid phase, the capture antibody may be immobilized on the solid phase. The molecule that specifically binds to the capture antibody is exemplified by protein A, protein G, and antibody (secondary antibody) that specifically recognizes the capture antibody. The capture antibody and the solid phase may alternatively be bound, making use of combination of substances interposed between the capture antibody and the solid phase. Such combination of substances is exemplified by combination of biotins and avidins, or, hapten and anti-hapten antibody. In an exemplary case where the capture antibody is preliminarily DNP-modified, use of the solid phase, having an anti-DNP antibody immobilized thereon, can immobilize thereon such capture antibody.
Material for composing the solid phase is selectable typically from organic polymer compound, inorganic compound, and biopolymer. The organic polymer compound is exemplified by latex, polystyrene, polypropylene, styrene-methacrylate copolymer, styrene-glycidyl (meth)acrylate copolymer, styrene-styrene sulfonate salt copolymer, methacrylate polymer, acrylate polymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride-acrylate ester copolymer, and polyvinyl acetate-acrylate copolymer. The inorganic compound is exemplified by magnetic substance (iron oxide, chromium oxide, cobalt, ferrite, etc.), silica, alumina and glass. The biopolymer is exemplified by insoluble agarose, insoluble dextran, gelatin and cellulose. Two or more of them may be used in combination.
Shape of the solid phase is exemplified by, but not specifically limited to, particle, microplate, microtube and test tube. Among them, particle and microplate are preferred, and magnetic particle is particularly preferred. The solid phase, when given in the form of particle, may be used while being suspended in liquid, as the solid for formation of the immune complex. The solid phase, when given in the form of container such as a microplate, the immune complex may be formed in the container as the solid phase. When using the magnetic particles capable of immobilizing thereon the capture antibody, the measurement method of this embodiment may be conducted with a commercially available fully automated immunoassay system such as HISCL (registered trademark) series (Sysmex Corporation).
Next, a signal generated by the label contained in the immune complex is detected. The label is owned by the labeled antibody bound to the target substance. The signal generated by the label in the immune complex therefore corresponds to the amount of the target substance. In this specification, the phrase “detecting a signal” encompasses qualitatively detecting presence or absence of a signal, quantifying the signal intensity, and semi-quantitatively detecting signal intensity. The phrase “semi-quantitatively detecting signal intensity” means that the signal intensity is detected stepwise as “no signal”, “weak”, “medium” and “strong”. Preferably, the intensity of the signal generated by the label contained in the immune complex is quantified to obtain a measured value.
Results of detection of the signal may be used as results of measurement of the target substance in the sample. When quantifying the signal intensity, the measured value of the signal intensity per se, or a value acquired from the measured value, may be used as the measured value of the target substance. The value acquired from the measured value of the signal intensity is exemplified by a value obtained by subtracting a measured value of a negative control, or a background value, from the measured value. The negative control is properly selectable, which is exemplified by a buffer solution free of the target substance.
The measured value of the signal intensity may be fitted on an analytical curve, to determine the amount or concentration of the target substance in the sample. The analytical curve may be created on the basis of measured values of multiple calibrators. The measured values of the calibrators are obtainable by measuring the calibrators according to the measurement method of this embodiment, in the same way as for the sample. The analytical curve may be prepared by plotting the measured values of the plurality of calibrators on an X-Y chart, having concentration of the target substance in the calibrator scaled on the X-axis, and the measured value (signal intensity, for example) scaled on the Y-axis, and by fitting thereto a straight line or a curve according to a known technique such as the least square method. The calibrators may be prepared typically by adding any known concentrations of an isolated or synthesized target substance into a buffer free of the target substance. The calibrator free of the target substance may be a buffer per se which is free of the target substance.
The measurement method of this embodiment may be conducted with a commercially available fully automated immunoassay system. The fully automated immunoassay system is an instrument that automatically starts, upon sample setting and entry of measurement start command by the user, preparation and immunoassay of a measurement sample, and outputs measurement results of the target substance. This sort of fully automated immunoassay system is exemplified by those in HISCL Series (Sysmex Corporation), such as HISCL (registered trademark)-5000, and HISCL-2000i. Measurement with the HISCL Series system relies upon the sandwich ELISA method, with use of a magnetic particle as the solid phase.
In this embodiment, B/F (bound/free) separation for removing any free component that remains unreacted may be interposed between formation of the sandwich immune complex and the signal detection. The free component that remains unreacted refers to a component that does not constitute the immune complex. This is exemplified by excessive capture antibody and detection antibody of this embodiment, not having been bound to the target substance. Technique for the B/F separation is not specially limited. With the solid phase given a particle form, the B/F separation may be enabled by centrifugation, by which only the solid having the immune complex captured thereon is collectable. With the solid phase given as a container such as a microplate or a microtube, the B/F separation may rely upon removal of a liquid that contains the unreacted free component. With the solid phase given as a magnetic particle, the B/F separation may rely upon removal of a liquid that contains the unreacted free component, under suction through a nozzle, while magnetically restraining the magnetic particle with use of a magnet. The method is preferred from the viewpoint of automatization. After removing the unreacted free component, the solid phase having the immune complex captured thereon may be washed with a suitable aqueous medium, such as PBS.
With the label of the labeled antibody given as a fluorescent dye or fluorescent protein, the measurement method of this embodiment may rely upon a flow cytometer. The “flow cytometer” in this specification encompasses an imaging flow cytometer (IFC). The IFC is a sort of flow cytometer equipped with an imaging unit such as a CCD camera. A flow cytometer not equipped with such imaging unit is structured to irradiate light on the individual formed elements in a liquid that flow through a flow cell, and to detect scattered light and/or fluorescence emitted from the individual formed elements as an optical signal. With the formed element given as cells, the IFC can typically measure the size of each cell, and distribution of the amount of molecules on the cell surface or inside the cells. The IFC is an instrument capable of acquiring images of the individual formed elements in the liquid that flow through the flow cell. The IFC can quantitatively measure the formed elements within a time as short as several seconds to several minutes, by acquiring fluorescence signal, scattered light signal, fluorescence image, or bright field image (also referred to as a transmitted light image) from each of several thousands to several millions of formed elements. The image processing also enables extraction of information from the individual formed elements.
The measurement method of this embodiment, with use of the flow cytometer, may be targeted at the formed element that contains the substance recognizable by the labeled antibody. When measured with the flow cytometer, the immune complex may be formed by mixing the sample that presumably contains the target substance, and the labeled antibody. In this way, the labeled antibody can bind to the target substance that resides on the formed element, thereby forming the immune complex on the formed element. Conditions for mixing, such as temperature and incubation time, are as described above.
The flow cytometer takes part in detection of a signal ascribed to the label contained in the immune complex. That is, the signal is detected by the flow cytometer, upon introduction of the immune complex into the flow cell of the flow cytometer. In the flow cytometer, each of the formed element having the immune complex formed thereon is irradiated with light, while passing through the flow cell. The flow cytometer then detects fluorescence signal emitted from the formed element. Since the measurement method of the present embodiment with use of the flow cytometer employs the fluorescent dye or the fluorescent protein as the label, so that detectable is a fluorescent signal emitted from the fluorescent dye or the fluorescent protein. Fluorescence information is then acquired according to the detected fluorescence signal. A scattered light signal may alternatively be detected from the formed element as necessary, and scattered light information may be acquired on the basis of the scattered light signal. The scattered light is exemplified by forward scattered light (scattered light typically at a light receiving angle of 0° to approximately 20°), and side scattered light (scattered light typically at a light receiving angle of approximately 200 to approximately 90°).
Light source of the flow cytometer is not particularly limited, and is typically selectable from those having a wavelength suitable for exciting the fluorescent dye or the fluorescent protein. The light source usable here may typically be semiconductor laser source, argon laser source, He—Ne laser light source, or mercury lamp.
The fluorescence information is exemplified by fluorescence pulse height, pulse width, pulse area, transmittance, Stokes shift, ratio, temporal change, and values correlated thereto. The scattered light information is exemplified by scattered light pulse height, pulse width, pulse area, transmittance, Stokes shift, ratio, temporal change, and values correlated thereto.
With the flow cytometer given as an IFC, the fluorescence information may typically be a fluorescent signal-based value acquired from an image captured by the IFC. In a fluorescence image of the particle captured by the IFC, each pixel that constitutes an area demonstrating the fluorescence signal has a pixel value that corresponds to the fluorescence signal intensity. Hence, a value based on the optical signal, acquired from the image, may typically be a value based on the pixel that constitutes the area demonstrating the fluorescence signal. Such value is exemplified by fluorescence intensity, maximum fluorescence intensity, total fluorescence signal intensity, and fluorescence signal area value. The “fluorescence intensity” is an average value of the pixel values of the pixels that constitute the area demonstrating the fluorescence signal, in the fluorescence image of the formed element that contains the immune complex. The “maximum fluorescence intensity” is the maximum value from among the pixel values of the pixels that constitute the area demonstrating the fluorescence signal, in the fluorescence image of the formed element that contains the immune complex. The “total fluorescence intensity” is the total of the pixel values of the pixels that constitute the area demonstrating the fluorescence signal, in the fluorescence image of the formed element that contains the immune complex. The “fluorescence signal area value” is the number of pixels that constitute the area demonstrating the fluorescence signal, in the fluorescence image of the formed element that contains the immune complex.
The formed element that contains the immune complex may also be detected, on the basis of the acquired fluorescence information. With the fluorescence information given as information indicated by numerical values such as the pulse peak of fluorescence, or the fluorescence intensity acquired from the fluorescence image, the fluorescence information of the formed element that contains the immune complex will have a value larger than the value of the fluorescence information of the formed element having no immune complex formed thereon. Hence, the measurement data of the formed element that contains the immune complex may be extracted, referring to a result of comparison between the value of the acquired fluorescence information and a predetermined threshold value. For example, a formed element whose value of the acquired fluorescence information is equal to or larger than the predetermined threshold value, may be detected as the formed element that contains the immune complex. The predetermined threshold value may be freely determined without special limitation. For example, a value of fluorescence information may be acquired by measuring a buffer solution free of the target substance, or a sample that contains the formed element already known to contain no target substance, by the measurement method of this embodiment with use of the flow cytometer. The thus obtained value may be used as the predetermined threshold.
The present invention will be further detailed referring to EXAMPLES, to which the present invention is by no means limited.
Materials and methods used in Test Examples described later will be described below. Details of the method will also be described in the individual Test Examples.
1. Antibodies
(1.1) Preparation of Q-Tag-Attached Anti-p24 Fab Antibody
(1.1.1) Establishment of Anti-p24 Antibody (sF001-5, sF028-22 and sF001-25)-Producing Hybridoma
An anti-p24 antibody (sF001-5, sF028-22, sF001-25)-producing hybridoma was established, while using a mouse (ddY; 5 weeks old, female) as a host, and recombinant HIV p24 protein as an antigen. That is, a mouse (ddY; 5 weeks old, female) was immunized with recombinant HIV p24 protein, with use of Freund's complete adjuvant (FCA) and incomplete Freund's adjuvant (FIA) as adjuvants. The mouse, after confirming increase in the blood antibody titer, was euthanized, and the spleen was then extracted by necropsy. The extracted spleen cells were fused with mouse myeloma cells by the PEG method, and hybridomas were selected on a HAT medium. The antibody secreted into the culture supernatant of each hybridoma was assayed by ELISA, to establish hybridomas that produce each of sF001-5, sF028-22 and sF001-25, as an anti-p24 antibody.
(1.1.2) Preparation of sF001-5, sF028-22 and sF001-25
sF001-5, sF028-22 and sF001-25 derived from the mouse hybridomas were prepared. That is, the thus established sF001-5-, sF028-22-, and sF001-25-producing hybridoma cells were cultured in an SFM medium to induce secretion of sF001-5, sF028-22, and sF001-25 into the medium, respectively. The culture supernatant, filtered through a filter to remove the cells, was subjected to column chromatography with use of Protein G carrier, to obtain each of purified sF001-5, sF028-22, and sF001-25 antibodies.
(1.1.3) Preparation of Q-Tag-Attached Anti-p24 Fab Antibodies
Chimeric sF001-5 (abbreviated as “csF001-5”, hereinafter), chimeric sF028-22 (abbreviated as “csF028-22”, hereinafter), and chimeric sF001-25 (abbreviated as “csF001-25”, hereinafter) Fab antibodies were obtained by linking the hypervariable regions of sF001-5, sF028-22, and sF001-25, derived from the mouse hybridoma, with the constant region of human IgG1 κ isotype Fab. A Q-tag sequence was attached to the heavy chain C terminal of each of these chimeric Fab antibodies, to obtain tagged anti-p24 Fab antibodies (also referred to as “csF001-5Fab-Qtag”, “csF028-22Fab-Qtag”, and “csF001-25Fab-Qtag”, hereinafter). Amino acid sequences of the constant region including the inserted Q-tag sequence, and base sequences that encode the same are listed below (with the Q-tag sequence underlined).
Polynucleotides that encode the light chains of csF001-5Fab-Qtag, csF028-22Fab-Qtag, and csF001-25Fab-Qtag were obtained by individually amplifying the variable regions of the light chains of sF001-5, sF028-22, and sF001-25, and the constant region of the human IgG light chain K, and then by linking the individual fragments by overlap-PCR. The polynucleotides that encode the heavy chain was obtained by overlap-PCR, with use of the CHI region of human IgG1 as a template, in which fragments each having a Q-tag sequence attached to the C-terminal while mediated by a primer was coupled with fragments of the variable domains of the heavy chains of sF001-5, sF028-22, and sF001-25 amplified by PCR. Each of the thus prepared PCR products was introduced into pcDNA3.4 (Thermo Fisher Scientific) by TA cloning, to construct plasmids that encode csF001-5 LC (light chain in chimerized sF001-5), csF028-22_LC, and csF001-25_LC. Next, constructed were plasmids that encode csF001-5_HC_Fab-Qtag (chimeric sF001-5 whose Fab heavy chain has Qtag attached to the C-terminal), csF028-22_HC_Fab-Qtag, and csF001-25_HC_Fab-Qtag. Next, the plasmids that encode each of the heavy chain and the light chain were co-transfected into Expi-293 cell (Thermo Fisher Scientific), so as to express recombinant proteins csF001-5Fab-Qtag, csF028-22Fab-Qtag, and csF001-25Fab-Qtag in the culture supernatant. Each of csF001-5Fab-Qtag, csF028-22Fab-Qtag, and csF001-25 Fab-Qtag thus expressed in the supernatant was purified through Protein G column.
(1.2) Preparation of Q-Tag-Attached Anti-HBs628 Fab Antibody
(1.2.1) Establishment of Anti-HBs628 Antibody-Producing Hybridoma
A hybridoma that produces mouse anti-HBs628 antibody was produced according to the Kohler-Milstein method (Kohler G. and Milstein C., Nature, 256, 495-497 (1975)), with use of HBs628 antigen.
(1.2.2) Preparation of Q-Tag-Attached Anti-HBs628 Fab Antibody
Q-tag-attached HBs628 Fab antibody (also referred to as “HBs628Fab-Qtag”, hereinafter) was obtained by attaching the Q-tag sequence to the heavy chain C-terminal of the Fab moiety of HBs628 derived from mouse hybridoma. Amino acid sequences of the constant region including the inserted Q-tag sequence, and base sequences that encode the same are listed below (with the Q-tag sequence underlined).
A polynucleotide that encodes the light chain of HBs628Fab-Qtag was obtained by cloning from hybridoma RNA according to 5′-RACE, and then by amplifying it by PCR. A polynucleotide that encodes the heavy chain of HBs628Fab-Qtag was obtained by cloning from hybridoma RNA according to 5′-RACE, and a fragment having a Q-tag attached to the C-terminal mediated by a primer was amplified by PCR. Each of the thus prepared PCR products was introduced into pcDNA3.4 (Thermo Fisher Scientific) by TA cloning, to construct a plasmid that encodes HBs628Fab-Qtag. Next, the plasmid was transfected into Expi-293 cell (Thermo Fisher Scientific), so as to express recombinant protein HBs628Fab-Qtag in the culture supernatant. HBs628Fab-Qtag thus expressed in the supernatant was purified through CaptureSelect LC-Lambda (mur) affinity column (Thermo Fisher Scientific).
(1.3) Preparation of Q-Tag-Attached Anti-CD20 Fab Antibody
Q-tag-attached anti-CD20 Fab antibody (also referred to as “anti-CD20Fab-Qtag”, hereinafter) was obtained by attaching the Q-tag sequence to the heavy chain C-terminal of the Fab moiety of human anti-CD20 Fab. Amino acid sequences of the hypervariable region and the constant region that contains the Q-tag inserted thereto, and base sequences that encode them were listed below (with the Q-tag sequence underlined).
The polynucleotide that encodes the light chain Fab, and the polynucleotide having the Q-tag sequence attached to the heavy chain Fab C-terminal were individually inserted into pcDNA3.4 (Thermo Fisher Scientific), to construct, by total synthesis, expression plasmids that encode anti-CD20Fab-LC and anti-CD20Fab-HC-Qtag, respectively. Next, the plasmids were transfected into Expi-293 cell (Thermo Fisher Scientific), so as to express recombinant protein anti-CD20Fab-Qtag in the culture supernatant. Anti-CD20Fab-Qtag thus expressed in the supernatant was purified through CaptureSelect (registered trademark) Kappa XL pre-packed column (Thermo Fisher Scientific).
2. PEG Linker Having Amino Group and Thiol Group (SH-PEG-NH2 linker) SH-PEG-NH2 linkers purchased include (a) HS-PEG2K—NH2, HCl salt, average Mn 2000 (Sigma-Aldrich), (b) SH-PEG-NH2, MW2K (Biopharma PEG Scientific Inc.), (c) HS-PEG-NH2, MW2K (Creative PEGWorks), (d) HS-PEG-NH2, HCl salt, average Mn 3500 (Sigma-Aldrich), (e) HS-PEG-NH2, HCl salt, average Mn 5000 (Sigma-Aldrich), (f) Thiol PEG-NH2, average Mn 400 Da (Nanocs Inc.) and (g) HS-PEG-NH2, MW 1 kDa (Creative PEGWorks). Also purchased was HS-PEG2K-NH2, HCl salt, average Mn 2000 (Sigma-Aldrich), but from a lot different from the linker (a) (also referred to as linker (a′), hereinafter). The linkers (a), (a′), (b) and (c) were sold as products with a molecular weight indication of 2000. Each of the linkers (a), (a′), (b) and (c) will also be referred to as “SH-PEG-NH2 linker (2K)”. The linker (d) was sold as a product with a molecular weight indication of 3500. The linker (d) will also be referred to as “SH-PEG-NH2 linker (3.5K)”. The linker (e) was sold as a product with a molecular weight indication of 5000. The linker (e) will also be referred to as “SH-PEG-NH2 linker (5K)”. The linker (f) was sold as a product with a molecular weight indication of 400. The linker (f) will also be referred to as “SH-PEG-NH2 linker (400 Da)”. The linker (g) was sold as a product with a molecular weight indication of 1000. The linker (g) will also be referred to as “SH-PEG-NH2 linker (1K)”. The linkers (a), (a′), (b), (d), and (e) were available as hydrochlorides, and the linkers (c), (f), and (g) were available in the free form. Structure of the individual linkers are listed below.
Fab antibody having the SH-PEG-NH2 linker bound thereto will also be referred to as “SH-PEG-Fab”. In particular, each Fab antibody having any of the SH-PEG-NH2 linkers (a), (a′), (b), (c) bound thereto will also be referred to as “SH-PEG(2K)-Fab”. Meanwhile, The Fab antibodies having the SH-PEG-NH2 linkers (d), (e), (f) and (g) bound thereto will also be referred to as “SH-PEG(3.5K)-Fab”, “SH-PEG(5K)-Fab”, “SH-PEG(400 Da)-Fab” and “SH-PEG(1K)-Fab”, respectively.
3. Crosslinking Agent Having Maleimide Group and NHS Ester (EMCS Reagent)
EMCS reagent (Product name: EMCS, CAS No.: 55750-62-5) was purchased from Dojindo Laboratories. The molecular formula and molecular weight were C14H16N2O6 and 308.29, respectively, with a structure given below.
4. Labeling
(4.1) Biotin Having Maleimide Group
Biotin-PEAC5-maleimide (product name: Biotin-PEAC5-maleimide, CAS No.: 374592-98-0) was purchased from Dojindo Laboratories. The molecular formula and molecular weight were C26H41ClN6OSS and 585.16, respectively, with a structure given below.
(4.2) Fluorescent Dye Having Maleimide Group
Alexa 488-maleimide (product name: Alexa Fluor (registered trademark) 488 C5 maleimide, CAS No.: 500004-82-0) was purchased from Thermo Fisher Scientific. The molecular formula and molecular weight were C30H25N4NaO12S2 and 720.66, respectively, with a structure given below.
(4.3) R-Phycoerythrin (R-PE)
R-PE (product name: OBI) was purchased from One Biotech.
(4.4) Alkaline Phosphatase (ALP)
ALP derived from bovine small intestine (product name: ALP-55) was purchased from Oriental Yeast Co., Ltd.
5. Transglutaminase (TG)
TG employed herein was Activa (registered trademark) KS-CT (Ajinomoto Corporation), which is a microorganism-derived transglutaminase, used after purified. The thus purified enzyme will also be referred to as “BTGase”, hereinafter.
6. Mass Spectrometry Based on Infusion Scheme (Infusion MS)
The aforementioned SH-PEG-NH2 linkers (a), (a′), (b), (c), (d) and (e) were analyzed by Infusion MS, in order to find the molecular weight distributions and occurrence of dimerization. An MS analyzer employed herein was Q Exactive (Thermo Fisher Scientific). The ionization mode was set to positive. The measurement sample was prepared by dissolving each of the aforementioned SH-PEG-NH2 linkers in a BTGase reaction solution (50 mM Tris, 2 mM EDTA, pH 8.2; or 20 mM Tris, 2 mM EDTA, 150 mM NaCl, pH 8.2), followed by 100-fold dilution with a 30% acetonitrile solution containing 0.1% formic acid. Result of mass analysis for each SH-PEG-NH2 linker having a repeating structure was examined to find an isotope peak whose intensity is highest of all peaks having an S/N of 5 or larger, and from the molecular weight and intensity of such most intense peak, the weight average molecular weight of each linker was determined. The MS peak was also used to determine the distribution range of molecular weight.
7. Quantification of Target Protein
The individual Fab antibodies and derivatives thereof were quantified on the basis of Abs280 peak area in SEC. Reference used herein was a recombinant human interleukin 6 (rhIL-6) (absorption coefficient 0.43).
8. Analysis of Non-Reducing SDS-PAGE
The individual Fab antibodies and derivatives thereof were analyzed by non-reducing SDS-PAGE under the conditions below.
(1) Binding of SH-PEG-NH2 Linker (2K) with Fab Antibodies
The aforementioned (a) SH-PEG-NH2 linker (2K) (Sigma-Aldrich) was bound to each of csF001-5Fab-Qtag, csF028-22Fab-Qtag, csF001-25Fab-Qtag and HBs628Fab-Qtag, while catalyzed by BTGase. Also the aforementioned (a′) SH-PEG-NH2 linker (2K) (Sigma-Aldrich) was bound to anti-CD20Fab-Qtag. Operational details were as follows. csF001-5Fab-Qtag or HBs628Fab-Qtag was dissolved in MES buffer solution (50 mM MES, 2 mM EDTA, pH 7.0) so as to adjust the concentration to 10 μM, and the solution was incubated with 50 equivalents of SH-PEG-NH2 linker (2K) and 0.1 U/mL of BTGase at 25° C. for 3 hours. The activity of BTGase was assayed by the hydroxamate method (Folk J. E. and Cole P. W., J. Biol. Chem. 241, 5518-5525 (1966)). Progress of the reaction was monitored by SEC.
Enhancement of introduction efficiency of the SH-PEG-NH2 linker (2K) was examined in a Tris buffer (20 mM Tris, 2 mM EDTA, 150 mM NaCl, pH 8.2 or pH 8.5), by adding 50 equivalents of the linker to 10 μM of each of csF001-5Fab-Qtag, csF028-22Fab-Qtag, and csF001-25Fab-Qtag, and by reacting the mixture at room temperature for 5 hours. Results of the SEC analysis of the reaction products obtained at pH 8.2 and pH 8.5, and the reaction product obtained at pH 7.0 were then compared.
The reaction condition (pH 8.2 or 8.5) aimed at enhanced introduction efficiency was applied to all reactions between the Fab-Qtags and the SH-PEG-NH2 linkers. Among the reaction solutions, the reaction solution of csF001-5Fab-Qtag was desalted and concentrated with Amicon 10K (Merck), to remove the SH-PEG-NH2 linker. The reaction solution of csF028-22Fab-Qtag was fractionated with Superdex 200 Increase 10×300 mm, and concentrated with Amicon 10K (Merck). The reaction solution of HBs628Fab-Qtag was prepared through Superdex 75 Increase 10×300 mm, and concentrated with Amicon 10K (Merck). The reaction solutions of csF001-25Fab-Qtag and anti-CD20Fab-Qtag were desalted and purified through Protein G column, and then concentrated with Amicon 10K (Merck).
(2) SEC Analysis
The reaction solutions of csF001-5Fab-Qtag, csF028-22Fab-Qtag, csF001-25Fab-Qtag, each reacted with the SH-PEG-NH2 linker (2K) (Sigma-Aldrich), were analyzed by SEC. The reaction solution of csF028-22Fab-Qtag and SH-PEG-NH2 linker (2K) was also fractionated. Conditions were as follows.
(3) SEC Analysis and Fractionation
The reaction solution of HBs628Fab-Qtag and the SH-PEG-NH2 linker (2K) (Sigma-Aldrich) was analyzed and isolated by fractionation according to the conditions below.
(4) Fractionation of SH-PEG-Fab Through Protein G
SH-PEG (2K)-Fab derived from each of csF001-25 Fab-Qtag and anti-CD 20Fab-Qtag was isolated by fractionation under the following conditions.
(1) Biotin Labeling of SH-PEG-Fab
SH-PEG(2K)-Fab from HBs628Fab-Qtag was labeled with biotin, as described below. The BTGase-catalyzed reaction solution of HBs628Fab-Qtag and the (a) SH-PEG-NH2 linker (2K) (Sigma-Aldrich) was desalted and concentrated with Amicon 10K (Merck), to obtain a concentrated solution that contains SH-PEG-Fab. In a 50 mM MES-2 mM EDTA buffer solution (pH 7.0), added was Biotin-PEAC5-maleimide, whose amount was adjusted to 100 equivalents relative to 10 μM of SH-PEG-Fab, and the mixture was reacted at 5° C. overnight. The reaction solution was purified by SEC. The reaction product of SH-PEG-Fab and maleimide-labeled biotin will also be referred to as “biotin-PEG-Fab”, hereinafter. The biotin-PEG-Fab was found to have the structure represented by formula (I) (wherein X represents ethylene, Y represents a PEG chain, Z represents biotin, and L represents a spacer).
(2) SEC Analysis and Fractionation of Biotin-PEG-Fab
Biotin-PEG-Fab derived from HBs628Fab-Qtag was analyzed and fractionated.
(3) Analysis by Western Blotting (WB)
HBs628Fab-Qtag, SH-PEG(2K)-Fab thereof, and biotin-PEG-Fab were separated by non-reducing SDS-PAGE, and then analyzed by WB as follows. Fab (5 to 10 ng equivalent) was separated by SDS-PAGE (10 to 20% polyacrylamide gel), and then transferred to a PVDF membrane (Invitrogen) with use of iBlot2 dry blotting system (Thermo Fisher Scientific). The PVDF membrane after the transfer was blocked with 1% skim milk in TBST (10 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.4) at room temperature for one hour. The PVDF membrane after the blocking was washed with TBST (10 min, three times). HRP-conjugated streptavidin (Thermo Fisher Scientific) diluted 1:20,000 with 1% skim milk-containing TBST was added to the PVDF membrane, and the membrane was left still at room temperature for one hour for reaction. The PVDF membrane was then washed with TBST (10 min, three times). ECL Prime Western Blotting Detection Reagent (Cytiva) was added to the PVDF membrane, and the presence or absence of biotin labeling was detected on Amersham Imager 680 (Cytiva).
(4) LC-MS Analysis
SH-PEG-Fab derived from HBs628Fab-Qtag was analyzed by MS according to two types of method below. That is, non-reduced and reduced samples were analyzed. The samples were reduced by adding an excessive amount of 85 mM TCEP (tris(2-carboxyethyl) phosphine) to 10 μg of Fab, the mixture was left overnight at 5° C., and then subjected to LC-MS analysis. Analytical conditions were as follows.
(1) Maleimide Modification of ALP
ALP was modified with maleimide, aiming at use for ALP labeling of SH-PEG(2K)-Fab derived from each of csF001-5Fab-Qtag, csF028-22Fab-Qtag, and HBs628Fab-Qtag. ALP was dissolved in a 25 mM triethanolamine buffer (1 mM MgCl2, 0.1 mM ZnCl2, 150 mM NaCl, pH 7.0), so as to adjust the concentration to 10 μM. Twenty equivalents of EMCS reagent was then added to the ALP solution, and the mixture was incubated at 37° C. for one hour. The reaction solution was desalted through PD10 column (Cytiva), and then concentrated with Amicon 10K.
(2) ALP Labeling of SH-PEG(2K)-Fab
(2.1) Labeling of SH-PEG(2K)-Fab Derived from csF001-5Fab-Qtag
SH-PEG(2K)-Fab derived from csF001-5Fab-Qtag was labeled with ALP, as described below. Twenty micromoles of SH-PEG(2K)-Fab and 10 μM of maleimide-modified ALP were incubated in a 25 mM triethanolamine buffer (1 mM MgCl2, 0.1 mM ZnCl2, 150 mM NaCl, pH7.0) at 5° C. overnight. After analyzing the reaction solution by HPLC with use of Superdex 200 Increase (Cytiva), each of ALP-labeled Fab having Fab bound to one maleimide group owned by one ALP molecule (referred to as “Fab-PEG-ALP”, hereinafter); ALP-labeled Fab having Fab bound to each of two maleimide groups owned by one ALP molecule (referred to as “(Fab-PEG)2-ALP”, hereinafter); and ALP-labeled Fab having Fab bound to each of three maleimide groups owned by one ALP molecule (referred to as “(Fab-PEG)3-ALP”, hereinafter) were individually purified and fractionated. Note that Fab-PEG-ALP was a complex of one ALP molecule and one Fab molecule, (Fab-PEG)2-ALP was a complex of one ALP molecule and two Fab molecules, and (Fab-PEG)3-ALP was a complex of one ALP molecule and three Fab molecules. Each of Fab-PEG-ALP, (Fab-PEG)2-ALP, and (Fab-PEG)3-ALP was found to have the structure represented by formula (I) (wherein X represents ethylene, Y represents a PEG chain, Z represents ALP, and L represents a spacer).
(2.2) Labeling of SH-PEG(2K)-Fab Derived from csF028-22Fab-Qtag
SH-PEG(2K)-Fab derived from csF028-22Fab-Qtag was labeled with ALP, as described below. SH-PEG(2K)-Fab was concentrated with Amicon 10K (Merck), and the buffer was exchanged to a 25 mM triethanolamine buffer (1 mM MgCl2, 0.1 mM ZnCl2, 150 mM NaCl, pH 7.0). Twenty micromoles of the thus obtained reaction solution and 6.7 μM of maleimide-modified ALP were incubated in a 25 mM triethanolamine buffer (1 mM MgCl2, 0.1 mM ZnCl2, 150 mM NaCl, pH7.0) at 5° C. overnight. After analyzing the reaction solution by HPLC with use of Superdex 200 Increase (Cytiva), Fab-PEG-ALP, (Fab-PEG)2-ALP, and (Fab-PEG)3-ALP were individually purified and fractionated. Each ALP-labeled Fab was quantified according to the aforementioned quantification method for the target protein. BSA (Proliant) was added to the solution of each ALP-labeled Fab, so as to adjust the final concentration to 0.1%.
(2.3) Labeling of SH-PEG(2K)-Fab Derived from HBs628Fab-Qtag
SH-PEG(2K)-Fab derived from HBs628Fab-Qtag was labeled with ALP, as described below. SH-PEG(2K)-Fab and the maleimide-modified ALP (also referred to as “(Mal)n-ALP”, hereinafter) were dissolved according to the molar proportion listed in Table 1 into a 25 mM triethanolamine buffer (1 mM MgCl2, 0.1 mM ZnCl2, 150 mM NaCl, pH7.0), and incubated at 5° C. overnight. After analyzing the reaction solution by HPLC with use of Superdex 200 Increase (Cytiva), Fab-PEG-ALP, (Fab-PEG)2-ALP, and (Fab-PEG)3-ALP were individually purified and fractionated. Note that (Mal)n-ALP represents one ALP molecule to which n (n is an integer of 1 or larger) maleimide groups are added.
(3) SEC Analysis and Fractionation of Fab-PEG-ALP, (Fab-PEG)2-ALP and (Fab-PEG)3-ALP
(3.1) Analytical Conditions for ALP-PEG-Fab Derived from SH-csF001-5Fab-Qtag, and HBs628Fab-Qtag
(3.2) Conditions for Analysis and Fractionation of Fab-PEG-ALP, (Fab-PEG)2-ALP and (Fab-PEG)3-ALP
(1) Maleimide Modification of ALP
Intended to be used for ALP labeling of Fab′ free of Q-tag, ALP was modified with maleimide as follows. ALP was dissolved in D-PBS(−) (pH 7.4), so as to adjust the concentration to 20 μM. Thirty equivalents of EMCS reagent was then added to the ALP solution, and the mixture was incubated at 37° C. for one hour. The reaction solution was desalted through PD10 column (Cytiva), and the buffer was exchanged to a 0.1 M triethanolamine buffer (1 mM MgCl2, 0.1 mM ZnCl2, 150 mM NaCl, pH 7.0). The solution was then concentrated with Amicon 50K.
(2) Preparation of F(Ab′)2 from Full-Length Antibody, and SEC Fractionation
sF001-5 and pepsin (Sigma) were dissolved into McIlvaine buffer (pH 3.8) so as to adjust the concentrations to 6.67 μM and 0.95 μM, respectively, and the mixture was incubated at 37° C. for 3 hours. Then 10 v % of 1 M Tris-HCl (pH 8.5) was added for neutralization, thereby terminating the reaction. The product was concentrated with Amicon 10K, and F(ab′)2 was then purified and fractionated through Superdex 200 Increase (Cytiva). The thus obtained F(ab′)2 solution was again concentrated with use of Amicon 10K. Note that the reactivity of the antibody, per unit amount of substance, was not found to decline, even after thus processed. Conditions for fractionation were as follows.
(3) Preparation of Fab′ from F(Ab′)2, and SEC Fractionation
F(ab′)2 thus prepared in (2) above was dissolved in a 0.1 M phosphate buffer (1 mM EDTA 2Na, pH 6.0), so as to adjust the concentration to 20 μM. To the F(ab′)2 solution, 1500 equivalents of 2-mercaptoethylamine reagent (Nacalai Tesque, Inc.) was added, and the mixture was incubated at 37° C. for 90 minutes. Only a fraction that contains the produced sF001-5Fab′ was fractionated and purified through Superdex 200 Increase (Cytiva), and concentrated with Amicon 10K. Conditions for fractionation were as follows.
(4) ALP Labeling of Fab′ (Random Labeling)
Fab′ was labeled with ALP, by coupling of the cysteine residue in Fab′, with maleimide-modified ALP. Operational details were as follows. Forty-four micromoles of sF001-5Fab′ and 8.8 μM of maleimide-modified ALP were incubated in a 0.1 M triethanolamine buffer (1 mM MgCl2, 0.1 mM ZnCl2, 150 mM NaCl, pH7.0) at 5° C. overnight. The thus produced coupled product (referred to as “(Fab′)n-ALP”, hereinafter) was then fractionated and purified through Superdex 200 Increase (Cytiva). Conditions for fractionation were as follows. Note that (Fab′)n-ALP represents a complex in which Fab′ is bound to each of n (n is an integer of 1 or more) maleimide groups owned by ALP.
The sample that contains purified (Fab′)-ALP was analyzed by non-reducing SDS-PAGE and SEC. Analytical conditions for SEC were as follows.
(1) Coupling of SH-PEG-Fab with Alexa 488-Maleimide
SH-PEG(2K)-Fab derived from csF001-25Fab-Qtag was labeled with a fluorescent dye, as described below. SH-PEG(2K)-Fab was concentrated with Amicon 10K (Merck), and the buffer was exchanged to a 50 mM sodium phosphate buffer (2 mM EDTA, pH 7.0). Six micromoles of reaction solution and 120 μM of Alexa 488-maleimide were incubated in the 50 mM sodium phosphate buffer (2 mM EDTA, pH 7.0) overnight at 5° C. The Alexa 488-labeled Fab (Alexa 488-PEG-Fab) in the reaction solution was fractionated and analyzed by HPLC with use of Superdex 200 Increase (Cytiva). The reaction product of SH-PEG-Fab and Alexa 488-maleimide will also be referred to as “Alexa 488-PEG-Fab”, hereinafter. The Alexa 488-PEG-Fab was found to have the structure represented by formula (I) (wherein X represents ethylene, Y represents a PEG chain, Z represents Alexa 488, and L represents a spacer).
(2) Conditions for SEC Fractionation
(3) Analytical Conditions for SEC
(4) LC-MS Analysis
SH-PEG(2K)-Fab derived from csF001-25Fab-Qtag, and Alexa 488-PEG-Fab were analyzed by LC-MS as described below. The samples were measured by LC-MS, after reduced with 200 equivalents of TCEP in 1 M Tris (pH 7.5) at 37° C. for 2 hours. Analytical conditions were as follows.
(1) Maleimide Modification of R-PE
R-PE was modified with maleimide, aiming at use for R-PE labeling of SH-PEG(2K)-Fab derived from each of csF001-25Fab-Qtag and anti-CD20Fab-Qtag. R-PE was dialyzed against a 50 mM sodium phosphate buffer (2 mM EDTA, pH 7.0) to replace the buffer. Then, 400 μM of EMCS reagent was added to the 5 μM R-PE solution, and the mixture was incubated at 37° C. for one hour. The reaction solution was desalted through PD10 column (Cytiva), and then concentrated with Amicon 3K. The thus obtained maleimide-modified R-PE is also referred to as “(Mal)n-R-PE”, hereinafter. (Mal)n-R-PE represents one R-PE molecule to which n (n is an integer of 1 or larger) maleimide groups are added.
(2) Coupling of SH-PEG-Fab with Maleimide-Modified R-PE
SH-PEG(2K)-Fab derived from each of csF001-25Fab-Qtag and anti-CD20Fab-Qtag was labeled with a fluorescent dye, as described below. SH-PEG(2K)-Fab was concentrated with Amicon 10K (Merck), and the buffer was exchanged to a 50 mM sodium phosphate buffer (2 mM EDTA, pH 7.0). Six micromole of the reaction solution and 2 μM of (Mal)n-R-PE were incubated in a 50 mM sodium phosphate buffer (2 mM EDTA, pH 7.0) overnight at 5° C. L-cysteine was added to the reaction solution to adjust the final concentration to 0.1 mM, so as to cap the unreacted maleimide groups. The reaction solution was then analyzed by HPLC with Superdex 200 Increase (Cytiva). R-PE-labeled Fab having Fab bound to one maleimide group owned by one R-PE molecule (referred to as “Fab-PEG-R-PE”, hereinafter); R-PE-labeled Fab having Fab bound to each of two maleimide groups owned by one R-PE molecule (referred to as “(Fab-PEG)2-R-PE”, hereinafter); and R-PE-labeled Fab having Fab bound to each of three maleimide groups owned by one R-PE molecule (referred to as “(Fab-PEG)3-R-PE”, hereinafter) were individually purified and fractionated. These fractions were also analyzed by SEC. Conditions for the fractionation and analysis were as follows. Note that Fab-PEG-R-PE was a complex of one R-PE molecule and one Fab molecule, (Fab-PEG)2-R-PE was a complex of one R-PE molecule and two Fab molecules, and (Fab-PEG)3-R-PE was a complex of one R-PE molecule and three Fab molecules. Each of Fab-PEG-R-PE, (Fab-PEG)2-R-PE, and (Fab-PEG)3-R-PE was found to have the structure represented by formula (I) (wherein X represents ethylene, Y represents a PEG chain, Z represents R-PE, and L represents a spacer).
(3) Conditions for SEC Fractionation
(4) Analytical Conditions for SEC
ALP activity of Fab-PEG-ALP, (Fab-PEG)2-ALP and (Fab-PEG)3-ALP derived from csF028-22Fab-Qtag prepared in Test Example 3 were measured as follows. The ALP activity of the calibrator (ALP-55 (Oriental Yeast Co., Ltd.) whose activity value was assigned with use of biochemistry automatic analyzer; Hitachi 7170), and the individual ALP-labeled antibody samples whose concentrations were adjusted within a range of analytical curve, were measured, while using CDP-star (Thermo Fisher Scientific) as a substrate. The ALP activity of the individual ALP-labeled antibodies was calculated by multiplying the measured ALP activity of the individual measurement samples by the dilution rate. Specific activities relative to unmodified ALP were compared.
HIV p24 antigen was assayed by immunological assay that individually uses, as the detection antibodies, Fab-PEG-ALP derived from csF001-5 Fab-Qtag prepared in Test Example 3, and (Fab′)n-ALP prepared in Test Example 4. Measured results were compared to examine the performance of the reagents that individually contain Fab-PEG-ALP and (Fab′)n-ALP. The measurement employed a fully automated immunoassay system HISCL (registered trademark) 2000i (Sysmex Corporation).
(1) Preparation of Reagents
Reagents used here were R1 reagent (biotin-labeled antibody), R2 reagent (streptavidin-immobilized magnetic particle), R4 reagent (chemiluminescent substrate diluent), and R5 reagent (chemiluminescent substrate), all contained in HIV-1 p24 antigen/HIV antibody kit “HISCL (registered trademark) HIV Ag+Ab Reagent” (Sysmex Corporation); or reagents prepared by a method according to the method for producing these reagents, while excluding R3 reagent which is a detection antibody reagent. The R3 reagent was prepared as follows, with use of Fab-PEG-ALP or (Fab′)n-ALP. Each of Fab-PEG-ALP and (Fab′)n-ALP was diluted with a 0.1 M triethanolamine buffer (3% BSA, 0.5% sodium caseinate, 1 mM MgCl2, 0.1 mM ZnCl2, 150 mM NaCl, pH 6.5), and the concentration was adjusted to the level summarized in Table 2. Each antibody solution was filtered through Millex-GS 0.22 μm (Merck) to obtain R3 reagent. The ALP activity of each antibody was estimated in the same way as described in Test Example 7.
(2) Preparation of Samples
The HIV p24 antigen (Abcam plc) was diluted with each of D-PBS (0.1% BSA, pH 7.4) and human pool serum (Nissui Pharmaceutical Co., Ltd.), to prepare samples with antigen concentrations of 10 pg/mL, 100 pg/mL and 1000 pg/mL. Also D-PBS (0.1% BSA, pH 7.4) and human pooled serum per se were used as antigen-free (0 pg/mL) samples.
(3) Assay
The R1 to R5 reagents described in (1) above were set on HISCL-2000i (Sysmex Corporation), and eight types of sample prepared in (2) above were assayed. Measurement procedures with use of HISCL-2000i were as follows. The sample (20 μL) and R1 reagent (50 μL) were mixed, and reagent R2 (30 μL) was then added. The magnetic particle in the resultant mixed liquid was magnetically collected to remove the supernatant, and the magnetic particle was washed by adding HISCL washing solution (300 μL). The supernatant was removed, and R3 reagent (100 μL) was added to the magnetic particle, followed by mixing. The magnetic particle in the resultant mixed liquid was magnetically collected to remove the supernatant, and the magnetic particle was washed by adding HISCL washing solution (300 μL). The supernatant was removed, R4 reagent (50 μL) and R5 reagent (100 μL) were added to the magnetic particle, and chemiluminescence intensity was then measured.
Antigen binding capacity of SH-PEG(2K)-Fab derived from csF001-25Fab-Qtag, and Alexa 488-PEG-Fab, prepared in Test Example 5, was measured by surface plasmon resonance (SPR) analysis with use of Biacore (registered trademark) T200 (Cytiva). For comparison, also csF001-25 Fab-Qtag was measured. Operational details were as follows. Human Fab Binder attached to Human Fab Capture Kit (Cytiva) was immobilized on a sensor chip CM5 (Cytiva), by amine coupling. SH-PEG(2K)-Fab, Alexa 488 PEG-Fab, and unlabeled Fab (csF001-25Fab-Qtag) were individually bound as ligands. Recombinant protein HIV-1 p24 (Prospec) was reacted as an analyte. The interaction was analyzed according to a 1:1 binding reaction model, with use of Biacore T 200 Evaluation software. Apparatus, reagents and reaction conditions employed here were as follows.
Antigen binding capacity of Fab-PEG-R-PE, (Fab-PEG)2-R-PE, and (Fab-PEG)3-R-PE derived from csF001-25Fab-Qtag prepared in Test Example 6 was measured by ELISA, as follows. Each of Fab-PEG-R-PE, (Fab-PEG)2-R-PE, and (Fab-PEG)3-R-PE was diluted so as to give equivalent fluorescence intensity of R-PE, to prepare each detection reagent. His-tagged recombinant HIV-1 p24, used as an antigen (Prospec), was inoculated in a black 96-well microplate having anti-His tag antibody immobilized thereon, and left for reaction at room temperature for one hour. Also anti-His tag antibody-immobilized microplate was prepared for comparison, without adding the antigen. Each well was then washed with a washing solution (0.05% Tween 20 in saline). Each detection reagent was added to each well of the microplate, and left for reaction at room temperature for one hour. After washing each well with the washing solution, the R-PE-labeled antibody in the microplate well was detected with a fluorescence plate reader (excitation wavelength: 488 nm, fluorescence wavelength: 578 nm).
CD20-expressing cell was measured by flow cytometry (FCM), while using each of Fab-PEG-R-PE, (Fab-PEG)2-R-PE, and (Fab-PEG)3-R-PE derived from anti-CD20 Fab-Qtag prepared in Test Example 6, as a detection antibody. Measurement results were compared to examine performances of the reagents that contain the individual R-PE-labeled antibodies. Specific procedures for measurement were as follows. Each of Fab-PEG-R-PE, (Fab-PEG)2-R-PE, and (Fab-PEG)3-R-PE was diluted with a diluting buffer (2% FBS, 2 mM EDTA/D-PBS (pH 7.4)), so as to give equivalent fluorescence intensity of R-PE, to prepare each detection reagent. CD20-expressing Ramos cell was suspended in each detection reagent, and left for reaction at 4° C. for 30 minutes. The cell was pelletized by centrifugation, and then washed with a dilution buffer. Each R-PE-labeled antibody bound to CD20 on the surface of Ramos cell was detected with BD Accuri (registered trademark) C6 Plus flow cytometer (Becton, Dickinson and Company).
(1) Coupling of SH-PEG-NH2 Linker (3.5K) and SH-PEG-NH2 Linker (5K) with Fab Antibody
Each of (d) SH-PEG-NH2 linker (3.5K) (Sigma-Aldrich), and (e) SH-PEG-NH2 linker (5K) (Sigma-Aldrich) was bound to csF001-25Fab-Qtag, while catalyzed by BTGase. Operational details were as follows. csF001-25Fab-Qtag was dissolved in Tris buffer (20 mM Tris, 2 mM EDTA, 150 mM NaCl, pH 8.2) so as to adjust the concentration to 10 μM, and the solution was incubated with 100 equivalents of SH-PEG-NH2 linker (3.5K) or SH-PEG-NH2 linker (5K), and with 0.1 U/mL of BTGase at 5° C. overnight. Activity of BTGase was assayed by the hydroxamate method. Progress of the reaction was monitored by SEC. The reaction solutions of the individual linkers were desalted and purified through Protein G column, and then concentrated with Amicon 10K (Merck). Analytical conditions for SEC were as follows.
(2) Analytical Conditions for SEC
(1) Fluorescence Labeling of SH-PEG-Fab
SH-PEG(3.5K)-Fab and SH-PEG(5K)-Fab derived from csF001-25Fab-Qtag were labeled with a fluorescent dye, as described below. Each SH-PEG-Fab was concentrated with Amicon 10K (Merck), and the buffer was exchanged to a 25 mM triethanolamine buffer (1 mM MgCl2, 0.1 mM ZnCl2, 150 mM NaCl, pH 7.0). Six micromoles of reaction solution and 120 μM of Alexa 488-maleimide were incubated in a 25 mM triethanolamine buffer (1 mM MgCl2, 0.1 mM ZnCl2, 150 mM NaCl, pH7.0) at 5° C. overnight. Alexa 488-PEG-Fab in the reaction solution was assayed by HPLC with use of Superdex 200 Increase (Cytiva). Analytical conditions for SEC were as follows.
(2) Analytical Conditions for SEC
(1) Coupling of SH-PEG-NH2 Linker (400 Da) with Fab Antibody
The aforementioned (f) SH-PEG-NH2 linker (400 Da) (Nanocs Inc.) was bound to csF001-5Fab-Qtag, while catalyzed by BTGase. Operational details were as follows. csF001-5Fab-Qtag was dissolved in Tris buffer (20 mM Tris, 2 mM EDTA, 150 mM NaCl, pH 8.2) so as to adjust the concentration to 10 μM, and the solution was incubated with 50 equivalents of SH-PEG-NH2 linker (400 Da) and 0.1 U/mL of BTGase, at 5° C. overnight. Activity of BTGase was assayed by the hydroxamate method. The reaction solution was desalted and purified through Superdex 200 Increase (Cytiva), and concentrated with Amicon 10K (Merck).
(2) ALP Labeling of SH-PEG (400 Da)-Fab
Ten micromoles of SH-PEG(400 Da)-Fab and 5 μM of maleimide-modified ALP were incubated in a 25 mM triethanolamine buffer (1 mM MgCl2, 0.1 mM ZnCl2, 150 mM NaCl, pH7.0) at 5° C. overnight. The reaction solution was analyzed by HPLC with Superdex 200 Increase (Cytiva). Analytical conditions for SEC were as follows.
(3) Analytical Conditions for SEC
(1) Coupling of SH-PEG-NH2 Linker (1K) with Fab Antibody
The aforementioned (g) SH-PEG-NH2 linker (1K) (Creative PEGWorks) was bound to HBs628Fab-Qtag, while catalyzed by BTGase. Operational details were as follows. HBs628Fab-Qtag was dissolved in MES buffer solution (50 mM MES, 2 mM EDTA, pH 7.0) so as to adjust the concentration to 10 μM, and the solution was incubated with 50 equivalents of SH-PEG-NH2 linker (1K) and 0.1 U/mL of BTGase at 25° C. for 3 hours. Progress of the reaction was monitored by SEC. Activity of BTGase was assayed by the hydroxamate method. The reaction solution was desalted, concentrated, and purified with Amicon 10K (Merck).
(2) ALP Labeling of SH-PEG(1K)-Fab
Ten micromoles of SH-PEG(1K)-Fab and 5 μM of maleimide-modified ALP were incubated in a 25 mM triethanolamine buffer (1 mM MgCl2, 0.1 mM ZnCl2, 150 mM NaCl, pH7.0) at 5° C. overnight. The reaction solution was analyzed by HPLC with Superdex 200 Increase (Cytiva). Analytical conditions for SEC were as follows.
(3) Analytical Conditions for SEC
Results
Results of the above tests will be described below.
1. Analytical Results of SH-PEG-NH2 Linker (2K) by Infusion MS
Analytical results of the individual SH-PEG-NH2 linkers (2K) (a), (a′), (b) and (c) obtained by Infusion MS were shown in FIGS. 2A, 2B, 2C and 2D. In
In all linkers, the PEG chain moiety, exclusive of the functional groups (SH—CH2CH2— and —NH2) was found to have a molecular weight of 1100 or larger. The weight-average molecular weight of the PEG chain moiety, subtracted the weight of the functional groups, was found to be 1300 or larger. The SH-PEG-NH2 linker (2K) after adding the BTGase reaction solution (no NaCl added, pH 8.2) was not found to dimerize while interposed by a disulfide (S—S) bond, and only the MS spectrum of the monomer, correlated with the molecular weight distribution of the PEG chain, was detected. The results demonstrated that none of the SH-PEG-NH2 linkers (2K) (a), (a′), (b) and (c) caused dimerization in the BTGase reaction solution.
2. BTGase-Catalyzed Modification of csF001-5Fab-Qtag with SH-PEG-NH2 Linker (2K) (Test Example 1)
SEC analytical results of a BTGase-catalyzed reaction product formed between csF001-5Fab-Qtag and the (a) SH-PEG-NH2 linker (2K) (Sigma-Aldrich) were shown in
3. BTGase-Catalyzed Modification of csF028-22Fab-Qtag with SH-PEG-NH2 Linker (2K) (Test Example 1)
An SEC analytical result of a product of the BTGase-catalyzed reaction between csF028-22Fab-Qtag and the aforementioned (a) SH-PEG-NH2 linker (2K) (Sigma-Aldrich), under conditions aimed at enhanced introduction efficiency (pH 8.2 or 8.5), was shown in
4. BTGase-Catalyzed Modification of csF001-5Fab-Qtag with SH-PEG-NH2 Linker (3.5K) (Test Example 12)
An SEC analytical result of a product of the BTGase-catalyzed reaction between csF001-5Fab-Qtag and the aforementioned (d) SH-PEG-NH2 linker (3.5K) (Sigma-Aldrich), under conditions aimed at enhanced introduction efficiency, was shown in
5. ALP Labeling of SH-PEG-Fab Derived from csF001-5Fab-Qtag (Test Example 3)
(1) SEC Analysis of Coupling Reaction Solution
Analytical results of SH-PEG(2K)-Fab derived from csF001-5Fab-Qtag, maleimide-modified ALP ((Mal)1-ALP), and a coupling reaction solution of them, analyzed by SEC and non-reducing SDS-PAGE were shown in
(2) SEC Fractionation of Coupling Reaction Peaks, and SEC Analysis of Preparative Fractions
A coupling reaction solution that contains SH-PEG(2K)-Fab derived from csF001-5Fab-Qtag, and the maleimide-modified ALP was fractioned by SEC, and the preparative fractions were analyzed by non-reducing SDS-PAGE and SEC. Results were shown in
6. ALP Labeling of SH-PEG-Fab Derived from csF028-22Fab-Qtag (Test Example 3)
(1) SEC Analysis of Coupling Reaction Solution
Analytical results of SH-PEG(2K)-Fab derived from csF028-22Fab-Qtag, maleimide-modified ALP ((Mal)n-ALP), and a coupling reaction solution of them, analyzed by SEC were shown in
(2) SEC Fractionation of Coupling Reaction Peaks, and SEC Analysis of Preparative Fractions
A coupling reaction solution that contains SH-PEG(2K)-Fab derived from csF028-22Fab-Qtag, and the maleimide-modified ALP ((Mal)n-ALP) was fractioned by SEC, and the preparative fractions were analyzed by non-reducing SDS-PAGE and SEC. Results were shown in
7. Coupling Reaction Between sF001-5 Fab′ and Maleimide-Modified ALP (Test Example 4)
A chromatogram obtained in SEC fractionation of the coupling reaction solution that contains sF001-5 Fab′ and (Mal)n-ALP, and analytical results of the preparative individual fractions analyzed by non-reducing SDS-PAGE were shown in
Although ALP normally exists as a 150-kDa dimer having two 75-kDa molecules combined therein, SDS-PAGE now demonstrated ALP as a monomer, with a band appeared at around 70 kDa, as shown in
8. Measurement of ALP Activity of ALP-Labeled Antibody (Test Example 7)
Specific ALP activity of ALP molecule per se; and Fab-PEG-ALP, (Fab-PEG)2-ALP and (Fab-PEG)3-ALP derived from csF028-22Fab-Qtag, were summarized in Table 3. As summarized in Table 3, the individual ASP-labeled antibodies did not decrease the specific activity even with increase in the number of binding of (Fab-PEG)s to the ALP molecule. The preparation method of this embodiment was thus proved to prepare the ALP-labeled polypeptide with the ALP activity well maintained. The specific activities exceeding 100% were presumably ascribed to influences of purity of the fractionated samples.
9. Performance Evaluation of Reagents that Contain Fab-PEG-ALP and (Fab′)n-ALP (Test Example 8)
(1) Reactivity of R3 Reagent
Samples in D-PBS (0.1% BSA, pH 7.4) were analyzed with use of each of Fab-PEG-ALP from csF001-5Fab-Qtag and (Fab′)n-ALP obtained by random labeling as R3 reagent, and the analytical results were shown in
(2) Suppression of Increase of Background Due to Serum-Derived Component
For comparison of the background, D-PBS (0.10% BSA, pH 7.4) and human pool serum were analyzed with use of each of Fab-PEG-ALP derived from csF001-5Fab-Qtag, and (Fab′)n-ALP as R3 reagent, and the analytical results were shown in
10. Preparation of SH-PEG-Fab Derived from HBs628Fab-Qtag, and Biotin Labeling Thereof (Test Examples 1 and 2)
(1) BTGase-Catalyzed Modification of HBs628Fab-Qtag with SH-PEG-NH2 Linker (2K)
SEC analytical results of a BTGase-catalyzed reaction product formed between HBs628Fab-Qtag and the (a) SH-PEG-NH2 linker (2K) (Sigma-Aldrich) were shown in
(2) Biotin Labeling of SH-PEG-Fab Derived from HBs628Fab-Qtag
Analytical results of fractions of a reaction solution that contains csF001-5Fab-Qtag and the (a) SH-PEG-NH2 linker (2K) (Sigma-Aldrich), collected from a peak at a retention time of 11.5 minutes in SEC analysis; and, analytical results of a reaction solution that contains SH-PEG-Fab-containing concentrated solution and Biotin-PEAC5-maleimide fractioned from a peak in SEC analysis, were shown in
In fact, even if Fab was kept with the SH-PEG-NH2 linker (2K) in a reaction solution in the absence of BTGase, the Fab did not cause reduction of the S—S bond between the H chain and the L chain (data not shown). As seen in
Referring now to
11. LC-MS Analysis of SH-PEG Fab Derived from HBs628Fab-Qtag, and Biotin-PEG-Fab (Test Example 2)
(1) Results of LC-MS Analysis of HBs628Fab-Qtag
LC-MS spectra of non-reduced and reduced samples of HBs628Fab-Qtag were shown in
(2) Results of LC-MS Analysis of SH-PEG Fab and Biotin-PEG-Fab
LC-MS spectra of HBs628Fab-Qtag, SH-PEG-Fab thereof, and biotin-PEG-Fab were shown in
LC-MS spectra of reduced samples of HBs628Fab-Qtag and SH-PEG-Fab thereof were shown in
12. ALP Labeling of SH-PEG-Fab Derived from HBs628Fab-Qtag (Test Example 3)
A coupling reaction solution that contains SH-PEG(2K)-Fab derived from HBs628Fab-Qtag, and the maleimide-modified ALP ((Mal)n-ALP) was fractioned by SEC, and the preparative fractions were analyzed by SEC. Results were shown in
13. Preparation of SH-PEG-Fab Derived from csF001-25Fab-Qtag, and Fluorescence Labeling Thereof (Test Examples 1 and 5)
(1) BTGase-Catalyzed Modification of csF001-25Fab-Qtag with SH-PEG-NH2 Linker (2K)
An SEC analytical result of a reaction solution, desalted and concentrated after the BTGase-catalyzed reaction between csF001-25Fab-Qtag and the aforementioned (a) SH-PEG-NH2 linker (2K) (Sigma-Aldrich), under conditions aimed at enhanced introduction efficiency, was shown in
(2) Fluorescence Labeling of SH-PEG-Fab with Alexa 488-Maleimide
Results of SEC analysis of a reaction solution that contains SH-PEG(2K)-Fab derived from csF001-25Fab-Qtag, and Alexa 488-maleimide were shown in
14. LC-MS Analysis of SH-PEG Fab Derived from csF001-25Fab-Qtag, and Alexa488-PEG-Fab (Test Example 5)
LC-MS spectra of reduced samples of csF001-25Fab-Qtag, SH-PEG-Fab thereof, and Alexa 488-PEG-Fab were shown in
15. BTGase-Catalyzed Modification of Anti-CD20 Fab-Qtag with SH-PEG-NH2 Linker (2K) (Test Example 1)
An SEC analytical result of a reaction solution, desalted and concentrated after the BTGase-catalyzed reaction between anti-CD20 Fab-Qtag and the aforementioned (a′) SH-PEG-NH2 linker (2K) (Sigma-Aldrich), under conditions aimed at enhanced introduction efficiency, was shown in
16. R-PE Labeling of SH-PEG-Fab (Test Example 6)
(1) Maleimide Modification of R-PE
Analytical results of a reaction solution that contains R-PE and EMCS reagent, analyzed by SEC and reverse-phase HPLC were shown in
(2) Coupling Reaction of SH-PEG-Fab, Derived from csF001-25Fab-Qtag, and Maleimide-Modified R-PE
Analytical results of SH-PEG(2K)-Fab derived from sF001-25Fab-Qtag, (Mal)1-R-PE, and a coupling reaction solution of them, analyzed by SEC were shown in
(3) SEC Fractionation of Coupling Reaction Peaks, and SEC Analysis of Preparative Fraction
A coupling reaction solution that contains SH-PEG(2K)-Fab derived from sF001-25Fab-Qtag, and the maleimide-modified R-PE was fractioned by SEC, and the preparative fractions were analyzed by SEC. Results were shown in
(4) Coupling Reaction of SH-PEG-Fab Derived from Anti-CD20 Fab-Qtag, with Maleimide-Modified R-PE
Analytical results of SH-PEG(2K)-Fab derived from anti-CD20 Fab-Qtag, (Mal)n-R-PE, and a coupling reaction solution of them, analyzed by SEC were shown in
(5) SEC Fractionation of Coupling Reaction Peaks, and SEC Analysis of Preparative Fraction
A reaction solution of coupling of SH-PEG(2K)-Fab derived from anti-CD20 Fab-Qtag with the maleimide-modified R-PE was fractioned by SEC, and the preparative fractions were analyzed by SEC. Results were shown in
17. Evaluation of Antigen Binding Capacity of Alexa 488-Labeled Antibody (Test Example 9)
Interaction with antigen of csF001-25Fab-Qtag, SH-PEG(2K)-Fab thereof, and Alexa 488-PEG-Fab was measured with use of Biacore (registered trademark) T200 (Cytiva). Results were summarized and shown in Table 4 and
18. Antigen-Binding Capacity of R-PE-Labeled Antibody (Test Example 10)
Antigen binding capacity of Fab-PEG-R-PE, (Fab-PEG)2-R-PE, and (Fab-PEG)3-R-PE derived from csF001-25Fab-Qtag was measured by ELISA. Results were shown in
19. Performance Evaluation of Reagent that Contains R-PE-Labeled Antibody (Test Example 11)
CD20-expressing cell (Ramos cell) was measured by FCM, with use of Fab-PEG-R-PE, (Fab-PEG)2-R-PE, and (Fab-PEG)3-R-PE derived from anti-CD20 Fab-Qtag, individually as the detection antibody. Results were shown in
20. Results of Infusion MS of SH-PEG-NH2 Linker (3.5K) and SH-PEG-NH2 Linker (5K)
The aforementioned (d) SH-PEG-NH2 linker and (e) SH-PEG-NH2 linker (Sigma-Aldrich) were analyzed by Infusion MS, and results were shown in
Each SH-PEG-NH2 linker, after addition of the BTGase reaction solution (no NaCl added, pH 8.2), was confirmed to cause almost no dimerization by a disulfide (S—S) bond, while only demonstrating an MS spectrum of the monomer correlated to the molecular weight distribution of the PEG chain. Hence, the results demonstrated that none of the (d) SH-PEG-NH2 linker and (e) SH-PEG-NH2 linker caused dimerization.
21. Fluorescence Labeling of SH-PEG-Fab with Alexa 488-Maleimide (Test Examples 12 and 13)
(1) BTGase-Catalyzed Modification of csF001-5Fab-Qtag with SH-PEG-NH2 Linker (3.5K/5K)
Analytical results of SEC and non-reducing SDS-PAGE of reaction solutions, desalted and concentrated after the BTGase-catalyzed reaction of csF001-25Fab-Qtag, with each of the aforementioned (d) SH-PEG-NH2 linker (3.5K) (Sigma-Aldrich) and the aforementioned (e) SH-PEG-NH2 linker (5K) (Sigma-Aldrich), were shown in
(2) Fluorescence Labeling of SH-PEG-Fab with Alexa 488-Maleimide
Results of SEC analysis of a reaction solution that contains SH-PEG(3.5K)-Fab derived from csF001-25Fab-Qtag, and Alexa 488-maleimide were shown in
As seen in
As seen in
22. Modification of Fab Antibody with SH-PEG-NH2 Linker (400 Da) (Test Example 14)
(1) BTGase-Catalyzed Modification of csF001-5Fab-Qtag with SH-PEG-NH2 Linker (400 Da)
Analytical results of BTGase-catalyzed reaction solution that contains csF001-5Fab-Qtag and the aforementioned (f) SH-PEG-NH2 linker (400 Da)(Nanocs Inc.), analyzed (fractionated) by SEC, were shown in
(2) ALP Labeling of SH-PEG (400 Da)-Fab
Analytical results of SH-PEG(400 Da)-Fab derived from csF001-5Fab-Qtag, maleimide-modified ALP ((Mal)1-ALP), and a coupling reaction solution of them, analyzed by SEC were shown in
23. Modification of Fab Antibody with SH-PEG-NH2 Linker (1K) (Test Example 15)
(1) BTGase-Catalyzed Modification of HBs628Fab-Qtag with SH-PEG-NH2 Linker (1K)
SEC analytical results of BTGase-catalyzed reaction solution that contains HBs628Fab-Qtag and the aforementioned (g) SH-PEG-NH2 linker (1K) (Creative PEGWorks) were shown in
(2) ALP Labeling of SH-PEG(1K)-Fab
Analytical results of SH-PEG(1K)-Fab derived from HBs628Fab-Qtag, maleimide-modified ALP ((Mal)n-ALP), and a coupling reaction solution of them, analyzed by SEC were shown in
These experimental results proved that the present invention excels in the efficiency of introducing the label into polypeptide, as compared with the case of using the SH-PEG-NH2 linker (400 Da) or the SH-PEG-NH2 linker (1K).
| Number | Date | Country | Kind |
|---|---|---|---|
| 2022-099159 | Jun 2022 | JP | national |
| 2023-012581 | Jan 2023 | JP | national |
