Increased polypeptide production yields of butyrylcholinesterase polypeptides for therapeutic use
11473070 · 2022-10-18
Assignee
Inventors
Cpc classification
C12Y301/01008
CHEMISTRY; METALLURGY
C07K2319/30
CHEMISTRY; METALLURGY
International classification
Abstract
The presently-disclosed subject matter describes fusion proteins comprising butyrylcholinesterase (BChE) having an improved production yield and biological half-life and nucleotides encoding the same.
Claims
1. A fusion polypeptide, comprising: an Fc polypeptide joined to an N- or C-terminal end of a butyrylcholinesterase (BChE) polypeptide, wherein (a) the Fc polypeptide is joined to the BChE polypeptide via a linker, the linker comprising a sequence selected from the sequences of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 19, and SEQ ID NO: 37; (b) the Fc polypeptide has the sequence of SEQ ID NO: 8, or a fragment thereof, wherein the Fc polypeptide or fragment thereof includes 3 to 8 amino acid substitutions at 3 to 8 of residues selected from 1, 6, 12, 15, 24, 38, 40, 42, 58, 69, 80, 98, 101, 142, and 144; and (c) the fusion polypeptide has an improved production yield and biological half-life compared to a wild-type BChE polypeptide which is not fused.
2. The fusion polypeptide of claim 1, wherein the Fc polypeptide is a fragment wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids are removed from the N-terminus of SEQ ID NO: 8.
3. The fusion polypeptide of claim 1, wherein the Fc polypeptide is selected from SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34.
4. The fusion polypeptide of claim 1, wherein the BChE polypeptide has the sequence of SEQ ID NO: 10 or a fragment thereof, wherein the BChE polypeptide or fragment thereof includes 3 to 8 amino acid substitutions at 3 to 8 of residues chosen from 199, 227, 285, 286, 287, 328, 332, and 441.
5. The fusion polypeptide of claim 4, wherein the BChE polypeptide has a group of amino acid substitutions selected from A199S, F227A, F227S, F227Q, F227I, F227G, F227V, F227I, F227L, F227S, F227T, F227M, F227C, P285A, P285S, P285Q, P285I, P285G, P285M, P285N, P285E, S287G, A328W, Y332G, E441D, and combinations thereof.
6. The fusion polypeptide of claim 5, wherein the BChE polypeptide is a fragment wherein from 1 to 116 amino acids are removed from the N-terminus of SEQ ID NO: 10.
7. The fusion polypeptide of claim 5, wherein the BChE polypeptide is a fragment wherein from 1 to 432 amino acids are removed from the C-terminus of SEQ ID NO: 10.
8. The fusion polypeptide of claim 7, wherein the BChE polypeptide has a group of amino acid substitutions selected from A199S, F227A, F227S, F227Q, F227I, F227G, F227V, F227I, F227L, F227S, F227T, F227M, F227C, P285A, P285S, P285Q, P285I, P285G, P285M, P285N, P285E, S287G, A328W, Y332G, E441D, and combinations thereof.
9. The fusion polypeptide of claim 1, wherein the transient expression level of the polypeptide is at least 9 times higher than a reference BChE polypeptide that does not include the Fc polypeptide and linker.
10. The fusion polypeptide of claim 1 of SEQ ID NO: 35.
11. The fusion polypeptide of claim 1, wherein the BChE polypeptide is selected from: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15.
12. The fusion polypeptide of claim 11, wherein the Fc polypeptide is selected from SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18.
13. The fusion polypeptide of claim 1, wherein the transient expression level of the polypeptide is about 9 times higher than a reference BChE polypeptide that does not include the Fc polypeptide and linker.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The presently-disclosed subject matter will be better understood, and features, aspects and advantages other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such detailed description makes reference to the following drawings, wherein:
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(7) While the disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and are herein described below in detail. It should be understood, however, that the description of specific embodiments is not intended to limit the disclosure to cover all modifications, equivalents and alternatives falling within the spirit and scope of the disclosure as defined by the appended claims.
DESCRIPTION OF EXEMPLARY EMBODIMENTS
(8) The details of one or more embodiments of the presently-disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom. In case of conflict, the specification of this document, including definitions, will control.
(9) While the terms used herein are believed to be well understood by those of ordinary skill in the art, certain definitions are set forth to facilitate explanation of the presently-disclosed subject matter.
(10) Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong.
(11) All patents, patent applications, published applications and publications, GenBank sequences, databases, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety.
(12) Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.
(13) As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, Biochem. (1972) 11(9):1726-1732).
(14) Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently-disclosed subject matter, representative methods, devices, and materials are described herein.
(15) Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a biomarker” includes a plurality of such biomarkers, and so forth.
(16) Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.
(17) As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, width, length, height, concentration or percentage is meant to encompass variations of in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.
(18) As used herein, ranges can be expressed as from “about” one particular value, and/or to “about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
(19) As used herein, “optional” or “optionally” means that the subsequently described event or circumstance does or does not occur and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally variant portion means that the portion is variant or non-variant.
(20) As used herein, the term “subject” refers to a target of administration. The subject of the herein disclosed methods can be a mammal. Thus, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. A “patient” refers to a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects.
(21) As used herein, the term “BChE polypeptide” can refer to various mutations and truncations of the BChE protein including the mutations that are characterized by cocaine hydrolase (CoCH). BChE polypeptide for example includes, but it not limited to, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15.
EXAMPLES
(22) The fusion proteins as disclosed herein were designed in view of a number of considerations. For example, they make use of a protein that is normally expressed in a high level as the N-terminal fusion partner to improve the expression of protein of interest [27, 28]. It was contemplated that the N-terminal fusion partner could “fool” the cellular process into expressing the fusion protein at a high level [28]. For another example, human IgG has a very long biological half-life (t.sub.1/2). The fragment crystallizable (Fc) region of IgG binds to the neonatal Fc receptor (FcRn) in the acidic environment of the endosome and later is transported to the cell surface where, upon exposure to a neutral pH, IgG is released back into the main bloodstream [29]. In addition, IgG is the most common type of antibody found in the circulation, and can be expressed in CHO cells with a yield of more than 1 g/L [30].
(23) The present inventors sought to design a long-acting CocH form which has not only a prolonged biological half-life without affecting the catalytic activity, but also an improved expression level in CHO cells. For this purpose, exemplary embodiments were prepared for testing, starting from CocH3 (the A199S/F227A/S287G/A328W/Y332G mutant [9] of human BChE) (SEQ ID NO: 12), a IL-2 signal peptide followed by Fc(M3) (the A1V/D142E/L144M mutant [31] of Fc)(SEQ ID NO: 16), which was fused with the N-terminal of CocH3 (SEQ ID NO: 12). Then the tetramerization domain (amino-acid residues 530 to 574) of CocH3 was deleted to minimize the possibility of affecting the correct folding of Fc(M3) or CocH3. On the other hand, it was contemplated that the presence of Fc(M3) might break the tetramer structure, resulting in a long and flexible peptide, which could be proteolyzed easily. In addition, according to computational modeling (data not shown), directly fusing Fc(M3) with the N-terminal of CocH3 could affect the entrance of substrate to the active site of CocH3, thus affecting the catalytic activity of CocH3. Hence, several types of linkers were selected and inserted between Fc(M3) and CocH3. In this way, various Fc(M3)-linker-CocH3 entities were prepared and tested for their catalytic activity against cocaine, protein expression yields in CHO cells, and pharmacokinetic profile (for the most promising entity), leading to identification of a promising Fc(M3)-linker-CocH3 entity, as discussed below.
(24) Materials and Methods
(25) Materials
(26) Q5® Site-Directed Mutagenesis Kit was ordered from New England Biolabs (Ipswich, Mass.). All oligonucleotides were synthesized by Eurofins MWG Operon (Huntsville, Ala.). Chinese Hamster Ovary-suspension (CHO-S) cells, FreeStyle™ CHO Expression Medium, Fetal Bovine Serum (FBS), 4-12% Tris-Glycine Mini Protein Gel, and SimpleBlue SafeStain were obtained from Invitrogen (Grand Island, N.Y.). TransIT-PRO® Transfection Kit was purchased from Minis (Madison, Wis.). The rmp Protein A Sepharose Fast Flow was from GE Healthcare Life Sciences (Pittsburgh, Pa.). (−)-Cocaine was provided by the National Institute on Drug Abuse (NIDA) Drug Supply Program (Bethesda, Md.); and [.sup.3H](−)-Cocaine (50 Ci/mmol) was obtained from PerkinElmer (Waltham, Mass.). All other materials were from Sigma-Aldrich (St Louis, Mo.) or Thermo Fisher Scientific (Waltham, Mass.).
(27) Preparation of Gene Fusion Constructs in pCMV-MCS
(28) Q5® Site-Directed Mutagenesis Kit was used to introduce each linker between Fc(M3) and CocH3. The pCMV-Fc(M3)-CocH3, constructed in a previous study [32] to encode N-terminal Fc-fused CocH3 without a linker, was used as the template. PCR reactions with Q5 hot start high-fidelity DNA polymerase along with primers listed in Table 1 were utilized to create insertions. Then 1 μl of each PCR product was incubated with Kinase-Ligase-DpnI enzyme mix for 15 minutes at room temperature. These steps allowed for rapid circulation of the PCR product and removal of the template DNA. 5 μl of final product was added to 50 μl of chemically-competent E. coli cells for transformation. All obtained plasmid encoding different Fc-fused CocH3 were confirmed by DNA sequencing.
(29) TABLE-US-00003 TABLE 1 Examples of primers for inserting various linkers Linker Primer name Primer sequence EAAAK EAAAK-F 5′-G TCT CCG GGT AAA GAG GCT GCC GCC AAG GAA GAT GAC ATC A-3′ (SEQ ID NO: 20) EAAAK-R 5′-CTT GGC GGC AGC CTC TTT ACC CGG AGA CAG GGA GAG-3′ (SEQ ID NO: 21) PAPAP PAPAP-F 5′-G TCT CCG GGT AAA CCT GCT CCA GCC CCG GAA GAT GAC ATC A-3′ (SEQ ID NO: 22) PAPAP-R 5′-CGG GGC TGG AGC AGG TTT ACC CGG AGA CAG GGA GAG-3′ (SEQ ID NO: 23) GGGSGGGS (G3S).sub.2-F 5′-G TCT CCG GGT AAA GGT GGA GGT TCC GGT GGA GGT TCC GAA GAT GAC ATC A-3′ (SEQ ID NO: 24) (G3S).sub.2-R 5′- GGA ACC TCC ACC GGA ACC TCC ACC TTT ACC CGG AGA CAG GGA GAG-3′ (SEQ ID NO: 25) PAPAPPAPAP (PAPAP).sub.2-F 5′-G TCT CCG GGT AAA CCT GCT CCA GCC CCG CCT GCT CCA GCC CCG GAA GAT GAC ATC A-3′ (SEQ ID NO: 26) (PAPAP).sub.2-R 5′- CGG GGC TGG AGC AGG CGG GGC TGG AGC AGG TTT ACC CGG AGA CAG GGA GAG-3′ (SEQ ID NO: 27)
(30) Expression and Purification
(31) CHO-S cells were grown under the condition of 37° C. and 8% CO.sub.2 in a humidified atmosphere. Once cells grown to a density of ˜1.0×10.sup.6 cells/ml, cells were transfected with plasmids encoding various proteins using TransIT-PRO® transfection kit. The culture medium was harvested 7 days after transfection. Enzyme secreted in the culture medium was purified by protein A affinity chromatography described previously [31]. Briefly, pre-equilibrated rmp Protein A Sepharose Fast Flow was mixed with cell-free medium, and incubated overnight at 6° C. with occasional stirring. Then the suspension was packed in a column, washed with 20 mM Tris.HCl (pH 7.4), and eluted by adjustment of salt concentration and pH. The eluate was concentrated and dialyzed in storage buffer (50 mM HEPES, 20% sorbitol, 1 M glycine, pH 7.4). Purified proteins were analyzed by native PAGE electrophoresis.
(32) In Vitro Activity Assay Against (−)-Cocaine.
(33) A radiometric assay based on toluene extraction of [.sup.3H](−)-cocaine labeled on its benzene ring was used to determine the catalytic activity of proteins [9, 11, 33]. Reactions were initiated by adding 150 μl enzyme solution (100 mM phosphate buffer, pH 7.4) to 50 μl [.sup.3H](−)-cocaine solution with varying concentration. Then 200 μl of 0.1 M HCl was added to stop each reaction and neutralize the liberated benzoic acid while ensuring a positive charge on the residual (−)-cocaine. [.sup.3H]Benzoic acid was extracted by 1 ml of toluene and measured by scintillation counting. Catalytic rate constant (k.sub.cat) and Michaelis-Menten constant (K.sub.m) were determined by fitting the substrate concentration-dependent data using Michaelis-Menten kinetics.
(34) Determination of Relative Expression Level of Proteins
(35) Cells were grown in 12-well plates to a density of ˜1.0×10.sup.6 cells/ml. Then cells were transfected with plasmids encoding different proteins using the same method described above. The test was tripled for each protein, occupying 3 out of 12 wells in a plate. Medium was collected from each well 3 days post the transfection. Cells were removed by centrifuge at 4000 rpm for 15 min, and the catalytic activity of each sample against cocaine was determined using radiometric assay described above. Protein concentration was calculated by dividing the catalytic activity by the k.sub.cat (determined by using the aforementioned purified protein) for each specific protein.
(36) Determination of Biological Half-Life in Rats
(37) Male Sprague-Darley rats (220-250 g) were ordered from Harlan (Harlan, Indianapolis, Ind.), and housed initially as one or two rats per cage. All rats were allowed ad libitum access to food and water and maintained on a 12 h light/12 h dark cycle, with the lights on at 8:00 a.m. at a room temperature of 21-22° C. Experiments were performed in a same colony room in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. The animal procedure was approved by the IACUC (Institutional Animal Care and Use Committee) as part of the animal protocol 2010-0722 on Jun. 21, 2016 at the University of Kentucky. Rats were injected with the purified Fc(M3)-(PAPAP).sub.2—CocH3 protein via tail vein (0.075 mg/kg). Blood samples were collected from saphenous vein puncture. Approximately 100 μl blood was collected by using heparin-treated capillary tube at various time points after enzyme administration. Collected samples were centrifuged for 15 min at a speed of 5000 g to separate the plasma, which was kept at 4° C. before analysis. Radiometric assay using 100 μM (−)-cocaine was carried out to measure the active enzyme concentration in plasma.
(38) Results and Discussion
(39) Optimization of Fc-Fused CocH3 Entity with a Linker
(40) Four different linkers, including flexible linkers (GGGSGGGS) (SEQ ID NO: 6) (GGGGSGGGGS)(SEQ ID NO: 36), (GGGGGGSGGGGGGS)(SEQ ID NO: 37) and three rigid linkers (EAAAK—SEQ ID NO: 19), (PAPAP—SEQ ID NO: 4), and (PAPAPPAPAP-SEQ ID NO: 2), were utilized in this study. Previous studies reported in literature indicated that a linker similar to these could separate carrier protein and functional protein effectively and lead to improved biological activity of the fusion proteins with a linker. The four fusion proteins (see
(41) To optimize the construct of Fc(M3)-CocH3, the above mentioned four linkers were used to eliminate the negative effects of N-terminal Fc portion on the catalytic activity of C-terminal CocH3. As seen in Table 2 and
(42) TABLE-US-00004 TABLE 2 Kinetic parameters determined for (-)-cocaine hydrolysis catalysed by the fusion proteins Protein k.sub.cat/K.sub.M # Enzyme K.sub.m (μM) k.sub.cat (min.sup.-1) (min.sup.-1 M.sup.-1) 1* Fc(M3)CocH3(529) 3.9 ± 0.5 1835 ± 61 4.7 × 10.sup.8 (SEQ ID NO: 16-SEQ ID NO: 13) 2* Fc(M3)-G.sub.6S-CocH3 4.4 ± 0.6 5694 ± 238 1.3 × 10.sup.9 (SEQ ID NO: 16-SEQ ID NO: 37-SEQ ID NO: 13) 3 Fc(M3)-EAAAK-CocH3(529) 4.3 ± 0.4 5684 ± 148 1.3 × 10.sup.9 (SEQ ID NO: 16-SEQ ID NO: 19-SEQ ID NO: 13) 4 Fc(M3)-PAPAP-CocH3(529) 4.2 ± 0.5 6078 ± 202 1.4 × 10.sup.9 (SEQ ID NO: 16-SEQ ID NO: 4-SEQ ID NO: 13) 5 Fc(M3)-(G.sub.3S).sub.2-CocH3(529) 3.7 ± 0.4 3579 ± 104 9.6 × 10.sup.8 (SEQ ID NO: 16-SEQ ID NO: 6-SEQ ID NO: 6-SEQ ID NO: 13) 6 Fc(M3)-(PAPAP).sub.2-CocH3(529) 4.5 ± 0.5 5666 ± 148 1.3 × 10.sup.9 (SEQ ID NO: 16-SEQ ID NO: 2-SEQ ID NO: 13) *The k.sub.cat and K.sub.M of the enzymes against (-)-cocaine were reported in ref. [32]
(43) Effects of the Linker on the Expression of Fc(M3)-Fused CocH3 Protein
(44) Fc(M3)-EAAAK-CocH3, Fc(M3)-PAPAP-CocH3, and Fc(M3)-(PAPAP).sub.2—CocH3 were further expressed together with Fc(M3)-CocH3 and the unfused CocH3 for comparison of relevant protein expression levels. All five proteins were expressed in the same plate under the same conditions at the same time. Cells in each well transfected using the same method and cultured under the same conditions after the transfection. All media were collected 3 days after the transfection. Protein expression level in each well was determined using the radiometric assay using 100 μM [.sup.3H](−)-cocaine. As seen in Table 3, the expression of the unfused CocH3 was 0.5 mg/L 3 days after the transient transfection. Usually, inserting the Fc portion at the N-terminal of the target protein could significantly improve the protein expression level. In this study, directly fusing Fc to the N-terminal of CocH3 increased the protein expression level by ˜2-fold. However, as Fc(M3)-CocH3 protein had only ˜30% catalytic activity against cocaine as compared to the unfused CocH3 [32]. As Fc(M3) domain sterically interferes with the CocH3 domain activity and lowers its catalytic activity against cocaine, it is also possible that this steric interference affects the efficiency of the protein folding. Therefore, an appropriate linker capable of avoiding such steric interference may not only improve the catalytic activity against cocaine, but also increase the protein expression level. As shown in Table 3, the protein expression yields of Fc(M3)-EAAAK-CocH3 and Fc(M3)-PAPAP-CocH3 was 4.8, and 5.2 mg/L, respectively. Linkers EAAAK, and PAPAP improved the yield of Fc(M3)-CocH3 protein expression by ˜9 and ˜10 folds, respectively. Among all fusion proteins constructed in this study, Fc(M3)-(PAPAP).sub.2—CocH3 has the highest protein expression yield. The linker (PAPAPPAPAP) increased the yield of protein expression by ˜10 fold compared to the corresponding fusion protein without a linker. Further, compared to the corresponding unfused protein (CocH3), Fc(M3)-(PAPAP).sub.2-CocH3 had a ˜21-fold improved yield of protein expression.
(45) TABLE-US-00005 TABLE 3 Transient expression levels of fusion proteins, in comparison of CocH3 Expression Enzyme level (mg/L) Ratio CocH3 (SEQ ID NO: 13) 0.5 ± 0.1 1 Fc(M3)-CocH3 (SEQ ID NO: 1.1 ± 0.2 2 16-SEQ ID NO: 13) Fc(M3)-EAAAK-CocH3 4.8 ± 0.6 9 (SEQ ID NO: 16-SEQ ID NO: 19-SEQ ID NO: 13) Fc(M3)-PAPAP-CocH3 5.2 ± 0.6 10 (SEQ ID NO: 16-SEQ ID NO: 4-SEQ ID NO: 13) Fc(M3)-(PAPAP).sub.2-CocH3 10.9 ± 1.1 21 (SEQ ID NO: 16-SEQ ID NO: 2-SEQ ID NO: 13)
(46) It should be pointed out that the transient expression method (with the protein expression within only three days) in this study was used only for the purpose of comparing the relative expression levels of various fusion proteins and unfused protein under the same conditions. So, the key results of this study are the relative protein expression levels, rather than the absolute protein expression levels. The absolute protein expression levels are expected to significantly increase when the stable CHO cell lines are developed and used to express the same proteins; of course, development of a stable cell line is a very time-consuming process. For example, using a lentivirus-based repeated-transduction method which was established in a previous study [24], the protein expression yield of the unfused CocH3 reached ˜10 mg/L in a flask-based culture. Thus, one would reasonably expect that an appropriately developed stable CHO cell line might be able to express ˜200 mg/L Fc(M3)-(PAPAP)2-CocH3 protein by using the same lentivirus-based repeated-transduction method. The protein expression yield could be improved further by optimizing of the culture conditions, such as cell density, medium, and culture temperature.
(47) Biological Half-Life of Fc(M3)-(PAPAP).sub.2—CocH3 in Rats
(48) Pharmacokinetic testing was carried out to determine biological half-life of Fc(M3)-(PAPAP).sub.2-CocH3. Rats (n=3) were administered IV with 0.075 mg/kg of the purified protein. The blood was collected at 1 hr, 4 hr, 8 hr, 12 hr, 1 day and once each day within 14 days after the enzyme injection. Depicted in
(49) TABLE-US-00006 TABLE 4 Summary of biological half-life of BChE or mutants in mice or rats In vivo Protein form half-life (h) BChE purified from human plasma 43.sup.a BChE produced in transgenic goat 2.7.sup.b BChE mutant produced in transgenic plant 0.2.sup.c BChE mutant produced in CHO cells 7.3.sup.d CocH3-Fc(M3) produced in CHO cells 107.sup.e (SEQ ID NO: 13-SEQ ID NO: 16) Fc(M3)-(PAPAP).sub.2-CocH3 produced in CHO cells 105 (SEQ ID NO: 16-SEQ ID NO: 2-SEQ ID NO: 13) .sup.aBiological half-life of enzyme was reported in ref. [26]. .sup.bBiological half-life of enzyme was reported in ref. [21]. .sup.cBiological half-life of enzyme was reported in ref. [19]. .sup.dData from ref. [24]. .sup.eData from ref. [31].
(50) A previously reported study [31] demonstrated that a single injection of CocH-Fc(M3) was able to accelerate cocaine metabolism in rats after 20 days and, thus, block cocaine-induced physiological and toxic effects for a long period [31]. The CocH3-Fc(M3) protein was expected to allow dosing once every 2-4 wk, or longer, for treating cocaine addiction in humans. Given the facts that Fc(M3)-(PAPAP).sub.2-CocH3 has the similarly long biological half-life in rats and same catalytic activity against cocaine, it is reasonable to expect that Fc(M3)-(PAPAP).sub.2-CocH3 may also be able to provide the similar efficacy and duration for the cocaine addiction treatment.
(51) It has been a significant challenge to efficiently express BChE polypeptides with both a long biological half-life comparable to the native BChE purified from human plasma and a high yield of protein expression. In this study, it has been demonstrated that an exemplary polypeptides including a BChE polypeptide molecule have not only a long biological half-life, but also an improved yield of protein expression compared to CocH3 (e.g., ˜105±7 hr in rats and ˜21-fold improved yield for Fc(M3)-(PAPAP)2-CocH3).
(52) In a further example of the present invention:
(53) BChE or BChE(574) refers to the wild-type human butyrylcholinesterase (full-length protein, with 574 amino acids) (SEQ ID NO:10). BChE(xxx) refers to a trucated fragment (with only the first xxx amino acids) of human butyrylcholinesterase (SEQ ID NO:10).
(54) BChE-Fc refers to a fusion protein in which the C-terminus of human BChE (SEQ ID No: 10) is fused to the N-terminus of the Fc portion of human IgG-1 (SEQ ID NO: 8) or (SEQ ID NO 10-SEQ ID NO: 8). BChE(xxx)-Fc refers to a fusion protein in which the C-terminus of BChE(xxx) fragment fused to the N-terminus of the Fc portion of human IgG-1. CocH is a BChE polypeptide with specific mutations. CocH-LAF generally represents a cocaine hydrolase (CocH) in a long-acting form (LAF).
(55) TABLE-US-00007 CocH-LAF1 (in which ″1″ means the first version) refers to the (SEQ ID NO: 13-SEQ ID NO: 16) A199S/F227A/S287G/A328W/Y332G/A530V/D671E/L673M mutant of BChE(529)-Fc. CocH-LAF4 refers to the (SEQ ID NO: 13-SEQ ID NO: 17) A199S/F227A/S287G/A328W/Y332G/A530V/M567Y/D671E/ L673M mutant of the BChE(529)-Fc protein. CocH-LAF6 refers to the ((SEQ ID NO: 13-SEQ ID NO: 18) A199S/F227A/S287G/A328W/Y332G/A530V/M567Y/S569T/ T571E/D671E/L673M mutant of BChE(529)-Fc. CocH-LAF7 refers to the (SEQ ID NO: 14-SEQ ID NO: 18) A199S/F227A/P285A/S287G/A328W/Y332G/A530V/M567Y/ S569T/T571E/D671E/L673M mutant of BChE(529)-Fc. CocH-LAF8 refers to the (SEQ ID NO: 15-SEQ ID NO: 18) A199S/F227A/P285Q/S287G/A328W/Y332G/A530V/M567Y/ S569T/T571E/D671E/L673M mutant of BChE(529)-Fc.
(56) TABLE-US-00008 TABLE 5 Transient expression levels of BChE-Fc and BChE(529)-Fc Expression Protein level (mg/L) Ratio BChE-Fc (SEQ ID NO: 11-SEQ 0.55 1 ID NO: 8) BChE(529)-Fc (SEQ ID NO: 3.78 6.9 11-SEQ ID NO: 8)
(57) According to the data in Table 5, BChE(529)-Fc can be expressed with a significantly improved yield (about ˜7 fold), compared BChE-Fc. Both the Fc fusion and BChE fragmentation did not significantly change the catalytic activity of BChE.
(58) In light of the production data in Table 5, a further designed mutants of BChE(529)-Fc with an improved binding affinity with neonatal Fc receptor (FcRn) at pH 6 in order to further prolong the biological half-life (t.sub.1/2) in addition to the protein expression yield (see Table S2).
(59) TABLE-US-00009 TABLE 6 Binding affinity of BChE(529)-Fc and its mutants with human FcRn and their biological half-life (t.sub.1/2), along with the protein expression levels in stably transfected CHO cells. K.sub.d (nM) with t.sub.1/2 (hours) Expression Protein FcRn at pH 6 in rats level.sup.a BChE(529)-Fc 2500 to 4000 86 ± 6 >100 mg/L (SEQ ID NO: 11-SEQ ID NO: 8) CocH3-LAF1 992 107 ± 6 >1 g/L (SEQ ID NO: 13-SEQ ID NO: 16) CocH3-LAF4 327 195 ± 10 >1 g/L (SEQ ID NO: 13-SEQ ID NO: 17) CocH3-LAF6 43 206 ± 7 >200 mg/L (SEQ ID NO: 13-SEQ ID NO: 18) CocHl-LAF7 43 206 ± 7 >1 g/L (SEQ ID NO: 14-SEQ ID NO: 18) CocH2-LAF8 43 206 ± 7 >200 mg/L (SEQ ID NO: 15-SEQ ID NO: 18) .sup.aThe protein expression level is also affected by the culture conditions. Listed here is the lower end of the protein expression level.
(60) To illustrate the approach to the rational design and discovery of BChE(529)-Fc mutants with improved binding affinity with FcRn and prolonged biological half-lives, depicted in
(61) CocH3 represents the A199S/F227A/S287G/A328W/Y332G mutant of human butyrylcholinesterase (BChE). The full-length BChE or CocH3 has 574 amino-acid residues. CocH3(xxx) refers to the fragment (with only the first xxx amino acids) of the A199S/F227A/S287G/A328W/Y332G mutant of human butyrylcholinesterase.
(62) TABLE-US-00010 TABLE 7 Transient expression levels of fusion proteins Expression Enzyme level (mg/L) CocH3(574) (SEQ ID NO: 12) 0.51 Fc(M3)-CocH3(529) 1.05 (SEQ ID NO: 16-SEQ ID NO: 13) Fc(M3)-G.sub.3S-CocH3(529) 3.10 (SEQ ID NO: 16-SEQ ID NO: 6-SEQ ID NO: 13) Fc(M3)-G.sub.4S-CocH3(529) 3.69 (SEQ ID NO: 16-SEQ ID NO: 36- SEQ ID NO: 13) Fc(M3)-G.sub.6S-CocH3(529) 5.14 (SEQ ID NO: 16-SEQ ID NO: 37- SEQ ID NO: 13) Fc(M3)-EAAAK-CocH3(529) 4.76 (SEQ ID NO: 16-SEQ ID NO: 19- SEQ ID NO: 13) Fc(M3)-PAPAP-CocH3(529) 5.15 (SEQ ID NO: 16-SEQ ID NO: 4-SEQ ID NO: 13) CocH3(529)-(G.sub.3S).sub.2-Fc(M3) 7.86 (SEQ ID NO: 13-SEQ ID NO: 6-SEQ ID NO: 6-SEQ ID NO: 16) Fc(M3)-(PAPAP).sub.2-CocH3(529) 10.87 (SEQ ID NO: 16-SEQ ID NO: 2-SEQ ID NO: 13) CocH3(574)-Fc(M3) 4.11 (SEQ ID NO: 12-SEQ ID NO: 16) CocH3(574)-G.sub.6S-Fc(M3) 3.96 (SEQ ID NO: 12-SEQ ID NO: 37- SEQ ID NO: 16) CocH3(529)-Fc(M3) 5.75 (SEQ ID NO: 13-SEQ ID NO: 16) CocH3(529)-G.sub.6S-Fc(M3) 6.39 (SEQ ID NO: 13-SEQ ID NO: 37- SEQ ID NO: 16) CocH3(529)-Fc(M6) 3.89 (SEQ ID NO: 13-SEQ ID NO: 18)
(63) It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the subject matter disclosed herein. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.
(64) All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference, including the references set forth in the following list:
(65) TABLE-US-00011 SEQUENCE LISTING SEQ ID NO: 1 Nucleotide Encoding SEQ ID NO: 2 CCGGCGCCGGCGCCGCCGGCGCCGGCGCCG SEQ ID NO: 2 PAPAPPAPAP SEQ ID NO: 3 Nucleotide encoding SEQ ID NO: 4 CCGGCGCCGGCGCCG SEQ ID NO: 4 PAPAP SEQ ID NO: 5 Nucleotide encoding SEQ ID NO: 6 GGCGGCGGCAGCGGCGGCGGCAGC SEQ ID NO: 6 GGGSGGGS SEQ ID NO: 7-Nucleotide encoding Wild type Fc polypeptide GCA GAG CCT AAG TCC TGC GAC AAA ACT CAC ACA TGC CCA CCG TGC CCA GCA CCT GAA CTC CTG GGG GGA CCG TCA GTC TTC CTC TTC CCC CCA AAA CCC AAG GAC ACC CTC ATG ATC TCC CGG ACC CCT GAG GTC ACA TGC GTG GTG GTG GAC GTG AGC CAC GAA GAC CCT GAG GTC AAG TTC AAC TGG TAC GTG GAC GGC GTG GAG GTG CAT AAT GCC AAG ACA AAG CCG CGG GAG GAG CAG TAC AAC AGC ACG TAC CGT GTG GTC AGC GTC CTC ACC GTC CTG CAC CAG GAC TGG CTG AAT GGC AAG GAG TAC AAG TGC AAG GTC TCC AAC AAA GCC CTC CCA GCC CCC ATC GAG AAA ACC ATC TCC AAA GCC AAA GGG CAG CCC CGA GAA CCA CAG GTG TAC ACC CTG CCC CCA TCC CGG GAC GAG CTG ACC AAG AAC CAG GTC AGC CTG ACC TGC CTG GTC AAA GGC TTC TAT CCC AGC GAC ATC GCC GTG GAG TGG GAG AGC AAT GGG CAG CCG GAG AAC AAC TAC AAG ACC ACG CCT CCC GTG CTG GAC TCC GAC GGC TCC TTC TTC CTC TAC AGC AAG CTC ACC GTG GAC AAG AGC AGG TGG CAG CAG GGG AAC GTC TTC TCA TGC TCC GTG ATG CAC GAG GCT CTG CAC AAC CAC TAC ACG CAG AAG AGC CTC TCC CTG TCT CCG GGT AAA SEQ ID NO: 8-Wild type Fc polypeptide AEPKSCDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK SEQ ID NO: 9-Nucleotide encoding Wild type BChE GAA GAT GAC ATC ATA ATT GCA ACA AAG AAT GGA AAA GTC AGA GGG ATG AAC TTG ACA GTT TTT GGT GGC ACG GTA ACA GCC TTT CTT GGA ATT CCC TAT GCA CAG CCA CCT CTT GGT AGA CTT CGA TTC AAA AAG CCA CAG TCT CTG ACC AAG TGG TCT GAT ATT TGG AAT GCC ACA AAA TAT GCA AAT TCT TGC TGT CAG AAC ATA GAT CAA AGT TTT CCA GGC TTC CAT GGA TCA GAG ATG TGG AAC CCA AAC ACT GAC CTC AGT GAA GAC TGT TTA TAT CTA AAT GTA TGG ATT CCA GCA CCT AAA CCA AAA AAT GCC ACT GTA TTG ATA TGG ATT TAT GGT GGT GGT TTT CAA ACT GGA ACA TCA TCT TTA CAT GTT TAT GAT GGC AAG TTT CTG GCT CGG GTT GAA AGA GTT ATT GTA GTG TCA ATG AAC TAT AGG GTG GGT GCC CTA GGA TTC TTA GCT TTG CCA GGA AAT CCT GAG GCT CCA GGG AAC ATG GGT TTA TTT GAT CAA CAG TTG GCT CTT CAG TGG GTT CAA AAA AAT ATA GCA GCC TTT GGT GGA AAT CCT AAA AGT GTA ACT CTC TTT GGA GAA AGT GCA GGA GCA GCT TCA GTT AGC CTG CAT TTG CTT TCT CCT GGA AGC CAT TCA TTG TTC ACC AGA GCC ATT CTG CAA AGT GGT TCC TTT AAT GCT CCT TGG GCG GTA ACA TCT CTT TAT GAA GCT AGG AAC AGA ACG TTG AAC TTA GCT AAA TTG ACT GGT TGC TCT AGA GAG AAT GAG ACT GAA ATA ATC AAG TGT CTT AGA AAT AAA GAT CCC CAA GAA ATT CTT CTG AAT GAA GCA TTT GTT GTC CCC TAT GGG ACT CCT TTG TCA GTA AAC TTT GGT CCG ACC GTG GAT GGT GAT TTT CTC ACT GAC ATG CCA GAC ATA TTA CTT GAA CTT GGA CAA TTT AAA AAA ACC CAG ATT TTG GTG GGT GTT AAT AAA GAT GAA GGG ACA GCT TTT TTA GTC TAT GGT GCT CCT GGC TTC AGC AAA GAT AAC AAT AGT ATC ATA ACT AGA AAA GAA TTT CAG GAA GGT TTA AAA ATA TTT TTT CCA GGA GTG AGT GAG TTT GGA AAG GAA TCC ATC CTT TTT CAT TAC ACA GAC TGG GTA GAT GAT CAG AGA CCT GAA AAC TAC CGT GAG GCC TTG GGT GAT GTT GTT GGG GAT TAT AAT TTC ATA TGC CCT GCC TTG GAG TTC ACC AAG AAG TTC TCA GAA TGG GGA AAT AAT GCC TTT TTC TAC TAT TTT GAA CAC CGA TCC TCC AAA CTT CCG TGG CCA GAA TGG ATG GGA GTG ATG CAT GGC TAT GAA ATT GAA TTT GTC TTT GGT TTA CCT CTG GAA AGA AGA GAT AAT TAC ACA AAA GCC GAG GAA ATT TTG AGT AGA TCC ATA GTG AAA CGG TGG GCA AAT TTT GCA AAA TAT GGG AAT CCA AAT GAG ACT CAG AAC AAT AGC ACA AGC TGG CCT GTC TTC AAA AGC ACT GAA CAA AAA TAT CTA ACC TTG AAT ACA GAG TCA ACA AGA ATA ATG ACG AAA CTA CGT GCT CAA CAA TGT CGA TTC TGG ACA TCA TTT TTT CCA AAA GTC TTG GAA ATG ACA GGA AAT ATT GAT GAA GCA GAA TGG GAG TGG AAA GCA GGA TTC CAT CGC TGG AAC AAT TAC ATG ATG GAC TGG AAA AAT CAA TTT AAC GAT TAC ACT AGC AAG AAA GAA AGT TGT GTG GGT CTC SEQ ID NO: 10-Wild type BChE Polypeptide EDDIIIATKNGKVRGMNLTVFGGTVTAFLGIPYAQPPLGRLRFKKPQSLTKWSDIWNATK YANSCCQNIDQSFPGFHGSEMWNPNTDLSEDCLYLNVWIPAPKPKNATVLIWIYGGGFQ TGTSSLHVYDGKFLARVERVIVVSMNYRVGALGFLALPGNPEAPGNMGLFDQQLALQ WVQKNIAAFGGNPKSVTLFGESAGAASVSLHLLSPGSHSLFTRAILQSGSFNAPWAVTSL YEARNRTLNLAKLTGCSRENETEIIKCLRNKDPQEILLNEAFVVPYGTPLSVNFGPTVDG DFLTDMPDILLELGQFKKTQILVGVNKDEGTAFLVYGAPGFSKDNNSIITRKEFQEGLKIF FPGVSEFGKESILFHYTDWVDDQRPENYREALGDVVGDYNFICPALEFTKKFSEWGNNA FFYYFERRSSKLPWPEWMGVMHGYEIEFVFGLPLERRDNYTKAEEILSRSIVKRWANFA KYGNPNETQNNSTSWPVFKSTEQKYLTLNTESTRIMTKLRAQQCRFWTSFFPKVLEMTG NIDEAEWEWKAGFHRWNNYMMDWKNQFNDYTSKKESCVGL SEQ ID NO: 11-Truncated (only the first 529 amino acids of wild type BChE) BchE (529) EDDIIIATKNGKVRGMNLTVFGGTVTAFLGIPYAQPPLGRLRFKKPQSLTKWSDIWNATK YANSCCQNIDQSFPGFHGSEMWNPNTDLSEDCLYLNVWIPAPKPKNATVLIWIYGGGFQ TGTSSLHVYDGKFLARVERVIVVSMNYRVGALGFLALPGNPEAPGNMGLFDQQLALQ WVQKNIAAFGGNPKSVTLFGESAGAASVSLHLLSPGSHSLFTRAILQSGSFNAPWAVTSL YEARNRTLNLAKLTGCSRENETEIIKCLRNKDPQEILLNEAFVVPYGTPLSVNFGPTVDG DFLTDMPDILLELGQFKKTQILVGVNKDEGTAFLVYGAPGFSKDNNSIITRKEFQEGLKIF FPGVSEFGKESILFHYTDWVDDQRPENYREALGDVVGDYNFICPALEFTKKFSEWGNNA FFYYFERRSSKLPWPEWMGVMHGYEIEFVFGLPLERRDNYTKAEEILSRSIVKRWANFA KYGNPNETQNNSTSWPVFKSTEQKYLTLNTESTRIMTKLRAQQCRFWTSFFPKV SEQ ID NO: 12-CoCH3 Full length (574) EDDIIIATKNGKVRGMNLTVFGGTVTAFLGIPYAQPPLGRLRFKKPQSLTKWSDIWNATK YANSCCQNIDQSFPGFHGSEMWNPNTDLSEDCLYLNVWIPAPKPKNATVLIWIYGGGFQ TGTSSLHVYDGKFLARVERVIVVSMNYRVGALGFLALPGNPEAPGNMGLFDQQLALQ WVQKNIAAFGGNPKSVTLFGESSGAASVSLHLLSPGSHSLFTRAILQSGSANAPWAVTSL YEARNRTLNLAKLTGCSRENETEIIKCLRNKDPQEILLNEAFVVPYGTPLGVNFGPTVDG DFLTDMPDILLELGQFKKTQILVGVNKDEGTWFLVGGAPGFSKDNNSIITRKEFQEGLKI FFPGVSEFGKESILFHYTDWVDDQRPENYREALGDVVGDYNFICPALEFTKKFSEWGNN AFFYYFEHRSSKLPWPEWMGVMHGYEIEFVFGLPLERRDNYTKAEEILSRSIVKRWANF AKYGNPNETQNNSTSWPVFKSTEQKYLTLNTESTRIMTKLRAQQCRFWTSFFPKVLEMT GNIDEAEWEWKAGFHRWNNYMMDWKNQFNDYTSKKESCVGL SEQ ID NO: 13-Truncated CoCH3 (only the first 529 amino acids of Full length CoCH3)(529) EDDIIIATKNGKVRGMNLTVFGGTVTAFLGIPYAQPPLGRLRFKKPQSLTKWSDIWNATK YANSCCQNIDQSFPGFHGSEMWNPNTDLSEDCLYLNVWIPAPKPKNATVLIWIYGGGFQ TGTSSLHVYDGKFLARVERVIVVSMNYRVGALGFLALPGNPEAPGNMGLFDQQLALQ WVQKNIAAFGGNPKSVTLFGESSGAASVSLHLLSPGSHSLFTRAILQSGSANAPWAVTSL YEARNRTLNLAKLTGCSRENETEIIKCLRNKDPQEILLNEAFVVPYGTPLGVNFGPTVDG DFLTDMPDILLELGQFKKTQILVGVNKDEGTWFLVGGAPGFSKDNNSIITRKEFQEGLKI FFPGVSEFGKESILFHYTDWVDDQRPENYREALGDVVGDYNFICPALEFTKKFSEWGNN AFFYYFEHRSSKLPWPEWMGVMHGYEIEFVFGLPLERRDNYTKAEEILSRSIVKRWANF AKYGNPNETQNNSTSWPVFKSTEQKYLTLNTESTRIMTKLRAQQCRFWTSFFPKV SEQ ID NO: 14-CoCH1 EDDIIIATKNGKVRGMNLTVFGGTVTAFLGIPYAQPPLGRLRFKKPQSLTKWSDIWNATK YANSCCQNIDQSFPGFHGSEMWNPNTDLSEDCLYLNVWIPAPKPKNATVLIWIYGGGFQ TGTSSLHVYDGKFLARVERVIVVSMNYRVGALGFLALPGNPEAPGNMGLFDQQLALQ WVQKNIAAFGGNPKSVTLFGESSGAASVSLHLLSPGSHSLFTRAILQSGSANAPWAVTSL YEARNRTLNLAKLTGCSRENETEIIKCLRNKDPQEILLNEAFVVPYGTALGVNFGPTVDG DFLTDMPDILLELGQFKKTQILVGVNKDEGTWFLVGGAPGFSKDNNSIITRKEFQEGLKI FFPGVSEFGKESILFHYTDWVDDQRPENYREALGDVVGDYNFICPALEFTKKFSEWGNN AFFYYFEHRSSKLPWPEWMGVMHGYEIEFVFGLPLERRDNYTKAEEILSRSIVKRWANF AKYGNPNETQNNSTSWPVFKSTEQKYLTLNTESTRIMTKLRAQQCRFWTSFFPKV SEQ ID NO: 15-CoCH2 EDDIIIATKNGKVRGMNLTVFGGTVTAFLGIPYAQPPLGRLRFKKPQSLTKWSDIWNATK YANSCCQNIDQSFPGFHGSEMWNPNTDLSEDCLYLNVWIPAPKPKNATVLIWIYGGGFQ TGTSSLHVYDGKFLARVERVIVVSMNYRVGALGFLALPGNPEAPGNMGLFDQQLALQ WVQKNIAAFGGNPKSVTLFGESSGAASVSLHLLSPGSHSLFTRAILQSGSANAPWAVTSL YEARNRTLNLAKLTGCSRENETEIIKCLRNKDPQEILLNEAFVVPYGTQLGVNFGPTVDG DFLTDMPDILLELGQFKKTQILVGVNKDEGTWFLVGGAPGFSKDNNSIITRKEFQEGLKI FFPGVSEFGKESILFHYTDWVDDQRPENYREALGDVVGDYNFICPALEFTKKFSEWGNN AFFYYFEHRSSKLPWPEWMGVMHGYEIEFVFGLPLERRDNYTKAEEILSRSIVKRWANF AKYGNPNETQNNSTSWPVFKSTEQKYLTLNTESTRIMTKLRAQQCRFWTSFFPKV SEQ ID NO: 16-Fc(M3) VEPKSCDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK SEQ ID NO: 17-Fc(M4) VEPKSCDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTYIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK SEQ ID NO: 18-Fc(M6) VEPKSCDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTYIT REPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK SEQ ID NO: 19-EAAAK Polypeptide EAAAK SEQ ID NO: 20-EAAAK-Forward Primer G TCT CCG GGT AAA GAG GCT GCC GCC AAG GAA GAT GAC ATC A SEQ ID NO: 21-EAAAK-Reverse Primer CTT GGC GGC AGC CTC TTT ACC CGG AGA CAG GGA GAG SEQ ID NO: 22-PAPAP-Forward Primer G TCT CCG GGT AAA CCT GCT CCA GCC CCG GAA GAT GAC ATC SEQ ID NO: 23-PAPAP-Reverse Primer CGG GGC TGG AGC AGG TTT ACC CGG AGA CAG GGA GAG SEQ ID NO: 24-(G3S)2-Forward Primer G TCT CCG GGT AAA GGT GGA GGT TCC GGT GGA GGT TCC GAA GAT GAC ATC A SEQ ID NO: 25-(G3S)2-Reverse Primer GGA ACC TCC ACC GGA ACC TCC ACC TTT ACC CGG AGA CAG GGA GAG SEQ ID NO: 26-(PAPAP)2-Forward Primer G TCT CCG GGT AAA CCT GCT CCA GCC CCG CCT GCT CCA GCC CCG GAA GAT GAC ATC SEQ ID NO: 27-(PAPAP)2-Reverse Primer CGG GGC TGG AGC AGG CGG GGC TGG AGC AGG TTT ACC CGG AGA CAG GGA GAG SEQ ID NO: 28-Fc(M8) VEPKSCDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPQVK FNWYVDGVQV HNAKTKPREQ QYNSTYRVVS VLTVLHQNWL DGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK SEQ ID NO: 29-Fc(M5) QEPKSSDKTH TSPPSPAPEL LGGSSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK SEQ ID NO: 30-Fc(M4′) VEPKSCDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS REPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK SEQ ID NO: 31-Fc(M5′) VEPKSCDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLYIT RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK SEQ ID NO: 32-Fc(M6′) QEPKSSDKTH TSPPSPAPEL LGGSSVFLFP PKPKDTLYIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK SEQ ID NO: 33-Fc(M7) QEPKSSDKTH TSPPSPAPEL LGGSSVFLFP PKPKDTLYIT RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK SEQ ID NO: 34-Fc(M8′) QEPKSSDKTH TSPPSPAPEL LGGSSVFLFP PKPKDTLYIT REPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK SEQ ID NO: 35-wild type Fc-Truncated wild type BChE-fusion polypeptide AEPKSCDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVVDVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGKEDDIIIATKNGKVRGMNLTVFGGTVTAFLGIPYAQPPLGRLRFKKPQSLTKWSDIWN ATKYANSCCQNIDQSFPGFHGSEMWNPNTDLSEDCLYLNVWIPAPKPKNATVLIWIYGG GFQTGTSSLHVYDGKFLARVERVIVVSMNYRVGALGFLALPGNPEAPGNMGLFDQQLA LQWVQKNIAAFGGNPKSVTLFGESAGAASVSLHLLSPGSHSLFTRAILQSGSFNAPWAV TSLYEARNRTLNLAKLTGCSRENETEIIKCLRNKDPQEILLNEAFVVPYGTPLSVNFGPTV DGDFLTDMPDILLELGQFKKTQILVGVNKDEGTAFLVYGAPGFSKDNNSIITRKEFQEGL KIFFPGVSEFGKESILFHYTDWVDDQRPENYREALGDVVGDYNFICPALEFTKKFSEWG NNAFFYYFEHRSSKLPWPEWMGVMHGYEIEFVFGLPLERRDNYTKAEEILSRSIVKRWA NFAKYGNPNETQNNSTSWPVFKSTEQKYLTLNTESTRIMTKLRAQQCRFWTSFFPKV SEQ ID NO: 36-G.sub.4S GGGGS GGGGS SEQ ID NO: 37-G.sub.6S GGGGGGS GGGGGGS
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