Patent Grant
10106785
The Sequence Listing in ASCII text file format of 70,213 bytes in size, created on Oct. 25, 2017, with the file name “2017-10-25SequenceListing_MAZOR2A,” filed in the U.S. Patent and Trademark Office on Oct. 25, 2017, is hereby incorporated herein by reference.
The present invention generally relates to fusion polypeptides comprising fragments of human Acetylcholinesterase conjugated to the Fc region of an immunoglobulin. The present invention also relates to methods of preparing these polypeptide constructs and to uses thereof as scavenging agents of organophosphate compounds.
References considered to be relevant as background to the presently disclosed subject matter are listed below:
Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.
Organophosphorus (OP) compounds are a diverse group of chemicals that include, among others, insecticides, antihelmintics (drugs that are used for killing parasitic worms) and nerve gases.
Organophosphates inhibit the enzyme acetylcholinesterase (AChE) by phosphorylating the serine hydroxyl residue in the AChE active site. AChE is critical for nerve function, so the irreversible blockage of this enzyme upon exposure to organophosphates (OP poisoning), causes acetylcholine accumulation, and results in muscle overstimulation that may lead to death.
An arsenal of OP compounds was developed as chemical warfare agents, for example tabun, sarin, soman and agent VX. In addition to the warfare context, organophosphorus pesticide self-poisoning is also a serious clinical problem in rural regions of the developing world, and kills an estimated 200,000 people every year (1).
Current treatment of OP poisoning includes a pretreatment with carbamates to protect AChE from inhibition by OP compounds and post-exposure treatments with anti-cholinergic drugs that act to counteract the effects of excess acetylcholine and reactivate AChE. While some OP poisoning antidotes are effective at preventing lethality from OP poisoning, current treatment lacks the ability to prevent post-exposure incapacitation, performance deficits, or permanent brain damage.
Choline esterases, such as AChE or Butyrylcholinesterase (BChE), were used for development of OP scavengers. Such enzyme scavengers are being developed as a pretreatment to sequester highly toxic OPs before they can reach their physiological targets and prevent the toxic effects from occurring (2, 3).
Since recombinant choline esterases have short half lives in the circulation system, with a mean retention time of 60 minutes (3), various conjugates of choline esterases were developed for preventing their rapid clearance from the circulation. For example, the publication WO 02/087624 (4) describes a circulatory long-lived cholinesterase, which is coupled with a non-antigenic polymer.
Cholinesterases which are covalently fused to another protein that naturally has a long circulating half-life (including human IgG1) were described for example in US 2009/0249503 (5) and in WO 03/061562 (6).
In a first of its aspects, the present invention provides a fusion polypeptide comprising:
In one embodiment, said AChE polypeptide component is covalently linked through its C-terminus to said Fc domain.
In another embodiment, said AChE polypeptide component is covalently linked through its N-terminus to said Fc domain.
In certain embodiments, said Fc domain of human IgG is an Fc domain of IgG1 or of IgG2.
In certain specific embodiments, said Fc domain comprises an amino acid sequence that is at least 70% identical to the amino acid sequence denoted by SEQ ID NO: 15 and wherein said Fc domain retains its functional activity.
In one embodiment, said fusion polypeptide comprises a dimer of two identical monomers, wherein each one of the identical monomers comprises an acetylcholinesterase (AChE) polypeptide component and an Fc domain of human IgG.
In another embodiment, said fusion polypeptide comprises a dimer of a first and second monomers, wherein said first monomer comprises an AChE polypeptide component and an Fc domain of human IgG and the second monomer comprises an Fc domain of human IgG.
In certain embodiments, said fusion polypeptide further comprises a spacer covalently linking the AChE polypeptide component and the Fc domain.
In one specific embodiment, said spacer comprises the amino acid sequence ASEAP denoted by SEQ ID NO: 9.
In one specific embodiment, said spacer consists of the amino acid sequence ASEAP denoted by SEQ ID NO: 9.
In one embodiment, said modified human AChE polypeptide comprises an amino acid sequence that is at least 70% identical to the amino acid sequence denoted by SEQ ID NO: 8 and wherein said human AChE polypeptide component retains the functional activity of human AChE.
In other embodiments, said modified human AChE polypeptide comprises an amino acid substitution in at least one position of SEQ ID NO: 8 and wherein said human AChE polypeptide component retains the functional activity of human AChE.
In a specific embodiment, said modified human AChE polypeptide comprises the amino acid Ala at a position corresponding to position 338 of the amino acid sequence denoted by SEQ ID NO: 8.
In another specific embodiment, said modified human AChE polypeptide consists of the amino acid sequence denoted by SEQ ID NO: 8.
In another specific embodiment, said fusion polypeptide comprises the amino acid sequence denoted by SEQ ID NO: 17.
In another specific embodiment, said fusion polypeptide consists of the amino acid sequence denoted by SEQ ID NO: 17.
In another one of its aspects, the present invention provides an isolated nucleic acid construct comprising a nucleic acid sequence encoding the fusion polypeptide of the invention.
In one embodiment, said nucleic acid construct further comprises a sequence encoding a secretion signal situated at the 5′ end of said nucleic acid sequence.
In a specific embodiment, said secretion signal is a kappa-leader sequence having the amino acid sequence denoted by SEQ ID NO: 11 or the native signal peptide of human AChE having the amino acid sequence denoted by SEQ ID NO: 12.
In another specific embodiment, said nucleic acid construct is of the nucleic acid sequence denoted by SEQ ID NO: 18 or by SEQ ID NO: 19.
The invention also provides an expression vector comprising the isolated nucleic acid construct of the invention, as well as an isolated host cell comprising the nucleic acid construct or the expression vector as described above.
In another one of its aspects, the present invention provides a method of producing the fusion polypeptide of the invention, comprising culturing the host cell under conditions suitable for expression of the fusion polypeptide in the host cell and recovering the fusion polypeptide thereby produced.
In another aspect, the present invention provides a pharmaceutical composition comprising the fusion polypeptide of the invention and a pharmaceutically acceptable carrier.
In certain embodiments, said pharmaceutical composition further comprises an additional therapeutic agent.
In another aspect, the fusion polypeptide of the invention, or the pharmaceutical composition of the invention are for use in prophylaxis of organophosphate poisoning.
In another aspect, the present invention provides a method of prophylaxis of organophosphate poisoning comprising administering an effective amount of the fusion polypeptide or the pharmaceutical composition of the invention to a patient in need thereof.
In one embodiment, said method further comprises administering at least one additional therapeutic agent.
In certain embodiments, said at least one additional therapeutic agent is selected from atropine, glycopyrrolate, benzodiazepines, pralidoxime and native (non-fused) cholinesterase(s).
In one specific embodiment, said additional therapeutic agent is administered after exposure to organophosphate poisoning.
In another one of its aspects, the invention provides a method of increasing the circulatory half-life of AChE, said method comprising preparing a fusion polypeptide comprising:
In still another one of its aspects, the invention provides a kit comprising:
(i) at least one fusion polypeptide comprising:
In some embodiments the kit further comprises at least one additional therapeutic agent.
In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:
The present invention is based on the construction of a chimeric recombinant molecule of human acetylcholinesterase (AChE) coupled to the Fc region of human IgG1 (also termed herein AChE-Fc fusion protein) that is compatible with biotechnological production and purification, maintains the catalytic activity of the human AChE enzyme and has a significantly longer half-life as compared to the half-life of free human AChE.
The novel fusion product was shown to have a bioscavenging reactivity toward the organophosphate-AChE inhibitors BW284c5, propidium, soman and VX. Owing to the ease of production, reactivity toward nerve agents and its optimized pharmacokinetics characteristics, AChE-Fc emerges as a promising next-generation bioscavenger.
The present invention thus provides a fusion polypeptide comprising:
Acetylcholinesterase is an enzyme of cardinal importance in neurotransmission systems, and is responsible for rapid termination of impulse transmission at cholinergic synapses by hydrolysis of the neurotransmitter acetylcholine (7). Acetylcholinesterase is a primary target of inhibition by organophosphorus compounds.
Thus the term “acetylcholinesterase” (also termed AChE, acetylhydrolase or EC 3.1.1.7) as herein defined, refers to an enzyme that hydrolyzes the neurotransmitter acetylcholine. The amino acid sequence of human acetylcholinesterase is denoted for example by SEQ ID NO: 21.
As described below, an N-terminal and C-terminal truncated version of human AChE was prepared for use in conjugation. This truncated version lacks both the N and C termini of the full length human AChE, namely the native signal sequence of AChE and the C-terminal 40 amino acids of the AChE “tail”. The resulting truncated polypeptide is termed herein “modified human AChE” or “modified AChE”.
It was previously reported that the full length human AChE may assemble into tertramers of catalytic subunits that are disulfide-linked to a filamentous tail unit, which is remote from the AChE catalytic subunit (8). In order to circumvent the formation of AChE-Fc conjugate multi-polypeptide complexes, the tail unit of the full length human AChE was deleted, as detailed below. As shown for example in
Thus the term “AChE enzyme tail” as used herein refers to the unit that forms a collagen-like structure at the C-terminal end of the AChE polypeptide, distal to the catalytic subunit. For example, the term AChE enzyme tail refers to amino acid residues DTLDEAERQWKAEFHRWSSYMVHWKNQFDHYSKQDRCSDL at positions 575 to 614 (in the N- to C-terminus direction) in the sequence of full length human AChE denoted by SEQ ID NO: 21 (Table 2 below).
Therefore, the term “modified human AChE” (also referred to herein as “modified AChE”) as used herein refers to a polypeptide fragment of the full length human AChE enzyme, which lacks the N-terminal signal peptide and at least 4 amino acid residues at the C-terminal tail of the full length human AChE enzyme. In certain embodiments the modified AchE enzyme lacks the N-terminal signal peptide and between about 4 and about 40 amino acid residues at the C-terminal tail of the full length human AChE enzyme. In a specific embodiment, the modified AchE enzyme lacks the N-terminal signal peptide and 40 amino acid residues at the C-terminal tail of the full length human AChE enzyme.
In some embodiments the amino acid sequence of the modified human AChE polypeptide as herein defined is denoted by SEQ ID NO: 8. Methods for preparing the modified human AChE polypeptide as herein defined based on the disclosed amino acid sequence thereof are well known in the art. For example, the modified human AChE polypeptide as herein defined may be prepared as described in Example 1 below. The modified AChE as well as the fusion polypeptide comprising the modified AChE retain the functional activity of human AChE.
The high reactivity of acetylcholinesterase towards Organophosphorus (OP) compounds renders exogenous acetylcholinesterase an effective scavenging agent in the prophylaxis of OP-poisoning. However, the use of acetylcholinesterase as a scavenging agent in the prophylaxis of OP-poisoning depends on the retention of the enzyme in the circulation for sufficiently long periods of time. As indicated above, recombinant choline esterases have rather short half lives in the circulation system, with a mean retention time of 60 minutes (3, 7).
As demonstrated in appended
Thus, as indicated above, the present invention provides a fusion polypeptide comprising an AChE polypeptide component (element) comprising a modified human AChE polypeptide and a fragment crystallizable (Fc) domain of human IgG.
The term “fusion polypeptide” in the context of the present invention concerns a sequence of amino acids, predominantly (but not necessarily) connected to each other by peptidic bonds. The term “fused” in accordance with the fusion polypeptide of the present invention refers to the fact that the amino acid sequences of at least two different origins, namely, the modified AChE as herein defined and the Fc domain of human IgG, are linked to each other by covalent bonds either directly or via an amino acid linker or spacer, joining (bridging, conjugating, covalently binding) the amino acid sequences. The fusion may be performed by chemical conjugation or by genetic engineering methods that are well known in the art, for example using the procedure to described below.
The term “polypeptide” as used herein refers to amino acid residues, connected by peptide bonds. A polypeptide sequence is generally reported from the N-terminal end containing free amino group to the C-terminal end containing free carboxyl group. A polypeptide may also be termed amino acid sequence, peptide, or protein and can be modified, for example, by manosylation, glycosylation, amidation, carboxylation or phosphorylation.
By the term “covalently linked” or “covalently linking” it is meant that the indicated domains are connected or linked by covalent bonds.
Fusion polypeptides based on the fragment crystallizable (Fc) domain of human IgG (Fc) are composed of an immunoglobulin Fc domain that is directly or indirectly linked to another peptide. It was previously reported that the presence of the Fc domain markedly increases the plasma half-life of the resulting fusion polypeptide, owing to its interaction with the salvage neonatal Fc-receptor (9). In the present invention, the Fc domain is directly or indirectly linked to the modified AChE.
In some embodiments the AChE polypeptide component as herein defined is covalently linked through its C-terminus to the Fc domain of human IgG. Namely, in some embodiments, in the N- to C-terminal direction, the fusion polypeptide according to the invention comprises the AChE polypeptide component and the Fc domain component.
In other embodiments the AChE polypeptide component as herein defined is covalently linked through its N-terminus to the Fc domain of human IgG. Namely, in some embodiments, in the N- to C-terminal direction, the fusion polypeptide of the invention comprises the Fc domain component and the AChE polypeptide component.
The term “fragment crystallizable (Fc) domain” (or Fc fragment) of human immunoglobulins G (IgG) as herein defined refers to the tail region of a human IgG antibody and encompasses native Fc and Fc variant molecules and sequences as defined herein below.
Human immunoglobulins are a group of structurally and functionally similar glycoproteins that confer humoral immunity in humans. As known in the art, the immunoglobulin protein “backbone” consists of two identical “heavy” and two identical “light” chains. Five classes of immunoglobulins (IgG, IgA, IgM, IgD, and IgE) have been distinguished. Human IgG subclasses are glycoproteins composed of two heavy and two light chains linked together by inter-chain disulfide bonds. The human IgG subclasses are further divided to IgG 1, 2, 3 and 4, which differ one from the other in their hinge region.
The term “Fc domain” includes molecules in a monomeric or a dimeric form (for example as in Immunoglobulin G) that may be digested from a whole antibody or produced by other means. In structural terms, the term Fc refers to a polypeptide that includes the hinge region, the heavy chain constant region 2 (CH2 domain) and the heavy chain constant region 3 (CH3 domain) of an immunoglobulin in an N-terminal to C-terminal direction.
In specific embodiments the Fc domain of human IgG is a monomeric polypeptide comprising the hinge region, the heavy chain constant region 2 (CH2 domain) and the heavy chain constant region 3 (CH3 domain) of an immunoglobulin in the N-terminal to C-terminal direction.
In some embodiments, the Fc domain is a native Fc domain of human IgG.
The term “native Fe” refers to a molecule or sequence comprising the amino acid sequence of a non-antigen-binding fragment resulting from digestion of a whole IgG antibody, whether in monomeric or dimeric form, at which a peptide may be added or conjugated by being covalently bound, directly or indirectly through a linker or a spacer, to the hinge region of the Fc domain of human IgG.
In some embodiments the fusion polypeptide according to the invention is wherein the Fc domain of human IgG is an Fc domain of IgG1 or of IgG2.
As indicated above, the present invention also encompasses variants of the modified AChE and variants of the Fc domain of human IgG.
By the term “variant” it is meant sequences of amino acids or nucleotides that are different from the sequences specifically identified herein, in which one or more amino acid residues or nucleotides are deleted, substituted or added, without affecting the functional activity of the original molecule (for example the functional activity of the human AChE or the functional activity of the Fc domain).
It should be appreciated that by the term “added”, as used herein it is meant any addition(s) of amino acid residues to the sequences described herein. For example, the variant antibodies of the invention may be extended at their N-terminus and/or C-terminus with various identical or different amino acid residues.
Variants also encompass various amino acid substitutions. An amino acid “substitution” is the result of replacing one amino acid with another amino acid which has similar or different structural and/or chemical properties Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
Variants also encompass conservative amino acid substitutions. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
Each of the following eight groups contains other exemplary amino acids that are conservative substitutions for one another:
1) Alanine (A), Glycine (G);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine (T); and
8) Cysteine (C), Methionine (M).
Conservative nucleic acid substitutions are nucleic acid substitutions resulting in conservative amino acid substitutions as defined above.
As used herein, the term “amino acid” or “amino acid residue” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
Variant sequences refer to amino acid or nucleic acid sequences that may be characterized by the percentage of the identity of their amino acid or nucleotide sequences to the amino acid or nucleotide sequences described herein (namely the amino acid sequence of or the nucleotide sequence encoding the modified AChE and Fc domain herein described).
In some embodiments, variant sequences as herein defined refer to nucleic acid sequences that encode the polypeptides as herein defined (namely the modified AChE or the Fc domain of human IgG), each having a sequence of nucleotides with at least 70% or 75% of sequence identity, around 80% or 85% of sequence identity, around 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of sequence identity when compared to the nucleic acid sequences that encode the modified AChE or the Fc domain of human IgG described herein.
In some embodiments, variant sequences as herein defined refer to the amino acid sequences of the polypeptides as herein defined (namely the modified AChE or the Fc domain of human IgG), each having a sequence of amino acid residues with at least 70% or 75% of sequence identity, around 80% or 85% of sequence identity, around 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of sequence identity when compared to the amino acid sequences of the modified AChE or the Fc domain of human IgG described herein.
In some embodiments the modified human AChE polypeptide as herein defined comprises an amino acid sequence that is at least 70% identical to the amino acid sequence denoted by SEQ ID NO: 8, wherein said human AChE polypeptide component retains the functional activity of human AChE.
In other embodiments the modified human AChE polypeptide as herein defined comprises an amino acid substitution in at least one position of SEQ ID NO: 8, wherein said human AChE polypeptide component retains the functional activity of human AChE.
An aging-resistant organophosphate bioscavenger based on polyethylene glycol-conjugated F338A human Acetylcholinesterase was previously reported (10). Therefore in further embodiments the modified human AChE polypeptide as herein defined comprises the amino acid Ala at a position corresponding to position 338 of the amino acid sequence denoted by SEQ ID NO: 8. In other words, in specific embodiments the amino acid Ala replaces the amino acid Phe at position 338 of the amino acid sequence denoted by SEQ ID NO: 8.
In yet further embodiments the modified human AChE polypeptide as herein defined consists of the amino acid sequence denoted by SEQ ID NO: 8.
By the term “the AChE polypeptide component retains the functional activity of human AChE” it is meant that the fusion polypeptide as herein defined is capable of hydrolyzing acetylcholine to a level comparable to that of human AChE. In other words, by this term it is meant that the fusion polypeptide as herein defined maintains to cholinesterase activity of free human AChE. Assays for determining cholinesterase activity are well known in the art. For example, the functional activity of fusion polypeptides prepared as herein described may be determined by an ELISA assay using Acetyl-thio-cholin (ATC) as substrate, as exemplified below, while comparing the enzymatic activity of the fusion polypeptide described herein to that of free human AChE.
By the term “a level comparable to that of human AChE” it is meant that the fusion polypeptides as herein defined retains at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% of the functional activity of free human AChE acetylcholine.
As indicated above, the Fc domain of human IgG also encompasses Fc variants of the Fc domain of human IgG.
The term “Fc variant” refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn. In some embodiments the term “Fc variant” encompasses a molecule or sequence that is humanized from a native Fc domain of a non-human IgG. The term “Fc variant” also contemplates a molecule or sequence that lacks one or more native Fc sites or residues that affect or are involved in (i) disulfide bond formation, (ii) incompatibility with a selected host cell (iii) N-terminal heterogeneity upon expression in a selected host cell, (iv) glycosylation, (v) interaction with complement, (vi) binding to an Fc receptor other than a salvage receptor, or (vii) antibody-dependent cellular cytotoxicity (ADCC).
Determining whether an Fc variant still comprises a binding site for the salvage receptor, FcRn may be performed by methods known to a person skilled in the art, for example by measuring its binding to recombinant FcRn molecules using ELISA, Octet or surface plasmon resonance (SPR).
In some embodiments the fusion polypeptide according to the invention is wherein the Fc domain comprises an amino acid sequence that is at least 70% identical to the amino acid sequence denoted by SEQ ID NO: 15 and wherein said Fc domain retains its functional activity (namely its binding to the salvage receptor).
As demonstrated by
Thus the fusion polypeptide according to the invention forms a dimer of two identical or different monomers, obtained by expression of the polypeptides as monomers that subsequently form stable dimers by non-covalent interactions. Thereby, a structure that is similar to the structural configuration of an antibody is obtained. A schematic presentation of a dimer of two identical monomers, where each one of the monomers comprises both the AChE and the Fc domain components, is demonstrated in
In some embodiments, the fusion polypeptide according to the invention is wherein said fusion polypeptide comprises a dimer of two identical monomers, wherein each one of the identical monomers comprises an acetylcholinesterase (AChE) polypeptide component and an Fc domain of human IgG.
The fusion polypeptide according to the invention in the form of a dimer of two identical monomers may be prepared using any method known in the art, for example, as detailed below.
While it is essential that the Fc domain will be configured in a dimeric form, the AChE polypeptide component may reside in the resulting fusion protein as a monomer.
Therefore in other embodiments the fusion polypeptide according to the invention is wherein said fusion polypeptide as herein defined comprises a dimer of a first monomer and a second monomer, wherein said first monomer comprises an AChE polypeptide component and an Fc domain of human IgG and the second monomer comprises an Fc domain of human IgG.
The fusion polypeptide according to the invention comprising a dimer in which the first monomer comprises an AChE polypeptide component and an Fc domain of human IgG and the second monomer comprises an Fc domain of human IgG may be prepared by expressing the fusion polypeptide according to the invention alongside with a free Fc (in the same host cell), allowing the formation of a protein consisting of two FC arms with one AChE covalently linked to one of the arms.
As indicated above, the present invention provides a fusion polypeptide in which the AChE polypeptide component and the Fc domain of human IgG are linked to each other by covalent bonds either directly or via an amino acid linker or spacer. In other words, the fusion polypeptide in the context of the present invention may also optionally comprise at least one linker or spacer covalently joining the different domains of the polypeptide protein construct.
Therefore in some embodiments the fusion polypeptide according to the invention further comprises a spacer covalently linking the AChE polypeptide component and the Fc domain of human IgG.
The term “spacer” in the context of the invention concerns an amino acid sequence of from about 4 to about 20 amino acid residues positioned between the modified AChE and the Fc domain of human IgG and covalently joining them together. For example, a spacer in accordance with the invention may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids long. Spacers are often composed of flexible amino acid residues, for example but not limited to glycine and serine so that the adjacent protein domains are free to move relative to one another. The term “spacer” can be interchangeably used with the term “linker”.
The design of a spacer that enables proper folding of the various domains of a protein is well known in the art. A non-binding example of a spacer is the amino acid sequence ASEAP (Ala-Ser-Glu-Ala-Pro) as denoted by SEQ ID NO. 9. This sequence was used in the Examples below to construct the fusion proteins. An additional example for a spacer that may be used in accordance with the invention is the spacer GGGS×n (where n can be 1, 2, 3, 4 or 5 depending on the desired linker length; corresponding to residues 1-4, residues 1-8, residues 1-12, residues 1-16, and residues 1-10 of SEQ ID NO:26, respectively).
The use of a spacer or a linker is optional and not mandatory. In the present invention, a spacer having the amino acid sequence ASEAP (denoted by SEQ ID NO: 9) was added to the C terminus of the modified human AChE polypeptide, thereby replacing the “tail unit” of AChE, in order to facilitate the secretion of AChE.
Therefore in some embodiments the spacer as herein defined comprises the amino acid sequence ASEAP denoted by SEQ ID NO: 9. In other embodiments the spacer as herein defined consists of the amino acid sequence ASEAP denoted by SEQ ID NO: 9.
In fact, when the modified human AChE polypeptide is linked to the Fc domain through the C-terminal end of modified AChE, a spacer is less needed since the hinge region of the Fc domain serves as a flexible linker. However, when the modified human AChE polypeptide is fused to the Fc domain through the N-terminal end of modified AChE, a longer linker will be necessary in order to obtain a more flexible fusion polypeptide.
In some further embodiments the spacer as herein defined is of the amino acid sequence GGGS×n (wherein n can be 1, 2, 3, 4 or 5 depending on the desired linker length; corresponding to residues 1-4, residues 1-8, residues 1-12, residues 1-16, and residues 1-10 of SEQ ID NO:26, respectively).
In specific embodiments the fusion polypeptide as herein defined comprises, in the N- to C terminus direction, an acetylcholinesterase (AChE) polypeptide component (or variant thereof), a spacer and an Fc domain of human IgG (or variant thereof). Such fusion protein may be prepared as detailed below and is schematically presented in
As shown in Example 3, a fusion polypeptide comprising the modified human AChE polypeptide, the spacer ASEAP and the Fc domain of human IgG was active, based on its ability to hydrolyze the Acetyl-thio-cholin (ATC) substrate (
Therefore in some specific embodiments the fusion polypeptide according to the invention comprises the amino acid sequence denoted by SEQ ID NO: 17.
In other specific embodiments the fusion polypeptide according to the invention consists of the amino acid sequence denoted by SEQ ID NO: 17.
In some embodiments the fusion polypeptide according to the present invention is an isolated or purified fusion polypeptide.
In another one of its aspects, the present invention provides an isolated nucleic acid construct comprising a nucleic acid sequence encoding the fusion polypeptide according to the invention. One of skill will appreciate that, utilizing the sequence information provided for the various regions of the fusion polypeptide as herein defined, nucleic acids encoding these sequences may be obtained using any methods well known in the art. For example nucleic acids constructs in accordance with the present invention may be prepared using the recombinant procedures described below.
The isolated nucleic acid constructs according to the invention may further comprise additional elements, for example promoters, regulatory and control elements (for example a signal peptide or a leader peptide), translation, expression and other signals, operably linked to the nucleic acid sequence encoding the fusion polypeptide of the invention.
By the term “operably linked” is meant that a nucleic acid sequence and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s).
The term “nucleic acid” or “nucleic acid construct” as herein defined refers to polymer of nucleotides, which may be either single- or double-stranded, which is a polynucleotide such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The terms should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides. The term DNA used herein also encompasses cDNA, i.e. complementary or copy DNA produced from an RNA template by the action of reverse transcriptase (RNA-dependent DNA polymerase). A nucleic acid sequence as well known in the art is given in the 5′ to 3′ direction.
In some embodiments the nucleic acid construct according to the invention is an isolated or purified nucleic acid construct.
As indicated above, the isolated nucleic acid constructs according to the invention may further comprise additional elements such as regulatory and control elements.
As detailed in Example 1 below, the AChE-Fc fusion protein described herein was prepared by adding a sequence encoding the Kappa-leader sequence (also referred to herein as “K”), having the amino acid sequence of MDMRAHVHLLGLLLLWLPGAKC (denoted by SEQ ID NO. 11, Table 2) to the 5′ end of the nucleic acid sequence encoding the modified AChE fusion polypeptide.
As detailed in Example 2 below, an additional nucleic acid construct encoding the AChE-Fc fusion protein was prepared by adding a sequence encoding the signal peptide of the full length human AChE (also referred to herein as “SP”), having the amino acid sequence of MRPPQCLLHTPSLASPLLLLLLWLLGGGVGA (denoted by SEQ ID NO. 12, Table 2) to the 5′ end of the nucleic acid sequence encoding the modified AChE fusion polypeptide.
The addition of a leader is mandatory for the secretion of the protein. Without wishing to be bound by theory, different leaders may affect the amount of protein secreted but will not affect its structure or activity.
Therefore, in some embodiments the isolated nucleic acid construct as herein defined further comprises a sequence encoding a secretion signal situated at the 5′ end of the nucleic acid sequence encoding the fusion polypeptide according to the invention.
The term “secretion signal” as herein defined refers to a signal peptide (also referred to as a signal sequence, targeting signal, localization signal, localization sequence, transit peptides leader sequence or leader peptide) which is a short (5-30 amino acids long) peptide present at the N-terminus of the majority of newly synthesized proteins that are destined towards the secretory pathway.
In some embodiments the isolated nucleic acid construct according to the invention comprises a secretion signal which is a kappa-leader sequence having the amino acid sequence denoted by SEQ ID NO: 11. In other embodiments the secretion signal is the native signal peptide of human AChE having the amino acid sequence denoted by SEQ ID NO: 12.
In some further embodiments the isolated nucleic acid construct according to the invention is of the nucleic acid sequence denoted by SEQ ID NO: 18. In still further embodiments the nucleic acid construct according to the invention is of the nucleic acid sequence denoted by SEQ ID NO: 19.
The present invention further provides an expression vector comprising the isolated nucleic acid construct as herein defined.
The term “expression vector”, also referred to as “expression vehicle” or “expression construct”, as used herein, encompasses vectors such as plasmids, viruses, bacteriophage, integratable DNA fragments, and other vehicles, which comprise nucleic acid sequences encoding the desired polypeptide and enable its expression in a host cell. Expression vectors are typically self-replicating DNA or RNA constructs containing the desired gene or its fragments, and operably linked genetic control elements that are recognized in a suitable host cell and effect expression of the desired genes. These control elements are capable of effecting expression within a suitable host. The expression vector in accordance with the invention may be competent with expression in bacterial, yeast, or mammalian host cells, to name but few.
For example, the fusion polypeptide according to the present invention was prepared by incorporating the nucleic acid construct encoding thereof into a mammalian expression vector, as detailed below. The mammalian expression vector (also referred to herein as the “plasmid”) comprising the nucleic acid sequence of the fusion polypeptide was transiently transfected to FreeStyle HEK293 cells and the supernatant was collected after seven days.
The present invention further provides an isolated host cell comprising the nucleic acid construct or the expression vector according to the present invention.
The term “host cells” as used herein refers to cells which are susceptible to the introduction of the isolated nucleic acid construct or the expression vector according to the invention. Preferably, said cells are mammalian cells, for example CHO cells, or HEK 293 cells.
Any of the well known procedures for introducing foreign nucleotide sequences into host cells (transfection) may be used.
As detailed in Example 1 below, the AChE-Fc fusion protein construct according to the invention was prepared by fusing a modified AChE, which lacks both its N-terminal signal peptide and its C-terminal tail, to the Fc domain of human IgG1 using the K-leader sequence as a secretion signal sequence. Example 2 described the preparation of a AChE-Fc fusion protein construct according to the invention prepared using the signal peptide sequence of native AChE.
The activity of the AChE-Fc fusion polypeptide prepared as herein described is evidenced from
In addition, the AChE fusion polypeptide as herein defined was shown to have similar kinetic parameters as those of the native AChE enzyme, in an in vitro kinetic analysis, as detailed in Example 5 below.
Therefore, in another one of its aspects the present invention provides a method of producing the fusion polypeptide as herein defined, comprising culturing the host cell as herein defined under conditions suitable for expression of the fusion polypeptide in the host cell and recovering the fusion polypeptide thereby produced.
Laboratory techniques for culturing host cells are well known in the art. Cells are generally grown and maintained at an appropriate temperature and gas mixture (typically, 37° C., 5% CO2 for mammalian cells) in a cell incubator. Culture conditions vary for each host cell type.
Any conditions suitable for expression of the fusion polypeptide in the host cell are encompassed by the present invention. As detailed below, the HEK293 cells transfected with the expression vector carrying the fusion polypeptide as herein defined were grown for 7 days under standard growth conditions.
Any of the well known procedures for recovering the fusion polypeptide as herein defined may be used. In some embodiments, cell-culture supernatants may be adsorbed to procainamide SEPHAROSE (a cross-linked, beaded form of agarose) 4B columns (4000 units/ml resin which are then rinsed with 50 mM sodium phosphate buffer, pH 8.0/1 mM EDTA and again with 50 mM sodium phosphate buffer, pH 8.0/0.4 M NaCl/1 mM EDTA. Elution of the fusion polypeptide as herein defined may be performed for example with decamethonium (0.02 M) in 50 mM sodium phosphate buffer, pH 8.0/1 mM EDTA.
In order to verify the activity and integrity of the obtained fusion polypeptide prepared as described herein, standard methods well-known in the art may be employed, as for example the ELISA assay and the kinetic assay described herein.
The present invention further provides a pharmaceutical composition comprising the fusion polypeptide according to the invention and a pharmaceutically acceptable carrier.
The term “pharmaceutical composition” as herein defined comprises the fusion polypeptide according to the invention as the active agent and a buffering agent, an agent which adjusts the osmolarity of the composition and optionally, one or more pharmaceutically acceptable carriers, excipients and/or diluents as known in the art. Supplementary active ingredients can also be incorporated into the compositions, e.g. additional prophylaxis or therapeutic agents.
Any known pharmaceutically acceptable carrier may be used for preparing the pharmaceutical composition according to the invention. For example, the term “pharmaceutically acceptable carrier, excipient or diluent” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents and the like, as known in the art. The carrier can be solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the subject. Except as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated.
As indicated above, organophosphate poisoning (OP poisoning) results from exposure to organophosphorus (OP) compounds, which cause the inhibition of acetylcholinesterase (AChE), leading to the accumulation of acetylcholine (ACh) in the body. The health effects associated with organophosphate poisoning are a result of excess acetylcholine (ACh) present at different nerves and receptors in the body. For example, accumulation of ACh at motor nerves causes overstimulation of nicotinic expression at the neuromuscular junction.
Organophosphorus (OP) compounds (organic compounds that contain a carbon-phosphorus bond) are a diverse group of chemicals that include, among others, insecticides, antihelmintics (drugs that are used for killing parasitic worms) and nerve gases. For example, organophosphorus (OP) compounds are, but not limited to insecticides (for example malathion, parathion, diazinon, fenthion, etc.), nerve gases (for example soman, sarin, tabun, VX, etc.), ophthalmic agents (for example echothiophate, isoflurophate, etc.), antihelmintics such as trichlorofon and herbicides (for example tribufos, merphos, etc.).
A possible strategy to prevent the toxic manifestations of OP poisoning is to sequester OP compounds in the circulation using exogenously administered AChE, thereby detoxifying them before they can inhibit the endogenous AChE. However, and as indicated above recombinant choline esterases have short half lives in the circulation system.
Surprisingly, as described below, the circulatory half-life of the fusion polypeptide AChE-Fc as herein defined was extremely longer when compared to the half-life of free AChE (Table 3 below and
Furthermore, the reactivity of the AChE-Fc fusion polypeptide, prepared as herein described, against the organophosphorus compounds VX and Sarin was verified as detailed in Table 4 below.
Therefore in another one of its aspects the present invention provides the fusion polypeptide or the pharmaceutical composition according to the invention for use in prophylaxis of organophosphate poisoning.
The term “prophylaxis” as herein defined refers to acting in a protective manner, to defend against or prevent from organophosphate poisoning.
In a further aspect the present invention provides a method of prophylaxis of organophosphate poisoning comprising administering an effective amount of the fusion polypeptide or the pharmaceutical composition according to the invention to a subject in need thereof.
Exposure to OP compounds may occur on a daily basis through inhalation, absorption, and ingestion, most commonly of food that has been treated with an organophosphate herbicide or insecticide. Exposure to OP compounds may also occur during war.
Therefore, the term “subject in need thereof” in the context of the present invention means warm-blooded animals, such as for example household animals or farm animals (e.g. dogs, cats, cattle, sheep, horses etc) and humans at risk of being exposed to OP compounds or anyone who is at a risk of coming in contact with OP compounds, for example farmers, agronomists, laboratory professionals and military personnel.
In specific embodiments the fusion polypeptide, the pharmaceutical composition comprising thereof or its use in a method according to the present invention is wherein said fusion polypeptide comprises the amino acid sequence denoted by SEQ ID NO: 17.
In further specific embodiments the fusion polypeptide, the pharmaceutical composition comprising thereof or its use in a method according to the present invention is wherein said fusion polypeptide consists of the amino acid sequence denoted by SEQ ID NO: 17.
Administration according to the present invention may be performed by any of the following routes: oral administration, intravenous, intramuscular, intraperitoneal, intratechal or subcutaneous injection, intrarectal administration, intranasal administration, ocular administration or topical administration. In preferred embodiments the administration is performed by intravenous or intramuscular injection.
In specific embodiments the fusion polypeptide or the pharmaceutical composition according to the invention is administered to the subject between about 30 days to about 1 minute before potential exposure to OP compounds.
In some embodiments the method of prophylaxis of organophosphate poisoning according to the invention further comprises administering an effective amount of at least one additional therapeutic agent as herein defined. In other specific embodiments the fusion polypeptide or the pharmaceutical composition as herein defined is administered with at least one additional therapeutic agent.
In some embodiments the fusion polypeptide or pharmaceutical composition comprising same as herein defined is administered concomitantly with the at least one additional therapeutic agent as herein defined. In other embodiments the fusion polypeptide or pharmaceutical composition comprising same as herein defined is administered before the administration of the at least one additional therapeutic agent as herein defined.
Currently, the standard medical therapy administered after exposure to OP compounds includes a muscarinic antagonist (usually atropine), an acetylcholinesterase reactivator (for example pralidoxime, 2-PAM), and benzodiazepines (for example diazepam).
Therefore in some embodiments the at least one additional therapeutic agent is selected from atropine, glycopyrrolate, benzodiazepines, pralidoxime and native cholinesterase(s).
The term “atropine” as known in the art refers to an agent indicated for temporary blockade of severe or life threatening muscarinic effects, e.g., as an antisialagogue, an antivagal agent, an antidote for organophosphorus or muscarinic mushroom poisoning, and to treat bradyasystolic cardiac arrest.
The term “glycopyrrolate” as known in the art refers to an anticholinergic agent.
Benzodiazepines as known in the art enhance the effect of the neurotransmitter gamma-aminobutyric acid (GABA) at the GABA receptor, resulting in sedative, hypnotic (sleep-inducing), anxiolytic (anti-anxiety), anticonvulsant, and muscle relaxant properties. These properties make benzodiazepines useful in treating anxiety, insomnia, agitation, seizures, muscle spasms, alcohol withdrawal and as a premedication for medical or dental procedures.
“Pralidoxime” (2-pyridine aldoxime methyl chloride or 2-PAM) belongs to a family of compounds called oximes that bind to organophosphate-inactivated acetylcholinesterase. It is known in the art for its use against poisoning by organophosphates or acetylcholinesterase inhibitors (nerve agents) in conjunction with atropine and diazepam.
The term “native cholinesterase” or “non fused cholinesterase” as herein defined refers to any native cholinesterase known in the art that may be used in conjunction with the fusion polypeptide as herein defined, for example but not limited to human AChE.
In some embodiments, administering at least one additional therapeutic agent directed against organophosphate compounds or counteracting their effect is performed as a further post exposure therapy step.
Thus, in specific embodiments, the additional therapeutic agent is administered after the exposure to organophosphates (or after exposure to organophosphate poisoning).
By the term “after the exposure to organophosphates” it is meant that the additional therapeutic agent as defined above is administered at least about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60 minutes or more after exposure to organophosphates. The at least one additional therapeutic agent as defined above may be formulated for self-administration.
The “effective amount” of the fusion polypeptide per se or comprised in the pharmaceutical composition as herein defined may be determined by a skilled person by considerations well known in the art.
The present invention further provides for a method of increasing the circulatory half-life of AChE, said method comprising preparing a fusion polypeptide comprising:
The term “circulatory half-life” with reference to AChE as herein defined refers to the time required for half of the AChE molecules administered to an organism to be metabolized or eliminated by normal biological processes.
The circulatory half-life of AChE or of the fusion polypeptide comprising AChE polypeptide component as herein defined may be measured using any method known to a person of skill in the art. For example and as exemplified herein below, the circulatory half-life of AChE may be measured in animals (for example mice) injected with the fusion polypeptide, where mice injected with the native AChE are used as control.
Blood samples are taken from these animals at different intervals, for example between 45 sec to 70 hours after injection. The blood samples withdrawn from the animals are processed and used for an ELISA assay to determine the presence of the fusion polypeptide comprising AChE polypeptide component as herein defined or the presence of native AChE.
By still another one of its aspects the present invention provides a fusion polypeptide as herein defined or a pharmaceutical composition comprising the fusion polypeptide as herein defined for preparing a medicament for the prophylaxis of organophosphate poisoning.
In specific embodiments the present invention provides a fusion polypeptide comprising the amino acid sequence denoted by SEQ ID NO: 17 or a pharmaceutical composition comprising said fusion polypeptide for preparing a medicament for the prophylaxis of organophosphate poisoning.
In further specific embodiments the present invention provides a fusion polypeptide consisting of the amino acid sequence denoted by SEQ ID NO: 17 or a pharmaceutical composition comprising said fusion polypeptide for preparing a medicament for the prophylaxis of organophosphate poisoning.
The present disclosure further provides a kit comprising:
(i) at least one fusion polypeptide comprising:
In some embodiments the kit as herein defined further comprises at least one additional therapeutic agent according to the present invention.
In some embodiments the kit as herein defined comprises the at least one fusion polypeptide in a first unit dosage form and the at least one additional therapeutic agent in a second unit dosage form.
The at least one fusion polypeptide and the at least one additional therapeutic agent may be administered to a subject in need thereof concomitantly or separately, before or after exposure of the subject to organophosphate poisoning.
It is appreciated that the term “purified” or “isolated” refers to molecules, such as amino acid or nucleic acid sequences, peptides, polypeptides or antibodies that are removed from their natural environment, isolated or separated. An “isolated fusion polypeptide”, “an isolated nucleic acid construct” or an “isolated host cell” is therefore a purified fusion polypeptide, nucleic acid construct or host cell, respectively. As used herein, the term “purified” or “to purify” also refers to the removal of contaminants from a sample.
The term “about” as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range.
When used in connection with an amino acid sequence, the term “comprising” means that a compound may include additional amino acid residues on either or both of the N- or C-termini of the given sequence.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present disclosure to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the claimed invention in any way.
Standard molecular biology protocols known in the art not specifically described herein are generally followed essentially as in Sambrook & Russell, 2001.
Standard medicinal chemistry methods known in the art not specifically described herein are generally followed essentially in the series “Comprehensive Medicinal Chemistry” by various authors and editors, published by Pergamon Press.
Primers
The primers designed and used for constructing the acetylcholinesterase (AChE) Fc-fusion protein described below are detailed in Table 1 below. All of the primers were synthesized by Integrated DNA Technologies (IDT).
Assembly PCR
In order to construct a fusion protein that comprises the human Acetylcholine esterase enzyme linked to the Fc portion of an antibody, several different components were amplified from different sources and then assembled together by PCR, as detailed below.
Human AChE was amplified from the AChE vector (7) using primers having the nucleic acid sequences denoted by SEQ ID NO: 1 and SEQ ID NO: 3 (Table 1 above). The nucleic acid sequence encoding the AChE enzyme was amplified from the AChE vector without the sequence encoding the AChE N-terminal signal peptide and without the sequence encoding the last (C-terminal) 40 amino acids that comprise the AChE enzyme “tail”. The AChE enzyme tail is involved in the formation of AChE tetramers (7). This procedure resulted in a nucleic acid sequence encoding a polypeptide having the amino acid sequence denoted by SEQ ID NO. 8 (Table 2), also termed herein “modified AChE”. In other words, the procedure described above resulted in a nucleic acid sequence encoding a polypeptide fragment of the human AChE enzyme, which does not include the N-terminal signal peptide and from which a 40 amino acid-long C-terminal tail was deleted.
Then, a nucleic acid sequence encoding a five amino acids spacer, namely ASEAP (denoted by SEQ ID NO. 9, Table 2) was added to the 3′ end of the nucleic acid construct encoding the modified AChE. This procedure resulted in a nucleic acid sequence encoding a polypeptide having the amino acid sequence denoted by SEQ ID NO. 10 (Table 2), namely the polypeptide resulting from fusing the peptide spacer of the amino acid sequence ASEAP to the C terminus of modified AChE.
A second PCR amplification was used to add a sequence encoding the Kappa-leader sequence (also referred to herein as “K”), having the amino acid sequence of MDMRAHVHLLGLLLLWLPGAKC (denoted by SEQ ID NO. 11, Table 2) to the 5′ end of the sequence encoding the modified AChE (which is fused at its C terminus to the spacer), using primers having the nucleic acid sequences denoted by SEQ ID NO: 2 and SEQ ID NO: 4 (Table 1 above). This step resulted in a nucleic acid sequence encoding a polypeptide having the amino acid sequence denoted by SEQ ID NO: 13 (Table 2).
Alternatively, a nucleic acid sequence encoding the signal peptide of native human AChE, having the amino acid sequence denoted by SEQ ID NO: 12 (Table 2) was added to the 5′ end of the sequence encoding the modified AChE (that in turn is conjugated to the spacer at its C terminus).
The hinge region of an IgG1 antibody (having the amino acid sequence defined by SEQ ID NO: 14, Table 2) was amplified from a mammalian (human) cDNA) full-length Ig expression vector designed by the inventors, using primers having the nucleic acid sequences denoted by SEQ ID NO: 5 and SEQ ID NO: 7 (Table 1). A cysteine residue that facilitates the covalent linkage at the hinge region between the heavy and the light chains of IgG1, was replaced by a serine, as detailed below, in order to prevent non-specific bonding.
The cysteine to serine substitution was performed by a point mutation inserted in the hinge region of IgG1 at position 1723 of the nucleic acid sequence denoted by SEQ ID NO: 18 (Table 2 and
The nucleic acid encoding the kappa-leader sequence followed by the modified AChE enzyme followed by the spacer (namely the nucleic acid sequence encoding the polypeptide denoted by SEQ ID NO: 13, Table 2) was then assembled 5′ to the IgG1 hinge region by PCR (under the conditions of a single cycle of 2 min at 95° C., 35 cycles of 1 min at 94° C., 30 sec at 57° C. and 1.5 min at 72° C. and a final single cycle of 5 min at 72° C.), using primers having the nucleic acid sequences denoted by SEQ ID NO: 6 and SEQ ID NO: 7 (Table 1). The primers were also designed to add appropriate restriction sites to the leader-enzyme sequence.
Cloning
A mammalian expression vector, containing the heavy-chain of human IgG1 (denoted by SEQ ID NO: 20, Table 2), under the control of the CMV promotor (designed by the inventors), was digested with the SacI/AleI restriction enzymes (Thermo scientific). This restriction removed the variable region, CH1 and hinge regions and the heavy chain leader. The nucleic acid sequence of the K-modified AChE-spacer-hinge (encoding the amino acid sequence denoted by SEQ ID NO: 13 fused N-terminal to the amino acid sequence denoted by SEQ ID NO: 14, Table 2) was also digested with the same restriction enzymes and then ligated to the digested vector, resulting in cloning (fusion) of the K-modified AChE-hinge N-terminal to the Fc portion (having the amino acid sequence denoted by SEQ ID NO: 15, Table 2). This step resulted in a nucleic acid having the sequence denoted by SEQ ID NO: 18 (Table 2 and
The nucleic acid encoding a fusion protein comprising the native signal peptide of human AChE, the modified AChE and the spacer unit and Fc domain described above is denoted by SEQ ID NO: 19.
Single-stranded DNA of the fusion construct was prepared using Big Dye (Applied Biosystems) and the PCR products were analyzed with ABI PRISM 310 Genetic Analyzer (Applied Biosystems) to verify the integrity of the construct. Sequencing of the nucleic acid of the construct encoding the fusion polypeptide comprising the K-modified AChE-spacer-Fc domain, also referred to herein as the “K-NL1 fusion protein” confirmed that it comprises all the desired portions (namely the Kappa-leader sequence, the modified AChE, the spacer and the Fc domain including the hinge region, as described above). ELISA assay was used to confirm that the AChE part of the protein is active, as described below.
Polypeptide Expression and Purification
The plasmid (40 μg) comprising the nucleic acid sequence of the K-NL1 fusion construct was transiently transfected to FreeStyle HEK293 cells (30 ml, 1×106 cells/ml) (Life technology) and the supernatant was collected after seven days. Cell-culture supernatants were adsorbed to procainamide SEPHAROSE (a cross-linked, beaded form of agarose) 4B columns (4000 units/ml resin) which were then rinsed with 50 mM sodium phosphate buffer, pH 8.0/1 mM EDTA and again with 50 mM sodium phosphate buffer, pH 8.0/0.4 M NaCl/1 mM EDTA. Enzyme elution was performed with decamethonium (Sigma, 0.02 M) in 50 mM sodium phosphate buffer, pH 8.0/1 mM EDTA. Leader sequences are cleaved off prior to secretion and therefore the resulting protein, named the “NL1 fusion protein”, comprised the modified AChE fused at the N-terminal to the Fc domain, where the modified AChE and the Fc domain are covalently linked by the spacer ASEAP. The amino acid sequence of the NL1 fusion protein is denoted by SEQ ID NO: 17 (Table 2 above). The concentration of each enzyme was determined using 7-(methylethoxyphosphinyloxy)-1-methylquinolinium iodide (MEPQ, prepared in-house as previously described (3)) titration. Briefly, active site titration of enzyme solutions was performed in the presence of 0.1 mg/ml BSA in 50 mM sodium phosphate buffer, pH 8.0, by adding various amounts of MEPQ Inhibition was allowed to proceed to completion and the residual activity was plotted against the concentration of inhibitor.
ELISA Assay
In order to assess the activity of the AChE after fusion to Fc (namely the NL1 fusion protein) and to verify that the obtained fusion AChE enzyme indeed comprises the Fc domain at the protein level, an ELISA assay was performed as described below. Maxisorp 96-well microtiter plates (Nunc) were coated with 2 μg/ml anti-human Fc antibody (50 μl/well, Goat anti-human IgG, FC specific, Sigma #I3391). The plates were then washed and blocked with PBST buffer (0.05% Tween 20, 2% BSA in PBS) at room temperature for one hour. NL1 fusion protein samples, directly obtained from the cell-culture supernatant or purified fractions (0.4-10 pM) were added to the wells and incubated for another hour. Elman's substrate mix (comprising 50 mM phosphate buffer pH 8, 0.1 mg/ml BSA, 1 mM Acetyl-thio-cholin (ATC), Sigma #A5751) and 0.6 mM dithiobisnitro-benzoate (DTNB, Sigma #D8130) was prepared and 100 μl were added to each well at the end of the incubation. This mix serves as a substrate for AChE and thus allows monitoring of its activity. The substrate hydrolysis was monitored by repeated spectrophotometric readings (412-650 nm) for 5 min, at 45 sec intervals using a spectrophotometer (VERSAmax microplate reader, Molecular Devices).
Western Blot Analysis
A sample of HEK293 cells expressing the NL1 fusion protein (namely cells transfected with the plasmid encoding the K-NL1 fusion protein), was boiled in 1× sample buffer (Bio-Rad) with or without β-mercaptoethanol, and loaded onto 4-12% pre-casted SDS-PAGE gel (Invitrogen). Antibodies used for detection: 1:100 mouse anti-HuAChE followed by 1:1000 rabbit anti-mouse IgG-AP (Sigma #A1902), or 1:1000 goat anti-human IgG (Fc specific) (Sigma #A9544).
Organophosphate Inhibitors
Sarin (O-isopropyl methylphosphonofluoridate) and VX (O-ethyl-S-(2-isopropylaminoethyl) methylphosphonothiolate, were prepared as previously described. (3). The purity of the OPs (>95%) was determined by 1H and 31P NMR spectroscopy. Stock solutions were kept at −20° C., and diluted in sodium phosphate buffer to the desired concentration, prior to use.
Kinetic Studies
AChE enzymatic activity was assayed as described before (11) in the presence of AChE substrate buffer (0.1 mg/ml BSA, 0.3 mM 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB), 50 mM sodium phosphate buffer (pH 8.0), and 0.5 mM Acetyl-thio-cholin (ATC, sigma) at 27° C. and monitored with a Thermomax microplate reader (Molecular Devices). Measurements of phosphorylation rates were carried out by monitoring residual activity (E) at various time points, following incubation of the enzyme in the presence of at least four different concentrations of an OP-inhibitor (I). The apparent bimolecular phosphorylation rate constants (ki) determined under pseudo first-order conditions were computed from the plot of slopes of ln(E) versus time at different inhibitor concentrations (12). Rate constants under second order conditions were determined from plots of ln {E/[I0−(E0−E)]} versus time.
Inhibition constants (KO by AChE and AChE-Fc were assayed as described before (3), by monitoring residual activity at various time points, after incubation of the enzymes in the presence of at least three different concentrations of propidium (3,8 diamino-5-3′-(trimethylammonium)propyl-6-phenylphenanthridniumiodide (Sigma) or BW284C51 (di(p-allyl-N-methylaminophenyl)pentan) (Sigma).
In Vivo Kinetics
Female outbred ICR mice (Charles River Laboratories) were maintained at 20-22° C. and a relative humidity of 50±10% on a 12-h light/dark cycle, fed with commercial rodent chow (Koffolk Inc.) and provided with tap water ad libitum. Treatment of animals was in accordance with regulations outlined in the USDA Animal Welfare Act and the conditions specified in Guide for Care and Use of Laboratory Animals (National Institute of Health, 1996). Animal studies were approved by the local ethical committee on animal experiments.
Pharmacokinetic experiments in mice (three mice, 26-28 gr, per enzyme sample) were carried out essentially as described previously (3). Briefly, mice were injected intravenously with native human AChE (HuAChE, to reach 30-fold increase over endogenous background levels) or with AChE-Fc in 0.2 ml PBS. At different time points, blood samples (5 μl) were drawn from the tail vein, diluted 20-fold in PBS, and centrifuged for three minutes at 3000 rpm for the removal of red blood cells. The levels of native HuAChE in each sample were determined as described above and expressed as the percent of the initial concentration at time zero (background levels of endogenous AChE activity were subtracted from all measurements). The levels of AChE-Fc in each sample were determined using captured ELISA, as follows: Maxisorp 96-well microtiter plates (Nunc, Roskilde, Denmark) were coated overnight with 5 μg/ml of anti-human Fc F(ab)2 fragments antibody (sigma; 50 μl/well) in NaHCO3 buffer (50 mM, pH 9.6), washed and blocked with PBST buffer at room temperature for one hour. Samples were serially diluted in PBST, added to the coated plates and incubated for one hour at 37° C. Plates were then washed with PBST, incubated with the AChE substrate buffer and color formation was monitored as described below. Values of AChE-Fc are expressed as the percent of the initial concentration at time zero. Pharmacokinetic parameters were calculated using the Prism software (GraphPad Software Inc., USA).
Preparation of an AChE-Fc Fusion Protein Using a K-Leader Sequence
As indicated above, choline esterases have short half lives in the human circulation system. In order to prolong the circulatory life-time of Acetylcholinesterase (AChE), an AchE was fused to the Fc domain of human IgG1, as described above.
First, a AChE-Fc fusion protein construct was prepared by fusing a modified AChE, which lacks both its N-terminal signal peptide and its C-terminal tail, to the Fc domain of human IgG1 using the K-leader sequence as a secretion signal sequence.
Briefly, a modified AChE was prepared by deleting the N-terminal signal peptide and the C-terminal 40 amino acid residues from the native human AChE enzyme (the amino acid of the native human AChE is denoted for example by SEQ ID NO: 21). The resulting modified AChE (denoted by SEQ ID NO: 8) was fused through its C-terminus to a short peptide spacer having the amino acid sequence ASEAP (denoted by SEQ ID NO: 9, Table 2), resulting in a polypeptide construct having the amino acid sequence denoted by SEQ ID NO: 10 (Table 2).
A sequence encoding the human Kappa-leader sequence having the amino acid sequence denoted by SEQ ID NO: 11 (Table 2) was then added to the 5′ end of the sequence encoding the modified AChE that is in turn linked to the spacer, thereby obtaining the polypeptide K-Modified AChE-spacer, the amino acid sequence of which is denoted by SEQ ID NO: 13 (Table 2).
Fusion of the above polypeptide to the Fc domain of human IgG1 was performed in a two-step procedure, as described above, resulting in a fusion polypeptide (also termed herein the “NL1 fusion protein”) that comprises from it N-terminal to it C-terminal end the modified AChE, and the Fc domain of human IgG1 linked via a spacer (ASEAP) situated between the modified AChE and the Fc domain of human IgG1.
The amino acid sequence of the above modified AChE-Fc fusion protein comprising the spacer is shown in
Sequencing of NL1 fusion protein confirmed that it comprises all of the desired portions (AChE, Fc).
As shown in
A schematic presentation (ribbon diagram) of the NL1 fusion protein is provided in
Preparation of an AChE-Fc Fusion Protein Using the Signal Peptide of Native AChE
A construct containing the NL1 protein, linked to the AChE native signal peptide instead of the Kappa leader (also termed herein the SP-NL1 fusion protein), was synthesized by Integrated DNA Technologies (IDT). In other words, the K-leader sequence was replaced by the native AChE signal peptide. The amino acid sequence of the AChE signal peptide is denoted by SEQ ID NO: 12.
The construct was cloned to the expression vector, as described above for K-NL1, and transiently transfected into HEK293 cells. The nucleic acid sequence construct encoding the SP-NL1 fusion protein is denoted by SEQ ID NO: 19 (Table 2) and is shown in
As demonstrated in
ELISA Activity Assay of the AChE-Fc Fusion Protein
First, in order to verify that the obtained fusion AChE enzyme indeed comprises the Fc domain at the protein level and that the resulting fusion polypeptide is active, an ELISA assay was performed as described above.
As shown in
Pharmacokinetic Analysis of the AChE Fusion Protein in Mice
The therapeutic use of the AChE-Fc fusion protein prepared as described above requires both an efficient enzymatic function as well as extended plasma half-life. In order to assess the pharmacokinetic characteristics (half-life) of the AChE fusion protein prepared as described above it was first necessary to establish a specific and sensitive assay that will enable monitoring the circulatory levels of the AChE-Fc with minimal interferences from endogenous AChE that naturally resides in the blood. To that end, a functional capture ELISA assay was designed, as detailed above.
Briefly, plates were coated with antibodies directed against the human Fc for immobilization of the AChE-Fc present in blood samples, as schematically shown in
The above functional capture ELISA assay was then used for assessing the AChE-Fc fusion protein blood levels in vivo, in mice. As detailed above, AChE-Fc, as well as HuAChE (the native AChE), were administered intravenously to mice, and their pharamacokinetic profiles were determined.
As shown in
aPresented data is average ± SEM of 3 mice for each enzyme.
bThe percentage each term contributes to the area under the curve.
The prolonged half-life of the AChE-Fc fusion protein prepared as described above provides a clear prophylactic potential for scavenging compounds targeting the AChE enzyme (e.g. organophosphate compounds) from the circulatory system for as long as 60 hours, or more, after its injection.
Kinetic Analysis of the AChE Fusion Protein
In order to verify that the AChE-Fc fusion protein has retained the catalytic activity of the native AChE enzyme, an in vitro kinetic analysis was performed as described above and it was found that both enzymes share the same kinetic hydrolysis parameters toward Acetyl-thio-cholin (ATC, Table 4).
In order to further evaluate whether the structure of the enzymatic moiety remained intact, the interactions of AChE-Fc with propidium, a peripheral anionic site ligand and with the bis-quaternary inhibitor BW284c51 whose binding site spans both the peripheral and the active-center gorge, were measured. The fusion protein displayed high affinity toward these two ligands, with similar values as the native enzyme (Table 4).
In addition, the bioscavenging potential of AChE-Fc toward various nerve agents was examined. To that end, the reactivity of the fusion protein towards sarin, a representative of the “G-agents” oragnophosphonates was determined. The apparent bimolecular rate constant (ki) of AChE-Fc towards sarin was found to be 11.5×105M−1 min, indicating that it retained its full bioscavenging activity as the HuAChE (Table 4). Similarly, both AChE-Fc and HuAChE exhibit similar inhibition rate constants toward VX, a charged oragnophosphonate of the “V-agents”, with ki of 400 and 450×105M−1 min−1, respectively (Table 4).
aEnzyme inhibition constant
bApparent bimolecular rate constant for phosphylation
Taken together the above results demonstrate that the AChE-Fc conjugate polypeptide maintained its reactivity towards various ligands and organophosphates and is a potential candidate as a prophylactic and scavenging agent against compounds targeting the AChE enzyme.
| Number | Name | Date | Kind |
|---|---|---|---|
| 20090249503 | Rosendahl | Oct 2009 | A1 |
| Number | Date | Country |
|---|---|---|
| 02087624 | Nov 2002 | WO |
| 03061562 | Jul 2003 | WO |
| WO 2011005621 | Jan 2011 | WO |
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| Number | Date | Country | |
|---|---|---|---|
| 20160289657 A1 | Oct 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62142188 | Apr 2015 | US |
