KR20140137211A - Compositions, methods and uses for alpha-1 antitrypsin fusion molecules - Google Patents

Abstract

The present invention relates to a recombinant α-1 antitrypsin (hereafter ′AAT′) composition. The recombinant AAT of the present invention can be purified easier than that of other types and has more increased anti-inflammatory activities and immune activities compared to natural AAT or other commercial AAT products. In addition, the recombinant AAT (rAAT) whose amount is ten times less than that of current AAT is used for preventing or treating disease as an alternative to the current AAT.

Description

FIELD OF THE INVENTION [0001] The present invention relates to compositions, methods and uses for alpha 1 antitrypsin fusion molecules,

The present invention relates to a composition of recombinant alpha-1 antitrypsin (hereinafter referred to as 'AAT'). The recombinant AAT of the present invention is more readily purified than other forms of AAT and has increased anti-inflammatory and anti-immunological activity compared to natural AAT or other commercial AAT preparations. Also, less than 10-fold less recombinant AAT (rAAT) may be used as an alternative to AAT in its current form for the prevention or treatment of disease.

In general, the normal serum level of AAT (alpha-1 antitrypsin) ranges from 1.3 to 3.5 mg / ml. Under certain conditions, AAT readily diffuses into the tissue space and forms a 1: 1 complex with the target protein, primarily neutrophil elastase. Other enzymes such as trypsin, chymotrypsin, cathepsin G, plasmin, thrombin, tissue kallikrein, and factor Xa may also act as substrates. The enzyme / inhibitor complex then binds to the SEC (serpin-enzyme complex) receptor and is removed from the circulatory system and metabolized in the liver and spleen.

Anti-inflammatory activity of AAT (alpha-1 antitrypsin) has traditionally been thought of as their ability to inhibit serine proteases, particularly neutrophil elastase. It is based on its use in the treatment of patients with AAT deficiency. AAT, which is currently used for human use, has been standardized by an anti-elastase unit, not for other AAT-related activities. These commonly available formulations are purified from the collected human plasma but these are not pure because they contain other serum proteins. Most studies on human AAT in in vivo models as well as in vivo have each depended on the use of these normally useful formulations according to the serine protease inhibitory activity approved for use in humans. In humans with multiple AAT deficiencies, AAT infusion is considered safe, but the role of contaminated proteins remains unknown. Some embodiments of the present invention report the rapid production of recombinant forms of AAT with high purity and high activity to overcome the problems of contaminated, co-purified serum proteins.

One drawback of AAT therapy is that normally useful AATs are isolated from human blood donors and thus are restricted to sera that may be available for supply. The use of therapeutic AAT is growing because its application is not limited to current availability such as chronic pulmonary obstructive disease (COPD) and AAT replacement therapy.

[Prior Patent Literature]

Korean Patent Publication No. 1020060010740

The present invention has been made in view of the above needs, and an object of the present invention is to provide a composition effective for treating AAT deficiency disorder or AAT reactive disease.

In order to accomplish the above object, the present invention provides a nucleic acid encoding alpha-1 antitrypsin (AAT); And a nucleic acid coating an immunoglobulin fragment comprising a modified hinge region.

In one embodiment of the present invention, the nucleic acid construct preferably has the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, but is not limited thereto.

In another embodiment of the present invention, the immunological fragment is preferably but not limited to a mutated or tranulated molecule of Fc or IgG2.

In another embodiment of the present invention, the construct is preferably, but not necessarily, characterized in that the AAT and its carboxy-terminal fragment are linked to the immunological fragment through its N-terminal end.

The present invention also relates to a method for the detection of mutations or tran- sients of (i) alpha-1 antitrypsin (AAT) or its last 80 or 36 amino acid residue fragments, (ii) peptide linkers, and (iii) Fc or IgG2, RTI ID = 0.0 > immunized fragment. ≪ / RTI >

In one embodiment of the present invention, the fusion polypeptide preferably has the amino acid sequence of SEQ ID NO: 3 or 4, but is not limited thereto.

The present invention also provides a recombinant cell clone comprising the nucleic acid construct or the fusion polypeptide of the present invention.

The present invention also provides a pharmaceutical composition comprising the fusion polypeptide of the present invention and a pharmaceutically acceptable carrier.

The present invention also provides a pharmaceutical composition comprising the nucleic acid construct of the present invention and a pharmaceutically acceptable carrier.

Hereinafter, the present invention will be described.

Some embodiments of the present invention report human alpha 1 antitrypsin in an effective recombinant form to treat AAT deficiency conditions or AAT reactive diseases similar to serum-derived AAT and are more effective than serum-derived AAT in some embodiments. In some embodiments, the compositions and methods of the invention relate to AATs (e.g., humans or other mammals) associated or fused with immune molecules or fragments thereof (e.g., IgGl, IgG2, IgG3 or IgG4, Fc, do. In other embodiments, the fusion proteins may comprise a truncated version of the AAT. According to these embodiments, some fusion polypeptides may be linked through the amino terminus of the AAT or the amino terminus of the fragment thereof. Some embodiments relate to constructs of AAT that are linked or fused with immunoglobulin molecules such as Fc. In some embodiments, the AAT or its C-terminal peptide fragment may be linked to an Fc derived from IgG1 or IgG2. Fc derived from IgG2 can be used, for example, because the Fc of human IgG1 is bound to the bone marrow cell complement receptor and IgG2 is not, thus avoiding this interaction with the complement receptor. In other embodiments, a linker can be used between an AAT or AAT peptide and an immunomolecule to produce a fusion polypeptide having from about 1 to about 20 additional amino acids.

Three distinct types of Fc-gamma (g) receptors are possible: the indicated FcgRI, FcgRII, and FcgRIII are found on human leukocytes.

Fc IgG2 has 12 amino acids in the hinge region and is less flexible than Fc IgG1. The 12 amino acid sites of this IgG2 can be mutated, tran- cated, or extended in some fusion constructs of the invention to mutate this hinge site to produce superior molecules. In addition, Fc IgG2 is resistant to proteases and not only binds to high affinity FcgRI as described above, but also has a weak ability to activate complement. In some embodiments, the Fc used in the fusion protein may be derived from IgG1 or IgG2 or IgG3 or IgG4. In some embodiments, Fc molecules can be linked to AAT molecules to produce dimers of Fc-AAT linked by disulfide bonds, for example, through an immunomolecule. The molecules of the invention can be rapidly purified using, for example, Protein A columns, other affinity purification methods or other rapid purification methods.

In relation to other AAT activities other than serine protease inhibition, AAT performs anti-inflammatory properties by several mechanisms. In some embodiments, a plurality of recombinant tranques and mutant forms of native human AAT (e. G., 394 AA, M _r About 51,000 or other AAT formulations) are prepared to evaluate the anti-inflammatory properties of the molecule.

In some embodiments, the human AAT at the C-terminal region of the recombinantly produced fusion peptides is generated to inhibit caspase-1 activity, IL-1 beta production or toxic activity.

The current trend for obtaining therapeutic agent concentrates AAT is to obtain AAT from the blood donor's serum, which requires extensive purification steps to obtain a limited source and marketable product.

Some embodiments of the present invention are directed to recombinant methods having greater activity and greater functionality as compared to conventional preparations for use in known AAT methods or treatments AAT constructs.

The AAT molecule of the construct of the present invention is entirely natural alpha-1 antitrypsin, or a fragment thereof, or a derivative thereof, or a mutant form of AAT, an AAT molecule or its alleles that does not have significant serine protease inhibitor activity, Log or its fusion protein. According to these embodiments, the construct may comprise a dimeric AAT construct linked to an immunological fragment (e. G., An Fc fragment linked to the AAT of two molecules) wherein the Fc-AAT construct is linked by one or more disulfide bonds. See, for example, Figures 1 and 2. According to these embodiments, the recombinant AAT molecules of the invention can be used as a fusion polypeptide (e.g. in the form of a dimer or monomer) or after cleavage from its immunomolecule and can be used at reduced concentrations as compared to conventional preparations . In some instances, these molecules may be used in compositions to modulate immune and inflammatory function or inhibit cytokines as compared to a control.

In some embodiments, the subject is a mammal, and the mammal is a human.

Some embodiments relate to using a recombinant AAT or fusion protein or a C-terminal fusion molecule thereof to treat a subject in need of AAT therapy.

Also, it can be seen from the present invention that AAT not fused to Fc is not expressed in CHO cells, whereas only AAT-Fc of the present invention is expressed well in CHO.

Radiation protection and cancer

In some embodiments of the invention, the composition (e.g., an imbricate composition) and methods involve modulating the adverse effects of radiation on the subject. In some embodiments, the compositions and methods involve treating a subject having radiation therapy, for example, when the subject has cancer or is administered to a subject suspected of having a malignant tumor.

Some embodiments relate to the treatment of a subject to perform cancer therapy. According to these embodiments, the subject undergoing cancer therapy can be treated with a composition of the present invention to reduce or prevent the deleterious effects of treatment. Cancer treatment includes, but is not limited to, treatment of bladder cancer, breast cancer, kidney cancer, leukemia, lung cancer, bone cancer, tongue cancer, prostate cancer, gastric cancer, colon cancer, uterine cancer, melanoma, pancreatic cancer, brain cancer, skin cancer and other known cancers. Or more.

Some side effects may be radiation exposure and even side effects of radiation therapy or chemotherapy. Some embodiments of the present invention involve treating a subject with a composition of the invention to reduce or prevent these side effects in the subject. The compositions may comprise recombinant forms of AAT C and / or recombinant forms of AAT C-terminal peptides (e.g., 80 mer, 36 mer, etc.). Side effects of radiation therapy include, but are not limited to, cell damage, pain, edema, local inflammation, and the like.

Some embodiments of the invention relate to treating a subject suspected of having prostate cancer or who has developed prostate cancer. According to these embodiments, male males with suspected prostate cancer or males with prostate cancer may be treated with the composition of the present invention to reduce the side effects of treatment with di before, after, or during radiation therapy and / or chemotherapy.

Other embodiments of the invention relate to organ or cell preservation prior to implantation. For example, protection or other conservation methods during transport can be improved by exposing the organ, tissue or cells to the composition of the present invention.

In some embodiments of the invention, the compositions of the invention may further comprise a combination treatment. For example, combination therapies include, but are not limited to, one or more interferon, betaseron, interferon derivatives including beta-interferon, and the like.

Various Peptides  Construct

Embodiments of the present invention include a recombinant having one or more carboxy terminal peptides derived from AAT, such as a C terminal peptide of AAT found in the late 80 amino acids of AAT or a C terminal end of AAT found in the late 36 amino acids of AAT Peptides, etc.) or recombinant AAT.

In one embodiment of the invention, the composition may comprise a construct for treating a subject in need of AAT therapy, for example, administering a series of peptides comprising a carboxy terminal amino acid corresponding to AAT and its derivatives. These peptides are part of the fused peptides and fused peptides including FVFLM, FVFAM, FVALM, FVFLA, FLVFI, FLMII, FLFVL, FLFVV, FLFLI, FLFFI, FLMFI, FMLLI, FIIMI, FLFCI, FLFAV, FVYLI, FAFLM and AVFLM It may contain a combination thereof.

In other embodiments, the AAT peptides for use in the constructs, pharmaceutical compositions and methods comprise all of the natural AATs of the 394 amino acids linked to the C-terminal amino acid (the most common form is the M type with subtypes M1, M2, M3, etc.) RTI ID = 0.0 > AAT < / RTI > All AAT polypeptides of the invention include anti-inflammatory and / or immunomodulatory activities. A combination of consecutive amino acids promoting AAT or AAT-like activity may use amino acids such as the range 315-394, 325-384, 358-394, 340-380, and the like. A combination of consecutive amino acid sequences such as 5-mers, 10-mers, 15-mers, 20-mers, 25-mers, 30-mers and 35-mers at the carboxy terminus may also be used.

Some embodiments include contacting the peptide molecules derived from the entire AAT molecule or the carboxy terminal amino acid site of the AAT with an IgG (e.g., a truncated IgG molecule, Fc or mutant Fc to reduce the length of the hinge region or remove it entirely) To a recombinant fusion polypeptide or protein comprising linking to a fragment thereof. The general form of AAT is shown below. One construct of the invention is referred to, for example, as the entire AAT, leader sequence and Fc region of an immunoglobulin molecule. These constructions may be used in the form of dimers or monomers in the compositions of the present invention. According to these embodiments, a pharmaceutically acceptable composition may comprise a dimer of Fc-AAT or a monomer of Fc-AAT or a truncated AAT from Fc or a combination thereof and a pharmaceutically acceptable excipient. Point mutations can also be made in the Fc region to generate new Fc-AAT molecules and reduce the flexibility of the hinge region.

 EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFS LYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGF QELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQATTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK

In other embodiments, the AAT protein binding domains can be mutated to reduce or eliminate the protease function of the molecule and not to inhibit elastase activity; these molecules can be used in the constructs of the invention. In some embodiments, the mutated AAT can be used to generate an AAT construct by the methods of the present invention. In other embodiments, the mutated molecule retains its anti-inflammatory and / or immunomodulatory effects and may be used as an anti-inflammatory molecule in subjects in need of AAT therapy.

In each of these methods alpha -antitrypsin or its C-terminal peptide derivatives are used in the combinations of the present invention. These peptide derivatives include peptides containing amino acids derived from the late 80 C terminal of AAT, GITKVFSNGA, DLSGVTEEAP, LKLSKAVHKA, VLTIDEKGTE, AAGAMFLEAI, PMSIPPEVKF, NKPFVFLMIE, QNTKSPLFMG, KVVNPTQK, LEAIPMSIPPEVKFNKPFVFLM; And LEAIPMSIPPEVKFNKPFVF, GADLSGVTEEAPLKLSKAVHKA VLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK, or combinations thereof.

In some embodiments, compositions of AAT-derived C-terminal peptides or recombinant AATs capable of binding to SEC receptors or compositions having AAT-like activity may be administered to a subject in need thereof. In other embodiments, the AAT-derived peptides comprise AAT molecules up to 5-mers, 10-mers, 20-mers, 25-mers, 30-mers, 35-mers, 40-mers, Peptides that do not have prominent serine protease inhibitor activity can be used, for example, when treating a subject who is receiving radiation therapy from a C-terminus of AAT.

In one embodiment of the invention, the construct may comprise compounds that are associated with or involved in the SEC receptor. Also, 5-mers or 10-mers or a combination of 20-mers or 30-mers or more amino acids may be used. In the present invention, the C terminus is referred to as the C terminus. In some embodiments, the carboxy domain of AAT going from the C-terminus backwards is defined as amino acids that are mostly conserved among species differences and not involved in the protein binding domain of AAT. Also in other embodiments the AAT protease binding domain can be mutated to reduce or eliminate the protease function of the molecule and these molecules can be used in the compositions of the present invention. In other embodiments, the mutated AAT-related molecules may retain their anti-inflammatory and / or immunomodulatory effects. The carboxy domain of the present invention is a non-protease binding domain having another AAT activity.

In each of the methods described above, the compositions of the present invention comprise a carboxy-derived peptide of AAT. In some embodiments, the AAT-linked molecules used in the methods and compositions of the invention may include, but are not limited to, native AAT, other AAT M-types types or other AAT molecules.

Commercially available preparations for comparison with recombinants of the fusion molecules of the present invention and / or controls include, but are not limited to, Aralast ^™ (Baxter), Zemaira ^™ (Aventis Behring), Prolastin ^™ or Prolastin ^™ (Talecris) ^TM or ^TM Trasylol (Bayer Corporation Pharmaceutical), Ulinistatin ^TM (Ono Pharmaceuticals, Inc.), and the suction and / or injectable AAT (Kamada, Ltd., Israel) , or other commonly available including AAT composition or in a combination of can do.

Other embodiments relate to mutants of human AAT and the mutants have increased anti-inflammatory activity. Known techniques for generating mutants are possible. Some embodiments include using site-specific mutagenesis to produce hAAT without significant serine protease inhibitor activity (see examples). In some embodiments, the fusion molecules comprise an AAT mutant having one or more point mutations in one or more amino acids within the RCL (e. G., Amino acids 355-363 of native AAT) with FC (e. G., IgG1,2,3 or 4) Wherein the AAT mutant has no significant serine protease inhibitory activity and retains RCL inactivation.

Pharmaceutical composition

Embodiments of the invention provide administration of compositions to a subject in a biologically compatible form suitable for pharmaceutical administration in vivo. "Biologically compatible form suitable for pharmaceutical administration in vivo" means a form in which the therapeutic effect of the active ingredient is administered with an active ingredient (pharmaceutical compound, protein, gene, antibody, etc.) which overwhelms the toxic effect. Administration of a therapeutically active amount of a therapeutic composition is defined as an effective amount in the period of time, dose, required to achieve the desired result. For example, the therapeutically active amount of a compound may vary with disease state, age, sex, and body weight.

A pharmaceutical composition comprising AAT or a peptide fragment thereof or an analog thereof or a mutant thereof or a functional derivative thereof may be administered to a subject by, for example, subcutaneous, intravenous, intracardial, intramuscular, oral, Application, vaginal application topical application, intranasal or rectal administration. Depending on the route of administration, the active compound may be coated with a material to protect the compound from degradation by natural conditions, acids, enzymes that inactivate the compound. In a preferred embodiment, the compound can be administered orally. In another preferred embodiment the compound can be administered intravenously. In certain embodiments, the composition may be intranasal, such as inhalation.

The compounds (e. G., Peptides, proteins, fusion proteins or mixtures thereof) may be administered to a subject with a suitable carrier or diluent. In the present invention, the term "pharmaceutically acceptable carrier" may include diluents such as saline and aqueous buffer solutions.

Pharmaceutical compositions suitable for injectable use may be administered by methods known in the art. Sterile aqueous solutions or suspensions and sterile powders for the preparation of, for example, sterile injectable solutions or suspensions may be used.

The sterile injectable solution may contain, if necessary, an active compound (e. G., A compound that reduces serine protease activity) in the required amount in one or more of the above components, or a combination thereof, Mixing and filtering sterilization.

The water-soluble composition may contain an effective amount of the therapeutic compound, peptide epitopic core site, enhancer, inhibitor and the like, dissolved or dispersed in a water-soluble medium or a pharmaceutically acceptable carrier.

The formulations can be readily administered in a variety of dosage forms, such as the types of injectable solutions described above. Sustained-release capsules, timed-release microcapsules, and the like can be employed.

The active therapeutic ingredients may be formulated to contain about 0.0001 to 1.0 mg per dose or about 0.001 to 1.0 or even 1 to 10 grams per dose in the mixture. The exact dose and duration of treatment can be determined formally using known testing protocols or testing of the composition in a known model system. The dose varies with the severity of the disease. In some embodiments, the composition ranges from 10 to 75 mg / kg daily or weekly to the subject. A therapeutically effective amount of alpha -antitrypsin, peptide or alpha-antitrypsin or a drug having an activity similar to a peptide may be measured in molar concentrations and may range from about 1 nM to about 2 mM.

In another embodiment, intranasal solution or spray, aerosol or inhalant may be used for delivery of the compound of interest.

Liposomes or microcapsules may be prepared according to known laboratory techniques and used as therapeutic agent delivery systems.

In some embodiments, the pharmaceutical construct compositions do not have significant serine protease inhibitor activity but are associated with constructs derived from AAT molecules that have other alpha-antitrypsin activities. For example, the fusion polypeptide of the present invention may be a fusion polypeptide having no significant protease inhibitory activity.

In some embodiments, the compositions of the present invention may be administered orally or systemically via an implant or sustained release composition.

Expression of proteins and constructs

Once a target gene or a portion of a gene has been determined, the gene can be inserted into an appropriate expression system. The gene may be expressed in several different recombinant DNA expression systems to produce large amounts of the polypeptide product and then purified and used in the compositions and methods of the present invention.

Examples of expression systems, known to those skilled in the art, include E. coli Yeast such as the bacterium Pichia pastoris , baculovirus, and mammalian expression systems such as Cos or CHO cells.

The gene fragment or AAT gene encoding the fusion polypeptide can be inserted into the expression vector by standard subcloning methods. An E. coli expression vector can be used to produce a recombinant polypeptide as a fusion protein. Examples of such fusion protein expression systems include the glutathione S-transferase system (Pharmacia, Piscataway, NJ), the maltose binding protein system (NEB, Beverley, Mass.), The FLAG system (IBI, New Haven, CT) Qiagen, Chatsworth, Calif.).

"Engineered" and "recombinant" cells in the present invention refer to a cell into which a foreign DNA fragment or gene has been inserted through a human hand.

To express recombinantly encoded proteins or peptides, whether mutant or wild-type, one skilled in the art can construct an expression vector comprising one isolated nucleic acid operably linked to or under control with one or more promoters.

Cell types useful for expression include, but are not limited to, recombinant bacteriophage DNA, plasmid DNA, or bacteria such as E. coli and B. subtilis transformed with a cosmid expression vector.

Examples of prokaryotes include E. coli strain RR1, E. coli LE392, E. coli B, E. coli X 1776 (ATCC No. 31537), E. coli W3110 (F-, lambda-, prototrophic, ATCC No. 273325); Bacillus subtilis ; Salmonella typhimurium , Serratia marcescens , and several Pseudomonas species.

The most commonly used promoters in the construction of recombinant DNA include the beta-lactamase (penicillinase), lactose and tryptophan (trp) promoter systems.

For expression in Saccharomyces , for example plasmid YRp7 is commonly used (Stinchcomb et < RTI ID = 0.0 > al . , 1979; Kingsman et al . , 1979; Tschemper et al . , 1980). Suitable promoter sequences in yeast vectors include 3-phosphoglycerate kinase (Hitzeman et < RTI ID = 0.0 > al . , 1980).

Where appropriate constructing plasmids are constructed, the termination sequence associated with these genes is linked to expression vector 3 'of the sequence desired to be expressed to provide polyadenylation and termination of mRNA.

In addition to microorganisms, multicellular organism-derived cell culture may be used as a host. In addition to mammalian cells, insect cell systems and plant cell systems are also possible.

Examples of useful mammalian host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell line, W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines.

The promoters may be mammalian cell genomes (e. G., Metallothionein promoter) or mammalian viral genomic (e. G., Adenovirus late promoter; the vaccinia virus 7.5K promoter).

Certain start signals may be required for effective translation of the isolated nucleic acid coding sequence. These signals include the ATG start codon and adjacent sequences.

In some embodiments, the eukaryotic host cell is capable of expressing the desired polypeptide. They can be obtained by insertion of DNA molecules into cells, preferably in the form of expression cassettes of the invention. Selection of cells containing the DNA of the target molecule can be performed by selecting the selectable marker polypeptide using conventional methods.

In some embodiments, the DNA molecule of interest is inserted into the nucleotide of the eukaryotic host cell. Selection for the presence of a selectable marker polypeptide, and thus, during the initial acquisition of the cells for expression. In some embodiments, the selection material is present in the culture medium during the incubation period at a concentration sufficient or at a lower concentration to select cells expressing the selectable marker polypeptide.

Conditions for culturing or propagating the cells (see, for example, Tissue Culture, Academic Press, Kruse and Paterson, editors (1973)) and conditions for expression of recombinant products are known to those skilled in the art. The actual techniques, principles and protocols that generally maximize the productivity of mammalian cell culture are described in Mammalian Cell Biotechnology: a Practical Approach (M. Butler, ed., IRL Press, 1991).

In some embodiments, the expressed protein is collected (separated) from the cell or culture medium, or both. It can be further purified using known methods such as filtration, column chromatography.

A number of selection systems including, but not limited to, herpes simplex virus thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase, and adenine phosphoribosyltransferase genes in tk-, hgprt- or aprt- cells, respectively, can be used.

The constructs of the invention produced using immunomolecule molecules (e. G. Fc portions) can be separated using several affinity columns or affinity matrices (e. G. Protein A). In some embodiments, the purification of the constructs of the invention can be performed using minimal steps to preserve anti-inflammatory or immunomodulatory activity or other AAT-related activities from degradation and the like. According to these embodiments, purification of the constructs of the invention can be by a single step (e. G. Protein A column purification of Fc-AAT molecules) (see, for example, Kin-Ming et al. Protein Engineering vol. no.6 pp.495-500, 1998; expression / Fc / Fc-X / fusion protein; and diabody technologies).

Nucleic acids encoding proteins or peptides that can be reversibly bound (e. G., Disulfide bonds or other bonds) to themselves may be used to prepare the AAT constructs of the present invention. According to some of these embodiments, the fragment or portion of the immunomolecule linked to the AAT or carboxy terminal fragment may be essentially non-immunogenic if increased immunity is not required for therapeutic purposes. The constructs of the invention can be used to induce beneficial immune or inflammatory effects; Or to reduce or eliminate adverse immunological or inflammatory effects, for example, by reducing the presence of some proinflammatory cytokines.

Separated proteins

In some embodiments, the native polypeptide is mutated or otherwise cleaved to remove or reduce serine protease inhibitor activity and then used for constructs of the invention. In certain embodiments, serine protease inhibitor activity is reduced without significant activity. In yet another embodiment, the polypeptides of the invention are produced primarily by recombinant DNA technology. Or can be synthesized using standard peptide synthesis techniques. Also, for example, the AAT composition may be treated to reduce or eliminate the serine protease inhibitor activity, or the AAT polypeptide with or without significant serine protease inhibitor activity may be isolated. These AAT molecules can then be used in the constructs of the invention for rapid production and purification.

 A "isolated " or" purified "protein, or a biologically active site thereof, is substantially free of cellular material or other contaminating proteins from cells (e.g., clones) In the case there is substantially no other chemicals.

In some embodiments, the nucleotide encoding the polypeptide can be inserted into any construct known to produce the peptide or protein. These polypeptides may comprise polypeptides having consecutive amino acid sequences corresponding to all or part of the late 80 carboxy terminal amino acids of AAT. These polypeptides may be used in constructs of the invention (e. G., Immunomolecule linked constructs (Fc)).

Biologically active sites of the polypeptides of the present invention include polypeptides derived from amino acid sequences of the protein or comprising sufficiently identical amino acid sequences. The biologically active portion of the present invention can be, for example, a polypeptide having an amino acid length of 5, 10, 20, 30, 40 or more. Some of these polypeptides may be linked to immunomolecules and the peptides may retain anti-inflammatory and / or anti-immunological activity. The constructs of the invention may have reduced or truncated linker moieties (e.g., IgG2, IgG1, IgG4, IgG3, IgM, or IgD, etc.) depending on the desired function of the construct. Reduced flexibility is desirable in some implementations.

Variants of AAT molecules that do not have significant serine protease activity can be generated by mutation, such as, for example, point mutations or tran- sition. For example, a point mutation still retains a reactive center loop (RCL), while the ability of the AAT or peptide to bind serine protease binding is inhibited or inhibited, but its ability to regulate radiation adverse effects is generated in the conserved AAT or its derivatives .

fusion Polypeptides

In some embodiments, a material such as AAT and / or an analog or peptide derivative or fragment thereof may be part of a fusion polypeptide (AAT-related molecules). In some embodiments, the fusion polypeptide may comprise an AAT (e. G., A native mammalian alpha-antitrypsin) or a peptide or other amino acid sequence associated with or related to an AAT-related molecule, such as a peptide fragment or an IgG fragment .

In another embodiment, the AAT polypeptide or peptide fusion protein may be a GST fusion protein fused to the C terminus of the GST sequence. Fusion expression vectors and purification and detection means are known in the art.

Clones

Other embodiments include separate cloning and generation of fusion or recombinant molecules of the invention. In some embodiments, the clones of the invention may be derived from an Fc-AAT molecule (e.g., full-length AAT or a peptide fragment thereof) having a modified linker site that has increased activity compared to the control AAT. In another embodiment, another clone is associated with an IgG2 molecule linked to AAT or a fragmented derivative thereof.

Kit

In a further embodiment, kits for convenient use with the above-described methods, compositions, constructions (e. G. Recombinant and / or fusion molecules) are possible. Kits may be constructed from AAT fusion or recombinant constructs (e.g., Fc-AAT; Fc-mutant AAT, Fc-AAT peptide fragments, IgG2 mutants linked to carboxy terminal derivatives of AAT or AAT), constructs of one or more peptides derived from AAT , Mutant AAT construct compositions, mutant AAT molecules associated with gene therapy delivery systems, or other combinations. The kits may comprise suitable container means (e. G. Vials, tubes, etc.), proteins or peptides or analogues and optionally one or more additional substances.

In some embodiments, the kit may comprise a composition comprising, but not limited to, constructs of AAT, AAT fragments, or AAT analogs or polypeptides that do not have significant serine protease inhibitor activity.

As can be seen from the present invention, the recombinant AAT of the present invention is more readily purified than other forms of AAT and has increased anti-inflammatory and anti-immunological activity compared to natural AAT or other commercial AAT preparations. Also, less than 10-fold less recombinant AAT (rAAT) may be used as an alternative to AAT in its current form for the prevention or treatment of disease.

Figure 1 shows the Fc-AAT construct,
Figure 2 is a schematic diagram of AAT-Fc in dimer form connected by disulfide bonds.
Figure 3 shows an anti-inflammatory model of the effect of the fusion protein described for cytokine expression.
Figure 4 shows a histogram diagram depicting the concentration range of the construct of the invention for stimulation of anti-inflammatory molecules, IL-1 receptor antagonist (IL-1Ra).
Figure 5 shows that AAT-Fc inhibits hyperglycemia in a diabetic STZ-induced mouse model.
Figures 6 and 7 show dose-dependent inhibition of TNF [alpha] -induced IL-6 production in human PBMCs. Figure 6 shows that both AAT-Fc and AAT fail to inhibit LPS-induced IL-6 production, and Figure 7 shows that AAT-Fc inhibits TNF [alpha] -induced IL-6 production in a dose- Fc completely inhibited IL-6 production at 1000 ng / ml, but the same amount of plasma-derived AAT showed no effect on IL-6 induction.

The present invention will now be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the invention and the scope of the invention is not to be construed as being limited by the following examples.

Generation of expression plasmids for recombinant human AAT production

Example 1

In one exemplary method, Fc-AAT constructs can be prepared as described in FIG. For example, human AAT sequences can be inserted into an expression vector, pCAGGS. In this exemplary method, a 1260 bp human AAT cDNA was isolated from a human liver library and inserted into pCAGGS as described in FIG. Other AAT molecules or peptide fragments thereof can be used at positions of the 1260 bp sequence if desired. In this example, Chinese Hamster Ovarian (CHO) cells were transfected with plasmids to produce cells capable of producing the construct. Using limited dilution, AAT clones were selected and cultured in serum-free medium. The supernatant was collected, collected and analyzed for AAT fusion protein. Using an antibody against human AAT, a band of about 55 kDa was observed on the western blot (data not shown). In this example, the human IgGl Fc receptor was used to purify recombinant AAT using Protein A. The clones producing the fusion protein were identified and the aliquots were frozen for further use.

Example 2

In another exemplary method, the constructs depicted in Figures 1 and 2 can be used in a treatment or method for a disease associated with a purified and conventional AAT composition or other known AAT compositions. In one embodiment, an AAT fragment or AAT dimer linked to an immunomolecule may be used.

In one exemplary method, human Fc IgG plasmids (e.g., IgG1, IgG2, IgG3, IgG4, and IgD, etc.) were purchased from Qiagen. The human cDNA was clipped and inserted into the human Fc vector via PCR cloning. In-frame sequences were performed for confirmation. The plasmid was transfected into CHO cells and a few diluted clones were isolated to obtain a single clone. The stabilized clone was expanded and selected using serum-free medium. Large scale cell culture was performed and the supernatant was collected and collected.

The supernatant construct was purified using a single step Protein A matrix. Human Fc-AAT was eluted with glycine (pH 2.4) and then rapidly neutralized to pH 7.4. SDS PAGE showed a single band under reducing conditions. In one exemplary method, the purified Fc-AAT was compared to the conventional Aralast ^TM for inhibition of elastase activity. Clones with Fc-AAt constructs were identified and proliferated and aliquots were frozen and stored for later use.

Purification of human AAT Fc: The western blot of the fusion protein represents a band (about 170 kDa) representing the inact dimer of the two Fc-AAT molecules. On the Western blot, another lane shows that all disulfide bonds in the fusion protein are cleaved to form FC-AAT of the two monomer molecules. Both the reducing gel as well as the non-reducing gel exhibited high levels of AAT structure purification. And thus can be purified in a single step from the mammalian cell culture supernatant using protein A chromatography.

Figure 3 shows an anti-inflammatory model of the effect of the fusion protein described for cytokine expression. In this example, IL-8 expression was measured in the presence or absence of LPS.

This model is a well-known model for measuring inflammatory activity in cells using promoter LPS. Human blood neutrophils (3 x ¹⁰⁶ cells / ml) were incubated alone or in combination with LPS (10 ng / ml), Fc-AAT (rAAT) 10 micrograms / ml or LPS and Fc-AAT for 6 hours. IL-8 levels were measured in culture supernatants (N = 3). Neutrophils treated with LPS in the presence of Fc-AAT significantly reduced IL-8 levels compared to LPS alone.

In one embodiment, the Fc-AAT construct may have decreased or insignificant serine protease inhibitory activity. Other fusion proteins of the present invention include serine protease inhibitory activity.

Example 3

Figure 4 shows a histogram diagram depicting the concentration range of the construct of the invention for stimulation of anti-inflammatory molecules, IL-1 receptor antagonist (IL-1Ra). In this exemplary method, neutrophil cells were cultured with a reduced concentration of a fusion protein of the invention (e.g., Fc-AAT with a reduced linker site of an IgG2 immunomolecule). Control samples were used for comparison. These molecules have an activity of greater than about 100 than the corresponding capacity of a conventional preparation (e.g., Aralast ^TM ). In this experiment, N = 2.

Example 4

Fc - The AAT teureon of Kate Construct variants .

In some exemplary embodiments, the protease cleavage of AAT can be a simple insertion of a protease site, such as, for example, the tobacco mosaic virus protease within the AAT sequence. Insertion of the protease recognition site produces a truncated C terminus of AAT. This site is upstream from the carboxy-36-terminal peptide of the native AAT (see, e.g., Figure 5): SIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK

These tranated AAT molecules can inhibit LPS-induced IL-1 [beta], IL-6 and TNF [alpha]. The bivalent tranated fused molecule may be superior to its own peptide in terms of increased plasma half-life. The C-36 peptide reduced LPS-induced IL-1β at concentrations ranging from 120 μg / mL to 30 μg / mL for 18 hours (data not shown).

In some embodiments, the peptide size (about 4 kDa) may have a short half-life and thus will be formulated with a carrier. Thus, due to the ability to bind to the complement receptor (FcR), the Fc region of human IgG1, termed the Fc domain, can be used. One advantage of the fusion protein is that it extends the half-life in the subject ' s circulatory system. Here, there are at least two unique advantages to Fc fusion proteins: 1) easy purification using Protein A affinity chromatography of crude cell supernatant; And 2) Fc fusion proteins can be used to treat a number of diseases in humans and have an established safety record on them. As described above, the effect of the other truncated versions of Fc-AAT can be readily evaluated. For example, protease cleavage with the tobacco mosaic virus protease will be performed in the CHO cell supernatant. The mixture is then applied to a Protein A column and the fractions are eluted in an acid buffer and rapid neutralization will be performed. Some truncated Fc-AAT will not have anti-elastase activity. For example, the 36-C peptide does not contain this activity, so the activity of these molecules may be anything other than serine protease inhibitory activity. The truncated molecules may be C-80 or C-60, etc., which are larger than the C-36 molecule as described in FIG.

Cutting of the Fc domain .

Another cleavage site can be of the Fc itself to remove AAT-derived peptides or AAT-derived Fc fragments when necessary. This site produces an AAT or tranated AAT derived monomer. However, the enzyme for Fc-IgG1 differs from Fc-IgG2 or other IgG molecules.

The N-terminal fusion protein

Construction of the N-terminal AAT is a novel concept based on data indicating that the anti-inflammatory (or anti-immune) properties of AAT are independent of the elastase inhibiting properties of AAT. Thus, the use of the N-terminus for inframe construction allows the formation of molecules with bi-valent C-termini. For each construct, expression in CHO is necessary because glycosylation of the molecule is an important component of the molecule. Thus, CHO cells will be used for expression of wild-type as well as transcribed Fc-AAT.

Tranquilized Fc - AAT Tablets and essays

In the case of the protease insertion site, a protease for cleaving a molecule can be inserted first, and a protein A for separating only the next fragment can be inserted. It will produce an almost pure form of the product. In some methods, the molecule is purified on Protein A as in the case of the Fc cleavage site, and the Fc cleavage can be removed on Protein A, which remains in the pure or residual protein after the addition of the Fc cleavage protease.

Table 1 is a table showing cytokine stimulation by the present invention and control constructs

<110> YbdY <120> COMPOSITIONS, METHODS AND USES FOR ALPHA-1 ANTITRYPSIN FUSION          MOLECULES <160> 4 <170> Kopatentin 1.71 <210> 1 <211> 1977 <212> DNA <213> Artificial Sequence <220> <223> AAT-Fc2 <400> 1 gaattcgcca ccatgccgtc ttctgtctcg tggggcatcc tcctgctggc aggcctgtgc 60 tgcctggtcc ctgtctccct ggctgaggat ccccagggag atgctgccca gaagacagat 120 acatcccacc acgatcagga tcacccaacc ttcaacaaga tcacccccaa cctggctgag 180 ttcgccttca gcctataccg ccagctggca caccagtcca acagcaccaa tatcttcttc 240 tccccagtga gcatcgctac agcctttgca atgctctccc tggggaccaa ggctgacact 300 cacgatgaaa tcctggaggg cctgaatttc aacctcacgg agattccgga ggctcagatc 360 catgaaggct tccaggaact cctccgtacc ctcaaccagc cagacagcca gctccagctg 420 accaccggca atggcctgtt cctcagcgag ggcctgaagc tagtggataa gtttttggag 480 gatgttaaaa agttgtacca ctcagaagcc ttcactgtca acttcgggga caccgaagag 540 gccaagaaac agatcaacga ttacgtggag aagggtactc aagggaaaat tgtggatttg 600 gtcaaggagc ttgacagaga cacagttttt gctctggtga attacatctt ctttaaaggc 660 aaatgggaga gaccctttga agtcaaggac accgaggaag aggacttcca cgtggaccag 720 gcgaccaccg tgaaggtgcc tatgatgaag cgtttaggca tgtttaacat ccagcactgt 780 aagaagctgt ccagctgggt gctgctgatg aaatacctgg gcaatgccac cgccatcttc 840 ttcctgcctg atgaggggaa actacagcac ctggaaaatg aactcaccca cgatatcatc 900 accaagttcc tggaaaatga agacagaagg tctgccagct tacatttacc caaactgtcc 960 attactggaa cctatgatct gaagagcgtc ctgggtcaac tgggcatcac taaggtcttc 1020 agcaatgggg ctgacctctc cggggtcaca gaggaggcac ccctgaagct ctccaaggcc 1080 gtgcataagg ctgtgctgac catcgacgag aaagggactg aagctgctgg ggccatgttt 1140 ttagaggcca tacccatgtc tatccccccc gaggtcaagt tcaacaaacc ctttgtcttc 1200 ttaatgattg aacaaaatac caagtctccc ctcttcatgg gaaaagtggt gaatcccacc 1260 caaaaaacgc gtgagcccaa atcttgtgac aaaactcaca catgcccacc gtgcccagca 1320 cctgaactcc tggggggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc 1380 atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct 1440 gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg 1500 cgggaggagc agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag 1560 gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc 1620 atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg 1680 cccccatccc gggatgagct gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc 1740 ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac 1800 aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctacag caagctcacc 1860 gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgat gcatgaggct 1920 ctgcacaacc actacacgca gaagagcctc tccctgtctc cgggtaaatg aggatct 1977 <210> 2 <211> 1950 <212> DNA <213> Artificial Sequence <220> <223> AAT-Fc3 <400> 2 gaattcgcca ccatgccgtc ttctgtctcg tggggcatcc tcctgctggc aggcctgtgc 60 tgcctggtcc ctgtctccct ggctgaggat ccccagggag atgctgccca gaagacagat 120 acatcccacc acgatcagga tcacccaacc ttcaacaaga tcacccccaa cctggctgag 180 ttcgccttca gcctataccg ccagctggca caccagtcca acagcaccaa tatcttcttc 240 tccccagtga gcatcgctac agcctttgca atgctctccc tggggaccaa ggctgacact 300 cacgatgaaa tcctggaggg cctgaatttc aacctcacgg agattccgga ggctcagatc 360 catgaaggct tccaggaact cctccgtacc ctcaaccagc cagacagcca gctccagctg 420 accaccggca atggcctgtt cctcagcgag ggcctgaagc tagtggataa gtttttggag 480 gatgttaaaa agttgtacca ctcagaagcc ttcactgtca acttcgggga caccgaagag 540 gccaagaaac agatcaacga ttacgtggag aagggtactc aagggaaaat tgtggatttg 600 gtcaaggagc ttgacagaga cacagttttt gctctggtga attacatctt ctttaaaggc 660 aaatgggaga gaccctttga agtcaaggac accgaggaag aggacttcca cgtggaccag 720 gcgaccaccg tgaaggtgcc tatgatgaag cgtttaggca tgtttaacat ccagcactgt 780 aagaagctgt ccagctgggt gctgctgatg aaatacctgg gcaatgccac cgccatcttc 840 ttcctgcctg atgaggggaa actacagcac ctggaaaatg aactcaccca cgatatcatc 900 accaagttcc tggaaaatga agacagaagg tctgccagct tacatttacc caaactgtcc 960 attactggaa cctatgatct gaagagcgtc ctgggtcaac tgggcatcac taaggtcttc 1020 agcaatgggg ctgacctctc cggggtcaca gaggaggcac ccctgaagct ctccaaggcc 1080 gtgcataagg ctgtgctgac catcgacgag aaagggactg aagctgctgg ggccatgttt 1140 ttagaggcca tacccatgtc tatccccccc gaggtcaagt tcaacaaacc ctttgtcttc 1200 ttaatgattg aacaaaatac caagtctccc ctcttcatgg gaaaagtggt gaatcccacc 1260 caaaaaacgc gtacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 1320 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 1380 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 1440 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 1500 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 1560 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1620 gggcagcccc gagaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 1680 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1740 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1800 gcggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1860 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1920 ctctccctgt ctccgggtaa atgaggatct 1950 <210> 3 <211> 652 <212> PRT <213> Artificial Sequence <220> <223> AAT-Fc2 <400> 3 Met Pro Ser Ser Val Ser Trp Gly Ile Leu Leu Leu Ala Gly Leu Cys   1 5 10 15 Cys Leu Val Pro Val Ser Leu Ala Glu Asp Pro Gln Gly Asp Ala Ala              20 25 30 Gln Lys Thr Asp Thr Ser His His Asp Gln Asp His Pro Thr Phe Asn          35 40 45 Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala Phe Ser Leu Tyr Arg Gln      50 55 60 Leu Ala His Gln Ser Asn Ser Thr Asn Ile Phe Phe Ser Pro Val Ser  65 70 75 80 Ile Ala Thr Ala Phe Ala Met Leu Ser Leu Gly Thr Lys Ala Asp Thr                  85 90 95 His Asp Glu Ile Leu Glu Gly Leu Asn Phe Asn Leu Thr Glu Ile Pro             100 105 110 Glu Ala Gln Ile His Glu Gly Phe Gln Glu Leu Leu Arg Thr Leu Asn         115 120 125 Gln Pro Asp Ser Gln Leu Gln Leu Thr Thr Gly Asn Gly Leu Phe Leu     130 135 140 Ser Glu Gly Leu Lys Leu Val Asp Lys Phe Leu Glu Asp Val Lys Lys 145 150 155 160 Leu Tyr His Ser Glu Ala Phe Thr Val Asn Phe Gly Asp Thr Glu Glu                 165 170 175 Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu Lys Gly Thr Gln Gly Lys             180 185 190 Ile Val Asp Leu Val Lys Glu Leu Asp Arg Asp Thr Val Phe Ala Leu         195 200 205 Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp Glu Arg Pro Phe Glu Val     210 215 220 Lys Asp Thr Glu Glu Glu Asp Phe His Val Asp Gln Ala Thr Thr Val 225 230 235 240 Lys Val Pro Met Met Lys Arg Leu Gly Met Phe Asn Ile Gln His Cys                 245 250 255 Lys Lys Leu Ser Ser Trp Val Leu Leu Met Lys Tyr Leu Gly Asn Ala             260 265 270 Thr Ala Ile Phe Leu Pro Asp Glu Gly Lys Leu Gln His Leu Glu         275 280 285 Asn Glu Leu Thr His Asp Ile Ile Thr Lys Phe Leu Glu Asn Glu Asp     290 295 300 Arg Arg Ser Ala Ser Leu His Leu Pro Lys Leu Ser Ile Thr Gly Thr 305 310 315 320 Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu Gly Ile Thr Lys Val Phe                 325 330 335 Ser Asn Gly Ala Asp Leu Ser Gly Val Thr Glu Glu Ala Pro Leu Lys             340 345 350 Leu Ser Lys Ala Val His Lys Ala Val Leu Thr Ile Asp Glu Lys Gly         355 360 365 Thr Glu Ala Gla Ala Met Phe Leu Glu Ala Ile Pro Met Ser Ile     370 375 380 Pro Pro Glu Val Lys Phe Asn Lys Pro Phe Val Phe Leu Met Ile Glu 385 390 395 400 Gln Asn Thr Lys Ser Pro Leu Phe Met Gly Lys Val Val Asn Pro Thr                 405 410 415 Gln Lys Thr Arg Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro             420 425 430 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe         435 440 445 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val     450 455 460 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 465 470 475 480 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro                 485 490 495 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr             500 505 510 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val         515 520 525 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala     530 535 540 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 545 550 555 560 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly                 565 570 575 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro             580 585 590 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser         595 600 605 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln     610 615 620 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 625 630 635 640 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys                 645 650 <210> 4 <211> 643 <212> PRT <213> Artificial Sequence <220> <223> AAT-Fc3 <400> 4 Met Pro Ser Ser Val Ser Trp Gly Ile Leu Leu Leu Ala Gly Leu Cys   1 5 10 15 Cys Leu Val Pro Val Ser Leu Ala Glu Asp Pro Gln Gly Asp Ala Ala              20 25 30 Gln Lys Thr Asp Thr Ser His His Asp Gln Asp His Pro Thr Phe Asn          35 40 45 Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala Phe Ser Leu Tyr Arg Gln      50 55 60 Leu Ala His Gln Ser Asn Ser Thr Asn Ile Phe Phe Ser Pro Val Ser  65 70 75 80 Ile Ala Thr Ala Phe Ala Met Leu Ser Leu Gly Thr Lys Ala Asp Thr                  85 90 95 His Asp Glu Ile Leu Glu Gly Leu Asn Phe Asn Leu Thr Glu Ile Pro             100 105 110 Glu Ala Gln Ile His Glu Gly Phe Gln Glu Leu Leu Arg Thr Leu Asn         115 120 125 Gln Pro Asp Ser Gln Leu Gln Leu Thr Thr Gly Asn Gly Leu Phe Leu     130 135 140 Ser Glu Gly Leu Lys Leu Val Asp Lys Phe Leu Glu Asp Val Lys Lys 145 150 155 160 Leu Tyr His Ser Glu Ala Phe Thr Val Asn Phe Gly Asp Thr Glu Glu                 165 170 175 Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu Lys Gly Thr Gln Gly Lys             180 185 190 Ile Val Asp Leu Val Lys Glu Leu Asp Arg Asp Thr Val Phe Ala Leu         195 200 205 Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp Glu Arg Pro Phe Glu Val     210 215 220 Lys Asp Thr Glu Glu Glu Asp Phe His Val Asp Gln Ala Thr Thr Val 225 230 235 240 Lys Val Pro Met Met Lys Arg Leu Gly Met Phe Asn Ile Gln His Cys                 245 250 255 Lys Lys Leu Ser Ser Trp Val Leu Leu Met Lys Tyr Leu Gly Asn Ala             260 265 270 Thr Ala Ile Phe Leu Pro Asp Glu Gly Lys Leu Gln His Leu Glu         275 280 285 Asn Glu Leu Thr His Asp Ile Ile Thr Lys Phe Leu Glu Asn Glu Asp     290 295 300 Arg Arg Ser Ala Ser Leu His Leu Pro Lys Leu Ser Ile Thr Gly Thr 305 310 315 320 Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu Gly Ile Thr Lys Val Phe                 325 330 335 Ser Asn Gly Ala Asp Leu Ser Gly Val Thr Glu Glu Ala Pro Leu Lys             340 345 350 Leu Ser Lys Ala Val His Lys Ala Val Leu Thr Ile Asp Glu Lys Gly         355 360 365 Thr Glu Ala Gla Ala Met Phe Leu Glu Ala Ile Pro Met Ser Ile     370 375 380 Pro Pro Glu Val Lys Phe Asn Lys Pro Phe Val Phe Leu Met Ile Glu 385 390 395 400 Gln Asn Thr Lys Ser Pro Leu Phe Met Gly Lys Val Val Asn Pro Thr                 405 410 415 Gln Lys Thr Arg Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly             420 425 430 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met         435 440 445 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His     450 455 460 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 465 470 475 480 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr                 485 490 495 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly             500 505 510 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile         515 520 525 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val     530 535 540 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 545 550 555 560 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu                 565 570 575 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro             580 585 590 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val         595 600 605 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met     610 615 620 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 625 630 635 640 Pro Gly Lys            

Claims (9)

A nucleic acid encoding alpha-1 antitrypsin (AAT); And a nucleic acid that coats an immunoglobulin fragment comprising a modified hinge region. The nucleic acid construct according to claim 1, wherein the nucleic acid construct has the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. 2. The nucleic acid construct of claim 1, wherein said immunological fragment is a mutated or tranulated molecule of Fc or IgG2. 2. The nucleic acid construct of claim 1, wherein the construct is linked to the AAT and its carboxy-terminal fragment through its N-terminal end to the immunological fragment. Starting from the N-terminus, (i) alpha-1 antitrypsin (AAT) or its last 80 or 36 amino acid residue fragments, (ii) peptide linker, and (iii) mutated or tran- &Lt; / RTI &gt; The fusion polypeptide according to claim 5, wherein the fusion polypeptide has the amino acid sequence of SEQ ID NO: 3 or 4. A recombinant cell clone comprising the nucleic acid construct of claim 1 or the fusion polypeptide of claim 5. A pharmaceutical composition comprising the fusion polypeptide of claim 5 and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising the nucleic acid construct of claim 1 and a pharmaceutically acceptable carrier.

Images