MXPA06010445A - Methods and constructs for expressing polypeptide multimers in eukaryotic cells using alternative splicing - Google Patents

Abstract

The invention provides a method of producing multiple polypeptides, such as antibodies or antibody fragments, in a eukaryotic cell using a single expression vector. The expression vector is engineered to comprise two or more expression cassettes under the control of a single promoter wherein the expression cassettes have splice sites which allow for their alternative splicing and expression as two or more independent gene products at a desired ratio. Use of the vector for the efficient expression of recombinant antibodies in eukaryotic host cells is disclosed as well as the use of such antibodies in diagnostic and therapeutic applications.

Description

METHODS AND CONSTRUCTS TO EXPRESS POLYPEPTIDE MULTIMERS IN EUCARIAN CELLS USING ALTERNATIVE SPLICE Field of the Invention Certain embodiments of the invention described herein relate to vectors and methods for expressing polypeptide multimers in eukaryotic cells, both in vitro and in vivo, using alternative splicing. Methods for producing cells containing these vectors are included, as well as the use of these vectors and the polypeptides expressed therein for the treatment of a disease and for the efficient production in vivo or in vitro of such multimeric proteins.

Background of the Invention Polypeptide multimers are assembled from two or more polypeptides that together form a complex. The polypeptides that make the complex are usually different. The antibodies are a typical polypeptide multimer which are comprised of two antibody light chain polypeptides and two antibody heavy chain polypeptides that together form a tetrameric complex. Expression of polypeptide multimers in host cells is a challenging process in which the expression of each different polypeptide that makes the multimer of REF: i75741 polypeptide should be carefully coordinated. For example, to express an antibody in eukaryotic cells, a first gene encoding the light chain antibody and a second gene encoding a heavy chain antibody should be introduced into the cell and expressed within an acceptable range of ratios. The expression of an unacceptable ratio of light to heavy chain antibody within the same cell or culture system can result in a highly inefficient production of the desired multimeric complex or in toxicity of the cell or organism. Previous approaches for expressing polypeptide multimers in eukaryotic cells include introducing two or more vectors, each vector separately encoding each of the different polypeptides that make the polypeptide multimer. Each vector typically carries a promoter that drives the expression of a polypeptide of the complex, and at least one vector typically encodes a selection marker. The vectors are then, in series or together, introduced into the cell (usually by transfection) and the cells are co-selected for the expression of both selection markers. In another approach, a sequence encoding together with a promoter for each polypeptide making the polypeptide complex, is engineered into a single vector. This approach eliminates the need to work with multiple vectors, but still does not eliminate the potential for promoter competition between each coding sequence. Also, this approach may not typically solve the problem of expressing the individual polypeptides comprising the protein multimer in an acceptable ratio to result in efficient expression of the protein multimer. Thus, in any of the above approaches, a consistent ratio of two products may not always be obtained in the host cell. This may be due to factors such as a promoter difference activity, promoter competition for cellular factors required for optimal expression, efficiency of transcription and / or translation of the multimeric protein component polypeptides, and / or a difference in the number of copies for each of the vectors introduced into the cell. The splice vectors use a simple splice donor and a splice acceptor has also been developed. The Patent E.U.A. No. 5,043,270 describes a minigene expressing a selectable marker, e.g., DHFR, and has an intron that contains a gene encoding the protein of interest. La- Patent E.U.A. No. 5,561,053 describes the reverse situation, in which the gene encoding the protein of interest contains a 5 'intron for the coding sequence. This intron contains the gene encoding the selectable marker linked by the splice donor and acceptor. This type of intron expression vector is. also describes in Lukas, B.K., Nucleic Acids Res. 24: 1114-1119 (1996). The application for publication of Patent E.U.A. ?or. 2005/0019925 Al describes similar intronic vectors with a selectable fusion marker. The use of two pairs of splice donors and splice acceptors for the expression of more than one protein of interest is also described. All these published constructs, however, depend on pairs of splice donors and splice acceptors, that is, they have a splice donor aligned to a single-splice acceptor. Each of these constructs depends on a highly efficient splice at all sites for their effectiveness. There is no reference to using a single splice donor to activate the alternative splice from more than one splice acceptor to express multiple polypeptides. Furthermore, there is no suggestion in wishing to express the polypeptides at different ratios, or the substitution of different splice acceptors to control the relative expression of the polypeptides. Accordingly, there is a need for a construct that links the expression of two or more genes in a consistent relationship, such that the resulting gene products are produced and assembled efficiently.

Brief Description of the Invention The invention solves the above problems of expressing the polypeptide multimer in a host cell using an expression vector by ligating the expression of two or more genes through the use of alternative splicing. Accordingly, a simple vector having a promoter can be used to drive the expression of a pre-mRNA that can be spliced into two or more different mRNA transcripts in such a way that the two or more mRNA transcripts encode different polypeptides. In this way, the relative expression of the two or more products is not influenced by the differential activity of independent promoters, competition of the promoter, or number of copies of the vector. In particular, the invention provides a method for introducing a simple expression vector into a eukaryotic cell using a simple promoter to drive the transcription of a single pre-AR? M that is then spliced alternately into two or more different gene products that can be then translate into two or more different polypeptides. In one embodiment, the gene products encode the polypeptide subunits of a multimeric protein. In a further embodiment, the gene products are light and heavy chain antibodies that comprise an antibody.

Accordingly, the invention has several advantages including, but not limited to, the following: providing a vector for expression of multiple polypeptides, in particular, heteromeric polypeptides such as antibodies or antibody fragments; - an efficient method of producing multiple polypeptides, e.g., heavy and light chain polypeptides of antibodies, in a eukaryotic cell such as an animal cell or a yeast cell; - an efficient method of production of recombinant antibodies for use in diagnostic or therapeutic applications; and - recombinant antibodies produced by the method, to treat a subject in need of recombinant antibody therapy. Accordingly, in one embodiment, the invention provides an expression vector comprising, in a 5 'to 3' direction or in the 3 'direction, a promoter, for example, the CMV promoter; a non-translated region (UTR) that provides, for example, a closing signal; a splice donor; an intron; a first splice acceptor; a first exon encoding a first polypeptide; a second splice acceptor; and a second exon encoding a second polypeptide, wherein the promoter is operably linked to the first and second exons. The splice sites (ie, donors and acceptors) can be naturally occurring splice sites, designed splice sites, for example, synthetic splice sites, canonical or consensus splice sites, or non-canon splice sites, by example, cryptic splice sites. Each exon may also comprise a polyadenylation signal. Here, the term "first" or "second" as applied to a genetic element such as an exon, intron, any splice site, etc., is used primarily to identify and distinguish several elements from each other and does not refer to the linear placement of the momentum or numbering of elements within a gene or the order in which the pre-mRNA molecules encoding polypeptide components separated from a protein multimer are expressed. In another embodiment, the vector contains a splice donor and more than one splice acceptor, for example, two, three, four, five, six, seven, eight, nine, ten or more splice acceptors. Each splice acceptor is associated with an exon located just in the 3 'direction of the splice acceptor. Therefore, the vector contains more than one exon, for example, two, three, four, five, six, seven, eight nine, ten or more exons. In one embodiment, the first polypeptide is the heavy chain of an antibody and the second polypeptide is the light chain of the antibody or, alternatively, the first polypeptide is the light chain of an antibody and the second polypeptide is the heavy chain of the antibody. In a related mode, the light and / or heavy chain is murine, chimeric, humanized, or human. The light or heavy chains may contain amino acid alterations such as the introduction or ablation of glycosylation sites in, for example, the Fc region. In another embodiment, the first and second polypeptides are expressed in a ratio of about 20: 1 to about 1:20, about 15: 1 to about 1:15, about 12: 1 to about 1:12. , about 10: 1 to about 1:10, about 9: 1 to about 1: 9, about 8: 1 to about 1: 8, about 7: 1 to about 1: 7, about from 6: 1 to about 1: 6, about 5: 1 to about 1: 5, about 4: 1 to about 1: 4, about 3: 1 to about 1: 3, about 2 : 1 to about 1: 2, or about 1: 1. In particular embodiments, the first and second polypeptides are expressed in a ratio of about 20: 1, about 19: 1, about 18: 1, about 17: 1, about 16: 1, about 15: 1 , around 14: 1, around 13: 1, around 12: 1, around 11: 1, around 10: 1, around 9: 1, around 8: 1, around 7: 1, around of 6: 1, around 5: 1 ,. about 4: 1, about 3: 1, about 2: 1, about 1: 1, about 1: 2, about 1: 3, about 1: 4, about 1: 5, about 1: 6, about 1: 7, about 1: 8, about 1: 9, about 1:10, about 1:11, about 1:12, about 1:13, about 1: 14, around 1:15, around 1:16, around 1:17, around 1:18, around 1:19 and / or around 1:20. The determination of the ratio is commonly determined using techniques recognized in the art such as reverse transcriptase polymerase chain reaction (RT-PCR) or Northern blot. (for measurement of relative amounts of transcripts) or immunostaining or enzyme linked immunosorbent assay (ELISA) techniques (for measurement of relative amounts of polypeptides). In one embodiment, the vector comprises SEQ ID N0: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, or 28. In a related embodiment, the vector comprises SEQ ID NO: 29. In another embodiment, the invention provides a eukaryotic cell that contains the above vector capable of expressing two or more splice gene products alternatively. In one embodiment, the alternative splicing vector of the present invention is integrated into the chromosomal DNA of the cell, and in yet another embodiment, the vector is episomal.

In related embodiments, the alternative splice cassette or construct of the present invention is integrated into the chromosomal DNA of the cell. In another related embodiment, the alternative splice cassette or construct of the present invention is episomal. In still other related embodiments, the alternative splicing vector of the present invention comprises a viral vector. In another embodiment, the eukaryotic cell containing the vector of the invention is a mammalian cell, such as, for example, a newborn hamster kidney cell, a fibroblast, a myeloma cell, an NSO cell, a cell of PER.C6, or CHO cell, or, alternatively, an insect cell, such as, for example, a Spodoptera frugiperda (Sf9) cell, a Tricoplusia ni (TnHigh-Five) cell, or a cell of Bombyx mori (BMN). Alternatively, the eukaryotic cell containing the vector of the invention is a yeast cell, for example, ~ a Saccharomyces cell, Schizosaccharomyces cell, or a Pichia cell. In another embodiment, the invention provides a method of producing polypeptides, the method comprising cultivating the above cell containing the splice vector or expression cassette or alternative construct of the present invention followed by isolation of the first and second polypeptides of the invention. cell culture.

In one embodiment of the method, the first and second polypeptides form a polypeptide multimer such as an antibody. In another embodiment, the invention provides the above peptide multimer, produced by the method for the manufacture of a medicament for the treatment or prevention of a disease or disorder. In another embodiment, the invention provides for the delivery of the alternative splice vector or alternative splice cassette or construct for an in vivo patient. In a further embodiment, the invention provides the delivery of the alternative splice vector or alternative splice cassette or construct to cells of the patient or tissue ex vivo. In related embodiments, the present invention also provides for the return of cells or tissues from patients comprising the alternative splice vector or the alternative splice cassette or construct to the patient's body. Other features and advantages of the invention will be apparent to one of ordinary skill in the art of the following detailed description and claims.

Brief Description of the Figures Figure 1 shows a schematic of the structural and functional aspects of an alternative splice vector of the invention that is designed to allow a single promoter to drive the transcription of a single pre-mRNA which can then alternatively be spliced into two or more transcripts. The two more O-transcripts are then translated into two or more corresponding polypeptides. Figure 2 shows a schematic of an expression vector of the invention when used to express a light and heavy chain antibody that is then assembled to form a mature tetrameric antibody. Figure 3 is a plasmid map of an alternative splice vector of the invention designated to express a light and heavy chain antibody of a single promoter for the production of an antibody in eukaryotic cells. The plasmid map also indicates the position and orientation of splice sites, cloning sites, polyadenylation sequences, and markers for selection in eukaryotic cells (DHFR) and propagation in prokaryotic cells (beta-lactamase). See also SEQ ID? O: 29, which provides the sequence of the vector column. Figure 4 shows the sequence of the splice sites of the first splice acceptors tested, intron and exon regions (respectively, lower case and upper case), and the corresponding plasmid designations and sequence identifiers.

Detailed Description of the Invention In order to provide a clear understanding of the specification and claims, the following definitions are conveniently provided below.

Definitions The terms "first polypeptide" or "second polypeptide" refer to polypeptides whose coexpression is desired. These include, for example, "chains" of multimeric protein polypeptides comprising multiple polypeptide subunits. These multimeric proteins can be those found in nature, for example, those described in the art. Of course, the terms "first polypeptide" and "second polypeptide" may also be used in the future to describe multimeric proteins that have not yet been described in the art. These multimeric proteins can also be artificial, for example, comprising polypeptides that are not normally associated in nature. In addition, the polypeptides may not be combined as a whole, but may be coexpressed for some functional purpose, for example, the co-expression of a selectable marker and a polypeptide of interest. In addition, the polypeptides may be native or mutated sequences, such as by the addition, removal, or substitution of amino acids. A person of ordinary skill in the art would quickly recognize the viability of the vectors of the present invention for a broad spectrum of polypeptides and would be able to adapt the vectors for use with these polypeptides using standard molecular biology techniques. The term "antibody" or "antibody fragment" refers to an assembly of polypeptides or antibody fragments, which have binding activity for a target or receptor polypeptide or have the desired effector function. Typically, such assemblies include at least the variable region of an antibody light chain and heavy chain, for example a Fab fragment, or two antibody heavy chains and two antibody heavy chains, the four chains together form a tetrameric antibody (L: H: H: L) the variable regions from which they can bind an antigen. The antibodies of the invention can be in any manner known in the art, for example, murine, chimeric, humanized, human, or synthetic. The antibodies of the invention can also be modified to have other characteristics such as altered glycosylation sites or Fc regions. The term "UTR" means the untranslated region and refers to a segment of a nucleic acid sequence that is transcribed into a non-translated region of the pre-mRNA and mature mRNA. A 5 'UTR typically serves as the 5' end of the transcript that is modified or "capped" with a 7-guano, 7-methylguanosine cap that initiates the translation of the mRNA transcript into a polypeptide. The term "expressed in a relation" refers to the production ratio of a gene product expressed either as a transcript or polypeptide. The determination of the ratio is typically determined using art recognized techniques art such as chain reaction reverse transcriptase polymerase (RT-PCR) or techniques Northern (to measure the relative amounts of transcripts) or immunoblotting techniques or linked immunosorbent assay the enzyme (ELISA) (to measure relative amounts of polypeptides). In certain embodiments, the first and second polypeptides are expressed in a ratio of about 20: 1 to about 1:20, about 15: 1 to about 1:15, about 12: 1 to about 1:12. , around 10: 1 to around-1:10, around 9: 1 to about 1: 9, about 8: 1 to about 1: 8, about 7: 1 to about 1: 7, about 6: 1 to about 1: 6, about 5: 1 to about 1: 5, about from 4: 1 to about 1: 4, about 3: 1 to about 1: 3, about 2: 1 to about 1: 2, or about 1: 1. In particular embodiments, the first and second-polypeptides are expressed in a ratio of about 20: 1, about 19: 1, about 18: 1, about 17: 1, about 16: 1, about 15: 1, about 14: 1, about 13: 1, about 12: 1, about 11: 1, about 10: 1, about 9: 1, about 8: 1, about 7: 1, about 6: 1, about 5: 1, about 4: 1, about 3: 1, about 2: 1, about 1: 1, about 1: 2, about 1: 3, about 1: 4, about 1: 5, about 1: 6, about 1: 7, about 1: 8, about 1: 9, about 1:10, about 1:11, about 1: 12, around 1:13, around 1:14, around 1:15, around 1:16, around 1:17, around 1:18, around 1:19 and / or about 1: twenty. The term "first exon" refers to an encoded sequence or nucleic acid sequence that encodes a polypeptide or polypeptide region and the term "second exon" refers to a second, different coding sequence or nucleic acid sequence encoding a second polypeptide region. The first and second exons of the present invention also comprise a 5 'splice acceptor sequence. The term "host cell" or "eukaryotic host cell" refers to any eukaryotic cell that produces or expresses the gene products of the first and second exons, using the expression system of the invention. This includes, for example, mammalian cells such as baby hamster kidney cells, fibroblasts, myeloma cells (e.g., NSO cells), human PER.C6 cells, or Chinese hamster ovary (CHO) cells. Insect cells useful for expression include, for example, Spodoptera frugiperda (Sf9) cells, Tricoplusia ni cells (Tn. TnHigh-five), or Bombyx mori (BMN) cells. Yeast cells useful for expression include, for example, Saccharomyces cells, Schizoeaccharomyces cells and Pichia cells. Such cells are readily accessible from public and commercial sources, such as the American Type Culture Collection (ATCC, Manassas, VA). The term "intron" refers to a segment of a nucleic acid sequence that is transcribed and present in the pre-mRNA but is excised by the splicing machinery based on the sequences of the splice donor and the splice acceptors and therefore it does not appear in the mature mRNA transcript. The term "operably linked" refers to a juxtaposition wherein the components are in a relationship that allows them to function in their intended manner (e.g., functionally linked). The term "polyadenylation signal" refers to a nucleic acid sequence present in the RNA transcript that allows the transcript, when in the presence of the polyadenyl transferase enzyme, to be polyadenylated. Many polyadenylation signals are known in the art and are useful for the present invention. Examples include the human variable growth hormone polyadenylation signal, the SV40 late polyadenylation signal and the bovine growth hormone polyadenylation signal. The term "promoter" refers to a minimum sequence sufficient to direct transcription, preferably in a eukaryotic cell. Promoters for use in the invention include, for example, viral, mammalian, insect and yeast promoters that provide high levels of expression, eg, mammalian cytomegalovirus or CMV promoter, SV40 promoter, or any promoter known in the appropriate art. for the expression in eukaryotic cells. The term "splice site" refers to specific nucleic acid sequences that are capable of being recognized by the splicing machinery of a eukaryotic cell as is appropriate to be cut and / or ligated to the corresponding splice site. The splice sites allow the removal of introns present in a pre-mRNA transcript. Typically the 5 'portion of the splice site is referred to as the splice donor and the corresponding 3' splice site is referred to as the splice acceptor site. The term splice site includes, for example, naturally occurring splice sites, engineered splice sites, for example, synthetic splice sites, canonical or consensus splice sites, and / or non-canon splice sites, eg, splice sites cryptic. The term "spliced with" refers to the splice donor that interacts with the splice acceptor to allow splicing of the transcript by the splicing machinery (e.g., the spliceosome). As described above, splicing is the removal of a portion of the transcript (the intron) linked by the splice donor and the splice acceptor. For each transcript, the splice donor is spliced only with a splice acceptor. For alternative splicing, within the group of transcripts the splice donor is spliced with more than one splice acceptor. For example, the splice donor can be spliced with a first splice acceptor for a transcript, but in another transcript, the splice donor can be spliced with a second splice acceptor, which generates a heterogeneous group of transcripts. The term "spliced transcript" refers to an RNA transcribed from the alternative splice vector of the invention comprising a first or a second exon and which undergoes a splice between the splice donor and any of the first or second splice acceptors.

The term "vector" refers to a nucleic acid molecule (either DNA or RNA) capable of conferring the expression of a gene product when introduced into a host cell or host cell extract. This term is interchangeable with "alternative splicing vector", "expression vector", "expression cassette" or "construct". Such expression vectors or cassettes or constructs may comprise the alternative splice elements of the present invention as well as additional sequences for vector propagation in cells, vector entry into cells and subsequent expression, selectable markers, or any other functional elements. . Such elements are well known in the art and can be exchanged as necessary using standard molecular biology techniques. The term "viral vector" or variations thereof (such as "adenoviral vector") refers to a lightened or replication-deficient stained glass particle comprising the alternative splice vector or expression cassette or construct of the invention. As described in more detail below, such viral vectors are useful for inserting the alternative splice vector or expression cassette or construct of the invention into host cells.

Detailed Description of the Invention 1. Introduction The invention provides, in part, a method for expressing two or more gene products in a eukaryotic cell by providing an expression vector or cassette or construct comprising a single promoter that drives the expression of two. or more exons that have been engineered to alternatively splice into two or more expressible transcripts. In this manner, the expression vector or cassette or construct is suitable for expressing two or more polypeptides, and in particular, polypeptide multimers, for example antibodies (or antibody fragments) that are typically an assembly of two-chain polypeptide antibodies. light and two heavy chains. On the other hand, because the expression vector or cassette of the invention uses a simple promoter to drive the expression of a pre-mRNA, which is then alternatively spliced into two or more gene products, the invention avoids the use of multiple vectors, promoter competition of the use of multiple promoters, or differential activity of independent promoters. Additionally, through the use of multiple splice acceptors, the invention has the advantage of providing for the expression of multiple gene products in a eukaryotic cell at a desired ratio such that, for example, the resulting polypeptide assembly, for example a tetrameric antibody, is produced efficiently. In addition, the present invention is provided to alter the expression ratio of the encoded polypeptides by changing the sequence of the. splice elements, particularly the first splice acceptor. The ability to alternate the ratio of the expressed polypeptides allows a more efficient multimerization or other functional aspect of the polypeptides by providing the polypeptides in optimal amounts. In this manner, the expression vector or cassette of the present invention allows the expression of the polypeptides in sufficient amounts to produce one or more desired proteins. In this manner, the invention also provides vectors suitable for the expression of polypeptide multimers, eg, antibodies, using alternative splicing as well as cells comprising the vector and antibodies produced from such cells, and their use in, for example, prognosis, diagnosis, prevention, alleviation or treatment of a disorder or disease in a subject, for example, a human patient. In addition, the invention provides vectors suitable for the expression of the polypeptides in vivo, either by administering the vector to the subject directly or through ex vivo techniques. 2. Vector Design or Expression Casete or construct _ to_Express Polypeptide Multimers The methods of the invention employ the use of a vector comprising (at 5 'to 3' or downstream) a promoter; a 5 'untranslated region (UTR) (which may or may not include a coding sequence); a 5 'simple splice donor; an intron that ends with a 3 'splice acceptor that is used with preferably at an efficiency of about 5 to 95% (depending on the desired ratio of products) (exemplary, the 3' splice acceptor efficiently includes, but does not is limited to, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80 %, 85%, 90% and 95%); an exon containing the first gene and, optionally, a polyadenylation signal; an intron sequence terminating with the 3 'splice acceptor that is greater than 50% efficiency (in preferred embodiments, the 3' splice acceptor is greater than 75% efficiency (eg, 80%, 85%, 90 %, 95% or 100%), greater than 85% efficiency (eg, 90%, 95% or 100%), greater than 90% efficiency (eg, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99% or 100%), or, in a highly preferred embodiment, greater than 95% efficiency (eg, 96%, 97%, 98%, 99% or 100%)); and an exon containing the second gene, and optionally, a polyadenylation signal (see Fig. 1). The vector, expression cassette or construct of the present invention can be introduced into a host cell where it alternatively produces spliced mRNA transcripts. These two or more transcripts of .RNAs encode different polypeptides, which are then expressed in sufficient amounts to produce one or more desired multimeric proteins. The ratio of the two or more products can then, if desired, be altered through the selection of appropriate splice sites, for example, a weaker or stronger splice acceptor. In particular embodiments, the relationship of the two or more products can be altered through the selection of only the first splice acceptor. Typically, the splicing of pre-mRNA involves the precise removal of intron sequences and is partly based on the recognition of specific sequences, that is, donor and acceptor splice sites, at intron-exon boundaries by the splicing machinery , for example the splenyosome. These boundary sequences typically place the consensus sequences of MAG / GURAGU (SEQ ID NO: 30) for the 5 'end of the intron and YIQ? CAG / G (SEQ ID NO: 31) for the 3' end of the intron (where M = A or C, non-variant nucleotides are underlined, Y? 0 = 10 consecutive nucleotides C or T, and? = Any nucleotide). The sequences within the introns and exons as well as the size of the intron have also been shown to play a role in the efficiency of the splice (see, for example, Chabot, B. Trends in Genetics, 12, 472-478. )). In certain embodiments of the present invention, alternative splicing can regulate the expression of each polypeptide encoded by the exons of the expression vector or cassette or construct of the invention. For each individual spliced transcript, the splice donor is spliced only with one splice acceptor or none at all. However, the alternative splice forms a heterogeneous group of transcripts as the splice donor can be spliced with the first splice acceptor in one transcript, and spliced with a second splice acceptor in another transcript. In this manner, the splicing of a single splice donor to multiple splice acceptors will regulate the levels of spliced transcripts of the first and second (or more) exons generated from an expression vector or cassette or construct of the present invention in a cell. The relative amounts of differentially spliced transcript levels will depend on how frequently the splice donor joins with a particular splice acceptor, which is referred to as the efficiency of the splicing event. The more frequently a particular splice event occurs, then the splice donor and splice acceptor associated with such splice event are the most efficient or strong.

In certain embodiments of the present invention, it is preferable that the splice donor and the 3 'terminal splice acceptor (e.g., the second splice acceptor) are highly efficient so that the general splice is highly efficient and the strength of the first splice splice acceptor controls the relative levels of spliced exon transcripts. The translation of the non-spliced transcript is typically very inefficient, so that the maximum splice promotes the expression of the polypeptide. For example, if the splice donor and the second splice acceptor are highly efficient, the majority of the incipient mRNA will be spliced. If the first splice acceptor is strong (that is, efficient), then relatively high levels of spliced transcripts comprising the first exon within the cell will be present, leading to a high ratio of the expression of the first polypeptide compared to the second. polypeptide. If the first splice acceptor is weaker, then relatively low levels of spliced transcripts comprising the first exon will be present within the cell, which leads to a low ratio of the expression of the first polypeptide compared to the second polypeptide. Because the translation is highly dependent on the levels of spliced transcript comprising a given exon, expression of the polypeptide encoded by the first or second exon depends on the splice levels using the splice acceptor immediately upstream of that exon. Therefore, in certain embodiments, because the splice affects the levels of mature transcripts comprising each exon encoded by a vector or expression cassette or construct of the invention, the alternative splice is used to regulate the expression of the encoded polypeptides. for the exons.

Splicing donors and splicing acceptors are well known in the art and can be used in the present invention. These elements can be found, inter alia, in the art or derived from consensus sequences, either empirically when inserting, deleting or replacing nucleotides, or by using software capable of predicting a splicing sequence, such as Netgene2 version 2.4. Such splicing elements may be tested to be suitable in the present invention, such as by the use of the methods described in the examples. In part, the present invention incorporates and improves such sequences to perform, through genetic engineering, the alternative splicing of two or more desirable recombinant gene products in eukaryotic cells. The expression vector or cassette of the invention is useful for expressing a variety of heteromultimeric proteins. Examples include, but are not limited to, heterodimers such as glycoprotein hormones (e.g., chorionic gonadotropin (CG), thyrotropin (TSH), lutropin (LH), and follitropin (FSH)) or members of the integrin family. Heterotetramers consisting of two pairs of identical subunits may also be used. Examples of suitable heterotetramers include antibodies, the insulin receptor (alpha2 beta2) and the TFIIE transcription initiation factor (alpha2 beta2). In addition, by using a pair of splice acceptors that generate a 2: 1 expression ratio, the expression vector or cassette or construct can be used for the expression of heterotrimers such as lymphotoxin alfalbeta2. By altering the expression ratio by combining different splice acceptors, vectors or expression cassettes or constructs capable of expressing multimers in different ratios can be generated, allowing efficient expression of many different heteromultimeric proteins. In certain embodiments, expression relationships are predicted based on the sequence of the splice donor and / or signals from the acceptor. In other modalities, the relations of expression are determined empirically. On the other hand, the genetic sequences useful for producing polypeptide multimers, -for example, antibodies, using the alternative splicing system of the invention, can be obtained from a number of different sources. For example, a variety of human protein genes are available that are available in the form of publicly accessible gene sequences and / or plasmid deposits, clones, cells, and the like. Alternatively, the cell lines that produce proteins can be selected and cultured using techniques recognized in the art. In other modalities, genetic sequences are obtained through access to databases by subscription. One of ordinary skill in the art would know many appropriate methods for obtaining genetic sequence information. For example, the RNA encoding the antibody can be isolated from the hybridoma cells that produce the original antibody or from other transformed cells by standard techniques, such as guanidinium isothiocyanate extraction and precipitation followed by centrifugation or chromatography. Wherever desired, the mRNA can be isolated from the AT? total by standard techniques such as chromatography on oligo dT cellulose. Appropriate techniques for these purposes are familiar to someone of ordinary skill in the art. The AD? C encoding the light and heavy chains of the antibody can be made, either simultaneously or separately, using reverse transcriptase and AD polymerase. in accordance with well-known methods. It can be initiated by consensus constant region primers or by more specific primers based on, for example, the AD? of light and heavy chain of published antibody and amino acid sequences. As discussed above, PCR can also be used to isolate the DNA clones encoding the light and heavy chains of the antibody. In this case, collections can be separated by exclusion by consensus primers or large homologous probes, such as mouse constant region probes. Alternatively, antibodies and antibody fragments can be synthesized using sequences derived from well-known computer modeling techniques. Such modeling techniques can be used to predict antibody sequences which, in the context of a given antibody structure defined by the conservative amino acid sequences, would link a predicted ligand structure. By using this known relationship, the person skilled in the art can design the amino acid sequence of the desired antibody or antibody fragment, then synthesize the nucleic acid molecules encoding the desired polypeptides. Such designated antibody or antibody fragments are referred to as "synthetic". The compositions and inventive methods of the present invention are suitable for any antibody, or indeed any multi-chain or multimeric protein. Oligonucleotide synthesis techniques compatible with this embodiment of the invention are well known to one of ordinary skill in the art, and can be carried out using any of several commercially available automated synthesizers. In addition, the DNA sequences encoding various types of heavy and light chains established herein can be obtained through the services of commercial DNA vendors. The genetic material obtained using any of the above methods can then be altered or modified to provide antibodies compatible with the present invention and the desired use of such antibodies. A variety of different types of antibodies can be expressed according to the invention. For example, antibody or antibody fragments with an immunoreactive activity specific for an antigen, for example, an antigen associated with tumor, pathogen, or autoantigen involved in an autoimmune disease. The antibody (or fragment thereof), it can be modified in such a way that one or more constant regions are deleted or otherwise altered to provide the desired functional activity such as serum half-life, or effector function. Many such antibodies are described in Kuby, J. Immunology, 3rd ed., .H. Freeman and Co. (1997). Antibodies suitable for expression in a eukaryotic cell using the method of the invention include the five distinct classes of antibody: IgA, IgD, IgG, IgE, and IgM. Although all five classes are within the scope of the present invention, the following discussion is generally directed to the class of IgG molecules. One of ordinary skill in the art will readily be able to adapt the following discussion to the other classes of immunoglobulins. The IgG molecules typically comprise two identical antibody light chains of a molecular weight of approximately 23 kD each, and two identical antibody heavy chains of a molecular weight of 53-70 kD each. The disulfide bonds between chains, in a configuration as shown in Fig. 2, joins the four chains. In addition, both the light and heavy chains of the antibody are divided into regions of functional and structural homology. The terms "constant" and "variable" e use functionally. In this regard, it will be appreciated by one of ordinary skill in the art that the variable domains of both the light (VL) and heavy (VH) chains of the antibody determine the recognition and specificity of the antigen. Conversely, the constant domains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3) - confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement linkage, and other effector functions . Light chains are classified as either kappa or lambda chains. Each heavy chain class can be linked to any kappa or lambda light chain. In general, the light and heavy chains are covalently linked to each other, and the "tail" portions of the two heavy chains are linked to each other by covalent disulfide bonds when the immunoglobulins are generated either by hybridomas, cells B, or host cells produced by genetic engineering (see Fig. 2). In terminal N it is a variable region and in terminal C it is a constant region. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, with some subclasses between them. It is the nature of this chain that determines the "class" of the antibody as IgA, IgD, IgE IgG, or IgM. The subclasses of immunoglobulin (isotypes) for example, IgGl, IgG2, IgG3, IgG4, IgAl are well characterized and are known to confer functional specialization. It is understood that the first exon or the second exon of the present invention can be used to alternatively encode the antibody chain either light or heavy so that the resulting vector encodes a light chain and a heavy chain. The antigen binding site is defined by three complementarily determining regions (CDRs) in each of the VH and VL chains. The six CDRs present in each monomeric antibody are short, noncontiguous, amino acid sequences that are specifically placed to form the antigen binding site as the antibody assumes its three-dimensional configuration in an aqueous environment. The rest of the heavy and light variable domains show less molecular variability in the amino acid sequence and are called the column regions. The framework regions widely adopt a beta sheet conformation and the CDRs form curls connected to, and in some cases forming part of, the beta sheet structure. In this way, the structure regions act to form a scaffold that is provided to place the six CDRs in the correct orientation by non-covalent, inter-chain interactions. The binding site of the antigen formed by placing the CDRs defines a surface complementary to the epitope in the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to the immunoreactive antigen epitope. The antibody fragments are suitable for expression using the alternative splice vectors or expression cassettes or constructs of the invention. Such fragments are any portion or portions of a desired antibody, and may include, for example, Fab fragments, F (ab ') 2 fragments, and Fc fragments. In addition, the antibody fragments may include single chain antibodies or other antibody derived polypeptides comprising less than the full length tetramer antibody protein.

It will be appreciated by someone of ordinary experience in the It is understood that "the expressed antibodies using the alternative splice vectors or expression cassettes or constructs of the invention can comprise any type of variable region that provides the association of the antibody with the selected antigen. In this regard, the variable region can comprise or be derived from any type of mammal that can be induced to support a humoral response and generate immunoglobulins against the desired antigen. As such, the variable region of the modified antibodies can be, for example, murine, non-human or human primate. When derived from a different species, commonly a murine variable region fused to human constant regions, the antibody is referred to as a chimeric antibody. In preferred embodiments, both the variable and the constant regions of the antibodies are human. In other selected modalities the variable regions of compatible antibodies (usually derived from a non-human source) can be designed or adapted specifically to increase the binding properties (eg, affinity maturation) or reduce the immunogenicity of the molecule. In this regard, the variable regions useful in the present invention can be humanized or otherwise altered through the inclusion of imported DNA or amino acid sequences. Such human antibodies, which have the CDRs grafted from other species, are referred to as humanized antibodies. In addition, the variable regions can be designed, or synthetic, as previously described. Any of the above antibodies can be further modified to have altered glycosylation sites, sites suitable for pegylation, and / or sites that confer an altered effector function to the antibody, eg, altered complement binding, altered Fc receptor binding, and / or altered immune cell interaction activity. For the purposes of this invention, numerous alternative splice expression vector systems may be employed. For example, the alternative splice vector or expression cassette or construct of the invention may contain DNA elements which are derived from animal viruses such as a human or bovine papilloma virus, a polyoma virus, an adenovirus, a virus of vaccinia, a baculovirus, a retrovirus (eg, HIV), a cytomegalovirus, or an SV40 virus. Additionally, cells that can integrate the alternative splice vector or expression cassette or construct within their chromosomes or maintain the expression vector or cassette or construct episomally can be selected by introducing one or more markers that allow selection of the cells transfected hosts. The selectable marker gene can be ligated directly to the DNA sequences to be expressed, or introduced into the same cell by cotransfection. Suitable host cells for introduction of the expression vectors or cassettes or constructs of the invention are discussed below. 3. Expression of Polypeptide Multimers in Cells Eukaryotic in Cultivation. The alternative splice vector or expression cassette or construct of the invention can be introduced into an appropriate host cell using technologies that are well known to one of ordinary skill in the art. These include, for example, transfection (which includes electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and virus infection (see, for example, Ridgway, AAG "" Mammalian Expression Vectors "Chapter 24.2, pp. 470-472 Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass. 1988) As mentioned above, the expression vectors or cassettes of the invention can be integrated into the chromosome of the host cell, maintained episomally, or expressed transiently. The transformed cells grow under conditions suitable for the production of the encoded polypeptides contained therein, eg, the antibody, light or heavy chains, and tested for polypeptide synthesis. Techniques tested for identification and quantification of polypeptide synthesis include, for example, enzyme-linked immunosorbent assay (ELISA), fluorescence resonance energy transfer (FRET), radioimmunoassay (RIA), analysis, fluorescence activated cell sorter. (FACS), and immunohistochemistry. The host cell line used for the expression of protein is preferably of eukaryotic origin, for example of mammalian origin or, alternatively, a yeast or insect. Exemplary host cell lines include, for example, Chinese Hamster Ovary (CHO), HeLA (human cervical carcinoma), CVl (monkey kidney line), COS (a CVl derivative with SV40 T antigen), R1610 (Chinese hamster fibroblast), BALBC / 3T3 (mouse fibroblast), HAK (hamster kidney line), SP2 / 0 (mouse myeloma), NSO (myeloma), (bovine endothelial cells), RAJI (lymphocyte) human), 293 (human kidney), PER.C6 (human), Spodoptera frugiperda (Sf9) (insect), Tricoplusia ni (Tn. TnHigh-Five) (insect), Bombyx mori (BMN) (insect), Saccharomyces (yeast) ), Schizosaccharomyces (yeast), and Pichia (yeast). Host cell lines are commonly available from commercial services, such as the American Tissue Culture Collection or published literature.

In vitro production allows scaling to give large amounts of the desired polypeptide produced using the alternative splicing system of the present invention, preferably an antibody. Techniques for eukaryotic culture, e.g., culture of mammalian cells or yeast under tissue culture conditions are well known to those of ordinary skill in the art and include homogeneous suspension culture, e.g., in an air transport reactor or in a continuous agitator reactor, or cell culture immobilized or entrapped, for example, in hollow fibers, microcapsules, in agarose microbeads, ceramic cartridges or in thermistors. For isolation and recovery of the multimeric proteins produced according to the invention, in particular with respect to antibodies, the proteins (for example, immunoglobulins) in the culture supernatants can first be concentrated, for example, by precipitation with ammonium sulfate, dialysis against hygroscopic material such as PEG and filtration through selective membranes. If necessary and / or desired, the concentrated solutions of the multimeric proteins (eg, multivalent antibodies) are purified by traditional chromatography methods, for example, gel filtration, ion exchange chromatography, chromatography on DEAE cellulose, or chromatography. of immunoaffinity (for example, Protein A or Protein G). The invention further contemplates the expression of any antibody light and heavy chain sequence which when expressed using the alternative splicing system of the invention, is associated to produce a functional antibody, for example, one that specifically binds to a target antigen. , such as an antigen associated with tumor, pathogen, or the same antigen or have a desired effector function. Importantly, the number of copies of the antibody light and heavy chain genes in the splicing construct can alternatively be selected such that the preferred light / heavy chain ratio is obtained. In certain embodiments, the light chain is expressed in levels which are commonly in the range of about 10/1, about 5/1, about 3/1 or about 1/1 relative to the heavy chain. In related embodiments the light and heavy polypeptides are expressed in a ratio of about 20: 1 to about 1:20, about 15: 1 to about 1:15, about 12: 1 to about 1:12, about 10: 1 to about 1:10, about 9: 1 to about 1: 9, about 8: 1 to about 1: 8, about 7: 1 to about 1: 7, about 6: 1 to about 1: 6, about 5: 1 to about 1: 5, about 4: 1 to about 1: 4, about 3: 1 to about 1: 3, about 2: 1 to about 1: 2, or about 1: 1. In particular embodiments, the light and heavy polypeptides are expressed in a ratio of about 20: 1, about 19: 1, about 18: 1, about 17: 1, about 16: 1, about 15: 1 , around 14: 1, around 13: 1, around 12: 1, around 11: 1, around 10: 1, around 9: 1, around 8: 1, around 7: 1, around of 6: 1, about 5: 1, about 4: 1, about 3: 1, about 2: 1, about 1: 1, about 1: 2, about 1: 3, about 1 : 4, about 1: 5, about 1: 6, about 1: 7, about 1: 8, about 1: 9, about 1:10, about 1:11, about 1:12 , around 1:13, around 1:14, around 1:15, around 1:16, around 1:17, around 1:18, around 1:19 and / or around 1:20 . The invention also provides a method for selection of any desired ratio by exclusion separation vectors having different splice sites such that, for example, in a given cell line, the desired ratio is achieved (see, for example, Example 2). This is especially critical when an antibody is efficiently expressed because it has been observed that certain levels of expression of the light chain can be instrumental in the direction of the proper assembly of the heavy and light chains of antibodies, and the excessive odd heavy chain can induce cell toxicity. Moreover, the light chain is also critical in the direction of the multiplication of the antibody heavy and light chains assembled to produce an antibody that binds functional antigen in the endoplasmic reticulum. Accordingly, in certain embodiments, the antibody light chain is commonly expressed from about 10/1 to 1/7 relative to the antibody heavy chain (exemplarily light / heavy chain ratios include, but are not limited to, around of 10/1, 9/1, 8/1, 7/1, 6/1, 5/1, 4/1, 3/1, 2/1 and 1/1). In certain embodiments, an antibody that is expressed according to the subject expression system may be specific to any desired antigen. Preferably, the antibody will be a functional antibody that produces a therapeutic effect, such as an antibody useful for treating an autoimmune, inflammatory, infectious, allergic or neoplastic disease. The antibody can be combined with other therapeutic agents for synergistic effects. For example, the antibody can be combined with, for example, other antibodies, small molecules, or a radioactive source for use as a cancer chemotherapeutic agent.

In certain embodiments, the iYOS alternating splice vectors or expression cassettes or constructs of the present invention are used in combination with other expression vectors or cassettes or constructs that do not intentionally depend on the alternative splice to express multiple polypeptides. 4. Pharmaceutical compositions. The invention also provides, inter alia, therapeutic compositions comprising multimeric proteins expressed using methods and / or expression vectors or cassettes or constructs of the present invention for the treatment of a subject or patient in need thereof. The compositions of the present invention can be used to treat a subject (i.e., a patient) in need thereof, via administration of therapeutic polypeptides produced by the methods of the invention or by gene therapy comprising the alternative splicing vector or expression cassette or construct of the invention. A subject in need of this is a subject who suffers, or is at risk of suffering, from a disease, disorder or condition that can be treated or prevented by administering a composition of this invention. That subject can be a mammalian subject. A preferred subject is a human subject.

In certain embodiments, the therapeutic compositions do not include the multimeric proteins in a pharmaceutically acceptable carrier. In preferred embodiments, the therapeutic compositions include at least one recombinant antibody or antibody fragment produced according to the invention in a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" refers to at least one component of a pharmaceutical preparation that is normally used for administration of active ingredients. As such, a carrier can contain any pharmaceutical excipient used in the art and any form of vehicle for administration. The compositions can be, for example, injectable solutions, aqueous suspensions or solutions, non-aqueous suspensions or solutions, solid and liquid oral formulations, ointments, gels, ointments, intradermal patches, creams, lotions, tablets, capsules, sustained release formulations, and similar. Additional excipients may include, for example, colorants, flavor modification agents, solubility aids, suspending agents, compression agents, enteric coatings, sustained release aids, and the like. The agents of the invention are often administered as pharmaceutical compositions comprising an active therapeutic agent and a variety of other pharmaceutically acceptable components. See Remington's Pharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pennsylvania (1980)). The preferred form depends on the projected mode of administration and the therapeutic application. The compositions may also include, depending on the formulation, pharmaceutically acceptable non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for administration to animals or humans. The diluent is selected such that it does not affect the biological activity of the combination. Examples of such diluents include, but are not limited to, distilled water, physiological phosphate buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other non-toxic, non-therapeutic, non-immunogenic carriers, adjuvants or stabilizers. Commonly, the compositions are administered in therapeutically effective amounts, which are an amount sufficient to produce a beneficial effect preferably medically detectable in a subject or patient suffering or at risk of suffering from a disease, disorder or condition amenable to treatment with the compositions of the invention. Multimeric proteins produced in accordance with the present invention can be administered in the form of an injection or implant preparation, which can be formulated in such a way as to allow a sustained release of the active ingredient. In a preferred embodiment, the multimeric protein is an antibody. An exemplary composition comprises monoclonal antibody at 5 mg / mL, formulated in aqueous buffer solution consisting of 50 mM L-histidine, 150 mM NaCl, adjusted to pH 6.0 with HCl. Commonly, the compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution, or suspension in liquid vehicles before injection may also be prepared. The preparation can also be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above (see, Langer, Science 249: 1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28 : 97 (1997)).

. Prophylactic and therapeutic methods. In certain embodiments, the present invention is directed to the production of appropriate proteins for the prevention, amelioration or treatment of any disease, disorder or condition. The disorders - or diseases include cancer, precancerous conditions, and disease or genetic condition such as muscular dystrophy. A disease, disorder or condition susceptible to treatment with the proteins produced by the methods of the present invention includes conditions such as genetic diseases (ie, disease condition that is attributable to one or more genetic defects), acquired pathologies (this is, a pathological condition that is not attributable to a birth defect) and prophylactic processes (that is, prevention of a disease or unwanted medical condition). An acquired pathology can be a disease or syndrome manifested by an 'abnormal, physiological, biochemical, cellular, structural or molecular biological state. A disease, disorder or condition susceptible to being treated with the proteins produced by the methods of the present invention can be an infection, including viral and bacterial infection, a hyperproliferative disease or disorder, including cancer or precancerous conditions, immune disorders, such as rheumatoid arthritis, genetic immunodeficiency conditions, such as hyper-IgM syndrome, primary or combined immunodeficiency conditions, including conditions characterized by neutropenia, in addition to neurological disorders, cardiovascular disorders, such as ischemia, and endocrine disorders, such as diabetes, thyroid disorders and infertility.

A disease, disorder or condition susceptible to be treated with the proteins produced by the methods of the present invention may be hyperproliferative diseases or disorders, including cancer. The diseases or disorders can involve any cell, tissue or organ, including brain, lung, squamous cells, bladder, stomach, pancreas, breast, head, neck, liver, kidney, ovary, prostate, colon, rectum, esophagus, nasopharynx, thyroid and skin. The cancer can be melanoma, lymphoma, leukemia, multiple myeloma, sarcoma or carcinoma. The cancer can be solid tumors or it can involve a body fluid such as blood. A disease, disorder or condition susceptible to treatment with the proteins produced by the methods of the present invention can be genetically inherited diseases, such as Huntington's disease, bipolar disorder, Parkinson's disease, Carpio tunnel syndrome, cystic fibrosis, Pelizaeus-Merzbacher disease, multiple sclerosis or Muscular Dystrophy of Duchenne. A disease, disorder or condition susceptible to treatment with the proteins produced by the methods of the present invention may be an infectious disease, such as tuberculosis, malaria, yellow fever, or a disease caused by infection by hepatitis B virus, virus of herpes, human_dg_ virus, etc. In particular embodiments, the present invention is also directed, inter alia, to the production of antibodies or antibody fragments appropriate for the prevention or treatment of a disorder or disease, for example, a disorder or disease of the immune system. Accordingly, in certain embodiments, the antibodies or antibody fragments of the present invention are useful in the prevention or treatment of immune disorders including, for example, glomerulonephritis, scleroma, cirrhosis, multiple sclerosis, lupus nephritis, atherosclerosis, inflamed bowel, allergies or rheumatoid arthritis. In another embodiment, the antibody or antigen-binding fragments of the invention can be used to treat or prevent inflammatory diseases, including, but not limited to, Alzheimer's, severe asthma, atopic dermatitis., cachexia, CHF ischemia, coronary restinosis, Crohn's disease, diabetic neuropathy, lymphoma, psoriasis, induced b brosis / radiation, juvenile arthritis, stroke, inflammation of the brain or central nervous system due to trauma, and ulcerative colitis. Other inflammatory disorders which can be prevented or treated with antibodies or antibody fragments produced according to the invention include inflammation due to corneal transplantation, chronic obstructive pulmonary disease, hepatitis C, multiple myeloma, and osteoarthritis. In another embodiment, the antibodies or fragments that bind antigens of the invention can be used to prevent or treat neoplasia, which includes, but is not limited to, bladder cancer, breast cancer, head and neck cancer, Kaposi's sarcoma, melanoma , ovarian cancer, small cell lung cancer, stomach cancer, leukemia / lymphoma, and multiple myeloma. Conditions of additional neoplasia include, cervical cancer, colorectal cancer, endometrial cancer, kidney cancer, non-squamous cell lung cancer, and prostate cancer.

In another embodiment, the antibodies or antibody fragments of the invention can be used to prevent or treat neurodegenerative disorders, including, but not limited to Alzheimer's, stroke, traumatic brain or central nervous system lesions. Additional neurodegenerative disorders include ALS / motor neuron disease, diabetic peripheral neuropathy, diabetic retinopathy, Huntington's disease, macular degeneration, and Parkinson's disease. In clinical applications, a subject is identified as having or is at risk of developing one of the aforementioned conditions by exhibiting at least one sign or symptom of the disease or disorder. At least one antibody or antibody fragment of these of the invention or compositions comprising at least one antibody or fragment that binds antigen of these of the invention is administered in an amount sufficient to treat at least one symptom of a disease or disease. disorder, for example, as mentioned above. Accordingly, a protein of the invention is suitable for administration as a therapeutic reagent for a subject under conditions that generate a beneficial therapeutic response in a subject, for example, for the prevention or treatment of a disease or disorder, for example, as described in the present. The therapeutic agents of the invention are commonly substantially pure of unwanted contaminants. This means that an agent is commonly at least about 50% w / w (weight / weight) of purity, in addition to being substantially free of interfering proteins and contaminants. Sometimes the agents are at least about 80% w / w purity and, more preferably at least 90% or about 95% w / w purity. However, using conventional protein purification techniques, for example, as described herein, homogeneous peptides of at least 99% w / w can be obtained.

The methods can be used in asthmatic subjects and in those that commonly show symptoms of disease. The antibodies used in such methods can be human, humanized, chimeric or non-human antibodies, or fragments thereof (eg, antigen-binding fragments) and can be monoclonal or polyclonal. • In another embodiment, the invention features the administration of an antibody produced according to a method of the invention, with a pharmaceutical carrier as a pharmaceutical composition. Alternatively, the antibody can be administered to a subject by administration of a polynucleotide encoding at least one antibody chain. The polynucleotide is expressed to produce the antibody chain in the subject. Accordingly, the polynucleotide encodes the heavy and light chains of the antibody. The polynucleotide is expressed to produce the heavy and light chains in the subject. In exemplary embodiments, the subject is monitored for the level of antibodies administered in the subject's blood. 6. Gene administration The present invention also encompasses gene therapy by means of which nucleic acids comprising the alternative splicing vector are provided to a patient requiring them. They can be treated. same diseases, disorders and conditions can be improved or prevented as described above for the treatment with polypeptides produced by the methods of the invention. Various methods are described for transferring or administering DNA to cells for the expression of the gene product of the protein, referred to elsewhere as gene therapy, as described in Gene Transfer into Mammalian Somatic Cells in vivo, N. Yang, Crit. Rev. Biotechn. 12 (4): 335-356 (1992) which is incorporated herein by reference. Gene therapy includes the incorporation of DNA sequences into somatic cells or germline cells for use either ex vivo or in vivo therapy. Gene therapy works to replace the genes, increase the function of the normal or abnormal gene, and fight infectious diseases and other pathologies. Strategies to treat these medical problems with gene therapy include therapeutic strategies such as identifying the defective gene and then adding a functional gene to replace the function of the defective gene or to slightly increase a functional gene; or prophylactic strategies, such as adding a gene for the product protein that will treat the condition or that will make the tissue or organ more susceptible to a treatment regimen. Alternatively, genes conferring immunity can be transferred by providing antibodies against a particular antigen in the alternative splicing vector of the invention. Methods for transferring genes in gene therapy fall into three broad physical categories (eg, electroporation, direct gene transfer and particle bombardment), chemical (carriers with lipid base or other non-viral vectors) and biological (vector derived from of virus and receptor uptake). For example, non-viral vectors that include liposomes that complex with DNA can be used. Such liposome / DNA complexes can be injected directly intravenously into the patient. It is believed that the liposome complexes / AD? Are they concentrated in the liver where they release AD? to macrophages and Kupffer cells. These cells have a long life span, thus providing a long term expression of the AD? released. Additionalment-e, vectors or the "AD?" of the gene can be injected directly into the desired organ, tissue or tumor for targeted release of the AD? therapy. Gene therapy methodologies can also be described through the release site. The fundamental mode for release genes includes ex vivo gene transfer, gene transfer in vivo, and in vi tro gene transfer. In the ex vivo gene transfer, cells are taken from the patient and growth in the cell culture. The DNA is transfected into the cells, the transfected cells are expanded in number and then reimplanted in the patient. In the transfer of the in vi tro gene, the method is the same, but the transfected cells are cells growing in the culture, such as tissue culture cells, and not the cells of the individual patient. In vivo gene transfer involves introducing the DNA into the patient's cells when the cells are inside the patient. The methods include using virally mediated gene transfer using a non-infectious virus for the release of the gene in the patient or by injecting the discovered DNA at a site in the patient and the AD? It is absorbed. by a percentage of cells in which the gene of the product protein is expressed. Additionally, the other methods described herein, such as mechanical or chemical methods can be used for the in vivo insertion of the nucleic acids of the invention. Mechanical methods of DNA delivery include the direct injection of AD ?, such as the microinjection of AD? in somatic or germ cells, particles coated with AD? pneumatically supplied, such as the golden particles used in a "gene gun", and inorganic chemical methodologies such as transfection of calcium phosphate. Another method, ligand-mediated gene therapy involves forming DNA complexes with the specific ligands to form DNA-ligand conjugates, for DNA to a specific cell or tissue. Chemical methods of gene therapy may involve a chemical to bind to the cell and / or transport the DNA through the cell membrane, such as fusogenic lipid vesicles such as liposomes or other vesicles for membrane fusion, lipid particles of J &DN that incorporate cationic lipids such as lipofectin, or polylysine-mediated transfer of DNA. Lipofectins or cytofectins, positive ions based on lipids that bind to AD? negatively charged, form a complex that can cross the cell membrane and provide the AD? inside the cell. Another chemical method uses endocytosis based on the receptor, which involves binding a specific ligand to a cell surface receptor by wrapping it and transporting it through the cell membrane. The ligand binds to AD? and the entire complex is transported in the cell. The ligand gene complex is injected into the bloodstream and then to target cells that have the receptor that will specifically bind to the ligand and transport the DNA-ligand complex to the cell.Many gene therapy methodologies employ viral vectors to insert genes into cells and can be genetically engineered to comprise the alternative splicing vector of the present invention. For example, altered retrovirus vectors have been used in ex vivo methods to introduce genes into lymphocytes, hepatocytes, epidermal cells, or other peripheral somatic cells that infiltrate the tumor. These altered cells are then introduced into the patient to provide the gene product of the inserted DNA. Viral vectors have also been used to insert genes into cells using in vivo protocols (see, for example, Eck, SX., And J.M. ilson, "Gene Based Therapy", Goodman &; Gilman's The Pharmacological Basis of Therapeutics, 9th edition, pp. 77-101, McGraw-Hill, New York (1996)). To direct . tissue-specific expression of foreign genes, promoters or cis-acting regulatory elements that are known to be a specific tissue can be used. Alternatively, this can be achieved by using the in situ release of DNA or viral vectors to specific anatomical sites in vivo. For example, the transfer of the gene to the blood vessels in vivo was achieved by implanting transduced endothelial cells in vi tro at the selected sites in the arterial walls. The virus infects the surrounding cells, which also express the gene product. A viral vector can be released directly to the site in vivo, by means of a catheter, for example, allowing only certain areas to be infected by the virus, and providing expression of the gene at the specific site in the long term. Gene transfer in vivo using retroviral vectors has also been demonstrated in the breast tissue and in the liver tissue by injecting the altered virus into the blood vessels leading to the organs. Viral vectors that have been used for gene therapy protocols include, but are not limited to, retroviruses, other RNA viruses such as poliovirus or Sindbis virus, adenovirus, adeno-associated virus, herpes virus, SV 40, vaccinia and other DNA viruses. Retroviral vectors of defective replication murine and adenoviral vectors are the widely used gene transfer vectors. Murine leukemia retroviruses are composed of a single strand of RNA that forms complexes with a nuclear core protein and polymerase enzymes (pol), embedded by a protein center (gag) and surrounded by an envelope (env) of glycoprotein (env) that determines the range of the host. The genomic structure of retroviruses includes the gag, pol and env genes attached by the long terminal 5 'and 3' end repeats (LTR). The retroviral vector systems take advantage of the fact that a minimum vector containing the 5 'and 3' LTR and the packaging signal are sufficient to allow packaging of the vector, infection and integration into the target cells. those that provide the viral structural proteins are delivered in trans in the packaging cell line. The fundamental advantages of retroviral vectors for gene transfer include efficient infection and gene expression in most cell types, the precise integration of the simple copy of the vector into the chromosomal DNA of the target cell and the easy manipulation of the genome. retroviral The adenovirus is composed of linear double-stranded DNA that forms complexes with the core proteins and surrounded with capsid proteins. Advances in molecular virology have led to the use of the biology of these organisms to create vectors capable of transducing novel genetic sequences in target cells in vivo. The adenovirus-based vectors will express the peptides of the gene product at high levels. Adenoviral vectors have high infectivity efficiencies, even at low virus concentrations. Additionally, the virus is totally infectious as a cellular free virion in such a way that the production of cell lines is not necessary. Another potential advantage of adenoviral vectors is the ability to achieve long-term expression of heterologous genes in vivo in some cell types.

Adenoviral vectors are derived from the incompetent replication of adenoviruses typically containing a deletion in the El gene. Such vectors are transfected into the cells, such as the human embryonic kidney cell line 293, which allows the replication of the deleted adenoviruses El. After transfection, the adenoviral vector is allowed to replicate in specialized helper cells and form infectious particles that they are gathered and purified. These particles are capable of infecting a wide range of host cells for the expression of the transgene, for example, the alternative splicing vector of the present invention, but it is not capable of replicating without the addition of additional viral factors. To reduce the likelihood of adenovirus-competent replication that contaminates the adenoviral preparation, adenoviral vectors with additional deletions or mutations in the viral genome can be used, such as deleted vectors E1 / E3 or "squeamish" that have had all or most of the inactivated viral genes. Alternatively, an adenoviral vector containing an inverted protein pIX gene can be used, such as described in U.S. App. Nos. 60/621, 782 and 60 / 631,246. It has been found that injecting the plasmid DNA into muscle cells produces a high percentage of cells that are transfected and that have sustained the expression of marker genes. The plasmid DNA may or may not be integrated into the genome of the cells. The non-integration of the transfected DNA would allow transfection and expression of the product protein gene in terminally differentiated nonproliferative tissues over a prolonged period of time without fear of mutational insertions, deletions or alterations in the mitochondrial or cellular genome. In the long term, but not necessarily permanently, the transfer of therapeutic genes into specific cells can provide treatments for genetic diseases or for prophylactic use. The DNA could be reinjected periodically to maintain the level of the gene product without mutations that occur in the genomes of the recipient cells. The non-integration of exogenous DNAs may allow the presence of several different exogenous DNA constructs within a cell with all the constructs expressing several or multiple products of the gene. Particle-mediated gene transfer methods were first used in the transformation of plant tissue. With a particle bombardment device, or "gene gun", generates a driving force to accelerate the high density particles coated with DNA (such as gold or tungsten) at a high speed that allows the penetration of target organs, tissues or cells. Bombardment of the particles can be used in in vitro systems, or with ex vivo or in vivo techniques to introduce DNA into cells, tissues or organs. Electroporation for gene transfer uses an electric current to make cells or tissues susceptible to gene transfer mediated by electroporation. A short electric impulse with a given field strength is used to increase the permeability of a membrane in such a way that DNA molecules can penetrate the cells. This technique can be used in in vitro systems, or with ex vivo or in vivo techniques to introduce DNA into cells, tissues or organs. The transfer of the gene mediated by the carrier in vivo can be used to transfect the foreign DNA in the cells. The carrier DNA complex can be suitably introduced into fluids of the body or into the bloodstream and then into the site specifically targeting the targeted tissue or organ in the body. Both liposomes and polycations can be used such as polylysine, lipofectins or cytofectins. The liposomes can be developed in a specific cell or specific organ and thus the foreign DNA directed by the liposomes will absorb the target cells. The injection of immunoliposomes that target a specific receptor in certain cells can be used as a suitable method to insert the DNA into the cells that carry the receptor. Another carrier system that has been used is the conjugated system of asialoglycoprotein / polylysine to carry the DNA to the hepatocytes for gene transfer in vivo. The transfected DNA can also be formed with complexes together with other types of carriers for the DNA to be transported to the receptor cell and then reside in the cytoplasm or nucleoplasm. DNA can be coupled to transport nuclear proteins in specifically designed vesicle complexes and transported directly into the nucleus. For example, tumor cells removed from a patient can be transfected with a vector of the present invention that expresses the antitumor polypeptides, and reintroduce them to the patient. Transfected tumor cells * produce the levels of the protein in the patient that inhibit tumor growth. Patients can be human or non-human animals. The cells can also be transfected by physical or chemical methods known in the art such as electroporation, ionoporation, or via a "gene weapon". Additionally, the nucleic acids comprising the alternative splicing vector of the invention can be injected directly, without the aid of a carrier, into a patient. In particular, the vector DNA can be injected into the skin, muscle or blood.

The gene therapy protocol for the transfection of the vector of the invention in a patient can be either through the integration of the vector DNA into the genome of the cells, into the minichromosomes or as a DNA construct of replication or non-replication individual in the cytoplasm or nucleoplasm of the cell. The expression of the protein may continue for a long period of time or may be reinjected periodically to maintain a desired level of the protein in the cell, tissue or organ or a given blood level. 7. Dosages and Treatment Regimens In prophylactic applications, the pharmaceutical compositions or medicaments are administered to a subject suffering from a disorder treatable with a recombinant protein of the invention, for example, a disorder of the immune system, in an amount sufficient to eliminate or reduce the risk, reduce the severity, or delay the onset of the disorder, including the biochemical, histological and / or behavioral symptoms of the disorder, its complications and intermediate pathological phenotypes that occur during the development of the disorder. In therapeutic applications, compositions or medicaments are administered to a subject suspected or suffering from such disorder in an amount sufficient to cure, or at least partially suspend, the symptoms of the disorder (biochemical, histological and / or --- eonduG-te-ual -) -, including. , .sus __ complications and intermediate pathological phenotypes in the development of the disorder. The polypeptides of the invention are particularly useful for modulating the biological activity of a cell surface antigen residing in the blood, wherein the disease to be treated or prevented is caused at least in part, due to the abnormally high biological activity. or low antigen. In some methods, the administration of compositions of the present invention reduces or eliminates the immune disorder, for example, inflammation. An adequate amount to achieve prophylactic or therapeutic treatment is defined as a dose prophylactically or therapeutically effective. In both prophylactic and therapeutic regimens, the agents are normally administered in several dosages until a sufficient immune response has been obtained. The effective doses of the compositions of the present invention, for the treatment of the described conditions The previous ones vary depending on different factors, including means of administration, target site, physiological state of the subject, if the subject is human or an animal, other medications administered, and if the treatment is 'prophylactic or therapeutic. Normally, the subject is a human but non-human mammal that includes mammals can be treated. The polypeptides and proteins expressed by the alternative splice vector or expression cassette or construct of the invention described above can be provided as substantially purified and isolated proteins and protein fragments in pharmaceutically acceptable formulations using the formulation methods known to those skilled in the art. in art. These formulations can be administered by normal routes. In general, the combinations can be administered by a topical, transdermal, intraperitoneal, intracranial, intracerebroventricular, intracerebral, intravaginal, intrauterine, oral, rectal or parenteral route (e.g., intravenous, intraspinal, hypodermic or intramuscular). In addition, the polypeptides can be incorporated into biodegradable polymers that allow sustained release of the compound, the polymers that are implanted in the vicinity where it is desired to release the drug, for example, at the site of a tumor or implanted so that the polypeptides are slowly released systemically. Osmotic minipumps can also be used to provide controlled release of high concentrations of the polypeptides through a cannula at the site of interest., such as directly in a metastatic growth or in the vascular supply for that tumor. Biodegradable polymers and their use are described, for example, in detail in Brem et al., J. Neurosurg. 747AI-446 (1991). The dosage of the polypeptides of the present invention will depend on the state of the disease or condition to be treated and other clinical factors such as the weight and condition of the human or animal and the route of administration of the compound. For treating humans or animals, between about 0.5 mg / kilograms to 500 mg / kilograms of the polypeptides can be administered. Depending on the half-life of the polypeptides in the particular or human animal, the polypeptides may be administered several times a day to once a week. It will be understood that the present invention has application for human and veterinary use. The methods of the present invention contemplate single or multiple administrations, given simultaneously or over an extended period of time. For passive immunization with an antibody, the dosage ranges from about 0.0001 to 100 mg / kg, more usually 0.01 to 20 mg / kg, of the host body weight. For example, the dosages may be 1 mg / kg of body weight or 10 mg / kg of body weight or within the range of 1-10 mg / kg, preferably at least 1 pg / kg. The subjects can be given such dosages daily, on alternating days, weekly or according to any other scheme determined by the analysis, empirical .__ An exemplary treatment brings with it the administration in multiple dosages over a prolonged period, for example, of at least six months Additional exemplary treatment regimens involve administration once every two weeks or once a month, or once every 3 to 6 months. Exemplary dosage schedules include 1-10 mg / kg or 15 mg / kg on consecutive days, 30 mg / kg on alternate days or 60 mg / kg weekly. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated. The antibody is usually administered on multiple occasions. The intervals between simple dosages can be weekly, monthly or annually. In some methods, the dosage is adjusted to achieve a plasma antibody concentration of 1-1000 μg / ml and in some methods of 25-300 μg / ml. Alternatively, the antibody can be administered as a sustained release formulation in which case, less frequent administration is required. Dosage and frequency vary depending on the antibody's half-life in the subject. In general, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies and non-human antibodies. The dosage and frequency of administration may vary, depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, compositions containing the antibodies present or in a cocktail thereof are administered to a subject that is no longer in the disease state to improve the subject's resistance. Such amount is defined as being an "effective prophylactic dose". In this use, the precise amounts depend again on the state of health of the subject and general immunity, but generally they are in the range of 0.1 to 25 mg per dose, especially from 0.5 to 2.5 mg per dose. A relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some subjects continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage (for example, approximately 1 to 200 mg of antibodies per dose, with dosages of 5 to 25 mg are the most commonly used) at relatively short intervals is sometimes required until the progress of the disease is reduced or finished, and preferably until the subject shows partial or complete improvement of the symptoms of the disease. After this, the patient can be given a prophylactic regimen. The doses for the nucleic acids encoding the antibodies range from about 10 ng to 1 g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg of DNA per subject. Doses for infectious viral vectors will vary depending on the type of viral vector used, but will be between lxlO5 to lxlO20 virions per dose in vivo. For in vitro doses, approximately 0.5 to 100 virions per cell will generally be used. The therapeutic agents can be administered by parenteral, topical, intravenous, oral, hypodermic, intraarterial, intracranial, intraperitoneal, intranasal or intramuscular means for prophylactic treatment and / or therapeutic treatment. The most typical route of administration of a drug of the protein is intravascular, hypodermic or intramuscular, although other routes can be effective. In some methods, agents are injected directly into a particular tissue where the deposits have increased, for example intracranial injection. In some methods, the antibodies are administered as a sustained release composition or device, such as a Medipad ™ device. The drug of the p-rotein can also be administered via the respiratory tract, for example, using a dry powder inhalation device.

The agents of the invention can optionally be administered in combination with other agents that are at least partly effective in the treatment of disorders directed by the present invention. The following examples are included for the purposes of illustration and should not be construed as limiting the invention. The contents of any patent, patent applications, patent publications (national and international), (specifically, even the sequence listings) and references cited throughout this specification are hereby incorporated by reference in their entirety.

Exemplification Throughout the examples, the following materials and methods were used unless otherwise stated.

Materials and Methods In general, the practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology, recombinant DNA technology and oncology, neurology and immunology, especially, for example, antibody technology . For example, See Sambrook, Fritsch and. aniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press 1989); j &ntibody Engineering Protocols (Methods in Molecular Biology), 510, Paul, S., Humana Pr (1996); Antibody Engineering: A Practical Approach (Practical Approach Series, 169), McCafferty, Ed., M Pr (1996); Antibodies: A Laboratory Manual, Harlow et al., C.S.H.L. Press, Pub. (1999); and Current Protocols in Molecular Biology, eds. Ausubel et al, John Wiley & Sons (1992).

Introduction of Alternative Splicing Vectors into Eukaryotic Cells For the expression of the polypeptides in eukaryotic cells, the vectors of the invention were typically introduced into CHO cells by electroporation in 0.8 ml of HEBS (20 nM Hepes pH 7.05, 137 mm NaCl, 5 mm KCl, 0.7 mm Na2HP04, 6 mM dextrose) using a 0.4 cm cell (BioRad, Hercules, CA) at 0.28 kV and 950 uF. Approximately 5 x 106 cells for each electroporation. After electroporation, the cells were allowed to incubate in the tub for 5-10 min at room temperature and then transferred to a centrifuge tube containing 10 ml of free CHO serum in 10 ml of free serum from the medium CHO, were seeded in T75 flasks and incubated at 36 ° C with 5% Co2 in a humidified incubator. Three of the five days after transfection, the conditioned medium was harvested and the antibody titer was determined by ELISA. Transfections were performed in duplicate.

Expression Analysis of the Multimer Polypeptide The expression analysis was typically carried out using a selection of D The stably transfected cells were grown in a CHO-free medium lacking nucleosides for approximately two weeks after transfection. Transfected cells without DNA (negative control) were killed considering that the cells containing the selectable marker grew. The productivity of cell-specific polypeptide multimers was evaluated by swapping the media, plating the cells at 2 × 10 5 to 3 × 10 5 viable cells / ml, allowing the cells to grow for two to three days after the concentration of the antibody and the cell densities were determined. All titrations were determined using a FRET assay or an ELISA specific for the variable region of the antibody and / or an ELISA specific for the Fc region of the antibody. Briefly, the plates were covered with 50 μl / well of the Fc? Fragment. Goat anti-human IgG AffiniPure (Cat. No. 109-005-098, Jackson Immunoresearch, West Grove, PA) or specific antigen at 10 μg / ml in PBS and incubate overnight at 4 ° C. Before use, the solutions of cover were removed and washed 3x with PBS, 0.05% Tween 20, then blocked for at least 1 hr with 200 PBS μl / well, 0.05% Tween 20, 1% BSA. { PBST / BSA). The blocking solutions were removed and the samples analyzed against a normal curve. Both standard and known were typically diluted in PBST / BSA, they were incubated for 2 hr. at room temperature, and then washed as described above. An aliquot of 50 μl / well peroxidase conjugated with human anti-lgG donkey (H + L) (Jackson Immunoresearch, Cat. No. 709-035-149) was used to detect the presence of antibody. The conjugate was diluted 1: 10,000 in PBST / BSA and incubated for 1.5 hr at room temperature, then removed and washed as above. An aliquot of 100 μl / well substrate (420 mM tetramethyl benzidine and 0.05% hydrogen peroxide in 0.1 M sodium acetate buffer solution pH 4.9) was then added to resolve the antigen binding by incubation with the substrate for 2 min, followed by fixation with a buffer buffer of 100 μl / well 2 sulfuric acid. The resulting absorbance was read at 450 nm in a plate reader of a SpectraMax Plus Molecular Device using Softmax software (Molecular Devices, Sunnyvale, CA). For the FRET assay, 50 μL of conditioned medium samples were mixed with 75 μL of 1.67 μg / mL LanceTM Eu-IDEC-152 (a monoclonal IgGl antibody labeled with europium) Perkin Elmer (Boston, MA) diluted in PBS with 1 % BSA (saline solution of a Dulbecco phosphate buffer, cat. do not. 9280, Irvine Scientific, Santa Ana, CA; BSA, the Sigma, cat. do not . CA-7906). After addition of the labeled antibody, 75 μL of the test mixture is added containing 6.67 μg / mL of the conjugate (APC) (specific Fc) -Xl Goat Phycolink® anti-human IgG Aloophocyanin (Cat. No. PJ253, Prozyme San Leandro , CA) diluted in PBS with 1% BSA. The additional mixture of the samples was incubated in a 96 well Black plate not treated with lids, (cat # 237105, NalgeNunc International, Rochester, NY) and shaken for more than 15 minutes using a titration plate shaker ( cat. No. 4625 (VWR # 57019-600), Barnstead / Lab-line, Meirose Park, IL). The plates were read using a multiple label counter 1420 (Wallac, Gaithersburg, MD) for a time that resolves the fluorescence mode with excitation at 337 μM and emission at 665 μM. The data was analyzed using Softmax software (Molecular Devices).

EXAMPLES Example 1 Methods for genetically designing a vector expressing multiple polypeptides using alternative splices. The following example describes the methods for constructing a suitable vector to be expressed in eukaryotic cells, two or more products of the gene by an alternative splicing.

The expression vectors described herein are derivatives of the expression vector pV80 described in the U.S. Application Ser. No. 10 / 237,067 containing a native CMV intron with the native CMV splice donor and the splice acceptor. Briefly, a DNA construct was constructed by comprising (in a 5 'to 3' or ascending) immediate cytomegalovirus (CMV IEl) immediate promoter 1 including the 5 'untranslated region preceding the IE1 CMV intron (strain AD169 of the human cytomegalovirus) and the 5 'half of the CMV intron IEl, which includes the sequence of the native splice donor (SD). The modified 3 'portion of the intron I CMV included in a cloning poliligadura (Swal-BstBI) for the alternative splice acceptors (SAI) that are to be exchanged conveniently. In addition, a cloning site (Ascl) for the first coding sequence in which a humanized light chain gene was cloned, was added precisely in the downstream direction of SAI together with a human variant in the polyadenylation region of the hormone of growth (hGH). In addition to the downstream direction, the 3 'portion of the CMV intron IE1, which includes the native splice acceptor sequence (SA2), was incorporated into the vector. A cloning site (BamHI) for the second coding sequence in which a humanized IgGl heavy chain gene was cloned and added just in the downstream direction of SA2 together with the human variant in the polyadenylation region (hGH) of the growth hormone. At a single location in the vector, the selectable marker of dihydrofolate reductase (DHFR) was introduced. The selectable marker is derived from pSI (Genbank access # U47121, Promega, Madison, Wl) and transcriptionally controlled by the SV40 promoter / enhancer, an artificial intron (separated from the alternative splice vector) and the late SV40 polyadenylation sequence. To clone and propagate the alternative splice vector in the prokaryotic cells, sequences derived from pUC19 including the beta lactamase gene were added. The resulting vector. { that is, pHL005) of the above genetically engineered steps is shown in Fig. 3 (see also SEQ ID NO: 29 which provides the sequence of the vector structure without the sequences encoding the exon inserted into the Ascl sites and BamHI). In this example, the heavy and light chain sequences of the humanized antibody were cloned into the Ascl and BamHI sites, respectively. This vector also allows the insertion of a variety of splice acceptors at the restriction sites (Swal-BstBl) just in the upward direction of the first coding sequence a, optionally, the change in the proportion of the expression of the heavy and light chain.

Additional expression vectors were made in the portions of the intron just 5 'of the splice acceptor, were removed to generate the vector structure vHLP005. The intron sequence of pHLP005 contains a PflMI site and a BspEI site of approximately 310 base pairs and 110 base pairs 5 'of an Svral site, respectively. To generate these deletions, the plasmid pHLP005 was linearized by partial digestion with PfIMI or BspEI and then completely digested with BspEI and gel purified. To generate expression vectors with different first splice acceptors (SAI), oligonucleotides with compatible sites PfIMI and BspEI or BspEI and BspEI (SEQ ID NOs: 1-28) were ligated into the respective pHLP005 digested vector and given a new designation of the vector (Fig. 4). All the constructs were then confirmed by the analysis of DNA sequences. The resulting vectors contain, in a 5 'to 3' direction or in a downward orientation, a native CMV splice donor (SD), a first splice acceptor (SAI) selected from SEQ ID NOS: 1-28, a first restriction site (Ascl) for the insertion of the first polypeptides, a second splice acceptor (SA2) which is a native CMV splice acceptor and a second restriction site (BamHI) for the introduction of the second polypeptide The heavy chain sequences and light of the antibody were prepared using a primer-based polymerase chain reaction (PCR) and the resulting products were introduced into the vector using standard genetic engineering techniques. The primers used in the restriction sites contained adjacent to the coding regions for insertion into the Ascl and BamHI sites. For example, the 5 'primer could be TTTTGGCGCGCCATGN (20) (SEQ ID NO: 32) for the light chain and TTTTGGATCCATGN (20) (SEQ ID NO: 33) for the heavy chain. The 3 'primer could be GCACGGCGCGCCCTAN (20) (SEQ ID NO: 34) for the light chain and GCAGGGATCCTCAN (20) (SEQ ID NO: 35) for the heavy chain. For these primers, N (20) represents the nucleotide sequence specific for the DNA encoding the desired heavy chain or light chain.

Example 2 Methods to determine the efficiency of alternative splicing. In this example, methods and constructs are described to determine the efficacy of using alternative splices to express two different gene products encoded by exons adjacent to different splice acceptors. Although splicing is mediated by consensus sequences at the exon / intron junctions, an accurate prediction of efficient splicing can not be determined by just these sequences. For this reason, an empirical methodology is necessary to identify splice acceptor sequences that would generate the appropriate proportion of products, which can be measured here as the total amount of multimeric protein produced. Accordingly, the invention provides suitable vectors for efficient exclusion separation combinations at desirable splice sites in any given cell line. To generate a variety of expression vectors with alternative splice sites, oligonucleotides with a blunt-ended end and a compatible BstBI end were generated for insertion into the Swal and BstBI sites in the vector pHLP005. All constructs were confirmed by a DNA sequence analysis. The sequences of the splice sites tested in Fig. 4 and in the sequence listing (ie, SEQ ID NOS: 1-28). The vectors generated with the different splice acceptors were compared using a transient or stable transfection by electroporation. The host cell used for the transfections was the Chinese hamster ovary cell line DG44 (CHO) deficient in dihydrofolate reductase (DHFR) (Urlaub et al, Cell 33, 405-412 (198-3)). All DNA was prepared using the Megaprep kit (Qiagen, Valencia, CA). Prior to transfection, the DNA was precipitated in ethanol (EtOH), washed in 70% EtOH, dried, resuspended in HEBS, and quantified prior to transfection. The negative controls did not contain DNA transfection and were used as transfection controls. Both the mAb-specific ELISA and the IgG-specific ELISA measure the total antibody secreted within the cell culture medium. The levels of antibody expression for transiently transfected cells are given in Table 1. The results demonstrate that certain splice sites, eg, pHLP0015 and pHLPOOld, are more efficient than others, by generating higher levels of total antibody, when they are tested, transiently, in CHO cells.

Table 1. Transient Transfection Antibody Concentrations Example 3 Expression of Alternately Gene-Linked Gene Products in Eukaryotic Cells In this example, the expression of alternatively spliced gene products, in particular, assembled IgG antibodies, is described. Briefly, CHO cells were transfused with the alternative splice vectors of the invention by electroporation and allowed to recover in selective media, as described above, for about two weeks to generate stably transfected cells. After 3-5 days the conditioned media were harvested and the antibody concentration was determined by ELISA. In a first experiment, 50 μg of DNA from the separate vectors encoding the light chain and heavy chain, 50 μg of alternatively spliced vectors encoding both light and heavy chains of antibodies, or no DNA at all was introduced into eukaryotic cells (CHO) and the amount of functional polypeptide multimers produced by the cells was determined as a function of the antibody binding activity (Table 2).

Table 2. Specific Productivity of Stable Accumulations The results of the first experiment are proof of the principle that it is possible to generate a functional polypeptide multimer, for example, a monoclonal antibody product, of an alternative splicing vector.

In additional experiments, 25 μg of DNA from separate vectors encoding antibody light and heavy chain, 50 μg of alternatively spliced vectors encoding both light and heavy chains of antibodies, or no DNA at all, were introduced into eukaryotic cells (CHO ) and the amount of functional polypeptide multimers produced by the cells as a function of the antibody binding activity was determined (Tables 3-6). The results of the experiments demonstrate that it is possible to generate a functional polypeptide multimer, for example, a monoclonal antibody product, from an alternative splicing vector. The data suggest that pHLP015 is a better alternative splicing vector than others tested because of its ability to produce high levels of functional antibody.

Table 3. Transient Transfection Antibody Concentrations Table 4. Specific Productivity of Stable Accumulations Table 5. Transient Transfection Antibody Concentrations Table 6. Specific Productivity of Stable Accumulations Thus, it was concluded that a functional polypeptide multimer, e.g., an antibody, can be produced by using an alternative splicing vector both transiently and stably in eukaryotic cells. Therefore, the ease and utility of a system of a vector expressing more than one polypeptide through alternative splicing was demonstrated.

Example 4 Demonstration that the vector can produce cell lines with good expression potential The vector of the present invention was designed to optimize the ratio of light and heavy chains for the expression of antibodies. Immunoglobulin heavy chains are not secreted unless they are assembled with light chains, while most light chains can be secreted as free molecules. Thus, excessive amounts of free light chain would be indicative of the need to further optimize the vector to increase the production ratio of. heavy to light chain. In order to evaluate the production of free light chain, cell lines containing one of the vectors of the invention were generated and their conditioned media, which contain the recombinant free light chain secreted and / or assembled products of antibody multimers (Table 7).

Generation of Vectors A vector based on pHLP015 was prepared as described in Examples 1 and 2. This vector was inserted into cells CHO and was selected for stable transfection as previously described. The amplified cell lines were derived with targeting of the industry standard of a specific productivity in excess of 10 picograms of protein produced per cell per day as measured by the FRET assay as previously described. The results provided below demonstrate the usefulness of this expression vector in the preparation of cell lines acceptable for production of antibodies. The results of RT-PCR confirm the predicted splice sites. Reverse transcription of cellular RNA followed by the polymerase chain reaction of the cDNA (RT-PCR) was performed to confirm the splicing junctions of the heavy and light chain mRNAs against the predicted junctions of the expression vector design. A stably transfected cell line was isolated from one of the transfections by using the plasmid pHLP015 and the total RNA was prepared (R? Awiz, Ambion, Austin, TX). By using a primer complementary to the approximate center of the heavy chain mRNA, a cDNA was generated using the reverse transcriptase (Superscript III reverse transcriptase, Invitrogen, Carlsbad, CA). The cDNA was then used as a template for PCR (AD Vent? Polymerase? Ew England Biolabs, Beverly, MA) by using a primer in the CMV 5 'untranslated region (5' of the splice donor) and a primer in the coding region (3 'of the splice acceptor in the heavy chain coding sequence). This PCR product was sequenced and the predicted splicing product was confirmed between the splice donor in the CMV intron and the splice acceptor just 5 'of the heavy chain coding sequence. When using oligo (dT) 15 as a primer, was an AD? C of the AR generated? cellular when using reverse transcriptase (reverse transcription system, Promega, Madison, Wl).

This AD? C was then used as a template for PCR (Vent polymerase AD ?,? ew England Biolabs, Beverly, MA) when using a primer in the untranslated region of CMV '(51 of the splice donor) and a primer in the coding region (3' of the splice acceptor in the light chain coding sequence). This product by PCR was formed into sequences and the splicing product predicted between the splice donor in the CMV intron and the splice acceptor just 5 'of the light chain coding sequence was confirmed, indicating that the expression vector It was working by alternate splicing as expected.

SDS-PAGE analysis of conditioned media A stably transfected cell line was isolated from a transfection by using the plasmid pHLP015 (mAb # 1-l in Table 7) of Examples 1-3. In addition, a number of stably transfected cell lines were generated by using another vector based on pHLP015 (as generated in this Example, mAb # 2-l to 5 in Table 7). To purify both the whole mAb and the free light chains, approximately 50 mL of conditioned medium was applied to a 1.3 mL column of L-agarose protein (cat # P3351, Sigma-Aldrich, St. Louis, MO), the column was washed with 15 mL 3X PBS, then 5 mL of IX PBS and eluted with 100 mM NaH2P04, pH 2.8 in aliquots of 0.3 mL and neutralized immediately with 75 μL of 1M Hepes, pH 8. The protein peak was localized by UV absorbance at 280 nm and the protein concentration was determined by using an extinction coefficient of 1.5 A280 / mg / mL. An appropriate volume of the peak sample was diluted in sample buffer to charge 1.5 μg of protein in 15 μL. The 4X stock solution of sample buffer based on Laemmli (0.25 M Tris-HCl, pH 6.8, 8% SDS, 40% glycerol, and 0.01% bromophenol blue) was prepared with 100 mM fresh NEM for non-reducing gels. The samples were heated for 5 min at 100 ° C and 15 μL were loaded on PAGEr Gold Precast gels with 4-20% tris-glycine (cat # 59517, Cambrex, East Rutherford, NJ). The gels were run at 45 mA for 40 min in Laemmli buffer. After the run, the gels were stained with approximately 100 mL of Coomassie Cleveland blue stain (50% methanol, 10% acetic acid, 0.1% Coomassie blue) by microwave for 1 min and shaking for 10 min. The gels were then destained in approximately 100 mL of 10% methanol, 10% acetic acid by microwave for 2 min and stirred overnight with absorbent foam. The gels were scanned on a Biorad GS-800 calibrated densitometer and the whole antibody and free light chain bands were quantified with a Biorad Quantity One software. The amount of free light chain as a percentage of the total secreted protein (complete antibody + free light chain) is shown in Table 7.

Table 7. Evaluation of the relative Quantities of Whole Antibody and Free Light Chain in Conditioned Media Cell lines based on pHLP015 Cell line% LC free mAb # ll not detected mAb # 2-l 14 mAb # 2-2 14 mAb # 2- 3 16 mAb # 2-4 not detected mAb # 2-5 not detected As shown in Table 7, high levels of free light chain were not detected which indicates that the vector is well balanced in the expression of the light chain with relation to the heavy chain.

Example 5 Multimeric proteins that produce vectors The vectors and cassettes or expression constructs of the present invention can be used to express any multimeric protein, including, but not limited to, the multimeric proteins described in the Detailed Description supra. Many such proteins are well known in the art and are suitable for use in the vectors and cassettes or expression constructs of the present invention. The number of polypeptides to be expressed may vary. For example, two polypeptides can be expressed as previously described. Additional polypeptides can be expressed by inserting additional exons encoding the polypeptides in the vector, preferably between the first exon and second splice acceptor. Each of the additional exons would have a 5 'splice acceptor to the exon to regulate transcription when splicing occurs between the single splice donor and the individual splice acceptors. Thus, more than one polypeptide can be expressed by using the same splice donor and different splice acceptors to express the polypeptides in ratios to optimize the expression of the desired protein or proteins. A vector or construct or expression cassette is prepared as described in the above examples containing splice acceptors that allow splicing and subsequent translation of the polypeptide chains in ratios that optimize the expression of the multimeric protein. First, the genes encoding the polypeptide chains are cloned or amplified using standard techniques, such as RT-PCR by using specific primers for the chains and an appropriate collection containing the genes expressing the desired polypeptides. The genes are then inserted into a vector or construct or expression cassette of the invention capable of expressing the polypeptides in suitable ratios sufficient to express high levels of the multimeric protein.

This vector or construct or expression cassette may have convenient restriction sites to facilitate insertion of the splice sites, exons and other desired elements. For example, pHLP0015 can be used. Alternatively, the genes encoding the polypeptides are inserted into more than one expression vector or construct or cassette, each with a different first splice acceptor, to select a splice acceptor that allows for the appropriate level of expression, as described in Examples 2 and 3. Finally, the vector or expression cassettes or constructs encoding the polypeptides are inserted into a suitable cell line, eg, mammalian cells, insect cells or yeast cells, and expressed. The multimeric protein can then be harvested from the cell culture medium if it is secreted, or from the lysed cells if the protein is intracellular or bound to the membrane. All references cited herein are hereby incorporated by reference in their entirety.

Equivalents For someone of ordinary skill in the art, by using no more than routine experimentation, there are many equivalent to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (46)

Claims Having described the invention as above, the content of the following claims is claimed as property.

1. An expression vector comprising, a promoter; a 5 'UTR; a simple splice donor; an intron; a first splice acceptor; a first exon encoding a first polypeptide; a second splice acceptor; and a second exon encoding a second polypeptide, characterized in that the promoter is operably linked to the first and second exons, where upon entry into a cell, the single splice donor is spliced with the first splice acceptor, forming a spliced transcript which allows the transcription of the first exon, and the second splice acceptor forming a spliced transcript > . which allows the transcription of the second exon, and wherein the first polypeptide and the second polypeptide are expressed from the spliced transcripts.

2. The vector according to claim 1, characterized in that the promoter is a CMV promoter.

3. The vector according to claim 1, characterized in that it also comprises a polyadenylation signal operatively linked to the first exon or the second exon.

4. The vector according to claim 1, characterized in that the vector further comprises one or more of an additional additional splice and acceptor acceptor encoding an additional polypeptide, wherein upon entry into a cell, the single splice donor is spliced with the additional splice acceptor, forming an additional spliced transcript which allows for 'transcription of the additional exon, and wherein the additional polypeptide is expressed from the spliced additional transcript.

5. The vector according to claim 1, characterized in that the first or second exons or both encode a selectable marker.

6. The vector according to claim 1, characterized in that the first and second polypeptides form a multimer.

7. The vector according to claim 6, characterized in that the multimeric protein is a heterodimer, a heterotrimer, or a heterotetramer.

8. The vector according to claim 1, characterized in that the first polypeptide is expressed at a frequency of about 10: 1 to about 1:10 relative to the second polypeptide.

9. The vector according to claim 1, characterized in that the first polypeptide is expressed at a frequency of about 3: 1 to about 1: 3 relative to the second polypeptide.

10. The vector according to claim 1, characterized in that the first polypeptide is expressed at a frequency of about 1: 1 relative to the second polypeptide.

11. The vector according to claim 1, characterized in that the first or second splice acceptor comprises any of the sequences selected from the group consisting of SEQ ID NOS: 1-28.

12. The vector according to claim 11, characterized in that the splice donor and the second splice acceptor are derived from CMV and the first splice acceptor comprises any of the sequences selected from the group consisting of SEQ ID NOS: 1-28.

13. The vector according to claim 1, characterized in that the vector is a viral vector.

14. The vector according to claim 1, characterized in that it comprises SEQ ID NO: 29.

15. A eukaryotic cell characterized in that it contains the vector according to claim 1.

16. The cell according to claim 15, characterized in that the vector is integrated into the chromosomal DNA of the cell.

17. The cell according to claim 15, characterized in that the vector is episomal.

18. The cell according to claim 15, characterized in that the cell is a mammalian cell or a yeast cell.

19. The cell according to claim 18, characterized in that the cell is selected from the group comprising: a newborn hamster kidney cell, a fibroblast, a myeloma cell, an NSO cell, a PER.C6 cell, or a cell CHO.

20. The cell according to claim 19, characterized in that the cell is a CHO cell.

21. A polypeptide production method, the method characterized in that it comprises culturing a cell of claim 15 in a culture and isolating the first polypeptide and the second polypeptide from the culture.

22. A first polypeptide and a second characterized polypeptide | or which is produced by the method according to claim 21.

23. A composition characterized in that it comprises the first polypeptide and second polypeptides according to claim 22, further comprising a pharmaceutically acceptable carrier.

24. A method of treating a patient in need thereof, characterized in that it is with the composition according to claim 23.

25. An expression vector comprising, a promoter; a 5 'UTR; a simple splice donor; an intron; a first splice acceptor; a first exon encoding a first antibody polypeptide or fragment thereof; a second splice acceptor; and a second exon encoding a second antibody polypeptide or fragment thereof, characterized in that the promoter is operably linked to the first and second exons, where upon entry into a cell, the single splice donor is spliced with the first acceptor of splice, forming a spliced transcript which allows the transcription of the first exon, and the second splice acceptor, forming a spliced transcript which allows the transcription of the second exon, and where the first antibody polypeptide or fragment thereof and the second antibody polypeptide or fragment thereof are expressed from the spliced transcripts and associate to form an antibody or antibody fragment.

26. The vector according to claim 25, characterized by the first exon or the second exon or both, encode an antibody fragment.

27. The vector according to claim 25, characterized in that the first polypeptide encodes an antibody heavy chain or a fragment thereof and the second polypeptide is a light chain of antibodies or a fragment thereof.

28. The vector according to claim 25, characterized in that the first polypeptide is a light chain of antibodies or a fragment thereof and the second polypeptide is an antibody heavy chain or a fragment thereof.

29. The vector according to claim 27, characterized in that the light or heavy chain or both is murine, chimeric, humanized, human or synthetic.

30. The vector according to claim 28, characterized in that the light or heavy chain or both is murine, chimeric, humanized, human or synthetic.

31. The vector according to claim 25, characterized in that the first antibody polypeptide or fragment thereof is expressed at a frequency of about 3: 1 to about 1: 3 relative to the second antibody polypeptide or fragment thereof.

32. The vector according to claim 25, characterized in that the first antibody polypeptide or fragment thereof is at a frequency of about 1: 1 relative to the second antibody polypeptide or fragment thereof.

33. The vector according to claim 25, characterized in that the first splice acceptor or the second splice acceptor comprises any of the sequences selected from the group consisting of SEQ ID NOS: 1-28.

34. The vector according to claim 33, characterized in that the splice donor and the second splice acceptor are derived from CMV and the first splice acceptor comprises any of the sequences selected from the group consisting of SEQ ID NOS: 1- 28

35. The vector according to claim 25, characterized in that the vector is a viral vector.

36. The vector according to claim 25, characterized in that it comprises SEQ ID NO: 29.

37. A eukaryotic cell characterized in that it contains the vector according to claim 25.

38. The cell according to claim 37, characterized in that the vector is integrated into the chromosomal DNA of the cell.

39. The cell according to claim 37, characterized in that the vector is episomal.

40. The cell according to claim 37, characterized in that the cell is a mammalian cell or a yeast cell.

41. The cell according to claim 40, characterized in that the cell is selected from the group comprising: a newborn hamster kidney cells, a fibroblast, a myeloma cell, an NSO cell, a PER.C6 cell, or a CHO cell.

42. The cell according to claim 41, characterized in that the cell is a CHO cell.

43. A method of producing antibodies or fragments of antibodies, the method characterized in that it comprises culturing a cell according to claim 37 in a culture, and isolating the first polypeptide and the second polypeptide from the culture.

44. An antibody or fragment of antibodies characterized in that it is produced by the method according to claim 43.

45. A composition characterized in that it comprises an antibody or antibody fragment according to claim 44, further comprising a pharmaceutically acceptable carrier.

46. A method of treating a patient in need thereof characterized in that it comprises the composition according to claim 45.