MXPA00008312A - Enhancing the circulating half-life of antibody-based fusion proteins - Google Patents

Abstract

Disclosed are methods for the genetic construction and expression of antibody-based fusion proteins with enhanced circulating half-lives. The fusion proteins of the present invention lack the ability to bind to immunoglobulin Fc receptors, either as a consequence of the antibody isotype used for fusion protein construction, or through directed mutagenesis of antibody isotypes that normally bind Fc receptors. The fusion proteinsof the present invention may also contain a functional domain capable of binding an immunoglobulin protection receptor.

Description

METHODS TO IMPROVE AVERAGE LIFE IN CIRCULATION PROTEINS OF FUSION BASED ON ANTIBODY CROSS REFERENCE TO THE RELATED APPLICATION This is incorporated by reference, and claims priority and benefit of the provisional patent application of E.U.A. series number 60 / 075,887, which was presented on February 25, 1998.

FIELD OF THE INVENTION The present invention relates generally to fusion proteins. More specifically, the present invention relates to methods for improving the circulating half-life of antibody-based fusion proteins.

BACKGROUND OF THE INVENTION The use of antibodies for the treatment of human diseases is well established and has become more sophisticated with the introduction of genetic engineering. Several techniques have been developed to improve the usefulness of antibodies. These include: (1) the generation of monoclonal bodies through cell fusion to create "hybridomas", or through molecular cloning of the heavy (H) and light (L) antibody chains from antibody-producing cells; (2) the conjugation of other molecules to antibodies to deliver them to preferred sites in vivo, eg, radio isotopes, toxic drugs, protein toxins and cytosines; (3) the manipulation of antibody effector functions to improve or decrease biological activity; (4) the binding of another protein such as toxins and cytokines with antibodies at the genetic level to produce antibody-based fusion proteins; and (5) the binding of one or more antibody combining groups at the genetic level to produce bi-specific antibodies. When proteins are linked together through either chemical or genetic manipulation, it is usually difficult to predict what properties the final product will retain from the source molecules. With chemical conjugation, the binding process can occur at different sites on the molecules, and generally results in molecules with varying degrees of modification that can affect the function of one or both proteins. The use of genetic fusions, on the other hand, makes the binding process more consistent, and results in the production of consistent final products that retain the function of both component proteins. See, for example, Gillies et al., PROC. NATL. ACAD. SCI. USA 89: 1428-1432 (1992); and patent of E.U.A. No. 5,650,150. However, the utility of recombinantly produced antibody-based fusion proteins can be limited by their rapid elimination in vivo from the circulation. Antibody-cytosine fusion proteins, for example, have been shown to have a significantly lower in vivo circulating half life than free antibody. When testing a variety of antibody-cytosine fusion proteins, Gillies et al. Reported that all fusion proteins tested had a half-life of (distribution phase) less than 1.5 hours. In fact, most of the antibody-based fusion proteins were removed at 10% of the serum concentration of the free antibody for 2 hours. See, Gillies and others, BIOCONJ. CHEM. 4: 230-235 (1993). Therefore, there is a need in the art for methods to improve the in vivo circulating half-life of antibody-based fusion proteins. COMPENDIUM OF THE INVENTION A novel aspect to improve the in vivo circulating half life of antibody-based fusion proteins has now been discovered. Specifically, the present invention provides methods for the production of fusion proteins between an immunoglobulin with a reduced binding affinity for an Fc receptor, and a second protein that is not immunoglobulin. The antibody-based fusion proteins with reduced binding affinity for Fc receptors have significantly longer life in vivo in circulation than the second protein which is not unbound immunoglobulin. IgG molecules interact with three classes of Fc receptors (FcR) specific for the IgG class of antibody, mainly Fc? RI, Fc? RII and Fc? RIII. In preferred embodiments, the immunoglobulin component, (Ig) of the fusion protein has at least a portion of the constant region of an IgG having a reduced binding affinity for at least one of FcγRI, FcγRII or Fc? RIII. In one aspect of the invention, the binding affinity of fusion proteins for Fc receptors is reduced using heavy chain isotopes as fusion standards that have reduced the binding affinity for Fc receptors in cells. For example, both human I gG 1 and lgG3 have been reported to bind to FcR? L with a high affinity, while IgG 4 binds 10 times less well and IgG2 does not bind at all. The sequences important for the binding of IgG to the Fc receptors have been reported as localized in the CH2 domain. Thus, in a preferred embodiment, an antibody-based fusion protein with improved in vivo circulating half-life is obtained by attaching at least the CH2 domain of IgG2 or IgG4 to a second non-immunoglobulin protein. In another aspect of the invention, the binding affinity of fusion proteins for Fc receptors is reduced by introducing a genetic modification of one or more amino acids into the constant region of the heavy chains of I gG 1 or lgG 3 which reduces the binding affinity of these isotypes for Fc receptors. Such modifications include alterations of residues necessary to make contact with Fc receptors or alter others that alter the contacts between the heavy chain residues and Fc receptors through induced conformational changes. Thus, in a preferred embodiment, an antibody-based fusion protein with improved in vivo circulating half-life is obtained by introducing a mutation, deletion or insertion in the constant region of IgG 1 into one or more amino acids selected from Leu234 , Leu235, Gly236, Gly237, Asn297, and Pro33 ?, and then binding the resulting immunoglobulin, or portion thereof, to a second protein that is not immunoglobulin. In an alternative preferred embodiment, the mutation, or deletion or insertion is introduced into the constant region IgG3 into one or more amino acids selected from Leu281, Leu282, Gly283, Gly284, Asn3 4, and Pro3 8, and the resulting immunoglobulin, or portion of it binds to a second protein that is not immunoglobulin. The resulting antibody-based fusion proteins have a longer in vivo circulating half-life than the second non-immunoglobulin, unbound protein. In a preferred embodiment, the second non-immunoglobulin component of the fusion protein is a cytosine. The term "cytosine" is used herein to describe proteins, analogs thereof, and fragments thereof, which are produced and excreted by a cell, and which produce a specific response in a cell that has a receptor for that cell. cytosine Preferably, the cytosines include interleukins such as interleukin-2 (IL-2), hematopoietic factors such as granulocyte-macrophage colony stimulation factor (GM-CSF), tumor necrosis factor (TNF), such as TNFa, and lymphokines such as lymphotoxin. Preferably, the antibody-cytosine fusion protein of the present invention exhibits a cytosine biological activity. In an alternative preferred embodiment, the second non-immunoglobulin component of the fusion protein is a ligand binding protein with biological activity. Such ligand binding proteins can, for example, (1) block receptor-ligand interactions on the surface of the cell; or (2) neutralize the biological activity of a molecule (eg, a cytosine) in the fluid phase of the blood, thus preventing it from reaching its cell target. Preferably, the ligand-binding proteins include CD4, CTLA-4, TNF receptors, or interleukin receptors such as L-1 and IL-4 receptors. Preferably, the antibody-receptor fusion protein of the present invention exhibits the biological activity of the ligand binding protein. In another alternative preferred embodiment, the second non-immunoglobulin component of the fusion protein is a protein toxin. Preferably, the antibody-toxin fusion protein of the present invention exhibits the toxicity activity of the protein toxin. In a preferred embodiment, the antibody-based fusion protein comprises a variable region specific for a target antigen and a constant region linked through a peptide bond to a second non-immunoglobulin protein. The constant region may be the constant region normally associated with the variable region or a different one, for example, the variable and constant regions of different species. The heavy chain can include CH1, CH2 and / or CH3 domains. Also encompassed within the term "fusion protein" are constructs that have a binding domain that comprises framework regions and variable regions (i.e. regions of complementarity determination) of different species, as described by Winter, and others, GB 2,188,638. antibody-based fusion proteins comprising a variable region preferably exhibit specific antigen-binding character. In another preferred embodiment, the antibody-based fusion protein further comprises a light chain. The invention in this manner provides fusion proteins wherein the specific character of antigen binding and the activity of an antibody are combined with the potent biological activity of a second non-immunoglobulin protein, such as a cytosine. A fusion protein of the present invention can be used to selectively deliver the second non-immunoglobulin protein to a target cell in vivo, so that the second non-immunoglobulin protein can exert a localized biological effect. In a preferred alternative embodiment, the antibody-based fusion protein comprises a heavy chain constant region linked through a peptide bond to a second protein that is not immunoglobulin, but does not comprise a heavy chain variable region. The invention further provides fusion proteins that retain the potent biological activity of a second protein that is not immunoglobulin, but which lacks the specific character of antigen binding and activity of an antibody. In preferred embodiments, the antibody-based fusion proteins of the present invention further comprise sequences necessary for binding to Fc protection receptors (FcRp), such as the neonatal intestinal transport receptor containing beta-2-microglobulin (FcRn). In preferred embodiments, the fusion protein comprises two chimeric chains comprising at least a portion of a heavy chain and a second non-immunoglobulin protein that are linked through a disulfide bond. The invention also modalizes DNA constructs that encode the fusion proteins described above, and cell lines, eg, myelomas, transfected with these constructs. These and other objects, together with advantages and aspects of the invention described herein, will be more apparent from the description, drawings, and claims that follow.

BRIEF DESCRIPTION OF THE DRAWINGS The foregoing objects, aspects and advantages and others of the present invention, as well as the same invention, can be more easily understood from the following description of the preferred embodiments, when read together with the accompanying drawings, in which: Figure 1 is a homology alignment of the amino acid sequences of the constant region of C 1 and C 3, aligned to maximize the identity of the amino acid and where the non-conserved amino acids are identified through boxes; Figure 2 is a homology alignment of the amino acid sequences of the C? 1, C? 2 and C? 4 constant region, aligned to maximize the identity of the amino acid, and where the non-conserved amino acids are identified by boxes; Figure 3 is a diagrammatic representation of a map of the genetic construct encoding an antibody-based fusion protein showing the important restriction sites; Figure 4 is a bar graph showing the binding of antibody, hu-KS-1/4 and the antibody-based fusion proteins, hu-KS? 1-IL2 and hu-KS? 4-IL2, to receptors Fc in cells Mouse J774 in the presence (solid bars) or absence (dotted bars) of an excess of mouse IgG; Figure 5 is a line graph illustrating the in vivo plasma concentration of the total antibody (free antibody and fusion protein) of hu-KS? 1-IL2 (closed diamond) and hu-KS? 4-IL2 (closed triangle) ) of an intact fusion protein of hu-KS? 1-IL2 (open diamond) and hu-KS? 4-IL2 (open triangle) as a function of time; Figure 6 is a diagrammatic representation of the protocol for constructing an antibody-based fusion protein with a mutation that reduces binding affinity to Fc receptors; and Figure 7 is a line graph illustrating the in vivo plasma concentration of the intact fusion protein of hu-KS? 1-IL2 (0); mutated hu-KS? 1-IL2 (D) and hu-KS? 4-IL2 (?) as a function of time.

DETAILED DESCRIPTION OF THE INVENTION It has now been discovered that fusion of a second protein, such as a cytosine, to an immunoglobulin can alter the structure of the antibody, resulting in an increase in binding affinity for one or more of the Fc receptors bound to the cell and driving to a rapid elimination of the antibody-based fusion protein of the circulation. The present invention describes antibody-based fusion proteins with improved, in vivo, circulating half-lives and involves producing, through recombinant DNA technology, antibody-based fusion proteins with reduced binding affinity for one or more receptors. Fc.

First, the antibody-based fusion protein with improved in vivo circulating half-life can be obtained by constructing an isotope fusion protein having reduced binding affinity for an Fc receptor, and avoiding the use of antibody isotype sequences that bind to Fc receivers. For example, of the four known IgG isotypes, IgG 1 (C? 1) and IgG3 (C? 3) are known to bind to FcR? L with high affinity, while IgG4 (C? 4) has a reduction affinity. 10 times smaller and, IgG2 (C? 2) does not bind to FcR? L. In this way, an antibody-based fusion protein with reduced binding affinity for an Fc receptor can be obtained by constructing a fusion protein with a C? 2 constant region (Fc region) or a C? 4 region, and avoiding constructions with a C? 1 region or a C? 3Fc region. Second, an antibody-based fusion protein with improved in vivo circulating half-life can be obtained by modifying sequences necessary for binding to Fc receptors in isotypes that have binding affinity for an Fc receptor, in order to reduce or eliminate the union. As mentioned above, IgG molecules interact with three classes of Fc receptors (FcR), mainly Fc? RI, Fc? RII, and Fc? RUI. C? 1 and C? 3 bind to FcR? L with high affinity, while C? 4 and C? 2 have reduced binding affinity or no affinity for FcR? 1. A comparison of C? 1 and C? 3 indicates that, with the exception of a hinge segment extended at C? 3, the amino acid sequence homology between these two isotypes is very high. This is true even in those regions that have shown to interact with the C1q fragment of the complement and the various classes of FcγR. Figure 1 provides an alignment of the amino acid sequences of C 1 and C 3. The other two isotypes of human IgG (C? 2 and C? 4) have sequence differences which have been associated with the binding of FcR. Figure 2 provides an alignment of the amino acid sequences of C 1, C 2 and C 4. The sequences important for the binding of Fc? R are Leu-Leu-Gly-Gly (residues 234 to 237 in C?), Located in the CH2 domain adjacent to the hinge. Canfield and Morrison, J. EXP. MED. 173: 1483-1491 (1991). These sequence motifs are conserved in C 1 and C 3, according to their similar biological properties, and possibly refer to the similarity of pharmacokinetic behavior when used to construct IL-2 fusion proteins. Many mutational analyzes have been performed to demonstrate the effect of specific mutations in the binding of FcR, including those in residues 234-237, as well as the binding residue close to the Pro331 hinge which is replaced by Ser in IgG4. Another important structural component necessary for the effective binding of FcR is the presence of an N-linked carbohydrate chain covalently linked to Asn297. The enzymatic removal of this structure or mutation of the Asn residue effectively removes, or at least dramatically reduces, binding to all classes of Fc? R. Brumbell and others postulated the existence of a protective receptor (FcRp) that could reduce the rate of catabolism of circulating antibodies through binding to the Fc portion of antibodies and, following their pinocytosis to cells, could return to address back to circulation. Brumbell, et al., NATURE 203: 1352-1355 (1964). The neonatal intestinal transport receptor containing beta-2-microglobulin (FcRn) has recently been identified as an FcRp. See, Junghans and others, PROC. NATL. ACAD. SCI. USA 93: 5512-5516 (1996). The sequences necessary for binding to this receptor are conserved in the four classes of human IgG and are located on the abutting surface between the CH2 and CH3 domains. See, Medesan et al., J. IMMUNOL. 158: 2211-2217 (1997). It has been reported that these sequences are important for the in vivo circulating half life of antibodies. See, international PCT publication WO 97/34631. In this manner, the preferred antibody-based fusion proteins of the present invention will have the necessary sequence for binding to FcRp. Methods for synthesizing useful embodiments of the invention are described, as well as assays useful for testing their pharmacokinetic activities, both in vitro and in preclinical, in vivo animal models. The preferred gene construct encoding a chimeric chain includes, in the 5 'to 3' orientation, a DNA segment encoding at least a portion of an immunoglobulin and the DNA encoding a second non-immunoglobulin protein. An alternative preferred gene construct includes, in the 5 'to 3' orientation, a DNA segment encoding a second non-immunoglobulin protein and the DNA encoding an immunoglobulin portion. The fused gene is assembled into or inserted into an expression vector for transfection of the appropriate recipient cells when they are expressed. The invention will be further illustrated through the following non-limiting examples: Example 1 Improvement of the in vivo circulating half-life of an antibody-IL2 fusion protein through commutation of constant regions class of C? 1 to C? 4 IgG. In accordance with the present invention, antibody-based fusion proteins with improved in vivo circulating half-lives can be obtained by constructing antibody-based fusion proteins using sequence of antibody isotypes having reduced binding affinity or having no binding affinity. binding affinity for Fc receptors. In order to analyze whether the in vivo circulating half-life of the antibody-based fusion protein can be improved using sequence of antibody isotypes with reduced binding affinity or no affinity for Fc receptors, an IL2 antibody fusion protein with a human C? 1 constant region (Fc region) was compared with an antibody-IL2 fusion protein with a human C? 4 region. 1.1 Construction of antibody-IL2 fusion proteins with a constant region of C? 4 IgG. The construction of antibody-IL2 fusion proteins with a C? 1 constant region has been described in the prior art. See, for example, Gillies, and others, PROC. NATL. ACAD. SCI. USA 89: 1428-1432 (1992); and patent of E.U.A. No. 5,650,150, the description of which is incorporated herein by reference. To construct antibody-IL2 fusion proteins with a C? 4 constant region, a plasmid vector, capable of expressing a humanized antibody-IL2 fusion protein with variable regions (v) specific for a human pancarcinoma antigen (KSA) and the human C? 1 heavy chain fused to human IL-2 was modified to remove the C? 1 gene fragment and replace it with the corresponding sequence of the C? 4 gene. A map of some of the important restriction sites and the insertion site of the C? 4 gene fragment is provided in Figure 3. These plasmid constructs contain the cytomegalovirus (CMV) anterior promoter for transcription of the mRNA encoding the variable regions (V) of light chain (L) and heavy chain (H) derived from mouse antibody KS-1/4. The mouse V regions were humanized through standard methods and their coding DNA sequences were chemically synthesized. A functional splice donor site was added to the end of each 5 'region so that it could be used in vectors containing H and L chain constant region genes. The human Ck light chain gene was inserted downstream of the cloning site for the VL gene and was followed by its endogenous 3 'untranslated region and the poly adenylation site. This transcription unit was followed by a second independent transcription unit for the heavy chain fusion protein-IL2. It is also run by a CMV promoter. The VH coding sequence was inserted upstream of the DNA encoding the heavy chain gene C? of choice, fused to human IL-2 coding sequences. Such genes C? contain splice acceptor sites for the first heavy chain exon (CH1), just downstream from Hind III single common to all C genes? humans. A 3 'non-translated site and polyadenylation of the SV40 virus was inserted at the end of the IL-2 coding sequence. The remainder of the bacterial plasmid DNA tube vector necessary for propagation in E. coli and a selectable marker (dihydrofolate-dhfr reductase) for the selection of mammalian cell transfectants. The exchange of the C? 1 and C? 4 fragments was achieved through the digestion of plasmid DNA containing original C? 1 with Hind III and Xho I and purifying the large 7.8 kb fragment through agarose gel electrophoresis . A second plasmid DNA containing the C? 4 gene was digested with Hind III and Nsi I and the 1.75 kb fragment was purified. A third plasmid containing the human IL-2 cDNA and the SV40 poly A site, fused to the carboxyl terminus of the human C? 1 gene, was digested with Xho I and Nsi and the small 470 bp fragment was purified. The three fragments were ligated together in approximately equal molar amounts and the ligation product was used to transform competent E. coli. The product was used to transform competent E. coli and colonies were selected through growth containing ampicillin. Properly assembled recombinant plasmids were identified through restriction analysis of plasmid DNA preparations from isolated transformants and Fsp I digestion was used to discriminate between C? 1 gene grafts (without Fsp I) and C? 4 ( a place). The final vector, containing the heavy chain replacement of C? 4-IL2, was introduced into mouse myeloma cells and tranfectants were selected through growth in a medium containing methotrexate (0.1 μM). Cell clones expressing high levels of the antibody-IL2 fusion protein were expanded and the fusion protein was purified from the culture supernatants using protein A Sepharose chromatography. The purity and integrity of the fusion protein C? 4 were determined through SDS-polyacrylamide gel electrophoresis. The activity of IL-2 was measured in a T cell proliferation assay and found to be identical to that of the C? 1 construct. 1. 2 Fc receptor binding through antibody and antibody-IL2 fusion proteins with the constant region of IgG of C? 1 and C? 4. Several mouse and human cell lines express one or more Fc receptors. For example, the mouse macrophage J774 cell line expresses FcR? L which is capable of binding the mouse or human IgG of the appropriate subclasses. Likewise, the human K562 erythroleukemic cell line expresses FcR? Ll but not FcR? L. In order to determine the potential contribution of Fc receptor binding to the removal of antibody-based fusion proteins from the circulation, the binding affinities of an antibody, a C? 1 -IL2 fusion protein, and a protein d C? 4-IL2 fusion for FcR? l were compared in the mouse J774 cell line. The two IL2-antibody proteins described in Example 1, hu-KS? 1-IL2 and hu-KS? 4-IL2, were diluted at 2μg / ml in PBS containing 0.1% bovine serum albumin (BSA), together with 2x105 J774 in a final volume of 0.2 ml. After incubation on ice for 20 minutes, an anti-human IgG Fc antibody conjugated with FITC (Fab2) was added and the incubation continued for a further 30 minutes. The unbound bodies were removed through two washes with PBS-BSA, and the cells were analyzed by a fluorescence activated cell sorter (FACS). The control reactions contained the same cells mixed with the secondary antibody labeled with FITC or with the humanized KS? L antibody (without IL-2). As expected, the binding of the C? 4-IL2 fusion protein to the J774 cells was significantly lower than the binding to the C? 1-IL2 fusion protein. See Figure 4. Unexpectedly, however, both the C? 1-IL2 and C? 4-IL2 fusion proteins had a significantly higher binding to J774 cells than the KS? 1 antibody (without IL-2). This suggests that the fusion of a second protein, such as a cytosine to an immunoglobulin can alter the structure of the antibody, resulting in an increase in binding affinity for one or more of the Fc receptors attached to cells, thus leading to a rapid elimination of circulation. In order to determine if the highest binding observed with IL-2 fusion proteins was due to the presence of IL-2 receptors or FcR? I receptors in the cells, an excess of mouse IgG (mlgG) was used to compete with the binding in the Fc receptors. As illustrated in Figure 4, the above levels of binding were observed with the antibody and both antibody-IL2 fusion proteins in the presence of a molar excess of 50 times more than mlgG. This suggests that the high signal binding of the antibody-IL2 fusion proteins was due to increased binding to the Fc receptor. Cell lines expressing Fc receptors are useful for testing the binding affinities of candidate fusion proteins to Fc receptors in order to identify antibody-based fusion proteins with improved half-lives in vivo. Antibody-based candidate fusion proteins can be tested through the methods described above. The candidate antibody-based fusion proteins with a substantially reduced binding affinity for an Fc receptor will be identified as antibody-based fusion proteins with improved half-lives in vivo. 1.3 Measurement of the circulating half-life of antibody-IL2 fusion proteins with the constant region of C? 4 IgG. In order to determine whether using the Fc region of an IgG isotype having reduced affinity for Fc receptors will improve the circulating half-life in vivo, fusion proteins containing the heavy chain of the C? 1 isotype were compared (ie, hu-KS ? 1-IL2) with fusion proteins containing the heavy chain of the C? 4 isotype (ie, hu-KS? 4-IL2). The purified humanized KS-1/4-IL2 fusion proteins containing the heavy chain of the C? 1 or C? 4 isotype were changed in their pH regulator through diafiltration to pH regulated saline with phosphate (PBS) and they were further diluted to a concentration of approximately 100μg / ml. About 20μg of the antibody-based fusion protein (0.2ml) was injected into 6-8 week old Balb / c mice in the tail vein using a slow thrust. Per group, four mice were injected. At several time points, small blood samples were taken through retro-orbital bleeding of the anesthetized animals and collected in tubes containing citrate pH regulator to prevent coagulation. The cells were removed by centrifugation in an overhead table centrifuge at high speed for 5 minutes. The plasma was removed with a micro pipette and frozen at -70 ° C. The concentration of human antibody determinants in the mouse blood was measured by ELISA. A capture antibody specific for the human H and L antibody chains was used to capture the fusion proteins from the diluted plasma samples. After a two hour incubation in 96 well plates covered with antibody, the unbound material was removed through three washes with ELISA pH buffer (0.01% Tween 80 in PBS). A second incubation step used either an anti-human Fc antibody (for the detection of fusion protein both antibody and intact), and an anti-human IL2 antibody (for the detection of only the intact fusion protein). Both antibodies were conjugated to horseradish peroxidase (HRP). After one hour of incubation, the unbound detection antibody was removed by washing with an ELISA pH regulator and the amount of bound HPR was determined by incubation with substrate and measuring in a spectrophotometer. As illustrated in Figure 5, the half-life of phase a of the hu-KS? 4-IL2 fusion protein was significantly longer than the half-life of phase a of the hu-KS? 1-IL2 fusion protein. The increased half-life is best illustrated through the significantly higher concentrations of the hu-KS? 4-IL2 fusion protein (3.3 μg / ml) compared to the hu-KS? 1-IL2 fusion protein (60 ng / ml). ml) found in mice after 24 hours. The protein hu-KS? 1-IL2 had a phase (a) of rapid distribution followed by a slower (?) Catabolic phase, as reported previously for the 14.18-IL2 fusion protein. Chimerical See, Gillies, and others BIOCONJ. CHEM. 4: 230-235 (1993). In the study by Gillies, et al., Only the antibody determinants were measured, so that the elimination represented by the removal of the intact fusion protein or the removal of the antibody component from the fusion protein was not evident. In the example herein, samples were analyzed using both (1) a specific antibody ELISA assay, and (2) a specific fusion protein ELISA assay (i.e., an ELISA assay that requires both the antibody and the of the IL-2 components that are physically linked). As illustrated in Figure 5, in animals injected with the hu-KS? 1-IL2 fusion protein, the amount of circulating protein was less than the total amount of antibody in circulation, especially at the 24-hour time point. . This suggests that the fusion protein is being proteolytically cleaved in vivo and that the released antibody continues to circulate. Surprisingly, in animals injected with the hu-KS? 4-IL2 fusion protein, there were no significant differences between the amount of fusion protein in circulation and the total amount of antibody in circulation. This suggests that the hu-KS? 4-IL2 fusion protein was not proteolytically cleaved in these animals during the 24-hour period measured. As discussed above, C? 1 and C? 3 have binding affinity for Fc receptors, while C? 4 has a reduced binding affinity and C? 2 has no binding affinity for Fc receptors. The example herein describes methods for producing antibody-based fusion proteins using the C? 4 Fc region, an isotype of IgG having reduced affinity for Fc receptors, and it was established that said antibody-based fusion proteins have a life improved in vivo circulation media. Therefore, one skilled in the art can use these methods to produce antibody-based fusion proteins with the C? 2 Fc region, instead of the C? 4 Fc region, in order to improve the circulating half-life of the proteins of fusion. A Hu-KS-IL2 fusion protein utilizing the human C? 2 region can be constructed using the same restriction fragment replacement and the methods described above for the C? 4-IL2 fusion protein and tested using the methods described in the present to demonstrate an increased average life in circulation. The fusion proteins based on antibody with the C? 2 Fc region, or any other Fc region having reduced binding affinity or lacking binding affinity for an Fc receptor, will have an improved in vivo circulating half-life, as compared to with the antibody-based fusion proteins having binding affinity for an Fc receptor.

Example 2. Mutation of the human C? 1 or C? 3 gene in antibody-based fusion protein constructs to improve their half-life in circulation, in vivo. The IgG molecules interact with several molecules in the circulation, including members of the protein complement system (for example, the C1q fragment), as well as the three FcR classes. The residues important for the binding of C1q are the residues Glu318, Lys320, and Lys322 which are located in the CH2 domain of human heavy chains. Tao, and others, EXP. MED. 178: 661-667 (1993). In order to discriminate between the binding of FcR and C1q as mechanisms for rapid elimination, the element closer to the hinge C? 2 more drastically altered in the heavy chain of C? 2 was replaced. It is expected that this mutation affects the binding of FcR but not the complement fixation. The mutation was achieved by cloning and adapting the small region between the hinge and the beginning of the CH2 exon of the germline C? 1 gene using overlapping polymerase chain reactions (PCR). The PCR primers were designed to replace the new sequence at the junction of two adjacent PCR fragments by extending a Pst to Drd I fragment (see Figure 6). In the first step, two separate PCR reactions with primers 1 and 2 (SEQ ID NOS: 5 and 6, respectively), or primers 3 and 4 (SEQ ID NOS: 7 and 8, respectively), were prepared using the C? 1 gene as the template. Cycle conditions for the primary PCR were 35 cycle of 94 ° C for 45 seconds, heating and then cooling to 48 ° C for 45 seconds, and primer extension at 72 ° C for 45 seconds. The products of each PCR reaction were used as a template for the second step of the binding reaction. One tenth of each primary reaction was mixed together and combined with primers 1 and 4 to amplify only the combined product of the two initial PCR products. The conditions for secondary PCR were: 94 ° C for 1 minute, heating and rapid cooling at 51 ° C for 1 minute, and extension of initiator at 72 ° C for 1 minute. The union occurred as a result of the overlap between the two individual fragments, which are paired with the other end, following the denaturation and heating and rapid cooling. The fragments that form hybrids are extended through Taq polymerase, and the mutated, complete product was selectively amplified by the initiation of the external primers, as shown in Figure 6. The final PCR product was cloned into a plasmid vector and its sequence verified through DNA sequence analysis. The assembly of the mutated gene was performed in multiple steps. In the first step, a cloning vector containing the C? 1 gene was digested with Pst I and Xho I to remove the non-mutated hinge-CH2-CH3 coding sequences. A fragment of Drd I to Xho I encoding part of CH2, all CH3 and the fused human IL-2 coding sequences was prepared from the vector C? 1-IL2, as described above. A third fragment was prepared from the subcloned PCR product through digestion with Pst I and Drd I. The three fragments were purified through agarose gel electrophoresis and ligated together in a reaction mixture. The ligation product was used to transform competent E. coli and colonies were selected through growth on plates containing ampicillin. The correctly assembled recombinant plasmids were identified through restriction analysis of plasmid DNA preparations from isolated transformants and mutated genes that were confirmed through DNA sequence analysis. The Hind III to Xho I fragment of the mutated C? 1-IL2 gene was used to resemble the complete hu-KS IL-2 antibody fusion protein expression vector. In order to analyze the improvement of the in vivo circulating half-life induced by a mutation of an important amino acid for the binding of FcR, and to discriminate the binding between FcR and C1q as mechanisms of rapid elimination, the in vivo plasma concentration of hu-KS1-IL2 was compared to the plasma concentration of hu-KS1-IL2 at various specific times. As illustrated in Figure 7, the in vivo elimination regimens of mutated hu-KS1-IL2 were significantly lower than the elimination regimen of hu-KS1-IL2. These results suggest that an antibody-based fusion protein with improved in vivo circulating half-life can be obtained by modifying sequences necessary for binding to Fc receptors in isotypes that have binding affinity for an Fc receptor. In addition, the results suggest that the mechanisms for rapid elimination involve FcR binding instead of C1q binding. Those skilled in the art will understand, from the teachings of the present invention, that various other mutations can be introduced into the C? 1 or C? 3 genes in order to reduce the binding to FcR and improve the half-life in circulation. in vivo of an antibody-based fusion protein. In addition, mutations in the C? 4 gene can also be introduced in order to further reduce the binding of C? 4 fusion proteins to FcR. For example, additional possible mutations include mutations in the amino acid residues near the hinge, mutating Pro331, or through mutation of the individual N-linked glycosylation site in all regions of IgG Fc. The latter is located in Asn297 as part of the canonical sequence: Asn-X-Thr / Ser, where the second position can be any amino acid (with the possible exception of Pro), and the third position is either Thr or Ser. A conservative mutation to the amino acid Gln, for example, could have a small effect on the protein, but could prevent the binding of any carbohydrate side chain.

A strategy to mutate this residue can follow the general procedure, already described, for the region next to the hinge. Methods for generating point mutations in cloned DNA sequences are well established in the art and established in the art and commercial equipment is available from various vendors for this purpose.

Example 3. Increase in the average life in circulation of fusion proteins based on receptor-antibody. Several references have been reported that the Fc portion of human IgG can serve as a useful carrier for many ligand-binding proteins or receptors, with biological activity. Some of these ligand binding proteins have been fused to the N-terminus of the Fc portion of an Ig such as CD4, CTLA-4, and TNF receptors. See, for example, Capón, et al., NATURE 337: 525-531 (1989); Linsley, et al., J. EXP. MED. 174: 561-569 (1991); Wooley, and others, J. IMMUNOL. 151: 6602-6607 (1993). Increasing the circulating half-life of receptor-antibody fusion proteins can allow the ligand-binding protein pattern (ie, the second non-Ig protein) to more effectively block (1) drug interactions. receptor-ligand on the surface of the cell; or (2) neutralize the biological activity of a molecule (eg, a cytosine) in the fluid phase of the blood, thus allowing it to reach its cellular target. In order to determine if the reduction of the ability of the receptor-antibody-based fusion proteins to bind to IgG receptors will improve their circulating half-life in vivo, the receptor-antibody-based fusion proteins with C regions? 1 human Fc are compared with antibody-based fusion proteins with human C? 4 Fc regions. To construct CD4-antibody-based fusion proteins, the ectodomain of the human CD4 cell surface receptor is cloned using PCR from human peripheral blood monocytic cells (PBMC). The cloned CD4 receptor includes compatible restriction sites and splice donor sites described in Example 1. The expression vector contains a unique Xba I cloning site downstream of the CMV anterior promoter and the human C? 1 or C? 4 gene downstream of its endogenous Hind III site. The rest of the plasmid contains bacterial genetic information for propagation at the E. coli site, as well as a selectable dhfr marker gene. Ligated DNAs were used to transform competent bacteria and recombinant plasmids were identified from restriction analysis of individual bacterial colonies. Two plasmid DNA constructs were obtained: CD4-C? 1 and CD4-C? 4. The expression plasmids are used to transfect mouse myeloma cells through electroporation and the transfectants were selected through growth in a culture medium containing methotrexate (0.1 μM). The transfectants expressing the fusion proteins were identified through the ELISA analyzes and expanded in the culture in order to generate the fusion protein for purification by binding to and elision of protein A Sepharose. The purified proteins in the chromatography elution pH buffer were diafiltered in PBS and diluted to a final concentration of 100 μg / ml. Balb / c mice were injected with 0.2 ml (20 μg) of the fusion protein of either CD4-C? 1 or CD4-C? 4 and the pharmacokinetics were tested as described in Example 1.3. The CD4-C? 4 fusion protein has a significantly longer half-life than the CD4-C? 1 fusion protein.

EQUIVALENTS The invention can be modalized in other specific forms without departing from the spirit or its essential characteristics. The foregoing modalities, therefore, should be considered in all illustrative aspects rather than limitations of the invention described herein. The scope of the invention in this manner is indicated by the appended claims rather than by the foregoing description, and all changes within the meaning and scale of equivalence of the claims are intended to be encompassed here.

LIST OF SEQUENCES < 110 > GILLIES, Stephan D LO, Kin-Ming LAN, Yan WESOLOWSKY, John < 120 > Methods to Improve the Average Life in Circulation of Antibody-based Fusion Proteins < 130 > LEX-003PC < 140 > < 141 > < 150 > US 60 / 075,887 < 151 > 1998-02-25 < 160 > 8 < 170 > Patentln Ver. 2.0 < 210 > 1 < 211 > 447 < 212 > PRT < 213 > Homo sapiens < 220 > < 223 > CHAIN REGION IGG-1 < 400 > 1 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Wing Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Wing Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Wing Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Val Val Thr Val Pro Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr He Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Pro Wing Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met He Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val ASD Gly Val Glu Val His Asn Ala Lys 275 28'0 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lvs 305 310 315 320 Cys Lys Val Ser Asr. Lys Ala Leu Pro Ala Pro He Glu Lys Thr He 325 330 335 Be Lys Wing Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp He Wing Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser C 420? Yís Ser Val Met His Glu Ala Leu 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 < 21 0 > 2 < 21 1 > 443 < 21 2 > P RT < 21 3 > Homo sa piens < 220 > < 223 > REG I O N C D E CADE NA I GG-2 < 400 > 2 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Wing Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Wing Val Leu Gln Ser 165 170 175 Ser Glv Leu Tyr Ser Leu Ser Val Val Thr Val Pro Ser Ser Asn 180 185 190 Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val ASD Lys Thr Val Glu Arg Lys Cys Cvs Val Glu Cys Pro 210. "215 220 Pro Cys Pro Pro Wing Pro Val Wing Gly Pro Ser Val Phe Leu Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Thr Leu Met He Ser Arg Thr Pro Glu Val Thr 245 250 255 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn 260 265 270 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val 290 295 300 Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 305 310 315 320 Asn Lys Gly Leu Pro Wing Pro He Glu Lys Thr He Ser Lys Thr Lys 325 330 335 Gly Gln Pro Arg Glu Pro Gln Val Tyr- Thr Leu Pro Pro Ser Arg Glu 340 345 350 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365 Tyr Pro Ser Asp He Wing Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375 380 Asn Asn Tyr Lys Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe 385 390 395 400 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 405 410 415 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425 430 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 < 210 > 3 < 211 > 494 < 212 > PRT < 213 > Homo sapiens < 220 > < 223 > REG I O N C D E CADE NA I GG-3 < 400 > 3 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Ala Ser Thr Lys Giy Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Wing Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Val Val Thr Val Pro Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr 210 215 220 Thr His Thr Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro 225 230 235 240 Pro Pro Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro 245 250 255 Pro Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys Asp Pro Pro Pro 260 265 270 Cys Pro Arg Cys Pro Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 275 280 285 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met He Ser Arg Thr Pro 290 295 300 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu ASD Pro Glu Val 305 310 315"320 Gln Phe Lys Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 325 330 335 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val 340 345 350 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 355 360 365 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro He Glu Lys Thr He Ser 370 375 380 Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 385 390 395 400 Be Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 405 410 415 Lys Gly Phe Tyr Pro Ser Asp He Wing Val Glu Trp Glu Ser Ser Gly 420 425 430 Gln Pro Glu Asn Asn Tyr Asn Thr Pro Pro Pro Met Leu Asp Ser Asp 435 440 445 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 450 455 460 Gln Gln Gly Asn He Phe Ser Cys Ser Val Met His Glu Ala Leu His 465 470 475 480 Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 485 490 < 210 > 4 < 211 > 444 < 212 > P RT < 213 > Homo sa pie ns < 220 > < 223 > C D E CADE REG ION NA I G G-4 < 400 > 4 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Wing Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Wing Val Leu Gln Ser 165 170 175 Ser Gly Leu Tryyrr Ser Leu Ser Val Val Thr Val Pro Ser Ser L80 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Ser Cys Pro Wing Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met He Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trs Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Be He Glu Lys Thr He Ser Lys Wing 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gl 355 360 365 Phe Tyr Pro Ser Asp He Wing Val Glu Trp Glu Ser Asn Gly Gln Pro 3 0 375 380 Glu Asn Asn Tyr Lys Thr Pro Pro Val Leu Asp Ser Asp Gl Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 < 210 > 5 < 211 > 17 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of Artificial Sequence: initiator 1 < 400 > 5 catcggtctt ccccctg 17 < 210 > 6 < 211 > 35 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: initiator 2 < 400 > 6 cggtcctgcg acgggaggtg ctgaggaaga gatgg 35 < 210 > 7 < 211 > 45 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: initiator 3 < 400 > 7 tcttcctcag cacctcccgt cgcaggaccg tcagtcttcc tcttc 45 < 210 > 8 < 211 > 17 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: initiator 4 < 400 > 8 gaggcgtggt cttgtag 17

Claims (26)

1. CLAIMS 1. An antibody-based fusion protein with an improved circulating half-life, comprising at least a portion of an immunoglobulin heavy chain (Ig) having a substantially reduced binding affinity for an Fc receptor, said portion of heavy chain being linked to a second non-Ig protein, the antibody-based fusion protein having a longer in vivo circulating half life than a second non-unbound Ig protein.
2. 2. The antibody protein based on claim 1, wherein said heavy chain portion comprises at least the CH2 domain of a constant region of IgG2 or IgG4.
3. 3. The antibody-based fusion protein according to claim 1, wherein said heavy chain portion comprises at least a portion of an IgG 1 constant region having a mutation or deletion in one or more amino acids selected from the group which consists of Leu234, Leu235, Gly236, Gly23, Asn297, and Pro33 ?.
4. 4. The antibody fusion protein according to claim 1, wherein said heavy chain protein comprises at least a portion of a constant region of IgG3 having a mutation or deletion in one or more amino acids selected from the group consisting of of Leu281, Leu282, Gly283, Gly284, Asn3 4, and Pro378.
5. 5. The antibody-based fusion protein according to claim 1, wherein said heavy chain portion further has binding affinity for an immunoglobulin protection receptor.
6. 6. The antibody-based fusion protein according to claim 1, wherein said heavy chain portion has a substantially reduced binding affinity for an Fc receptor selected from the group consisting of Fc? RI, Fc? RII, Fc ? RIII.
7. 7. The antibody-based fusion protein according to claim 1, wherein the second non-Ig protein is selected from the group consisting of a cytosine, a ligand binding protein, and a protein toxin.
8. 8. The antibody-based fusion protein according to claim 1, wherein said cytosine is selected from the group consisting of a tumor necrosis factor, an interleukin, and a lymphokine.
9. 9. The antibody-based fusion protein according to claim 8, wherein the tumor necrosis factor is an alpha tumor necrosis factor.
10. 10. The antibody-based fusion protein according to claim 8, wherein interleukin-2 interleukin.
11. 11. The antibody-based fusion protein according to claim 8, wherein said lymphokine is a lymphotoxin or a colony stimulation factor.
12. 12. The antibody-based fusion protein according to claim 11, wherein said colony stimulation factor is a granulocyte-macrophage colony stimulation factor.
13. 13. The antibody-based fusion protein according to claim 1, wherein said ligand binding protein is selected from the group consisting of CD4, CTLA-4, TNF receptor, and an interleukin receptor.
14. 14. A method for increasing the average circulating vine of an antibody-based fusion protein, comprising the step of binding at least a portion of an Ig heavy chain to a second non-Ig protein, said portion of heavy chain having a substantially reduced binding affinity for an Fc receptor, thus forming an antibody-based fusion protein having a circulating half-life, in vivo, greater than a second non-unbound Ig protein.
15. 15. The method according to claim 14, wherein the portion of the heavy chain comprises at least the CH2 domain of a constant region of IgG2 or IgG4.
16. 16. A method for increasing the circulating half-life of an antibody-based fusion protein, comprising the steps of: (a) introducing a mutation or deletion into one or more amino acids of a constant region I gG 1, said amino acids are selected from the group consisting of Leu234, Leu235, Gly236, Gly237, Asn297, and Pro331, thus producing an Ig heavy chain having a substantially reduced binding affinity for an Fc receptor.; and (b) joining at least a portion of the heavy chain from step (a) to a second non-Ig protein, thereby forming an antibody-based fusion protein having a circulating half-life, greater than in vivo second protein that is not Ig, not bound.
17. 17. A method for increasing the circulating half-life of an antibody-based fusion protein, comprising the steps of: (a) introducing a mutation or deletion into one or more amino acids of a constant region of Leu281, Leu282, Gly283, Gly284, Asn34, and Pro378, thus producing an Ig heavy chain having a substantially reduced binding affinity for an Fc receptor; and (b) joining at least a portion of the Ig heavy chain from step (a) to a second non-Ig protein, thereby forming an antibody-based fusion protein having a longer in vivo circulating half-life than a second protein that is not Ig, not bound.
18. 18. The method according to claim 14, 16 or 17, wherein said heavy chain portion further has an immunoglobulin protection binding affinity.
19. 19. The method according to claim 14, 16 or 17, wherein the portion of the heavy chain has a substantially reduced binding affinity for an Fc receptor selected from the group consisting of Fc? RI, Fc? RII, Fc? RIII.
20. The method according to claim 14, 16 or 17, wherein the second non-Ig protein is selected from the group consisting of a cytosine, a ligand binding protein, and a protein toxin.
21. The method according to claim 14, 16 or 17, wherein said cytosine is selected from the group consisting of a tumor necrosis factor, an interleukin and a lymphokine.
22. 22. The method according to claim 21, wherein said tumor necrosis factor is an alpha tumor necrosis factor.
23. 23. The method according to claim 21, wherein said interleukin is interleukin-2.
24. 24. The method according to claim 21, wherein the lymphokine is a lymphotoxin or a colony stimulation factor.
25. 25. The antibody-based fusion protein according to claim 24, wherein said colony stimulation factor is a granulocyte-macrophage colony stimulation factor.
26. 26. The method according to claim 14, 16 or 17, wherein the ligand binding protein is selected from the group consisting of CD4, CTLA-4, TNF receptor, and an interleukin receptor.