AU758240B2 - Enhancing the circulating half-life of antibody-based fusion proteins - Google Patents

Description

WO 99/43713 PCT/US99/03966 ENHANCING THE CIRCULATING HALF-LIFE OF ANTIBODY-BASED FUSION PROTEINS Cross Reference to Related Application This incorporates by reference, and claims priority to and the benefit of, U.S. Provisional Patent Application Serial Number 60/075,887 which was filed on February 25, 1998.

Field of the Invention The present invention relates generally to fusion proteins. More specifically, the present invention relates to methods of enhancing the circulating half-life of antibody-based fusion proteins.

Background of the Invention The use of antibodies for treatment human disease is well established and has become more sophisticated with the introduction of genetic engineering. Several techniques have been developed to improve the utility of antibodies. These include: the generation of monoclonal antibodies by cell fusion to create "hyridomas", or by molecular cloning of antibody heavy (H) and light chains from antibody-producing cells; the conjugation of other molecules to antibodies to deliver them to preferred sites in vivo, radioisotopes, toxic drugs, protein toxins, and cytokines; the manipulation of antibody effector functions to enhance or diminish biological activity; the joining of other protein such as toxins and cytokines with antibodies at the genetic level to produce antibody-based fusion proteins; and the joining of one or more sets of antibody combining regions at the genetic level to produce bi-specific antibodies.

When proteins are joined together through either chemical or genetic manipulation, it is often difficult to predict what properties that the end product will retain from the parent molecules. With chemical conjugation, the joining process may 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 joining process more consistent, and results in the production of consistent end 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 U.S. Patent No. 5,650,150.

SUBSTITUTE SHEET (RULE 26) WO 99/43713 PCT/US99/03966 -2- However, the utility of recombinantly-produced antibody-based fusion proteins may be limited by their rapid in vivo clearance from the circulation. Antibody-cytokine fusion proteins, for example, have been shown to have a significantly lower in vivo circulating half-life than the free antibody. When testing a variety of antibody-cytokine fusion proteins, Gillies et al. reported that all of the fusion proteins tested had an ct phase (distribution phase) half-life of less than hour. Indeed, most of the antibody-based fusion protein were cleared to 10% of the serum concentration of the free antibody by two hours. See, Gillies et al., BIOCONJ. CHEM. 4: 230-235 (1993). Therefore, there is a need in the art for methods of enhancing the in vivo circulating halflife of antibody-based fusion proteins.

Summary of the Invention A novel approach to enhancing 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 non-immunoglobulin protein. Antibody-based fusion proteins with reduced binding affinity for Fc receptors have a significantly longer in vivo circulating half-life than the unlinked second non-immunoglobulin protein.

IgG molecules interact with three classes of Fc receptors (FcR) specific for the IgG class of antibody, namely FcyRI, FcyRII and FcyRIII. In preferred embodiments, the immunoglobulin (Ig) component of the fusion protein has at least a portion of the constant region of an IgG that has a reduced binding affinity for at least one of FcyRI, FcyRII or FcyRIII.

In one aspect of the invention, the binding affinity of fusion proteins for Fc receptors is reduced by using heavy chain isotypes as fusion partners that have reduced binding affinity for Fc receptors on cells. For example, both human IgGI and IgG3 have been reported to bind to FcRyI with high affinity, while IgG4 binds 10-fold less well, and IgG2 does not bind at all. The important sequences for the binding of IgG to the Fc receptors have been reported to be located in the CH2 domain. Thus, in a preferred embodiment, an antibody-based fusion protein with enhanced in vivo circulating half-life is obtained by linking 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 acid in the constant SUBSTITUTE SHEET (RULE 26) WO 99/43713 PCT/US99/03966 -3region of the IgG 1 or IgG3 heavy chains that reduces the binding affinity of these isotypes for Fc receptors. Such modifications include alterations of residues necessary for contacting Fc receptors or altering others that affect the contacts between other heavy chain residues and Fc receptors through induced conformational changes. Thus, in a preferred embodiment, an antibody-based fusion protein with enhanced in vivo circulating half-life is obtained by first introducing a mutation, deletion, or insertion in the IgGI constant region at one or more amino acid selected from Leu2 34 Leu 235 Gly 236 Gly 237 Asn2 97 and Pro33, and then linking the resulting immunoglobulin, or portion thereof, to a second non-immunoglobulin protein. In an alternative preferred embodiment, the mutation, deletion, or insertion is introduced in the IgG3 constant region at one or more amino acid selected from Leu 28 1 Leu, 8 2 Gly 28 3 Gly 284 Asn 344 and Pro 37 8 and the resulting immunoglobulin, or portion thereof, is linked to a second non-immunoglobulin protein. The resulting antibody-based fusion proteins have a longer in vivo circulating half-life than the unlinked second non-immunoglobulin protein.

In a preferred embodiment, the second non-immunoglobulin component of the fusion protein is a cytokine. The term "cytokine" is used herein to describe proteins, analogs thereof, and fragments thereof which are produced and excreted by a cell, and which elicit a specific response in a cell which has a receptor for that cytokine. Preferably, cytokines include interleukins such as interleukin-2 hematopoietic factors such as granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor (TNF) such as TNFa, and lymphokines such as lymphotoxin. Preferably, the antibody-cytokine fusion protein of the present invention displays cytokine 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 may, for example, block receptor-ligand interactions at the cell surface; or neutralize the biological activity of a molecule a cytokine) in the fluid phase of the blood, thereby preventing it from reaching its cellular target. Preferably, ligand-binding proteins include CD4, CTLA-4, TNF receptors, or interleukin receptors such as the IL-1 and IL-4 receptors. Preferably, the antibody-receptor fusion protein of the present invention displays the biological activity of the ligand-binding protein.

SUBSTITUTE SHEET (RULE 26) WO 99/43713 PCT/US99/03966 -4- In yet 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 displays 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 nonimmunoglobulin protein. The constant region may be the constant region normally associated with the variable region, or a different one, variable and constant regions from different species. The heavy chain can include a CH1, CH2, and/or CH3 domains. Also embraced within the term "fusion protein" are constructs having a binding domain comprising framework regions and variable regions complementarity determining regions) from different species, such as are disclosed by Winter, et al., GB 2,188, 638. Antibody-based fusion proteins comprising a variable region preferably display antigen-binding specificity. In yet another preferred embodiment, the antibody-based fusion protein further comprises a light chain. The invention thus provides fusion proteins in which the antigen-binding specificity and activity of an antibody are combined with the potent biological activity of a second non-immunoglobulin protein, such as a cytokine. A fusion protein of the present invention can be used to deliver selectively the second non-immunoglobulin protein to a target cell in vivo so that the second nonimmunoglobulin protein can exert a localized biological effect.

In an alternative preferred embodiment, the antibody-based fusion protein comprises a heavy chain constant region linked through a peptide bond to a second non-immunoglobulin protein, but does not comprise a heavy chain variable region. The invention thus further provides fusion proteins which retain the potent biological activity of a second nonimmunoglobulin protein, but which lack the antigen-binding specificity 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 beta-2 microglobulin-containing neonatal intestinal transport receptor (FcRn).

In preferred embodiments, the fusion protein comprises two chimeric chains comprising at least a portion of a heavy chain and a second, non-Ig protein are linked by a disulfide bond.

SUBSTITUTE SHEET (RULE 26) WO 99/43713 PCT/US99/03966 The invention also features DNA constructs encoding the above-described fusion proteins, and cell lines, myelomas, transfected with these constructs.

These and other objects, along with advantages and features of the invention disclosed herein, will be made more apparent from the description, drawings, and claims that follow.

Brief Description of the Drawings The foregoing and other objects, features, and advantages of the present invention, as well as the invention itself, may be more fully understood from the following description of preferred embodiments, when read together with the accompanying drawings, in which: FIG. 1 is a homology alignment of the amino acid sequences of the constant region of Cyl and Cy3, aligned to maximize amino acid identity, and wherein non-conserved amino acids are identified by boxes; FIG. 2 is a homology alignment of the amino acid sequences of constant region of Cyl, Cy2, and Cy 4, aligned to maximize amino acid identity, and wherein non-conserved amino acids are identified by boxes; FIG. 3 is a diagrammatic representation of a map of the genetic construct encoding an antibody-based fusion protein showing the relevant restriction sites; FIG. 4 is a bar graph depicting the binding of antibody hu-KS-1/4 and antibody-based fusion proteins, hu-KSyl-IL2 and hu-KSy4-IL2, to Fc receptors on mouse J774 cells in the presence (solid bars) or absence (stippled bars) of an excess of mouse IgG; FIG. 5 is a line graph depicting the in vivo plasma concentration of total antibody (free antibody and fusion protein) of hu-KSyl-IL2 (closed diamond) and hu-KSy4-IL2 (closed triangle) and of intact fusion protein of hu-KSyl-IL2 (open diamond) and hu-KSy4-IL2 (open triangle) as a function of time; FIG. 6 is a diagrammatic representation of protocol for constructing an antibody-based fusion protein with a mutation that reduces the binding affinity to Fc receptors; FIG. 7 is a line graph depicting the in vivo plasma concentration of intact fusion protein of hu-KSyl-IL2 mutated hu-KSy -IL2 and hu-KSy4-IL2 as a function of time.

SUBSTITUTE SHEET (RULE 26) WO 99/43713 PCT/US99/03966 -6- Detailed Description of the Invention It has now been discovered that fusing a second protein, such as a cytokine, to an immunoglobulin may alter the antibody structure, resulting in an increase in binding affinity for one or more of the cell-bound Fc receptors and leading to a rapid clearance of the antibody-based fusion protein from the circulation. The present invention describes antibody-based fusion proteins with enhanced in vivo circulating half-lives and involves producing, through recombinant DNA technology, antibody-based fusion proteins with reduced binding affinity for one or more Fc receptor.

First, an antibody-based fusion protein with an enhanced in vivo circulating half-life can be obtained by constructing a fusion protein with isotypes having reduced binding affinity for a Fc receptor, and avoiding the use of sequences from antibody isotypes that bind to Fc receptors.

For example, of the four known IgG isotypes, IgGI (Cyl) and IgG3 (Cy3) are known to bind FcRyI with high affinity, whereas IgG4 (Cy4) has a 10-fold lower binding affinity, and IgG2 (Cy2) does not bind to FcRyI. Thus, an antibody-based fusion protein with reduced binding affinity for a Fc receptor could be obtained by constructing a fusion protein with a Cy2 constant region (Fc region) or a Cy4 Fc region, and avoiding constructs with a Cyl Fc region or a Cy3 Fc region.

Second, an antibody-based fusion protein with an enhanced 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 binding. As mentioned above, IgG molecules interact with three classes of Fc receptors (FcR), namely FcyRI, FcyRII, and FcyRIII. Cyl and Cy3 bind FcRyI with high affinity, whereas Cy4 and Cy2 have reduced or no binding affinity for FcRyI. A comparison of the Cyl and Cy3 indicates that, with the exception of an extended hinge segment in Cy3, the amino acid sequence homology between these two isotypes is very high. This is true even in those regions that have been shown to interact with the Clq fragment of complement and the various FcyR classes. FIG. 1 provides a alignment of the amino acid sequences of Cy and Cy3. The other two isotypes of human IgG (Cy2 and Cy4) have sequence differences which have been associated with FcR binding. FIG. 2 provides a alignment of the amino acid sequences of Cyl, Cy2, and Cy4. The important SUBSTITUTE SHEET (RULE 26) WO 99/43713 PCT/US99/03966 -7sequences for FcyR binding are Leu-Leu-Gly-Gly (residues 234 through 237 in Cyl), 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 Cyl and Cy3, in agreement with their similar biological properties, and possibly related to the similarity of pharmacokinetic behavior when used to construct IL-2 fusion proteins. Many mutational analyses have been done to demonstrate the effect of specific mutations on FcR binding, including those in residues 234-237 as well as the hinge-proximal bend residue Pro 33 that is substituted by Ser in IgG4. Another important structural component necessary for effective FcR binding is the presence of an N-linked carbohydrate chain covalently bound to Asn 2 97 Enzymatic removal of this structure or mutation of the Asn residue effectively abolish, or at least dramatically reduce, binding to all classes of FcyR.

Brumbell et al. postulated the existence of a protection receptor (FcRp) that would slow the rate of catabolism of circulating antibodies by binding to the Fc portion of antibodies and, following their pinocytosis into cells, would redirect them back into the circulation. Brumbell et al., NATURE 203: 1352-1355 (1964). The beta-2 microglobulin-containing neonatal intestinal transport receptor (FcRn) has recently been identified as a FcRp. See, Junghans et al., PROC.

NATL. ACAD. SCI. USA 93: 5512-5516 (1996). The sequences necessary for binding to this receptor are conserved in all four classes of human IgG and are located at the interface between the CH2 and CH3 domains. See, Medesan et al., J. IMMUNOL. 158:2211-2217 (1997). These sequences have been reported to be important for the in vivo circulating half-life of antibodies.

See, International PCT publication WO 97/34631. Thus, preferred antibody-based fusion proteins of the present invention will have the sequences necessary 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 pre-clinical in vivo animal models. The preferred gene construct encoding a chimeric chain includes, in 5' to 3' orientation, a DNA segment which encodes at least a portion of an immunoglobulin and DNA which encodes a second, non-immunoglobulin protein. An alternative preferred gene construct includes, in 5' to 3' orientation, a DNA segment which encodes a second, non-immunoglobulin protein and DNA which encodes at least a portion of an immunoglobulin. The fused gene is assembled in or inserted into an expression vector for transfection of the appropriate recipient cells where it is expressed.

SUBSTITUTE SHEET (RULE 26) WO 99/43713 PCT/US99/03966 -8- The invention is illustrated further by the following non-limiting examples: Example 1 Improving the in vivo circulating half-life of an antibody-IL2 fusion protein by class switching from Cyl to Cy4 IgG constant regions.

According to the present invention, antibody-based fusion proteins with enhanced in vivo circulating half-lives can be obtained by constructing antibody-based fusion proteins using sequences from antibody isotypes that have reduced or no binding affinity for Fc receptors.

In order to assess whether the in vivo circulating half-life of the antibody-based fusion protein can be enhanced by using sequences from antibody isotypes with reduced or no binding affinity for Fc receptors, an antibody-IL2 fusion protein with a human Cyl constant region (Fc region) was compared to an antibody-IL2 fusion protein with a human Cy4 Fc region.

1.1 Construction of antibody-IL2 fusion proteins with a Cy4 IgG constant region.

The construction of antibody-IL2 fusion proteins with a Cyl constant region has been described in the prior art. See, for example, Gillies et al., PROC. NATL. ACAD. SCL. USA 89: 1428-1432 (1992); and U.S. Patent No 5,650,150, the disclosure of which is incorporated herein by reference.

To construct antibody-IL2 fusion proteins with a Cy4 constant region, a plasmid vector, capable of expressing a humanized antibody-IL2 fusion protein with variable regions specific for a human pancarcinoma antigen (KSA) and the human Cyl heavy chain fused to human IL-2, was modified by removing the Cyl gene fragment and replacing it with the corresponding sequence from the human Cy4 gene. A map of some of the relevant restriction sites and the site of insertion of the Cy4 gene fragment is provided in FIG. 3. These plasmid constructs contain the cytomegalovirus (CMV) early promoter for transcription of the mRNA encoding the light (L) and heavy chain variable regions derived from the mouse antibody KS-1/4. The mouse V regions were humanized by standard methods and their encoding DNA sequences were chemically synthesized. A functional splice donor site was added at the end of each V 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 poly adenylation site. This transcription unit was followed by a second independent transcription unit for the heavy chain-IL2 fusion protein. It is SUBSTITUTE SHEET (RULE 26) WO 99/43713 PCT/US99/03966 -9also driven by a CMV promoter. The VH encoding sequence was inserted upstream of the DNA encoding the Cy heavy chain gene of choice, fused to human IL-2 encoding sequences. Such Cy genes contain splice acceptor sites for the first heavy chain exon (CHI), just downstream from a unique Hind III common to all human Cy genes. A 3' untranslated and polyadenylation site from SV40 virus was inserted at the end of the IL-2 encoding sequence. The remainder of the vector contained bacterial plasmid DNA necessary for propagation in E. coli and a selectable marker gene (dihydrofolate reductase dhfr) for selection of transfectants of mammalian cells.

The swapping of the Cyl and Cy4 fragments was accomplished by digesting the original Cyl-containing plasmid DNA with Hind III and Xho I and purifying the large 7.8 kb fragment by agarose gel electrophoresis. A second plasmid DNA containing the Cy4 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 SV40 poly A site, fused to the carboxyl terminus of the human Cy1 gene, was digested with Xho I and Nsi I and the small 470 bp fragment was purified. All three fragments were ligated together in roughly equal molar amounts and the ligation product was used to transform competent E. coli. The ligation product was used to transform competent E. coli and colonies were selected by growth on plates containing ampicillin. Correctly assembled recombinant plasmids were identified by restriction analyses of plasmid DNA preparations from isolated transformants and digestion with Fsp I was used to discriminate between the Cyl (no Fsp I) and Cy4 (one site) gene inserts. The final vector, containing the Cy4-IL2 heavy chain replacement, was introduced into mouse myeloma cells and transfectants were selected by growth in medium containing methotrexate (0.1 gM). Cell clones expressing high levels of the antibody-IL2 fusion protein were expanded and the fusion protein was purified from culture supernatants using protein A Sepharose chromatography. The purity and integrity of the Cy4 fusion protein was determined by SDS-polyacrylamide gel electrophoresis. IL-2 activity was measured in a T-cell proliferation assay and found to be identical to that of the Cy construct.

1.2 Binding to Fc receptors by antibody and antibody-IL2 fusion proteins with Cyl and Cy4 IgG constant region.

Various mouse and human cell lines express one or more Fc receptor. For example, the mouse J774 macrophage-like cell line expresses FcRyI that is capable of binding mouse or human IgG of the appropriate subclasses. Likewise, the human K562 erythroleukemic cell line SUBSTITUTE SHEET (RULE 26) WO 99/43713 PCT/US99/03966 expresses FcRyII but not FcRyI. In order to assess the potential contribution of Fc receptor binding to clearance of antibody-based fusion proteins from the circulation, the binding affinities of an antibody, a Cyl-IL2 fusion protein, and a Cy4-IL2 fusion protein for FcRyI were compared in the mouse J774 cell line.

The two antibody-IL-2 fusion proteins described in Example 1, hu-KSyl-IL2 and hu-KSy4-IL2, were diluted to 2 pg/ml in PBS containing 0.1% bovine serum albumin (BSA), together with 2x10 5 J774 cells in a final volume of 0.2 ml. After incubation on ice for 20 min, a FITC-conjugated anti-human IgG Fc antibody (Fab2) was added and incubation was continued for an additional 30 min. Unbound antibodies were removed by two washes with PBS-BSA, and the cells were analyzed in a fluorescence-activated cell sorter (FACS). Control reactions contained the same cells mixed with just the FITC-labeled secondary antibody or with the humanized KSyl antibody (without IL-2).

As expected, the binding of the Cy4-IL2 fusion protein to J774 cells was significantly lower than the binding of the Cyl-IL2 fusion protein. See FIG. 4. Unexpectedly, however, both the Cl1-IL2 and Cy4-IL2 fusion proteins had significantly higher binding to J774 cells than the KSyl antibody (without IL-2). This suggests that fusing a second protein, such as a cytokine, to an immunoglobulin may alter the antibody structure, resulting in an increase in binding affinity for one or more of the cell-bound Fc receptors, thereby leading to a rapid clearance from the circulation.

In order to determine whether the greater binding observed with IL-2 fusion proteins was due to the presence of IL-2 receptors or FcRyI receptors on the cells, excess mouse IgG (mIgG) was used to compete the binding at the Fc receptors. As illustrated in FIG. 4, background levels of binding were observed with the antibody and both antibody-IL2 fusion proteins in the presence of a 50-fold molar excess of mIgG. This suggests that the increased signal binding of 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 enhanced in vivo half lives. Candidate antibody-based fusion proteins can be tested by the above-described methods. Candidate antibody-based fusion proteins with substantially reduced binding affinity SUBSTITUTE SHEET (RULE 26) WO 99/43713 PCT/US99/03966 11 for an Fc receptor will be identified as antibody-based fusion proteins with enhanced in vivo half lives.

1.3 Measuring the circulating half-life of antibody-IL2 fusion proteins with Cyl and Cy4 IgG constant region.

In order to assess whether using the Fc region of an IgG isotype having reduced affinity for Fc receptors will enhance the in vivo circulating half-life, fusion proteins containing the Cy1 isotype heavy chain hu-KSy 1-IL2) were compared to fusion proteins containing the Cy4 isotype heavy chain hu-KSy4-IL2).

Purified humanized KS-1/4-IL2 fusion proteins containing either the Cyl or Cy4 isotype heavy chain were buffer-exchanged by diafiltration into phosphate buffered saline (PBS) and diluted further to a concentration of-100 Lg/ml. Approximately 20 .tg of the antibody-based fusion protein (0.2 ml) was injected into 6-8 week old Balb/c mice in the tail vein using a slow push. Four mice were injected per group. At various time points, small blood samples were taken by retro-orbital bleeding from anaesthetized animals and collected in tubes containing citrate buffer to prevent clotting. Cells were removed by centrifugation in an Eppendorf high-speed tabletop centrifuge for 5 min. The plasma was removed with a micropipettor and frozen at -70 0 C. The concentration of human antibody determinants in the mouse blood was measured by ELISA. A capture antibody specific for human H and L antibody chains was used for capture of the fusion proteins from the diluted plasma samples. After a two hour incubation in antibody-coated 96-well plates, the unbound material was removed by three washes with ELISA buffer (0.01% Tween 80 in PBS). A second incubation step used either an anti-human Fc antibody (for detection of both antibody and intact fusion protein), or an anti-human IL-2 antibody (for detection of only the intact fusion protein). Both antibodies were conjugated to horse radish peroxidase (HRP). After a one hour incubation, the unbound detecting antibody was removed by washing with ELISA buffer and the amount of bound HPR was determined by incubation with substrate and measuring in a spectrophotometer.

As depicted in FIG. 5, the a phase half-life of the hu-KSy4-IL2 fusion protein was significantly longer than the a phase half-life of the hu-KSyl-IL2 fusion protein. The increased half-life is best exemplified by the significantly higher concentrations of the hu-KSy4-IL2 fusion SUBSTITUTE SHEET (RULE 26) WO 99/43713 PCT/US99/03966 -12protein (3.3 ig/ml) compared to the hu-KSyl-IL2 fusion protein (60 ng/ml) found in mice after 24 hours.

The hu-KSyl-IL2 protein had a rapid distribution phase followed by a slower catabolic phase, as reported earlier for the chimeric 14.18-IL2 fusion protein. See, Gillies et al., BIOCONJ. CHEM. 4: 230-235 (1993). In the Gillies et al. study, only antibody determinants were measured, so it was not clear if the clearance represented the clearance of the intact fusion protein or the clearance of the antibody component of the fusion protein. In the present Example, samples were assayed using both an antibody-specific ELISA, and a fusion proteinspecific ELISA an ELISA that requires that both the antibody and IL-2 components be physically linked). As illustrated in FIG. 5, in animals injected with the hu-KSyl-IL2 fusion protein, the amount of circulating fusion protein was lower than the total amount of circulating antibody, especially at the 24 hr 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-KSy4-IL2 fusion protein, there was no significant differences between the amount of circulating fusion protein and the total amount of circulating antibody.

This suggests the hu-KSy4-IL2 fusion protein was not being proteolytically cleaved in these animals during the 24 hour period measured.

As discussed above, Cyl and Cy3 have binding affinity for Fc receptors, whereas while Cy4 has reduced binding affinity and Cy2 has no binding affinity for Fc receptors. The present Example described methods for producing antibody-based fusion proteins using the Cy4 Fc region, an IgG isotype having reduced affinity for Fc receptors, and established that such antibody-based fusion proteins have enhanced in vivo circulating half-life. Accordingly, a skilled artisan can use these methods to produce antibody-based fusion proteins with the Cy2 Fc region, instead of the Cy4 Fc region, in order to enhance the circulating half-life of fusion proteins. A Hu-KS-IL2 fusion protein utilizing the human Cy2 region can be constructed using the same restriction fragment replacement and the above-described methods for Cy4-IL2 fusion protein.

and tested using the methods described herein to demonstrate increased circulating half-life.

Antibody-based fusion proteins with the Cy2 Fc region, or any other Fc region having reduced binding affinity or lacking binding affinity for a Fc receptor will have enhanced in vivo SUBSTITUTE SHEET (RULE 26) WO 99/43713 PCTIUS99/03966 -13circulating half-life compared to antibody-based fusion proteins having binding affinity for a Fc receptor.

Example 2 Mutating the human Cyl or C 7 3 gene in antibody-based fusion protein constructs to improve their in vivo circulating half-life.

IgG molecules interact with several molecules in the circulation, including members of the complement system of proteins C q fragment), as well as the three classes of FcR. The important residues for Clq binding are residues Glu 3 1 8 Lys 320 and Lys 322 which are located in the CH2 domains of human heavy chains. Tao et al., J. Exp. MED. 178: 661-667 (1993). In order to discriminate between FcR and C q binding as mechanisms for rapid clearance, we substituted the more drastically altered Cy2 hinge-proximal segment into the Cyl heavy chain. This mutation is expected to affect FcR binding but not 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 germ line Cyl gene using overlapping polymerase chain reactions (PCR). The PCR primers were designed to substitute the new sequence at the junction of two adjacent PCR fragments spanning a Pst I to Drd I fragment (see FIG. 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 Cyl gene as the template. The cycle conditions for the primary PCR were 35 cycles of: 94°C for 45 sec, annealing at 480C for 45 seconds, and primer extension at 72°C for 45 sec. The products of each PCR reaction were used as template for the second, joining reaction step. 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 the secondary PCR were: 94°C for 1 min, annealing at 51 C for 1 min, and primer extension at 720C for 1 min. Joining occurs as a result of the overlapping between the two individual fragments which pairs with the end of the other, following denaturation and annealing. The fragments that form hybrids get extended by the Taq polymerase, and the complete, mutated product was selectively amplified by the priming of the outer primers, as shown in FIG. 6. The final PCR product was cloned in a plasmid vector and its sequence verified by DNA sequence analysis.

The assembly of the mutated gene was done in multiple steps. In the first step, a cloning vector containing the human Cyl gene was digested with Pst I and Xho I to remove the SUBSTITUTE SHEET (RULE 26) WO 99/43713 PCT/US99/03966 -14non-mutated hinge-CH2-CH3 coding sequences. A Drd I to Xho I fragment encoding part of CH2, all of CH3 and the fused human IL-2 coding sequences was prepared from the Cy1-IL2 vector, described above. A third fragment was prepared from the subcloned PCR product by digestion with Pst I and Drd I. All three fragments were purified by agarose gel electrophoresis and ligated together in a single reaction mixture. The ligation product was used to transform competent E. coli and colonies were selected by growth on plates containing ampicillin.

Correctly assembled recombinant plasmids were identified by restriction analyses of plasmid DNA preparations from isolated transformants and mutated genes were confirmed by DNA sequence analysis. The Hind III to Xho I fragment from the mutated Cyl-IL2 gene was used to reassemble the complete hu-KS antibody-IL2 fusion protein expression vector.

In order to assess the enhancement of the in vivo circulating half-life induced by a mutation of an important amino acid for FcR binding, and to discriminate between FcR and C 1 q binding as mechanisms for rapid clearance, the in vivo plasma concentration of the mutated hu-KSyl-IL2 was compared to the plasma concentration ofhu-KSyl-IL2 at various specified times. As illustrated in FIG. 7, the in vivo clearance rates of the mutated hu-KSyl-IL2 and hu-KSy4-IL2 were significantly lower than the clearance rate of hu-KSyl-IL2. These results suggests that an antibody-based fusion protein with enhanced 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. Further, the results suggests that the mechanisms for rapid clearance involve FcR binding rather than Clq binding.

The skilled artisan will understand, from the teachings of the present invention, that several other mutations to the Cyl or Cy3 genes can be introduced in order to reduce binding to FcR and enhance the in vivo circulating half-life of an antibody-based fusion protein. Moreover, mutations can also be introduced into the Cy4 gene in order to further reduce the binding of Cy4 fusion proteins to FcR. For example, additional possible mutations include mutations in the hinge proximal amino acid residues, mutating Pro 3 3 or by mutating the single N-linked glycosylation site in all IgG Fc regions. The latter is located at Asn 29 7 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 Gin, for example, would have little effect on the protein but would prevent the SUBSTITUTE SHEET (RULE 26) WO 99/43713 PCT/US99/03966 attachment of any carbohydrate side chain. A strategy for mutating this residue might follow the general procedure, just described, for the hinge proximal region. Methods for generating point mutations in cloned DNA sequences are well established in the art and commercial kits are available from several vendors for this purpose.

Example 3 Increasing the circulating half-life of receptor-antibody-based fusion proteins.

Several references have 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-terminal of the Fc portion of an Ig, such as CD4, CTLA-4, and TNF receptors. See, for example, Capon et al., NATURE 337: 525-531 (1989); Linsley et al., J. EXP. MED. 174: 561-569 (1991); Wooley et al., J. IMMUNOL. 151: 6602- 6607 (1993). Increasing the circulating half-life of receptor-antibody-based fusion proteins may permit the ligand-binding protein partner the second non-Ig protein) to more effectively block receptor-ligand interactions at the cell surface; or neutralize the biological activity of a molecule a cytokine) in the fluid phase of the blood, thereby preventing it from reaching its cellular target. In order to assess whether reducing the ability of receptor-antibodybased fusion proteins to bind to IgG receptors will enhance their in vivo circulating half-life, receptor-antibody-based fusion proteins with human Cyl Fc regions are compared to antibodybased fusion proteins with human Cy4 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 early promoter, and the human Cyl or Cy4 gene downstream of their endogenous Hind III site.

The remainder of the plasmid contains bacterial genetic information for propagation in E. coli, as well as a dhfr selectable marker gene. Ligated DNAs are used to transform competent bacteria and recombinant plasmids are identified from restriction analyses from individual bacterial colonies. Two plasmid DNA constructs are obtained: CD4-Cyl and CD4-Cy4.

The expression plasmids are used to transfect mouse myeloma cells by electroporation and transfectants are selected by growth in culture medium containing methotrexate (0.1 tM).

SUBSTITUTE SHEET (RULE 26) -16- Transfectants expressing the fusion proteins are identified by ELISA analyses and are expanded in culture in order to generate fusion protein for purification by binding to and elution from protein A Sepharose. Purified proteins in chromatography elution buffer are diafiltered into PBS and diluted to a final concentration of 100 pg/ml. Balb/c mice are injected with 0.2 ml (20 Pg) of either the CD4-Cyl or CD4-Cy4 fusion protein and the pharnnacokinetics are tested as described in Example 1.3. The CD4-Cy4 fusion protein has a significantly greater half-life than the CD4-Cyl fusion protein.

Equivalents The invention may be embodied in other specific forms without departing from the spirit 10 or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the :invention is thus indicated by the appended claims rather than by the foregoing.description, and all changes which come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group f integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

EDITORIAL NOTE APPLICATION NUMBER 27842/99 The following Sequence Listing pages numbered 1 to 6 are part of the Description.

WO 99/43713 <110> GILLIES, Stephen D LO, Kin-Ming LAN, Yan WESOLOWSKI, John PCTIUS99/03966 Antibody-based Fusion <130> LEX-003PC <140> <141> <150> US 60/075, 887 <151> 1998-02-25 <160> 8 <170> Patentln Ver. <210> 1 <211> 447 <212> PRT <213> Homno sapiens <220> <223> IGG-1 CHAIN C REGION <400> 1 Xaa Xaa Xaa Xaa Xaa Xaa Xa~ 1 5 Xaa Xaa Xaa Xaa Xaa Xaa Xa.

Xaa Xaa Xaa Xaa Xaa Xaa Xa Xaa Xaa Xaa Xaa Xaa Xaa Xa 5 Xaa Xaa Xaa Xaa Xaa Xaa Xa.

70 Xaa Xaa Xaa Xaa Xaa Xaa Xa Xaa Xaa Xaa Xaa Xaa Xaa Xa 100 Xaa Xaa Xaa Xaa Xaa Ala Se 115 Ala Pro Ser Ser Lys Ser Th 130 13 Leu Val Lys Asp Tyr Phe Pr 145 150 Gly Ala Leu Thr Ser Gly Va 165 Ser Gly Les Tyr Ser Leu Se 180 Leu Gly Thr Gin Thr Tyr Il 195 Thr Lys Val Asp Lys Lys ye 210 21 Thr Cys Pro Pro Cys Pro A] 225 230 Phe Leu Phe Pro Pro Lys P~ 245 Pro Glu Val Thr Cys Val V~ ting Half-life of Proteins aXaa Xaa a a a a a a r r 5 0 .1 .5 Xaa Xaa 40 Xaa Xaa Xaa Xaa Thr 120 Ser Glu His Ser Cys 200 Glu Pro Lys Val Xaa 25 Xaa Xaa Xaa Xaa Xaa 105 Lys Gly Pro Thr Val 185 As n Pro Giu Asp Asp Xaa 10 Xaa Xaa Xaa Xaa Xaa 90 Xaa Gly Gly Val Phe 170 Val Val Lys Leu Thr 250 Val aa Xaa Xaa Xaa Xaa Xaa Xaa X~aa Xaa X(aa 75 Xaa Xaa Pro Thr Thr 155 Pro Thr Asn Ser Leu 235 Leu Ser Xaa Xaa Xaa Xaa Xaa Xaa Ser Ala 140 Val Ala Val His Cys 220 Gly Met His Xaa Xaa Xaa Xaa Xaa Xaa Val 125 Ala Ser Val Pro Lys 205 Asp Gly Ile Gli.

Xaa Xaa Xaa Xaa Xaa Xaa 110 Phe Leu Trp Leu Ser 190 Pro Lys Pro Ser 1Asp Xaa X(aa Xa a Xaa Xaa Xaa Pro Gly Asn Gin 175 Ser Ser Thr Ser Arc 255 Pro Xaa Xaa Xaa Xaa Xaa Xaa Leu Cys Ser 160 Ser Ser Asn His Val 240 IThr Glu SUBSTITUTE SHEET (RULE 26) WO 99/43713 WO 9943713PCT/US99/03966 Val1 Thr Val 305 Cys Ser Pro Val1 Gly 385 Asp Trp L ys Lys 290 Leu Lys Lys Ser Lys 370 Gin Gly Gin Phe 275 Pro Thr Val1 Ala Arg 355 Gi y Pro Ser Gin 260 Asn Arg Val1 Ser Lys 340 Asp Phe Glu Phe Gly 420 Trp Tyr Val Glu Leu Asn 325 Gly Glu Tyr Asn Phe 405 Asn Glu His 310 Lys Gin Leu Pro As n 390 Leu Val Gin 295 Gin Al a Pro Thr Ser 375 Tyr Tyr Phe 265 Asp Gly 280 Tyr Asn Asp Trp Leu Pro Arg Glu 345 Lys Asn 360 Asp Ile Lys Thr Ser Lys Ser Cys 425 Val Ser Leu Al a 330 Pro Gin Ala Thr Leu 410 Ser Giu Thr Asn 315 Pro Gin Vai Val Pro 395 Thr Val1 Val1 Tyr 300 Gly Ile Val Ser Glu 380 Pro Val1 Met His 285 Arg Lys Glu Tyr Leu 365 T rp Vai Asp His 270 Asn Val1 Glu Lys Thr 350 Thr Glu Leu Lys Glu 430 Al a Val Tyr Thr 335 Leu Cys Ser Asp Ser 415 Ala Lys Ser Lys 320 Ile Pro Leu Asn Ser 400 Ar g Leu His Asn His 435 Tyr Thr Gin Lys Ser 440 Leu Ser Leu Ser Pro Gly Lys 445 <210> 2 <211> 443 <212> PRT <213> Homo sapiens <220> <223> IGG-2 CHAIN C REGION <400> 2 Xaa Xaa 1 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Aia Pro 130 Leu Val 145 Xaa Xaa Xaa Xaa Xaa Xaa Xa a Xaa 115 Cys Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 Xaa Ser Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ar g Tyr Xaa Xaa Xaa Xaa Xaa 70 Xaa Xaa Ala Ser Phe 150 Xaa Xaa Xaa Xaa 55 Xaa Xaa Xaa Ser Thr 135 Pro Val1 Xaa Xaa Xaa 40 Xaa Xaa Xaa Xaa Thr 120 Ser Glu His Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa 105 Lys Giu Pro Thr Xa a Xaa Xaa Xaa Xaa Xaa 90 Xaa Gly Ser Vai Phe 170 Xaa Xaa Xaa Xaa Xaa 75 Xaa Xaa Pro Thr Thr 155 Pro Xaa Xaa Xaa Xaa 60 Xaa Xaa Xaa Ser Ala 140 Val Ala Xaa Xaa Xaa 45 Xaa Xaa Xaa Xaa Val 125 Al a Ser Val Xaa Xaa 30 Xaa Xaa Xaa Xaa Xaa 110 Phe Leu Trp Leu Xaa Xaa Xaa Xaa Xaa Xaa Pro Gly As n Gin 175 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu C ys Ser 160 Ser Gly Ala Leu Thr Ser Giy 165 SUBSTITUTE SHEET (RULE 26) WO 99/43713 WO 9/4713PCT/US99/03966' Ser Phe Thr Pro 225 Pro Cys Trp Giu Val 305 Asn Gly Giu Tyr Asn 385 Phe Asn Gly Gly Lys 210 Cys Lys Val Tyr Giu 290 His Lys Gin Met Pro 370 Asn Leu Val1 Leu Thr 195 Val1 Pro Pro Val Val1 275 Gin Gin Gly Pro Thr 355 Ser Tyr Tyr Phe Tyr 180 Gin Asp Ala Lys Val 260 Asp Phe Asp Leu Arq 340 Lys Asp Lys Ser Ser 420 Ser Leu Thr Tyr Lys Thr Pro Pro 230 Asp Thr 245 Asp Vai Gly Val Asn Ser Trp Leu 310 Pro Ala 325 Glu Pro Asn Gin Ile Ala Thr Thr 390 Lys Leu 405 Cys Ser Ser Thr Val1 215 Val1 Leu Ser Glu Thr 295 Asn Pro Gin Val Vali 375 Pro Thr Val1 Ser Cys 200 Giu Ala Met His Val 280 Phe Gly Ile Val Se r 360 Giu Pro Val1 Met Val 185 Asn Arg Gly Ile Giu 265 His Arg Lys Giu Tyr 345 Leu T rp Met Asp His 425 Val1 Vai Lys Pro Ser 250 Asp Asn Vai Giu Lys 330 Thr Thr Giu Le u Lys 410 Thr Asp Cys Ser 235 Arg Pro Ala Val1 Tyr 315 Thr Leu Cys Ser Asp 395 Ser Val His Cys 220 Val Thr Giu Lys Ser 300 Lys Ile Pro Leu As n 380 Ser Arg Pro Lys 205 Val Phe Pro Val1 Thr 285 Vai Cys Ser Pro Val 365 Gly Asp T rp Ser 190 Pro Giu Leu Glu Gin 270 Lys Leu Lys Lys Ser 350 Lys Gin Gly Gin Ser Asn Ser Asn Cys Pro Phe Pro 240 Val Thr 255 Phe Asn Pro Arq Thr Vai Val Ser 320 Thr Lys 335 Arg Giu Giy Phe Pro Giu Ser Phe 400 Gin Gly 415 Giu Ala Leu His Asn His Tyr 430 Thr Gin Lys 435 Ser Leu Ser Leu Pro Gly Lys <210> 3 <211> 494 <212> PRT <213> Homo sapiens <220> <223> TGG-3 CHAIN C REGION <400> 3 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5-1 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 40 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 55 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 70 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 75 SUBSTITUTE SHEET (RULE 26) \WOf 00/41711 PCT/II S9/03966 vJ 7/J 4 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 90 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 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 Ala Val Leu Gin Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gin 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 Thr Pro Pro Pro 260 265 270 Cys Pro Arg Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 275 280 285 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 290 295 300 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 305 310 315 320 Gin Phe Lys Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 325 330 335 Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Phe Arg Val Val Ser Val 340 345 350 Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 355 360 365 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 370 375 380 Lys Thr Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro 385 390 395 400 Ser Arg Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val 405 410 415 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly 420 425 430 Gin Pro Glu Asn Asn Tyr Asn Thr Thr 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 Gin Gin Gly Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His 465 470 475 480 Asn Arg Phe Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 485 490 SUBSTITUTE SHEET (RULE 26) WO 99/437 13 PTU9/36 PCTIUS99/03966 <210> 4 <211> 444 <212> PRT <213> Homo sapiens <220> <223> IGG-4 CHAIN C REGION <400> 4 Xaa Xaa 1 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala Pro 130 Leu Vai 145 Gly Ala Ser Gly Leu Gly Thr Lys 210 Ser Cys 225 Pro Pro Thr Cys Asn Trp Arg Giu 290 Val Leu 305 Ser Asn (aa (aa (a a (a a Xaa X(aa Xa a Xaa 115 Cys Lys Leu Leu Thr 195 Val1 Pro Lys Val Tyr 275 Glu His~ L y, Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 Xaa Ser Asp Thr Tyr 180 Lys Asp Ala Pro Val1 260 Val Gir G1r Gi' Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Tyr Ser 165 Ser Thr *Lys Pro Lys 245 *Val -Asp SPhe i Asp y Leu 325 ~aa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa (aa (aa (aa (a a 70 Xaa Xa a Al a Ser Phe 150 Gly Leu Tyr Arq Glu 230 Asp Asp Gi Asr Try 31( Prc Xaa X( Xaa Xaa 55 Xaa Xaa Xaa Ser Thr 135 Pro Val Ser Thr Val 215 Phe Thr Val Val Ser 295 Leu Ser a a ~aa 40 (aa (aa (aa (a a rhr 120 Ser G1u His Se r Cys 200 Glu Leu Leu Ser GlL 28( Thi Asi Se~ Xaa X 25 Xaa Xaa Xaa Xaa Xaa 105 Lys Giu Pro Thr Val1 185 Asn *Ser *Gly Met Gin 265 Val Tyr SGly r Ile :aa aa ~aa a a (a a 90 (a a Gly Ser Val1 Phe 170 Val1 Val Lys Giy Ile 25C Glu Arc Ly~ Gl 33~ Xaa Xaa Xaa Xaa 75 Xaa Xaa Pro Thr Thr 155 Pro Thr Asp Tyr Pro 235 Ser Asp Asn ;Vai s Giu 315 u Lys 0 ~aa (aa (aa <aa Kaa Xaa Ser Al a 140 Val Al a Val His Gly 220 Ser Arc Prc Alz Va 30( Ty~ Th: Xaa Xaa Xaa Xaa Xaa Xaa Val1 125 Al a Ser Val1 Pro Lys 205 Pro Val Thr Giu Lys 285 L Ser 0 Lys Ile Xaa Xaa Xaa Xaa Xaa 110 Phe Leu T rp Leu Ser 190 Pro Pro Phe Pro Val 270 Thr Val Cy3 Se2 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Leu Gly Cys Asn Ser 160 Gin Ser 175 Ser Ser Ser Asn Cys Pro Leu Phe 240 Giu Val 255 Gin Phe *Lys Pro *Leu Thr Lys Val 320 Lys Ala 335 Lys Gly Gin Pro Arg Giu Pro Gin Val Tyr Thr Leu Pro Pro Ser Gin 340 345 350 SUBSTITUTE SHEET (RULE 26) Glu Phe Glu 385 Phe Gly Tyr WO 99/437 Glu Met 355 Tyr Pro 370 Asn Asn Phe Leu Asn Val Thr Gin 435 13 Thr Lys Asn Gin Ser Asp Ile Ala 375 Tyr Lys Thr Thr 390 Tyr Ser Arg Leu 405 Phe Ser Cys Ser 420 Lys Ser Leu Ser -6- Ser Leu Thr Glu Trp Glu Pro Val Leu 395 Val Asp Lys 410 Met His Glu 425 Ser Leu Gly PCT/US99/03966 Cys Leu Val Lys Gly 365 Ser Asn Gly Gin Pro 380 Asp Ser Asp Gly Ser 400 Ser Arg Trp Gin Glu 415 Ala Leu His Asn His 430 Lys <210> <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer 1 <400> catcggtctt ccccctg <210> 6 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer 2 <400> 6 cggtcctgcg acgggaggtg ctgaggaaga gatgg <210> 7 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer 3 <400> 7 tcttcctcag cacctcccgt cgcaggaccg tcagtcttcc tcttc <210> 8 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer 4 <400> 8 gaggcgtggt cttgtag SUBSTITUTE SHEET (RULE 26)

Claims (4)

17- THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1 I. An antibody-based fusion protein with an enhanced circulating half-life, comprising at 2 least a portion of an immunoglobulin (Ig) heavy chain having substantially reduced 3 binding affinity for an Fc receptor, said portion of heavy chain being linked to a second 4 non-Ig protein, said antibody-based fusion protein having a longer circulating half-life in vivo than an unlinked second non-Ig protein. 1 2. The antibody-based fusion protein of claim 1. wherein said portion of heavy chain 2 comprises at least the CH2 domain of an IgG2 or IgG4 constant region. 1 3. The antibody-based fusion protein of claim 1, wherein said portion of heavy chain 2 comprises at least a portion of an IgGI constant region having a mutation or a deletion at 3 one or more amino acid selected from the group consisting of Leu, 34 Leu, 35 Gly 2 3 6 4 Gly 2 3 7 Asn 2 9 7 and Pro 3 3 1 4. The antibody-based fusion protein of claim 1, wherein said portion of heavy chain 2 comprises at least a portion of an IgG3 constant region having a mutation or a deletion at 3 one or more amino acid selected from the group consisting of Leu 28 Leu 2 2 Gly 2 83 4 Gly 2 4, Asn 3 4 4 and Pro 37 1 5. The antibody-based fusion protein of claim 1, wherein said portion of heavy chain further 2 has binding affinity for an immunoglobulin protection receptor. 1 6. The antibody-based fusion protein of claim 1, wherein said portion of heavy chain has 2 substantially reduced binding affinity for a Fc receptor selected from the group consisting 3 of FcyRI, FcyRII and FcyRIII. 1 7. The antibody-based fusion protein of claim 1, wherein said second non-Ig protein is 2 selected from the group consisting of a cytokine. a ligand-binding protein, and a protein 3 toxin. 1 8. The antibody-based fusion protein of claim 7, wherein said cytokine is selected from the 2 group consisting of a tumor necrosis factor, an interleukin, and a lymphokine. 1 9. The antibody-based fusion protein of claim 8. wherein said tumor necrosis factor is tumor 2 necrosis factor alpha. 1 10. The antibody-based fusion protein of claim 8. wherein said interleukin is interleukin-2.

18- 1 11. The antibody-based fusion protein of claim 8. wherein said lymphokine is a lymphotoxin 2 or a colony stimulating factor. 1 12. The antibody-based fusion protein of claim 11, wherein said colony stimulating factor is 2 a granulocyte-macrophage colony stimulating factor. 1 13. The antibody-based fusion protein of claim 7, wherein said ligand-binding protein is 2 selected from the group consisting of CD4, CTLA-4, TNF receptor, and an interleukin 3 receptor. 1 14. A method of increasing the circulating half-life of an antibody-based fusion protein, 2 comprising the step of linking at least a portion of an Ig heavy chain to a second non-Ig 3 protein, said portion of heavy chain having substantially reduced binding affinity for an S 4 Fc receptor, thereby forming an antibody-based fusion protein having a longer circulating 5 half-life in vivo than an unlinked second non-Ig protein. 1 15. The method of claim 14, wherein said portion of heavy chain comprises at least the CH2 S2 domain of an IgG2 or IgG4 constant region. 1 16. A method of increasing the circulating half-life of an antibody-based fusion protein, 2 comprising the steps of: 3 introducing a mutation or a deletion at one or more amino acid of an IgG 1 4 constant region, said amino acid selected from the group consisting of Leu23., S* 5 Leu 2 3 Gly 2 3 6 Gly,,, Asn 97 and Pro 33 1 thereby producing an Ig heavy chain 6 having substantially reduced binding affinity for an Fc receptor; and 7 linking at least a portion of the heavy chain of step to a second non-Ig protein, 8 thereby forming an antibody-based fusion protein having a longer circulating half-life in 9 vivo than an unlinked second non-Ig protein. 1 17. A method of increasing the circulating half-life of an antibody-based fusion protein. 2 comprising the steps of: 3 introducing a mutation or a deletion at one or more amino acid of an IgG3 4 constant region, said amino acid selected from the group consisting of Leus,, Leu 2 Gly 2 83 Gly 2 4, Asn 3 4 and Pro, 37 thereby producing an Ig heavy chain 6 having substantially reduced binding affinity for an Fc receptor; and

19- 7 linking at least a portion of the Ig heavy chain of step to a second non-Ig 8 protein, 9 thereby forming an antibody-based fusion protein having a longer circulating half-life in vivo than an unlinked second non-Ig protein. 1 18. The method of claim 14, 16 or 17, wherein said portion of heavy chain further has 2 binding affinity for an immunoglobulin protection receptor. 1 19. The method of claim 14, 16 or 17, wherein said portion of heavy chain has substantially 2 reduced binding affinity for a Fc receptor selected from the group consisting of FcyRI. 3 FcyRII and FcyRIII. 1 20. The method of claim 14, 16 or 17, wherein said second non-Ig protein is selected from 2 the group consisting of a cytokine, a ligand-binding protein, and a protein toxin. 1 21. The method of claim 20, wherein said cytokine is selected from the group 2 consisting of a tumor necrosis factor, an interleukin, and a lymphokine.

22. The method of claim 21, wherein said tumor necrosis factor is tumor necrosis factor 2 alpha. 1 23. The method of claim 21, wherein said interleukin is interleukin-2. 1 24. The method of claim 21, wherein said lymphokine is a lymphotoxin or a colony S 2 stimulating factor. 1 25. The method of claim 24, wherein said colony stimulating factor is 2 a granulocyte-macrophage colony stimulating factor. S 1 26. The method of claim 20, wherein said ligand-binding protein is selected from 1 26. 2 the group consisting of CD4, CTLA-4, TNF receptor, and an interleukin receptor. Dated this 8 th day of January 2003. Lexigen Pharmaceuticals Corporation By its Patent Attorneys Davies Collison Cave