Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/55—IL-2
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/70514—CD4
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

• 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.
• 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: (1) the generation of monoclonal antibodies by cell fusion to create "hyridomas", or by molecular cloning of antibody heavy (H) and light (L) chains from antibody-producing cells; (2) the conjugation of other molecules to antibodies to deliver them to preferred sites in vivo, e.g., radioisotopes, toxic drugs, protein toxins, and cytokines; (3) the manipulation of antibody effector functions to enhance or diminish biological activity; (4) the joining of other protein such as toxins and cytokines with antibodies at the genetic level to produce antibody-based fusion proteins; and (5) the joining of one or more sets of antibody combining regions at the genetic level to produce bi-specific antibodies.
• 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.
• 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 Fc ⁇ RI, Fc ⁇ RII or Fc ⁇ RIII.
• 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.
• heavy chain isotypes as fusion partners that have reduced binding affinity for Fc receptors on cells.
• both human IgGl and IgG3 have been reported to bind to FcR ⁇ l 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.
• 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.
• 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 - 3 - region of the IgGl or IgG3 heavy chains that reduces the binding affinity of these isotypes for Fc receptors.
• 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.
• an antibody-based fusion protein with enhanced in vivo circulating half-life is obtained by first introducing a mutation, deletion, or insertion in the IgGl constant region at one or more amino acid selected from Leu 234 , Leu 235 , Gly 236 , Gly 237 , Asn 297 , and Pro 331 , and then linking the resulting immunoglobulin, or portion thereof, to a second non-immunoglobulin protein.
• the mutation, deletion, or insertion is introduced in the IgG3 constant region at one or more amino acid selected from Leu 28) , Leu 282 , Gly 283 , Gly 284 , Asn 344 , and Pro 378 , 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.
• the second non-immunoglobulin component of the fusion protein is a cytokine.
• 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.
• cytokines include interleukins such as interleukin-2 (IL-2), hematopoietic factors such as granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor (TNF) such as TNF ⁇ , and lymphokines such as lymphotoxin.
• IL-2 interleukin-2
• GM-CSF granulocyte-macrophage colony stimulating factor
• TNF tumor necrosis factor
• lymphokines such as lymphotoxin.
• the antibody-cytokine fusion protein of the present invention displays cytokine biological activity.
• the second non-immunoglobulin component of the fusion protein is a ligand-binding protein with biological activity.
• ligand-binding proteins may, for example, (1) block receptor-ligand interactions at the cell surface; or (2) neutralize the biological activity of a molecule (e.g., a cytokine) in the fluid phase of the blood, thereby preventing it from reaching its cellular target.
• ligand-binding proteins include CD4, CTLA-4, TNF receptors, or interleukin receptors such as the IL-1 and IL-4 receptors.
• the antibody-receptor fusion protein of the present invention displays the biological activity of the ligand-binding protein.
• the second non-immunoglobulin component of the fusion protein is a protein toxin.
• the antibody-toxin fusion protein of the present invention displays the toxicity activity of the protein toxin.
• 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, e.g., variable and constant regions from different species.
• the heavy chain can include a CHI, CH2, and/or CH3 domains.
• fusion protein also embraced within the term "fusion protein” are constructs having a binding domain comprising framework regions and variable regions (i.e., 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.
• 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 non- immunoglobulin protein can exert a localized biological effect.
• 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 non- immunoglobulin protein, but which lack the antigen-binding specificity and activity of an antibody.
• 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).
• 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.
• - 5 The invention also features DNA constructs encoding the above-described fusion proteins, and cell lines, e.g., myelomas, transfected with these constructs.
• FIG. 1 is a homology alignment of the amino acid sequences of the constant region of C ⁇ l and C ⁇ 3, 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 C ⁇ l, C ⁇ 2, and C ⁇ 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-KS ⁇ l-IL2 and hu-KS ⁇ 4-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-KS ⁇ l-IL2 (closed diamond) and hu-KS ⁇ 4-IL2 (closed triangle) and of intact fusion protein of hu-KS ⁇ l-IL2 (open diamond) and hu-KS ⁇ 4-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-KS ⁇ l-IL2 (0); mutated hu-KS ⁇ l-IL2 (ffl) and hu-KS ⁇ 4-IL2 ( ⁇ ) as a function of time. - 6 - Detailed Description of the Invention
• 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.
• 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.
• IgGl C ⁇ l
• IgG3 C ⁇ 3
• IgG4 C ⁇ 4
• IgG2 C ⁇ 2
• an antibody-based fusion protein with reduced binding affinity for a Fc receptor could be obtained by constructing a fusion protein with a C ⁇ 2 constant region (Fc region) or a C ⁇ 4 Fc region, and avoiding constructs with a C ⁇ l Fc region or a C ⁇ 3 Fc region.
• 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.
• IgG molecules interact with three classes of Fc receptors (FcR), namely Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII.
• FcR Fc receptors
• C ⁇ l and C ⁇ 3 bind FcR ⁇ l with high affinity
• C ⁇ 4 and C ⁇ 2 have reduced or no binding affinity for FcR ⁇ l.
• a comparison of the C ⁇ l and C ⁇ 3 indicates that, with the exception of an extended hinge segment in C ⁇ 3, the amino acid sequence homology between these two isotypes is very high.
• FIG. 1 provides a alignment of the amino acid sequences of C ⁇ l and C ⁇ 3.
• the other two isotypes of human IgG (C ⁇ 2 and C ⁇ 4) have sequence differences which have been associated with FcR binding.
• FIG. 2 provides a alignment of the amino acid sequences of C ⁇ l, C ⁇ 2, and C ⁇ 4.
• the important - 7 - sequences for Fc ⁇ R binding are Leu-Leu-Gly-Gly (residues 234 through 237 in C ⁇ l), located in the CH2 domain adjacent to the hinge.
• 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.
• FcRp protection receptor
• 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.
• 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. - 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 C ⁇ l to C ⁇ 4 IgG constant regions.
• 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.
• an antibody-IL2 fusion protein with a human C ⁇ l constant region was compared to an antibody-IL2 fusion protein with a human C ⁇ 4 Fc region.
• a plasmid vector capable of expressing a humanized antibody-IL2 fusion protein with variable (V) regions specific for a human pancarcinoma antigen (KSA) and the human C ⁇ l heavy chain fused to human IL-2, was modified by removing the C ⁇ l gene fragment and replacing it with the corresponding sequence from the human C ⁇ 4 gene.
• V variable
• KSA pancarcinoma antigen
• These plasmid constructs contain the cytomegalovirus (CMV) early promoter for transcription of the mRNA encoding the light (L) and heavy (H) chain variable (V) 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.
• the VH encoding sequence was inserted upstream of the DNA encoding the C ⁇ heavy chain gene of choice, fused to human IL-2 encoding sequences.
• Such C ⁇ genes contain splice acceptor sites for the first heavy chain exon (CHI), just downstream from a unique Hind III common to all human C ⁇ genes.
• CHI first heavy chain exon
• 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 C ⁇ l and C ⁇ 4 fragments was accomplished by digesting the original C ⁇ l -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 C ⁇ 4 gene was digested with Hind III and Nsi I and the 1.75 kb fragment was purified.
• mouse and human cell lines express one or more Fc receptor.
• the mouse J774 macrophage-like cell line expresses FcR ⁇ l that is capable of binding mouse or human IgG of the appropriate subclasses.
• the human K562 erythroleukemic cell line - 10 - expresses FcR ⁇ ll but not FcR ⁇ l.
• the binding affinities of an antibody, a C ⁇ l-IL2 fusion protein, and a C ⁇ 4-IL2 fusion protein for FcR ⁇ l were compared in the mouse J774 cell line.
• FACS fluorescence-activated cell sorter
• 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 - 11 - for an Fc receptor will be identified as antibody-based fusion proteins with enhanced in vivo half lives.
• fusion proteins containing the C ⁇ l isotype heavy chain i.e., hu-KS ⁇ l-IL2
• fusion proteins containing the C ⁇ 4 isotype heavy chain were compared to fusion proteins containing the C ⁇ 4 isotype heavy chain (i.e., hu-KS ⁇ 4-IL2).
• KS-1/4-IL2 fusion proteins containing either the C ⁇ l or C ⁇ 4 isotype heavy chain were buffer-exchanged by diafiltration into phosphate buffered saline (PBS) and diluted further to a concentration of -100 ⁇ g/ml.
• PBS phosphate buffered saline
• 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°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.
• HRP horse radish peroxidase
• the ⁇ phase half-life of the hu-KS ⁇ 4-IL2 fusion protein was significantly longer than the ⁇ phase half-life of the hu-KS ⁇ l-IL2 fusion protein.
• the increased half-life is best exemplified by the significantly higher concentrations of the hu-KS ⁇ 4-IL2 fusion - 12 - protein (3.3 ⁇ g/ml) compared to the hu-KS ⁇ l-IL2 fusion protein (60 ng/ml) found in mice after 24 hours.
• the hu-KS ⁇ l-IL2 protein had a rapid distribution ( ⁇ ) phase followed by a slower catabolic ( ⁇ ) phase, as reported earlier for the chimeric 14.18-IL2 fusion protein.
• ⁇ rapid distribution
• ⁇ catabolic
• samples were assayed using both (1) an antibody-specific ELISA, and (2) a fusion protein- specific ELISA (i.e., an ELISA that requires that both the antibody and IL-2 components be physically linked).
• a fusion protein-specific ELISA i.e., an ELISA that requires that both the antibody and IL-2 components be physically linked.
• C ⁇ l and C ⁇ 3 have binding affinity for Fc receptors, whereas while C ⁇ 4 has reduced binding affinity and C ⁇ 2 has no binding affinity for Fc receptors.
• the present Example described methods for producing antibody-based fusion proteins using the C ⁇ 4 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 C ⁇ 2 Fc region, instead of the C ⁇ 4 Fc region, in order to enhance the circulating half-life of fusion proteins.
• a Hu-KS-IL2 fusion protein utilizing the human C ⁇ 2 region can be constructed using the same restriction fragment replacement and the above-described methods for C ⁇ 4-IL2 fusion protein, and tested using the methods described herein to demonstrate increased circulating half-life.
• Antibody-based fusion proteins with the C ⁇ 2 Fc region, or any other Fc region having reduced binding affinity or lacking binding affinity for a Fc receptor will have enhanced in vivo - 13 - circulating half-life compared to antibody-based fusion proteins having binding affinity for a Fc receptor.
• Example 2 Mutating the human C ⁇ l or C ⁇ 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 (e.g., Clq fragment), as well as the three classes of FcR.
• the important residues for Clq binding are residues Glu 318 , Lys 320 , and Lys 322 which are located in the CH2 domains of human heavy chains.
• 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 C ⁇ l 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. 6).
• 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 ⁇ l gene as the template.
• the cycle conditions for the primary PCR were 35 cycles of: 94°C for 45 sec, annealing at 48°C for 45 seconds, and primer extension at 72°C for 45 sec.
• each PCR reaction was 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 72°C 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.
• a cloning vector containing the human C ⁇ l gene was digested with Pst I and Xho I to remove the - 14 - non-mutated hinge-CH2-CH3 coding sequences.
• CH2 all of CH3 and the fused human IL-2 coding sequences was prepared from the C ⁇ l-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 C ⁇ l-IL2 gene was used to reassemble the complete hu-KS antibody-IL2 fusion protein expression vector.
• the in vivo plasma concentration of the mutated hu-KS ⁇ l-IL2 was compared to the plasma concentration of hu-KS ⁇ l-IL2 at various specified times. As illustrated in FIG. 7, the in vivo clearance rates of the mutated hu-KS ⁇ l-IL2 and hu- S ⁇ 4-IL2 were significantly lower than the clearance rate of hu-KS ⁇ l-IL2.
• mutations to the C ⁇ l or C ⁇ 3 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.
• mutations can also be introduced into the C ⁇ 4 gene in order to further reduce the binding of C ⁇ 4 fusion proteins to FcR.
• additional possible mutations include mutations in the hinge proximal amino acid residues, mutating Pro 331 , or by mutating the single N-linked glycosylation site in all IgG Fc regions.
• the latter is located at Asn 297 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 would have little effect on the protein but would prevent the - 15 - 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.
• 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
• ligand-binding protein partner i.e., the second non-Ig protein
• the ligand-binding protein partner i.e., the second non-Ig protein
• the biological activity of a molecule e.g., a cytokine
• receptor-antibody-based fusion proteins with human C ⁇ l Fc regions are compared to antibody- based fusion proteins with human C ⁇ 4 Fc regions.
• 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).
• PBMC peripheral blood monocytic cells
• the expression vector contains a unique Xba I cloning site downstream of the CMV early promoter, and the human C ⁇ l or C ⁇ 4 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-C ⁇ l and CD4-C ⁇ 4.
• 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 ⁇ M).
• - 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 ⁇ g/ml.
• Balb/c mice are injected with 0.2 ml (20 ⁇ g) of either the CD4-C ⁇ l or CD4-C ⁇ 4 fusion protein and the pharmacokinetics are tested as described in Example 1.3.
• the CD4-C ⁇ 4 fusion protein has a significantly greater half-life than the CD4-C ⁇ l fusion protein.

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