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WO2005082004A2 - Anticorps rationnellement concus presentant un squelette a domaine echange - Google Patents

Anticorps rationnellement concus presentant un squelette a domaine echange Download PDF

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Publication number
WO2005082004A2
WO2005082004A2 PCT/US2005/005879 US2005005879W WO2005082004A2 WO 2005082004 A2 WO2005082004 A2 WO 2005082004A2 US 2005005879 W US2005005879 W US 2005005879W WO 2005082004 A2 WO2005082004 A2 WO 2005082004A2
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WO
WIPO (PCT)
Prior art keywords
antibody
domain
fragment
exchanged
peptide
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PCT/US2005/005879
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English (en)
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WO2005082004A3 (fr
Inventor
Katherine S. Bowdish
Shana Frederickson
Mark Renshaw
Toshiaki Maruyama
Cecilia Orencia
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Alexion Pharmaceuticals, Inc.
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Publication of WO2005082004A2 publication Critical patent/WO2005082004A2/fr
Publication of WO2005082004A3 publication Critical patent/WO2005082004A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/005—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
    • 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
    • C07K14/524—Thrombopoietin, i.e. C-MPL ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2318/00—Antibody mimetics or scaffolds
    • C07K2318/10—Immunoglobulin or domain(s) thereof as scaffolds for inserted non-Ig peptide sequences, e.g. for vaccination purposes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present disclosure relates to antibodies and biologically active peptides as diagnostic and therapeutic reagents. More specifically, the present disclosure relates to recombinant antibodies that include a biologically active peptide engineered into a domain-exchanged antibody scaffold.
  • Antibodies are produced by B lymphocytes and defend against infection. Antibodies are produced in millions of forms, each with a different amino acid sequence. Antibody molecules are composed of two identical light chains and two identical heavy chains. When digested by the enzyme papain, two identical Fab fragments are produced along with one Fc fragment. When digested with the enzyme pepsin one F(ab') 2 fragment is produced. Light and heavy chains consist of constant and variable regions. Within the variable regions are hypervariable regions (aka complementarity determining regions (CDRs)) which form the antigen binding site. The remaining parts of the variable regions are referred to as framework regions.
  • CDRs complementarity determining regions
  • Important biological functions such as receptor binding, activation and enzymatic activity, are often attributable to discrete regions of larger protein molecules, comprising a limited number of amino acid residues.
  • Peptides displaying binding, activation or enzymatic activity have also been discovered by screening libraries of peptides generated by the random linking of amino acid residues. These peptides may not correspond to a linear arrangement of amino acids in a larger protein molecule exhibiting similar biological activity and are referred to as discontinuous peptide epitopes or mimotopes.
  • Certain peptide mimetics have been described and cloned. See, e.g., U.S. Pat. NO: 6,083,913 (thrombopoietin (TPO) mimetic), U.S. Pat.
  • TPO Thrombopoietin
  • grafting two TPO mimetic peptides into a typical antibody Fab framework creates a c- Mpl receptor agonist which promotes receptor dimerization and activation as described in published international application WO 02/46238 A2.
  • a technical hurdle of this system involves presentation of the grafted peptides in an optimal conformation on the Fab scaffold. Multiple combinations are described in WO 02/46238 A2, including grafting two TPO peptides into different positions chosen from LC-CDR1 , LC-CDR2, HC-CDR2, and HC-CDR3 on the Fab framework.
  • Improving how any grafted peptide is displayed on the surface of the Fab requires optimal spacing between the two peptides grafted into different CDR loops and correct directionality of the grafted peptides relative to one another.
  • the spacing can be adjusted to some degree within the confines of the typical Fab scaffold, however the distances are limited by the dimensions of the Fab fragment.
  • the directionality of the peptides is invariant in a typical antibody Fab framework in that there is no way to shift the orientation of the two peptides relative to each other from a parallel to an antiparallel mode.
  • Recombinant antibodies in accordance with this disclosure include a domain-exchanged antibody or fragment thereof having a peptide of interest inserted into a complementarity determining region (CDR) of the domain-exchanged antibody.
  • CDR complementarity determining region
  • the peptide optionally replaces all or part of the amino acids of a CDR region, or may be added to an existing CDR as defined by either of the two accepted schemes (see, Kabat et al., Sequences of Proteins of Immunologies Interest, 5 th ed (1991), NIH Publication 91-3242 and Chothia et al., J. Mol. Bio., volume 227, pages 776-98 (1992).).
  • One suitable antibody scaffold is the domain-exchanged antibody 2G12.
  • the biologically active peptide when substituted for all or part of a CDR region or added into a CDR region, can have one, two or more additional flanking amino acid residues proximate to the amino and/or the carboxyl termini of the peptide.
  • Methods for screening of libraries of domain-exchanged antibodies having a common peptide substituted for or into a CDR region but different flanking amino acid residues are also described herein.
  • nucleic acid molecules encoding the present recombinant antibody molecules or fragments thereof in which a biologically active peptide is substituted for or into one or more CDR regions of a domain-exchanged antibody are also described herein.
  • nucleic acid molecules can be present in an expression vector, which can be introduced (e.g., transfected) into a recombinant host cell for expression of these molecules.
  • methods of producing an antibody molecule or fragment thereof containing a biologically active peptide comprising culturing a recombinant host cell under conditions such that the nucleic acid contained within the cell is expressed.
  • compositions that contain 1) a domain-exchanged antibody or fragment thereof which has a biologically active peptide substituted for or into one or more CDR regions and 2) a pharmaceutically acceptable carrier.
  • the methods encompass inserting a nucleic acid molecule encoding a biologically active peptide in place of at least a portion of a CDR region of a nucleic acid molecule encoding a domain- exchanged antibody heavy or light chain or adding the molecule to the native CDR sequence; and expressing the nucleic acid molecule encoding the antibody heavy or light chain variable domain.
  • this disclosure provides methods for creation of a library of monoclonal antibodies that can be screened for a desired activity.
  • These methods of making a library include the steps of inserting a nucleic acid molecule encoding a biologically active peptide into, or in place of at least a portion of, one or more CDR regions of a nucleic acid molecule encoding a domain-exchanged antibody heavy or light chain, providing one or more randomizing trinucleotides or sets of randomizing trinucleotides on either side of the inserted nucleic acid molecule, and expressing a library of monoclonal antibodies.
  • the library of monoclonal antibodies thus produced can then be screened for a desired activity.
  • CDRs complementarity determining regions
  • Figure 1 shows a typical structure of an antibody Fab domain.
  • Figures 2A and B show a model of a 2G12 antibody with TPO peptides grafted into HC-CDR2.
  • Figure 3 is a model of a 2G12 dimer with TPO peptides grafted into the HC-CDR3 regions.
  • Figure 4 shows the nucleic acid sequence (SEQ. ID NO: 11) encoding a 2G ' 12
  • FIG. 1 shows the nucleic acid sequence (SEQ. ID NO: 13) encoding a 2G12 Heavy Chain codon optimized variant referred to herein as 2G12-HCwt and the encoded amino acid sequence (SEQ. ID NO: 14).
  • Figure 6 shows the nucleic acid sequence (SEQ. ID NO: 16) encoding a peptide grafted 2G12 heavy chain codon optimized variant (2G12HCvarH2) and the encoded amino acid sequence (SEQ. ID NO: 17).
  • Figure 7 shows the nucleic acid sequence (SEQ.
  • FIG. ID NO: 22 encoding a peptide grafted 2G12 heavy chain codon optimized variant (2G12HCvarH3) and the encoded amino acid sequence (SEQ. ID NO: 23).
  • Figure 8 shows the nucleic acid sequence (SEQ. ID NO: 37) encoding the heavy chain of Fab clone X4b and the encoded amino acid sequence (SEQ. ID NO: 38).
  • Figure 9 shows the nucleic acid sequence (SEQ. ID NO: 39) encoding the light chain of Fab clone X4b and the encoded amino acid sequence (SEQ. ID NO: 40).
  • Recombinant antibodies in accordance with this disclosure include a domain- exchanged antibody or fragment thereof having a peptide of interest inserted into a complementarity determining region (CDR) of the domain-exchanged antibody.
  • the domain-exchanged antibody serves as a scaffold for presentation of the peptide.
  • antibody refers to an entire immunoglobulin molecule or molecules that contain immunologically active portions of whole immunoglobulin molecules and includes Fab, F(ab') 2 , scFv, Fv, heavy chain variable regions and light chain variable regions. Any peptide that exhibits a useful property is suitable for insertion in a domain- exchanged antibody framework.
  • Peptide activities and uses include, but are not limited to, binding a receptor, binding a membrane bound surface molecule, binding a ligand, binding an enzyme or structural protein, activating or inhibiting a receptor, targeted drug delivery, or any enzymatic activity.
  • Those peptides whose utility can be increased from the enhanced stability conferred upon them when presented in the context of a domain-exchanged antibody are usually selected.
  • biological activity includes any activity associated with a molecule having activity in a biological system, including, but not limited to, the stimulatory or inhibitory activity triggered by protein-protein interactions as well as the kinetics surrounding such interactions including the stability of a protein-protein complex.
  • Enhancing or increasing "biological activity” herein is meant to include an increase in overall activity or an increase in any component of overall activity. It should be understood that a peptide may exhibit one biological activity (such as, e.g., simply binding to a target) before insertion into the domain-exchanged antibody framework, and a different or enhanced biological activity (such as, e.g., agonist activity) after insertion into the antibody framework. Many peptides which could benefit from display in the context of an antibody have been identified and are known to those who practice the art, e.g., EPO and TPO mimetic peptides.
  • peptides that bind to receptors which are activated by ligand-induced homo-dimerization including active fragments displaying G-CSF activity, GHR activity and prolactin activity as described in Whitty and Borysenko, Chem Biol., (1999) Apr 6(4):R107-18;
  • suitable peptides include a nerve growth factor mimetic from the CD loop as described in Zaccaro et al., Med. Chem. (2000) 43(19); 3530-40; an IL-2 mimetic as described in Eckenberg, et al., J. Immunol. (2000) 165(8):4312-8; glucagon-like peptide-1 as described in Evans et al., Drugs R.D.
  • tetrapeptide I D-lysine-L-asparaginyl-L-prolyl-L- tyrosine which stimulates mitogen activated B cell proliferation as described in Gagnon et al., Vaccine (2000) 18(18):1886-92.
  • Peptides which exhibit receptor antagonistic activity are also contemplated.
  • N-terminal peptide of vMIP- II as an antagonist of CXCR4 for HIV therapy as described in Luo et al., Biochemistry (2000) 39(44):13545-50; antagonist peptide ligand (AFLARAA (SEQ ID NO: _)) of the thrombin receptor for antithrombotic therapy as described in Pakala et al., Thromb. Res. (2000) 100(1): 89-96; peptide CGRP receptor antagonist CGRP (8-37) for attenuating tolerance to narcotics as described in Powell et al., Br. J. Pharmacol.
  • biologically active peptide which can be incorporated into antibodies or antibody fragments in accordance with this disclosure include proteins secreted by the heart as part of the body's response to congestive heart failure, such as, for example, human brain natriuretic peptide (hBNP) as described in Mukoyama, et al., J. Clin. Invest. 87(4): 1402-12 (1991) and Clemens, et al., J. Pharmacol. Exp. Ther. 287(1): 67-71 (1998).
  • hBNP human brain natriuretic peptide
  • the peptide replacing the amino acids of a domain-exchanged antibody CDR is a TPO mimetic peptide, such as a peptide having at least the sequence IEGPTLRQWLAARA (SEQ. ID. NO: 1).
  • Another biologically active peptide that can replace the amino acid residues of a domain-exchanged antibody CDR is an EPO mimetic peptide, such as a peptide having at least the amino acid sequence DYHCRMGPLTWVCKPLGG (SEQ. ID. NO: 2).
  • the peptide replacing the amino acids of at least one domain- exchanged antibody CDR is a human brain natriuretic peptide (hBNP).
  • hBNP-32 One such peptide is hBNP-32 which has at least the sequence FGRKMDRISSSSGLG (SEQ. ID.
  • the peptide replacing the amino acids of at least one domain-exchanged antibody CDR is a peptide involved in insulin production.
  • Such peptides include exendin -4, GLP-1 (7-36), GLP-2 (1-34), glucagon and PACAP-
  • GLP-2 (SEQ. ID NO: 6)
  • Glucagon (SEQ. ID NO: 7) HSQGTFTSDYSKYLDSRRAQDRVQWLMNT
  • PACAP-38 (SEQ. ID NO: 8) HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK
  • the peptide replacing the amino acids of at least one domain-exchanged antibody CDR is an adipocyte-specific secretory protein.
  • One such peptide is Adiponectin (Acrp30).
  • the globular region of Acrp30 has at least a functional portion of the sequence: FSVGLETYVTIPNMPIRFTKIFYNQQNHYDGSTGKFHCNIPGLYYFAYHITVYM KDVKVSLFKKDKAMLFTYDQYQENNVDQASGSVLLHLEVGDQVWLQVYGEGE RNGLYADNDNDSTFTGFLLYHDTN (SEQ. ID. NO: 9).
  • a peptide that can be incorporated into an antibody scaffold is a peptide that represents a ligand-binding domain of liver/lymph node-specific intercellular adhesion molecule-3 grabbing integrin ("L-SIGN”, also sometimes referred to as “DC-SIGN-R,” “DC-SIGN2” or “CD209L”), such as a peptide having the amino acid sequence: RYWNSGEPNNSGNEDCAEFSGSGWNDNRCDVDN (SEQ ID NO: 10).
  • a domain-exchanged antibody serves as a scaffold sequence into which the peptide of interest is inserted.
  • domain-exchanged antibody means an antibody wherein at least one domain of the heavy chain variable region is positioned away from the light chain variable region.
  • a “typical” antibody is composed of two identical light chains and two identical heavy chains, with the hypentariable regions (aka complementarity determining regions (CDRs)) of the light and heavy chains forming the antigen binding site.
  • CDRs complementarity determining regions
  • the heavy chain variable region is closely associated with the variable region of the light chain.
  • the variable region of the heavy chain is positioned away from the light chain, as shown in Fig. 2A.
  • the domain-exchanged antibody into which the peptide of interest is inserted can be a complete domain-exchanged antibody or any fragment thereof containing a CDR, such as, for example, an Fab or F(ab') 2 fragment or other portion thereof containing at least one hypentariable region of either a heavy chain or a light chain.
  • the structure of a domain-exchanged antibody spontaneously forms a dimer, for example where the heavy chains of two Fab fragments swap with each other forming a heavy chain cross-dimerization motif. Using molecular modeling, the distance and the orientation of the grafted peptides can be approximated.
  • a domain-exchanged antibody allows for more optimal receptor dimerization.
  • the directionality of the peptides relative to each other can be advantageous with a domain-exchanged antibody scaffold compared to prior art antibody scaffolds as the symmetry within the dimer can generate peptides in an anti-parallel orientation relative to each other when they are grafted into the same CDR loop.
  • the peptides are grafted into HC-CDR2, they are presented in a bidentate fashion with a 2-fold symmetrical presentation generated by the cross-dimerization of domain-exchanged antibody Fab fragments.
  • the domain-exchanged antibody can be naturally occurring (as is the case with the 2G12 antibody discussed below) or can be artificially created by making one or more modifications to a "typical" antibody thereby producing a domain-exchanged antibody. As disclosed in Calarese, et al.
  • the modifications that will convert a "typical" antibody into a domain-exchanged antibody can include one or more: 1) modifications that weaken the VH/VL interface contacts (closed interface); 2) modifications that provide an unusual sequence and structure of the elbow region connecting the VH and CR1 domains (hinge loop); and 3) modifications that create a favorable VH/VH' interface (open interface).
  • the modifications can be made as specific point mutations or as more general randomization (e.g. within the hinge loop of the starting, "typical” antibody). Techniques for making such modifications are within the purview of one skilled in the art.
  • a "typical” antibody which has previously had a biologically active peptide inserted into or in place of at least a portion of at least one CDR is converted into a domain-exchanged antibody.
  • a domain-exchanged antibody To determine whether a domain-exchange has been achieved, the existence of a domain-swapped dimer is confirmed. Any known technique for detecting the presence of the dimer can be used.
  • sedimentation equilibrium analytical ultracentrifugation, gel filtration, native gel electrophoresis, sedimentation coefficients and/or negative-stain electron microscopy can be employed to confirm the formation of a domain-swapped dimer consistent with the presence of a domain-exchanged antibody.
  • domain-exchanged antibody is the human monoclonal antibody 2G12 deposited under ECACC Ace. Nr. 93091517 or a variation of a 2G12 antibody.
  • variant of a 2G12 antibody or “2G12 variant” means an antibody having at least 80% identity, and preferably at least 90% identity to the aforementioned 2G12 antibody deposited under ECACC Ace. Nr.
  • 2G12 The structure of 2G12 is disclosed in Calarese, et al., supra.
  • a particularly useful variation of a 2G12 antibody is a 2G12 antibody that has been codon optimized for bacterial expression, such as the 2G12 variant having a light chain and heavy chain having the sequence shown in Figures 4 and 5.
  • the 2G12 antibody reportedly contains point mutations that cause one domain of the heavy chain to swing away from the light chain (see, Figures 2A and 2B). This structure spontaneously forms a 2G12 dimer where the heavy chains of two Fab fragments swap with each other forming a heavy chain cross-dimerization motif. As seen in Fig.
  • TPO peptides grafted into the HC-CDR2 regions would result in peptides in close proximity to one another in an anti-parallel fashion. This orientation more closely approximates the C-terminus to C-terminus peptide fusion of a TPO mimetic that was shown to be 4000 fold more potent than its respective monomer by Cwirla et al. in Science, Vol. 276 pages 1696-1699 (1997).
  • a library of domain-exchanged antibodies can have one or more heavy and/or light chain CDRs replaced with a desired peptide. The resulting library can then be screened to identify antibodies having a desired activity. It should be understood that randomization within the substituted peptide can also be provided to generate an antibody library.
  • grafting of the DNA sequence of the peptide of choice into a domain- exchanged antibody so as to replace the CDR(s) of an antibody with the peptide sequence is carried out using recombinant DNA techniques within the purview of those skilled in the art.
  • methods which can be utilized to graft a desired peptide having biological activity in place of a CDR region include, but are not limited to, PCR overlap, restriction enzyme site cloning, site specific mutagenesis and completely synthetic means.
  • Site specific mutagenesis can be accomplished in several ways. One is based on dut/ung Kunkel mutagenesis (Kunkel, T.A., Proc. Natl.
  • the Muta-Gene in Vitro Mutagenesis kit is available from BioRad based on this methodology (cat. # 170-3581 or 170-3580).
  • Several PCR amplification based mutagenesis approaches are also commercially available such as Stratagene's QuickChange Site-Directed Mutagenesis Kit and the ExSite PCR-based Site-Directed Mutagenesis Kit.
  • Another non-PCR method is available from Promega as the GeneEditor in vitro Site-Directed Mutagenesis System.
  • Completely synthetic means are also well-known and described, e.g., in Deng, et al., Methods Mol. Biol.
  • flanking sequences may be added to the carboxyl and/or amino terminal ends of the biologically active peptide. Flanking sequences can be useful to reduce structural constraints on the grafted peptide to allow it to more easily adopt a conformation necessary for biological activity.
  • a flanking region can be generated by randomizing amino acid positions on each side of the peptide graft in order to determine the best sequence. In this manner, a library having members with multiple varied sequences can be generated. The resulting constructs are then tested for biological activity as described below by, e.g., panning techniques. Recombinant proteins can be generated that have random amino acids at specific positions. This can be accomplished by modifying the encoding DNA.
  • N may be A, C, G, or T (nominally equimolar)
  • K is G or T (nominally equimolar)
  • x is typically 1 to 3 but can be as high as 8 or more, thereby producing antibody libraries with randomized flanking sequences of any desired number of amino acids.
  • the third position may also be G or C, designated "S”.
  • NNK or NNS code for all the amino acids, (ii) code for only one stop codon, and (iii) reduce the range of codon bias from 6:1 to 3:1.
  • Other alternatives include, but are not limited to: (NNN) ⁇ which would provide all possible amino acids and all stops; (NNY) ⁇ eliminates all stops and still covers 14 of 20 amino acids; (NNR) ⁇ covers 14 of 20 amino acids; and (NNS) ⁇ covers all 20 amino acids and only one stop.
  • the third nucleotide position in the codon can be custom engineered using any of the known degenerate mixtures.
  • the group NNK, NNN, NNY, NNR, NNS cover the most commonly used doping strategies and the ones used herein.
  • the collection of engineered domain-exchanged antibodies that are created during this process can be surveyed for those that exhibit desirable properties. Suitable techniques for screening antibodies are within the purview of one skilled in the art, including but not limited to the use of phage displayed antibodies, essentially as has been described in Barbas, CF, III, Kang, A.S, Lerner R.A, and Benkovic, S.J, Assembly of combinatorial antibody libraries on phage surfaces: the gene III site, Proc. Natl. Acad. Sci.
  • error prone PCR can introduce random mutations into nucleic acid sequences (See, e.g., Hawkins et al, J. Mol. Biol, (1992) 226(3): 889-96). Briefly, PCR is run under conditions which compromise the fidelity of replication, thus introducing random mutations in sequences as those skilled in the art would accomplish. After generation of such random mutants, they can be placed into phage display formats, panned and thus evaluated for activity.
  • the presentation of the peptide can be altered by point mutations and randomization strategies, which affect how the peptide is presented. For example, if the peptide is grafted into HC-CDR3, modeling of the structure shows that the histidine residue at position 32 comes into close contact with the peptide ( Figure 3). Randomly mutating this position in conjunction with randomly mutating the flanking positions of the peptide can lead to an optimized presentation of the peptides.
  • libraries having repertoires of multiple constructs resulting from such randomization can be generated and assayed. Selection involves isolating from the library the best candidates that specifically bind to the peptide's target molecule and display biological activity. Methods for whole cell panning have been described previously (Siegel, D.L, Chang, T.Y, Russell, S.L, and Bunya, V.Y. 1997. J. Immunol. Methods 206:73-85 incorporated herein by reference). Other techniques for selection which can be applied include fluorescent activated cell sorting (FACs). Alternative methods for selection using libraries include, but are not limited to, ribosome display and plaque hybridization to a labeled antigen.
  • FACs fluorescent activated cell sorting
  • bioassays for functional screens of agonist antibodies can be carried out. Once binding antibodies are identified by panning or another selection method, the individual clones, each expressing a unique antibody on the phage surface are tested for proliferation, differentiation, activation or survival effects on target cells. In addition, activity of the selected antibodies is examined in bioassays, such as the assays described below:
  • Biological Assays for Screening for TPO-like Activity 1. Colony formation assays - Megakaryocytic colonies from bone marrow (Megacult C Kit from Stem Cell Technologies Inc., Vancouver BC, Canada). 2. Proliferation assays - proliferation of Ba/F3 cells (Cwirla et al. 1997, Science, Vol. 276 pages 1696-1699). The Ba/F3-mpl cell line was established (F. de Sauvage et al., Nature, 369:533 (1994)) by introduction of the cDNA encoding the entire cMpl receptor into the IL-3 dependent murine lymphoblastoid cell line Ba/F3.
  • Biological Assays for Screening for hBNP-like Activity Functional screening of isolated clones is conducted using a cell based assay system for the evaluation of human brain natriuretic peptide (hBNP) activity on natriuretic peptide receptor type A (NPRA)-bearing cells (e.g., neuroblastoma cell line, SK-N-SH).
  • hBNP human brain natriuretic peptide
  • NPRA natriuretic peptide receptor type A
  • Biological Assays for Screening for GLP-1 -like or Exendin-like Activity Functional screening of insulinotropic activity of isolated clones can be conducted by: 1. Rat pancreas perfusion experiments or stimulation of cyclic AMP production evaluated using cultured RINm ⁇ F insulinoma cells using the methods described in Watanabe, et al, J Endocrinol. Jan;140(1):45-52 (1994); Gallwitz et al, J Mol Endocrinol.
  • HASMCs human aortic smooth muscle cells
  • HAECs human aortic endothelial cells
  • the coding regions for both the light and heavy chains, or fragments thereof can be separately cloned out of a bacterial vector and into mammalian vector(s).
  • a single vector system can be used to clone both light and heavy chain cassettes into the same plasmid.
  • dual expression vectors where heavy and light chains are produced by separate plasmids can be used.
  • Mammalian signal sequences need to be either already present in the final vector(s) or appended to the 5' end of the light and heavy chain DNA inserts. This can be accomplished by initial transfer of the chains into a shuttle vector(s) containing the proper mammalian leader sequences.
  • the light chain and heavy chain regions, or fragments thereof are introduced into final vector(s) where the remaining constant regions for lgG1 are provided either with or without introns.
  • primer design for PCR amplifying the light and heavy chain variable regions out of expression vectors may need to include exon splice donor sites in order to get proper splicing and production of the antibodies in mammalian cells.
  • vector expression system single or dual plasmid
  • the production of antibody heavy and light chains can be driven by promoters that work in mammalian cells such as, but not limited to, CMV, SV40, or IgG promoters.
  • the vector(s) will contain a selectable marker for growth in bacteria (such as, but not limited to, ampicillin, chloramphenicol, kanamycin, or zeocin resistance).
  • selectable markers for mammalian cells such as, but not limited to, DHFR, GS, gpt, Neomyocin, or hygromyocin resistance
  • IgG vector(s) may also be present in the IgG vector(s), or could be provided on a separate plasmid by co-transfection.
  • yeast, mammalian, insect, and plants Carlson, J.R. and Weissman, I.L, Mol. Cell.
  • Detectable markers include radioactive and non-radioactive labels and are well-known to those with skill in the art. Common non-radioactive labels include detectable enzymes such as horseradish peroxidase, alkaline phosphatase and fluorescent molecules.
  • Fluorescent molecules absorb light at one wavelength and emit it at another, thus allowing visualization with, e.g., a fluorescent microscope.
  • Spectrophotometers, fluorescence microscopes, fluorescent plate readers and flow sorters are well-known and are often used to detect specific molecules which have been made fluorescent by coupling them covalently to a fluorescent dye.
  • Fluorochromes such as green flurorescent protein, red shifted mutants of green fluorescent protein, amino coumarin acetic acid (AMCA), fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), Texas Red, Cy3.0 and Cy ⁇ .O are examples of useful labels.
  • the molecules can be used in cell isolation strategies such as fluorescence- activated cell sorting (FACS) if fluorescent markers are used.
  • FACS fluorescence- activated cell sorting
  • cells tagged with fluorescent molecules are sorted electronically on a flow cytometer such as a Becton-Dickinson (San Jose, California) FACS IV cytometer or equivalent instrument.
  • the fluorescent molecules are antibodies that recognize specific cell surface antigens.
  • the antibodies are conjugated to fluorescent markers such as fluorescein isothiocyanate (FITC) or Phycoerythrin (PE).
  • FITC fluorescein isothiocyanate
  • PE Phycoerythrin
  • radiolabeled antibodies can be used for diagnostic purposes.
  • Antibodies and fragments thereof disclosed herein are useful for the amplification of a variety of clinically relevant cell types.
  • Treatment can be in vivo or ex vivo.
  • agonist antibodies are useful to treat patients suffering from a deficiency in a cell population caused by disease, disorder or treatment related to for example suppression of hematopoiesis where less than the normal number of cells of a given lineage or lineages are present in a patient.
  • Those who practice the art will be able to identify other diseases and conditions that can be treated with the antibodies containing biologically active peptides disclosed herein.
  • the molecules encompassed by the present disclosure can also be used for ex vivo proliferation and differentiation of cells. This is useful for gene therapy purposes, for example for traditional viral vector approaches, and for autologous bone marrow transplants.
  • certain antibodies in accordance with the present disclosure can be radiolabeled for radioimmunotherapy or conjugated to toxins to deliver such toxins to specific cell types and result in the killing of those cells.
  • a biologically active c-mpl agonist antibody capable of stimulating proliferation, differentiation and maturation of hematopoietic cells may be used in a sterile pharmaceutical preparation or formulation to stimulate megakaryocytopoietic or thrombopoietic activity in patients suffering from thrombocytopenia due to impaired production, sequestration, or increased destruction of platelets.
  • Thrombocytopenia- associated bone marrow hypoplasia may be effectively treated with the disclosed antibodies as well as disorders such as disseminated intravascular coagulation (DIG), immune thrombocytopenia (including HIV-induced ITP and non HIV-induced ITP), chronic idiopathic thrombocytopenia, congenital thrombocytopenia, myelodysplasia, thrombotic thrombocytoenia, chronic liver disease, and lupus.
  • DIG disseminated intravascular coagulation
  • immune thrombocytopenia including HIV-induced ITP and non HIV-induced ITP
  • chronic idiopathic thrombocytopenia congenital thrombocytopenia
  • myelodysplasia myelodysplasia
  • thrombotic thrombocytoenia chronic liver disease, and lupus.
  • c-Mpl agonist antibodies may be used to stimulate platelet production prior to platelet harvesting by apheresis for autologous or allogeneic transfusion, or for the mobilization of Peripheral Blood Progenitor Cells for autologous or allogeneic transplantation.
  • the antibodies disclosed herein containing the TPO mimetic peptide may be used in the same way and for the same indications as thrombopoietin (TPO).
  • TPO Thrombopoietin
  • EPO mimetic antibodies herein stimulate hematopoiesis in a manner similar to naturally occurring EPO.
  • this disclosure contemplates the treatment of congestive heart failure (CHF), either prophylactically or during CHF.
  • CHF congestive heart failure
  • Domain-exchanged antibody antibodies having hBNP incorporated therein in accordance with this disclosure can be administered intravenously into acutely decompensated CHF patients, to exert diuresis, natriuresis, and vasodilatation in a dose dependent manner.
  • the present disclosure contemplates methods of treating diabetes by administering antibodies having biologically active peptides incorporated therein.
  • the domain-exchanged antibody is engineered to contain the glucagon-like peptide (GLP)-1 , a potent insulinotropic hormone.
  • GLP glucagon-like peptide
  • the present disclosure contemplates methods to halt or delay the progressive deterioration of the diabetic state associated with type 2 diabetes by administering a domain-exchanged antibody containing GLP-1 to a patient afflicted with type 2 diabetes.
  • the Adiponectin- domain-exchanged antibody described herein can also be used as a diabetes treatment. Methods of increasing systemic insulin sensitivity by administering adiponectin antibodies are also contemplated.
  • adiponectin antibodies prepared in accordance with the present disclosure decrease hepatic glucose output.
  • reduction of insulin resistance and hyperinsulinemia can be treated by administering the present adiponectin-containing antibodies, thereby reducing obesity and the development of diabetes.
  • Acrp30(C39S) or wild-type Acrp30 are engineered into domain-exchanged antibodies in accordance with the present disclosure.
  • the route of antibody administration is in accord with known methods, e.g., injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, subcutaneous, intraocular, intraarterial, intrathecal, inhalation or intralesional routes, topical or by sustained release systems as noted below.
  • the antibody is preferably administered continuously by infusion or by bolus injection.
  • One may administer the antibodies in a local or systemic manner.
  • the antibodies in accordance with this disclosure may be prepared in a mixture with a pharmaceutically acceptable carrier. Techniques for formulation and administration of the compounds of the instant application may be found in "Remington's Pharmaceutical Sciences,” Mack Publishing Co, Easton, PA, latest edition.
  • This therapeutic composition can be administered intravenously or through the nose or lung, preferably as a liquid or powder aerosol (lyophilized).
  • the composition may also be administered parenterally or subcutaneously as desired.
  • the therapeutic composition should be sterile, pyrogen- free and in a parenterally acceptable solution having due regard for pH, isotonicity, and stability.
  • the antibody formulation When used for in vivo administration, the antibody formulation must be sterile and can be formulated according to conventional pharmaceutical practice. These conditions are known to those skilled in the art.
  • Pharmaceutical compositions suitable for use include compositions wherein one or more recombinant domain-exchanged antibodies are contained in an amount effective to achieve their intended purpose.
  • a therapeutically effective amount means an amount of antibody effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. Therapeutically effective dosages may be determined by using in vitro and in vivo
  • a typical daily dosage might range from about 1 ⁇ g/kg to up to 100 mg/kg or more, depending on the factors mentioned above.
  • the clinician will administer the molecule until a dosage is reached that achieves the desired effect.
  • the progress of this therapy is easily monitored by conventional assays.
  • the present antibodies can also be used in diagnostic assays, e.g., for detecting expression of certain proteins in specific cells, tissues, or serum.
  • diagnostic assay techniques known in the art may be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogeneous phases (Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. (1987) pp. 147-158).
  • the antibodies used in the diagnostic assays can be labeled with a detectable moiety.
  • the detectable moiety should be capable of producing, either directly or indirectly, a detectable signal.
  • the detectable moiety may be a radioisotope, such as 3 H, 14 C, 32 P, 35 S, or 125 l, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase. Any method known in the art for conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter et al.
  • the present antibodies also are useful for the affinity purification of proteins from recombinant cell culture or natural sources.
  • the antibodies are immobilized on a suitable support, such a Sephadex resin or filter paper, using methods well known in the art.
  • the immobilized antibody then is contacted with a sample containing the protein to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the protein, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent that will release the protein from the antibody.
  • Example 1 Library Construction Of TPO Mimetic Sequences Grafted Into The Heavy Chain CDR2 of a Domain-Exchanged Antibody An agonist TPO mimetic-peptide IEGPTLRQWLAARA (SEQ. ID. NO: 1) was grafted into the heavy chain CDR2 (HCDR2) position (Kabat et. al., 1991 , U.S.
  • the final library will have the following in the CDR2-postions: SIXXIEGPTLRQWLAARAXX (SEQ. ID. NO: 15), where the X's represent the randomized amino acids flanking the TPO mimetic. See Fig. 6.
  • the 2G12 Fab is amplified as two fragments. Fragment A is amplified using the forward primer lead VH having the sequence 5' GCT GCC CAA CCA GCC ATG GCC 3' (SEQ. ID.
  • the Expand High Fidelity PCR System from Roche Diagnostics GmbH Indianapolis, IN was used for the extension PCR, following the PCR program: 94° 4', then 10 cycles of 94° 30", 56° 30", and 72° 2', followed by an extension period of 10' at 72° and then a 4° hold.
  • the Lead VH and Ndp-bsiWI primers were then added and the above program run for an additional 30 cycles to amplify the overlap extension PCR product. After generation, the fragment is gel isolated and digested with Xho I and Apa I.
  • HC CDR3 position is prepared. See, Fig. 7.
  • This peptide replaces the entire original CDR3 (Kabat et. al., 1991 , U.S. Department of Health, NIH, Publication number 91- 3242) of a 2G12 heavy chain (namely the heavy chain of 2G12-HCwt (synthesized by Blue Heron Biotechnology, Bothell, WA, USA) shown in Fig. 5).
  • the TPO mimetic peptide is again flanked by two random amino acids on either side to create a library from which to select for optimal presentation of the peptide.
  • the histidine residue (H32) in the 2G12 HC CDR 1 is randomized.
  • This HC CDR1 histidine residue position is randomized in order to select for any amino acid that interacts with and stabilizes the TPO mimetic peptide in the HC CDR3 position.
  • the library is made step-wise using overlap extension PCR.
  • the histidine within the HC CDR1 is randomized using overlap extension PCR.
  • Fragment A is generated using the forward primer lead VH (SEQ. ID NO: 18) and the reverse primer 2G12HtoXR (5' ACG ACG TAC CCA GTT CAT AGT MNN TGC AGA AAT ACG GAA ATT AGA 3') ( SEQ. ID.
  • Fragment B is created using the forward primer 2G12HtoXF (5' ACT ATG AAC TGG GTA CGT CGT GTA CCA 3') (SEQ. ID. NO: 25) which anneals to the region encoding the last three amino acids of the HC CDR1 and the first six amino acids region of framework 2, and the reverse primer Ndp-bsiWI (SEQ. ID NO: 21).
  • the new CDR coding primers contain a complementary 21 base overlap region.
  • the vector specific primers Lead VH and Ndp-bsiWI are used for the overlap PCR protocol (see Example 1). After generation, the fragment is gel isolated and digested with Xho I and Apa I. The insert is again gel purified and ligated into the similarly digested pRL5 -Bsi Wl vector which already contains a 2G12 light chain (See, Fig. 4). The DNA is precipitated and transformed into TOP 10 F' bacteria for the growth for plasmid isolation as a library pool for continuation into step 2.
  • step 2 the TPO mimetic peptide is placed into the HC CDR 3 using overlap extension PCR similar to above, with the exception that fragment A is generated using the reverse primer 2G12H3TPOR (5' CGC CAG CCA CTG GCG CAG GGT CGG GCC TTC AAT MNN MNN ACG TGC GCA GTA GTA AAT AGC 3') (SEQ. ID. NO: 26), which binds to the end of framework 3 of the 2G12 heavy chain. Fragment B is created using the forward primer 2G12H3TPOF (5' CCG ACC CTG CGC CAG TGG CTG GCG GCG CGC GCG NNK NNK TGG GGT CCA GGT ACC GTA GTA 3') (SEQ. ID.
  • This peptide replaces the entire original CDR3 (Kabat et. al., 1991 , U.S. Department of Health, NIH, Publication number 91-3242) of the 2G12 heavy chain.
  • the BNP mimetic peptide (SEQ. ID NO: 3) is flanked by two random amino acids on either side to create a library from which to select for optimal presentation of the peptide.
  • the histidine residue (H32) in the 2G12 HC CDR 1 is randomized in order to select for any amino acid that interacts with and stabilizes the BNP mimetic peptide in the HC CDR3 position.
  • the library is made step-wise using overlap extension PCR as follows.
  • Fragment A is generated using the forward primer lead VH (SEQ. ID NO: 18) and the reverse primer 2g12HtoXR (5' ACG ACG TAC CCA GTT CAT AGT MNN TGC AGA AAT ACG GAA ATT AGA 3') ( SEQ. ID. NO: 24) which anneals to the region encoding the last amino acid of framework 1 , the HC CDR1 and the first four amino acids of framework 2 of the heavy chain.
  • Fragment B is created using the forward primer 2G12HtoXF (5' ACT ATG AAC TGG GTA CGT CGT GTA CCA 3') (SEQ. ID.
  • step 2 the BNP mimetic peptide is placed into the HC CDR 3 using overlap extension PCR similar to above, with the exception that fragment A is generated using the reverse primer 2G12H3BNPR (5' CTG GAG GAG CTG ATC CGG
  • Fragment B is created using the forward primer 2G12H3BNPF (5' AGA TGG ACC GGATCA GCT CCT CCA GTG GCC TGG GCT GCN NKN NKT GGG GTC CAG GTA
  • CCG TAG TA 3' (SEQ. ID. NO: 29), which binds to the beginning of framework 4.
  • the fragments are gel isolated and combined for overlap extension PCR as detailed above.
  • the fragment is again gel isolated and then digested with BsmBI and Apa I.
  • a library is created wherein ANP mimetic peptide is placed in the HC CDR3 position of a 2G12 Antibody. This peptide replaces the entire original CDR3 (Kabat et. al, 1991) of the heavy chain of the 2G12 antibody shown in Figure 5.
  • the ANP mimetic peptide (CFGGRMDRIGAQSGLGC (SEQ. ID NO: 30)) is flanked by two random amino acids on either side to create a library from which to select for optimal presentation of the peptide.
  • the histidine residue (H32) in the 2G12 HC CDR 1 is randomized in order to select for any amino acid that interacts with and stabilizes the ANP mimetic peptide in the HC CDR3 position.
  • the library is made step-wise using overlap extension PCR.
  • the histidine within the HC CDR1 is randomized using overlap extension PCR.
  • Fragment A is generated using the forward primer lead VH (SEQ. ID NO: 18) and the reverse primer 2G12HtoXR (5' ACG ACG TAC CCA GTT CAT AGT MNN TGC AGA AAT ACG GAA ATT AGA 3') (SEQ. ID. NO: 24) which anneals to the region encoding the last amino acid of framework 1 , the HC CDR1 and the first four amino acids of framework 2 of the heavy chain.
  • Fragment B is created using the forward primer 2G12HtoXF (5' ACT ATG AAC TGG GTA CGT CGT GTA CCA 3') (SEQ. ID. NO: 25) which anneals to the region encoding the last three amino acids of the HC CDR1 and the first six amino acids region of framework 2, and the reverse primer Ndp-bsiWI (SEQ. ID NO: 21).
  • the fragments are generated they are gel isolated and combined for overlap extension PCR.
  • the new CDR coding primers contain a complementary 21 base overlap region.
  • the vector specific primers Lead VH and Ndp-bsiWI are used for the overlap PCR protocol (as detailed previously). After generation, the fragment is gel isolated and digested with Xho I and Apa I.
  • step 2 the ANP mimetic peptide is placed into the HC CDR 3 using overlap extension PCR similar to above, with the exception that fragment A is generated using the reverse primer 2G12H3ANPR (5' CTC TGG GCT CCA ATC CTG TCC ATC CTG CCC CCG AAG CAM NNM NNA CGT GCG CAG TAG TAA ATA GC 3') (SEQ. ID. NO: 31), which binds to the end of framework 3 of the 2G12 heavy chain.
  • Fragment B is created using the forward primer 2G12H3ANPF (5' GGA TGG ACA GGA TTG GAG
  • a library is made wherein GLP-1 mimetic peptides are placed in the HC CDR3 position. This peptide replaces the entire original CDR3 (Kabat et. al, 1991) of the 2G12 heavy chain shown in Fig. 5.
  • the GLP-1 mimetic peptide (SEQ. ID NO: 5) is flanked by two random amino acids on either side to create a library from which to select for optimal presentation of the peptide.
  • the histidine residue (H32) in the 2G12 HC CDR 1 is randomized in order to select for any amino acid that interacts with and/or stabilizes the GLP-1 mimetic peptide in the HC CDR3 position.
  • the library is made step-wise using overlap extension PCR.
  • Fragment A is generated using the forward primer lead VH (SEQ. ID NO: 18) and the reverse primer 2G12HtoXR (5' ACG ACG TAC CCA GTT CAT AGT MNN TGC AGA AAT ACG GAA ATT AGA 3') (SEQ. ID. NO: 24) which anneals to the region encoding the last amino acid of framework 1 , the HC CDR1 and the first four amino acids of framework 2 of the heavy chain.
  • Fragment B is created using the forward primer 2G12HtoXF (5' ACT ATG AAC TGG GTA CGT CGT GTA CCA 3') (SEQ. ID.
  • step 2 the GLP-1 mimetic peptide is placed into the HC CDR 3 using overlap extension PCR similar to above, with the exception that fragment A is generated using the reverse primer 2G12H3GLP-1 R (5' TTC CAA ATA AGA ACT TAC ATC ACT GGT AAA GGT CCC TTC AGC ATG MNN MNN ACG TGC GCA GTA GTA AAT AGC 3') (SEQ. ID.
  • GLP-1 int-R (5' CAA TGA ATT CCT TGG CAG CTT GGC CTT CCA AAT AAG AAC TTA CAT CAC TG 3') (SEQ. ID. NO: 34), which allows the extension of the amplified product.
  • 2G12H3GLP-1 R and GLP-1 int-R primers are mixed 1 :10 in the PCR reaction.
  • Fragment B is created using the forward primer 2G12H3GLP-1 F (5' GGC CAA GCT GCC AAG GAA TTC ATT GCT TGG CTG GTG AAA GGC CGA NNK NNK TGG GGT CCA GGT ACC GTA GTA 3') (SEQ. ID. NO: 35), which binds to the beginning of framework 4, and GLP-1 int-F (5' CAG TGA TGT AAG TTC TTA TTT GGA AGG CCA AGC TGC CAA GGA ATT CAT TG 3') (SEQ. ID. NO: 36), which also allows the extension of the amplified product and is complement reverse to the GLP-1 int-R primer.
  • 2G12H3GLP-1 F 5' GGC CAA GCT GCC AAG GAA TTC ATT GCT TGG CTG GTG AAA GGC CGA NNK NNK TGG GGT CCA GGT ACC GTA GTA 3'
  • GLP-1 int-F 5' CAG TGA TGT A
  • 2G12H3GLP-1 F and GLP-1 int-F primers are mixed 1 :10 in the PCR reaction.
  • the fragments are gel isolated and combined for overlap extension PCR as detailed above.
  • the fragment is again gel isolated and then digested with BsmBI and Apa I.
  • This fragment is gel isolated and ligated into the pooled library vector plasmid prep in which the H32 was randomized (as described above), that has been similarly digested with BsmBI and Apa I.
  • the DNA is precipitated and transformed into bacteria for expression of the 2G12 CDR3 library.
  • Example 6 An antibody Fab fragment containing a single peptide within a HC CDR region conferring binding to a single cell surface receptor, is converted to a potential therapeutic reagent capable of binding, dimerizing and activating two receptors by conversion of the normal Fab scaffold to a domain exchanged scaffold structure.
  • This domain exchanged structure contains two light chains, and two heavy chains each of which contains a binding peptide.
  • the domain exchanged molecule contains two binding peptides.
  • Conversion of the scaffold structure is accomplished by changing Ala113 to Pro (Kabat numbering system).
  • the cMpl-R binding Fab clone X4b is modified to achieve a domain exchanged scaffold.
  • Fab clone X4b see Figs.
  • the first step in the conversion process is to convert the alanine in X4b at amino acid position 113 (Kabat numbering system) to a proline. This is accomplished through use of overlap PCR.
  • Fragment A is created using the forward primer Lead VH (5'-GCT GCC CAA CCA GCC ATG GCC-3') (SEQ. ID. NO: 18) and the reverse primer DomainXR (5'-GGA GAC GGT GAC CGT GGT CCC TTG GCC-3') (SEQ. ID. NO: 41).
  • Fragment B is created using the forward primer DomainXF ( ⁇ '-CAA GGG ACC ACG GTC ACC GTC TCC CCG GCC TCC ACC AAG GGC CCA TCG GTC-3') (SEQ. ID. NO: 42) and the reverse primer N-dp (5'-AGC GTA GTC CGG AAC GTC GTA CGG-3' ) (SEQ. ID. NO: 43).
  • the fragments are gel purified and combined for overlap extension PCR.
  • a 24 bp overlap is provided in the DomainXF and R primers.
  • the Lead VH and N-dp primers are used in the overlap extension PCR to generate the new heavy chain.
  • the fragment is gel isolated and then digested with Xho I and Age I. This fragment is again gel isolated and ligated into the pRL5-X4b vector which is similarly digested and gel isolated, thereby replacing the original heavy chain with the modified one. Additionally, further changes are introduced to help stabilize the domain exchanged structure. Changes along the interface between the light chain and the exchanged heavy chain, which stabilize their interaction, are made.
  • the Lys, Ala and Thr at positions 19, 57 and 77, respectively, can be randomized to stabilize the domain exchanged structure.
  • These changes are made using ovelap extension PCR in a manner similar to that described above.
  • bacterial supernatants containing the antibody product from the converted clones are run on isoelectric focusing gels and compared to the non- converted clones. Conversion to a domain exchanged structure doubles the molecular weight of the antibody and alters its mobility on an isoelectric focusing gel. Additionally, size exclusion chromatography is used to detect the increased molecular weight.
  • a medium such as Bio-Gel 100 commercially available from Bio- Rad is used to separate a domain exchanged Fab structure with a molecular weight of approximately 100Kd from a standard Fab structure with a molecular weight of 50 Kd.
  • Commercially available molecular weight standards are used during the chromatography to confirm the molecular weights of the Fab products. Mutations which induce a domain exchanged structure result in the production of the higher molecular weight (100Kd) antibody form. Although some normal monomeric Fab (MW 50Kd) or individual light and heavy chains (MW 25Kd) may still be observed, an increased molecular weight form (100Kd) is detectable.
  • polypeptides or antibodies having a degree of identity greater than 70% to the specific antibodies described herein are within the scope of this disclosure.
  • antibodies having an identity greater than about 80% to the specific antibodies described herein are contemplated.
  • antibodies having an identity greater than about 90% to the specific antibodies described herein are contemplated. Therefore, the above description should not be construed as limiting, but merely as exemplifications of preferred embodiments. Those skilled in the art will envision other modifications within the scope and spirit of this disclosure.

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Abstract

L'invention concerne des anticorps à domaine échangé présentant des régions CDR remplacées ou fusionnées par des peptides biologiquement actifs. Des séquences flanquantes peuvent éventuellement être fixées à une extrémité carboxyterminale ou à une extrémité aminoterminale, ou aux deux extrémités du peptide en association covalente avec les régions de structures adjacentes. Des compositions contenant de tels anticorps à domaine échangé modifiés sont utiles dans des modalités thérapeutiques et diagnostiques.
PCT/US2005/005879 2004-02-24 2005-02-22 Anticorps rationnellement concus presentant un squelette a domaine echange WO2005082004A2 (fr)

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WO2006084327A1 (fr) * 2005-02-09 2006-08-17 Apollo Life Sciences Limited Molécule et molécules chimères de celle-ci
EP2172481A1 (fr) * 2008-10-06 2010-04-07 Novoplant GmbH Formats d'anticorps stables de manière protéolytique
WO2010040545A1 (fr) * 2008-10-06 2010-04-15 Novoplant Gmbh Formats d’anticorps stables à la protéolyse
WO2010033229A3 (fr) * 2008-09-22 2010-11-25 Calmune Corporation Procédés et vecteurs de présentation de molécules et molécules présentées et collections
WO2011035205A3 (fr) * 2009-09-18 2011-11-10 Calmune Corporation Anticorps dirigés contre candida, leurs collectes et procédés d'utilisation
US10441649B2 (en) 2015-02-02 2019-10-15 The University Of Birmingham Targeting moiety peptide epitope complexes having a plurality of T-cell epitopes
EP3553082A1 (fr) 2018-04-12 2019-10-16 Bayer Aktiengesellschaft Anticorps greffés de peptide natriurétique du cerveau
EP3553079A1 (fr) 2018-04-12 2019-10-16 Bayer Aktiengesellschaft Anticorps greffés de peptide natriurétique de type c
EP3553081A1 (fr) 2018-04-12 2019-10-16 Bayer Aktiengesellschaft Anticorps greffés de peptide natriurétique auriculaire
RU2822433C2 (ru) * 2018-04-12 2024-07-05 Байер Акциенгезельшафт Антитела с привитым предсердным натрийуретическим пептидом

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US20030232972A1 (en) * 2000-12-05 2003-12-18 Bowdish Katherine S. Rationally designed antibodies

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030232972A1 (en) * 2000-12-05 2003-12-18 Bowdish Katherine S. Rationally designed antibodies

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WO2006084327A1 (fr) * 2005-02-09 2006-08-17 Apollo Life Sciences Limited Molécule et molécules chimères de celle-ci
WO2010033229A3 (fr) * 2008-09-22 2010-11-25 Calmune Corporation Procédés et vecteurs de présentation de molécules et molécules présentées et collections
EP2172481A1 (fr) * 2008-10-06 2010-04-07 Novoplant GmbH Formats d'anticorps stables de manière protéolytique
WO2010040545A1 (fr) * 2008-10-06 2010-04-15 Novoplant Gmbh Formats d’anticorps stables à la protéolyse
WO2011035205A3 (fr) * 2009-09-18 2011-11-10 Calmune Corporation Anticorps dirigés contre candida, leurs collectes et procédés d'utilisation
US10441649B2 (en) 2015-02-02 2019-10-15 The University Of Birmingham Targeting moiety peptide epitope complexes having a plurality of T-cell epitopes
WO2019197477A1 (fr) 2018-04-12 2019-10-17 Bayer Aktiengesellschaft Anticorps greffés de peptides natriurétiques de type c
JP2021520797A (ja) * 2018-04-12 2021-08-26 バイエル・アクチエンゲゼルシヤフト Ｃ型ナトリウム利尿ペプチド移植抗体
EP3553081A1 (fr) 2018-04-12 2019-10-16 Bayer Aktiengesellschaft Anticorps greffés de peptide natriurétique auriculaire
WO2019197470A1 (fr) 2018-04-12 2019-10-17 Bayer Aktiengesellschaft Anticorps à greffe de peptides natriurétiques atriaux
EP3553082A1 (fr) 2018-04-12 2019-10-16 Bayer Aktiengesellschaft Anticorps greffés de peptide natriurétique du cerveau
WO2019197475A1 (fr) 2018-04-12 2019-10-17 Bayer Aktiengesellschaft Anticorps greffés de peptides natriurétiques cérébraux
CN112236196A (zh) * 2018-04-12 2021-01-15 拜耳公司 心房利钠肽嫁接抗体
CN112236197A (zh) * 2018-04-12 2021-01-15 拜耳公司 脑利钠肽嫁接抗体
CN112292182A (zh) * 2018-04-12 2021-01-29 拜耳公司 C型利钠肽嫁接抗体
EP3553079A1 (fr) 2018-04-12 2019-10-16 Bayer Aktiengesellschaft Anticorps greffés de peptide natriurétique de type c
JP2021520803A (ja) * 2018-04-12 2021-08-26 バイエル・アクチエンゲゼルシヤフト 脳性ナトリウム利尿ペプチド移植抗体
JP2021520801A (ja) * 2018-04-12 2021-08-26 バイエル・アクチエンゲゼルシヤフト 心房性ナトリウム利尿ペプチド移植抗体
RU2822433C2 (ru) * 2018-04-12 2024-07-05 Байер Акциенгезельшафт Антитела с привитым предсердным натрийуретическим пептидом
RU2828786C2 (ru) * 2018-04-12 2024-10-21 Берингер Ингельхайм Интернациональ Гмбх Антитела с привитым натрийуретическим пептидом с-типа
IL277867B1 (en) * 2018-04-12 2025-01-01 Bayer Ag Brain natriuretic peptide conjugated antibodies
IL277865B1 (en) * 2018-04-12 2025-03-01 Bayer Ag C-type natriuretic peptide conjugated antibodies
IL277868B2 (en) * 2018-04-12 2025-04-01 Bayer Ag Atrial natriuretic peptide conjugated antibodies

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AL Designated countries for regional patents

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122 Ep: pct application non-entry in european phase