US20120251541A1 - Dual Variable Region Antibody-Like Binding Proteins Having Cross-Over Binding Region Orientation - Google Patents

Dual Variable Region Antibody-Like Binding Proteins Having Cross-Over Binding Region Orientation Download PDF

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US20120251541A1
US20120251541A1 US13/433,033 US201213433033A US2012251541A1 US 20120251541 A1 US20120251541 A1 US 20120251541A1 US 201213433033 A US201213433033 A US 201213433033A US 2012251541 A1 US2012251541 A1 US 2012251541A1
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antibody
amino acid
length
acid residues
binding protein
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Nicolas Baurin
Christian Beil
Carsten Corvey
Christian Lange
Danxi Li
Vincent Mikol
Anke Steinmetz
Ercole Rao
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Sanofi SA
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Sanofi SA
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Priority to US13/433,033 priority Critical patent/US20120251541A1/en
Assigned to SANOFI reassignment SANOFI ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BEIL, CHRISTIAN, LANGE, CHRISTIAN, MIKOL, VINCENT, BAURIN, NICOLAS, CORVEY, CARSTEN, LI, DANXI, RAO, ERCOLE, STEINMETZ, ANKE
Publication of US20120251541A1 publication Critical patent/US20120251541A1/en
Priority to US13/826,217 priority patent/US20130345404A1/en
Priority to US13/826,126 priority patent/US9181349B2/en
Priority to US13/804,965 priority patent/US9221917B2/en
Priority to US14/947,791 priority patent/US20160200811A1/en
Priority to US15/836,810 priority patent/US20180155450A1/en
Priority to US17/135,529 priority patent/US20210189011A1/en
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Definitions

  • the invention relates to antibody-like binding proteins comprising four polypeptide chains that form four antigen binding sites, wherein each pair of polypeptides forming the antibody-like binding protein possesses dual variable domains having a cross-over orientation.
  • the invention also relates to methods for making such antigen-like binding proteins.
  • IgG antibodies are bivalent and monospecific. Bispecific antibodies having binding specificities for two different antigens can be produced using recombinant technologies and are projected to have broad clinical applications. It is well known that complete IgG antibody molecules are Y-shaped molecules comprising four polypeptide chains: two heavy chains and two light chains. Each light chain consists of two domains, the N-terminal domain being known as the variable or V L domain (or region) and the C-terminal domain being known as the constant (or C L ) domain (constant kappa (CK) or constant lambda (C ⁇ ) domain). Each heavy chain consists of four or five domains, depending on the class of the antibody.
  • the N-terminal domain is known as the variable (or V H ) domain (or region), which is followed by the first constant (or C H1 ) domain, the hinge region, and then the second and third constant (or C H2 and C H3 ) domains.
  • V H variable
  • C H1 constant
  • C H2 and C H3 constant domains
  • the V L and V H domains associate together to form an antigen binding site.
  • the C L and C H1 domains associate together to keep one heavy chain associated with one light chain.
  • the two heavy-light chain heterodimers associate together by interaction of the C H2 and C H3 domains and interaction between the hinge regions on the two heavy chains.
  • Fab and Fab2 fragments of the whole antibody can exhibit antigen binding activity.
  • Antibody fragments can also be produced recombinantly. Fv fragments, consisting only of the variable domains of the heavy and light chains associated with each other may be obtained. These Fv fragments are monovalent for antigen binding. Smaller fragments such as individual variable domains (domain antibodies or dABs; Ward et al., 1989 , Nature 341(6242): 544-46), and individual complementarity determining regions or CDRs (Williams et al., 1989 , Proc. Natl. Acad. Sci. U.S.A. 86(14): 5537-41) have also been shown to retain the binding characteristics of the parent antibody, although most naturally occurring antibodies generally need both a V H and V L to retain full binding potency.
  • Single chain variable fragment (scFv) constructs comprise a V H and a V L domain of an antibody contained in a single polypeptide chain wherein the domains are separated by a flexible linker of sufficient length (more than 12 amino acid residues), that forces intramolecular interaction, allowing self-assembly of the two domains into a functional epitope binding site (Bird et al., 1988 , Science 242(4877): 423-26).
  • These small proteins (MW ⁇ 25,000 Da) generally retain specificity and affinity for their antigen in a single polypeptide and can provide a convenient building block for larger, antigen-specific molecules.
  • An advantage of using antibody fragments rather than whole antibodies in diagnosis and therapy lies in their smaller size. They are likely to be less immunogenic than whole antibodies and more able to penetrate tissues. A disadvantage associated with the use of such fragments is that they have only one antigen binding site, leading to reduced avidity. In addition, due to their small size, they are cleared very fast from the serum, and hence display a short half-life.
  • BsAbs bispecific antibodies
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • Bispecific antibodies were originally made by fusing two hybridomas, each capable of producing a different immunoglobulin (Milstein et al., 1983 , Nature 305(5934): 537-40), but the complexity of species (up to ten different species) produced in cell culture made purification difficult and expensive (George et al., 1997, THE ANTIBODIES 4: 99-141 (Capra et al., ed., Harwood Academic Publishers)). Using this format, a mouse IgG2a and a rat IgG2b antibody were produced together in the same cell (e.g., either as a quadroma fusion of two hybridomas, or in engineered CHO cells).
  • the two parental antibodies Because the light chains of each antibody associate preferentially with the heavy chains of their cognate species, three major species of antibody are assembled: the two parental antibodies, and a heterodimer of the two antibodies comprising one heavy/light chain pair of each, associating via their Fc portions.
  • the desired heterodimer can be purified from this mixture because its binding properties to Protein A are different from those of the parental antibodies: rat IgG2b does not bind to Protein A, whereas the mouse IgG2a does.
  • the mouse-rat heterodimer binds to Protein A but elutes at a higher pH than the mouse IgG2a homodimer, and this makes selective purification of the bispecific heterodimer possible (Lindhofer et al., 1995 , J. Immunol. 155(1): 219-25).
  • the resulting bispecific heterodimer is fully non-human, hence highly immunogenic, which could have deleterious side effects (e.g., “HAMA” or “HARA” reactions), and/or neutralize the therapeutic.
  • HAMA or “HARA” reactions
  • BsAb formats with minimal molecular mass, termed sc-BsAbs or Ta-scFvs (Mack et al., 1995 , Proc. Natl. Acad. Sci. U.S.A. 92(15): 7021-25; Mallender et al., 1994 , J. Biol. Chem. 269(1): 199-206).
  • BsAbs have been constructed by genetically fusing two scFvs to a dimerization functionality such as a leucine zipper (Kostelny et al., 1992 , J. Immunol. 148(5): 1547-53; de Kruif et al., 1996 , J. Biol. Chem. 271(13): 7630-34).
  • Diabodies are small bivalent and bispecific antibody fragments.
  • the fragments comprise a V H connected to a V L on the same polypeptide chain, by using a linker that is too short (less than 12 amino acid residues) to allow pairing between the two domains on the same chain.
  • the domains are forced to pair intermolecularly with the complementary domains of another chain and create two antigen-binding sites.
  • dimeric antibody fragments, or “diabodies” are bivalent and bispecific (Holliger et al., 1993 , Proc. Natl. Acad. Sci. U.S.A. 90(14): 6444-48).
  • Diabodies are similar in size to a Fab fragment.
  • sc-BsAb and diabody-based constructs display interesting clinical potential, it was shown that such non-covalently associated molecules are not sufficiently stable under physiological conditions.
  • the overall stability of a scFv fragment depends on the intrinsic stability of the V L and V H domains as well as on the stability of the domain interface. Insufficient stability of the V H -V L interface of scFv fragments has often been suggested as a main cause of irreversible scFv inactivation, since transient opening of the interface, which would be allowed by the peptide linker, exposes hydrophobic patches that favor aggregation and therefore instability and poor production yield (Worn et al., 2001 , J. Mol. Biol. 305(5): 989-1010).
  • variable domains of two different antibodies are expressed in a tandem orientation on two separate chains (one heavy chain and one light chain), wherein one polypeptide chain has two times a V H in series separated by a peptide linker (V H1 -linker-V H2 ) and the other polypeptide chain consists of complementary V L domains connected in series by a peptide linker (V L1 -linker-V L2 ).
  • variable domains of two different antibodies are expressed in a tandem orientation on two separate polypeptide chains (one heavy chain and one light chain), wherein one polypeptide chain has two times a V H in series separated by a peptide linker (V H1 -linker-V H2 ) and the other polypeptide chain consists of complementary V L domains connected in series by a peptide linker in the opposite orientation (V L2 -linker-V L1 ).
  • V H1 -linker-V H2 peptide linker
  • V L2 -linker-V L1 complementary V L domains connected in series by a peptide linker in the opposite orientation
  • PPC Polyvalent protein complexes
  • PPCs comprise two polypeptide chains generally arranged laterally to one another.
  • Each polypeptide chain typically comprises three or four “v-regions,” which comprise amino acid sequences capable of forming an antigen binding site when matched with a corresponding v-region on the opposite polypeptide chain. Up to about six “v-regions” can be used on each polypeptide chain.
  • the v-regions of each polypeptide chain are connected linearly to one another and may be connected by interspersed linking regions. When arranged in the form of the PPC, the v-regions on each polypeptide chain form individual antigen binding sites.
  • the complex may contain one or several binding specificities.
  • U.S. Patent Application Publication No. US 2010/331527 A1 describes a bispecific antibody based on heterodimerization of the C H3 domain, introducing in one heavy chain the mutations H95R and Y96F within the C H3 domain. These amino acid substitutions originate from the C H3 domain of the IgG3 subtype and will heterodimerize with an IgG1 backbone. A common light chain prone to pair with every heavy chain is a prerequisite for all formats based on heterodimerization though the C H3 domain. A total of three types of antibodies are therefore produced: 50% having a pure IgG1 backbone, one-third having a pure H95R and Y96F mutated backbone, and one-third having two different heavy chains (bispecific).
  • the desired heterodimer can be purified from this mixture because its binding properties to Protein A are different from those of the parental antibodies: IgG3-derived C H3 domains do not bind to Protein A, whereas the IgG1 does. Consequently, the heterodimer binds to Protein A, but elutes at a higher pH than the pure IgG1 homodimer, and this makes selective purification of the bispecific heterodimer possible.
  • U.S. Pat. No. 7,612,181 describes a Dual-Variable-Domain IgG (DVD-IgG) bispecific antibody that is based on the Dual-Fv format described in U.S. Pat. No. 5,989,830. A similar bispecific format was also described in U.S. Patent Application Publication No. US 2010/0226923 A1.
  • the addition of constant domains to respective chains of the Dual-Fv (C H1 -Fc to the heavy chain and kappa or lambda constant domain to the light chain) led to functional bispecific antibodies without any need for additional modifications (i.e., obvious addition of constant domains to enhance stability).
  • Some of the antibodies expressed in the DVD-Ig/TBTI format show a position effect on the second (or innermost) antigen binding position (Fv2).
  • Fv2 the second (or innermost) antigen binding position
  • this antibody domain displays a reduced affinity to its antigen (i.e., loss of on-rate in comparison to the parental antibody).
  • the linker between V L1 and V L2 protrudes into the CDR region of Fv2, making the Fv2 somewhat inaccessible for larger antigens.
  • the second configuration of a bispecific antibody fragment described in U.S. Pat. No. 5,989,830 is the cross-over double head (CODH), having the following orientation of variable domains expressed on two chains:
  • V L1 -linker-V L2 for the light chain
  • V H2 -linker-V H1 for the heavy chain
  • the '830 patent discloses that a bispecific cross-over double-head antibody fragment (construct GOSA.E) retains higher binding activity than a Dual-Fv (see page 20, lines 20-50 of the '830 patent), and further discloses that this format is less impacted by the linkers that are used between the variable domains (see page 20-21 of the '830 patent).
  • the invention provides an antibody-like binding protein comprising four polypeptide chains that form four antigen binding sites, wherein two polypeptide chains have a structure represented by the formula:
  • the invention also provides an antibody-like binding protein comprising two polypeptide chains that form two antigen binding sites, wherein a first polypeptide chain has a structure represented by the formula:
  • V L1 is a first immunoglobulin light chain variable domain
  • V L2 is a second immunoglobulin light chain variable domain
  • V H1 is a first immunoglobulin heavy chain variable domain
  • V H2 is a second immunoglobulin heavy chain variable domain
  • C L is an immunoglobulin light chain constant domain
  • C H1 is the immunoglobulin C H1 heavy chain constant domain
  • L 1 , L 2 , L 3 , and L 4 are amino acid linkers
  • first and second polypeptides form a cross-over light chain-heavy chain pair.
  • the invention further provides a method of making an antibody-like binding protein comprising four polypeptide chains that form four antigen binding sites, comprising identifying a first antibody variable domain that binds a first target antigen and a second antibody variable domain that binds a second target antigen, each containing a V L , and a V H ; assigning either the light chain or the heavy chain as template chain; assigning the V L of the first antibody variable domain or the second antibody variable domain as V L1 ; assigning a V L2 , a V H1 , and a V H2 according to formulas [I] and [II] below:
  • V L1 is a first immunoglobulin light chain variable domain
  • V L2 is a second immunoglobulin light chain variable domain
  • V H1 is a first immunoglobulin heavy chain variable domain
  • V H2 is a second immunoglobulin heavy chain variable domain
  • C L is an immunoglobulin light chain constant domain
  • C H1 is the immunoglobulin C H1 heavy chain constant domain
  • Fc is the immunoglobulin hinge region and C H2 , C H3 immunoglobulin heavy chain constant domains;
  • L 1 , L 2 , L 3 , and L 4 are amino acid linkers
  • polypeptides of formula I and the polypeptides of formula II form a cross-over light chain-heavy chain pair.
  • the invention further provides a method of making an antibody-like binding protein comprising four polypeptide chains that form four antigen binding sites, comprising identifying a first antibody variable domain that binds a first target antigen and a second antibody variable domain that binds a second target antigen, each containing a V L , and a V H ; assigning either the light chain or the heavy chain as template chain; assigning the V L of the first antibody variable domain or the second antibody variable domain as V L1 ; assigning a V L2 , a V H1 , and a V H2 according to formulas [I] and [II] below:
  • V L1 is a first immunoglobulin light chain variable domain
  • V L2 is a second immunoglobulin light chain variable domain
  • V H1 is a first immunoglobulin heavy chain variable domain
  • V H2 is a second immunoglobulin heavy chain variable domain
  • C L is an immunoglobulin light chain constant domain
  • C H1 is the immunoglobulin C H1 heavy chain constant domain
  • L 1 , L 2 , L 3 , and L 4 are amino acid linkers
  • polypeptides of formula I and the polypeptides of formula II form a cross-over light chain-heavy chain pair.
  • FIG. 1 Schematic representation of the antigen binding domains Fv1 and Fv2 within the dual V region configuration and arrangement of their respective peptide linkers L L and L H in the TBTI format.
  • FIG. 2 Schematic diagram (2D) of the antigen binding domains Fv1 (anti-IL4) and Fv2 (anti-IL13) within the cross-over dual variable (CODV) configuration and the arrangement of their respective peptide linkers.
  • FIG. 3 Schematic representation of the Fv anti-IL4 and Fab anti-IL13 showing one possible spatial arrangement obtained by protein-protein docking of Fv of anti-IL4 and the Fv of anti-IL13.
  • FIG. 4 Assessment of tetravalent and bispecific binding ability of the CODV protein in a BIACORE assay by injecting the two antigens sequentially or simultaneously over a DVD-Ig protein-coated chip.
  • the maximal signal observed by sequential injection can be obtained by co-injection of both antigens, demonstrating saturation of all binding sites.
  • FIG. 5 Schematic diagram (2D) of the antigen binding domains within the CODV configuration and arrangement of their respective peptide linker L L (L 1 and L 2 ) and L H (L 3 and L 4 ).
  • L L L 1 and L 2
  • L H L 3 and L 4
  • panel A the light chain is kept in a “linear or template” alignment, whereas the heavy chain is in the “cross-over” configuration.
  • panel B the heavy chain is kept in a “linear or template” alignment and the light chain is in the “cross-over” configuration.
  • FIG. 6 Schematic representation of CODV-Ig design based on whether the light chain or heavy chain is used as “template.”
  • FIG. 7 Comparison of TBTI/DVD-Ig or CODV-Ig molecules incorporating anti-IL4 and anti-IL13 sequences.
  • FIG. 8 Comparison of CODV-Fab and B-Fab formats in a cytotoxic assay using NALM-6 cells.
  • the invention provides antibody-like binding proteins comprising four polypeptide chains that form four antigen binding sites, wherein each pair of polypeptides forming an antibody-like binding protein possesses dual variable domains having a cross-over orientation.
  • the invention also provides methods for making such antigen-like binding proteins.
  • antibody-like binding protein constructs were prepared in which constant domains were attached to the CODH configuration to form antibody-like binding proteins comprising four polypeptide chains that form four antigen binding sites, wherein each pair of polypeptides forming an antibody-like binding protein possesses dual variable domains having a cross-over orientation (i.e., CODH-Ig).
  • CODH-Ig molecules are expected to possess significantly improved stability as compared with CODH molecules (as DVD-Ig/TBTI possessed improved stability over Dual-Fv molecules).
  • CODH-Ig molecule was prepared using the anti-IL4 and anti-IL13 antibody sequences described in U.S. Patent Application Publication No. US 2010/0226923 A1.
  • the CODH-Ig molecule differed from the CODH molecule of US 2010/0226923 with respect to the lengths of amino acid linkers separating the variable domains on the respective polypeptide chains.
  • the CODH-Ig molecules were expressed in cells following transient transfection and were then purified by Protein A chromatography.
  • This protocol was based on protein-protein docking of homology and experimental models of the FvIL4 and FvIL13 regions, respectively, inclusion of the Fc1 domain the model, and construction of appropriate linkers between the FvIL4 and FvIL13 regions and between the Fv and constant Fc1 regions.
  • Standard recombinant DNA methodologies are used to construct the polynucleotides that encode the polypeptides which form the antibody-like binding proteins of the invention, incorporate these polynucleotides into recombinant expression vectors, and introduce such vectors into host cells. See e.g., Sambrook et al., 2001, M OLECULAR C LONING : A L ABORATORY M ANUAL (Cold Spring Harbor Laboratory Press, 3rd ed.). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein.
  • polynucleotide refers to single-stranded or double-stranded nucleic acid polymers of at least 10 nucleotides in length.
  • the nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide.
  • Such modifications include base modifications such as bromuridine, ribose modifications such as arabinoside and 2′,3′-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate.
  • base modifications such as bromuridine, ribose modifications such as arabinoside and 2′,3′-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate.
  • polynucleotide specifically includes single-stranded and double-stranded forms of DNA.
  • isolated polynucleotide is a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the isolated polynucleotide: (1) is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, (2) is linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.
  • isolated polypeptide is one that: (1) is free of at least some other polypeptides with which it would normally be found, (2) is essentially free of other polypeptides from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is not associated (by covalent or noncovalent interaction) with portions of a polypeptide with which the “isolated polypeptide” is associated in nature, (6) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature.
  • Such an isolated polypeptide can be encoded by genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination thereof.
  • the isolated polypeptide is substantially free from polypeptides or other contaminants that are found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).
  • human antibody as used herein includes antibodies having variable and constant regions substantially corresponding to human germline immunoglobulin sequences.
  • human antibodies are produced in non-human mammals, including, but not limited to, rodents, such as mice and rats, and lagomorphs, such as rabbits.
  • rodents such as mice and rats
  • lagomorphs such as rabbits.
  • human antibodies are produced in hybridoma cells.
  • human antibodies are produced recombinantly.
  • Naturally occurring antibodies typically comprise a tetramer.
  • Each such tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one full-length “light” chain (typically having a molecular weight of about 25 kDa) and one full-length “heavy” chain (typically having a molecular weight of about 50-70 kDa).
  • the terms “heavy chain” and “light chain” as used herein refer to any immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen.
  • the amino-terminal portion of each light and heavy chain typically includes a variable domain of about 100 to 110 or more amino acids that typically is responsible for antigen recognition.
  • the carboxy-terminal portion of each chain typically defines a constant domain responsible for effector function.
  • a full-length heavy chain immunoglobulin polypeptide includes a variable domain (V H ) and three constant domains (C M , C H2 , and C H3 ), wherein the V H domain is at the amino-terminus of the polypeptide and the C H3 domain is at the carboxyl-terminus, and a full-length light chain immunoglobulin polypeptide includes a variable domain (V L ) and a constant domain (C L ), wherein the V L domain is at the amino-terminus of the polypeptide and the C L domain is at the carboxyl-terminus.
  • Human light chains are typically classified as kappa and lambda light chains, and human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4.
  • IgM has subclasses including, but not limited to, IgM1 and IgM2.
  • IgA is similarly subdivided into subclasses including, but not limited to, IgA1 and IgA2.
  • variable and constant domains typically are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids.
  • the variable regions of each light/heavy chain pair typically form an antigen binding site.
  • the variable domains of naturally occurring antibodies typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs.
  • both light and heavy chain variable domains typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • native Fc refers to a molecule comprising the sequence of a non-antigen-binding fragment resulting from digestion of an antibody or produced by other means, whether in monomeric or multimeric form, and can contain the hinge region.
  • the original immunoglobulin source of the native Fc is preferably of human origin and can be any of the immunoglobulins, although IgG1 and IgG2 are preferred.
  • Native Fc molecules are made up of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association.
  • the number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2).
  • class e.g., IgG, IgA, and IgE
  • subclass e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2
  • One example of a native Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG.
  • native Fc as used herein is generic to the monomeric, dimeric, and multimeric forms.
  • Fc variant refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn (neonatal Fc receptor). Exemplary Fc variants, and their interaction with the salvage receptor, are known in the art. Thus, the term “Fc variant” can comprise a molecule or sequence that is humanized from a non-human native Fc. Furthermore, a native Fc comprises regions that can be removed because they provide structural features or biological activity that are not required for the antibody-like binding proteins of the invention.
  • Fc variant comprises a molecule or sequence that lacks one or more native Fc sites or residues, or in which one or more Fc sites or residues has be modified, that affect or are involved in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).
  • ADCC antibody-dependent cellular cytotoxicity
  • Fc domain encompasses native Fc and Fc variants and sequences as defined above. As with Fc variants and native Fc molecules, the term “Fc domain” includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by other means.
  • antibody-like binding protein refers to a non-naturally occurring (or recombinant) molecule that specifically binds to at least one target antigen, and which comprises four polypeptide chains that form four antigen binding sites, wherein two polypeptide chains have a structure represented by the formula:
  • antibody-like binding protein as used herein also refers to a non-naturally occurring (or recombinant) molecule that specifically binds to at least one target antigen, and which comprises two polypeptide chains that form two antigen binding sites, wherein a first polypeptide chain has a structure represented by the formula:
  • V L1 is a first immunoglobulin light chain variable domain
  • V L2 is a second immunoglobulin light chain variable domain
  • V H1 is a first immunoglobulin heavy chain variable domain
  • V H2 is a second immunoglobulin heavy chain variable domain
  • C L is an immunoglobulin light chain constant domain
  • C H1 is the immunoglobulin C H1 heavy chain constant domain
  • L 1 , L 2 , L 3 , and L 4 are amino acid linkers
  • a “recombinant” molecule is one that has been prepared, expressed, created, or isolated by recombinant means.
  • One embodiment of the invention provides antibody-like binding proteins having biological and immunological specificity to between one and four target antigens. Another embodiment of the invention provides nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form such antibody-like binding proteins. Another embodiment of the invention provides expression vectors comprising nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form such antibody-like binding proteins. Yet another embodiment of the invention provides host cells that express such antibody-like binding proteins (i.e., comprising nucleic acid molecules or vectors encoding polypeptide chains that form such antibody-like binding proteins).
  • “swapability” as used herein refers to the interchangeability of variable domains within the CODV format and with retention of folding and ultimate binding affinity. “Full swapability” refers to the ability to swap the order of both V H1 and V H2 domains, and therefore the order of V L1 and V L2 domains, in a CODV-Ig (i.e., to reverse the order) or CODV-Fab while maintaining full functionality of the antibody-like binding protein as evidenced by the retention of binding affinity. Furthermore, it should be noted that the designations V H and V L within a particular CODV-Ig or CODV-Fab refer only to the domain's location on a particular protein chain in the final format.
  • V H1 and V H2 could be derived from V L1 and V L2 domains in parent antibodies and placed into the V H1 and V H2 positions in the antibody-like binding protein.
  • V L1 and V L2 could be derived from V H1 and V H2 domains in parent antibodies and placed in the V H1 and V H2 positions in the antibody-like binding protein.
  • the V H and V L designations refer to the present location and not the original location in a parent antibody. V H and V L domains are therefore “swappable.”
  • an “isolated” antibody-like binding protein is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody-like binding protein, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • the antibody-like binding protein will be purified: (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated antibody-like binding proteins include the antibody-like binding protein in situ within recombinant cells since at least one component of the antibody-like binding protein's natural environment will not be present.
  • substantially pure or substantially purified refer to a compound or species that is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition).
  • a substantially purified fraction is a composition wherein the species comprises at least about 50% (on a molar basis) of all macromolecular species present.
  • a substantially pure composition will comprise more than about 80%, 85%, 90%, 95%, or 99% of all macromolar species present in the composition.
  • the species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.
  • antigen or “target antigen” as used herein refers to a molecule or a portion of a molecule that is capable of being bound by an antibody-like binding protein, and additionally is capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen.
  • a target antigen may have one or more epitopes. With respect to each target antigen recognized by an antibody-like binding protein, the antibody-like binding protein is capable of competing with an intact antibody that recognizes the target antigen.
  • a “bivalent” antibody-like binding protein, other than a “multispecific” or “multifunctional” antibody-like binding protein, is understood to comprise antigen binding sites having identical antigenic specificity.
  • a bispecific or bifunctional antibody typically is an artificial hybrid antibody having two different heavy chain/light chain pairs and two different binding sites or epitopes.
  • Bispecific antibodies may be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of F(ab′) fragments.
  • a F(ab) fragment typically includes one light chain and the V H and C H1 domains of one heavy chain, wherein the V H -C H1 heavy chain portion of the F(ab) fragment cannot form a disulfide bond with another heavy chain polypeptide.
  • a F(ab) fragment can also include one light chain containing two variable domains separated by an amino acid linker and one heavy chain containing two variable domains separated by an amino acid linker and a C H1 domain.
  • a F(ab′) fragment typically includes one light chain and a portion of one heavy chain that contains more of the constant region (between the C H1 and C H2 domains), such that an interchain disulfide bond can be formed between two heavy chains to form a F(ab) 2 molecule.
  • biological property in reference to an antibody-like binding protein of the invention are used interchangeably herein and include, but are not limited to, epitope affinity and specificity, ability to antagonize the activity of the antigen target (or targeted polypeptide), the in vivo stability of the antibody-like binding protein, and the immunogenic properties of the antibody-like binding protein.
  • Other identifiable biological properties or characteristics of an antibody-like binding protein include, for example, cross-reactivity, (i.e., with non-human homologs of the antigen target, or with other antigen targets or tissues, generally), and ability to preserve high expression levels of protein in mammalian cells.
  • immunoglobulin fragment refers to a polypeptide fragment that contains at least the CDRs of the immunoglobulin heavy or light chains from which the polypeptide fragment was derived.
  • An immunologically functional immunoglobulin fragment is capable of binding to a target antigen.
  • a “neutralizing” antibody-like binding protein as used herein refers to a molecule that is able to block or substantially reduce an effector function of a target antigen to which it binds.
  • substantially reduce means at least about 60%, preferably at least about 70%, more preferably at least about 75%, even more preferably at least about 80%, still more preferably at least about 85%, most preferably at least about 90% reduction of an effector function of the target antigen.
  • epitope includes any determinant, preferably a polypeptide determinant, capable of specifically binding to an immunoglobulin or T-cell receptor.
  • epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics and/or specific charge characteristics.
  • An epitope is a region of an antigen that is bound by an antibody or antibody-like binding protein.
  • an antibody-like binding protein is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.
  • an antibody-like binding protein is said to specifically bind an antigen when the equilibrium dissociation constant is ⁇ 10 ⁇ 8 M, more preferably when the equilibrium dissociation constant is ⁇ 10 ⁇ 9 M, and most preferably when the dissociation constant is ⁇ 10 ⁇ 10 M.
  • the dissociation constant (K D ) of an antibody-like binding protein can be determined, for example, by surface plasmon resonance.
  • surface plasmon resonance analysis measures real-time binding interactions between ligand (a target antigen on a biosensor matrix) and analyte (an antibody-like binding protein in solution) by surface plasmon resonance (SPR) using the BIAcore system (Pharmacia Biosensor; Piscataway, N.J.).
  • SPR surface plasmon resonance
  • Surface plasmon analysis can also be performed by immobilizing the analyte (antibody-like binding protein on a biosensor matrix) and presenting the ligand (target antigen).
  • K D refers to the dissociation constant of the interaction between a particular antibody-like binding protein and a target antigen.
  • the term “specifically binds” as used herein refers to the ability of an antibody-like protein or an antigen-binding fragment thereof to bind to an antigen containing an epitope with an Kd of at least about 1 ⁇ 10 ⁇ 6 M, 1 ⁇ 10 ⁇ 7 M, 1 ⁇ 10 ⁇ 8 M, 1 ⁇ 10 ⁇ 9 M, 1 ⁇ 10 ⁇ 10 M, 1 ⁇ 10 ⁇ 11 M, 1 ⁇ 10 ⁇ 12 M, or more, and/or to bind to an epitope with an affinity that is at least two-fold greater than its affinity for a nonspecific antigen.
  • linker refers to one or more amino acid residues inserted between immunoglobulin domains to provide sufficient mobility for the domains of the light and heavy chains to fold into cross over dual variable region immunoglobulins.
  • a linker is inserted at the transition between variable domains or between variable and constant domains, respectively, at the sequence level.
  • the transition between domains can be identified because the approximate size of the immunoglobulin domains are well understood.
  • the precise location of a domain transition can be determined by locating peptide stretches that do not form secondary structural elements such as beta-sheets or alpha-helices as demonstrated by experimental data or as can be assumed by techniques of modeling or secondary structure prediction.
  • the linkers described herein are referred to as L 1 , which is located on the light chain between the N-terminal V L1 and V L2 domains; L 2 , which is also on the light chain is located between the V L2 and C-terminal C L domains.
  • the heavy chain linkers are known as L 3 , which is located between the N-terminal V H2 and V H1 domains; and L 4 , which is located between the V H1 and C H1 -Fc domains.
  • the linkers L 1 , L 2 , L 3 , and L 4 are independent, but they may in some cases have the same sequence and/or length.
  • vector refers to any molecule (e.g., nucleic acid, plasmid, or virus) that is used to transfer coding information to a host cell.
  • the term “vector” includes a nucleic acid molecule that is capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double-stranded DNA molecule into which additional DNA segments may be inserted.
  • viral vector Another type of vector, wherein additional DNA segments may be inserted into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • vectors can be integrated into the genome of a host cell upon introduction into the host cell and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”).
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and “vector” may be used interchangeably herein, as a plasmid is the most commonly used form of vector.
  • the invention is intended to include other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.
  • flanking sequence operably linked is used herein to refer to an arrangement of flanking sequences wherein the flanking sequences so described are configured or assembled so as to perform their usual function.
  • a flanking sequence operably linked to a coding sequence may be capable of effecting the replication, transcription, and/or translation of the coding sequence.
  • a coding sequence is operably linked to a promoter when the promoter is capable of directing transcription of that coding sequence.
  • a flanking sequence need not be contiguous with the coding sequence, so long as it functions correctly.
  • intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.
  • recombinant host cell refers to a cell into which a recombinant expression vector has been introduced.
  • a recombinant host cell or host cell is intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but such cells are still included within the scope of the term “host cell” as used herein.
  • host cell expression systems can be used to express the antibody-like binding proteins of the invention, including bacterial, yeast, baculoviral, and mammalian expression systems (as well as phage display expression systems).
  • a suitable bacterial expression vector is pUC19.
  • a host cell is transformed or transfected with one or more recombinant expression vectors carrying DNA fragments encoding the polypeptide chains of the antibody-like binding protein such that the polypeptide chains are expressed in the host cell and, preferably, secreted into the medium in which the host cells are cultured, from which medium the antibody-like binding protein can be recovered.
  • transformation refers to a change in a cell's genetic characteristics, and a cell has been transformed when it has been modified to contain a new DNA.
  • a cell is transformed where it is genetically modified from its native state.
  • the transforming DNA may recombine with that of the cell by physically integrating into a chromosome of the cell, or may be maintained transiently as an episomal element without being replicated, or may replicate independently as a plasmid.
  • a cell is considered to have been stably transformed when the DNA is replicated with the division of the cell.
  • transfection refers to the uptake of foreign or exogenous DNA by a cell, and a cell has been “transfected” when the exogenous DNA has been introduced inside the cell membrane.
  • transfection techniques are well known in the art. Such techniques can be used to introduce one or more exogenous DNA molecules into suitable host cells.
  • non-naturally occurring refers to the fact that the object can be found in nature and has not been manipulated by man.
  • a polynucleotide or polypeptide that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man is naturally-occurring.
  • non-naturally occurring refers to an object that is not found in nature or that has been structurally modified or synthesized by man.
  • the twenty conventional amino acids and their abbreviations follow conventional usage.
  • Stereoisomers e.g., D-amino acids
  • unnatural amino acids such as ⁇ -, ⁇ -disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for the polypeptide chains of the antibody-like binding proteins of the invention.
  • Examples of unconventional amino acids include: 4-hydroxyproline, ⁇ -carboxyglutamate, ⁇ -N,N,N-trimethyllysine, ⁇ -N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, ⁇ -N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline).
  • the left-hand direction is the amino terminal direction and the right-hand direction is the carboxyl-terminal direction, in accordance with standard usage and convention.
  • Naturally occurring residues may be divided into classes based on common side chain properties:
  • Conservative amino acid substitutions may involve exchange of a member of one of these classes with another member of the same class.
  • Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid residues. Non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class.
  • a skilled artisan will be able to determine suitable variants of the polypeptide chains of the antibody-like binding proteins of the invention using well-known techniques. For example, one skilled in the art may identify suitable areas of a polypeptide chain that may be changed without destroying activity by targeting regions not believed to be important for activity. Alternatively, one skilled in the art can identify residues and portions of the molecules that are conserved among similar polypeptides. In addition, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.
  • patient includes human and animal subjects.
  • disorder is any condition that would benefit from treatment using the antibody-like binding proteins of the invention.
  • disorder and condition are used interchangeably herein and include chronic and acute disorders or diseases, including those pathological conditions that predispose a patient to the disorder in question.
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures.
  • Those in need of treatment include those having the disorder as well as those prone to have the disorder or those in which the disorder is to be prevented.
  • composition or “therapeutic composition” as used herein refer to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.
  • pharmaceutically acceptable carrier or “physiologically acceptable carrier” as used herein refers to one or more formulation materials suitable for accomplishing or enhancing the delivery of an antibody-like binding protein.
  • a therapeutically effective amount when used in reference to a pharmaceutical composition comprising one or more antibody-like binding proteins refer to an amount or dosage sufficient to produce a desired therapeutic result. More specifically, a therapeutically effective amount is an amount of an antibody-like binding protein sufficient to inhibit, for some period of time, one or more of the clinically defined pathological processes associated with the condition being treated. The effective amount may vary depending on the specific antibody-like binding protein that is being used, and also depends on a variety of factors and conditions related to the patient being treated and the severity of the disorder.
  • the antibody-like binding protein is to be administered in vivo, factors such as the age, weight, and health of the patient as well as dose response curves and toxicity data obtained in preclinical animal work would be among those factors considered.
  • factors such as the age, weight, and health of the patient as well as dose response curves and toxicity data obtained in preclinical animal work would be among those factors considered.
  • the determination of an effective amount or therapeutically effective amount of a given pharmaceutical composition is well within the ability of those skilled in the art.
  • One embodiment of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of an antibody-like binding protein.
  • the antibody-like binding proteins comprise four polypeptide chains that form four antigen binding sites, wherein two polypeptide chains have a structure represented by the formula:
  • the antibody-like binding proteins comprise two polypeptide chains that form two antigen binding sites, wherein a first polypeptide chain has a structure represented by the formula:
  • V L1 is a first immunoglobulin light chain variable domain
  • V L2 is a second immunoglobulin light chain variable domain
  • V H1 is a first immunoglobulin heavy chain variable domain
  • V H2 is a second immunoglobulin heavy chain variable domain
  • C L is an immunoglobulin light chain constant domain
  • C H1 is the immunoglobulin C H1 heavy chain constant domain
  • L 1 , L 2 , L 3 , and L 4 are amino acid linkers
  • first and second polypeptides form a cross-over light chain-heavy chain pair.
  • the antibody-like binding proteins of the invention may be prepared using domains or sequences obtained or derived from any human or non-human antibody, including, for example, human, murine, or humanized antibodies.
  • the length of L 3 is at least twice the length of L 1 .
  • the length of L 4 is at least twice the length of L 2 .
  • the length of L 1 is at least twice the length of L 3 .
  • the length of L 2 is at least twice the length of L 4 .
  • L 1 is 3 to 12 amino acid residues in length
  • L 2 is 3 to 14 amino acid residues in length
  • L 3 is 1 to 8 amino acid residues in length
  • L 4 is 1 to 3 amino acid residues in length.
  • L 1 is 5 to 10 amino acid residues in length
  • L 2 is 5 to 8 amino acid residues in length
  • L 3 is 1 to 5 amino acid residues in length
  • L 4 is 1 to 2 amino acid residues in length.
  • L 1 is 7 amino acid residues in length
  • L 2 is 5 amino acid residues in length
  • L 3 is 1 amino acid residues in length
  • L 4 is 2 amino acid residues in length.
  • L 1 is 1 to 3 amino acid residues in length
  • L 2 is 1 to 4 amino acid residues in length
  • L 3 is 2 to 15 amino acid residues in length
  • L 4 is 2 to 15 amino acid residues in length
  • L 1 is 1 to 2 amino acid residues in length
  • L 2 is 1 to 2 amino acid residues in length
  • L 3 is 4 to 12 amino acid residues in length
  • L 4 is 2 to 12 amino acid residues in length.
  • L 1 is 1 amino acid residue in length
  • L 2 is 2 amino acid residues in length
  • L 3 is 7 amino acid residues in length
  • L 4 is 5 amino acid residues in length.
  • L 1 , L 3 , or L 4 may be equal to zero. However, in antibody-like binding proteins wherein L 1 , L 3 , or L 4 is equal to zero, the corresponding transition linker between the variable region and constant region or between the dual variable domains on the other chain cannot be zero.
  • L 1 is equal to zero and L 3 is 2 or more amino acid residues
  • L 3 is equal to zero and L 1 is equal to 1 or more amino acid residues
  • L 4 is equal to 0 and L 2 is 3 or more amino acid residues.
  • At least one of the linkers selected from the group consisting of L 1 , L 2 , L 3 , and L 4 contains at least one cysteine residue.
  • linkers include a single glycine (Gly) residue; a diglycine peptide (Gly-Gly); a tripeptide (Gly-Gly-Gly); a peptide with four glycine residues (Gly-Gly-Gly-Gly; SEQ ID NO: 25); a peptide with five glycine residues (Gly-Gly-Gly-Gly-Gly; SEQ ID NO: 26); a peptide with six glycine residues (Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO: 27); a peptide with seven glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO: 28); a peptide with eight glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO: 29).
  • amino acid residues may be used such as the peptide Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 30) and the peptide Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 31).
  • linkers include a single Ser, and Val residue; the dipeptide Arg-Thr, Gln-Pro, Ser-Ser, Thr-Lys, and Ser-Leu; Thr-Lys-Gly-Pro-Ser (SEQ ID NO: 52), Thr-Val-Ala-Ala-Pro (SEQ ID NO: 53), Gln-Pro-Lys-Ala-Ala (SEQ ID NO: 54), Gln-Arg-Ile-Glu-Gly (SEQ ID NO: 55); Ala-Ser-Thr-Lys-Gly-Pro-Ser (SEQ ID NO: 48), Arg-Thr-Val-Ala-Ala-Pro-Ser (SEQ ID NO: 49), Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 50), and His-Ile-Asp-Ser-Pro-Asn-Lys (SEQ ID NO: 51).
  • linkers comprising randomly selected amino acids selected from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate, asparagine, glutamine, glycine, and proline have been shown to be suitable in the antibody-like binding proteins of the invention (see Example 12).
  • the identity and sequence of amino acid residues in the linker may vary depending on the type of secondary structural element necessary to achieve in the linker. For example, glycine, serine, and alanine are best for linkers having maximum flexibility. Some combination of glycine, proline, threonine, and serine are useful if a more rigid and extended linker is necessary. Any amino acid residue may be considered as a linker in combination with other amino acid residues to construct larger peptide linkers as necessary depending on the desired properties.
  • the antibody-like binding protein is capable of specifically binding one or more antigen targets.
  • the antibody-like binding protein is capable of specifically binding at least one antigen target selected from the group consisting of B7.1, B7.2, BAFF, BlyS, C3, C5, CCL11 (eotaxin), CCL15 (MIP-1d), CCL17 (TARC), CCL19 (MIP-3b), CCL2 (MCP-1), CCL20 (MIP-3a), CCL21 (MIP-2), SLC, CCL24 (MPIF-2/eotaxin-2), CCL25 (TECK), CCL26 (eotaxin-3), CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CD3, CD19, CD20, CD24, CD40, CD40L, CD80, CD86, CDH1 (E-cadherin), Chitinase, CSF1
  • the antibody-like binding protein is bispecific and capable of binding two different antigen targets or epitopes.
  • the antibody-like binding protein is bispecific and each light chain-heavy chain pair is capable of binding two different antigen targets or epitopes.
  • the antibody-like binding protein is capable of binding two different antigen targets that are selected from the group consisting of IL4 and IL13, IGF1R and HER2, IGF1R and EGFR, EGFR and HER2, BK and IL13, PDL-1 and CTLA-4, CTLA4 and MHC class II, IL-12 and IL-18, IL-1 ⁇ and IL-1 ⁇ , TNF ⁇ and IL12/23, TNF ⁇ and IL-12p40, TNF ⁇ and IL-113, TNF ⁇ and IL-23, and IL17 and IL23.
  • the antibody-like binding protein is capable of binding the antigen targets IL4 and IL13.
  • the antibody-like binding protein specifically binds IL4 with an on-rate of 2.97 E+07 and an off-rate of 3.30 E ⁇ 04 and specifically binds IL13 with an on-rate of 1.39 E+06 and an off-rate of 1.63 E ⁇ 04. In other embodiments of the invention, the antibody-like binding protein specifically binds IL4 with an on-rate of 3.16 E+07 and an off-rate of 2.89 E ⁇ 04 and specifically binds IL13 with an on-rate of 1.20 E+06 and an off-rate of 1.12 E ⁇ 04.
  • an antibody-like binding protein comprising four polypeptide chains that form four antigen binding sites is prepared by identifying a first antibody variable domain that binds a first target antigen and a second antibody variable domain that binds a second target antigen, each containing a V L , and a V H ; assigning either the light chain or the heavy chain as template chain; assigning the V L of the first antibody variable domain or the second antibody variable domain as V L1 ; assigning a V L2 , a V H1 , and a V H2 according to formulas [I] and [II] below:
  • V L1 is a first immunoglobulin light chain variable domain
  • V L2 is a second immunoglobulin light chain variable domain
  • V H1 is a first immunoglobulin heavy chain variable domain
  • V H2 is a second immunoglobulin heavy chain variable domain
  • C L is an immunoglobulin light chain constant domain
  • C H1 is the immunoglobulin C H1 heavy chain constant domain
  • Fc is the immunoglobulin hinge region and C H2 , C H3 immunoglobulin heavy chain constant domains;
  • L 1 , L 2 , L 3 , and L 4 are amino acid linkers
  • polypeptides of formula I and the polypeptides of formula II form a cross-over light chain-heavy chain pair.
  • an antibody-like binding protein comprising four polypeptide chains that form four antigen binding sites is prepared by identifying a first antibody variable domain that binds a first target antigen and a second antibody variable domain that binds a second target antigen, each containing a V L , and a V H ; assigning either the light chain or the heavy chain as template chain; assigning the V L of the first antibody variable domain or the second antibody variable domain as V L1 ; assigning a V L2 , a V H1 , and a V H2 according to formulas [I] and [II] below:
  • V L1 is a first immunoglobulin light chain variable domain
  • V L2 is a second immunoglobulin light chain variable domain
  • V H1 is a first immunoglobulin heavy chain variable domain
  • V H2 is a second immunoglobulin heavy chain variable domain
  • C L is an immunoglobulin light chain constant domain
  • C H1 is the immunoglobulin C H1 heavy chain constant domain
  • L 1 , L 2 , L 3 , and L 4 are an amino acid linkers; and wherein the polypeptides of formula I and the polypeptides of formula II form a cross-over light chain-heavy chain pair.
  • an antibody-like binding protein in which the first antibody variable domain and the second antibody variable domain are the same is prepared.
  • One embodiment of the invention provides a method for making an antibody-like binding protein, comprising expressing in a cell one or more nucleic acid molecules encoding polypeptides having structures represented by the formulas [I] and [II] below:
  • polypeptides of formula I and the polypeptides of formula II form a cross-over light chain-heavy chain pair.
  • Another embodiment of the invention provides a method for making an antibody-like binding protein, comprising expressing in a cell one or more nucleic acid molecules encoding polypeptides having structures represented by the formulas [I] and [II] below:
  • V L1 is a first immunoglobulin light chain variable domain
  • V L2 is a second immunoglobulin light chain variable domain
  • V H1 is a first immunoglobulin heavy chain variable domain
  • V H2 is a second immunoglobulin heavy chain variable domain
  • C L is an immunoglobulin light chain constant domain
  • C H1 is the immunoglobulin C H1 heavy chain constant domain
  • L 1 , L 2 , L 3 , and L 4 are amino acid linkers
  • polypeptide of formula I and the polypeptide of formula II form a cross-over light chain-heavy chain pair.
  • the antibody-like binding proteins of the invention can be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays for the detection and quantitation of one or more target antigens.
  • the antibody-like binding proteins will bind the one or more target antigens with an affinity that is appropriate for the assay method being employed.
  • antibody-like binding proteins can be labeled with a detectable moiety.
  • the detectable moiety can be any one that is capable of producing, either directly or indirectly, a detectable signal.
  • the detectable moiety can be a radioisotope, such as 3 H, 14 C, 32 P, 35 S, 125 I, 99 Tc, 111 In, or 67 Ga; a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase, ⁇ -galactosidase, or horseradish peroxidase.
  • the antibody-like binding proteins of the invention are also useful for in vivo imaging.
  • An antibody-like binding protein labeled with a detectable moiety can be administered to an animal, preferably into the bloodstream, and the presence and location of the labeled antibody in the host assayed.
  • the antibody-like binding protein can be labeled with any moiety that is detectable in an animal, whether by nuclear magnetic resonance, radiology, or other detection means known in the art.
  • the invention also relates to a kit comprising an antibody-like binding protein and other reagents useful for detecting target antigen levels in biological samples.
  • reagents can include a detectable label, blocking serum, positive and negative control samples, and detection reagents.
  • Therapeutic or pharmaceutical compositions comprising antibody-like binding proteins are within the scope of the invention.
  • Such therapeutic or pharmaceutical compositions can comprise a therapeutically effective amount of an antibody-like binding protein, or antibody-like binding protein-drug conjugate, in admixture with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration.
  • Acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed.
  • the pharmaceutical composition can contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition.
  • Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite, or sodium hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as serum albumin, gelatin, or immunoglobulins), coloring, flavoring and diluting agents, emuls
  • compositions will be determined by a skilled artisan depending upon, for example, the intended route of administration, delivery format, and desired dosage. Such compositions can influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the antibody-like binding protein.
  • the primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature.
  • a suitable vehicle or carrier for injection can be water, physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration.
  • Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.
  • Other exemplary pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can further include sorbitol or a suitable substitute.
  • antibody-like binding protein compositions can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents in the form of a lyophilized cake or an aqueous solution. Further, the antibody-like binding protein can be formulated as a lyophilizate using appropriate excipients such as sucrose.
  • compositions of the invention can be selected for parenteral delivery.
  • the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally.
  • the preparation of such pharmaceutically acceptable compositions is within the skill of the art.
  • the formulation components are present in concentrations that are acceptable to the site of administration.
  • buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.
  • the therapeutic compositions for use in this invention can be in the form of a pyrogen-free, parenterally acceptable, aqueous solution comprising the desired antibody-like binding protein in a pharmaceutically acceptable vehicle.
  • a particularly suitable vehicle for parenteral injection is sterile distilled water in which an antibody-like binding protein is formulated as a sterile, isotonic solution, properly preserved.
  • Yet another preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, that provides for the controlled or sustained release of the product which can then be delivered via a depot injection.
  • Hyaluronic acid can also be used, and this can have the effect of promoting sustained duration in the circulation.
  • Other suitable means for the introduction of the desired molecule include implantable drug delivery devices.
  • a pharmaceutical composition can be formulated for inhalation.
  • an antibody-like binding protein can be formulated as a dry powder for inhalation.
  • Antibody-like binding protein inhalation solutions can also be formulated with a propellant for aerosol delivery.
  • solutions can be nebulized.
  • antibody-like binding proteins that are administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules.
  • a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized.
  • Additional agents can be included to facilitate absorption of the antibody-like binding protein. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be employed.
  • Another pharmaceutical composition can involve an effective quantity of antibody-like binding proteins in a mixture with non-toxic excipients that are suitable for the manufacture of tablets.
  • excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.
  • compositions of the invention will be evident to those skilled in the art, including formulations involving antibody-like binding proteins in sustained- or controlled-delivery formulations.
  • Techniques for formulating a variety of other sustained- or controlled-delivery means such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art.
  • Additional examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules.
  • Sustained release matrices can include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate, or poly-D( ⁇ )-3-hydroxybutyric acid.
  • Sustained-release compositions can also include liposomes, which can be prepared by any of several methods known in the art.
  • compositions of the invention to be used for in vivo administration typically must be sterile. This can be accomplished by filtration through sterile filtration membranes. Where the composition is lyophilized, sterilization using this method can be conducted either prior to, or following, lyophilization and reconstitution.
  • the composition for parenteral administration can be stored in lyophilized form or in a solution.
  • parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the pharmaceutical composition can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder.
  • Such formulations can be stored either in a ready-to-use form or in a form (e.g., lyophilized) requiring reconstitution prior to administration.
  • kits for producing a single-dose administration unit can each contain both a first container having a dried protein and a second container having an aqueous formulation. Also included within the scope of this invention are kits containing single and multi-chambered pre-filled syringes (e.g., liquid syringes and lyosyringes).
  • an antibody-like binding protein pharmaceutical composition to be employed therapeutically will depend, for example, upon the therapeutic context and objectives.
  • dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication for which the antibody-like binding protein is being used, the route of administration, and the size (body weight, body surface, or organ size) and condition (the age and general health) of the patient. Accordingly, the clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.
  • a typical dosage can range from about 0.1 ⁇ g/kg to up to about 100 mg/kg or more, depending on the factors mentioned above.
  • the dosage can range from 0.1 ⁇ g/kg up to about 100 mg/kg; or 1 ⁇ g/kg up to about 100 mg/kg; or 5 ⁇ g/kg, 10 ⁇ g/kg, 15 ⁇ g/kg, 20 ⁇ g/kg, 25 ⁇ g/kg, 30 ⁇ g/kg, 35 ⁇ g/kg, 40 ⁇ g/kg, 45 ⁇ g/kg, 50 ⁇ g/kg, 55 ⁇ g/kg, 60 ⁇ g/kg, 65 ⁇ g/kg, 70 ⁇ g/kg, 75 ⁇ g/kg, up to about 100 mg/kg.
  • Dosing frequency will depend upon the pharmacokinetic parameters of the antibody-like binding protein in the formulation being used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect.
  • the composition can therefore be administered as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages can be ascertained through use of appropriate dose-response data.
  • the route of administration of the pharmaceutical composition is in accord with known methods, e.g., orally; through injection by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; by sustained release systems; or by implantation devices.
  • the compositions can be administered by bolus injection or continuously by infusion, or by implantation device.
  • composition can also be administered locally via implantation of a membrane, sponge, or other appropriate material onto which the desired molecule has been absorbed or encapsulated.
  • a membrane, sponge, or other appropriate material onto which the desired molecule has been absorbed or encapsulated.
  • the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration.
  • the cross-over dual variable region in an Fv format was described in U.S. Pat. No. 5,989,830 and was referred to as a cross-over double head (CODH) configuration.
  • CODH cross-over double head
  • Molecular modeling predicted that the cross-over double-head (CODH) design results in a complex with both binding sites facing in opposite directions, without the restraints suggested for the Dual-Fv configuration.
  • the CODH Fv format was examined to determine whether it could be converted into complete antibody-like molecules by adding a C L domain to the light chain and an Fc region to the heavy chain.
  • a similar conversion was successful for the corresponding dual variable domains (DVD-Ig) and TBTI as described in U.S. Pat. No. 7,612,181 and International Publication No. WO 2009/052081.
  • the arrangement of the variable regions in the CODH format is shown in the structures below, which indicate the amino to carboxyl orientation of the peptide chains:
  • amino to carboxyl terminal arrangement of the variable regions in (a) and (b) above can be distinguished from the arrangement in the Dual-Fv configuration shown in (c) and (d) below:
  • V H1 N L1 and V H2 /V L2 The main difference to note is the distinct placement of the corresponding light chain and heavy chain variable regions (V H1 N L1 and V H2 /V L2 ) with respect to each other in the two dual variable region configurations.
  • the corresponding V L1 and V H1 domains were both at the N-terminus of the light and heavy chains in dual variable region configuration.
  • one half of one pair of an antibody variable region was separated spatially within the protein chain in the cross-over configuration.
  • the V L1 domain would be at the N-terminus of the protein light chain but the pairing V H1 domain is at the C-terminus of the cross-over configuration heavy chain.
  • the spatial relationship between V L1 and V H1 found in the dual variable region configuration is the arrangement found in natural antibodies.
  • cross-over dual variable region molecules having a C L domain on the light chain and an Fc region on the heavy chain were designed and constructed.
  • the polypeptides that form these antibody-like proteins have the structures shown below, in which the amino to carboxyl terminal orientation of the polypeptide chains is indicated:
  • WO 2009/052081 which is incorporated herein by reference in its entirety.
  • the parental humanized anti-IL4 V H and V L , and parental humanized anti-IL13 V H and V L sequences were combined and arranged as shown in Table 1.
  • the shorthand codes in column one of Table 1 were created to simplify discussion of these antibody-like binding proteins.
  • the antibody-like binding proteins differ in the size of the linker inserted between the two variable regions as shown in Table 1.
  • DNA molecules encoding the polypeptides shown in Table 1 were generated from the back-translated parental anti-IL4 and anti-IL13 antibodies.
  • C H1 , C L , and Fc domains were obtained from IGHG1 (GenBank Accession No. 569F4) and IGKC (GenBank Accession No. Q502W4).
  • This molecular modeling protocol was based on protein-protein docking of homology models and experimental models of the Fv IL4 and Fv IL13 regions, respectively, in combination with appropriate linkers between the Fv IL4 and Fv IL13 regions and between the Fv and constant or Fc regions.
  • L 1 refers to the linker between N-terminal V L and the C-terminal V L on the light chain
  • L 2 refers to the linker between the C-terminal V L and C L on the light chain
  • L 3 refers to the linker between N-terminal V H and the C-terminal V H on the heavy chain
  • L 4 refers to the linker between the C-terminal V H and C H1 (and Fc) on the heavy chain.
  • V H and V L refer only to the domain's location on a particular protein chain in the final format.
  • V H1 and V H2 could be derived from V L1 and V L2 domains in parent antibodies and placed into the V H1 and V H2 positions in a CODV-Ig.
  • V L1 and V L2 could be derived from V H1 and V H2 domains in parent antibodies and placed into the V H1 and V H2 positions in a CODV-Ig.
  • V H and V L designations refer to present location and not the original location in a parent antibody.
  • a homology model of Fv IL4 was constructed on PDB entries 1YLD (light chain) and 1IQW (heavy chain).
  • the Fv IL4 dimer was recomposed on an in-house crystal structure of the IL13/anti-IL13 Fab IL13 complex and optimized.
  • the crystal structure of IL4 (1RCB.pdb) was docked to the homology model of Fv IL4 .
  • twenty-two putative models of the complex were generated that merited further consideration.
  • the models of the light chain suggested that the linker L 1 between the V L1 and V L2 domains and the linker L 2 between the V L2 and C L1 domains should be between one to three and zero to two glycine residues long, respectively.
  • Models of the heavy chain suggested that the linker L 3 between the V H2 and V H1 domains and the linker L 4 between the V H1 and C H1 domains should be between two to six and four to seven glycine residues long, respectively (see Table 3 and FIG. 2 ).
  • glycine was used as a prototypical amino acid for the linkers but other amino acid residues may also serve as linkers.
  • Nucleic acid molecules encoding the variable heavy and light chains of the six heavy chains and four light chains described in Table 4 were generated by gene synthesis at Geneart (Regensburg, Germany).
  • the variable light chain domains were fused to the constant light chain (IGKC, GenBank Accession No. Q502W4) by digestion with the restriction endonucleases ApaLI and BsiWI and subsequently ligated into the ApaLI/BsiWI sites of the episomal expression vector pFF, an analogon of the pTT vector described by Durocher et al., (2002 , Nucl. Acids Res. 30(2): E9), creating the mammalian expression plasmid for expression of the light chains.
  • variable heavy chain domains were fused to the “Ted” variant of the human constant heavy chain (IGHG1, GenBank Accession No. 569F4), or alternatively, to a 6 ⁇ His tagged C H1 domain from the human constant IGHG1 in order to create a bispecific Fab.
  • the V H domain was digested with the restriction endonucleases ApaLI and ApaI and then fused to the IGHG1 or His tagged C H1 domain respectively, by ligation into the ApaLI/ApaI sites of the episomal expression vector pFF, creating the mammalian expression plasmids for expression of the heavy chains (IgG1 or Fab respectively).
  • Plasmids used for transfection were prepared from E. coli using the Qiagen EndoFree Plasmid Mega Kit.
  • HEK 293-FS cells growing in Freestyle Medium were transfected with indicated LC and HC plasmids encoding the heavy chains and light chains shown in Table 4 using 293fectin (Invitrogen) transfection reagent as described by the manufacturer. After 7 days, cells were removed by centrifugation and the supernatant was passed over a 0.22 ⁇ m filter to remove particles.
  • CODV-IgG1 constructs were purified by affinity chromatography on Protein A columns (HiTrap Protein A HP Columns, GE Life Sciences). After elution from the column with 100 mM acetate buffer and 100 mM NaCl, pH 3.5, the CODV-IgG1 constructs were desalted using HiPrep 26/10 Desalting Columns, formulated in PBS at a concentration of 1 mg/mL and filtered using a 0.22 ⁇ m membrane.
  • Bispecific CODV Fab constructs were purified by IMAC on HiTrap IMAC HP Columns (GE Life Sciences). After elution from the column with a linear gradient (Elution buffer: 20 mM sodium phosphate, 0.5 M NaCl, 50-500 mM imidazole, pH 7.4), the protein containing fractions were pooled and desalted using HiPrep 26/10 Desalting Columns, formulated in PBS at a concentration of 1 mg/mL and filtered using a 0.22 ⁇ m membrane.
  • Protein concentration was determined by measurement of absorbance at 280 nm. Each batch was analyzed by SDS-PAGE under reducing and non-reducing conditions to determine the purity and molecular weight of each subunit and of the monomer.
  • a Nunc F96-MaxiSorp-Immuno plate was coated with goat anti-Human IgG (Fc specific) [NatuTec A80-104A].
  • the antibody was diluted to 10 ⁇ g/ml in carbonate coating buffer (50 mM sodium carbonate, pH 9.6) and dispensed at 50 ⁇ L per well.
  • the plate was sealed with adhesive tape, and stored overnight at 4° C.
  • the plate was washed three times with Wash buffer (PBS, pH 7.4 and 0.1% Tween20).
  • 150 ⁇ L of blocking solution 1% BSA/PBS was dispensed into each well to cover the plate. After 1 hour at room temperature, the plate was washed three times with Wash buffer.
  • the aggregation level was measured by analytical size-exclusion chromatography (SEC).
  • SEC analytical size-exclusion chromatography
  • Analytical SEC was performed on assembled pairs using an ⁇ KTA explorer 10 (GE Healthcare) equipped with a TSKgel G3000SWXL column (7.8 mm ⁇ 30 cm) and TSKgel SWXL guard column (Tosoh Bioscience). The analysis was run at 1 ml/min using 250 mM NaCl, 100 mM Na-phosphate, pH 6.7, with detection at 280 nm. 30 ⁇ L of protein sample (at 0.5-1 mg/ml) were applied onto the column. For estimation of the molecular size, the column was calibrated using a gel filtration standard mixture (MWGF-1000, SIGMA Aldrich). Data evaluation was performed using UNICORN software v5.11.
  • Table 5 shows the results of the first set of 24 different CODV-Ig molecules made using the anti-IL4 and anti-IL13 variable region combinations described in Table 4.
  • the codes assigned in Table 4 represent the adjacent structures shown in Table 4.
  • LC4 corresponds to the structure IL4 V L -(Gly)-IL13 V L -(Gly2)-C L1 having linker L 1 equal to 1, where a single amino acid residue separated the two V L domains of the dual variable region light chain.
  • LC4 had L 2 equal to 2, which contained a Gly-Gly dipeptide linker between the central V L and the C-terminal C H1 .
  • CODV-Ig antibody-like molecules corresponding to the LC4:HC4 and LC4:HC6 combinations described in Table 4 were chosen for assessment of a full kinetic analysis using surface plasmon resonance.
  • Two pairs of heavy and light chains were selected for full kinetic analysis.
  • Recombinant human IL13 and IL4 was purchased from Chemicon (USA).
  • Kinetic characterization of purified antibodies was performed using surface plasmon resonance technology on a BIACORE 3000 (GE Healthcare).
  • a capture assay using a species specific antibody e.g., human-Fc specific MAB 1302, Chemicon
  • the capture antibody was immobilized via primary amine groups (11000 RU) on a research grade CM5 chip (GE Life Sciences) using standard procedures.
  • the analyzed antibody was captured at a flow rate of 10 ⁇ L/min with an adjusted RU value that would result in maximal analyte binding of 30 RU.
  • Binding kinetics were measured against recombinant human IL4 and IL13 over a concentration range between 0 to 25 nM in HBS EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% Surfactant P20) at a flow rate of 30 ⁇ l/min. Chip surfaces were regenerated with 10 mM glycine, pH 2.5. Kinetic parameters were analyzed and calculated in the BIAevaluation program package v4.1 using a flow cell without captured antibody as reference.
  • Table 6 shows the comparison of kinetics of the parental BB13 (anti-IL13) and 8D4 (anti-IL4) antibodies (expressed as IgGs) with the respective domains within the CODV-Ig format (Table 4, Codes LC4:HC4 and LC4:HC6). As shown in Table 6, the CODV-Ig constructs did not exhibit decreased binding properties against the corresponding antigens, when compared to the parental anti-IL13 and anti-IL4 antibodies. The loss in the on-rate observed in the DVD-Ig/TBTI format using the same Fv sequences did not occur with the CODV-Ig configuration.
  • the opposite facing binding sites should allow for binding large antigens or bridging different cells with a bispecific antibody-like configuration, and would also be suitable for a wider selection of parental antibodies.
  • a further advantage of the CODV-Ig was that no linker residues protrude into the antigen binding site and reduce accessibility of the antigen.
  • the co-injection was done with 3.125 nM IL4/25 nM IL13 (and vice versa) and with a 1:1 mixture of 3.125 nM IL4 and 25 nM IL13.
  • a co-injection of HBS-EP buffer was done as a reference.
  • an identical binding level of 63 RU was achieved after injection of the antigen mixture or co-injection of the antigens, regardless of the co-injection sequence.
  • CODV-Ig protein had been saturated by the first antigen (IL4)
  • the second antigen (IL13) was injected and a second binding signal was observed. This observation was reproduced when the antigen injection sequence was reversed.
  • the CODV-Ig construct was able to bind both antigens simultaneously (i.e., exhibit bispecificity) saturating all binding sites (i.e., exhibited tetravalency).
  • CODV-Ig molecules having different combinations of linker lengths for L 1 , L 2 on the light chain and L 3 and L 4 on the heavy chain.
  • CODV-Ig constructs were generated with heavy chain linkers L 3 and L 4 varying between 1 through 8 residues for L 3 and either 0 or 1 residues for L 4 .
  • the heavy chain contained anti-IL4 as the N-terminal binding domain and anti-IL13 as the C-terminal binding domain followed by C H1 -Fc.
  • the light chain linkers L 1 and L 2 were varied from 3 to 12 residues for L 1 and from 3 to 14 residues for L 2 .
  • the light chain contained anti-IL13 as the N-terminal binding domain and anti-IL4 as the C-terminal binding domain followed by C L1 .
  • aggregation level was by analytical size-exclusion chromatography (SEC).
  • SEC analytical size-exclusion chromatography
  • Analytical SEC was performed using an ⁇ KTA explorer 10 (GE Healthcare) equipped with a TSKgel G3000SWXL column (7.8 mm ⁇ 30 cm) and TSKgel SWXL guard column (Tosoh Bioscience). The analysis was run at 1 ml/min using 250 mM NaCl, 100 mM Na-phosphate pH 6.7, with detection at 280 nm. 30 ⁇ L of protein sample (at 0.5-1 mg/ml) was applied onto the column. For estimation of the molecular size, the column was calibrated using a gel filtration standard mixture (MWGF-1000, SIGMA Aldrich). Data evaluation was performed using UNICORN software v5.11.
  • Recombinant human IL13 and IL4 were purchased from Chemicon (USA).
  • Recombinant human TNF- ⁇ was purchased from Sigma Aldrich (H8916-10 ⁇ g), recombinant human IL-1 ⁇ (201-LB/CF), recombinant human IL-23 (1290-IL/CF), recombinant human EGFR (344 ER), and recombinant human HER2 (1129-ER-50) were purchased from R&D Systems.
  • Biacore Kinetic binding analysis by Biacore was performed as follows. Surface plasmon resonance technology on a Biacore 3000 (GE Healthcare) was used for detailed kinetic characterization of purified antibodies. A capture assay using a species-specific antibody (e.g., human-Fc specific MAB 1302, Chemicon) for capture and orientation of the investigated antibodies was used. For determination of IL4 and IL13 binding kinetics, the corresponding CODV Fabs as in Example 10, Table 12 were captured using the anti-human Fab capture Kit (GE Healthcare). The capture antibody was immobilized via primary amine groups (11000 RU) on a research grade CM5 chip (GE Life Sciences) using standard procedures.
  • a species-specific antibody e.g., human-Fc specific MAB 1302, Chemicon
  • the analyzed antibody was captured at a flow rate of 10 ⁇ L/min with an adjusted RU value that would result in maximal analyte binding of 30 RU.
  • Binding kinetics were measured against recombinant human IL4 and IL13 over a concentration range between 0 to 25 nM in HBS EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Surfactant P20) at a flow rate of 30 ⁇ L/min. Chip surfaces were regenerated with 10 mM glycine pH 2.5.
  • Kinetic parameters were analyzed and calculated in the BIAevaluation program package v4.1 using a flow cell without captured antibody as a reference.
  • Binding affinities of CODV-Ig, CODV-Fab, and TBTI against EGFR and HER2 were measured using a Proteon XPR36 protein interaction array system (Biorad).
  • the antigens were immobilized by amine reactive coupling on GLC sensor chips (Biorad).
  • Dilution series of the bispecific antibody variants in PBSET buffer (Biorad) were analyzed in parallel in one-shot kinetics mode with double referencing. Data were analyzed using Proteon Manager Software v3.0 (Biorad) with either Langmuir 1:1 model with mass transfer or bivalent analyte model.
  • Table 7 summarizes the results for yield, aggregation (as measured by size exclusion chromatography), and binding affinity for CODV-Ig having different size combinations of linkers.
  • CODV-Ig linker lengths described above were found to be more sensitive to increases in 1 amino acid residue than increases in 2 amino acid residues.
  • Batch ID Nos. 103 and 104 differ by 1 amino acid residue in L 2
  • Batch ID No. 103 shows 6 fold more aggregation
  • Batch ID No. 104 displays less aggregation and twice the yield.
  • Batch ID Nos. 104 and 105 which differ by two residues in L 2 , displayed similar profiles with respect to yield, aggregation, and binding.
  • the optimal short linker sizes on the light chain suggested that the light chain was serving as a template by remaining in a linear arrangement and that larger linkers were required on the heavy chain in order for the heavy chain to fold properly into the cross-over configuration to conform to the template light chain (see FIG. 5 , Panel A). Whether the short linkers specifically placed on the heavy chain to maintain a liner arrangement on the heavy chain rendered the heavy chain the “template” chain, and whether the pattern would repeat itself and larger linkers would be required to allow the non-template chain to fold properly and accommodate the now template heavy chain was evaluated next (see FIG. 5 , Panel B).
  • FIG. 6 illustrates these principles of CODV-Ig design based on having either the light chain or the heavy chain as the “template.”
  • CODV-Ig constructs were generated with heavy chain linkers L 3 and L 4 varying between 1 through 8 residues for L 3 and either 0 or 1 residues for L 4 .
  • the heavy chain contained anti-IL4 as the N-terminal binding domain and anti-IL13 as the C-terminal binding domain followed by C H1 -Fc.
  • the light chain linkers L 1 and L 2 were varied from 3 to 12 residues for L 1 and from 3 to 14 residues for L 2 .
  • the light chain contained anti-IL13 as the N-terminal binding domain and anti-IL4 as the C-terminal binding domain followed by C L1 .
  • Table 8 summarizes the results for yield, aggregation (as measured by size exclusion chromatography), and binding affinity for CODV-Ig having different size combinations of linkers and where the heavy chain is maintained in a linear arrangement as the template chain and the light chain is allowed to fold in a cross-over configuration.
  • One exception was Batch ID No. 210, in which L 1 was 7, L 2 was 5, L 3 was 2, and L 4 was zero. This arrangement produced a sufficient amount of protein and had an acceptable level of aggregation and binding, which suggested that some combination of linker sizes could be found to compensate for a zero length linker at L 4 in some circumstances.
  • variable regions from numerous existing human and humanized antibodies having specificity for insulin-like growth factor 1 receptor (IGF1R(1)), a second antibody to insulin-like growth factor 1 receptor (IGF1R(2)), human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), tumor necrosis factor-alpha (TNF ⁇ ), Interleukin 12 and 23 (IL-12/23) and interleukin 1beta (IL-1 ⁇ ) were incorporated into the CODV-Ig format (see Table 10).
  • Heavy Chains (N to C terminal) HC10 IGF1R(1) V H -(Gly)-HER2 V H -(Gly2)-C H1 -Fc 32 HC11 HER2 V H -(Gly)-IGF1R(1) V H -(Gly2)-C H1 -Fc 33 HC12 IGF1R(2) V H -(Gly)-EGFR V H -(Gly2)-C H1 -Fc 34 HC13 EGFR V H -(Gly)-IGF1R(2) V H -(Gly2)-C H1 -Fc 35 HC14 TNF ⁇ V H -(Gly)-IL12/23 V H -(Gly2)-C H1 -Fc 36 HC15 IL12/23 V H -(Gly)-TNF ⁇ V H -(Gly2)-C H1 -Fc 36 HC15 IL12/23 V H -(Gly)-TNF ⁇ V H -(G
  • the antibody variable regions from known human and humanized antibodies were used to test the universal applicability of the CODV-Ig format in designing bispecific antibody-like binding proteins.
  • the possibility of positional effects with regard to the placement of certain antibody variable regions either N-terminal or C-terminal on either the heavy chain or the light chain was examined.
  • the identical antibody sequences for anti-IL4 and anti-IL13 were incorporated into either the TBTI/DVD-Ig or CODV-Ig formats for a direct comparison of these configurations, the positioning of the linkers, and affinities of the resulting molecules.
  • the parental affinity of each of the antibodies was maintained in the CODV format.
  • the variable regions were placed in the TBTI/DVD-Ig format, a drop in affinity of the IL4 antibody positioned at the inner Fv2 position was manifested as a reduction of the on-rate of antibody binding to the antigen.
  • there was no loss in affinity for the CODV-Ig format as compared to the parental antibodies (see FIG. 7 , lower panel).
  • Fab fragments were expressed by transient transfection as described previously. Seven days post-transfection, cells were removed by centrifugation, 10% vol/vol 1M Tris-HCl, pH 8.0, was added and the supernatant was passed over a 0.22 ⁇ m filter to remove particles. The Fab proteins were captured using HisTrap High Performance columns (GE Healthcare) and eluted via imidazole gradient. The protein containing fractions were pooled and desalted using PD-10 or Sephadex columns. Concentrated and sterile filtered (0.22 ⁇ m) protein solutions were adjusted to 1 mg/ml and kept at 4° C. until use.
  • Binding protein constructs from Batch ID Nos. 401-421 directly compared antibody-like proteins in which antibody variable regions were arranged as in CODV-Ig molecules with the heavy chain as the template (401, 402, 406, and 407), CODV Fab-like fragments (402, 408, 413, 418, and 421), four domain antibody-like molecules in TBTI/DVD-Ig format (404, 409, 414, and 419), and CODV-Ig with no linkers (405, 410, 415, and 420).
  • CODV-Fab bispecific CODV Fab-like binding proteins having a TCR binding site (CD3epsilon) and a CD19 binding site were generated and compared to a bispecific Fab derived from the TBTI/DVD-Ig format (B-Fab).
  • B-Fab bispecific CODV Fab-like binding proteins
  • the binding proteins were characterized in a cytotoxic assay using NALM-6 (CD19 expressing) cells as target cells and primary human T-cells as effector cells.
  • CD3 positive cells were isolated from freshly prepared human PBMC's. Effector and target cells were mixed at a ratio of 10:1 and incubated for 20 hours with the indicated concentrations of bispecific binding proteins (see FIG. 8 ).
  • Apoptotic target cells were determined in a FACS-based assay using 7-Aminoactinomycin staining.
  • the B-Fab format in the configuration CD3-CD19 (1060) was shown to be active in inducing T-cell mediated cytotoxicity towards NALM-6 cells with an EC50 of 3.7 ng/ml.
  • a similarly high activity was observed for the CD19-CD3 CODV-Fab (1109) with an EC50 of 3.2 ng/ml (see FIG. 8 ).
  • a swap of the configuration of the B-Fab molecule (Fab of the TBTI/DVD-Ig format) to a CD19-CD3 orientation resulted in a significant loss of activity (see FIG. 8 ).
  • the swapped B-Fab molecule showed no activity at concentrations that were maximal for both orientations of CODV-Ig Fab and the other orientation of B-Fab.
  • a maximum response was observed (ranging between 1 and 100 ng/ml).
  • For the CD19-CD3 orientation of B-Fab even at the maximal concentration (30 ⁇ g/ml), the optimal cytotoxic response was not reached.
  • Linker lengths were set at 7, 5, 1, and 2 residues in length for L 1 , L 2 , L 3 , and L 4 , respectively (see Table 13).
  • Test sequences were derived from naturally occurring linkers at the transitions between natural antibody V H and C H1 domains or between antibody Fv and C L domains of kappa or lambda light chains.
  • the candidate sequences were ASTKGPS (SEQ ID NO: 48), which is derived from the V H and C H1 domain transition, RTVAAPS (SEQ ID NO: 49) and GQPKAAP (SEQ ID NO: 50), which were derived from the Fv and C L domain transitions of kappa and lambda light chains, respectively. Furthermore, one construct was generated with an arbitrary linker composition to show that any sequence can be potentially used in linkers L 1 to L 4 .
  • This linker composition was obtained by randomly distributing the amino acids valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate, asparagine, glutamine, glycine, and proline at the 15 positions of the four linkers.
  • the aromatic amino acids phenylalanine, tyrosine, and tryptophan, as well as the amino acids methionine and cysteine were deliberately excluded to avoid potential increases in aggregation.
  • a three-dimensional model of the construct for Batch ID No. 204 was generated to assure suitability or refine the choices of linker composition.
  • serine was chosen for linker L 3 as positively and negatively charged residues are observed nearby in the three-dimensional model.
  • the residues in linker L 4 were selected to be compatible with solvent exposure of these positions as suggested by the model.
  • no problems were anticipated or predicted for the linker compositions of L 1 and L 2 .
  • Three-dimensional models of selected proposals for linker composition were constructed.
  • linker composition may have a dramatic influence on yield. Sequences that were derived from lambda chain on L 1 (comparing Batch ID Nos. 505-507 with Batch ID Nos. 501-503) were more productive protein generators (up to 8 fold increase). Indeed, the linkers based on random generation also produced good yields, as shown in Table 13, Batch ID No. 508. Therefore, linker composition should be one parameter considered during CODV-Ig optimization.
  • HEK-Blue IL-4/IL-13 reporter cells are designed to monitor the activation of the STAT6 pathway by IL-4 or IL13. Stimulation of the cells with either cytokine results in production of the reporter gene secreted embryonic alkaline phosphatase (SEAP), which can be measured in the culture supernatant with the QUANTI-Blue assay.
  • SEAP embryonic alkaline phosphatase
  • the cytokines were pre-incubated for 1 hour with different concentrations of the antibodies and added to 50,000 HEK-Blue IL-4/IL-13 cells. Cytokine-mediated induction of SEAP was measured after 24 hours incubation in the cell culture supernatant with the QUANTI-Blue assay (InvivoGen).
  • each of the mutated CODV-Ig molecules containing additional cysteine residues had melting temperatures that were the same as the melting temperature for the CODV-Ig construct Batch ID No. 204.
  • cysteines were introduced at Kabat positions 100 for the light chain and 44 for the heavy chain on each of the variable domains as described in Brinkmann et al., 1993 , Proc. Natl. Acad. Sci. U.S.A. 90: 7538-42. These positions have been shown to be structurally conserved within antibody folds, and therefore tolerable of cysteine substitution without interfering with the integrity of the individual domains.
  • Tm Melting points (Tm) of CODV and TBTI variants were determined using differential scanning fluorimetry (DSF). Samples were diluted in D-PBS buffer (Invitrogen) to a final concentration of 0.2 ⁇ g/ ⁇ l and added to 2 ⁇ l of a 40 ⁇ concentrated solution of SYPRO-Orange dye (Invitrogen) in D-PBS in white semi-skirt 96-well plates. All measurements were done in duplicate using a MyiQ2 real time PCR instrument (Biorad). Tm values were extracted from the negative first derivative of the melting curves using iQ5 Software v2.1.
  • SEQ ID NO: 28 (SEQ ID NO: 26) 807 IL4 ⁇ IL13 S60C S123C GGGGGGG GGGGG G GG 7.5 27.9 2 202 66 (SEQ ID NO: 28) (SEQ ID NO: 26) 808 IL4 ⁇ IL13 S60C G124C GGGGGGG GGGGG C GG 1.1 5.8 5 70 64 (SEQ ID NO: 28) (SEQ ID NO: 26) 809 IL4 ⁇ IL13 G61C S122C GGGGGGG GGGGG G GG 8.7 6.9 6 81 65 (SEQ ID NO: 28) (SEQ ID NO: 26)

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Cited By (82)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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US9708400B2 (en) 2012-04-20 2017-07-18 Abbvie, Inc. Methods to modulate lysine variant distribution
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US9850320B2 (en) 2014-11-26 2017-12-26 Xencor, Inc. Heterodimeric antibodies to CD3 X CD20
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US20180155450A1 (en) * 2011-03-28 2018-06-07 Sanofi Dual Variable Region Antibody-Like Binding Proteins Having Cross-Over Binding Region Orientation
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US10064952B2 (en) 2014-10-09 2018-09-04 Genzyme Corporation Glycoengineered antibody drug conjugates
US10106624B2 (en) 2013-03-15 2018-10-23 Xencor, Inc. Heterodimeric proteins
US10131710B2 (en) 2013-01-14 2018-11-20 Xencor, Inc. Optimized antibody variable regions
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US10227410B2 (en) 2015-12-07 2019-03-12 Xencor, Inc. Heterodimeric antibodies that bind CD3 and PSMA
US10227411B2 (en) 2015-03-05 2019-03-12 Xencor, Inc. Modulation of T cells with bispecific antibodies and FC fusions
US10316088B2 (en) 2016-06-28 2019-06-11 Xencor, Inc. Heterodimeric antibodies that bind somatostatin receptor 2
WO2019147973A1 (en) 2018-01-26 2019-08-01 Genzyme Corporation Fc variants with enhanced binding to fcrn and prolonged half-life
US10428155B2 (en) 2014-12-22 2019-10-01 Xencor, Inc. Trispecific antibodies
US10487155B2 (en) 2013-01-14 2019-11-26 Xencor, Inc. Heterodimeric proteins
US10501543B2 (en) 2016-10-14 2019-12-10 Xencor, Inc. IL15/IL15Rα heterodimeric Fc-fusion proteins
US10519242B2 (en) 2013-03-15 2019-12-31 Xencor, Inc. Targeting regulatory T cells with heterodimeric proteins
US10526417B2 (en) 2014-11-26 2020-01-07 Xencor, Inc. Heterodimeric antibodies that bind CD3 and CD38
US10544187B2 (en) 2013-03-15 2020-01-28 Xencor, Inc. Targeting regulatory T cells with heterodimeric proteins
US10647777B2 (en) 2014-05-28 2020-05-12 Genzyme Corporation Methods of controlling the formation of disulfide bonds in protein solutions
US10738095B2 (en) 2015-06-03 2020-08-11 The Medical College Of Wisconsin, Inc. Engineered CCL20 locked dimer polypeptide
US10787518B2 (en) 2016-06-14 2020-09-29 Xencor, Inc. Bispecific checkpoint inhibitor antibodies
US10793632B2 (en) 2016-08-30 2020-10-06 Xencor, Inc. Bispecific immunomodulatory antibodies that bind costimulatory and checkpoint receptors
US10836813B2 (en) 2012-09-12 2020-11-17 Genzyme Corporation Fc containing polypeptides with altered glycosylation and reduced effector function
US10851178B2 (en) 2011-10-10 2020-12-01 Xencor, Inc. Heterodimeric human IgG1 polypeptides with isoelectric point modifications
US10858417B2 (en) 2013-03-15 2020-12-08 Xencor, Inc. Heterodimeric proteins
US10882922B2 (en) 2016-04-13 2021-01-05 Sanofi Trispecific and/or trivalent binding proteins
WO2021016571A2 (en) 2019-07-25 2021-01-28 Genzyme Corporation Methods of treating antibody-mediated disorders with fcrn antagonists
US20210047435A1 (en) * 2019-08-15 2021-02-18 Janssen Biotech, Inc. Materials and methods for improved single chain variable fragments
US10968276B2 (en) 2013-03-12 2021-04-06 Xencor, Inc. Optimized anti-CD3 variable regions
US10981992B2 (en) 2017-11-08 2021-04-20 Xencor, Inc. Bispecific immunomodulatory antibodies that bind costimulatory and checkpoint receptors
US10982006B2 (en) 2018-04-04 2021-04-20 Xencor, Inc. Heterodimeric antibodies that bind fibroblast activation protein
US10995148B2 (en) 2014-03-19 2021-05-04 Genzyme Corporation Site-specific glycoengineering of targeting moieties
US11008389B2 (en) 2011-03-16 2021-05-18 Sanofi Uses of a dual V region antibody-like protein
US11053316B2 (en) 2013-01-14 2021-07-06 Xencor, Inc. Optimized antibody variable regions
US11084863B2 (en) 2017-06-30 2021-08-10 Xencor, Inc. Targeted heterodimeric Fc fusion proteins containing IL-15 IL-15alpha and antigen binding domains
WO2021174034A1 (en) 2020-02-28 2021-09-02 Genzyme Corporation Modified binding polypeptides for optimized drug conjugation
US11129905B2 (en) 2015-10-25 2021-09-28 Sanofi Bivalent, bispecific binding proteins for prevention or treatment of HIV infection
US11186638B2 (en) 2011-09-12 2021-11-30 Genzyme Corporation Anti-αβTCR antibody
US11186649B2 (en) 2017-10-10 2021-11-30 Sanofi Anti-CD38 antibodies and methods of use
CN114269784A (zh) * 2019-06-07 2022-04-01 美国安进公司 具有选择性可切割接头的双特异性结合构建体
US11312770B2 (en) 2017-11-08 2022-04-26 Xencor, Inc. Bispecific and monospecific antibodies using novel anti-PD-1 sequences
US11319355B2 (en) 2017-12-19 2022-05-03 Xencor, Inc. Engineered IL-2 Fc fusion proteins
US11358999B2 (en) 2018-10-03 2022-06-14 Xencor, Inc. IL-12 heterodimeric Fc-fusion proteins
US11472890B2 (en) 2019-03-01 2022-10-18 Xencor, Inc. Heterodimeric antibodies that bind ENPP3 and CD3
US11505595B2 (en) 2018-04-18 2022-11-22 Xencor, Inc. TIM-3 targeted heterodimeric fusion proteins containing IL-15/IL-15RA Fc-fusion proteins and TIM-3 antigen binding domains
WO2022249146A1 (en) 2021-05-27 2022-12-01 Sanofi Fc VARIANT WITH ENHANCED AFFINITY TO Fc RECEPTORS AND IMPROVED THERMAL STABILITY
US11524991B2 (en) 2018-04-18 2022-12-13 Xencor, Inc. PD-1 targeted heterodimeric fusion proteins containing IL-15/IL-15Ra Fc-fusion proteins and PD-1 antigen binding domains and uses thereof
US11530268B2 (en) 2018-10-09 2022-12-20 Sanofi Trispecific anti-CD38, anti-CD28, and anti-CD3 binding proteins and methods of use for treating viral infection
US11571462B2 (en) 2015-06-03 2023-02-07 The Medical College Of Wisconsin, Inc. Engineered CCL20 locked dimer polypeptide
US11591401B2 (en) 2020-08-19 2023-02-28 Xencor, Inc. Anti-CD28 compositions
US11613576B2 (en) 2019-04-09 2023-03-28 Sanofi Trispecific binding proteins, methods, and uses thereof
EP4223783A2 (en) 2012-09-12 2023-08-09 Genzyme Corporation Fc containing polypeptides with altered glycosylation and reduced effector function
US11739144B2 (en) 2021-03-09 2023-08-29 Xencor, Inc. Heterodimeric antibodies that bind CD3 and CLDN6
WO2023227790A1 (en) 2022-05-27 2023-11-30 Sanofi Natural killer (nk) cell engagers binding to nkp46 and bcma variants with fc-engineering
US11859012B2 (en) 2021-03-10 2024-01-02 Xencor, Inc. Heterodimeric antibodies that bind CD3 and GPC3
US11919956B2 (en) 2020-05-14 2024-03-05 Xencor, Inc. Heterodimeric antibodies that bind prostate specific membrane antigen (PSMA) and CD3
US11932704B2 (en) 2016-04-13 2024-03-19 Sanofi Trispecific and/or trivalent binding proteins
WO2024089609A1 (en) 2022-10-25 2024-05-02 Ablynx N.V. Glycoengineered fc variant polypeptides with enhanced effector function

Families Citing this family (75)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BR112013027224A8 (pt) 2011-04-22 2018-08-14 Emergent Product Dev Seattle Proteínas de ligação de antígeno de membrana específico de próstata e composições e métodos relacionados
US10167335B2 (en) * 2012-02-13 2019-01-01 Agency For Science, Technology And Research IL-1β neutralizing human monoclonal antibodies
US9856319B2 (en) 2012-12-28 2018-01-02 Abbvie Inc. Monovalent binding proteins
WO2014106015A2 (en) * 2012-12-28 2014-07-03 Abbvie, Inc. Multivalent binding protein compositions
US9458244B2 (en) 2012-12-28 2016-10-04 Abbvie Inc. Single chain multivalent binding protein compositions and methods
JP2016515093A (ja) * 2013-02-05 2016-05-26 サノフイ 抗体−薬物コンジュゲート療法とともに使用するための免疫イメージング剤
US20140302037A1 (en) * 2013-03-15 2014-10-09 Amgen Inc. BISPECIFIC-Fc MOLECULES
MX357011B (es) * 2013-03-15 2018-06-22 Amgen Inc Anticuerpos biespecificos heterodimericos.
CN103509117B (zh) * 2013-05-06 2016-03-09 江苏匡亚生物医药科技有限公司 抗人her2和人igf-ir的双特异性抗体及其制备方法和用途
CA2918795A1 (en) 2013-07-25 2015-01-29 Cytomx Therapeutics, Inc. Multispecific antibodies, multispecific activatable antibodies and methods of using the same
CN106459990B (zh) * 2014-02-07 2021-06-01 麦克马斯特大学 三功能t细胞抗原偶联物及其制备方法和用途
CN104974258B (zh) * 2014-04-01 2019-06-14 三生国健药业（上海）股份有限公司 重组抗hgf/dll4双特异性抗体、其制备方法和应用
CN107076750B (zh) * 2014-07-18 2020-06-23 赛诺菲 用于预测疑似患有癌症的患者使用阿柏西普的治疗结果的方法
CA2955947A1 (en) 2014-07-25 2016-01-28 Cytomx Therapeutics, Inc. Anti-cd3 antibodies, activatable anti-cd3 antibodies, multispecific anti-cd3 antibodies, multispecific activatable anti-cd3 antibodies, and methods of using the same
WO2016094834A2 (en) 2014-12-12 2016-06-16 Alexion Pharmaceuticals, Inc. A method for treating a complement mediated disorder caused by an infectious agent in a patient
CA2966776C (en) * 2014-12-19 2021-05-04 Alkermes, Inc. Single chain fc fusion proteins
CN117467017A (zh) 2014-12-22 2024-01-30 西雅图免疫公司 双特异性四价抗体及其制备和使用方法
CN107428835B (zh) 2015-01-23 2021-11-26 赛诺菲 抗cd3抗体、抗cd123抗体和与cd3和/或cd123特异性结合的双特异性抗体
TWI733661B (zh) 2015-03-04 2021-07-21 美商健臻公司 以高親合性、結合性及特異性結合轉化生長因子－β１的經修飾ＩｇＧ抗體
TWI726870B (zh) 2015-03-04 2021-05-11 美商健臻公司 具有高親和性、結合性及專一性之結合轉形生長因子-β１的ｓｃＦｖ-Ｆｃ二聚體
MA42971A (fr) 2015-03-13 2018-08-15 Cytomx Therapeutics Inc Anticorps anti-pdl1, anticorps anti-pld1 activables, et leurs procédés d'utilisation
JP7133925B2 (ja) 2015-03-23 2022-09-09 アレクシオン ファーマシューティカルズ インコーポレイテッド ウイルス濾過
AR104368A1 (es) 2015-04-03 2017-07-19 Lilly Co Eli Anticuerpos biespecíficos anti-cd20- / anti-baff
EP3307776A1 (en) 2015-06-15 2018-04-18 Numab Innovation AG Hetero-dimeric multi-specific antibody format
EA201890360A1 (ru) 2015-07-23 2018-06-29 Бёрингер Ингельхайм Интернациональ Гмбх Соединение, нацеленное на ил-23a и фактор активации в-лимфоцитов (baff), и его применение
JP2018528218A (ja) 2015-09-11 2018-09-27 アンドリアン、ブルース 組換えグリコシル化エクリズマブおよびエクリズマブ変異体
SG10202002577XA (en) 2015-09-21 2020-04-29 Aptevo Res & Development Llc Cd3 binding polypeptides
US11203634B2 (en) 2015-10-07 2021-12-21 Alexion Pharmaceuticals, Inc. Methods of treating age-related macular degeneration in a patient comprising administering an anti-C5a antibody
EP3156417A1 (en) * 2015-10-13 2017-04-19 Affimed GmbH Multivalent fv antibodies
CN109400709B (zh) * 2015-11-13 2021-03-23 广东医科大学 双功能抗体及其用途
WO2017189964A2 (en) 2016-04-29 2017-11-02 Voyager Therapeutics, Inc. Compositions for the treatment of disease
US11326182B2 (en) 2016-04-29 2022-05-10 Voyager Therapeutics, Inc. Compositions for the treatment of disease
RU2018141451A (ru) 2016-05-27 2020-06-29 Алексион Фармасьютикалс, Инк. Способы лечения рефрактерной генерализованной миастении
US10335459B2 (en) 2016-06-22 2019-07-02 Alkermes, Inc. Compositions for modulating IL-10 immunostimulatory and anti-inflammatory properties
TWI790206B (zh) 2016-07-18 2023-01-21 法商賽諾菲公司 特異性結合至ｃｄ３和ｃｄ１２３的雙特異性抗體樣結合蛋白
CN109641949B (zh) 2016-08-26 2023-09-15 赛诺菲 促进选择性轻链配对的多特异性抗体
JP7173965B2 (ja) 2016-10-19 2022-11-16 アレクシオン ファーマシューティカルズ， インコーポレイテッド 試料中の非結合Ｃ５ａの定量化方法
ES2848206T3 (es) 2016-10-19 2021-08-05 Alexion Pharma Inc Un método de cuantificar C5 sin unir en una muestra
WO2018151841A1 (en) 2017-02-17 2018-08-23 Sanofi Multispecific binding molecules having specificity to dystroglycan and laminin-2
JP7304288B2 (ja) * 2017-02-17 2023-07-06 サノフイ ジストログリカンおよびラミニン２に対する特異性を有する多特異性結合性分子
US20200385472A1 (en) * 2017-04-28 2020-12-10 Elstar Therapeutics, Inc. Multispecific molecules comprising a non-immunoglobulin heterodimerization domain and uses thereof
KR20220147684A (ko) * 2017-05-10 2022-11-03 더 위스타 인스티튜트 오브 아나토미 앤드 바이올로지 최적화된 핵산 항체 작제물
BR112019025188A2 (pt) 2017-06-01 2020-06-23 Cytomx Therapeutics, Inc. Anticorpos anti-pdl1 ativáveis e métodos de uso dos mesmos
UY37758A (es) 2017-06-12 2019-01-31 Novartis Ag Método de fabricación de anticuerpos biespecíficos, anticuerpos biespecíficos y uso terapéutico de dichos anticuerpos
JP7227630B2 (ja) 2017-10-12 2023-02-22 マックマスター ユニバーシティー Ｙ１８２ｔ突然変異を有するｔ細胞－抗原カプラおよびその方法ならびに使用
CA3088107A1 (en) 2018-01-12 2019-07-18 Genzyme Corporation Methods for the quantitation of polypeptides
CN111566126A (zh) * 2018-01-12 2020-08-21 美国安进公司 Pac1抗体及其用途
US11110123B2 (en) 2018-07-17 2021-09-07 Triumvira Immunologics Usa, Inc. T cell-antigen coupler with various construct optimizations
US10640562B2 (en) 2018-07-17 2020-05-05 Mcmaster University T cell-antigen coupler with various construct optimizations
US20220002393A1 (en) 2018-11-20 2022-01-06 Alexion Pharmaceuticals, Inc. Methods for treatment of refractory generalized myasthenia gravis in pediatric patients
GB2583560A (en) 2018-12-11 2020-11-04 Admirx Inc Fusion protein constructs for complement associated disease
KR20220015369A (ko) 2018-12-24 2022-02-08 사노피 슈도fab-기반 다중특이성 결합 단백질
EP3674316A1 (en) 2018-12-24 2020-07-01 Sanofi Multispecific binding proteins with mutant fab domains
US11965030B2 (en) 2018-12-24 2024-04-23 Sanofi Multispecific binding proteins with mutant fab domains
WO2020210392A1 (en) * 2019-04-09 2020-10-15 Sanofi Trispecific binding proteins, methods, and uses thereof
US20210032370A1 (en) 2019-08-02 2021-02-04 Immatics Biotechnologies Gmbh Recruiting agent further binding an mhc molecule
DE102019121007A1 (de) 2019-08-02 2021-02-04 Immatics Biotechnologies Gmbh Antigenbindende Proteine, die spezifisch an MAGE-A binden
WO2021026160A1 (en) 2019-08-05 2021-02-11 Alexion Pharmaceuticals, Inc. Methods for treatment of refractory generalized myasthenia gravis with eculizumab
US20210123926A1 (en) 2019-10-25 2021-04-29 Sanofi Methods for analyzing chain mispairing in multispecific binding proteins
WO2021144020A1 (en) 2020-01-15 2021-07-22 Immatics Biotechnologies Gmbh Antigen binding proteins specifically binding prame
KR20220156912A (ko) 2020-03-25 2022-11-28 일라이 릴리 앤드 캄파니 다중특이적 결합 단백질 및 그의 개발 방법
JP2023519699A (ja) * 2020-03-30 2023-05-12 サノフイ スプリットｃｈ２ドメイン
EP4214231A1 (en) 2020-09-18 2023-07-26 Ichnos Sciences SA Cd47-cd38 bispecific antibodies
US11912781B2 (en) 2021-01-13 2024-02-27 Visterra, Inc. Humanized complement 5A receptor 1 antibodies and methods of use thereof
WO2022184805A1 (en) 2021-03-03 2022-09-09 Immatics Biotechnologies Gmbh Antigen binding proteins specifically binding sars-cov-2 antigenic peptides in complex with a major histocompatibility complex protein
WO2022219080A1 (en) 2021-04-14 2022-10-20 INSERM (Institut National de la Santé et de la Recherche Médicale) New method to improve nk cells cytotoxicity
WO2022219076A1 (en) 2021-04-14 2022-10-20 INSERM (Institut National de la Santé et de la Recherche Médicale) New method to improve the anti-tumoral activity of macrophages
TW202309071A (zh) 2021-05-05 2023-03-01 德商英麥提克生物技術股份有限公司 特異性結合ｐｒａｍｅ之抗原結合蛋白
WO2023274342A1 (zh) * 2021-06-30 2023-01-05 江苏恒瑞医药股份有限公司 特异性结合baff和il-12/23的抗原结合分子及用途
AU2022380477A1 (en) 2021-11-02 2024-05-16 Visterra, Inc. Fc variants with abolished binding to fcgammar and c1q
EP4198052A1 (en) 2021-12-15 2023-06-21 Eberhard Karls Universität Tübingen Medizinische Fakultät Peptides and antigen binding proteins for use in immunotherapy against fibrolamellar hepatocellular carcinoma (fl-hcc) and other cancers
WO2023166420A1 (en) * 2022-03-03 2023-09-07 Pfizer Inc. Multispecific antibodies and uses thereof
TW202400660A (zh) 2022-05-06 2024-01-01 瑞士商伊克諾斯科學公司 Cd3/bcma/cd38 三特異性抗體
WO2023222665A1 (en) 2022-05-18 2023-11-23 Sanofi Method, system and apparatus for predicting pk values of antibodies
WO2023237690A2 (en) 2022-06-08 2023-12-14 Sanofi Methods for purification of multi-specific antibodies

Family Cites Families (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2122732C (en) 1991-11-25 2008-04-08 Marc D. Whitlow Multivalent antigen-binding proteins
US5736137A (en) 1992-11-13 1998-04-07 Idec Pharmaceuticals Corporation Therapeutic application of chimeric and radiolabeled antibodies to human B lymphocyte restricted differentiation antigen for treatment of B cell lymphoma
JP3095175B2 (ja) 1992-11-13 2000-10-03 アイデック ファーマシューティカルズ コーポレイション Ｂ細胞リンパ腫の治療のためのヒトｂリンパ球限定分化抗原に対するキメラ抗体と放射能標識抗体の療法利用
JP3720353B2 (ja) * 1992-12-04 2005-11-24 メディカル リサーチ カウンシル 多価および多重特異性の結合タンパク質、それらの製造および使用
UA40577C2 (uk) * 1993-08-02 2001-08-15 Мерк Патент Гмбх Біспецифічна молекула, що використовується для лізису пухлинних клітин, спосіб її одержання, моноклональне антитіло (варіанти), фармацевтичний препарат, фармацевтичний набір (варіанти), спосіб видалення пухлинних клітин
US5989830A (en) * 1995-10-16 1999-11-23 Unilever Patent Holdings Bv Bifunctional or bivalent antibody fragment analogue
US6090382A (en) 1996-02-09 2000-07-18 Basf Aktiengesellschaft Human antibodies that bind human TNFα
BRPI9707379C8 (pt) 1996-02-09 2017-12-12 Abbott Biotech Ltd composições farmacêuticas compreendendo anticorpo humano recombinante geneticamente engenheirado, e anticorpo humano recombinante geneticamente engenheirado.
EP0894135B1 (en) 1996-04-04 2004-08-11 Unilever Plc Multivalent and multispecific antigen-binding protein
US6057098A (en) 1997-04-04 2000-05-02 Biosite Diagnostics, Inc. Polyvalent display libraries
NZ521540A (en) * 2000-04-11 2004-09-24 Genentech Inc Multivalent antibodies and uses therefor
US20030133939A1 (en) * 2001-01-17 2003-07-17 Genecraft, Inc. Binding domain-immunoglobulin fusion proteins
KR100927261B1 (ko) * 2001-01-17 2009-11-18 트루비온 파마슈티칼스, 인코포레이티드 결합 도메인-면역글로불린 융합 단백질
US20050003403A1 (en) 2003-04-22 2005-01-06 Rossi Edmund A. Polyvalent protein complex
US9963510B2 (en) * 2005-04-15 2018-05-08 Macrogenics, Inc. Covalent diabodies and uses thereof
US7612181B2 (en) 2005-08-19 2009-11-03 Abbott Laboratories Dual variable domain immunoglobulin and uses thereof
CN107226864A (zh) * 2007-06-21 2017-10-03 宏观基因有限公司 共价双抗体及其用途
CA2696263C (en) * 2007-08-15 2017-06-13 Bing Liu Monospecific and multispecific antibodies and method of use
EP2050764A1 (en) 2007-10-15 2009-04-22 sanofi-aventis Novel polyvalent bispecific antibody format and uses thereof
US20090162359A1 (en) * 2007-12-21 2009-06-25 Christian Klein Bivalent, bispecific antibodies
AU2009335798B2 (en) * 2008-12-19 2014-11-27 Macrogenics, Inc. Covalent diabodies and uses thereof
EP3916011A1 (en) 2009-06-26 2021-12-01 Regeneron Pharmaceuticals, Inc. Readily isolated bispecific antibodies with native immunoglobulin format
UY32808A (es) * 2009-07-29 2011-02-28 Abbott Lab Inmunoglobulinas como dominio variable dual y usos de las mismas
US9493578B2 (en) * 2009-09-02 2016-11-15 Xencor, Inc. Compositions and methods for simultaneous bivalent and monovalent co-engagement of antigens
EP3235508B1 (en) * 2011-03-16 2020-12-30 Sanofi Compositions comprising a dual v region antibody-like protein
TWI588156B (zh) 2011-03-28 2017-06-21 賽諾菲公司 具有交叉結合區定向之雙重可變區類抗體結合蛋白
US20140213772A1 (en) * 2012-12-28 2014-07-31 Abbvie, Inc. Cross-over dual variable domain immunoglobulin constructs
CN107428835B (zh) * 2015-01-23 2021-11-26 赛诺菲 抗cd3抗体、抗cd123抗体和与cd3和/或cd123特异性结合的双特异性抗体
CR20180539A (es) * 2016-04-13 2019-02-15 Sanofi Sa Proteínas de unión triespecíficas y/o trivalentes
US11186649B2 (en) * 2017-10-10 2021-11-30 Sanofi Anti-CD38 antibodies and methods of use
AU2019357467A1 (en) * 2018-10-09 2021-05-27 Sanofi Trispecific anti-CD38, anti-CD28, and anti-CD3 binding proteins and methods of use for treating viral infection
US11613576B2 (en) * 2019-04-09 2023-03-28 Sanofi Trispecific binding proteins, methods, and uses thereof

Cited By (135)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9493578B2 (en) 2009-09-02 2016-11-15 Xencor, Inc. Compositions and methods for simultaneous bivalent and monovalent co-engagement of antigens
US9605061B2 (en) 2010-07-29 2017-03-28 Xencor, Inc. Antibodies with modified isoelectric points
US11008389B2 (en) 2011-03-16 2021-05-18 Sanofi Uses of a dual V region antibody-like protein
US20180155450A1 (en) * 2011-03-28 2018-06-07 Sanofi Dual Variable Region Antibody-Like Binding Proteins Having Cross-Over Binding Region Orientation
US9505834B2 (en) 2011-04-27 2016-11-29 Abbvie Inc. Methods for controlling the galactosylation profile of recombinantly-expressed proteins
US9365645B1 (en) 2011-04-27 2016-06-14 Abbvie, Inc. Methods for controlling the galactosylation profile of recombinantly-expressed proteins
US11186638B2 (en) 2011-09-12 2021-11-30 Genzyme Corporation Anti-αβTCR antibody
US10851178B2 (en) 2011-10-10 2020-12-01 Xencor, Inc. Heterodimeric human IgG1 polypeptides with isoelectric point modifications
US9957318B2 (en) 2012-04-20 2018-05-01 Abbvie Inc. Protein purification methods to reduce acidic species
US9505833B2 (en) 2012-04-20 2016-11-29 Abbvie Inc. Human antibodies that bind human TNF-alpha and methods of preparing the same
US9708400B2 (en) 2012-04-20 2017-07-18 Abbvie, Inc. Methods to modulate lysine variant distribution
US9683033B2 (en) 2012-04-20 2017-06-20 Abbvie, Inc. Cell culture methods to reduce acidic species
US9512214B2 (en) 2012-09-02 2016-12-06 Abbvie, Inc. Methods to control protein heterogeneity
EP4223783A2 (en) 2012-09-12 2023-08-09 Genzyme Corporation Fc containing polypeptides with altered glycosylation and reduced effector function
US10836813B2 (en) 2012-09-12 2020-11-17 Genzyme Corporation Fc containing polypeptides with altered glycosylation and reduced effector function
US10738133B2 (en) 2013-01-14 2020-08-11 Xencor, Inc. Heterodimeric proteins
US10472427B2 (en) 2013-01-14 2019-11-12 Xencor, Inc. Heterodimeric proteins
US10487155B2 (en) 2013-01-14 2019-11-26 Xencor, Inc. Heterodimeric proteins
US9650446B2 (en) 2013-01-14 2017-05-16 Xencor, Inc. Heterodimeric proteins
US11718667B2 (en) 2013-01-14 2023-08-08 Xencor, Inc. Optimized antibody variable regions
US11053316B2 (en) 2013-01-14 2021-07-06 Xencor, Inc. Optimized antibody variable regions
US9701759B2 (en) 2013-01-14 2017-07-11 Xencor, Inc. Heterodimeric proteins
US11634506B2 (en) 2013-01-14 2023-04-25 Xencor, Inc. Heterodimeric proteins
US10738132B2 (en) 2013-01-14 2020-08-11 Xencor, Inc. Heterodimeric proteins
US10131710B2 (en) 2013-01-14 2018-11-20 Xencor, Inc. Optimized antibody variable regions
US9738722B2 (en) 2013-01-15 2017-08-22 Xencor, Inc. Rapid clearance of antigen complexes using novel antibodies
US10494439B2 (en) 2013-03-11 2019-12-03 Genzyme Corporation Hyperglycosylated binding polypeptides
WO2014164534A2 (en) 2013-03-11 2014-10-09 Genzyme Corporation Site-specific antibody-drug conjugation through glycoengineering
EP4063389A2 (en) 2013-03-11 2022-09-28 Genzyme Corporation Site-specific antibody-drug conjugation through glycoengineering
EP4098663A1 (en) 2013-03-11 2022-12-07 Genzyme Corporation Hyperglycosylated binding polypeptides
WO2014164503A1 (en) 2013-03-11 2014-10-09 Genzyme Corporation Hyperglycosylated binding polypeptides
US11807690B2 (en) 2013-03-11 2023-11-07 Genzyme Corporation Hyperglycosylated binding polypeptides
US11130816B2 (en) 2013-03-11 2021-09-28 Genzyme Corporation Site-specific antibody-drug conjugation through glycoengineering
US10214589B2 (en) 2013-03-11 2019-02-26 Genzyme Corporation Site-specific antibody-drug conjugation through glycoengineering
EP3424956A1 (en) 2013-03-11 2019-01-09 Genzyme Corporation Hyperglycosylated binding polypeptides
US10968276B2 (en) 2013-03-12 2021-04-06 Xencor, Inc. Optimized anti-CD3 variable regions
US9708399B2 (en) 2013-03-14 2017-07-18 Abbvie, Inc. Protein purification using displacement chromatography
US9499614B2 (en) 2013-03-14 2016-11-22 Abbvie Inc. Methods for modulating protein glycosylation profiles of recombinant protein therapeutics using monosaccharides and oligosaccharides
US10858417B2 (en) 2013-03-15 2020-12-08 Xencor, Inc. Heterodimeric proteins
US10106624B2 (en) 2013-03-15 2018-10-23 Xencor, Inc. Heterodimeric proteins
US9605084B2 (en) 2013-03-15 2017-03-28 Xencor, Inc. Heterodimeric proteins
US10519242B2 (en) 2013-03-15 2019-12-31 Xencor, Inc. Targeting regulatory T cells with heterodimeric proteins
US11814423B2 (en) 2013-03-15 2023-11-14 Xencor, Inc. Heterodimeric proteins
US10544187B2 (en) 2013-03-15 2020-01-28 Xencor, Inc. Targeting regulatory T cells with heterodimeric proteins
US11299554B2 (en) 2013-03-15 2022-04-12 Xencor, Inc. Heterodimeric proteins
US10287364B2 (en) 2013-03-15 2019-05-14 Xencor, Inc. Heterodimeric proteins
US10005835B2 (en) 2013-04-29 2018-06-26 Sanofi Anti-IL-4/anti-IL-13 bispecific antibody formulations
US20150071927A1 (en) * 2013-09-11 2015-03-12 The University Of Hong Kong Anti-her2 and anti-igf-ir bi-specific antibodies and uses thereof
US9481729B2 (en) * 2013-09-11 2016-11-01 The University Of Hong Kong Anti-HER2 and anti-IGF-IR BI-specific antibodies and uses thereof
US9598667B2 (en) 2013-10-04 2017-03-21 Abbvie Inc. Use of metal ions for modulation of protein glycosylation profiles of recombinant proteins
US9688752B2 (en) 2013-10-18 2017-06-27 Abbvie Inc. Low acidic species compositions and methods for producing and using the same using displacement chromatography
US9522953B2 (en) 2013-10-18 2016-12-20 Abbvie, Inc. Low acidic species compositions and methods for producing and using the same
US9499616B2 (en) 2013-10-18 2016-11-22 Abbvie Inc. Modulated lysine variant species compositions and methods for producing and using the same
US9550826B2 (en) 2013-11-15 2017-01-24 Abbvie Inc. Glycoengineered binding protein compositions
WO2015073884A2 (en) 2013-11-15 2015-05-21 Abbvie, Inc. Glycoengineered binding protein compositions
US11697690B2 (en) 2014-03-19 2023-07-11 Genzyme Corporation Site-specific glycoengineering of targeting moieties
EP4015535A1 (en) 2014-03-19 2022-06-22 Genzyme Corporation Site-specific glycoengineering of targeting moieties
US10995148B2 (en) 2014-03-19 2021-05-04 Genzyme Corporation Site-specific glycoengineering of targeting moieties
US9822186B2 (en) 2014-03-28 2017-11-21 Xencor, Inc. Bispecific antibodies that bind to CD38 and CD3
US11840579B2 (en) 2014-03-28 2023-12-12 Xencor, Inc. Bispecific antibodies that bind to CD38 and CD3
US10858451B2 (en) 2014-03-28 2020-12-08 Xencor, Inc. Bispecific antibodies that bind to CD38 and CD3
US10647777B2 (en) 2014-05-28 2020-05-12 Genzyme Corporation Methods of controlling the formation of disulfide bonds in protein solutions
US9884921B2 (en) 2014-07-01 2018-02-06 Pfizer Inc. Bispecific heterodimeric diabodies and uses thereof
US11680096B2 (en) 2014-09-03 2023-06-20 Boehringer Ingelheim International Gmbh Compound targeting IL-23A and TNF-alpha and uses thereof
US10793629B2 (en) 2014-09-03 2020-10-06 Boehringer Ingelheim International Gmbh Compound targeting IL-23A and TNF-alpha and uses thereof
WO2016036918A1 (en) 2014-09-03 2016-03-10 Boehringer Ingelheim International Gmbh Compound targeting il-23a and tnf-alpha and uses thereof
US10059763B2 (en) 2014-09-03 2018-08-28 Boehringer Ingelheim International Gmbh Compound targeting IL-23A and TNF-alpha and uses thereof
EP3189153A4 (en) * 2014-09-03 2018-02-28 Boehringer Ingelheim International GmbH Compound targeting il-23a and tnf-alpha and uses thereof
US11160874B2 (en) 2014-10-09 2021-11-02 Genzyme Corporation Glycoengineered antibody drug conjugates
US10064952B2 (en) 2014-10-09 2018-09-04 Genzyme Corporation Glycoengineered antibody drug conjugates
EP3799887A1 (en) 2014-10-09 2021-04-07 Genzyme Corporation Glycoengineered antibody drug conjugates
WO2016073406A1 (en) 2014-11-05 2016-05-12 Eli Lilly And Company Anti-tnf-/anti-il-23 bispecific antibodies
US9718884B2 (en) 2014-11-05 2017-08-01 Eli Lilly And Company Anti-TNF-/anti-IL-23 bispecific antibodies
US9850320B2 (en) 2014-11-26 2017-12-26 Xencor, Inc. Heterodimeric antibodies to CD3 X CD20
US10259887B2 (en) 2014-11-26 2019-04-16 Xencor, Inc. Heterodimeric antibodies that bind CD3 and tumor antigens
US11945880B2 (en) 2014-11-26 2024-04-02 Xencor, Inc. Heterodimeric antibodies that bind CD3 and tumor antigens
US11352442B2 (en) 2014-11-26 2022-06-07 Xencor, Inc. Heterodimeric antibodies that bind CD3 and CD38
US10913803B2 (en) 2014-11-26 2021-02-09 Xencor, Inc. Heterodimeric antibodies that bind CD3 and tumor antigens
US11859011B2 (en) 2014-11-26 2024-01-02 Xencor, Inc. Heterodimeric antibodies that bind CD3 and tumor antigens
US10889653B2 (en) 2014-11-26 2021-01-12 Xencor, Inc. Heterodimeric antibodies that bind CD3 and tumor antigens
US11225528B2 (en) 2014-11-26 2022-01-18 Xencor, Inc. Heterodimeric antibodies that bind CD3 and tumor antigens
US11673972B2 (en) 2014-11-26 2023-06-13 Xencor, Inc. Heterodimeric antibodies that bind CD3 and tumor antigens
US11111315B2 (en) 2014-11-26 2021-09-07 Xencor, Inc. Heterodimeric antibodies that bind CD3 and tumor antigens
US9856327B2 (en) 2014-11-26 2018-01-02 Xencor, Inc. Heterodimeric antibodies to CD3 X CD123
US10526417B2 (en) 2014-11-26 2020-01-07 Xencor, Inc. Heterodimeric antibodies that bind CD3 and CD38
US10428155B2 (en) 2014-12-22 2019-10-01 Xencor, Inc. Trispecific antibodies
US10227411B2 (en) 2015-03-05 2019-03-12 Xencor, Inc. Modulation of T cells with bispecific antibodies and FC fusions
US11091548B2 (en) 2015-03-05 2021-08-17 Xencor, Inc. Modulation of T cells with bispecific antibodies and Fc fusions
US11571462B2 (en) 2015-06-03 2023-02-07 The Medical College Of Wisconsin, Inc. Engineered CCL20 locked dimer polypeptide
US10738095B2 (en) 2015-06-03 2020-08-11 The Medical College Of Wisconsin, Inc. Engineered CCL20 locked dimer polypeptide
US11779651B2 (en) 2015-10-25 2023-10-10 Sanofi Bivalent, bispecific binding proteins for prevention or treatment of HIV infection
US11129905B2 (en) 2015-10-25 2021-09-28 Sanofi Bivalent, bispecific binding proteins for prevention or treatment of HIV infection
US10227410B2 (en) 2015-12-07 2019-03-12 Xencor, Inc. Heterodimeric antibodies that bind CD3 and PSMA
US11623957B2 (en) 2015-12-07 2023-04-11 Xencor, Inc. Heterodimeric antibodies that bind CD3 and PSMA
US11192960B2 (en) 2016-04-13 2021-12-07 Sanofi Trispecific and/or trivalent binding proteins
US10882922B2 (en) 2016-04-13 2021-01-05 Sanofi Trispecific and/or trivalent binding proteins
US11932704B2 (en) 2016-04-13 2024-03-19 Sanofi Trispecific and/or trivalent binding proteins
US11236170B2 (en) 2016-06-14 2022-02-01 Xencor, Inc. Bispecific checkpoint inhibitor antibodies
US10787518B2 (en) 2016-06-14 2020-09-29 Xencor, Inc. Bispecific checkpoint inhibitor antibodies
US11492407B2 (en) 2016-06-14 2022-11-08 Xencor, Inc. Bispecific checkpoint inhibitor antibodies
US10316088B2 (en) 2016-06-28 2019-06-11 Xencor, Inc. Heterodimeric antibodies that bind somatostatin receptor 2
US11225521B2 (en) 2016-06-28 2022-01-18 Xencor, Inc. Heterodimeric antibodies that bind somatostatin receptor 2
US10793632B2 (en) 2016-08-30 2020-10-06 Xencor, Inc. Bispecific immunomodulatory antibodies that bind costimulatory and checkpoint receptors
US10550185B2 (en) 2016-10-14 2020-02-04 Xencor, Inc. Bispecific heterodimeric fusion proteins containing IL-15-IL-15Rα Fc-fusion proteins and PD-1 antibody fragments
US10501543B2 (en) 2016-10-14 2019-12-10 Xencor, Inc. IL15/IL15Rα heterodimeric Fc-fusion proteins
US11084863B2 (en) 2017-06-30 2021-08-10 Xencor, Inc. Targeted heterodimeric Fc fusion proteins containing IL-15 IL-15alpha and antigen binding domains
WO2019027780A1 (en) 2017-08-02 2019-02-07 Eli Lilly And Company BISPECIFIC ANTI-TNF- / ANTI-IL-23 IGG ANTIBODIES
US11365261B2 (en) 2017-10-10 2022-06-21 Sanofi Anti-CD38 antibodies and methods of use
US11186649B2 (en) 2017-10-10 2021-11-30 Sanofi Anti-CD38 antibodies and methods of use
US11312770B2 (en) 2017-11-08 2022-04-26 Xencor, Inc. Bispecific and monospecific antibodies using novel anti-PD-1 sequences
US10981992B2 (en) 2017-11-08 2021-04-20 Xencor, Inc. Bispecific immunomodulatory antibodies that bind costimulatory and checkpoint receptors
US11319355B2 (en) 2017-12-19 2022-05-03 Xencor, Inc. Engineered IL-2 Fc fusion proteins
WO2019147973A1 (en) 2018-01-26 2019-08-01 Genzyme Corporation Fc variants with enhanced binding to fcrn and prolonged half-life
US10982006B2 (en) 2018-04-04 2021-04-20 Xencor, Inc. Heterodimeric antibodies that bind fibroblast activation protein
US11524991B2 (en) 2018-04-18 2022-12-13 Xencor, Inc. PD-1 targeted heterodimeric fusion proteins containing IL-15/IL-15Ra Fc-fusion proteins and PD-1 antigen binding domains and uses thereof
US11505595B2 (en) 2018-04-18 2022-11-22 Xencor, Inc. TIM-3 targeted heterodimeric fusion proteins containing IL-15/IL-15RA Fc-fusion proteins and TIM-3 antigen binding domains
US11358999B2 (en) 2018-10-03 2022-06-14 Xencor, Inc. IL-12 heterodimeric Fc-fusion proteins
US11530268B2 (en) 2018-10-09 2022-12-20 Sanofi Trispecific anti-CD38, anti-CD28, and anti-CD3 binding proteins and methods of use for treating viral infection
US11472890B2 (en) 2019-03-01 2022-10-18 Xencor, Inc. Heterodimeric antibodies that bind ENPP3 and CD3
US11613576B2 (en) 2019-04-09 2023-03-28 Sanofi Trispecific binding proteins, methods, and uses thereof
CN114269784A (zh) * 2019-06-07 2022-04-01 美国安进公司 具有选择性可切割接头的双特异性结合构建体
WO2021016571A2 (en) 2019-07-25 2021-01-28 Genzyme Corporation Methods of treating antibody-mediated disorders with fcrn antagonists
CN114258400A (zh) * 2019-08-15 2022-03-29 詹森生物科技公司 用于改进的单链可变片段的材料和方法
US11787875B2 (en) * 2019-08-15 2023-10-17 Janssen Biotech, Inc. Materials and methods for improved single chain variable fragments
US20210047435A1 (en) * 2019-08-15 2021-02-18 Janssen Biotech, Inc. Materials and methods for improved single chain variable fragments
WO2021174034A1 (en) 2020-02-28 2021-09-02 Genzyme Corporation Modified binding polypeptides for optimized drug conjugation
US11879004B2 (en) 2020-02-28 2024-01-23 Genzyme Corporation Modified binding polypeptides for optimized drug conjugation
US11919956B2 (en) 2020-05-14 2024-03-05 Xencor, Inc. Heterodimeric antibodies that bind prostate specific membrane antigen (PSMA) and CD3
US11591401B2 (en) 2020-08-19 2023-02-28 Xencor, Inc. Anti-CD28 compositions
US11919958B2 (en) 2020-08-19 2024-03-05 Xencor, Inc. Anti-CD28 compositions
US11739144B2 (en) 2021-03-09 2023-08-29 Xencor, Inc. Heterodimeric antibodies that bind CD3 and CLDN6
US11859012B2 (en) 2021-03-10 2024-01-02 Xencor, Inc. Heterodimeric antibodies that bind CD3 and GPC3
WO2022249146A1 (en) 2021-05-27 2022-12-01 Sanofi Fc VARIANT WITH ENHANCED AFFINITY TO Fc RECEPTORS AND IMPROVED THERMAL STABILITY
WO2023227790A1 (en) 2022-05-27 2023-11-30 Sanofi Natural killer (nk) cell engagers binding to nkp46 and bcma variants with fc-engineering
WO2024089609A1 (en) 2022-10-25 2024-05-02 Ablynx N.V. Glycoengineered fc variant polypeptides with enhanced effector function

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