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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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  • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
  • C07K1/08—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using activating agents
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  • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
  • C07K1/08—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using activating agents
  • C07K1/088—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using activating agents containing other elements, e.g. B, Si, As
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  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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  • C07—ORGANIC CHEMISTRY
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  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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  • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
  • C07K16/244—Interleukins [IL]
  • C07K16/247—IL-4
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  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
  • C07K16/244—Interleukins [IL]
  • C07K16/248—IL-6
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  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
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  • C07—ORGANIC CHEMISTRY
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  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
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  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/46—Hybrid immunoglobulins
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  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/46—Hybrid immunoglobulins
  • C07K16/461—Igs containing Ig-regions, -domains or -residues form different species
• • C—CHEMISTRY; METALLURGY
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  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/46—Hybrid immunoglobulins
  • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
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  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
  • C07K2317/526—CH3 domain
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

• the invention concerns antigen-binding proteins or antibodies having heterodimers of heavy chains, i.e., two immunoglobulin heavy chains that differ by at least one amino acid, that allows isolation of the antigen-binding protein based on a differential affinity of an immunoglobulin heavy chain and a modified or mutated immunoglobulin heavy chain toward an affinity reagent.
• the invention also concerns antigen-binding proteins (including bispecific antibodies) that have IgG CH2 and CH3 regions with different affinities with respect to Protein A that allows rapid isolation by differential binding of the IgG regions to Protein A.
• Antibodies are multifunctional molecules, carrying a unique binding specificity for a target antigen, as well as the capacity to interact with the immune system via mechanisms that are antigen-independent.
• Many currently used biological therapeutics for cancer are monoclonal antibodies directed against antigens that are typically overexpressed on the targeted cancer cell. When such antibodies bind tumor cells, they may trigger antibody- dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).
• ADCC antibody- dependent cellular cytotoxicity
• CDC complement-dependent cytotoxicity
• bispecific antibodies In recent years, efforts have been underway to develop antibody-like therapeutics that have more than one antigen binding specificity, e.g., bispecific antibodies.
• multi-specific formats could allow the possibility of using, e.g., one specificity to target the molecule to a tumor cell antigen, the other specificity to trigger a response that is not normally available to the immune system.
• Bispecific antibodies may also find use as surrogate ligands for two-component heterodimeric receptor systems that are normally activated by their natural ligand when it binds to and brings together both components.
• scFv single chain variable fragment
• An scFv consists of an antibody's light and heavy chain variable regions fused via a flexible linker, which generally can fold appropriately and so that the regions can bind the cognate antigen.
• a BiTE concatenates two scFv's of different specificities in tandem on a single chain (see FIG. 1A). This configuration precludes the production of molecules with two copies of the same heavy chain variable region.
• the linker configuration is designed to ensure correct pairing of the respective light and heavy chains.
• the BiTE format has several disadvantages. First, scFv molecules are notorious for their tendency to aggregate. And although the immunogenicity of scFv linkers is reputedly low, the possibility of generating antibodies against a BiTE cannot be ruled out. The absence of an Fc portion in the BiTE format also makes its serum half-life very short, and this necessitates the complication of frequent repeated administrations or continuous infusion via a pump. Finally, the absence of an Fc also implies the absence of Fc-mediated effector functions, which may be beneficial in some circumstances.
• a second format is a hybrid of a mouse and a rat monoclonal antibody, and relies on a modification of conventional Protein A affinity chromatography (see, e.g., Lindhofer, H. et al. (1995) J. Immunol. 155:219-225)).
• a mouse lgG2a and a rat lgG2b antibody are 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 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 easily purified from this mixture because its binding properties to Protein A are different from those of the parental antibodies: rat lgG2b does not bind to protein A, whereas the mouse lgG2a does. Consequently, the mouse-rat heterodimer binds to Protein A but elutes at a higher pH than the mouse lgG2a homodimer, and this makes selective purification of the bispecific heterodimer possible.
• this hybrid format has two monovalent antigen binding sites.
• the disadvantage of the mouse/rat hybrid is that because it is non-human, it is likely to provoke an immune response in the patient, which could have deleterious side effects (e.g. "HAMA” or "HARA” reactions), and/or neutralize the therapeutic.
• HAMA or "HARA” reactions
• [001O]A third format, referred to as "knobs-into-holes" (FIG. 1 C) has been discussed in the prior art as potentially useful for the production of bispecific antibodies (US Patent No. 7,183,076).
• the Fc portions of two antibodies are engineered to give one a protruding "knob”, and the other a complementary "hole.”
• the heavy chains are said to preferentially form heterodimers rather than homodimers, by association of the engineered "knobs” with the engineered "holes.” Issues of correct light- heavy chain pairing are addressed by choosing antibodies that have different specificities but employ identical light chains.
• FIG. 1 illustrates three bispecific antibody formats: (A) Bispecific T cell Engager
• FIG. 2 illustrates the Fc ⁇ Adp modification: (A) Alignment of Fc regions of human
• IgGI SEQ ID NO:1
• lgG3 SEQ ID NO:3
• FIG. 3 illustrates an alignment of human CH3 domains (employing IMGT exon numbering and EU numbering) of IgGI , lgG2, and lgG4, with and without the ⁇ Adp dipeptide modification, as well as lgG3.
• FIG. 4 shows a Protein A column trace for isolation of bispecific antibodies, showing an elution profile utilizing a step gradient.
• FIG. 5 shows IL4-Ra and IL-6Ra BIACORETM binding profile of eluted column fractions from the chromatographic separation shown in FIG. 4. Antibodies in fractions were captured with immobilized anti-Fc antibodies, and then soluble IL-4Ra or IL-6Ra were assayed for binding to the captured antibodies.
• FIG. 6 shows a pharmacokinetic profile of an Fc ⁇ Adp bispecific antibody (IL-6R ⁇ /IL-
• IL-6R ⁇ /IL-6R ⁇ an Fc ⁇ Adp homodimer
• IgGI antibody with wild-type CH3 sequence IL-4R/IL-4R
• control monospecific antibody IL-6R ⁇ /IL-6R ⁇
• FIG. 7 illustrates efficacy of a CD20xCD3 ⁇ Adp bispecific antibody in a Raji cell killing assay.
• FIG. 8 illustrates a bystander cell (293) killing assay with the CD20xCD3 ⁇ Adp bispecific antibody.
• FIG. 9 shows results for expression experiments using different mFc heterodimers.
• Panel A Western blot of pH separation of heterodimeric mlgG2a/mlgG2aPTTTK from homodimeric mlgG2a and homodimeric lgG2aPTTTK
• Panel B Western blot of pH separation of heterodimeric mlgG2a/mlgG2aTTTK from homodimeric mlgG2a and homodimeric lgG2aTTTK
• IP input
• FT flow through
• W2 second wash (1x PBS pH
• FIG. 10 illustrates preferential formation of heterodimers of mutant lgG2a over formation of heterodimers of mixed isotypes (e.g., mlgG2a andm IgGI), using IFNAR1 construct: I FNAR2 construct ratio of 4: 1.
• Lane 1 IFNAR1-lgG2a:IFNAR2-lgG1 ;
• Lane 2 IFNAR1 construct ratio of 4: 1.
• the invention is based at least in part on employing two immunoglobulin CH3 heavy chain constant domain sequences that differ by at least one amino acid in a bispecific antigen-binding protein.
• the at least one amino acid difference results in an improved ability to isolate the protein, because the difference results in a differential ability of the CH3 domain sequences to bind an affinity agent.
• an antigen-binding protein comprising a first and a second polypeptide, the first polypeptide comprising, from N-terminal to C-terminal, a first antigen- binding region that selectively binds a first antigen, followed by a constant region that comprises a first CH3 region of a human IgG selected from IgGI (SEQ ID NO:1), lgG2 (SEQ ID NO:3), lgG4 (SEQ ID NO:5), and a combination thereof; and, a second polypeptide comprising, from N-terminal to C-terminal, a second antigen-binding region that selectively binds a second antigen, followed by a constant region that comprises a second CH3 region of a human IgG selected from IgGI , lgG2, lgG4, and a combination thereof, wherein the second CH3 region comprises a modification that reduces or eliminates binding of the second CH3 domain to protein A.
• the second CH3 region comprises an 95R modification (by
• the second CH3 region further comprises a 96F modification (IMGT; 436F by EU).
• the second CH3 region is selected from SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6.
• the second CH3 region is from a modified human IgGI (SEQ ID NO: 1]
• the second CH3 region is from a modified human lgG2 (SEQ ID NO: 1]
• the second CH3 region is from a modified human lgG4 (SEQ ID NO: 1
• the CH3 domain is a chimeric domain that comprises sequences of two or more of human IgGI , human lgG2, human lgG3, and human lgG4.
• the CH3 domain is from human IgGI , human lgG2, or human lgG4, and the antigen-binding protein further comprises a CH1 domain and a CH2 domain, wherein the CH1 domain and the CH2 domain are independently selected from the group consisting of a human IgGI CH1 or CH2 domain, a human lgG2 CH1 or CH2 domain, or a chimeric human/human lgG1/lgG2 or a chimeric human/human lgG1/lgG3 or a chimeric human/human lgG2/lgG3 domain or a chimeric human/human lgG1/lgG4 or a chimeric lgG3/lgG4 or a chimeric lgG2/lgG4 domain.
• the chimeric lgG1/lgG2, lgG1/lgG3, lgG2/lgG3, lgG1/lgG4, lgG3/lgG4, and igG2/!gG4 domains are non- immunogenic or substantially non-immunogenic in a human.
• the antigen-binding protein further comprises an immunoglobulin light chain.
• the immunoglobulin light chain is selected from a human lambda and a human kappa light chain.
• the first and the second antigen-binding regions each comprise at least one CDR, in another embodiment, at least two CDRs, in another embodiment, each comprises three CDRs.
• the CDRs are from an immunoglobulin heavy chain.
• the heavy chain is a human heavy chain.
• the first antigen-binding region comprises a first immunoglobulin heavy chain variable domain
• the second antigen-binding region comprises a second immunoglobulin heavy chain variable domain.
• the first and the second immunoglobulin heavy chain variable domains are independently selected from a mouse, rat, hamster, rabbit, monkey, ape, and human domain.
• the first and the second immunoglobulin heavy chain variable domains independently comprise a human CDR, a mouse CDR, a rat CDR, a rabbit CDR, a monkey CDR, an ape CDR, and a humanized CDR.
• the CDR is human and is somatically mutated.
• the first and the second immunoglobulin heavy chain variable domain comprises a human framework region (FR).
• the human FR is a somatically mutated human FR.
• the first and/or the second antigen-binding regions are obtained by screening a phage library comprising antibody variable regions for reactivity toward an antigen of interest.
• the first and/or the second antigen-binding regions are obtained by immunizing a non-human animal such as a mouse, a rat, a rabbit, a monkey, or an ape with an antigen of interest and identifying an antibody variable region nucleic acid sequence encoding a variable region specific for the antigen of interest.
• the non-human animal comprises one or more human immunoglobulin variable region genes.
• the one or more human immunoglobulin variable region genes are present in the non-human animal extrachromosomally, as a replacement at an endogenous immunoglobulin locus, or as a transgene randomly integrated into the genome of the non-human animal.
• the first and/or the second antigen-binding regions are obtained from a hybridoma or a quadroma, in another embodiment from screening immune cells of an immunized non-human animal using cell sorting.
• the antigen-binding protein is a bispecific antibody.
• the bispecific antibody is a fully human bispecific antibody and has an affinity for each epitope, independently, in the micromolar, nanomolar, or picomolar range.
• the antigen-binding protein is non-immunogenic or substantially non-immunogenic in a human.
• the antigen-binding protein lacks a non-native human T-cell epitope.
• the modification of the CH3 region is non-immunogenic or substantially non-immunogenic in a human.
• the modification of the CH3 region does not result in a non-native human T-cell epitope.
• the antigen-binding protein comprises a heavy chain, wherein the heavy chain is non-immunogenic or substantially non-immunogenic in a human.
• the heavy chain has an amino acid sequence that does not contain a non- native T cell epitope.
• the heavy chain comprises an amino acid sequence whose proteolysis cannot form an amino acid sequence of about 9 amino acids that is immunogenic in a human.
• the human is a human being treated with the antigen-binding protein.
• the heavy chain comprises an amino acid sequence whose proteolysis cannot form an amino acid sequence of about 13 to about 17 amino acids that is immunogenic in a human.
• the human is a human being treated with the antigen-binding protein.
• a bispecific binding protein comprising a CH2 and/or CH3 modification as described herein is provided, wherein the bispecific binding protein comprises a first binding moiety that specifically recognizes an antigen on a B cell, and a second binding moiety that specifically recognizes an antigen on a T cell.
• the binding protein is a bispecific antibody.
• the bispecific antibody comprises a human IgGI heavy chain and a human IgGi ⁇ Adp heavy chain.
• the first binding moiety is a human heavy chain variable domain that specifically recognizes CD20.
• the second binding moiety is a human heavy chain variable domain that specifically recognizes CD3.
• the bispecific antibody exhibits an EC50 in a Raji killing assay of about 2.8-3.2 x 1 Cr 12 M, or about 2.8-3.0 x 10 "12 M, and exhibits no more than about 1-10%, or 1-5%, bystander killing in a bystander cell killing assay, wherein the bystander cell does not comprise a CD20 epitope.
• the bystander cell is a 293 cell.
• bystander cell killing in the assay is measured across a concentration of bispecific antibody of about 10 "8 M to about 10 "14 M.
• a method for making a bispecific antibody comprising: obtaining a nucleic acid sequence encoding a first immunoglobulin heavy chain comprising a first variable domain that recognizes a first epitope, wherein the first immunoglobulin heavy chain comprises an IgGI , lgG2, or lgG4 isotype constant domain, or a chimeric isotype constant domain thereof; obtaining a second nucleic acid sequence encoding a second immunoglobulin heavy chain comprising a second variable domain that recognizes a second epitope, wherein the second immunoglobulin heavy chain comprises an IgGI , lgG2, or lgG4 isotype constant domain, or a chimeric isotype constant domain thereof, that comprises a modification in its CH3 domain that eradicates or reduces binding to Protein A; obtaining a third nucleic acid sequence encoding an immunoglobulin a light chain that pairs with the first and the second immunoglobulin heavy chain; introducing
• the cell is selected from a CHO, COS, 293, HeLa, and a retinal cell expressing a viral nucleic acid sequence (e.g., a PERC.6TM cell).
• a bispecific antigen-binding protein comprises a first specificity that binds an antigen and a second specificity that activates a receptor, wherein the bispecific antigen-binding protein comprises a first polypeptide comprising a first IgGI , lgG2, or lgG4 CH3 domain that comprises a Protein A-binding determinant, and a second polypeptide comprising a second IgGI , lgG3, or lgG4 CH3 domain that lacks the Protein A- binding determinant.
• the second specificity that activates the receptor binds the receptor with a K 0 that is in the molar, millimolar, micromolar, nanomolar, or picomolar range.
• the second specificity binds a receptor selected from a G-protein coupled receptor, a receptor tyrosine kinase, an integrin, and a toll-like receptor.
• the second specificity contacts the receptor and causes the receptor or a subunit or a protein physically associated therewith to undertake phosphorylation of a serine, threonine, or tyrosine; cause cyclicization of a nucleotide (e.g., cAMP, cADP, or cGMP); cause production of a phosphatidylinositol or derivative thereof
• a nucleotide e.g., cAMP, cADP, or cGMP
• lipid second messenger e.g., diacylclycerol, ceramide, lysophosphatidic acid, an eicosanoid
• dephosphorylation e.g., phosphatase activity
• cause phosphorylation of a lipid to form a second messenger cause hydrolysis of a second messenger; cause proteolysis; cause redox signaling; cause translocation of a protein to a cellular organelle (e.g., to the nucleus); cause the receptor to aggregate (with itself) to form homo- or (with other receptors) to form heteromultimers; or cause the opening or closing of a transmembrane channel.
• a method for making a bispecific antibody comprising: isolating a bispecific antibody of interest from a quadroma, wherein the bispecific antibody of interest comprises a first heavy chain that is an IgGI , lgG2, or lgG4 isotype, a second heavy chain that is an IgGI , lgG2, or lgG4 isotype having a constant domain that comprises a modification in its CH3 domain that eradicates or reduces binding to Protein A, wherein the bispecific antibody of interest is isolated using Protein A.
• a method for making a bispecific antibody comprising a step of isolating from a disrupted cell or a mixture of antibodies a bispecific antibody having differentially modified IgGI , lgG2, or lgG4 CH3 domains, wherein the differentially modified
• CH3 domains are non-immunogenic or substantially non-immunogenic in a human, and wherein the modification results in a bispecific antibody with a heterodimeric heavy chain whose monomers have a differential affinity for Protein A, and the bispecific antibody is isolated from the disrupted cell or the mixture using Protein A.
• the bispecific antibody is isolated using a Protein A affinity support, wherein the bispecific antibody elutes at a pH between about 3.9 to about 4.4, about 4.0 to about 4.3, about 4.1 to about 4.2, or at about pH 4.2. In one embodiment, the bispecific antibody elutes at a pH of about 4, 4.1 , 4.2, 4.3, 4.4, or 4.5.
• the bispecific antibody is isolated using a Protein A affinity support and a pH gradient or step, wherein the pH gradient or step includes an ionic modifier.
• the ionic modifier is present at a concentration of about 0.5 to about 1.0 molar.
• the ionic modifier is a salt.
• the ionic modifier is selected from the group consisting of beryllium, lithium, sodium, and potassium salts of acetate; sodium and potassium bicarbonates; lithium, sodium, potassium, and cesium carbonates; lithium, sodium, potassium, cesium, and magnesium chlorides; sodium and potassium fluorides; sodium, potassium, and calcium nitrates; sodium and potassium phosphates; and calcium and magnesium sulfates.
• the ionic modifier is a halide salt of an alkaline metal or alkaline earth metal.
• the ionic modifier is sodium chloride.
• a binding protein comprising an Fc, wherein the Fc comprises a first CH3 domain that is modified as described herein and a second CH3 that is not modified, so as to form a heterodimeric Fc, wherein the differential modification results in the binding protein eluting from a protein A affinity material at 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.3, or 1.4 pH unit(s) higher than a corresponding binding protein that lacks the differential modification.
• the differentially modified binding protein elutes at a pH of about 4.2, whereas the unmodified binding protein elutes at a pH of about 3.
• the differentially modified binding protein elutes at a pH of about 4.5, whereas the unmodified binding protein elutes at a pH of about 3.5. In one embodiment, the differentially modified binding protein elutes at a pH of about 4, whereas the unmodified binding protein elutes at a pH of about 2.8-3.5, 2.8-3.2, or 2.8-3. In one embodiment, the differentially modified binding protein elutes at a pH of about 4.2, whereas the unmodified binding protein elutes at a pH of about 2.8. In one embodiment, the differentially modified binding protein elutes at a pH of about 4.4, whereas the unmodified binding protein elutes at a pH of about 3.6.
• unmodified refers to lack of a modification at 435 (EU numbering), or lack of a modification at 435 and 436 (EU numbering), on both of the CH3 domains.
• antibody includes immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
• Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region.
• the heavy chain constant region comprises three domains, CH1 , CH2 and CH3.
• Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region.
• the light chain constant region comprises one domain, CL.
• VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
• CDR complementarity determining regions
• FR framework regions
• Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1 , HCDR2 and HCDR3; light chain CDRs may be abbreviated as LCDR1 , LCDR2 and LCDR3.
• high affinity antibody refers to those antibodies having a binding affinity to their target of at least 10 "9 M, at leasti O “10 M; at least 10 "11 M; or at least 10 "12 M, as measured by surface plasmon resonance, e.g., BIACORETM or solution-affinity ELISA.
• antigen-binding protein includes a protein having at least one CDR and that is capable of selectively recognizing an antigen, i.e., is capable of binding an antigen with a K D that is at least in the micromolar range.
• Therapeutic antigen-binding proteins e.g., therapeutic antibodies
• Antigen-binding protein also includes a protein comprising a first and a second CH3 domain as described herein and a first protein or ligand recognition domain and a second protein or ligand recognition domain, wherein the first protein or ligand recognition domain and the second protein or ligand recognition domain each independently recognize the same protein or ligand, or together recognize the same protein or ligand, or each independently recognize a different protein or ligand.
• a protein comprising a fusion protein (hetero- or homo-) dimer wherein the polypeptides of the dimer are fusion polypeptides that comprise a receptor component or a ligand component, wherein the ligand component comprises an amino acid sequence that binds a receptor.
• bispecific antibody includes an antibody capable of selectively binding two or more epitopes.
• Bispecific antibodies generally comprise two different heavy chains, with each heavy chain specifically binding a different epitope — either on two different molecules (e.g., antigens) or on the same molecule (e.g., on the same antigen). If a bispecific antibody is capable of selectively binding two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope will generally be at least one to two or three or four orders of magnitude lower than the affinity of the first heavy chain for the second epitope, and vice versa.
• the epitopes recognized by the bispecific antibody can be on the same or a different target (e.g., on the same or a different protein).
• Bispecific antibodies can be made, for example, by combining heavy chains that recognize different epitopes of the same antigen.
• nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen can be fused to nucleic acid sequences encoding different heavy chain constant regions, and such sequences can be expressed in a cell that expresses an immunoglobulin light chain.
• a typical bispecific antibody has two heavy chains each having three heavy chain CDRs, followed by (N-terminal to C-terminal) a CH1 domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain that either does not confer antigen-binding specificity but that can associate with each heavy chain, or that can associate with each heavy chain and that can bind one or more of the epitopes bound by the heavy chain antigen-binding regions, or that can associate with each heavy chain and enable binding or one or both of the heavy chains to one or both epitopes.
• cell includes any cell that is suitable for expressing a recombinant nucleic acid sequence.
• Cells include those of prokaryotes and eukaryotes (single-cell or multiple- cell), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., etc.), mycobacteria cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P.
• the cell is a human, monkey, ape, hamster, rat, or mouse cell.
• the cell is eukaryotic and is selected from the following cells: CHO (e.g., CHO K1 , DXB-1 1 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cell, Vero, CV1 , kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21 ), Jurkat, Daudi, A431 (epidermal), CV-1 , U937, 3T3, L cell, C127 cell, SP2/0, NS-O, MMT 060562, Sertoli cell, BRL 3A cell, HT1080 cell, myeloma cell, tumor cell, and a cell line derived from an aforementioned cell.
• CHO e.g., CHO K1 , DXB-1 1 CHO, Veggie-CHO
• the cell comprises one or more viral genes, e.g. a retinal cell that expresses a viral gene (e.g., a PER.C6TM cell).
• a viral gene e.g., a PER.C6TM cell.
• CDR complementarity determining region
• a CDR can be encoded by, for example, a germline sequence or a rearranged or unrearranged sequence, and, for example, by a naive or a mature B cell or a T cell.
• CDRs can be encoded by two or more sequences (e.g., germline sequences) that are not contiguous (e.g., in an unrearranged nucleic acid sequence) but are contiguous in a B cell nucleic acid sequence, e.g., as the result of splicing or connecting the sequences (e.g., V-D-J recombination to form a heavy chain CDR3).
• heavy chain or “immunoglobulin heavy chain” includes an immunoglobulin heavy chain constant region sequence from any organism, and unless otherwise specified includes a heavy chain variable domain.
• Heavy chain variable domains include three heavy chain CDRs and four FR regions, unless otherwise specified. Fragments of heavy chains include CDRs, CDRs and FRs, and combinations thereof.
• a typical heavy chain has, following the variable domain (from N-terminal to C-terminal), a CH1 domain, a hinge, a CH2 domain, and a CH3 domain.
• a functional fragment of a heavy chain includes a fragment that is capable of specifically recognizing an antigen (e.g., recognizing the antigen with a K D in the micromolar, nanomolar, or picomolar range), that is capable of expressing and secreting from a cell, and that comprises at least one CDR.
• an antigen e.g., recognizing the antigen with a K D in the micromolar, nanomolar, or picomolar range
• the phrase "Fc-containing protein” includes antibodies, bispecific antibodies, immunoadhesins, and other binding proteins that comprise at least a functional portion of an immunoglobulin CH2 and CH3 region.
• a “functional portion” refers to a CH2 and CH3 region that can bind a Fc receptor (e.g., an Fc ⁇ R; or an FcRn, i.e., a neonatal Fc receptor), and/or that can participate in the activation of complement. If the CH2 and CH3 region contains deletions, substitutions, and/or insertions or other modifications that render it unable to bind any Fc receptor and also unable to activate complement, the CH2 and CH3 region is not functional.
• a Fc receptor e.g., an Fc ⁇ R; or an FcRn, i.e., a neonatal Fc receptor
• Fc-containing proteins can comprise modifications in immunoglobulin domains, including where the modifications affect one or more effector function of the binding protein (e.g., modifications that affect Fc ⁇ R binding, FcRn binding and thus half-life, and/or CDC activity).
• modifications affect one or more effector function of the binding protein (e.g., modifications that affect Fc ⁇ R binding, FcRn binding and thus half-life, and/or CDC activity).
• Such modifications include, but are not limited to, the following modifications and combinations thereof, with reference to EU numbering of an immunoglobulin constant region: 238, 239, 248, 249, 250, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 297, 298, 301 , 303, 305, 307, 308, 309, 311 , 312, 315, 318, 320, 322, 324, 326, 327, 328, 329, 330, 331 , 332, 333, 334, 335, 337, 338, 339, 340, 342, 344, 356, 358, 359, 360, 361 , 362, 373, 375, 376, 378, 380, 382, 383, 384, 386, 388, 389, 398, 414, 416, 419, 428,
• the binding protein is an Fc-containing protein and exhibits enhanced serum half-life (as compared with the same Fc-containing protein without the recited modification(s)) and have a modification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or a modification at 428 and/or 433 (e.g., L/R/SI/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification at 250 and/or 428; or a modification at 307 or 308 (e.g., 308F, V308F), and 434.
• a modification at position 250 e.g., E or Q
• 250 and 428 e.g., L or F
• 252 e.g
• the modification can comprise a 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I (e.g., V259I), and a 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y 1 254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Q and M428L); a 307 and/or 308 modification (e.g., 308F or 308P).
• a 428L e.g., M428L
• 434S e.g., N434S
• 428L, 259I e.g., V259I
• a 308F e.g., V308
• ionic modifier includes moieties that reduce the effect of, or disrupt, non-specific (i.e., non-affinity) ionic interactions between proteins.
• Ionic modifiers include, for example, salts, ionic combinations of Group I and Group Il metals with acetate, bicarbonate, carbonate, a halogen (e.g., chloride or fluoride), nitrate, phosphate, or sulfate.
• a non-limiting illustrative list of "ionic modifiers” includes beryllium, lithium, sodium, and potassium salts of acetate; sodium and potassium bicarbonates; lithium, sodium, potassium, and cesium carbonates; lithium, sodium, potassium, cesium, and magnesium chlorides; sodium and potassium fluorides; sodium, potassium, and calcium nitrates; sodium and potassium phosphates; and calcium and magnesium sulfates.
• Ionic modifiers include those moieties that affect ionic interactions that, upon addition to a pH gradient or step, or upon equilibration of a Protein A support in an "ionic modifier" and application of a pH step or gradient, results in a broadening of pH unit distance between elution of a homodimeric IgG and a heterodimeric IgG (e.g., a wild-type human IgG and the same IgG but bearing one or more modifications of its CH3 domain as described herein).
• a suitable concentration of an "ionic modifier” can be determined by its concentration employing the same column, pH step or gradient, with increasing concentration of "ionic modifier” until a maximal pH distance is reached at a given pH step or pH gradient.
• Light chain includes an immunoglobulin light chain constant region sequence from any organism, and unless otherwise specified includes human kappa and lambda light chains.
• Light chain variable (VL) domains typically include three light chain CDRs and four framework (FR) regions, unless otherwise specified.
• FR framework
• a full-length light chain includes, from amino terminus to carboxyl terminus, a VL domain that includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant domain.
• Light chains that can be used with this invention include those, e.g., that do not selectively bind either the first or second antigen selectively bound by the antigen-binding protein.
• Suitable light chains include those that can be identified by screening for the most commonly employed light chains in existing antibody libraries (wet libraries or in silico), where the light chains do not substantially interfere with the affinity and/or selectivity of the antigen-binding domains of the antigen-binding proteins. Suitable light chains include those that can bind one or both epitopes that are bound by the antigen-binding regions of the antigen-binding protein. [0069]The phrase "micromolar range” is intended to mean 1-999 micromolar; the phrase “nanomolar range” is intended to mean 1-999 nanomolar; the phrase “picomolar range” is intended to mean 1-999 picomolar.
• the phrase "somatically mutated” includes reference to a nucleic acid sequence from a B cell that has undergone class-switching, wherein the nucleic acid sequence of an immunoglobulin variable region (e.g., a heavy chain variable domain or including a heavy chain CDR or FR sequence) in the class-switched B cell is not identical to the nucleic acid sequence in the B cell prior to class-switching, such as, for example, a difference in a CDR or framework nucleic acid sequence between a B cell that has not undergone class- switching and a B cell that has undergone class-switching.
• an immunoglobulin variable region e.g., a heavy chain variable domain or including a heavy chain CDR or FR sequence
• Somatically mutated includes reference to nucleic acid sequences from affinity-matured B cells that are not identical to corresponding sequences in B cells that are not affinity-matured (i.e., sequences in the genome of germline cells).
• the phrase “somatically mutated” also includes reference to a nucleic acid sequence from a B cell after exposure of the B cell to an antigen of interest, wherein the nucleic acid sequence differs from the corresponding nucleic acid sequence prior to exposure of the B cell to the antigen of interest.
• variable domain includes an amino acid sequence of an immunoglobulin light or heavy chain (modified as desired) that comprises the following amino acid regions, in sequence from N-terminal to C-terminal (unless otherwise indicated): FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.
• a "variable domain” includes an amino acid sequence capable of folding into a canonical domain (VH or VL) having a dual beta sheet structure wherein the beta sheets are connected by a disulfide bond between a residue of a first beta sheet and a second beta sheet.
• Bispecific Antibodies With Modified IgG CH3 Regions The inventors have developed a novel format that combines a common light chain strategy with an implementation of a selective Protein A purification scheme that can be used with human antibody components.
• IgGI ⁇ A a single point mutation in IgGI should be sufficient to create the different binding affinities amenable to a new purification scheme.
• This modification will be referred to as IgGI ⁇ A, to denote its inability to bind Protein A (and, similarly, lgG2 ⁇ A and lgG4 ⁇ A — or more generally, Fc ⁇ A).
• the specified point mutation introduces a novel peptide sequence across the mutation, which could potentially be immunogenic.
• the point mutation could, in theory, be loaded onto an MHC class Il molecule and presented to T cells, and consequently elicit an immune response.
• a dipeptide mutation H435R/Y436F (EU numbering; H95R/Y96F by IMGT) can be used.
• the resulting sequence in the vicinity of the alteration is identical to that of lgG3 (see FIG. 2A), and would therefore be expected to be immunologically "invisible,” because there would be no non-native short peptides available for presentation to T cells. It has been reported that this double mutant still does not bind Protein A (Jendeberg, L.
• the dipeptide mutation does not include any of the residues that form the Fc dimer interface, so it is unlikely to interfere with the formation of heterodimers.
• This dipeptide mutation is designated as "IgGi ⁇ Adp" (and, similarly, lgG2 ⁇ Adp, lgG4 ⁇ Adp, and Fc ⁇ Adp). Placement of the dipeptide modification in IgGI , lgG2, and lgG4 are indicated in FIG.
• the Fc ⁇ Adp modification does not include any of the residues believed to form the Fc dimer interface, so the Fc ⁇ Adp modification is unlikely to interfere with the formation of heterodimers. Because the Fc ⁇ Adp is so minimal, it can likely be incorporated into other engineered Fc forms as well. lgG2 ⁇ Adp and lgG4 ⁇ Adp may be advantageous in situations in which the effector functions (or lack thereof) associated with each of the latter are desired. [0078] In summary, the bispecific antibody format described above includes two antibodies of different specificity that use the same light chain, wherein the Fc region of one of them is modified to the Fc ⁇ Adp format (see FIG. 2B).
• Bispecific Binding Proteins With Modified Mouse IgG CH Regions The inventors have devised a method for readily isolating a binding protein comprising an immunoglobulin heavy chain (or functional CH2 and CH3-containing fragment thereof) that is heterodimeric with respect to one or more amino acids in the CH3 domain.
• a careful selection of modifications of a mouse IgG CH domain and application of a particular separation technology confer the ability to readily isolate a binding protein comprising two differentially modified mouse CH regions from homodimers and from heterodimers that do not contain the modifications.
• Mouse IgGI which contains a proline at 247, a threonine at 252, 254, and 256, and a lysine at 258, binds only weakly to protein A.
• Mouse lgG2a and lgG2b contain different residues at those positions (with the exception of lgG2b positions 256 and 258), and mouse lgG2a and 2b bind well to protein A.
• Differentially modifying the CH regions of two mouse IgGs in a method for making an antibody that is heterodimeric for heavy chains would confer a differential protein A binding characteristic to such an antibody.
• a differential protein A isolation scheme is devised that allows for ready separation of a modified heterodimer from any mouse IgG homodimer, whether it is a homodimer of IgGI (which would bind only quite weakly, if at all, to protein A), or a homodimer of mouse lgG2a, a homodimer of mouse lgG2b, or a heterodimer of lgG2a/lgG2b.
• a bispecific antibody having two different heavy chain variable domains but the same isotype e.g., lgG2a
• lgG2a can be expressed in a suitable expression system that employs the heavy chain sequences, wherein only one of the lgG2a CH regions is modified to reduce or eliminate a protein A binding determinant.
• only one of the lgG2a CH regions will exhibit a substantial affinity for protein A, and any antibody formed from a dimer of an unmodified lgG2a and a modified lgG2a will be readily isolated from the modified heterodimer.
• an antibody wherein a single CH region of an Fc dimer comprises the modified CH region, whereas the other CH of the Fc dimer lacks them.
• the mouse IgG CH region is modified to comprise particular amino acids at particular positions (EU numbering), selected from the group consisting of: 252T, 254T, and 256T; 252T, 254T, 256T, and 258K; 247P, 252T, 254T, 256T, and 258K; 435R and 436F; 252T, 254T, 256T, 435R, and 436F; 252T, 254T, 256T, 258K, 435R, and 436F; 24tP, 252T, 254T, 256T, 258K, 435R, and 436F; and, 435R.
• a particular group of modifications is made, selected from the groups consisting of: M252T, S254T, S256T; M252T, S254T, S256T, I258K; I247P, M252T, S254T, S256T, I258K; H435R, H436F; M252T, S254T, S256T, H435R, H436F; M252T, S254T, S256T, I258K, H435R, H436F; I247P, M252T, S254T, S256T, I258K, H435R, H436F; and, H435R.
• Heterodimeric mouse IgG-based binding proteins can be used for a variety of applications. For example, they allow for a method of isolating bispecific antibodies with mouse constant domains, wherein the modifications do not interfere or do not substantially interfere with binding of the antibody to one or more mouse Fc receptors, such that the antibody can participate, e.g., in ADCC or CDC and also bind two or more epitopes on the same or different target.
• a method for isolating a binding protein comprising a first mouse IgG CH region and a second mouse IgG CH region, wherein the first IgG CH region is modified (but not the second IgG CH region) so as to reduce or eliminate protein A binding affinity of the first mouse IgG CH region but not the second mouse IgG CH region, and wherein the binding protein comprises a first binding moiety that binds a first epitope and a second binding moiety that binds a second epitope.
• the modification does not alter or does not substantially alter binding affinity of the binding protein to an Fc receptor.
• the binding protein comprises a modification that increases or decreases affinity for the binding protein to an Fc receptor.
• the modification does not alter or does not substantially alter the serum half-life of the binding protein in a mouse comprising native mouse Fc ⁇ R receptors and/or a native mouse FcRn, as compared with a corresponding binding protein that lacks the modification.
• the modification does not alter or does not substantially alter the serum half-life of the binding protein in a mouse that comprises a replacement of native mouse high and low affinity Fc ⁇ R receptors and/or an FcRn receptor, as compared with a corresponding binding protein that lacks the modification.
• first and the second epitope are different and are on different cells or on different proteins. In one embodiment, the first epitope and the second epitope are different and are on the same cell or the same protein.
• the Fc receptor is selected from a high affinity Fc receptor, a low affinity Fc receptor, and a FcRn.
• the Fc receptor is selected from one or more of a mouse FcRn, a mouse Fc ⁇ R, a mouse Fc ⁇ RIIB, a mouse Fc ⁇ RIII, a mouse
• the Fc receptor is selected form one or more of a human FcRn, a human Fc ⁇ R, a human Fc ⁇ RIIB, a human Fc ⁇ RIIC, a human Fc ⁇ RIMB, a human Fc ⁇ RIIIA, a human Fc ⁇ RIIA, and a combination thereof.
• One advantage of many embodiments of the invention is the ability to employ the modification(s) to make a bispecific antibody that is both readily isolable based on differential binding to Protein A and is also non-immunogenic or substantially non-immunogenic in a human.
• This feature makes such embodiments particularly useful in making bispecific antibodies for human therapeutic use, and in making immunoadhesins, e.g., that are non- immunogenic or substantially non-immunogenic (employing human binding moieties, i.e., human receptor components and/or human ligands).
• This feature is associated with bispecific antibodies having CH3 domains with the H95R/Y96F (IMGT numbering) modifications of IgGI , lgG2, and lgG3, and those CH3 domains that contain further modifications that result in the position being modified reflecting a wild-type sequence of a different IgG isotype.
• the modification is not found in nature associated with the particular IgG isotype, the modified sequence is locally identical with a wild-type sequence of a different IgG isotype, and the modification is not expected to be immunogenic or substantially immunogenic. It is also possible that a modification is non-immunogenic even if its sequence is not locally identical to any native sequence; such modifications would be equally useful.
• bispecific antibodies are provided that are non-immunogenic or substantially non-immunogenic in a human, with respect to their heavy chain constant domains, but nonetheless bear one or more differential modifications of the heavy chain constant domain, including a modification that results in a differential affinity of the heavy chain constant domains with respect to an affinity reagent (e.g., Protein A).
• affinity reagent e.g., Protein A
• the bispecific antibody that is non- immunogenic or substantially non-immunogenic in a human with respect to its CH3 domain, yet having differentially modified heavy chains is a human IgGI , lgG2, or lgG4 comprising a CH3 domain that comprises one of the following modifications (or, in another embodiment, consists essentially of one of the following modifications): H95R, or H95R and Y96F (IMGT numbering).
• bispecific antibodies are expected to be non-immunogenic, or substantially non- immunogenic, with respect to humans in whom tolerance to human IgGI , lgG2, and lgG4 isoforms has not been broken to any significant degree.
• the Fc ⁇ Adp modification is expected to be immunologically "invisible” because the binding groove of MHC class Il molecules accommodates a 9-mer that comprises the major determinant recognized by variable loops of the T cell receptor, so that peptides lacking any native 9-mer subsequence would appear unlikely to elicit an immune response.
• peptides longer than 9-mers are bound by MHC class II, and it is possible that protruding segments may potentially influence binding. Therefore, additional modifications (over the Fc ⁇ Adp modification) that eliminate longer non- native sequences may further reduce potential for immunogenicity.
• V422I EU; V82I by IMGT numbering
• L445P EU; L105P by IMGT numbering
• the binding site for Protein A overlaps with the binding site for the neonatal Fc receptor, FcRN, which is thought to be responsible for conferring a prolonged serum half-life to immunoglobulins. Modifications in the vicinity of the Protein A binding site, therefore, raise the possibility that the format proposed here could have a shorter serum half-life than those of IgGI , 2, and 4, given that human lgG3 has a shorter serum half-life (about 7 days) than the other IgG subclasses (about 21 days). Some Fc mutants affecting His435 have been shown not to bind FcRN, and have a shorter half-life in mice.
• the serum half-life of the lgG1 ⁇ A/lgG1 heterodimer is not appreciably different from that of the IgGI homodimer (see Example 2).
• the IgGi ⁇ Adp mutation has the advantage of ablating Protein A binding while still preserving the longer half-life of IgGl
• a bispecific antigen-binding protein that comprises a modification of a CH3 domain as described herein, wherein the antigen-binding protein displays a pharmacokinetic profile equivalent to the same bispecific antigen-binding protein that lacks the modification at the CH3 domain.
• a bispecific antibody is provided that comprises an lgG1 ⁇ A/lgG1 heterodimeric Fc, wherein the bispecific antibody has a serum half-life that is about 1.5-fold, about 2-fold, about 2.5-fold, or about 3 fold higher than a bispecific antibody that is otherwise identical but comprises an lgG3 CH3 domain, or that is otherwise identical but comprises at least one lgG3 heavy chain.
• a bispecific antibody comprising an lgG1 ⁇ A/lgG1 heterodimeric Fc, wherein the bispecific antibody exhibits a serum half-life that is about the same as that of the bispecific antibody without the IgGi ⁇ A modification (i.e., an IgGI homodimeric bispecific antibody).
• Immunoglobulin heavy chain variable regions that can be used to generate bispecific antibodies with desired characteristics (e.g., desired specificities, desired affinities, desired functionalities, e.g., blocking, non-blocking, inhibiting, activating, etc.) can be generated using any method known in the art.
• the desired heavy chains can then be constructed by cloning nucleic acid sequences containing the variable regions in constructs having the desired heavy chain constant regions described herein.
• the first heavy chain comprises a variable region that is encoded by a nucleic acid that is derived from the genome of a mature B cell of a first animal that has been immunized with a first antigen, and the first heavy chain specifically recognizes the first antigen.
• the second heavy chain comprises a variable region that is encoded by a nucleic acid that is derived from the genome of a mature B cell of a second animal that has been immunized with a second antigen, and the second heavy chain specifically recognizes the second antigen.
• the first animal and/or the second animal is a genetically modified animal comprising an unrearranged human immunoglobulin heavy chain variable region. In one embodiment, the first animal and/or the second animal is a genetically modified animal comprising an unrearranged human immunoglobulin heavy chain variable region and a human immunoglobulin constant region. In one embodiment, the first animal and/or the second animal is a genetically modified mouse that comprises an unrearranged human immunoglobulin heavy chain variable region.
• Immunoglobulin heavy chain variable region sequences can be obtained by any other method known in the art, e.g., by phage display, and sequences obtained thereby can be employed to make nucleic acid constructs to be joined to nucleic acids encoding any suitable heavy chain, e.g., heavy chains with modified CH3 domains as described herein, and placed in an expression construct and transferred to a cell that is capable of making the heavy chain, e.g., in the presence of a suitable light chain.
• suitable heavy chain e.g., heavy chains with modified CH3 domains as described herein
• Bispecific antibodies comprising two heavy chains that recognize two different epitopes (or two different antigens) are more easily isolated where they can pair with the same light chain (i.e., light chains having identical variable and constant domains).
• a variety of methods are known in the art for generating light chains that can pair with two heavy chains of differing specificity, while not interfering or not substantially interfering with the selectivity and/or affinity of the heavy chain variable domain with its target antigen.
• a light chain can be selected by surveying usage statistics for all light chain variable domains, identifying the most frequently employed light chain in human antibodies, and pairing that light chain with the two heavy chains of differing specificity.
• a light chain can be selected by observing light chain sequences in a phage display library (e.g., a phage display library comprising human light chain variable region sequences, e.g., a human ScFv library) and selecting the most commonly used light chain variable region from the library.
• a phage display library e.g., a phage display library comprising human light chain variable region sequences, e.g., a human ScFv library
• a light chain can be selected by assaying a phage display library of light chain variable sequences using the heavy chain variable sequences of both heavy chains as probes.
• a light chain that associates with both heavy chain variable sequences is selected as a light chain for the heavy chains and allows binding and/or activation with respect to both epitopes.
• a light chain can be selected by combining known light chains with desired heavy chains and assaying the resulting bispecific antibody for binding specificity, affinity, and/or activation ability.
• the light chain can be aligned with the heavy chains' cognate light chains, and modifications are made in the light chain to more closely match sequence characteristics common to the cognate light chains of both heavy chains.
• the modifications preferably result in sequences that are present in known human light chain sequences, such that proteolytic processing is unlikely to generate a T cell epitope based on parameters and methods known in the art for assessing the likelihood of immunogenicity (i.e., in silico as well as wet assays).
• compositions and methods are particularly useful in making human bispecific antibodies, i.e., bispecific antibodies comprising human constant and variable domains.
• human antibodies include those having heavy chain variable and heavy chain constant domains derived from human germline immunoglobulin sequences, in some embodiments derived from somatically mutated human immunoglobulin sequences (generated, e.g., in an animal that comprises human immunoglobulin gene sequences).
• the human variable and/or constant regions may include amino acid residues not encoded by human germline immunoglobulin sequences or encoded as the result of recombination and/or splicing for example in the CDRs and in particular CDR3.
• Human antibodies are not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Those antibodies are referred to as humanized or chimeric antibodies. Human antibodies do include those comprising mutations, e.g., introduced in vitro by random or site-specific mutagenesis, but the mutations are preferably non-immunogenic in a human.
• Chimeric antibodies are antibodies in which one of a heavy chain variable region or framework or CDR or heavy chain constant region or domain are from different species (e.g., human and mouse, or human and primate).
• chimeric antibodies include antibodies having a heavy chain variable region of non-human origin (e.g., mouse) and a heavy chain constant region of human origin.
• chimeric antibodies include antibodies having a heavy chain variable region of human origin and a heavy chain constant region of non-human (e.g., mouse) origin.
• regions of mouse origin are identical or substantially identical to a mouse immunoglobulin germline sequence with or without somatic hypermutations.
• Chimeric antibodies also include antibodies having a light chain constant region that is identical or substantially identical to a human immunoglobulin germline sequence and a non-human (e.g., mouse) heavy chain or chimeric human/nonhuman heavy chain.
• Chimeric antibodies include antibodies having a light chain constant domain that is identical or substantially identical to a non-human (e.g., mouse) immunoglobulin germline sequence and a human or chimeric nonhuman/human heavy chain.
• compositions and methods are for making an affinity-matured antibody.
• an affinity-matured antibody comprises one or more alterations in one or more CDRs that result in higher affinity (e.g., K 0 in the nanomolar or picomolar range) of the antibody for its target antigen as compared to a substantially identical antibody that lacks the alteration(s).
• Affinity-matured antibodies can be made by any suitable method known in the art, e.g., by random or site-directed mutagenesis of CDRs and/or framework regions followed by affinity screening, VH domain shuffling, efc.
• the antibodies are neutralizing antibodies.
• Neutralizing antibodies include antibodies capable of neutralizing, inhibiting, or preventing an antigen's biological activity.
• Neutralizing antibodies include those that, upon binding an antigen, prevent or reduce the antigen's ability to act on a natural target of the antigen in vivo and in vitro.
• Examples of neutralizing antibodies include an antibody to a protein ligand of a biological receptor that prevents the ligand from binding the receptor, or an antibody to a biological receptor that prevents the receptor from binding its ligand, where ligand binding in the absence of the antibody causes the receptor to effect a change inside of a cell. Determining whether an antibody is a neutralizing antibody generally entails conducting a functional assay wherein the antibody's effect on the biological activity of the antigen is measured.
• compositions of the invention are also useful in a variety of applications for antibodies and other binding proteins. A short description of some useful applications are provided here.
• Bispecific binding proteins that comprise binding specificity toward a tumor antigen and a T-cell antigen can be made that target an antigen on a cell, e.g., CD20, and also target an antigen on a T-cell, e.g., CD3.
• the bispecific antibody targets both a cell of interest in a patient (e.g., B cell in a lymphoma patient, via CD20 binding) as well as a T-cell of the patient.
• the bispecific antibody in various embodiments, is designed so as to activate the T-cell upon binding CD3, thus coupling T-cell activation to a specific, selected tumor cell.
• Bispecific binding proteins that comprise two binding moieties that are each directed to a binding partner (i.e., each directed to a different target) on the surface of the same cell can also be made.
• This design is particularly suited to targeting specific cells or cell types that express both targets on the surface of the same cell.
• targets might appear individually on other cells, the binding moieties of these binding proteins are selected such that each binding moiety binds its target with a relatively low affinity (e.g., low micromolar, or high nanomolar — e.g., over a hundred nanomolar KD, e.g., 500, 600, 700, 800 nanomolar). In this way, prolonged target binding is favored only in situations where the two targets are in proximity on the same cell.
• Bispecific binding proteins that comprise two binding moieties that bind the same target, each at a different epitope of the same target, can be made. This design is particularly suited for maximizing the probability of successfully blocking a target with binding protein. Multiple extracellular loops, e.g., of a transmembrane channel or a cell surface receptor, can be targeted by the same bispecific binding molecule.
• Bispecific binding proteins that comprise two binding moieties that cluster and activate negative regulators of immune signaling to result in immune suppression can be made. Repression in cis can be achieved where the targets are on the same cell; repression in trans can be achieved where the targets are on different cells. Repression in cis, e.g., can be achieved with a bispecific binding protein having an anti-lgGRIIb binding moiety and an anti-FelD1 binding moiety, such that the IgGRIIb is clustered only in the presence of FeIDI , in order to down-regulate an immune response to FeIDI .
• Repression in trans can be achieved with a bispecific binding protein having an anti-BTLA binding moiety and a binding moiety that specifically binds a tissue-specific antigen of interest, such that clustering of the inhibitory BTLA molecule occurs only in the selected target tissue, which potentially addresses auto-immune diseases.
• Bispecific binding proteins that activate multi-component receptors can be made.
• two binding moieties directed to two components of a receptor bind, cross-link the receptor, and activate signaling from the receptor.
• This can be done, e.g., using a bispecific binding protein with a binding moiety that binds IFNAR1 and a binding moiety that binds IFNAR2, where binding cross-links the receptor.
• a bispecific binding protein can provide an alternative to interferon treatment.
• Bispecific binding proteins that transport binding moieties across a semipermeable barrier e.g., the blood-brain barrier
• one binding moiety binds a target that can transit a particular selective barrier; the other binding moiety targets a molecule with a therapeutic activity, wherein the target molecule with therapeutic activity cannot normally traverse the barrier.
• This kind of bispecific binding protein is useful for bringing therapeutics to tissues that the therapeutic would not otherwise reach. Some examples include targeting the plGR receptor to transport a therapeutic into the gut or lung, or targeting the transferrin receptor to transport a therapeutic across the blood-brain barrier.
• Bispecific binding proteins that transport binding moieties into specific cells or cell types can be made.
• one binding moiety targets a cell surface protein (e.g., a receptor) that is readily internalized into the cell.
• the other binding moiety targets an intracellular protein, where binding of the intracellular protein results in a therapeutic effect.
• Bispecific binding proteins that bind a surface receptor of a phagocytic immune cell and a surface molecule of an infectious pathogen (e.g., a yeast or bacterium), to bring the infectious pathogen in the vicinity of a phagocytic immune cell to facilitate phagocytosis of the pathogen.
• An example of such a design would be a bispecific antibodiy that targets a CD64 or CD89 molecule and also a pathogen.
• Bispecific binding proteins that have an antibody variable region as one binding moiety and a non-lg moiety as a second binding moiety.
• the antibody variable region achieves targeting, whereas the non-lg moiety is an effector or a toxin linked to an Fc.
• the ligand e.g., an effector or toxin
• any two protein moieties can be brought in each other's vicinity in the context of the Fc.
• traps e.g., homo- or heterodimeric trap molecules.
• Nucleic acid sequences encoding monoclonal antibodies can be obtained by any suitable method known in the art. Examples of suitable methods for obtaining monoclonal antibodies (and their nucleic acid sequences) include, for example, by a hybridoma method (see, e.g., Kohler et al. (1975) Nature 256:495-497) or a phage antibody library (see, e.g., see Clackson et al. (1991) Nature 352:624-628).
• the immunoglobulin heavy chain variable domains are derived from nucleic acid sequences of a genetically modified animal or a transgenic animal.
• the regions are derived from an animal that comprises a human immunoglobulin minilocus.
• the regions are derived from mice comprising one or more extrachromosomal nucleic acids that comprise one or more nucleic acids encoding immunoglobulin sequences.
• the animal can have one or more unrearranged human immunoglobulin nucleic acid sequences.
• the animal comprises human light chain variable regions nucleic acid sequences, in some embodiments human heavy chain variable sequences, in some embodiments both heavy and light chain variable sequences, and in some embodiments further comprises human constant region sequences.
• the nucleic acid sequences are derived from a mouse in which endogenous mouse heavy chain variable gene segments and light chain variable gene segments have been replaced with human heavy chain variable gene segments and light chain variable gene segments.
• the nucleic acid sequences are derived from naive B or T cells of such an animal.
• the nucleic acid sequences are derived from B or T cells of an animal that has been immunized with an antigen of interest.
• the nucleic acid sequences are derived from cells by amplifying them with primers, including for example sets of degenerate primers, that comprise one or more FR, joining, or constant sequences.
• the immunoglobulin heavy chain variable domains are derived from nucleic acids of an animal that has been immunized with an antigen of interest.
• a non-human transgenic or genetically modified animal is immunized with the antigen of interest (by, e.g., exposing the animal to the antigen or a cell bearing the antigen or a nucleic acid encoding an expressible form of the antigen), allowing the animal to undergo an immune response, isolating immune cells (e.g., B cells) from the animal, optionally immortalizing the cells, and screening the cells to identify reactivity with the antigen and/or identifying and/or isolating a nucleic acid sequences that encode an immunoglobulin variable region that is capable of recognizing the antigen when placed in the context of an antibody.
• immune cells e.g., B cells
• the cell is a B cell.
• a B cell of the immunized animal is used to make a hybridoma, and a B cell expressing an antibody that specifically recognizes an epitope of the antigen is identified and nucleic acid sequences that encode a variable region amino acid sequence that recognizes the epitope is identified and/or isolated.
• the nucleic acids are derived from humans, non- human primates (e.g., apes such as chimpanzees), monkeys (e.g., cynomologous or rhesus), rodents (e.g., mice, rats, hamsters), donkeys, goats, sheep, etc.
• the heavy chains comprise sequences that are derived from human cells. For example, human fetal cells exposed in vitro to an antigen and placed in a suitable host animal (e.g., a SCID mouse), [00134]
• the nucleic acids are introduced into a cell using a vector.
• Vectors include, for example, plasmids, cosmids, retroviruses, adenoviruses, adeno- associated viruses, plant viruses, YACs, BACs, EBV-derived episomes.
• the nucleic acids are present in an expression vector or expression construct.
• the expression vector or construct is a vector that contains a promoter operably linked to the nucleic acid sequence of interest such that the nucleic acid sequence of interest is capable of being expressed under suitable conditions in a suitable cell.
• Expression vectors or constructs can include leader sequences, enhancers, promoter elements that enhance transcription or translation, transcription stop sequences, splicing sequences, transcription-enhancing introns, IRES elements, marker genes, selection sequences, recombinase recognition sites, homology arms, viral sequences, operators (e.g., prokaryotic operators) etc.
• the expression vectors comprise elements that allow inducible expression, for example, a prokaryotic operator operably linked to a eukaryotic promoter.
• expression is induced upon addition of an expression inducer.
• expression is induced upon removal of an expression inhibitor.
• expression is induced by a temperature change.
• one or more heavy chain nucleotide sequences are on the same vector.
• a heavy chain nucleic acid sequence and a light chain nucleic acid sequence herein are on the same vector.
• two heavy chain nucleic acid sequences and a light chain nucleic acid sequence are on the same vector.
• the nucleic acids are expressed in a cell that comprises one or more nucleic acids from a virus.
• the virus is selected from adenovirus, adeno-associated virus, SV-40, Epstein-Barr virus, a retrovirus, a lentivirus, baculovirus, coronavirus, herpes simplex virus, poliovirus, Semliki Forest virus, Sindbis virus, and Vaccinia virus.
• Host cells are cells that can be transformed to express a nucleic acid of interest.
• transformation includes changing the nucleic acid content of a cell such that it contains exogenous nucleic acids (e.g., a nucleic acid not found in the cell in nature, or one or more additional copies of a nucleic acid corresponding to a nucleic acid sequence found in the cell in nature).
• the nucleic acid content of a cell can be changed by any suitable method known in the art, e.g., by integrating the nucleic acid into the cell's genome or by placing it in the cell in an extra-chromosomal or extra-genomic form.
• the nucleic acid content of the cell can be changed such that the cell transiently expresses the nucleic acid of interest, or the nucleic acid content can be changed such that the cell stably expresses the nucleic acid of interest.
• the change in genetic content of the cell is inherited when the cell divides.
• the Lindhofer approach failed when applied to separating an IgGI homodimer from an IgGI heterodimer having two IgGI heavy chains that were identical but for the fact that one of the IgGI CH3 domains contained an H435R/Y436F dipeptide modification.
• the inventors found that in a linear pH gradient, the dipeptide-modified IgGI eluted at about pH 3.9, whereas the IgGI homodimer eluted at about pH 3.7. This pH difference was deemed insufficient to achieve a satisfactory separation of the heterodimer from the homodimer using the Lindhofer method. The difference was not reproducible in a predictable manner.
• a method for separating a bispecific antigen- binding protein comprising a heterodimeric IgG with one chain comprising a modification as described herein comprises a step of employing a pH gradient in the presence of an ionic modifier.
• the ionic modifier is present at a concentration sufficient to maximize the pH difference between elution from a Protein A support of an IgG homodimer and an IgG heterodimer as described herein (i.e., with CH3 modification(s)).
• the ionic modifier is present at a concentration of about 0.5 to about 1.0 molar. In another specific embodiment, the ionic modifier is present at a concentration of about 0.15 to about 0.5 molar.
• the ionic modifier is a salt.
• the ionic modifier is a salt of an alkaline metal or an alkaline earth metal and a halogen.
• the salt is a chloride salt of an alkaline metal or an alkaline earth metal, e.g., NaCI, KCI, LiCI, CaCI 2 , MgCI 2 .
• the salt is present at a molarity of about 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0.
• the pH gradient is from about pH 3.9 to about pH 4.5, in another embodiment from about pH 4.0 to about pH 4.4., and in another embodiment about pH 4.1 to about pH 4.3. In a specific embodiment, the gradient is a linear gradient.
• the pH gradient is a step gradient. In one embodiment, the method comprises applying to an equilibrated Protein A column (equilibrated, e.g., in PBS or another suitable buffer or liquid) a step of about pH 3.9, about pH 4.0, about pH 4.1 , about pH 4.2, about pH 4.3, or about pH 4.4. In a specific embodiment, the step is about pH 4.2.
• the bispecific antibody comprising the heterodimeric IgG
• CH3 domain elutes from the Protein A support in one or more fractions substantially free of non-heterodimeric IgG.
• the eluted bispecific antibody fraction(s) comprise less than about 1 %, 0.5%, or 0.1 % of total protein by weight that is non- heterodimeric antibody.
• IL-4Ra and one against IL-6Ra had light chains that differed by only four amino acids.
• Co-expression experiments revealed that the light chain of the anti-IL-4Ra antibody could be replaced with the light chain from the IL-6Ra and still maintain high affinity binding to !L-4Ra, thus making it feasible to produce a bispecific antibody using the anti-IL-4Ra heavy chain and the anti-IL-6Ra heavy chain and the same light chain.
• the heavy chain of the IL-6Ra antibody was modified to the Fc ⁇ Adp form ⁇ i.e., CH3 dipeptide modification H95R/Y96F, by IMGT exon numbering).
• the anti-IL-6Ra light chain was then co-expressed with the anti-IL4Ra/Fc and anti-IL6Ra/Fc ⁇ Adp heavy chains in CHO cells, and conditioned medium from these cells was subjected to Protein A chromatography. After loading the Protein A column with cell supernatants containing a mixture of homo- and heterodimers, the elution was performed with a pH step gradient produced by varying combinations of two buffers (A: 100 mM Na citrate, 15OmM NaCI, pH 6.0, and B: 100 mM Na citrate, 150 mM NaCI, pH 3.0; see FIG. 4) so as to produce three phases at pH 6.0, pH 4.2, and pH 3.0, respectively.
• A 100 mM Na citrate, 15OmM NaCI, pH 6.0
• B 100 mM Na citrate, 150 mM NaCI, pH 3.0; see FIG. 4
• IL-4R denotes anti-IL-4Ra
• IL-6R ⁇ denotes anti-IL- ⁇ Ra(lgGi ⁇ Adp).
• Indicated column fractions were assayed for binding to IL-6Ra and IL-4Ra proteins (see FIG. 5).
• a step elution was done, giving rise to one peak eluting at pH 4.2, and a second peak at pH 3.0 (FIG. 4).
• BIACORE TM analysis showed that the flow-through material could bind soluble IL- 6Ra, but not IL-4Ra, as expected (FIG. 5).
• Fractions corresponding to the pH 4.2 peak could bind approximately equal amounts of IL-6Ra and IL-4Ra, consistent with the heterodimer.
• the peak eluting at pH 3.0 could bind only IL-4Ra, and not IL-6Ra, corresponding to the expected anti-IL-4Ra homodimer. This establishes that a heterodimeric bispecific antibody could be effectively isolated using Protein A chromatography, with a simple pH step gradient.
• mice were injected with the purified heterodimeric species of anti-IL-4Ra/anti-IL-6Ra described above, and concentrations of human immunoglobulin in serum were measured over a period of 28 days (FIG. 6; Table 1).
• the serum half-life of the heterodimer was about 10 days, similar to that of the wild type. This establishes that the Fc ⁇ Adp modifications had no detectable effect on serum half-life.
• IL-2 (R & D Systems) was then added and the cells were incubated for an additional 24 hours. The cells were then split to a concentration of 0.5 x 10 6 /mL and an additional 3OU IL- 2 added. The cells were then incubated an additional 48 hours and used in the bioassay. On the day of the bioassay 2 x 10 ⁇ /mL CD20 expressing target cells (Raji) were labeled for 30 minutes with 8uM calcein-AM (Invitrogen). Washed target cells were added to the activated hPBMC cells at a 1 :10 ratio of targeteffector cells (220,000 total cells per well) in 200 microliters total volume with the indicated amount of antibody containing supernatant.
• CHO cell supernatants from transfections as described in Example 3 were subjected to Protein A affinity chromatography, utilizing a step gradient for elution.
• the step gradient was produced by varying combinations of two buffers (A: 20 mM Na citrate, 1 M NaCI, pH 5.2; B: 20 mM Na Citrate, 1 M NaCI, pH 2.7) so as to produce three phases at pH 5.2, pH 4.2, and pH 2.8, respectively.
• Protein from the peak eluting at pH 4.2 was then used in a cell killing assay as described in Example 3.
• Target cell killing was observed at an EC 50 of 3 pM (FIG. 8).
• An additional cytotoxicity assay was performed which examined the target specificity of the observed killing.
• target cells expressing CD20 (Raji) or without CD20 (293) were incubated with activated human PBMCs. Each target cell type was added to the assay either alone or in combination with unlabeled target cells of the other type. In all cases the CD20 expressing target cells were killed specifically with an EC 50 of 3 pM while the CD20 negative cell line was not killed.
• Example 5 Protein A Separation of a Differentially Modified hlgG2 Fc
• the elution started with an isocratic elution with 100% A in the first 20 min followed by a quick switch to 100% B at 20 min. A linear gradient to 33.5% C and 66.5% B was then initiated over the next 10 min; the concentration of 33.5% of C was maintained for 20 min until the complete elution of the first peak (Fc/ ⁇ AdpFc). A linear gradient from 33.5 % C to 100 % C was followed for 30 min. The flow rate was kept 250 microliters/min and the chromatograms were detected at 280 nm by a UV detector. The hlgG2 Fc/ ⁇ AdpFc eluted at pH 4.5, whereas the hlgG2 eluted at pH 3.5.
• Example 6 Protein A Separation of a Differentially Modified hlgG4 Fc
• Differentially modified heterodimeric human lgG4 (Fc/ ⁇ AdpFc) and an unmodified homodimeric lgG4 (Fc/Fc) were first enriched by a bind-and-wash process through a protein A column (rProtein A FF, GE).
• rProtein A FF, GE protein A column
• a step-gradient elution was performed using a SMARTTM system (GE) as follows.
• the solvent system consisted of solvent A (PBS, 1X), solvent B (20 mM sodium citrate and 1 M NaCI, pH 5.1) and solvent C (20 mM sodium citrate and 1 M NaCI, pH 2.8).
• solvent A PBS, 1X
• solvent B (20 mM sodium citrate and 1 M NaCI, pH 5.1
• solvent C (20 mM sodium citrate and 1 M NaCI, pH 2.8).
• the elution started with an isocratic elution with 100% A in the first 20 min followed by a quick switch to 100% B at 20 min.
• a linear gradient to 50% C and 50% B was then initiated over the next 10 min; the concentration of 50% of C was maintained for 20 min until the complete elution of the first peak (Fc/ ⁇ AdpFc).
• a linear gradient from 50 % C to 100 % C was followed for 30 min.
• the flow rate was kept 250 microliters/min and the chromatograms were detected at 280 nm by a UV detector.
• Example 7 Protein A Separation of a Differentially Modified hlgG1 CD3xCD20
• a step was used to attain a pH of 4.2, which eluted bispecific CD3*xCD20 IgGI (Fc/ ⁇ AdpFc), then a linear gradient from pH 4.2 to pH 2.8 eluted the homodimeric anti-hCD20 IgGl
• CM5 chip having an amine-coupled anti- penta-his mAb (Qiagen) was used to capture various constructs of human Fc receptors.
• the following his-tagged Fc receptor ectodomains were bound to the surfaces of different anti- penta-his-coated CM5 chips: Fc ⁇ RI, Fc ⁇ RIIA(R131 polymorph), Fc ⁇ RIIB, and Fc ⁇ RIIIB (each obtained from R&D Systems); and Rc ⁇ RIIA(H131 polymorph), Fc ⁇ RIIIA(V176 polymorph), and Rc ⁇ RIIIA(F176 polymorph) (each made at Regeneron).
• Antibodies were passed over the surface at three concentrations for the high affinity receptor Fc ⁇ RI ectodomain (25 nM, 50 nM, and 100 nM), and at between 5 micromolar to 39 nanomolar for the low affinity Fc ⁇ R receptor ectodomains, and association and dissociation rate constants (k a and k d ) values were determined and used to calculate equilibrium dissociation constants (K D s) for the antibodies. Binding studies were performed at room temperature using HBS-T buffer at pH 7.2 K D s were determined for a control antibody (hmAb), an anti-CD20, and anti-CD3 ⁇ dp modification, and a CD20xCD3 ⁇ dp bispecific antibody. K 0 values for the anti-CD3 ⁇ dp antibody revealed no significant differences in binding to any of the Fc receptors tested as compared with unmodified hlgG1 isotype antibodies (Table 2). [00169]
• Example 9 Pharmacokinetics of a Bispecific hlgGI ⁇ Adp in hFcRn Mice
• the pharmacokinetic clearance rate of bispecific anti-hCD3/hCD20 IgGI ⁇ Adp antibody and its antibody related controls were determined in wild-type (WT) mice and mice homozygous for a replacement of mouse FcRn with a hFcRn gene (hFcRn mice).
• Wild-type and hFcRn mice were from cross-bred strains with a background containing C57BL6 (75%) and 129Sv (25%).
• Cohorts contained 4 each of either WT or hFcRn mice, except in the case of one cohort of WT mice receiving an IgGI isotyped matched control antibody in which the cohort contained 3 mice.
• Mice received 1 mg/kg of an isotype-matched (hlgG1) control, anti- hCD3xCD20 IgGi ⁇ Adp bispecific, anti-hCD3 IgGI , or anti-hCD3 IgGi ⁇ Adp homodimer. All test articles were administered subcutaneously. Bleeds were collected at Oh, 6h, 1 d, 2d, 3d, 4d, 7d, 10d, 14d, 21d, and 3Od.
• Serum levels of human antibodies were determined by a sandwich ELISA. Briefly, a goat polyclonal anti-human IgG (Fc-specific) antibody (Jackson ImmunoResearch) was coated in 96-well plates at a concentration of one microgram/mL and incubated overnight at 4° C. After the plates were blocked with BSA, serum samples in six-dose serial dilutions and reference standards of the respective antibodies in 12-dose serial dilutions were added to the plate and incubated for one hour at room temperature.
• Fc-specific antibody Jackson ImmunoResearch
• a bispecific CD3xCD20 ⁇ Adp antibody was isolated according to the invention from a large-scale culture. Briefly, a CHO-K1 cell line expressing a bispecific anti- hCD3xCD20 ⁇ Adp (modification on the CD3 heavy chain) antibody were cultured in an 11- liter bioreactor. Cells bearing the bispecific antibody grew to a denisty of about 8.25 x 106 cells/mL, yielding about 250-350 mg antibody/L. In contrast, a control anti-hCD3 antibody yielded about 100-150 mg/L.
• Antibody was isolated on an MabSelect SuReTM resin (GE) (20 cm bed height, 1 cm ID) equilibrated with 10 mM sodium phosphate, 0.5 M NaCI, pH 7.2, clarified cell culture loaded to 19 g/L, and the column was washed with 3 column volumes of 10 mM sodium phosphate, 0.5 M NaCI, pH 7.2, followed by a wash of 2 column volumes of 20 mM sodium phosphate, pH 7.2 (no NaCI). Antibody was eluted with 40 mM acetate, pH 3.0. [00178] The monospecific anti-CD30 antibody eluted at pH 3.6, whereas the bispecific anti- hCD3xCD20DAdp eluted at pH 4.4
• Example 11 Selective Protein A EIution of Mouse Heterodimers with Mild pH
• CHO-K1 cells were transiently transfected with expression constructs for human IFNAR1 (hlFNAFM ) and human IFNAR2 (MFNAR2) extracellular domain fused with a wild type or a mutant (TTTK or PTTK) mlgG2a Fc.
• the ratio between hlFNAR1-mFc and hlFNAR2-mFc was held at 1 :1 by transfecting cells with equal amounts of the two expression plasmids.
• Culture medium was collected 4 days after transfection and subjected to Protein A purification using 0.2 ml_ NAb Protein A PlusTM spin columns (Thermo Scientific/Pierce). Briefly, the columns were equilibrated with 1x PBS, pH 7.2. One ml of CHO-K1 culture medium was incubated with the Protein A resin for 10 minutes at room temperature. The columns were then washed three times with 1x PBS 1 pH 7.2. Bound proteins were eluted with 20 mM sodium citrate buffer containing 1 M NaCI. Three elutions were carried out using 0.4 mL of elution buffer with decreasing pH. Proteins in the different fractions were detected by Western blot analysis.
• Example 12 Preferential Heterodimer Formation of mlgG2a Mutants over lsotype Heterodimers
• CHO-K1 cells were transiently transfected with the expression constructs.
• the ratio between IFNAR1-mFc and IFNAR2-mFc was held at 4:1 by transfecting cells with 4-fold more hlFNAR1-mFc expression plasmid (hlFNAR1-mlgG2a) than hlFNAR2-mFc (mlgG1 or mutant mlgG2a).
• Culture medium was collected 4 days after transfection and mFc proteins were detected by Western blot analysis.

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