Patent Description:
Co-administration of two or more antibody therapies requires multiple injections or, alternatively, a single injection of a co-formulation of two different antibody compositions. While multiple injections permit flexibility in dose and timing of administration, the inconvenience and discomfort associated with multiple injections may reduce patient compliance. While a co-formulation of multiple antibody agents would permit fewer injections, the difficulty and/or expense associated with designing a suitable pharmaceutical formulation that provides the necessary stability and bioavailability, for each antibody ingredient, may be prohibitive. Further, any treatment regime which entails administration of separate antibody agents will incur added manufacturing and regulatory costs associated with the development of each individual agent.

Bispecific antibodies (BsAbs) - single agents capable of binding to two distinct antigens or epitopes - have been proposed as a means for addressing the limitations attendant with co-administration or co-formulation of separate antibody agents. BsAbs integrate the binding activities of two separate antibody therapeutics into a single agent, thus providing a potential cost and convenience benefit to the patient. In some circumstances, BsAbs may also elicit synergistic or novel activities beyond what an antibody combination can achieve.

Recombinant DNA technologies have enabled the generation of multiple BsAb formats. For example, single chain Fv (scFv) fragments composed of antigen recognition domains (i.e., heavy chain variable (VH) and light chain variable (VL) domains) tethered by flexible or structured linkers, taken from existing monoclonal antibody (MAb) therapeutics or discovered by in vitro screening methodologies, have been used as building blocks for BsAb generation. In this context, an scFv fragment(s) which binds a particular antigen can be linked to another moiety, for example a separate scFv or an IgG MAb which binds a separate antigen, to form a multi-valent BsAb. However, a limitation with the use of scFvs is that they lack the archetypical Fab architecture which provides stabilizing interactions of the heavy chain (HC) and light chain (LC) constant domains (i.e., CH<NUM> and CL, respectively) which can improve thermal stability or solubility, or reduce the potential for aggregation.

The ability to generate bispecific antibodies retaining the full IgG antibody architecture has long been a challenge in the field of antibody engineering. One approach for generating fully IgG bispecific antibodies entails co-expression of nucleic acids encoding two distinct HC-LC pairs which, when expressed, assemble to form a single antibody comprising two distinct Fabs. However, to achieve efficiency in manufacturing, each of the expressed polypeptides of the distinct HC-LC pairs must assemble with its cognate polypeptide with good specificity to reduce generation of mis-matched Fab by-products. In addition, the two distinct HCs must heterodimerize in assembly to reduce generation of mono-specific antibody by-products. Fab interface designs which promote assembly of particular HC-LC pairs have recently been described. (See <NPL>; and Published <CIT> and <CIT>) In addition, procedures for directing assembly of particular HC-HC pairs by introducing modifications into regions of the HC-HC interface have also been disclosed in the art. (See <NPL>); <NPL>); <NPL>); <NPL>); and <NPL>)).

<CIT> relates to binding domain-immunoglobulin fusion proteins that feature a binding domain for a cognate structure such as an antigen, a counterreceptor or the like, a wild-type IgG1, IGA or IgE hinge region polypeptide or a mutant IgG1 hinge region polypeptide having either zero, one or two cysteine residues, and immunoglobulin CH2 and CH3 domains.

<CIT> relates to binding domain-immunoglobulin fusion proteins that feature a binding domain for a cognate structure such as an antigen, a counterreceptor or the like, a wild-type IgG, IGA or IgE hinge-acting region, i.e., IgE CH2, region polypeptide or a mutant IgGI hinge region polypeptide having either zero, one or two cysteine residues, and immunoglobulin CH2 and CH3 domains.

<CIT> describes scaffolds having heavy chains that are asymmetric in the various domains (e.g.CH2 and CH3) and said to accomplish selectivity between the various Fc receptors involved in modulating effector function, beyond those achievable with a natural homodimeric (symmetric) Fc molecule, and increased stability and purity of the resulting variant Fc heterodimers.

However, there remains a need for alternative methods for generating fully IgG BsAbs.

In accordance with the present invention, HC-HC interface designs and processes have been identified for improving assembly of fully IgG bispecific antibodies. The designs and processes of the present invention achieve improved heterodimerization of distinct heavy chains by introducing specific mutations in the CH<NUM> domain of IgG1, IgG2 or IgG4 constant regions, and may be combined with known methods for improving HC-LC specific assembly, thus facilitating assembly of fully IgG BsAbs. In particular, the present invention provides an IgG bispecific antibody as defined in the appended set of claims.

As additional particular embodiments of the present invention, IgG BsAbs are provided which comprise first and second heavy chains comprising human IgG1 or human IgG4 constant regions, wherein each of said human IgG1 or human IgG4 constant regions comprise CH<NUM>-CH<NUM> segments of a particular amino acid sequence.

The present invention also provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (VH) and a first human IgG1 constant region, wherein said first human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:<NUM>; (b) a first light chain, wherein said first light chain comprises a first variable domain (VL) and a first constant domain (CL); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (VH) and a second human IgG1 constant region, wherein said second human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:<NUM>; and (d) a second light chain, wherein said second light chain comprises a second variable domain (VL) and a second constant domain (CL).

The present invention also provides an IgG bispecific antibody comprising: (a) a first heavy chain, wherein said first heavy chain comprises a first variable domain (VH) and a first human IgG4 constant region, wherein said first human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:<NUM>; (b) a first light chain, wherein said first light chain comprises a first variable domain (VL) and a first constant domain (CL); (c) a second heavy chain, wherein said second heavy chain comprises a second variable domain (VH) and a second human IgG4 constant region, wherein said second human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:<NUM>; and (d) a second light chain, wherein said second light chain comprises a second variable domain (VL) and a second constant domain (CL).

As an even more particular embodiment, the present invention combines CH<NUM> domain designs in the IgG1, IgG2 or IgG4 constant regions with Fab designs as described in<CIT>. In particular, the present invention provides an IgG bispecific antibody according to any one of the afore-mentioned IgG bispecific antibodies, wherein: (a) one of said first or second heavy chains further comprises a variable domain (VH) comprising a lysine substituted at residue <NUM> (or a lysine at residue <NUM>) and a glutamic acid substituted at the residue (or a glutamic acid at the residue) which is four amino acids upstream of the first residue of HFR3 according to Kabat Numbering system, and a human IgG1 or human IgG4 CH<NUM> domain comprising an alanine substituted at residue <NUM> (or an alanine at residue <NUM>) and a glycine substituted at residue <NUM> (or a glycine at residue <NUM>); (b) one of said first or second light chains comprises a kappa variable domain (VL) comprising an arginine substituted at residue <NUM> (or an arginine at residue <NUM>) and an aspartic acid substituted at residue <NUM> (or an aspartic acid at residue <NUM>), and a constant domain (CL) comprising a tyrosine substituted at residue <NUM> (or a tyrosine at residue <NUM>) and a tryptophan substituted at residue <NUM> (or a tryptophan at residue <NUM>); (c) the other of said first or second heavy chains further comprises a variable domain (VH) comprising a tyrosine substituted at residue <NUM> (or a tyrosine at residue <NUM>) and a WT human IgG1 or human IgG4 CH<NUM> domain; and (d) the other of said first or second light chains comprises a variable domain (VL) comprising an arginine substituted at residue <NUM> (or an arginine at residue <NUM>) and a WT constant domain (CL), wherein the VH domain comprising a lysine substituted at residue <NUM> (or a lysine at residue <NUM>) and a glutamic acid substituted at the residue (or a glutamic acid at the residue) which is four amino acids upstream of the first residue of HFR3 according to Kabat and the human IgG1 or human IgG4 CH<NUM> domain comprising an alanine substituted at residue <NUM> (or an alanine at residue <NUM>) and a glycine substituted at residue <NUM> (or a glycine at residue <NUM>) together with the (VL) comprising an arginine substituted at residue <NUM> (or an arginine at residue <NUM>) and an aspartic acid substituted at residue <NUM> (or an aspartic acid at residue <NUM>) and the CL domain comprising a tyrosine substituted at residue <NUM> (or a tyrosine at residue <NUM>) and a tryptophan substituted at residue <NUM> (or a tryptophan at residue <NUM>) form a first Fab which directs binding to a first target; and the VH domain comprising a tyrosine substituted at residue <NUM> (or a tyrosine at residue <NUM>) and the WT human IgG1 or human IgG4 CH<NUM> domain together with the (VL) comprising an arginine substituted at residue <NUM> (or an arginine at residue <NUM>) and the WT CL domain form a second Fab which directs binding to a second target which is different from the first target.

Even more particular, the present invention combines the afore-mentioned CH<NUM> domain designs with Fab designs to provide an IgG bispecific antibody, wherein: (a) one of said first or second heavy chains further comprises a variable domain (VH) comprising a lysine substituted at residue <NUM> (or a lysine at residue <NUM>) and a glutamic acid substituted at the residue (or a glutamic acid at the residue) which is four amino acids upstream of the first residue of HFR3 according to Kabat Numbering system, and a human IgG1 or human IgG4 CH<NUM> domain comprising an alanine substituted at residue <NUM> (or an alanine at residue <NUM>) and a glycine substituted at residue <NUM> (or a glycine at residue <NUM>); (b) one of said first or second light chains comprises a kappa variable domain (VL) comprising an arginine substituted at residue <NUM> (or an arginine at residue <NUM>) and an aspartic acid substituted at residue <NUM> (or an aspartic acid at residue <NUM>), and a constant domain (CL) comprising a tyrosine substituted at residue <NUM> (or a tyrosine at residue <NUM>) and a tryptophan substituted at residue <NUM> (or a tryptophan at residue <NUM>) ; (c) the other of said first or second heavy chains further comprises a variable domain (VH) comprising a tyrosine substituted at residue <NUM> (or a tyrosine at residue <NUM>) and an arginine substituted at residue <NUM> (or an arginine at residue <NUM>) and a human IgG1 or human IgG4 CH<NUM> domain comprising a cysteine substituted at residue <NUM> (or a cysteine at residue <NUM>), an aspartic acid substituted at residue <NUM> (or an aspartic acid at residue <NUM>), and a glycine substituted at residue <NUM> (or a glycine at residue <NUM>); and (d) the other of said first or second light chains comprises a variable domain (VL) comprising an arginine substituted at residue <NUM> (or an arginine at residue <NUM>) and an aspartic acid substituted at residue <NUM> (or an aspartic acid at residue <NUM>), and a constant domain (CL) comprising a lysine substituted at residue <NUM> (or a lysine at residue <NUM>), wherein the VH domain comprising a lysine substituted at residue <NUM> (or a lysine at residue <NUM>) and a glutamic acid substituted at the residue (or a glutamic acid at the residue) which is four amino acids upstream of the first residue of HFR3 according to Kabat and the human IgG1 or human IgG4 CH<NUM> domain comprising an alanine substituted at residue <NUM> (or an alanine at residue <NUM>) and a glycine substituted at residue <NUM> (or a glycine at residue <NUM>) together with the (VL) comprising an arginine substituted at residue <NUM> (or an arginine at residue <NUM>) and an aspartic acid substituted at residue <NUM> (or an aspartic acid at residue <NUM>) and the CL domain comprising a tyrosine substituted at residue <NUM> (or a tyrosine at residue <NUM>) and a tryptophan substituted at residue <NUM> (or a tryptophan at residue <NUM>) form a first Fab which directs binding to a first target; and the VH domain comprising a tyrosine substituted at residue <NUM> (or a tyrosine at residue <NUM>) and an arginine substituted at residue <NUM> (or an arginine at residue <NUM>) and the human IgG1 or human IgG4 CH<NUM> domain comprising a cysteine substituted at residue <NUM> (or a cysteine at residue <NUM>), an aspartic acid substituted at residue <NUM> (or an aspartic acid at residue <NUM>), and a glycine substituted at residue <NUM> (or a glycine at residue <NUM>) together with the (VL) comprising an arginine substituted at residue <NUM> (or an arginine at residue <NUM>) and an aspartic acid substituted at residue <NUM> (or an aspartic acid at residue <NUM>) and the CL domain comprising a lysine substituted at residue <NUM> (or a lysine at residue <NUM>) form a second Fab which directs binding to a second target which is different from the first target.

The present invention also provides processes for preparing the IgG bispecific antibodies of the present invention as defined in the appended set of claims.

As particular embodiments to the processes of the present invention, processes for preparing IgG bispecific antibodies are provided, wherein the nucleic acids encoding the first and second heavy chains each encode human IgG1 constant regions comprising CH<NUM>-CH<NUM> segments of a particular amino acid sequence.

In another particular embodiment to the process for preparing an IgG bispecific antibody of the present invention (a) the first nucleic acid sequence encodes a first heavy chain, wherein said first heavy chain comprises a first variable domain (VH) and a first human IgG1 constant region, wherein said first human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:<NUM>; (b) the second nucleic acid encodes a first light chain, wherein said first light chain comprises a first variable domain (VL) and a first constant domain (CL); (c) the third nucleic acid encodes a second heavy chain, wherein said second heavy chain comprises a second variable domain (VH) and a second human IgG1 constant region, wherein said second human IgG1 constant region comprises an amino acid sequence as given by SEQ ID NO:<NUM>; and (d) the fourth nucleic acid sequence encodes a second light chain, wherein said second light chain comprises a second variable domain (VL) and a second constant domain (CL).

In another particular embodiment to the process for preparing an IgG bispecific antibody of the present invention (a) the first nucleic acid sequence encodes a first heavy chain, wherein said first heavy chain comprises a first variable domain (VH) and a first human IgG4 constant region, wherein said first human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:<NUM>; (b) the second nucleic acid encodes a first light chain, wherein said first light chain comprises a first variable domain (VL) and a first constant domain (CL); (c) the third nucleic acid encodes a second heavy chain, wherein said second heavy chain comprises a second variable domain (VH) and a second human IgG4 constant region, wherein said second human IgG4 constant region comprises an amino acid sequence as given by SEQ ID NO:<NUM>; and (d) the fourth nucleic acid sequence encodes a second light chain, wherein said second light chain comprises a second variable domain (VL) and a second constant domain (CL).

As an even more particular embodiment to the processes for preparing an IgG bispecific antibody of the present invention, the present invention further combines CH<NUM> domain designs in the IgG1, IgG2 or IgG4 constant regions with Fab designs as described in<CIT>) in the process. In particular, the present invention provides a process for preparing an IgG bispecific antibody according to any one of the afore-mentioned processes, wherein: (a) one of said first or third nucleic acid sequences encodes a heavy chain further comprising a variable domain (VH) comprising a lysine substituted at residue <NUM> (or a lysine at residue <NUM>) and a glutamic acid substituted at the residue (or a glutamic acid at the residue) which is four amino acids upstream of the first residue of HFR3 according to Kabat Numbering system, and a human IgG1 or human IgG4 CH<NUM> domain comprising an alanine substituted at residue <NUM> (or an alanine at residue <NUM>) and a glycine substituted at residue <NUM>; (b) one of said second or fourth nucleic acid sequences encodes a light chain comprising a kappa variable domain (VL) comprising an arginine substituted at residue <NUM> (or an arginine at residue <NUM>) and an aspartic acid substituted at residue <NUM> (or an aspartic acid at residue <NUM>), and a constant domain (CL) comprising a tyrosine substituted at residue <NUM> (or a tyrosine at residue <NUM>) and a tryptophan substituted at residue <NUM> (or a tryptophan at residue <NUM>); (c) the other of said first or third nucleic acid sequences encodes a heavy chain further comprising a variable domain (VH) comprising a tyrosine substituted at residue <NUM> (or a tyrosine at residue <NUM>) and a WT human IgG1 or human IgG4 CH<NUM> domain; and (d) the other of said second or fourth nucleic acid sequences encodes a light chain comprising a variable domain (VL) comprising an arginine substituted at residue <NUM> (or an arginine at residue <NUM>) and a WT constant domain (CL), wherein the IgG bispecific antibody recovered comprises: a first Fab comprising (i) the variable domain (VH) comprising a lysine substituted at residue <NUM> (or a lysine at residue <NUM>) and a glutamic acid substituted at the residue (or a glutamic acid at the residue) which is four amino acids upstream of the first residue of HFR3 according to Kabat Numbering system, and the human IgG1 or human IgG4 CH<NUM> domain comprising an alanine substituted at residue <NUM> (or an alanine at residue <NUM>) and a glycine substituted at residue <NUM> (or a glycine at residue <NUM>), together with (ii) the light chain comprising a kappa variable domain (VL) comprising an arginine substituted at residue <NUM> (or an arginine at residue <NUM>) and an aspartic acid substituted at residue <NUM> (or an aspartic acid at residue <NUM>), and a constant domain (CL) comprising a tyrosine substituted at residue <NUM> (or a tyrosine at residue <NUM>) and a tryptophan substituted at residue <NUM> (or a tryptophan at residue <NUM>); and a second Fab comprising (i) the variable domain (VH) comprising a tyrosine substituted at residue <NUM> (or a tyrosine at residue <NUM>) and a WT human IgG1 or human IgG4 CH<NUM> domain, together with (ii) the variable domain (VL) comprising an arginine substituted at residue <NUM> (or an arginine at residue <NUM>) and a WT constant domain (CL).

Even more particular to the processes for preparing an IgG bispecific antibody of the present invention, the present invention combines the afore-mentioned CH<NUM> domain designs with additional Fab designs in the process. Thus, the present invention provides a process for preparing an IgG bispecific antibody according to any one of the afore-mentioned processes, wherein: (a) one of said first or third nucleic acid sequences encodes a heavy chain further comprising a variable domain (VH) comprising a lysine substituted at residue <NUM> (or a lysine at residue <NUM>) and a glutamic acid substituted at the residue (or a glutamic acid at the residue) which is four amino acids upstream of the first residue of HFR3 according to Kabat Numbering system, and a human IgG1 or human IgG4 CH<NUM> domain comprising an alanine substituted at residue <NUM> (or an alanine at residue <NUM>) and a glycine substituted at residue <NUM> (or a glycine at residue <NUM>); (b) one of said second or fourth nucleic acid sequences encodes a light chain comprising a kappa variable domain (VL) comprising an arginine substituted at residue <NUM> (or an arginine at residue <NUM>) and an aspartic acid substituted at residue <NUM> (or an aspartic acid at residue <NUM>), and a constant domain (CL) comprising a tyrosine substituted at residue <NUM> (or a tyrosine at residue <NUM>) and a tryptophan substituted at residue <NUM> (or a tryptophan at residue <NUM>); (c) the other of said first or third nucleic acid sequences encodes a heavy chain further comprising a variable domain (VH) comprising a tyrosine substituted at residue <NUM> (or tyrosine at residue <NUM>) and an arginine substituted at residue <NUM> (or an arginine at residue <NUM>) and a human IgG1 or human IgG4 CH<NUM> domain comprising a cysteine substituted at residue <NUM> (or a cysteine at residue <NUM>), an aspartic acid substituted at residue <NUM> (or an aspartic acid at residue <NUM>), and a glycine substituted at residue <NUM> (or a glycine at residue <NUM>); and (d) the other of second or fourth nucleic acid sequences encodes a light chain comprising a variable domain (VL) comprising an arginine substituted at residue <NUM> (or an arginine at residue <NUM>) and an aspartic acid substituted at residue <NUM> (or an aspartic acid at residue <NUM>), and a constant domain (CL) comprising a lysine substituted at residue <NUM> (or a lysine at residue <NUM>), wherein the IgG bispecific antibody recovered comprises: a first Fab comprising (i) the variable domain (VH) comprising a lysine substituted at residue <NUM> (or a lysine at residue <NUM>) and a glutamic acid substituted at the residue (or a glutamic acid at the residue) which is four amino acids upstream of the first residue of HFR3 according to Kabat Numbering system, and the human IgG1 or human IgG4 CH<NUM> domain comprising an alanine substituted at residue <NUM> (or an alanine at residue <NUM>) and a glycine substituted at residue <NUM> (or a glycine at residue <NUM>), together with (ii) the light chain comprising a kappa variable domain (VL) comprising an arginine substituted at residue <NUM> (or an arginine at residue <NUM>) and an aspartic acid substituted at residue <NUM> (or an aspartic acid at residue <NUM>), and a constant domain (CL) comprising a tyrosine substituted at residue <NUM> (or a tyrosine at residue <NUM>) and a tryptophan substituted at residue <NUM> (or a tryptophan at residue <NUM>); and a second Fab comprising (i) the variable domain (VH) comprising a tyrosine substituted at residue <NUM> (or a tyrosine at residue <NUM>) and an arginine substituted at residue <NUM> (or an arginine at residue <NUM>) and a human IgG1 or human IgG4 CH<NUM> domain comprising a cysteine substituted at residue <NUM> (or a cysteine at residue <NUM>), an aspartic acid substituted at residue <NUM> (or an aspartic acid at residue <NUM>), and a glycine substituted at residue <NUM> (or a glycine at residue <NUM>) , together with (ii) the variable domain (VL) comprising an arginine substituted at residue <NUM> (or an arginine at residue <NUM>) and an aspartic acid substituted at residue <NUM> (or an aspartic acid at residue <NUM>), and a constant domain (CL) comprising a lysine substituted at residue <NUM> (or a lysine at residue <NUM>).

An IgG bispecific antibody can be produced accord to any one of the processes of the present invention.

In addition to the preparation of Fully IgG BsAbs, the methods described herein may also be employed in the preparation of other multi- or mono-valent antigen binding compounds. <FIG>, included herein, provides a schematic diagram of a Fully IgG BsAb, as well as other antigen binding compounds that one of skill in the art could prepare using the CH<NUM> domain designs, or the CH<NUM> domain designs plus Fab designs, as described herein.

Nucleic acid sequences encoding the first and second heavy chains and the first and second light chains of any of the IgG BsAbs of the present invention are described herein. In addition, vectors comprising nucleic acid sequences encoding the first heavy chain, the first light chain, the second heavy chain and/or the second light chain of any of the IgG BsAbs of the present invention are described herein. Further still, the present invention provides host cells comprising nucleic acid sequences encoding the first heavy chain, the first light chain, the second heavy chain and the second light chain of any of the IgG BsAbs of the present invention.

<FIG> provides a schematic diagram of antigen binding compounds that may be prepared using the CH3 domain designs, methods or procedures of the present invention, including Fully IgG BsAbs (A), One-arm antibodies (B), a Tandem Fab-Fc molecules (C), IgG-scFv-Fc molecules (D), and IgG-scFv molecules (E and F).

The general structure of an "IgG antibody" is very well-known. A wild type (WT) antibody of the IgG type is hetero-tetramer of four polypeptide chains (two identical "heavy" chains and two identical "light" chains) that are cross-linked via intra- and inter-chain disulfide bonds. Each heavy chain (HC) is comprised of an N-terminal heavy chain variable region ("VH") and a heavy chain constant region. The heavy chain constant region is comprised of three domains (CH<NUM>, CH<NUM>, and CH<NUM>) as well as a hinge region ("hinge") between the CH<NUM> and CH<NUM> domains. Each light chain (LC) is comprised of an N-terminal light chain variable region ("VL") and a light chain constant region ("CL"). The VL and CL regions may be of the kappa ("κ") or lambda ("λ") isotypes. Each heavy chain associates with one light chain via an interface between the heavy chain VH - CH<NUM> segment and the light chain VL - CL segment. The association of each VH - CH<NUM>/ VL - CL forms two identical antigen binding fragments (Fabs) which direct antibody binding to the same target or epitope. Each heavy chain associates with the other heavy chain via an interface between the hinge-CH<NUM>- CH<NUM> segments of each heavy chain, with the association between the CH<NUM>- CH<NUM> segments forming the Fc region of the antibody. Together, each Fab and the Fc form the characteristic "Y-shaped" architecture of IgG antibodies, with each Fab representing the "arms" of the "Y. " IgG antibodies can be further divided into subtypes, e.g., IgG1, IgG2, IgG3, and IgG4 which differ by the length of the hinge regions, the number and location of inter- and intra-chain disulfide bonds and the amino acid sequences of the respective HC constant regions.

The variable regions of each heavy chain - light chain pair associate to form binding sites. The heavy chain variable region (VH) and the light chain variable region (VL) can be subdivided into regions of hypervariability, termed complementarity determining regions ("CDRs"), interspersed with regions that are more conserved, termed framework regions ("FR"). 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. CDRs of the heavy chain may be referred to as "CDRH1, CDRH2, and CDRH3" and the <NUM> CDRs of the light chain may be referred to as "CDRL1, CDRL2 and CDRL3. " The FRs of the heavy chain may be referred to as HFR1, HFR2, HFR3 and HFR4 whereas the FRs of the light chain may be referred to as LFR1, LFR2, LFR3 and LFR4. The CDRs contain most of the residues which form specific interactions with the antigen.

As used herein, the terms "IgG bispecific antibody", "IgG BsAb", "fully IgG bispecific antibody" or "fully IgG BsAb" refer to an antibody of the typical IgG architecture comprising two distinct Fabs, each of which direct binding to a separate antigen (e.g., different target proteins or different epitopes on the same target protein), and composed of two distinct IgG heavy chains and two distinct light chains. The VH - CH<NUM> segment of one heavy chain associates with the VL- CL segment of one light chain to form a "first" Fab, wherein the VH and VL domains each comprise <NUM> CDRs which direct binding to a first antigen. The VH - CH<NUM> segment of the other heavy chain associates with the VL - CL segment of the other light chain to form a "second" Fab, wherein the VH and VL domains each comprise <NUM> CDRs which direct binding to a second antigen that is different than the first. More particularly, the terms "IgG bispecific antibody", "IgG BsAb", "fully IgG bispecific antibody" or "fully IgG BsAb" refer to antibodies wherein the HC constant regions are composed of CH<NUM>, CH<NUM>, and CH<NUM> domains of the IgG1, IgG2 or IgG4 subtype, and particularly the human IgG1, human IgG2 or human IgG4. Even more particular, the terms refer to antibodies wherein the HC constant regions are composed of CH<NUM>, CH<NUM>, and CH<NUM> domains of the IgG1 or IgG4 subtype, and most particularly the human IgG1 or human IgG4 subtype. In addition, as used herein, the terms "IgG bispecific antibody", "IgG BsAb", "fully IgG bispecific antibody" and "fully IgG BsAb" refer to an antibody wherein the constant regions of each HC of the antibody are of the same subtype (for example, each HC of the antibody has CH<NUM>, CH<NUM>, and CH<NUM> domains of the human IgG1 subtype, or each HC of the antibody has CH<NUM>, CH<NUM>, and CH<NUM> domains of the human IgG2 subtype, or each HC of the antibody has CH<NUM>, CH<NUM>, and CH<NUM> domains of the human IgG4 subtype.

The processes and compounds of the present invention comprise designed amino acid modifications at particular residues within the constant and variable regions of heavy chain and light chain polypeptides. As one of ordinary skill in the art will appreciate, various numbering conventions may be employed for designating particular amino acid residues within IgG constant and variable region sequences. Commonly used numbering conventions include the "Kabat Numbering" and "EU Index Numbering" systems. "Kabat Numbering" or "Kabat Numbering system", as used herein, refers to the numbering system devised and set forth by the authors in <NPL>) for designating amino acid residues in both variable and constant domains of antibody heavy chains and light chains. "EU Index Numbering" or "EU Index Numbering system", as used herein, refers to the numbering convention for designating amino acid residues in antibody heavy chain constant domains, and is also set forth in Kabat et al. Other conventions that include corrections or alternate numbering systems for variable domains include Chothia (<NPL>; <NPL>), IMGT (<NPL>), and AHo (<NPL>). These references provide amino acid sequence numbering schemes for immunoglobulin variable regions that define the location of variable region amino acid residues of antibody sequences. Unless otherwise expressly stated herein, all references to immunoglobulin heavy chain variable region (i.e., VH), constant region CH<NUM> and hinge amino acid residues (i.e. numbers) appearing in the Examples and Claims are based on the Kabat Numbering system, as are all references to the light chain VL and CL residues. All references to immunoglobulin heavy chain constant regions CH<NUM> and CH<NUM> are based on the EU Index Numbering system. With knowledge of the residue number according to Kabat Numbering or EU Index Numbering, one of ordinary skill can apply the teachings of the art to identify amino acid sequence modifications within the present invention, according to any commonly used numbering convention. Note, while the Examples and Claims of the present invention employ Kabat Numbering or EU Index Numbering to identify particular amino acid residues, it is understood that the SEQ ID NOs appearing in the Sequence Listing accompanying the present application, as generated by Patent In Version <NUM>, provide sequential numbering of amino acids within a given polypeptide and, thus, do not conform to the corresponding amino acid residue numbers as provided by Kabat Numbering or EU Index Numbering.

However, as one of skill in the art will also appreciate, CDR sequence length may vary between individual IgG molecules and, further, the numbering of individual residues within a CDR may vary depending on the numbering convention applied. Thus, to reduce ambiguity in the designation of amino acid residues within CDRs, the disclosure of the present invention first employs Kabat Numbering to identify the N-terminal (first) amino acid of the HFR3. The amino acid residue to be modified is then designated as being four (<NUM>) amino acid residues upstream (i.e. in the N-terminal direction) from the first amino acid in the reference HFR3. For example, a Fab design used in combination with the CH<NUM> domain designs of the present invention comprises the replacement of a WT amino acid in HCDR2 with a glutamic acid (E) (i.e., Fab Design AB2133(a) comprising R62E mutation). This replacement is made at the residue located four amino acids upstream of the first amino acid of HFR3, according to Kabat Numbering. In the Kabat Numbering system, amino acid residue X66 is the most N-terminal (first) amino acid residue of variable region heavy chain framework three (HFR3). One of ordinary skill can employ such a strategy to identify the first amino acid residue (most N-terminal) of heavy chain framework three (HFR3) from any human IgG1 or IgG4 variable region. Once this landmark is identified, one can then locate the amino acid four residues upstream (N-terminal) to this location and replace that amino acid residue (using standard insertion/deletion methods) with a glutamic acid (E) to achieve the design modification of the invention. Given any variable IgG1 or IgG4 immunoglobulin heavy chain amino acid query sequence of interest to use in the processes of the invention, one of ordinary skill in the art of antibody engineering would be able to locate the N-terminal HFR3 residue in said query sequence and then count four amino acid residues upstream therefrom to arrive at the location in HCDR2 that should be modified to glutamic acid (E).

As used herein, the phrase ". a/an [amino acid name] substituted at residue. ", in reference to a heavy chain or light chain polypeptide, refers to substitution of the parental amino acid with the indicated amino acid. For example, a heavy chain comprising "a lysine substituted at residue <NUM>" refers to a heavy chain wherein the parental amino acid sequence has been mutated to contain a lysine at residue number <NUM> in place of the parental amino acid. Such mutations may also be represented by denoting a particular amino acid residue number, preceded by the parental amino acid and followed by the replacement amino acid. For example, "Q39K" refers to a replacement of a glutamine at residue <NUM> with a lysine. Similarly, "<NUM>" refers to replacement of a parental amino acid with a lysine. One of skill in the art will appreciate, however, that as a result of the HC-HC interface design modifications of the present invention, fully IgG BsAbs (and processes for their preparation) are provided wherein the component HC amino acid sequences, or component HC and LC amino acid sequences, comprise the resulting or "replacement" amino acid at the designated residue. Thus, for example, a heavy chain which "comprises a lysine substituted at residue <NUM>" may alternatively be denoted as a heavy chain "comprising a lysine at residue <NUM>.

An IgG BsAb of the present invention may be derived from a single copy or clone (e.g. a monoclonal IgG BsAb antibody). Preferably, an IgG BsAb of the present invention exists in a homogeneous or substantially homogeneous population. In an embodiment, the IgG BsAb, or a nucleic acid encoding a component polypeptide sequence of the IgG BsAb, is provided in "isolated" form. As used herein, the term "isolated" refers to a protein, polypeptide or nucleic acid which is free or substantially free from other macromolecular species found in a cellular environment.

An IgG BsAb of the present invention can be produced using techniques well known in the art, such as recombinant expression in mammalian or yeast cells. In particular, the methods and procedures of the Examples herein may be readily employed. In addition, the IgG BsAbs of the present invention may be further engineered to comprise framework regions derived from fully human frameworks. A variety of different human framework sequences may be used in carrying out embodiments of the present invention. Preferably, the framework regions employed in the processes of the present invention, as well as IgG BsAbs of the present invention are of human origin or are substantially human (at least <NUM>%, <NUM>% or <NUM>% of human origin). The sequences of framework regions of human origin are known in the art and may be obtained from<NPL>.

Expression vectors capable of directing expression of genes to which they are operably linked are well known in the art. Expression vectors contain appropriate control sequences such as promoter sequences and replication initiation sites. They may also encode suitable selection markers as well as signal peptides that facilitate secretion of the desired polypeptide product(s) from a host cell. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide. Nucleic acids encoding desired polypeptides, for example the components of the IgG BsAbs prepared according to the processes of the present invention, may be expressed independently using different promoters to which they are operably linked in a single vector or, alternatively, the nucleic acids encoding the desired products may be expressed independently using different promoters to which they are operably linked in separate vectors. In addition, nucleic acids encoding a particular HC/LC pair of the IgG BsAbs of the present invention may expressed from a first vector, while the other HC/LC pair is expressed from a second vector. Single expression vectors encoding both HC and both LC components of the IgG BsAbs of the present invention may be prepared using standard methods. For example, a pE vector encoding a particular HC/LC pair may be engineered to contain a NaeI site <NUM> prime of a unique SalI site, outside of the HC/LC expression cassette. The vector may then be modified to contain an AscI site <NUM> prime of the SalI site using standard techniques. For example, the NaeI to SalI region may be PCR amplified using a <NUM>' primer containing the AscI site adjacent to the SalI site, and the resulting fragment cloned into the recipient pE vector. The expression cassette encoding a second HC/LC pair, may then be isolated from a second (donor) vector by digesting the vector at suitable restriction sites. For example, the donor vector may be engineered with MluI and SalI sites to permit isolation of the second expression cassette. This cassette may then be ligated into the recipient vector previously digested at the AscI and SalI sites (as AscI and MluI restriction sites have the same overlapping ends).

As used herein, a "host cell" refers to a cell that is stably or transiently transfected, transformed, transduced or infected with nucleotide sequences encoding a desired polypeptide product or products. Creation and isolation of host cell lines producing an IgG BsAb of the present invention can be accomplished using standard techniques known in the art.

Mammalian cells are preferred host cells for expression of the IgG BsAb compounds according to the present invention. Particular mammalian cells include HEK293, NS0, DG-<NUM>, and CHO cells. Preferably, assembled proteins are secreted into the medium in which the host cells are cultured, from which the proteins can be recovered and isolated. Medium into which a protein has been secreted may be purified by conventional techniques. For example, the medium may be applied to and eluted from a Protein A or G column using conventional methods. Soluble aggregate and multimers may be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, hydroxyapatite or mixed modal chromatography. Recovered products may be immediately frozen, for example at -<NUM>, or may be lyophilized. As one of skill in the art will appreciate, when expressed in certain biological systems, e.g. mammalian cell lines, antibodies are glycosylated in the Fc region unless mutations are introduced in the Fc to reduce glycosylation. In addition, antibodies may be glycosylated at other positions as well.

The following Examples further illustrate the invention and provide typical methods and procedures for carrying out various particular embodiments of the present invention. However, it is understood that the Examples are set forth the by way of illustration and not limitation, and that various modifications may be made by one of ordinary skill in the art.

Residues for initial modification at the symmetric CH<NUM>/ CH<NUM> dimer interface (i.e., Chain A CH<NUM> domain and Chain B CH<NUM> domain) were selected using a combination of computational and rational design strategies. First, using a crystal structure of the human IgG1 CH<NUM>- CH<NUM> domains (PDB ID 1L6X), trimmed of carbohydrate moieties that connect the CH<NUM> domains, the Rosetta software suite and related modeling applications were employed to computationally identify potential modifications that favor heterodimer (i.e., Chain A/Chain B) formation over homodimer (i.e., Chain A/Chain A or Chain B/Chain B) formation. (See, <NPL>; <NPL>; <NPL>; and <NPL>; <NPL>)). More than sixty discrete initial designs, falling into varying design paradigms (i.e., different amino acid substitutions and/or different amino acid residue positions) were identified, synthesized and tested for heterodimer formation and thermostability (as measured by UPLC, FRET and DSC, as described below. ) Select Chain A/Chain B mutation pairs were further optimized rationally and/or assessed for compatibility for combination, including inverted combinations where mutations in one chain of a discrete design pair (e.g. Chain B) are added to the mutations in the opposite chain (e.g. Chain A) of a separate discrete design pair. Optimized and/or combination designs were then assessed computationally and those exhibiting promising heterodimer formation potential, and destabilized homodimer potential, were also synthesized and tested for heterodimer formation (as measured by UPLC and FRET) and thermostability.

Briefly, Rosetta's multistate design module explores sequence space and for each sequence calculates an energy for each of several "states" based on a weighted sum of energy potentials treating phenomena such as van der Waals forces and hydrogen bonding forces, and then aggregates these energies to compute a fitness for that sequence. The states represent different combinations and conformations of protein chain species, e.g. different conformations of the Chain A/Chain B heterodimer, or different conformations of the Chain A/Chain A or Chain B/Chain B homodimer. The summations of the energy potentials are measured in units known as the Rosetta Energy Unit (REU). These values are interpreted as free energies, but do not directly translate into typical units of energy. Binding energies are computed as the difference between the energy of the bound complex and the energies of its separated components. Using a fitness function which favors the binding and stability of Chain A CH<NUM>/Chain B CH<NUM> heterodimers and disfavors the binding of CH<NUM>/ CH<NUM> homodimers (Chain A/Chain A or Chain B/Chain B), initial sequences for modification are identified.

The identified mutations are subjected to computational docking of the heterodimer and homodimer complexes using RosettaDock via RosettaScripts (see, <NPL> and <NPL>). This docking step allows the complexes to relax into more favorable conformations for their sequences and facilitates the comparison of binding energies for the homodimer and heterodimer complexes. Energies are calculated using a variation on Rosetta's standard score function, "Talaris2013" (<NPL>), where the atomic-interaction distance was extended to 9Å and the amino-acid specific reference energies had been refit using Rosetta's automated refitting procedure, OptE (<NPL>). Following docking, binding energies are calculated as the difference in energies between the complex and the sidechain-optimized, separated conformations as reported by Rosetta's InterfaceAnalyzer tool (see, <NPL>). The conformations resulting from the docking simulations of homodimers which display favorable binding energies are used as additional states in subsequent multistate design simulations to further guide the those simulations away from sequences which favor homodimer formation. The multistate-design-followed-by-redocking process is iterated until the binding energies calculated by multistate design match well with binding energies calculated following docking.

Select designs, including further optimized and/or combination designs, resulting from this iterative process and their calculated binding energies are provided in Table <NUM>.

To assess the heterodimer formation potential of Chain A CH<NUM> domain/Chain B CH<NUM> domain design pairs, "one-arm" antibody constructs incorporating design modifications in the CH<NUM>/ CH<NUM> dimer interface are prepared and tested. Unless otherwise indicated, "Chain A" of each construct contains a full heavy chain sequence (with or without CH<NUM> domain design modifications) and "Chain B" of each construct contains an Fc only portion (CH<NUM>- CH<NUM> segment plus HA tag) of the heavy chain (with or without CH3 domain design modifications. )
Molecular Biology: Variable heavy domain (VH) and variable light domain (VL) sequences of the anti-cMet clone 5D5 (see US Patent # <NUM>,<NUM>,<NUM> ) are synthesized. The VH domain-encoding sequence is cloned into a plasmid (pcDNA3. <NUM>(+) (Life Technologies)) containing sequences encoding a mouse kappa chain leader sequence and a complete human IgG1 heavy chain using HindIII/EcoR1 restriction sites. The VL domain-encoding sequence is cloned into a pEHK mammalian expression vector (Lonza) containing a sequence encoding a mouse kappa chain leader sequence and a <NUM>' kappa constant domain, using the BamHI and EcoRI restriction sites. An HA-tagged Fc construct, to provide the other member of the CH<NUM> dimer interface, is constructed by first PCR amplifying a human IgG1 Fc from a full heavy chain using a forward primer which introduced an HA tag plus a four residue linker at the N-terminus of the chain. The HA-tagged Fc-encoding construct is then cloned into a pcDNA3. <NUM>(+) plasmid containing a sequence encoding a mouse kappa chain leader sequence using the BamH1 and EcoR1 restriction sites. Nucleic acid sequence modifications encoding the CH<NUM> domain design pair mutations are introduced using methods known in the art such as Kunkel mutagenesis (See <NPL>), Quickchange mutagenesis (Agilent), or direct Geneblock cloning (Integrated DNA Technologies®, IDT) using restriction site cloning using EcoRI and an internal SacII site within the CH<NUM> domain. Mutant CH<NUM> designs were introduced by Kunkel mutagenesis (See <NPL>), Quickchange mutagenesis (Agilent), or direct cloning from FRET constructs containing CH<NUM> mutations, using restriction site cloning with EcoRI and an internal SacII site within the CH<NUM> domain. The parental protein sequences for the one-arm antibody constructs, prior to incorporation of the CH<NUM> domain design pair modifications (i.e., WT CH<NUM> domains), are provided in SEQ ID NOs: <NUM>-<NUM>.

The three plasmids (<NUM>µg anti-c-Met VH-human IgG1 HC (with or without CH<NUM> modifications), <NUM>µg HA-tagged human IgG1 Fc portion (with or without CH<NUM> modifications), and <NUM>µg anti-c-Met light chain) are transiently transfected into <NUM> of HEK293F cells. Transfected cells are grown at <NUM> in a <NUM>% CO<NUM> incubator while shaking at <NUM> rpm for <NUM> days. Secreted protein is harvested by centrifugation at <NUM> rpm for <NUM>. and recovery of the supernatant. Expressed protein is purified from the supernatant using PureProteome Protein G Magnetic Beads (EMD Millipore), a DynaMag Magnetic Particle Concentrator (Invitrogen), and Protein G wash and Elution Buffers (Biomiga), as per manufacturer instructions. Eluted samples are neutralized with <NUM> Tris pH9. <NUM> (Sigma) and filtered with an Ultrafree-MC-GV centrifugal filter (Millipore), per manufacturer instructions. UPLC Detection: <NUM>µl samples of expressed protein are added to Waters UPLC tubes, from which <NUM>µl is injected into a Waters Acquity UPLC with a BEH200 SEC column, equilibrated in PBS and run at <NUM>/min. A dilution series of purified MetMab is also run as a standard. Resulting A280 chromatogram peaks from the UPLC traces are deconvoluted and integrated using a custom set of GNU Octave scripts to quantify % heterodimerization by peak area. Tables <NUM> and <NUM> below provide heterodimer formation data, as determined by UPLC, for select designs, including further optimized or combination designs. The following provides experimental details of the treatment of the UPLC traces, including various characteristic peaks obtained, as well as procedures employed for curve fitting and data interpretation.

From run to run, the retention times for the various protein species may shift forward and backwards in time together so that if a Chain A HC/Chain B HC heterodimer were to elute at <NUM>, then the Chain A/Chain A homodimer would elute at <NUM>, but if the Chain A HC/Chain B HC heterodimer were to elute, for example, <NUM> minutes later at <NUM>, then the Chain A HC/Chain A HC homodimer would elute similarly later at <NUM>. A sharp peak between <NUM> and <NUM>, from a non-antibody species, is characteristic of the UPLC traces, with no recorded species appearing <NUM> before the peak. A linear baseline absorption is subtracted from all of the UPLC traces. The linear baseline is fit from two points taken as the average absorption between <NUM> and <NUM> and the average absorption between <NUM> and <NUM>+<NUM>, before the characteristic non-antibody peak at about <NUM>. Parameters for the Generalized Exponentially Modified Gaussian (GEMG) curve (<NPL>) are fit for each of the protein species' peaks observed in the traces using data where these peaks are cleanly observed.

The five parameters that describe the shape of the GEMG curves for each of the various species observed in the UPLC traces were fit using traces that unambiguously displayed those species, and then used as seed values for subsequent curve fittings. After the shapes of each of the species were fit, the remaining curves were fit automatically in Octave by scanning the data for peaks and attempting to place the Chain A HC/Chain B HC heterodimer peak in each one and shifting the other peaks with it, running Octave's fminunc routine to minimize the restrained sum-of-square residuals (SSR), which includes a restraint score on the GEMG-parameter deviations, and then, following optimization, picking the Chain A HC/Chain B HC heterodimer peak assignment that yields the best SSR. For many curves, however, the best SSR does not represent a reasonable interpretation of the data, and so the peak-placement of the Chain A HC/Chain B HC heterodimer is manually determined. The volumes for the peaks are integrated numerically and the molar percentages are then determined by correcting for the absorbance of each species. Otherwise, higher molecular weight contaminants will appear more prominent and lower molecular weight contaminants less prominent than they actually are on a molar basis.

To further assess the heterodimer formation potential of Chain A CH<NUM> domain/Chain B CH<NUM> domain design pairs, further constructs incorporating design modifications in the CH<NUM>/ CH<NUM> dimer interface are prepared and tested, as described below. Molecular Biology: The construction of vectors housing oligonucleotide sequences used to express proteins for FRET analysis is performed as follows. Annealed oligos (IDT) are used to introduce a Myc tag into an in-house vector containing a nucleic acid encoding the mouse kappa leader sequence and a wild type human IgG1 Fc. Two complementary Myc oligos that leave protruding <NUM>' or <NUM>' overhangs for ligation into a vector cut with the appropriate enzymes are designed. The oligos are annealed and ligated into the in-house vector containing the human IgG1 Fc-encoding sequence digested with the appropriate restriction enzymes. After sequence verification, the vector is digested with appropriate enzymes to then introduce the human EGFR Domain <NUM> (hEGFR D3)- or mouse VEGFR1 Domain <NUM> (mVEGFR D3)- encoding sequences.

The hEGFR D3 construct was designed using the crystal structures of the extracellular domains of hEGFR bound to cetuximab (PDB ID 1YY9, Structural basis for inhibition of the epidermal growth factor receptor by cetuximab (<NPL>)) and the D3 domain, specifically, bound to matuzumab (PDB ID 3C09, Matuzumab binding to EGFR prevents the conformational rearrangement required for dimerization. The hEGFR D3 nucleic acid construct was designed with appropriate restriction sites to enable cloning and was synthesized (IDT®). The hEGFR D3 nucleic acid construct is restriction digested, separated on a <NUM>% agarose gel and the DNA fragment is purified using a Gel Extraction Kit (Qiagen). The purified DNA fragment is ligated into the pcDNA <NUM> mammalian expression plasmid (Life Technologies) between the Myc tag and the human IgG1 Fc, respectively. The construct is then sequence verified for use in subsequent cloning of CH3 designs. A two amino acid, GS, linker is inserted between the EGFR or VEGFR1 D3 domains and the human IgG1-Fc.

The mVEGFR1 D3-encoding sequence is obtained from an in-house source and used as a PCR template. The mVEGFR1 D3 protein binds an in-house generated chimeric Mab (as determined by in-house testing). The mVEGFR1 D3-encoding DNA is amplified by PCR using oligonucleotide primers designed to add restriction sites to enable cloning into the vector (pcDNA containing the Myc tag and human IgG1 Fc encoding sequences). The PCR product is digested with the appropriate restriction enzymes and gel purified. The purified DNA PCR product is then ligated into the pcDNA <NUM> between the Myc tag- and the IgG1 Fc- encoding sequences, respectively. The construct is then sequence verified for use in subsequent cloning of CH<NUM> designs.

The sequence of the hEGFR D3-Fc protein containing the wild-type human IgG1 CH<NUM> domain is provided below in SEQ ID. The sequence of the mVEGFR1 D3-Fc protein containing the wild-type human IgG1 CH<NUM> domain is provided below in SEQ ID. Mutant CH<NUM> designs are introduced by direct Geneblock cloning (IDT®) using restriction sites BsrGI and EcoRI and/or Quickchange mutagenesis (Agilent).

Each plasmid is scaled-up by transformation into TOP10 E. coli, mixed with <NUM> luria broth in a <NUM> baffled flask, and shaken O/N at <NUM> rpm. Large scale plasmid purifications are performed using the BenchPro <NUM> (Life Technologies) or HiSpeed Plasmid Maxi Kit (Qiagen) according to the manufacturer's instructions. For protein production, plasmids harboring the Chain A and Chain B DNA sequences are transfected (<NUM>:<NUM> plasmid) into HEK293F cells using Freestyle transfection reagents and protocols provided by the manufacturer (Life Technologies). Transfected cells are grown at <NUM> in a <NUM>% CO2 incubator while shaking at <NUM> rpm for <NUM> days. Secreted protein is harvested by centrifugation at <NUM> rpm for <NUM>. Supernatants are passed through <NUM> filters (both large scale and small scale) for purification. FRET Detection: The Fab detection reagents for use in the FRET assay are generated as follows. The matuzumab human IgG1 MAb (anti-hEGFR D3) was constructed in-house as described previously (Lewis et al. , <NUM>) and a Fab generated from the matuzumab IgG1 MAb using papain digestion as described previously (<NPL>). The anti-mVEGFR1 D3 Fab protein is generated in house from published sequences (<CIT>). Fluorescent isothiocyanato-activated Europium-W1024 (Perkin Elmer Life Sciences) labeling of the anti-mVEGFR1 D3 and Matuzumab Fabs is performed according to the manufacturer's instructions. Fluorescent Cy5 (Amersham Pharmacia Biotech) labeling of the anti-mVEGFR1 D3 and Matuzumab Fabs is performed according to the manufacturer's instructions.

To test the CH<NUM> designs in the FRET assay, Europium(Eu)-labeled anti-mVEGFR1 D3 Fab, or Eu-labeled Matuzumab FAb (anti-hEGFR D3), is mixed with Cy5-labeled anti-mVEGFR1 D3, or Cy5-labeled Matuzumab Fab to final concentrations of <NUM>µg/mL Eu-reagent, <NUM>µg/mL Cy5-reagent in diluted HEK293F cell culture supernatants containing secreted protein resulting from co-expression of both EGFR-D3-Fc (Chain A) and VEGFR1-D3-Fc (Chain B). The cell culture supernatants are diluted <NUM>:<NUM> or <NUM>:<NUM> in PBS, <NUM>/mL BSA, <NUM>% Tween-<NUM> for <NUM> and <NUM> transient transfections, respectively, prior to the FRET measurements. These particular supernatant dilutions result in a roughly <NUM>-<NUM>µg/mL final Protein-Fc concentration optimal for measuring the homodimer/heterodimer ratios. Mixing of the Eu- and Cy5-labeled Matuzumab Fabs enables detection of EGFR-D3-Fc AA homodimer. Mixing Eu- and Cy5-labeled anti-VEGFR1-D3 Fabs enables detection of VEGFR1-D3-Fc BB homodimer. Mixing of Eu-labeled anti-VEGFR1-D3 Fab with Cy5-labeled anti-Cy5-labeled Matuzumab enables detection of EGFR-D3-Fc/VEGFR1-D3-Fc AB heterodimer. The simultaneous binding of Eu-labeled Fab and Cy5-labeled Fab to a single protein molecule (either homodimer-Fc or heterodimer-Fc depending on the Fab combinations) results in a time-resolved fluorescence resonance energy transfer (TR-FRET) from the Europium label to the Cy5 label. <NUM>-<NUM>/<NUM> well microtiter plates (black from Costar) containing the diluted supernatants and labeled Fabs are incubated for approximately <NUM> minutes at room temperature. Fluorescence measurements are carried out on a Wallac Envision <NUM> Multilabel Reader with a dual mirror (PerkinElmer Life Sciences) with the laser excitation of the Europium at wavelength at <NUM> and the emission filters Europium <NUM> and APC <NUM>. Delay between excitation and emission was <NUM>.

To assess the thermostability of Chain A CH<NUM> domain/Chain B CH<NUM> domain design pairs, Fc constructs incorporating design modifications in the CH<NUM>/ CH<NUM> dimer interface are prepared and tested. To generate Fcs for thermostability analysis, including dimers incorporating CH<NUM> domain design pair mutations, an HA-tagged human IgG1 Fc portion (Chain B CH<NUM>- CH<NUM> segment) and a human IgG1 Fc portion without an HA tag (Chain A CH<NUM>- CH<NUM> segment) are constructed. The sequence of the HA tagged-human IgG1 Fc portion containing the WT CH<NUM> domain sequence is provided in SEQ ID NO:<NUM>. The sequence of the human IgG1 Fc portion (without an HA tag) containing the WT CH<NUM> domain sequence is provided by SEQ ID NO:<NUM>.

The CH<NUM> design constructs for use in DSC analysis are made in one of two ways, shuttling from another construct containing a nucleic acid encoding the CH<NUM> design of interest, or site directed mutagenesis. When shuttling between existing constructs, restriction cloning of the CH<NUM> encoding fragment containing the desired mutations is employed. The nucleic acid encoding the CH<NUM> containing the desired mutations is inserted into the vector of interest by digesting both the donor vector and the recipient vector into which the design mutations will be inserted. Both the insert and recipient vector DNA's are purified using gel electrophoresis and the purified insert and receptor vector DNA fragments are then ligated. All ligation constructs are transformed into E. coli strain TOP <NUM> competent cells (Life Technologies). When site-directed mutagenesis is employed, the basic procedure utilizes a supercoiled double-stranded DNA vector containing the wild-type nucleotide sequence of interest and two synthetic oligonucleotide primers (IDT®) containing the desired mutation. The oligonucleotide primers, each complementary to opposite strands of the vector, are extended during thermal cycling by the DNA polymerase (HotStar HiFidelity Kit, Qiagen Cat. Incorporation of the oligonucleotide primers generates a mutated plasmid. Following temperature cycling, the product is treated with Dpn I enzyme. (New England BioLabs, Cat #R0176) The Dpn I enzyme cleaves only methylated parental DNA. The enzyme digested mutated plasmid is then transformed into E. coli strain TOP <NUM> competent cells (Life Technologies).

Sequenced plasmids are scaled-up for transfection as described above for the FRET constructs. Plasmids are transfected into 293F using the same protocol as described for the FRET constructs above. Secreted protein is harvested by centrifugation at <NUM> rpm for <NUM>. Supernatants are passed through <NUM> filters for purification. Purification is performed using protein A chromatography as described by <NPL>.

Following procedures as described above, CH<NUM> designs are incorporated into the Fc portions containing the WT CH<NUM> domain sequences (SEQ ID NO:<NUM> (Chain B) and SEQ ID NO:<NUM> (Chain A)). Differential scanning calorimetry (DSC) measurements are carried out as generally described in <NPL>with a scan rate of <NUM> deg. All DSC thermograms are fit using analysis software provided by the manufacturer (GE Healthcare).

Table <NUM> provides heterodimer formation results (as measured by UPLC and FRET) as well as thermostability data (as determined by DSC) for select designs.

Table <NUM> provides additional heterodimer formation results and thermostability data for select initial designs (e.g., Design <NUM>) as well as further optimized variants of Design <NUM> and the combination Design <NUM>.

The data is Tables <NUM> and <NUM> demonstrate that exemplified CH3 designs yield significant enhancement of Chain A/Chain B heterodimerization relative to dimer constructs which contain only wild-type CH3 domains. Select heterodimer-favoring designs are incorporated into complete IgG heavy chains and the assembly of particular IgG bispecific antibodies is assessed as described in Example <NUM>, below.

Four published antibodies (with published sequences) were chosen to generate three IgG BsAbs. The first MAb pair to be expressed as an IgG BsAb consists of pertuzumab (anti-HER-<NUM>) (see, <NPL>; and <NPL>) and BHA10 (anti-LTβR) (see, <NPL>; and <NPL>. ) The second MAb pair to be expressed as an IgG BsAb consists of a combination of MetMAb (anti-cMET) (see, <NPL>; and <CIT>) and matuzumab (anti-EGFR) (see, <NPL>; and <NPL>. ) Lastly, pertuzumab was paired with matuzumab to form a third set of IgG BsAbs. All BsAbs were tested for assembly using select CH<NUM> designs, as described in Example <NUM>, or WT CH<NUM> domain sequences. The CH<NUM> designs are incorporated into the CH<NUM> domains of each parental Mab pair, with each Mab CH<NUM> domain receiving one set of mutations of a particular design pair (i.e., either the A Chain or B Chain mutations), and the other Mab CH<NUM> domain receiving the other set of mutations of the design pair. In addition, each HC and LC prepared and tested included previously described mutations in the Fab region to promote proper HC-LC pairing as well. Matuzumab and BHA10 HCs and LCs contain Design H4WT (+ DR_CS), while the pertuzumab and MetMAb HCs and LCs contain Design AB2133(a), each as described in <CIT> (see also, <NPL>).

The plasmids for the IgG BsAbs are obtained in-house (see <NPL>). The construction of BsAbs with each set of CH<NUM> designs are done in one of the two following ways.

Oligonucleotide primers with <NUM> base pair extensions (<NUM>') that are complementary to the N-termini of the VH region (VH forward) and the C-terminus of the CH<NUM> region (CH<NUM> reverse) of the HC are used in a PCR reaction to generate recombinase-compatible inserts of the entire HC except the CH<NUM> domain. A second set of oligonucleotides are used to generate additionally inserts encoding the design-containing CH<NUM> domains. The templates for these additional primers are from the FRET, UPLC or DSC constructs described in Example <NUM>. The <NUM>' primers for the CH<NUM> domain are complementary to the junction between CH<NUM> and CH<NUM> (called CH<NUM> forward) and the <NUM>' primers are complementary to the C-terminus of the CH<NUM> region (CH<NUM> reverse). Both the <NUM>' and <NUM>' primers contain <NUM> base pair extensions to allow recombinase-based cloning. The PCR products are gel purified. The BsAb vector(s) are digested with <NUM> different restriction enzymes, removing the CH<NUM>, CH<NUM> and CH<NUM> domains. Recombinase-based cloning is performed using the In-Fusion protocol (Clontech Laboratories, Inc. ) to generate each clone for testing. The LC-containing plasmids are constant throughout the experiments.

Alternately, overlapping PCR is used to generate inserts containing the entire IgG constant domains (<NPL>). The resulting single inserts contain <NUM> base pair <NUM>' and <NUM>' overlaps to allow recombinase-based cloning as described above.

For each of these methods listed above, the new HC-containing vector is then transformed into Dam+ E. coli (Invitrogen One Shot Top10 Chemically Competent E. coli) and plated on LB + Carbenicillin plates <NUM> overnight. Colonies were picked and mutations were verified by sequence analysis. To generate IgG BsAb protein, four plasmids, each containing either a HC or a LC from two separate MAbs, are co-transfected into <NUM> cultures of HEK293F cells using transfection reagent from Life Technologies. The plasmids are transfected at <NUM>µg of each LC and <NUM>µg of each HC into <NUM> cultures. After <NUM> days of shaking incubation in a CO<NUM> incubator at <NUM>, the cell culture supernatants are collected and filtered through <NUM> filters. The supernatants are purified, prepared, and analyzed by high pressure liquid chromatography/mass spectrometry (LCMS) as described in <NPL>. One deviation was that the proteins are enzymatically deglycosylated after purification and neutralization to approximately pH <NUM> using <NUM> Tris, pH <NUM>-<NUM>. Each protein was deglycosylated by the addition of <NUM>µL N-Glycanase (Prozyme) for <NUM>-<NUM> hrs at <NUM> prior to being submitted for LCMS.

Select CH<NUM> heterodimer designs from Example <NUM> are constructed in the HCs listed in Table <NUM>. The designs for testing include <NUM>, <NUM>. <NUM>, <NUM>, <NUM>. <NUM>, <NUM>. <NUM>, and <NUM>. The HCs and LCs from each antibody pair (<NUM> chains total) are transfected into 293F, cultured for <NUM> days, purified using protein G capture, and analyzed by LCMS as described in the methods.

The results of the LCMS data indicate that the exemplified CH<NUM> heterodimer designs from Example <NUM> which were incorporated into the IgG BsAb format resulted in improved correct IgG BsAb assembly (Table <NUM>). Using the wild-type CH<NUM>, the average percentage of heterodimer is found to be <NUM>% - almost identical to the theoretical level expected if both HCs express equally well and there is no bias for heterodimer formation. When the designs are added to the CH<NUM> domain, similar percentages of heterodimer are observed by LCMS of the IgG BsAbs as the percentages found in Example <NUM> by UPLC and FRET using the MetMAb and FRET constructs.

The data in Table <NUM> clearly demonstrates that designs <NUM>, <NUM>. <NUM>, <NUM>, <NUM>. <NUM>, <NUM>. <NUM> and <NUM>. <NUM> improve the assembly of the desired heterotetrameric IgG BsAbs (i.e., HCA/LC1+HCB/LC2) over what was observed with the WT CH<NUM>. The strong correlation between the % heterodimer induced by each design described in Example <NUM> and the % heterodimer induced within the IgG BsAbs in Example <NUM> suggests that all of the exemplified designs from Example <NUM> that improved heterodimer formation based on the UPLC and FRET assays will improve the percentage of heterodimer within the IgG BsAbs format.

Using the same parental Mab pairs as described in Example <NUM>, complete IgG Bispecific Antibodies comprising select CH<NUM> designs and fully IgG4 constant domains in each heavy chain are constructed. As in Example <NUM>, the CH<NUM> designs are incorporated into the CH<NUM> domains of each parental Mab pair, with each Mab CH<NUM> domain receiving one set of mutations of a particular design pair (i.e., either the A Chain or B Chain mutations), and the other Mab CH<NUM> domain receiving the other set of mutations of the design pair. Each HC and LC prepared also included the previously described mutations in the Fab region to promote proper HC-LC pairing as well. Matuzumab and BHA10 HCs and LCs contain Design H4WT (+ DR_CS), while the pertuzumab and MetMAb HCs and LCs contain Design AB2133(a), each as described in <CIT> (see also, <NPL>). Further, to make the resulting IgG BsAb proteins more homogeneous and amenable to eventual LCMS analyses, serine <NUM> (Kabat Numbering) was mutated to proline (S241P) to reduce natural IgG4 half-antibody formation (see, <NPL>)). Additionally, asparagine <NUM> was mutated to glutamine (N297Q) to eliminate N-linked glycosylation. Lastly, the IgG4 lower hinge regions contain a double alanine mutation at positions <NUM> and <NUM> that have been previously described.

DNA encoding complete IgG4 constant domain regions, containing both the Fab (CH<NUM>) specificity designs and CH<NUM> hetero-dimerization designs, are constructed in separate pieces or "blocks" as follows. A DNA block coding for the human IgG4 CH<NUM> region is prepared which contains a <NUM>' region overlapping with an NheI restriction site located behind the variable domain-encoding regions in the expression cassette. A second DNA block coding for the human IgG4 CH<NUM>-CH<NUM> region, containing a BSU361 restriction site at the beginning of the CH<NUM> encoding region, a PshAI site at the <NUM>' end of the CH<NUM> encoding region, and a <NUM>' region overlapping within the EcoRI site of a template IgG1 expression cassette is also prepared. Two CH<NUM>-CH<NUM> DNA blocks are prepared for each heavy chain of each parental Mab, one containing the hetero-dimerization design <NUM>. <NUM> mutations (either the "A" or "B" side mutations) and the other containing the design <NUM>. <NUM> mutations (either the "A" or "B" side mutations). The pertuzumab and metMAb constructs are designed to contain the "AB2133a" encoding Fab (CH<NUM>) design mutations and the 'A' side mutations for either <NUM>. <NUM> or <NUM>. <NUM>, whereas the matuzumab and BHA10 constructs are designed to contain the "H4WT(+DR_CS)" encoding Fab (CH<NUM>) designs and the 'B' side mutations for either <NUM>. <NUM> or <NUM>. Overlapping PCR is performed with the CH<NUM> and the CH<NUM>-CH<NUM> DNA blocks to generate inserts containing the entire human IgG4 constant domains (<NPL>). The complete IgG4 constant domain constructs are then amplified prior to cloning into mammalian expression vectors.

Mammalian expression plasmids encoding human IgG1 heavy chains for each of pertuzumab, metMab, matuzumab, and BHA10, as previously described (<NPL>)), are cut at restriction sites (Nhel and EcoRI) at the <NUM>' and <NUM>' ends of the heavy chain constant domain coding region to allow excision of the IgG1 constant domain-encoding sequences. The linearized vectors are then purified using a DNA gel extraction kit (Qiagen, Cat. No. <NUM>) according to the manufacturer's protocol. The human IgG4 constant domain encoding constructs are then cloned into the previously cut expression plasmid using Gibson Assembly ®Master Mix (New England Biolabs). All constructs utilized a murine kappa leader signal sequence that is cleaved upon secretion. Ligated constructs are transformed into chemically competent Top <NUM> E. Coli cells (Life Technologies) for scale up. Colonies are selected using an ampicillin selection marker, cultured, and final plasmids are prepared (Qiagen Mini Prep Kit). Correct sequences are confirmed by in-house DNA sequencing.

Complete IgG BsAbs are expressed in HEK293F cells as described in Example <NUM> above and as provided in Lewis et al. , cited above. The heavy chain and light chain components of the complete IgG bispecific antibodies, constructed in IgG4 heavy chain backbones, and their corresponding sequences, are provide in Table <NUM> below.

After a five day culture, small scale purifications of prepared IgG BsAbs (in human IgG4 heavy chain backbones) from <NUM>µL mammalian cell culture supernatants are performed using a multidimensional Dionex UPLC system. A protein G column (POROS® G <NUM> Column, <NUM> x <NUM>, <NUM> part # <NUM>-<NUM>-<NUM>) is equilibrated with 1x PBS prior to sample load. <NUM>µL of each cell culture supernatant (filtered using <NUM> syringe filters, Millipore) are injected onto the protein G column. After washing with 1x PBS, the BsAbs are eluted with <NUM> sodium phosphate, pH <NUM> (<NUM> minutes at <NUM>/min). Titers are determined using the ultraviolet peak area at <NUM> upon elution, with calculations based upon a standard curve created with an in-house mAb. Protein G eluted peak samples are collected into vials in an autosampler held at ambient temperature.

Mass spectrometry is used to quantify bispecific antibody assembly from the purified samples. Experiments are performed using Q-ToF (Waters Technologies) mass spectrometer (MS) with a Xevo source. Samples are introduced into the MS using an Acquity UPLC system (Waters Technologies) connected in-line with a Reversed Phase column (ThermoScientific, Proswift™, RP-<NUM>, <NUM>×<NUM> i. ) at a flow rate of 200uL/min. To eliminate salts and non-volatile buffers not compatible with MS, gradient elution was performed using <NUM>% formic acid in H<NUM>O (Solvent A) and <NUM>% formic acid in acetonitrile (Solvent B). Mass spectrometry is accomplished in positive ion mode with <NUM>. 6kV capillary voltage at a <NUM> source temperature. Data processing and interpretation of LC-MS runs is done in BiopharmLynx (a MassLynx Software application manager) using spectral summation over the chromatographic elution profile of the antibody.

The peak areas of the deconvoluted mass spectra are used to calculate the percent of each species, with the expectation that each of the IgG4 BsAb proteins with a mass near <NUM> kDa (whether assembled correctly or misassembled) are ionized with a similar efficiency. Results are provided in Table <NUM>, below.

The data in Table <NUM> demonstrates that designs <NUM>. <NUM> and <NUM>. <NUM>, when applied to the human IgG4 constant domains, and paired with the Fab designs, induce predominantly correct assembly (><NUM>%) of the desired heterotetrameric IgG BsAbs (i.e., HCA/LC1+HCB/LC2) over the misassembled protein products. No LC mispairing (existence of two of the same LCs on a HC heterodimer) was observed for any of the IgG BsAbs in the human IgG4 heavy chain backbones. Small levels of homodimeric HC products were observed (either AA homodimer or BB homodimer), however, the clear main peak for each of the six BsAbs prepared was the desired IgG BsAb.

Claim 1:
An IgG bispecific antibody comprising:
a. a first heavy chain, wherein said first heavy chain comprises a first variable domain VH and a first human IgG1, human IgG2 or human IgG4 constant region, wherein said first human IgG1, human IgG2 or human IgG4 constant region comprises an alanine at residue <NUM>, a methionine at residue <NUM>, and an aspartic acid at residue <NUM> of the CH<NUM> domain, wherein the residues are numbered according to the EU Index Numbering system;
b. a first light chain, wherein said first light chain comprises a first variable domain VL and a first constant domain CL;
c. a second heavy chain, wherein said second heavy chain comprises a second variable domain VH and a second human IgG1, human IgG2 or human IgG4 constant region, wherein said second human IgG1, human IgG2 or human IgG4 constant region comprises a valine at residue <NUM>, a valine at residue <NUM>, and an arginine at residues <NUM> and <NUM> of the CH<NUM> domain, wherein the residues are numbered according to the EU Index Numbering system; and
d. a second light chain, wherein said second light chain comprises a second variable domain VL and a second constant domain CL.