WO2018026942A1 - Heteromeric polypeptides - Google Patents

Abstract

Proteins comprising CH3 variants for generation of heteromeric polypeptides are disclosed.

Description

HETEROMERIC POLYPEPTIDES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 62/370,015, filed August 2, 2016. The content of the aforementioned application is hereby incorporated by reference.

TECHNICAL FIELD

This application relates to heteromeric polypeptides. BACKGROUND

Antibodies have become the modality of choice within the biopharma industry because they possess several characteristics that are attractive to those developing therapeutic molecules. Along with the ability to target specific structures or cells, antibodies make their target susceptible to Fc-receptor cell-mediated phagocytosis and killing (Raghavan and Bjorkman 1996). Further, the antibody's ability to interact with neonatal Fc-receptor (FcRn) in a pH dependent manner confers it with extended serum half-life (Ghetie and Ward 2000).

Antibodies belong to the immunoglobulin class of proteins which includes IgG, IgA, IgE, IgM, and IgD. The most abundant immunoglobulin class in human serum is IgG. The IgG structure has four chains, two light and two heavy chains; each light chain has two domains and each heavy chain has four domains. The antigen binding site is located in the Fab region

(Fragment antigen binding) which contains a variable light (VL) and a variable heavy (VH) chain domain as well as constant light (LC) and constant heavy (CHI) chain domains. The CH2 and CH3 domain region of the heavy chain is called Fc (Fragment crystallizable). The IgG molecule can be considered as a heterotetramer having two heavy chains that are held together by disulfide bonds at the hinge region and two light chains. The Fc region alone can be thought of as a homodimer of heavy chains comprising CH2 and CH3 domains.

In certain instances, it is desirable to create a molecule that contains the Fc portion of an antibody but comprises a heterodimer. An important application of Fc heterodimeric molecules is the generation of bispecific antibodies (BsAbs). Bispecific antibodies refer to antibodies having specificities for at least two different antigens (Nolan and O'Kennedy 1990; de Leij,

Molema et al. 1998; Carter 2001). In one format, instead of having identical sequence in both the Fabs, bispecific antibodies bear different sequences in the two Fabs so that each arm of the Y- shaped molecule can bind to different antigens.

The use of bispecific antibodies for immunotherapy of cancer has been extensively reviewed in the literature (see, e.g., Nolan and O'Kennedy 1990; de Leij, Molema et al. 1998; Carter 2001).

The classical method of producing BsAbs by co-expressing two different IgGs in hybrid hybridomas leads to up to 10 possible combinations of heavy and light chains. This compromises the yield and imposes a purification challenge. Carter and co-workers engineered heavy chains for heterodimerization using a "knobs-into-holes" strategy (Ridgway, Presta et al. 1996; Atwell, Ridgway et al. 1997; Merchant, Zhu et al. 1998; Carter 2001). Carter and co-workers created a knob at the CH3 domain interface of the first chain by replacing a smaller amino acid side chain with a larger one (for example, T366Y); and a hole in the juxtaposed position at the CH3 interface of the second chain was created by replacing a larger amino acid side chain with a smaller one (for example, Y407T). The "knobs-into-holes" technique is disclosed in U.S. Pat. Nos. 5,731,168 and 7,183,076.

SUMMARY

This application discloses improved heterodimeric polypeptides, e.g., for use in creation of asymmetric molecules.

This application describes a strategy for altering the interaction of antibody domains, e.g., altering a CH3 domain to reduce the ability of the domain to interact with itself, i.e., form homodimers. In particular, one or more residues that make up the CH3-CH3 interface is replaced with a protuberance, whereas a contacting residue at the interface is replaced with a cavity. In certain aspects, the invention provides a method of preparing a heterodimeric protein. The heterodimer may comprise a first CH3 -containing polypeptide and a second CH3 -containing polypeptide that meet together to form an interface engineered to promote heterodimer formation. The first CH3 -containing polypeptide and second CH3 -containing polypeptide are engineered to comprise one or more charged amino acids within the interface that are

electrostatically unfavorable to homodimer formation but electrostatically favorable to heterodimer formation. Such methods may include culturing a host cell comprising nucleic acids encoding the first and second CH3 -containing polypeptides such that the polypeptides are co-expressed by the cell. In certain embodiments, the nucleic acids encoding the first and the second CH3-containing polypeptides are provided to the host cell at a ratio, for example 1: 1, 1:2, 2: 1, 1:3, 3: 1, 1:4, 4: 1, 1:5, 5: 1, 1:6, 6: 1, 1:7, 7: 1, 1:8, 8: 1, 1:9, 9: 1, 1: 10, 10: 1. It is contemplated that altering the ratio of nucleic acids may increase the production of heterodimeric molecules versus homodimeric molecules.

The heterodimeric molecules may be purified from the host-cell culture using standard techniques. For example, when the heterodimeric protein comprises an Fc, the protein may be purified using a Protein A column. The purification techniques include but are not limited to chromatographic methods such as size exclusion, ion exchange and affinity-based

chromatography and ultracentrifugation.

In certain embodiments, the CH3 -containing polypeptide comprises an IgG Fc region, preferably derived from a wild-type human IgG Fc region. By "wild-type" human IgG Fc it is meant a sequence of amino acids that occurs naturally within the human population. Of course, just as the Fc sequence may vary slightly between individuals, one or more alterations may be made to a wild-type sequence and still remain within the scope of the invention. For example, the Fc region may contain additional alterations that are not related to the present invention, such as a mutation in a glycosylation site or inclusion of an unnatural amino acid.

In certain embodiments, the polypeptide containing the CH3 region is an IgG molecule and further contains a CHI and CH2 domain. Exemplary human IgG sequences comprise the constant regions of IgGl (e.g., SEQ ID NO:3; CHl=amino acids 1-98, CH2=amino acids 111- 223, CH3=224-330), IgG2 (e.g., SEQ ID NO:4; CHl=amino acids 1-94, CH2=amino acids 111- 219, CH3=220-326), IgG3 (e.g., SEQ ID NO:5; CHl=amino acids 1-98, CH2=amino acids 161- 270, CH3=271-377), and IgG4 (e.g., SEQ ID NO:6; CHl=amino acids 1-98, CH2=amino acids 111-220, CH3=221-327). Exemplary human IgGl, IgG2, IgG3, and IgG4 CH3 regions are also provided as SEQ ID NOs: l l-14. In one embodiment, the first CH3 domain and second CH3 domain are each selected from the group consisting of a human IgGl CH3 domain, a human IgG2 CH3 domain, a human IgG3 CH3 domain, a human IgG4 CH3 domain, a human IgA CH3 domain, a human IgD CH3 domain, a human IgE CH3 domain, a human IgM CH3 domain. Those of skill in the art may differ in their understanding of the exact amino acids corresponding to the various domains of the IgG molecule. Thus, the N-terminus or C-terminus of the domains outlined above may extend or be shortened by 1, 2, 3, 4, 5, 6, 7, 8, 9, or even 10 amino acids. Also note that the numbering scheme used here to designate domains differ from the Eu numbering scheme of Kabat that is used in the rest of this patent application. For example, IgGl "CH3=224-330" corresponds to "CH3=341-447" in Eu numbering scheme.

The Fc region also may be comprised within the constant region of an IgA (e.g., SEQ ID NO:7), IgD (e.g., SEQ ID NO:8), IgE (e.g., SEQ ID NO:9), and IgM (e.g., SEQ ID NO: 10) heavy chain.

The polypeptide containing the CH3 region may be an antibody heavy chain and the host cell may further express one or more antibody light chains. In embodiments wherein more than one heavy chain and light chains are co-expressed (e.g., bivalent antibody), each heavy chain may comprise a mutation in the CH3 region and each light chain may comprise a mutation in the constant region to preferentially bind to each other but not bind to the other light or heavy chain, respectively.

Preferred embodiments of the invention include but are not limited to an antibody, a bispecific antibody, a monospecific monovalent antibody, a bispecific maxibody (maxibody refers to scFv-Fc), a monobody, a peptibody, a bispecific peptibody, a monovalent peptibody (a peptide fused to one arm of a heterodimeric Fc molecule), a ligand-Fc fusion protein, and a receptor-Fc fusion protein.

Examples of mammalian host cells that may be used include but are not limited to CHO, 293, and myeloma cell lines. The host cell may also be yeast or a prokaryote, such as E. coli.

The heterodimeric proteins may be particularly useful in therapeutic compositions. In certain embodiments, a heterodimeric protein may be formulated in a composition that includes one or more pharmaceutically acceptable buffer or excipient. Such therapeutic composition may be administered to a subject to treat a disease or may be given to prevent a disease or prevent the symptoms of a disease from progressing. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a line graph depicting Fc-gamma receptor IIIA binding of antibodies comprising FCLl, FCL27, and hFc wt.

FIG. 2 is a line graph depicting Fc-gamma receptor IIIB binding of antibodies comprising FCLl, FCL27, and hFc wt.

DETAILED DESCRIPTION

I. Definitions

In general, the following words or phrases have the indicated definitions when used in the description, examples, and claims:

A "heteromultimer" or "heteromultimeric polypeptide" is a molecule comprising at least a first polypeptide and a second polypeptide, wherein the second polypeptide differs in amino acid sequence from the first polypeptide by at least one amino acid residue. Preferably, the

heteromultimer has binding specificity for at least two different ligands or binding sites. The heteromultimer can comprise a "heterodimer" formed by the first and second polypeptide or can form higher order tertiary structures where polypeptides in addition to the first and second polypeptide are present. Exemplary structures for the heteromultimer include heterodimers (e.g. the bispecific immunoadhesin described by Dietsch et al., supra), heterotrimers (e.g. the Ab/Ia chimera described by Chamow et al., supra), heterotetramers (e.g. a bispecific antibody) and further oligomeric structures. In one aspect, a protein or polypeptide comprises a first CH3 domain paired with a non-identical second CH3 domain. In one embodiment, the first CH3 domain and second CH3 domain are present on separate polypeptides. In another embodiment, the first CH3 domain and second CH3 domain are present on the same polypeptide. In another embodiment, the protein or polypeptide comprises one or more antibody antigen binding domains, receptor ligand binding domains, or ligand domains.

As used herein, "polypeptide" refers generally to peptides and proteins having more than about ten amino acids. Preferably, mammalian polypeptides (polypeptides that were originally derived from a mammalian organism) are used, more preferably those which are directly secreted into the medium. Examples of bacterial polypeptides include, e.g., alkaline phosphatase and beta-lactamase. Examples of mammalian polypeptides include molecules such as renin, a growth hormone, including human growth hormone; bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha- 1 -antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor; anti-clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or human urine or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and -beta; enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); human macrophage inflammatory protein (MIP-1 -alpha); a serum albumin such as human serum albumin; Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;

prorelaxin; mouse gonadotropin-associated peptide; a microbial protein, such as beta-lactamase; DNase; inhibin; activin; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; integrin; protein A or D; rheumatoid factors; a neurotrophic factor such as bone- derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF-beta; platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta, including TGF-betal, TGF-beta2, TGF-beta3, TGF-beta4, or TGF-beta5; insulin-like growth factor-I and -II (IGF-I and IGF-II); des(l-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins; CD proteins such as CD-3, CD-4, CD- 8, and CD-19; erythropoietin; osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); an interferon such as interferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10;

superoxide dismutase; T-cell receptors; surface membrane proteins; decay accelerating factor; viral antigen such as, for example, a portion of the AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins; antibodies; and fragments of any of the above-listed polypeptides. Additional polypeptides may include ligands, receptors, or soluble portions of any of EGF, EGFR, HER2/neu, ErbB3 (HER3), IGF1R, EphA2, c-MET, EpCAM, clotting factor VIII, clotting factor IX, clotting factor IXa, clotting factor X, lymphocyte function-associated antigen (LFA3), TNFR1, TNFR2, cytotoxic T lymphocyte associated molecule-4 (CTLA-4), VEGF-A, VEGFR1, VEGFR2, IL-1R, IL-2R alpha, IL-3R alpha, IL-4R alpha, IL-6R, IL-21R, thrombopoeitin binding peptide, TIE2, TIE2 mimetic peptide, BAFF, GLP1, GLP1 peptide analog, erythropoietin, erythropoietin mimetic peptide, IL-la, IL-lb, IL-2, IL-4, IL-5, IL-6, IL-8, IL-12, IL-13, IL-17, IL-17A, IL-17F, IL-22, IL-23, IL-23 pl9, IL-33, GM-CSF, HGF/SF, CD28, B7-1 (CD80), B7-2 (CD86), CD40, CD40L, OX40 (CD134), OX40L, 4-lBB (CD137), 4-lBBL, ICOS, ICOSL, GITR, GITRL, TLT-2, B7-H3, PD-1, PD-L1, PD-L2, B7-H4, VISTA, LAG3, TIM-3, galectin-9, TRAIL, DR4, DR5, CD3, CD4, CD5, CD19, CD20, CD22, CD26, CD30, CD32B, CD33, CD37, CD38, CD44, CD47, CD52, CD70, CD79B, CD95, CD123, TNF-alpha, CD 16 A, angiopoietin 2, carcinoembryonic antigen (CEA), PSMA, CD64, HSG, GD2, gpA33, gplOO, DLL4, human serum albumin (HSA), amyloid beta, MUC1, MUC18, PCSK9, NGF, Tau, RANKL, DKK1, CXCR4, complement component 5 (C5), complement C3b, glucagon receptor (GCGR), carbonic anhydrase IX (CAIX), GD2 ganglioside, mesothelin, tissue factor (TF), transferrin receptor (TfR), BCMA, ICAM-1, myostatin, sclerostin, TACSTD-2, CCR5, tenascin C, CSF-1 receptor, HMW-MAA, Notchl, Jagged, CA125, Integrin alpha lib beta 3, integrin alpha4 beta7, c-Kit, BSG2, FGFR, FGFR3, GD3, IGF-2, phosphatidylserine, RON, TAG-72, TGF-beta, trophoblast glycoprotein (TPBG), glypican 3, HB-EGF, TWEAK, TWEAKR, TrkB, urokinase-type plasminogen activator receptor, alpha-synuclein, LRP6, A33, fibroblast activation protein (FAP), interferon gamma, IP-10, NRP1, PDGFRA, PDGFRB, VAP-1, CXCL13, CCR2, Nogo-A, prominin-1, apolipoprotein E, erythropoietin receptor, EphB2, EphB4, inuslin receptor, beta-secretase 1, P-cadherin, FcRn, angiopoietin-like protein 4, VEGI, properdin, FLT1, ROR1, BST2, TIM-3, ActRIIB, MIC-1, CLEC12A.

The "first polypeptide" is any polypeptide which is to be associated with a second polypeptide. The first and second polypeptide meet at an "interface" (defined below). In addition to the interface, the first polypeptide may comprise one or more additional domains, such as "binding domains" (e.g. an antibody variable domain, receptor binding domain, ligand binding domain or enzymatic domain) or antibody constant domains (or parts thereof) including CH2, CHI and CL domains. Normally, the first polypeptide will comprise at least one domain which is derived from an antibody. This domain conveniently is a constant domain, such as the CH3 domain of an antibody and can form the interface of the first polypeptide. Exemplary first polypeptides include antibody heavy chain polypeptides, chimeras combining an antibody constant domain with a binding domain of a heterologous polypeptide (i.e. an immunoadhesin, see definition below), receptor polypeptides (especially those which form dimers with another receptor polypeptide, e.g., interleukin-8 receptor [IL-8R] and integrin heterodimers [e.g. LFA-1 or GPIIIb/IIIa]), ligand polypeptides (e.g. nerve growth factor [NGF], neurotrophin-3 [NT-3], and brain-derived neurotrophic factor [BDNF]— see Arakawa et al. J. Biol. Chem. 269(45):

27833-27839 [1994] and Radziejewski et al. Biochem. 32(48): 1350 [1993]) and antibody variable domain polypeptides (e.g. diabodies). The preferred first polypeptide is selected from an antibody heavy chain and an immunoadhesin.

The "second polypeptide" is any polypeptide which is to be associated with the first polypeptide via an "interface". In addition to the interface, the second polypeptide may comprise additional domains such as a "binding domain" (e.g. an antibody variable domain, receptor binding domain, ligand binding domain or enzymatic domain), or antibody constant domains (or parts thereof) including CH2, CHI and CL domains. Normally, the second polypeptide will comprise at least one domain which is derived from an antibody. This domain conveniently is a constant region, such as the CH3 domain of an antibody and can form the interface of the second polypeptide. Exemplary second polypeptides include antibody heavy chain polypeptides, chimeras combining an antibody constant domain with a binding domain of a heterologous polypeptide (i.e. an immunoadhesin, see definition below), receptor polypeptides (especially those which form dimers with another receptor polypeptide, e.g., interleukin-8 receptor [IL-8R] and integrin heterodimers e[g. LFA-1 or GPIIIb/IIIa]), ligand polypeptides (e.g. nerve growth factor [NGF], neurotrophin-3 [NT-3], and brain-derived neurotrophic factor [BDNF]— see Arakawa et al. J. Biol. Chem. 269(45): 27833-27839 [1994] and Radziejewski et al. Biochem. 32(48): 1350 [1993]) and antibody variable domain polypeptides (e.g. diabodies). The preferred second polypeptide is selected from an antibody heavy chain and an immunoadhesin.

A "binding domain" comprises any region of a polypeptide which is responsible for selectively binding to a molecule of interest (e.g. an antigen, ligand, receptor, substrate or inhibitor). Exemplary binding domains include an antibody variable domain, receptor binding domain, ligand binding domain and an enzymatic domain.

The term "antibody" shall mean a polypeptide containing one or more domains capable of binding an epitope on an antigen of interest, where such domain(s) are derived from or homologous with the variable region of an antibody. Examples of antibodies include full length antibodies, antibody fragments, single chain molecules, bispecific or bifunctional molecules, diabodies, and chimeric antibodies (e.g. humanized and primatized antibodies). "Antibody fragments" include Fv, Fv', Fab, Fab', and F(ab')2 fragments. "Humanized" forms of non-human (e.g. rodent or primate) antibodies are specific chimeric immunoglobulins, immunoglobulin chains or fragments thereof which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, rabbit or primate having the desired specificity, affinity and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The humanized antibody includes a primatized antibody wherein the antigen-binding region of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest.

A "multispecific antibody" is a molecule having binding specificities for at least two different antigens. While such molecules normally will only bind two antigens (i.e. bispecific antibodies, BsAbs), antibodies with additional specificities such as trispecific antibodies are encompassed by this expression when used herein. Examples of BsAbs include those with one arm directed against a tumor cell antigen and the other arm directed against a cytotoxic trigger molecule such as anti-FcgammaRI/anti-CD15, anti-pl85HER2/FcgammaRIII (CD16), anti- CD3/anti-malignant B-cell (ID 10), anti-CD3/anti-pl85HER2, anti-CD3/anti-p97, anti-CD3/anti- renal cell carcinoma, anti-CD3/anti-OVCAR-3, anti-CD3/L-Dl (anti-colon carcinoma), anti- CD3/anti-melanocyte stimulating hormone analog, anti-EGF receptor/anti-CD3, anti-CD3/anti- CAMA1, anti-CD3/anti-CD19, anti-CD3/MoV18, anti-neural cell adhesion molecule

(NCAM)/anti-CD3, anti-folate binding protein (FBP)/anti-CD3, anti-pan carcinoma associated antigen (AMOC-31)/anti-CD3; BsAbs with one arm which binds specifically to a tumor antigen and one arm which binds to a toxin such as anti-saporin/anti-Id-1, anti-CD22/anti-saporin, anti- CD7/anti-saporin, anti-CD38/anti-saporin, anti-CEA/anti-ricin A chain, anti-interferon-alpha (IFN-alpha)/anti-hybridoma idiotype, anti-CEA/anti-vinca alkaloid; BsAbs for converting enzyme activated prodrugs such as anti-CD30/anti-alkaline phosphatase (which catalyzes conversion of mitomycin phosphate prodrug to mitomycin alcohol); BsAbs which can be used as fibrinolytic agents such as anti-fibrin/anti-tissue plasminogen activator (tPA), anti-fibrin/anti- urokinase-type plasminogen activator (uPA); BsAbs for targeting immune complexes to cell surface receptors such as anti-low density lipoprotein (LDL)/anti-Fc receptor (e.g. FcgammaRI, FcgammaRII or FcgammaRIII); BsAbs for use in therapy of infectious diseases such as anti- CD3/anti-herpes simplex virus (HSV), anti-T-cell receptor:CD3 complex/anti-influenza, anti- FcgammaR/anti-HIV; BsAbs for tumor detection in vitro or in vivo such as anti-CEA/anti-

EOTUBE, anti-CEA/anti-DPTA, anti-pl85HER2/anti-hapten; BsAbs as vaccine adjuvants (see Fanger et al., supra); and BsAbs as diagnostic tools such as anti-rabbit IgG/anti-ferritin, anti- horse radish peroxidase (HRP)/anti-hormone, anti-somatostatin/anti-substance P, anti-HRP/anti- FITC, anti-CEA/anti-beta-galactosidase (see Nolan et al., supra). Examples of trispecific antibodies include anti-CD3/anti-CD4/anti-CD37, anti-CD3/anti-CD5/anti-CD37 and anti- CD3/anti-CD8/anti-CD37.

As used herein, the term "immunoadhesin" designates antibody-like molecules which combine the "binding domain" of a heterologous protein (an "adhesin", e.g. a receptor, ligand or enzyme) with the effector functions of immunoglobulin constant domains. Structurally, the immunoadhesins comprise a fusion of the adhesin amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site (antigen combining site) of an antibody (i.e. is "heterologous") and an immunoglobulin constant domain sequence. The immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgGl, IgG2, IgG3, or IgG4 subtypes, IgA, IgE, IgD or IgM.

The term "ligand binding domain" as used herein refers to any native cell-surface receptor or any region or derivative thereof retaining at least a qualitative ligand binding ability, and preferably the biological activity of a corresponding native receptor. In a specific embodiment, the receptor is from a cell-surface polypeptide having an extracellular domain which is homologous to a member of the immunoglobulin supergene family. Other typical receptors, are not members of the immunoglobulin supergene family but are nonetheless specifically covered by this definition, are receptors for cytokines, and in particular receptors with tyrosine kinase activity (receptor tyrosine kinases), members of the hematopoietin and nerve growth factor receptor superfamilies, and cell adhesion molecules, e.g. (E-, L- and P-) selectins.

The term "receptor binding domain" is used to designate any native ligand for a receptor, including cell adhesion molecules, or any region or derivative of such native ligand retaining at least a qualitative receptor binding ability, and preferably the biological activity of a

corresponding native ligand. This definition, among others, specifically includes binding sequences from ligands for the above-mentioned receptors.

As used herein the phrase "multispecific immunoadhesin" designates immunoadhesins (as hereinabove defined) having at least two binding specificities (i.e. combining two or more adhesin binding domains). Multispecific immunoadhesins can be assembled as heterodimers, heterotrimers or heterotetramers, essentially as disclosed in WO 89/02922 (published 6 Apr. 1989), in EP 314,317 (published 3 May 1989), and in U.S. Pat. No. 5,116,964 issued 2 May 1992. Preferred multispecific immunoadhesins are bispecific. Examples of bispecific

immunoadhesins include CD4-IgG/TNFreceptor-IgG and CD4-IgG/L-selectin-IgG. The last mentioned molecule combines the lymph node binding function of the lymphocyte homing receptor (LHR, L-selectin), and the HIV binding function of CD4, and finds potential application in the prevention or treatment of HIV infection, related conditions, or as a diagnostic.

An "antibody-immunoadhesin chimera (Ab/Ia chimera)" comprises a molecule which combines at least one binding domain of an antibody (as herein defined) with at least one immunoadhesin (as defined in this application). Exemplary Ab/Ia chimeras are the bispecific CD4-IgG chimeras described by Berg et al., supra and Chamow et al., supra.

The "interface" comprises those "contact" amino acid residues (or other non-amino acid groups such as carbohydrate groups, NADH, biotin, FAD or haem group) in the first polypeptide which interact with one or more "contact" amino acid residues (or other non-amino acid groups) in the interface of the second polypeptide. The preferred interface is a domain of an

immunoglobulin such as a variable domain or constant domain (or regions thereof), however the interface between the polypeptides forming a heteromultimeric receptor or the interface between two or more ligands such as NGF, NT-3 and BDNF are included within the scope of this term. The preferred interface comprises the CH3 domain of an immunoglobulin which preferably is derived from an IgG antibody and most preferably an human IgGl antibody. A "protuberance" refers to at least one amino acid side chain which projects from the interface of the first polypeptide and is therefore positionable in a compensatory cavity in the adjacent interface (i.e. the interface of the second polypeptide) so as to stabilize the

heteromultimer, and thereby favor heteromultimer formation over homomultimer formation, for example. The protuberance may exist in the original interface or may be introduced synthetically (e.g. by altering nucleic acid encoding the interface). Normally, nucleic acid encoding the interface of the first polypeptide is altered to encode the protuberance. To achieve this, the nucleic acid encoding at least one "original" amino acid residue in the interface of the first polypeptide is replaced with nucleic acid encoding at least one "import" amino acid residue which has a larger side chain volume than the original amino acid residue. It will be appreciated that there can be more than one original and corresponding import residue. The upper limit for the number of original residues which are replaced is the total number of residues in the interface of the first polypeptide. The side chain volumes of the various amino residues are shown in the following table.

Table 1. Properties of Amino Acid Residues

Lysine (Lys) K 128.18 168.6 200

Methionine (Met) M 131.21 162.9 185

Phenylalanine (Phe) F 147.18 189.9 210

Proline (Pro) P 97.12 122.7 145

Serine (Ser) S 87.08 89.0 115

Threonine (Thr) T 101.11 116.1 140

Tryptophan (Trp) w 186.21 227.8 255

Tyrosine (Tyr) Y 163.18 193.6 230

Valine (Val) V 99.14 140.0 155

a, Molecular weight amino acid minus that of water. Values from Handbook of Chemistry and Physics, 43rd ed. Cleveland, Chemical Rubber Publishing Co., 1961.

b, Values from A. A. Zamyatnin, Prog. Biophys. Mol. Biol. 24: 107 123, 1972.

c, Values from C. Chothia, J. Mol. Biol. 105: 1 14, 1975.

The preferred import residues for the formation of a protuberance are generally naturally occurring amino acid residues and are preferably selected from arginine (R), phenylalanine (F), tyrosine (Y), isoleucine (I), leucine (L), valine (V), and tryptophan (W). Most preferred are phenylalanine, isoleucine, leucine, valine, and tryptophan. In the preferred embodiment, the original residue for the formation of the protuberance has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, or threonine.

A "cavity" refers to at least one amino acid side chain which is recessed from the interface of the second polypeptide and therefore accommodates a corresponding protuberance on the adjacent interface of the first polypeptide. The cavity may exist in the original interface or may be introduced synthetically (e.g. by altering nucleic acid encoding the interface). Normally, nucleic acid encoding the interface of the second polypeptide is altered to encode the cavity. To achieve this, the nucleic acid encoding at least one "original" amino acid residue in the interface of the second polypeptide is replaced with DNA encoding at least one "import" amino acid residue which has a smaller side chain volume than the original amino acid residue. It will be appreciated that there can be more than one original and corresponding import residue. The upper limit for the number of original residues which are replaced is the total number of residues in the interface of the second polypeptide. The side chain volumes of the various amino residues are shown in Table 1 above. The preferred import residues for the formation of a cavity are usually naturally occurring amino acid residues and are preferably selected from alanine (A), serine (S), and threonine (T). In the preferred embodiment, the original residue for the formation of the protuberance has a large side chain volume, such as tyrosine, arginine, phenylalanine or tryptophan.

An "original" amino acid residue is one which is replaced by an "import" residue which can have a smaller or larger side chain volume than the original residue. The import amino acid residue can be a naturally occurring or non-naturally occurring amino acid residue, but preferably is the former. "Naturally occurring" amino acid residues are those residues encoded by the genetic code. By "non-naturally occurring" amino acid residue is meant a residue which is not encoded by the genetic code, but which is able to covalently bind adjacent amino acid residue(s) in the polypeptide chain. Examples of non-naturally occurring amino acid residues are norleucine, ornithine, norvaline, homoserine and other amino acid residue analogues such as those described in Ellman et al., Meth. Enzym. 202:301-336 (1991), for example. To generate such non-naturally occurring amino acid residues, the procedures of Noren et al. Science 244: 182 (1989) and Ellman et al., supra can be used. Briefly, this involves chemically activating a suppressor tRNA with a non-naturally occurring amino acid residue followed by in vitro transcription and translation of the RNA. The method of the instant invention involves replacing at least one original amino acid residue, but more than one original residue can be replaced. Normally, no more than the total residues in the interface of the first or second polypeptide will comprise original amino acid residues which are replaced. The preferred original residues for replacement are "buried". By "buried" is meant that the residue is essentially inaccessible to solvent. The preferred import residue is not cysteine to prevent possible oxidation or mispairing of disulfide bonds.

The protuberance is "positionable" in the cavity which means that the spatial location of the protuberance and cavity on the interface of the first polypeptide and second polypeptide respectively and the sizes of the protuberance and cavity are such that the protuberance can be located in the cavity without significantly perturbing the normal association of the first and second polypeptides at the interface. Since protuberances such as Tyr, Phe and Trp do not typically extend perpendicularly from the axis of the interface and have preferred conformations, the alignment of a protuberance with a corresponding cavity relies on modeling the

protuberance/cavity pair based upon a three-dimensional structure such as that obtained by X-ray crystallography or nuclear magnetic resonance (NMR). This can be achieved using widely accepted techniques in the art.

By "original nucleic acid" is meant the nucleic acid encoding a polypeptide of interest which can be "altered" (i.e. genetically engineered or mutated) to encode a protuberance or cavity. The original or starting nucleic acid may be a naturally occurring nucleic acid or may comprise a nucleic acid which has been subjected to prior alteration (e.g. a humanized antibody fragment). By "altering" the nucleic acid is meant that the original nucleic acid is mutated by inserting, deleting or replacing at least one codon encoding an amino acid residue of interest. Normally, a codon encoding an original residue is replaced by a codon encoding an import residue. Techniques for genetically modifying a DNA in this manner have been reviewed in

Mutagenesis: a Practical Approach, M. J. McPherson, Ed., (IRL Press, Oxford, UK. (1991), and include site-directed mutagenesis, cassette mutagenesis and polymerase chain reaction (PCR) mutagenesis, for example.

The protuberance or cavity can be "introduced" into the interface of the first or second polypeptide by synthetic means, e.g. by recombinant techniques, in vitro peptide synthesis, those techniques for introducing non-naturally occurring amino acid residues previously described, by enzymatic or chemical coupling of peptides or some combination of these techniques.

According, the protuberance or cavity which is "introduced" is "non-naturally occurring" or "non-native", which means that it does not exist in nature or in the original polypeptide (e.g. a humanized monoclonal antibody).

"Isolated" heteromultimer means heteromultimer which has been identified and separated and/or recovered from a component of its natural cell culture environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the heteromultimer, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the heteromultimer will be purified (1) to greater than 95% by weight of protein as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. The heteromultimers of the present invention are generally purified to substantial homogeneity. The phrases "substantially homogeneous", "substantially homogeneous form" and "substantial homogeneity" are used to indicate that the product is substantially devoid of byproducts originated from undesired polypeptide combinations (e.g. homomultimers). Expressed in terms of purity, substantial homogeneity means that the amount of by-products does not exceed 10%, and preferably is below 5%, more preferably below 1%, most preferably below 0.5%, wherein the percentages are by weight.

The expression "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, a ribosome binding site, and possibly, other as yet poorly understood sequences. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is

accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accord with conventional practice. II. Preparation of the Heteromultimer

1. Preparation of the Starting Materials

As a first step, the first and second polypeptide (and any additional polypeptides forming the heteromultimer) are selected. Normally, the nucleic acid encoding these polypeptides needs to be isolated so that it can be altered to encode the protuberance or cavity, or both, as herein defined. However, the mutations can be introduced using synthetic means, e.g. by using a peptide synthesizer. Also, in the case where the import residue is a non-naturally occurring residue, the method of Noren et al., supra is available for making polypeptides having such substitutions. Additionally, part of the heteromultimer is suitably made recombinantly in cell culture and other part(s) of the molecule are made by those techniques mentioned above.

Techniques for isolating antibodies and preparing immunoadhesins follow. However, it will be appreciated that the heteromultimer can be formed from, or incorporate, other

polypeptides using techniques which are known in the art. For example, nucleic acid encoding a polypeptide of interest (e.g. a ligand, receptor or enzyme) can be isolated from a cDNA library prepared from tissue believed to possess the polypeptide mRNA and to express it at a detectable level. Libraries are screened with probes (such as antibodies or oligonucleotides of about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures as described in chapters 10-12 of Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).

(i) Antibody Preparation

Several techniques for the production of antibodies have been described which include the traditional hybridoma method for making monoclonal antibodies, recombinant techniques for making antibodies (including chimeric antibodies, e.g. humanized antibodies), antibody production in transgenic animals and the recently described phage display technology for preparing "fully human" antibodies. These techniques shall be described briefly below.

Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies using the hybridoma method first described by Kohler & Milstein, Nature 256:495 (1975) or may be made by recombinant DNA methods (Cabilly et al., U.S. Pat. No. 4,816,567). In the hybridoma method, a mouse or other appropriate host animal, such as hamster, is immunized as hereinabove described to elicit lymphocytes that produce, or are capable of producing, antibodies that will specifically bind to the protein used for immunization.

Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 [Academic Press, 1986]). The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells. Preferred myeloma cells are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 cells available from the American Type Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 [1984]; and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York, 1987). See, also, Boerner et al., J. Immunol., 147(l):86-95 (1991) and WO 91/17769, published Nov. 28, 1991, for techniques for the production of human monoclonal antibodies. Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen of interest. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson & Pollard, Anal. Biochem. 107:220 (1980). After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods. Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-104 (Academic Press, 1986). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal. The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

Alternatively, it is now possible to produce transgenic animals (e.g. mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production.

Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551-255 (1993) and Jakobovits et al., Nature 362:255-258 (1993).

In a further embodiment, antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552- 554 (1990), using the antigen of interest to select for a suitable antibody or antibody fragment. Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries.

Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Mark et al., Bio/Technol. 10:779-783 [1992]), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Acids Res., 21:2265-2266 [1993]). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of

"monoclonal" antibodies (especially human antibodies) which are encompassed by the present invention.

DNA encoding the antibodies of the invention is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences, Morrison et al., Proc. Nat. Acad. Sci. 81:6851 (1984). In that manner, "chimeric" antibodies are prepared that have the binding specificity of an anti-antigen monoclonal antibody herein. Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature 321:522-525 [1986]; Riechmann et al., Nature 332:323-327 [1988]; Verhoeyen et al., Science 239: 1534-1536 [1988]), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (Cabilly, supra), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues, and possibly some FR residues, are substituted by residues from analogous sites in rodent antibodies. It is important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three dimensional models of the parental and humanized sequences. Three dimensional immunoglobulin models are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and import sequence so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. For further details see WO 92/22653, published Dec. 23, 1992.

(ii) Immunoadhesin Preparation

Immunoglobulins (Ig) and certain variants thereof are known and many have been prepared in recombinant cell culture. For example, see U.S. Pat. No. 4,745,055; EP 256,654; Faulkner et al., Nature 298:286 (1982); EP 120,694; EP 125,023; Morrison, J. Immun. 123:793 (1979); Kohler et al., Proc. Natl. Acad. Sci. USA 77:2197 (1980); Raso et al., Cancer Res.

41:2073 (1981); Morrison et al., Ann. Rev. Immunol. 2:239 (1984); Morrison, Science 229: 1202 (1985); Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851 (1984); EP 255,694; EP 266,663; and WO 88/03559. Reassorted immunoglobulin chains also are known. See, for example, U.S. Pat. No. 4,444,878; WO 88/03565; and EP 68,763 and references cited therein.

Chimeras constructed from an adhesin binding domain sequence linked to an appropriate immunoglobulin constant domain sequence (immunoadhesins) are known in the art.

Immunoadhesins reported in the literature include fusions of the T cell receptor (Gascoigne et al., Proc. Natl. Acad. Sci. USA 84:2936-2940 [1987]); CD4 (Capon et al., Nature 337:525-531

[1989]; Traunecker et al., Nature 339:68-70 [1989]; Zettmeissl et al., DNA Cell Biol. USA 9:347-353 [1990]; and Byrn et al., Nature 344:667-670 [1990]); L-selectin (homing receptor) (Watson et al., J. Cell. Biol. 110:2221-2229 [1990]; and Watson et al., Nature 349: 164-167

[1991]); CD44 (Aruffo et al., Cell 61: 1303-1313 [1990]); CD28 and B7 (Linsley et al., J. Exp. Med. 173:721-730 [1991]); CTLA-4 (Lisley et al., J. Exp. Med. 174:561-569 [1991]); CD22 (Stamenkovic et al., Cell 66: 1133-1144 [1991]); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88: 10535-10539 [1991]; Lesslauer et al., Eur. J. Immunol. 27:2883-2886

[1991]; and Peppel et al., J. Exp. Med. 174: 1483-1489 [1991]); and IgE receptor alpha (Ridgway and Gorman, J. Cell. Biol. Vol. 115, Abstract No. 1448 [1991]).

The simplest and most straightforward immunoadhesin design combines the binding domain(s) of the adhesin (e.g. the extracellular domain [ECD] of a receptor) with the hinge and Fc regions of an immunoglobulin heavy chain. Ordinarily, when preparing the immunoadhesins of the present invention, nucleic acid encoding the binding domain of the adhesin will be fused C-terminally to nucleic acid encoding the N-terminus of an immunoglobulin constant domain sequence, however N-terminal fusions are also possible.

Typically, in such fusions the encoded chimeric polypeptide will retain at least functionally active hinge, CH2 and CH3 domains of the constant region of an immunoglobulin heavy chain. Fusions are also made to the C-terminus of the Fc portion of a constant domain, or immediately N-terminal to the CHI of the heavy chain or the corresponding region of the light chain. The precise site at which the fusion is made is not critical; particular sites are well known and may be selected in order to optimize the biological activity, secretion, or binding

characteristics of the la.

In a preferred embodiment, the adhesin sequence is fused to the N-terminus of the Fc domain of immunoglobulin Gl (IgGl). It is possible to fuse the entire heavy chain constant region to the adhesin sequence. However, more preferably, a sequence beginning in the hinge region just upstream of the papain cleavage site which defines IgG Fc chemically (i.e. residue 216, taking the first residue of heavy chain constant region to be 114), or analogous sites of other immunoglobulins is used in the fusion. In a particularly preferred embodiment, the adhesin amino acid sequence is fused to (a) the hinge region and CH2 and CH3 or (b) the CHI, hinge, CH2 and CH3 domains, of an IgGl, IgG2, or IgG3 heavy chain. The precise site at which the fusion is made is not critical, and the optimal site can be determined by routine experimentation.

For bispecific immunoadhesins, the immunoadhesins are assembled as multimers, and particularly as heterodimers or heterotetramers. Generally, these assembled immunoglobulins will have known unit structures. A basic four chain structural unit is the form in which IgG, IgD, and IgE exist. A four chain unit is repeated in the higher molecular weight immunoglobulins; IgM generally exists as a pentamer of four basic units held together by disulfide bonds. IgA globulin, and occasionally IgG globulin, may also exist in multimeric form in serum. In the case of multimer, each of the four units may be the same or different.

Alternatively, the adhesin sequences can be inserted between immunoglobulin heavy chain and light chain sequences, such that an immunoglobulin comprising a chimeric heavy chain is obtained. In this embodiment, the adhesin sequences are fused to the 3' end of an immunoglobulin heavy chain in each arm of an immunoglobulin, either between the hinge and the CH2 domain, or between the CH2 and CH3 domains. Similar constructs have been reported by Hoogenboom, et al., Mol. Immunol. 28: 1027-1037 (1991).

Although the presence of an immunoglobulin light chain is not required in the immunoadhesins of the present invention, an immunoglobulin light chain might be present either covalently associated to an adhesin-immunoglobulin heavy chain fusion polypeptide, or directly fused to the adhesin. In the former case, DNA encoding an immunoglobulin light chain is typically coexpressed with the DNA encoding the adhesin-immunoglobulin heavy chain fusion protein. Upon secretion, the hybrid heavy chain and the light chain will be covalently associated to provide an immunoglobulin-like structure comprising two disulfide-linked immunoglobulin heavy chain-light chain pairs. Methods suitable for the preparation of such structures are, for example, disclosed in U.S. Pat. No. 4,816,567, issued 28 Mar. 1989.

In a preferred embodiment, the immunoglobulin sequences used in the construction of the immunoadhesins of the present invention are from an IgG immunoglobulin heavy chain constant domain. For human immunoadhesins, the use of human IgGl and IgG3 immunoglobulin sequences is preferred. A major advantage of using IgGl is that IgGl immunoadhesins can be purified efficiently on immobilized protein A. In contrast, purification of IgG3 requires protein G, a significantly less versatile medium. However, other structural and functional properties of immunoglobulins should be considered when choosing the Ig fusion partner for a particular immunoadhesin construction. For example, the IgG3 hinge is longer and more flexible, so it can accommodate larger "adhesin" domains that may not fold or function properly when fused to IgGl. Another consideration may be valency; IgG immunoadhesins are bivalent homodimers, whereas Ig subtypes like IgA and IgM may give rise to dimeric or pentameric structures, respectively, of the basic Ig homodimer unit. For immunoadhesins designed for in vivo application, the pharmacokinetic properties and the effector functions specified by the Fc region are important as well. Although IgGl, IgG2 and IgG4 all have in vivo half-lives of 21 days, their relative potencies at activating the complement system are different. IgG4 does not activate complement, and IgG2 is significantly weaker at complement activation than IgGl. Moreover, unlike IgGl, IgG2 does not bind to Fc receptors on mononuclear cells or neutrophils. While IgG3 is optimal for complement activation, its in vivo half-life is approximately one third of the other IgG isotypes. Another important consideration for immunoadhesins designed to be used as human therapeutics is the number of allotypic variants of the particular isotype. In general, IgG isotypes with fewer serologically-defined allotypes are preferred. For example, IgGl has only four serologically-defined allotypic sites, two of which (Glm and 2) are located in the Fc region; and one of these sites, Glml, is non-immunogenic. In contrast, there are 12 serologically- defined allotypes in IgG3, all of which are in the Fc region; only three of these sites (G3 m5, 11 and 21) have one allotype which is nonimmunogenic. Thus, the potential immunogenicity of a gamma-3 immunoadhesin is greater than that of a gamma- 1 immunoadhesin.

Immunoadhesins are most conveniently constructed by fusing the cDNA sequence encoding the adhesin portion in-frame to an Ig cDNA sequence. However, fusion to genomic Ig fragments can also be used (see, e.g. Gascoigne et al., supra; Aruffo et al., Cell 61: 1303-1313 [1990]; and Stamenkovic et al., Cell 66: 1133-1144 [1991]). The latter type of fusion requires the presence of Ig regulatory sequences for expression. cDNAs encoding IgG heavy-chain constant regions can be isolated based on published sequences from cDNA libraries derived from spleen or peripheral blood lymphocytes, by hybridization or by polymerase chain reaction (PCR) techniques. The cDNAs encoding the "adhesin" and the Ig parts of the immunoadhesin are inserted in tandem into a plasmid vector that directs efficient expression in the chosen host cells.

2. Generating a Protuberance and/or Cavity

As a first step to selecting original residues for forming the protuberance and/or cavity, the three-dimensional structure of the heteromultimer is obtained using techniques which are well known in the art such as X-ray crystallography or NMR. Based on the three-dimensional structure, those skilled in the art will be able to identify the interface residues.

The preferred interface is the CH3 domain of an immunoglobulin constant domain. The interface residues of the CH3 domains of IgG, IgA, IgD, IgE and IgM are identified in FIG. 5, including those which are optimal for replacing with import residues. The interface residues of various IgG subtypes are illustrated in FIG. 6. "Buried" residues are also identified. The basis for engineering the CH3 interface is that X-ray crystallography has demonstrated that the intermolecular association between human IgGl heavy chains in the Fc region includes extensive protein/protein interaction between CH3 domains whereas the glycosylated CH2 domains interact via their carbohydrate (Deisenhofer, Biochem. 20:2361 [1981]). In addition there are two inter-heavy chain disulfide bonds which are efficiently formed during antibody expression in mammalian cells unless the heavy chain is truncated to remove CH2 and CH3 domains (King et al., Biochem. J. 281:317 [1992]). Thus, heavy chain assembly appears to promote disulfide bond formation rather than vice versa. Taken together these structural and functional data led to the hypothesis that antibody heavy chain association is directed by the CH3 domains. It was further speculated that the interface between CH3 domains might be engineered to promote formation of heteromultimers of different heavy chains and hinder assembly of corresponding

homomultimers. The experiments described herein demonstrated that it was possible to promote the formation of heteromultimers over homomultimers using this approach. Thus, it is possible to generate a polypeptide fusion comprising a polypeptide of interest and the CH3 domain of an antibody to form a first or second polypeptide. The preferred CH3 domain is derived from an IgG antibody, such as an human IgGl.

Those interface residues which can potentially constitute candidates for forming the protuberance or cavity are identified. It is preferable to select "buried" residues to be replaced. To determine whether a residue is buried, the surface accessibility program of Lee et al. J. Mol. Biol. 55: 379-400 (1971) can be used to calculate the solvent accessibility (SA) of residues in the interface. Then, the SA for the residues of each of the first and second polypeptide can be separately calculated after removal of the other polypeptide. The difference in SA of each residue between the monomer and dimer forms of the interface can then be calculated by: S A (dimer)-SA (monomer). This provides a list of residues which lose SA on formation of the dimer. The SA of each residue in the dimer is compared to the theoretical SA of the same amino acid in the tripeptide Gly-X-Gly, where X=the amino acid of interest (Rose et al. Science 229: 834-838 [1985]). Residues which (a) lost SA in the dimer compared to the monomer and (b) had an SA less than 26% of that in their corresponding tripeptide are considered as interface residues. Two categories may be delineated: those which have an SA<10% compared to their

corresponding tripeptide (i.e. "buried") and those which have 25%>SA>10% compared to their corresponding tripeptide (i.e. "partially buried").

The effect of replacing residues on the polypeptide chain structure can be studied using a molecular graphics modeling program such as the INSIGHT program (Biosym Technologies). Using the program, those buried residues in the interface of the first polypeptide which have a small side chain volume can be changed to residues having a larger side chain volume (i.e. a protuberance), for example. Then, the residues in the interface of the second polypeptide which are in proximity to the protuberance are examined to find a suitable residue for forming the cavity. Normally, this residue will have a large side chain volume and is replaced with a residue having a smaller side chain volume. In certain embodiments, examination of the three - dimensional structure of the interface will reveal a suitably positioned and dimensioned protuberance on the interface of the first polypeptide or a cavity on the interface of the second polypeptide. In these instances, it is only necessary to model a single mutant, i.e., with a synthetically introduced protuberance or cavity.

Once the preferred original/import residues are identified by molecular modeling, the amino acid replacements are introduced into the polypeptide using techniques which are well known in the art. Normally the DNA encoding the polypeptide is genetically engineered using the techniques described in Mutagenesis: a Practical Approach, supra.

Oligonucleotide-mediated mutagenesis is a preferred method for preparing substitution variants of the DNA encoding the first or second polypeptide. This technique is well known in the art as described by Adelman et al., DNA, 2: 183 (1983). Briefly, first or second polypeptide DNA is altered by hybridizing an oligonucleotide encoding the desired mutation to a DNA template, where the template is the single- stranded form of a plasmid or bacteriophage containing the unaltered or native DNA sequence of heteromultimer. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template that will thus incorporate the oligonucleotide primer, and will code for the selected alteration in the heteromultimer DNA.

Cassette mutagenesis can be performed as described Wells et al. Gene 34:315 (1985) by replacing a region of the DNA of interest with a synthetic mutant fragment generated by annealing complimentary oligonucleotides. PCR mutagenesis is also suitable for making variants of the first or second polypeptide DNA. While the following discussion refers to DNA, it is understood that the technique also finds application with RNA. The PCR technique generally refers to the following procedure (see Erlich, Science, 252: 1643-1650 [1991], the chapter by R. Higuchi, p. 61-70).

This invention also encompasses, in addition to the protuberance or cavity mutations, amino acid sequence variants of the heteromultimer which can be prepared by introducing appropriate nucleotide changes into the heteromultimer DNA, or by synthesis of the desired heteromultimer polypeptide. Such variants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequences of the first and second polypeptides forming the heteromultimer. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired antigen- binding characteristics. The amino acid changes also may alter post-translational processes of the heteromultimer, such as changing the number or position of glycosylation sites.

A useful method for identification of certain residues or regions of the heteromultimer polypeptides that are preferred locations for mutagenesis is called "alanine scanning

mutagenesis," as described by Cunningham and Wells, Science, 244: 1081-1085 (1989). Here, a residue or group of target residues are identified (e.g. charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell. Those domains demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at or for the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is

predetermined, the nature of the mutation per se need not be predetermined. Normally the mutations will involve conservative amino acid replacements in nonfunctional regions of the heteromultimer. Exemplary mutations are shown in the following table.

Table 2

Covalent modifications of the heteromultimer polypeptides are included within the scope of this invention. Covalent modifications of the heteromultimer can be introduced into the molecule by reacting targeted amino acid residues of the heteromultimer or fragments thereof with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues. Another type of covalent modification of the heteromultimer polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide. By altering is meant deleting one or more carbohydrate moieties found in the original heteromultimer, and/or adding one or more glycosylation sites that are not present in the original heteromultimer. Addition of glycosylation sites to the heteromultimer polypeptide is conveniently accomplished by altering the amino acid sequence such that it contains one or more N-linked glycosylation sites. The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the original heteromultimer sequence (for O- linked glycosylation sites). For ease, the heteromultimer amino acid sequence is preferably altered through changes at the DNA level, particularly by mutating the DNA encoding the heteromultimer polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids. Another means of increasing the number of carbohydrate moieties on the heteromultimer polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. These methods are described in WO 87/05330 published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981). Removal of carbohydrate moieties present on the heteromultimer may be accomplished chemically or enzymatically.

Another type of covalent modification of heteromultimer comprises linking the heteromultimer polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

Since it is often difficult to predict in advance the characteristics of a variant

heteromultimer, it will be appreciated that some screening of the recovered variant will be needed to select the optimal variant.

In one aspect, the first CH3 domain comprises an amino acid at EU position 354 or 364 that creates a protuberance, and an amino acid at EU position 407 that creates a cavity, and the second CH3 domain comprises at least one amino acid mutation at EU position 347, 349, 350, 351, 366, 368, 370, or 407. In another embodiment, a protein or polypeptide comprises a first CH3 domain paired with a non-identical second CH3 domain, wherein the first CH3 domain comprises an amino acid at EU position 349 that creates a cavity and an amino acid at EU position 366 that creates a protuberance, and the second CH3 domain comprises at least one amino acid mutation at EU position 351, 354, 357, 360, 364, 366, 368, or 407. In another embodiment, the amino acid at EU position 354 of the first CH3 domain is isoleucine (I) or leucine (L), and the amino acid at EU position 349 of the second CH3 domain is selected from the group consisting of cysteine (C), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), arginine (R), serine (S), threonine (T), valine (V), and tryptophan (W); and the amino acid at EU position 407 of the first CH3 domain is a valine (V), and the amino acid at EU position 366 of the second CH3 domain is selected from the group consisting of alanine (A), cysteine (C), glycine (G), lysine (K), leucine (L), methionine (M), arginine (R), serine (S), tryptophan (W), and tyrosine (Y).

In another embodiment, the amino acid at EU position 357 of the first CH3 domain is leucine (L), and the amino acid at EU position 370 of the second CH3 domain is selected from the group consisting of alanine (A), isoleucine (I), lysine (K), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), and tryptophan (W); and the amino acid at EU position 360 of the first CH3 domain is serine (S), the amino acid at EU position 347 of the second CH3 domain is selected from the group consisting of alanine (A), glutamic acid (E), phenylalanine (F), glycine (G), isoleucine (I), lysine (K), leucine (L), methionine (M), glutamine (Q), serine (S), and valine (V).

In another embodiment the amino acid at EU position 350 of the second CH3 domain is selected from the group consisting of alanine (A), aspartic acid (D), glycine (G), isoleucine (I), lysine (K), methionine (M), asparagine (N), proline (P), arginine (R), serine (S), threonine (T), and tyrosine (Y); the amino acid at EU position 351 of the second CH3 domain is selected from the group consisting of phenylalanine (F), histidine (H), lysine (K), leucine (L), methionine (M), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), and tyrosine (Y); the amino acid at EU position 368 of the second CH3 domain is selected from the group consisting of alanine (A), phenylalanine (F), isoleucine (I), leucine (L), methionine (M), asparagine (N), arginine (R), threonine (T), and valine (V); and the amino acid at EU position

407 of the second CH3 domain is selected from the group consisting of alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), histidine (H), isoleucine (I), leucine (L), asparagine (N), glutamine (Q), arginine (R), serine (S), valine (V), tryptophan (W), and tyrosine

(Y).

In another embodiment, the amino acid at EU position 349 of the first CH3 domain is an isoleucine (I), and the amino acid at EU position 354 of the second CH3 domain is selected from the group consisting of cysteine (C), phenylalanine (F), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), arginine (R), serine (S), threonine (T), and valine (V); and the amino acid at EU position 366 the first CH3 domain is a trypophan (W), and the amino acid at EU position 407 of the second CH3 domain is selected from the group consisting of alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), leucine (L), asparagine (N), proline (P), arginine (R), serine (S), valine (V), and tyrosine (Y).

In another embodiment the amino acid at EU position 347 of the first CH3 domain is a valine (V), and the amino acid at EU position 360 of the second CH3 domain is selected from the group consisting of cysteine (C), phenylalanine (F), glycine (G), isoleucine (I), lysine (K), leucine (L), asparagine (N), proline (P), arginine (R), serine (S), threonine (T), valine (V), and tyrosine (Y); the amino acid at EU position 368 of the first CH3 domain is a methionine (M), and the amino acid at EU position 364 of the second CH3 domain is selected from the group consisting of alanine (A), cysteine (C), phenylalanine (F), leucine (L), proline (P), arginine (R), serine (S), threonine (T), tryptophan (W), and tyrosine (Y); and the amino acid at EU position

370 position of the first CH3 domain is a threonine (T), and the amino acid at EU position 357 of the second CH3 domain of is selected from the group consisting of cysteine (C), phenylalanine (F), glycine (G), lysine (K), leucine (L), methionine (M), glutamine (Q), serine (S), threonine (T), valine (V), and tryptophan (W).

In another embodiment the amino acid at EU position 351 of the second CH3 domain is selected from the group consisting of alanine (A), phenylalanine (F), isoleucine (I), leucine (L), proline (P), glutamine (Q), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y); the amino acid at EU position 366 of the second CH3 domain is selected from the group consisting of alanine (A), aspartic acid (D), lysine (K), leucine (L), methionine (M), proline (P), arginine (R), serine (S), threonine (T), tryptophan (W), and tyrosine (Y); and the amino acid at EU position 368 of the second CH3 domain is selected from the group consisting of alanine (A), aspartic acid (D), glutamic acid (E), glycine (G), isoleucine (I), lysine (K), leucine (L), proline (P), serine (S), threonine (T), and valine (V).

In another aspect, a protein or polypeptide comprises a first CH3 domain paired with a non-identical second CH3 domain, wherein a) the first CH3 domain comprises an amino acid at EU position 351 selected from the group consisting of leucine (L), phenylalanine (F), isoleucine (I), and valine (V); an isoleucine (I) at EU position 354; an amino acid at EU position 364 selected from the group consisting of serine (S) and leucine (L); a serine (S) at EU position 366; an alanine (A) at EU position 368; and a valine (V) at EU position 407; and b) the second CH3 domain comprises an amino acid at EU position 347 selected from the group consisting of glycine (G), arginine (R), and glutamine (Q); an amino acid at EU position 350 selected from the group consisting of asparagine (N) and threonine (T); and a tryptophan (W) at EU position 366.

In one embodiment the second CH3 domain comprises an amino acid at EU position 347 selected from the group consisting of glycine (G), arginine (R), and glutamine (Q); an amino acid at EU position 349 selected from the group consisting of serine (S), glycine (G), valine (V), alanine (A), arginine (R), methionine, (M), and tryptophan (W); an amino acid at EU position 350 selected from the group consisting of asparagine (N) and threonine (T); an amino acid at EU position 351 selected from the group consisting of valine (V), glycine (G), methionine (M), alanine (A), tryptophan (W), and leucine (L); a tryptophan (W) at EU position 366; an amino acid at EU position 368 selected from the group consisting of alanine (A), glycine (G), cysteine (C), valine (V), histidine (H), and leucine (L); and an amino acid at EU position 370 selected from the group consisting of alanine (A), glycine (G), arginine (R), tryptophan (W), and valine (V).

In another embodiment, the second CH3 domain comprises an amino acid at EU position 347 selected from the group consisting of glycine (G) and arginine (R); an amino acid at EU position 349 selected from the group consisting of serine (S), glycine (G), valine (V), alanine (A), and arginine (R); an amino acid at EU position 350 selected from the group consisting of asparagine (N) and threonine (T); an amino acid at EU position 351 selected from the group consisting of valine (V), glycine (G), methionine (M), alanine (A), and tryptophan (W); a tryptophan (W) at EU position 366; an amino acid at EU position 368 selected from the group consisting of alanine (A), glycine (G), cysteine (C), valine (V), and histidine (H); and an amino acid at EU position 370 selected from the group consisting of alanine (A), glycine (G), arginine (R), and tryptophan (W).

In another embodiment, the second CH3 domain comprises a glycine (G) at EU position 347; a serine (S) at EU position 349; an asparagine (N) at EU position 350; a valine (V) at EU position 351; a tryptophan (W) at EU position 366; an alanine (A) at EU position 368; and an alanine (A) at EU position 370. In another embodiment, the second CH3 domain comprises a sequence selected from the group consisting of SEQ ID NOs: 15, 17, 21, 25, 27, 29, 35, 37, and 170.

In another aspect, a protein or polypeptide comprises a first CH3 domain paired with a non-identical second CH3 domain, wherein a) the first CH3 domain comprises an amino acid at EU position 351 selected from the group consisting of leucine (L), phenylalanine (F), isoleucine (I), and valine (V); an isoleucine (I) at EU position 354; an amino acid at EU position 364 selected from the group consisting of serine (S) and leucine (L); a serine (S) at EU position 366; an alanine (A) at EU position 368; and a valine (V) at EU position 407; and b) the second CH3 domain comprises an amino acid at EU position 347 selected from the group consisting of valine (V), leucine (L), and alanine (A); an amino acid at EU position 349 selected from the group consisting of isoleucine (I), threonine (T), and glycine (G); an amino acid at EU position 350 selected from the group consisting of lysine (K), isoleucine (I), and threonine (T); an amino acid at EU position 351 selected from the group consisting of glutamine (Q) and leucine (L); a tryptophan (W) at EU position 366; an amino acid at EU position 368 selected from the group consisting of isoleucine (I), methionine (M), arginine (R), and valine (V); and an amino acid at EU position 407 selected from the group consisting of tyrosine (Y), serine (S), tryptophan (W), and phenylalanine (F).

In one embodiment, the second CH3 domain comprises a valine (V) at EU position 347; an amino acid at EU position 349 selected from the group consisting of isoleucine (I) and threonine (T); an amino acid at EU position 350 selected from the group consisting of lysine (K), isoleucine (I), and threonine (T); an amino acid at EU position 351 selected from the group consisting of glutamine (Q) and leucine (L); a tryptophan (W) at EU position 366; an amino acid at EU position 368 selected from the group consisting of isoleucine (I) and methionine (M); and an amino acid at EU position 407 selected from the group consisting of tyrosine (Y), serine (S), tryptophan (W), and phenylalanine (F).

In another embodiment, the second CH3 domain comprises a valine (V) at EU position 347; an isoleucine (I) at EU position 349; a lysine (K) at EU position 350; a glutamine (Q) at EU position 351; a tryptophan (W) at EU position 366; an isoleucine (I) at EU position 368; and a tyrosine (Y) at EU position 407. In another embodiment, the second CH3 domain comprises a sequence selected from the group consisting of SEQ ID NOs: 39, 140, 142, 153, 156, 161, 162, 163, 165, and 172. In another embodiment, wherein the first CH3 domain comprises an amino acid at EU position 351 selected from the group consisting of leucine (L), phenylalanine (F), isoleucine (I), and valine (V); an isoleucine (I) at EU position 354; an amino acid at EU position 357 selected from the group consisting of leucine (L), methionine (M), serine (S), proline (P), tryptophan (W), glutamic acid (E), glutamine (Q), and glycine (G); an amino acid at EU position 360 selected from the group consisting of serine (S), isoleucine (I), asparagine (N), glycine (G), tyrosine (Y), methionine (M), lysine (K), and arginine (R); an amino acid at EU position 364 selected from the group consisting of serine (S) and leucine (L); a serine (S) at EU position 366; an alanine (A) at EU position 368; and a valine (V) at EU position 407.

In another embodiment, the first CH3 domain comprises a leucine (L) at EU position

351; an isoleucine (I) at EU position 354; an amino acid at EU position 357 selected from the group consisting of glutamine (Q), methionine (M), and glycine (G); a lysine (K) at EU position 360; a leucine (L) at EU position 364; a serine (S) at EU position 366; an alanine (A) at EU position 368; and a valine (V) at EU position 407.

In another embodiment, the first CH3 domain comprises an amino acid at EU position

351 selected from the group consisting of leucine (L), phenylalanine (F), isoleucine (I), and valine (V); an isoleucine (I) at EU position 354; an amino acid at EU position 357 selected from the group consisting of leucine (L), methionine (M), serine (S), proline (P), tryptophan (W), and glutamic acid (E); an amino acid at EU position 360 selected from the group consisting of serine (S), isoleucine (I), asparagine (N), glycine (G), tyrosine (Y), methionine (M), lysine (K), and arginine (R); an amino acid at EU position 364 selected from the group consisting of serine (S) and leucine (L); a serine (S) at EU position 366; an alanine (A) at EU position 368; and a valine (V) at EU position 407.

In another embodiment, the first CH3 domain comprises an amino acid at EU position 351 selected from the group consisting of leucine (L), phenylalanine (F), isoleucine (I), and valine (V); an isoleucine (I) at EU position 354; an amino acid at EU position 357 selected from the group consisting of leucine (L), methionine (M), serine (S), proline (P), and tryptophan (W); an amino acid at EU position 360 selected from the group consisting of serine (S), isoleucine (I), asparagine (N), glycine (G), tyrosine (Y), and methionine (M); an amino acid at EU position 364 selected from the group consisting of serine (S) and leucine (L); a serine (S) at EU position 366; an alanine (A) at EU position 368; and a valine (V) at EU position 407. In another embodiment, the first CH3 domain comprises an amino acid at EU position 351 selected from the group consisting of leucine (L), phenylalanine (F), isoleucine (I), and valine (V); an isoleucine (I) at EU position 354; an amino acid at EU position 357 selected from the group consisting of leucine (L), methionine (M), and serine (S); an amino acid at EU position 360 selected from the group consisting of serine (S), isoleucine (I), glycine (G), and tyrosine (Y); an amino acid at EU position 364 selected from the group consisting of serine (S) and leucine (L); a serine (S) at EU position 366; an alanine (A) at EU position 368; and a valine (V) at EU position 407.

In another embodiment, the first CH3 domain comprises an isoleucine (I) at EU position 354; a leucine (L) at EU position 357; a serine (S)at EU position 360; a serine (S) at EU position 366; an alanine (A) at EU position 368; and a valine (V) at EU position 407. In another embodiment, the first CH3 domain comprises an isoleucine (I) at EU position 354; an amino acid at EU position 357 selected from the group consisting of glutamine (Q), methionine (M), and glycine (G); a serine (S) at EU position 366; an alanine (A) at EU position 368; and a valine (V) at EU position 407. In another embodiment, the first CH3 domain comprises an isoleucine (I) at EU position 354; a glutamine (Q) at EU position 357; a serine (S) at EU position 366; an alanine (A) at EU position 368; and a valine (V) at EU position 407. In another embodiment, the first CH3 domain comprises a sequence selected from the group consisting of SEQ ID NOs: 40, 72, 74, 76, 78, 82, 84, 178, 179, 180, 181, 182, 183, 184, 185, 189, and 209. In another

embodiment, the protein or polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-209.

3. Expression of the Heteromultimer

Following mutation of the DNA as discussed in the preceding section, the DNA encoding the molecule is expressed using recombinant techniques which are widely available in the art.

Often, the expression system of choice will involve a mammalian cell expression vector and host so that the heteromultimer is appropriately glycosylated (e.g. in the case of heteromultimers comprising antibody domains which are glycosylated). However, the molecules can also be produced in the prokaryotic expression systems elaborated below.

Accordingly, in one aspect, a nucleic acid encoding any of the proteins or a polypeptides comprising a CH3 domain described herein is provided. In one embodiment, an expression vector comprising a nucleic acid as described herein is provided. In another embodiment, a cell comprising a nucleic acid or an expression vector as described herein is provided. In another embodiment, a method of producing a protein or polypeptide comprising culturing a cell comprising a nucleic acid or an expression vector as described herein under conditions wherein the protein or polypeptide is expressed and isolating the protein or polypeptide is provided.

Normally, the host cell will be transformed with DNA encoding both the first polypeptide and the second polypeptide and other polypeptide(s) required to form the heteromultimer, on a single vector or independent vectors. However, it is possible to express the first polypeptide and second polypeptide in independent expression systems and couple the expressed polypeptides in vitro.

The nucleic acid (e.g., cDNA or genomic DNA) encoding the heteromultimer is inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

The polypeptides of the heteromultimer may be produced as fusion polypeptides with a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or it may be a part of the DNA that is inserted into the vector. The heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells, the signal sequence may be substituted by a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion the native signal sequence may be substituted by, e.g., the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyveromyces alpha-factor leaders, the latter described in U.S. Pat. No. 5,010,182 issued 23 Apr. 1991), or acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179 published 4 Apr. 1990), or the signal described in WO 90/13646 published 15 Nov. 1990. In mammalian cell expression the native signal sequence (e.g., the antibody or adhesin presequence that normally directs secretion of these molecules from human cells in vivo) is satisfactory, although other mammalian signal sequences may be suitable as well as viral secretory leaders, for example, the herpes simplex gD signal. The DNA for such precursor region is ligated in reading frame to DNA encoding the polypeptides forming the heteromultimer.

Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Generally, in cloning vectors this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2u plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors (the SV40 origin may typically be used only because it contains the early promoter).

Expression and cloning vectors should contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli. One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen.

Examples of such dominant selection use the drugs neomycin (Southern et al., J. Molec. Appl. Genet. 1:327 [1982]), mycophenolic acid (Mulligan et al., Science 209: 1422 [1980]) or hygromycin (Sugden et al., Mol. Cell. Biol. 5:410-413 [1985]). The three examples given above employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid), or hygromycin, respectively.

Another example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the heteromultimer nucleic acid, such as DHFR or thymidine kinase. The mammalian cell transformants are placed under selection pressure that only the transformants are uniquely adapted to survive by virtue of having taken up the marker. Selection pressure is imposed by culturing the transformants under conditions in which the concentration of selection agent in the medium is successively changed, thereby leading to amplification of both the selection gene and the DNA that encodes heteromultimer. Increased quantities of heteromultimer are synthesized from the amplified DNA. Other examples of amplifiable genes include metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. An appropriate host cell when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity, prepared and propagated as described by Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216 (1980). The transformed cells are then exposed to increased levels of methotrexate. This leads to the synthesis of multiple copies of the DHFR gene, and, concomitantly, multiple copies of other DNA comprising the expression vectors, such as the DNA encoding the components of the heteromultimer This amplification technique can be used with any otherwise suitable host, e.g., ATCC No. CCL61 CHO-K1, notwithstanding the presence of endogenous DHFR if, for example, a mutant DHFR gene that is highly resistant to Mtx is employed (EP 117,060).

Alternatively, host cells (particularly wild-type hosts that contain endogenous DHFR) transformed or co-transformed with DNA sequences encoding heteromultimer, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3 '-phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Pat. No.

4,965,199.

A suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7 (Stinchcomb et al., Nature 282:39 [1979]; Kingsman et al., Gene 7: 141 [1979]; or Tschemper et al., Gene 10: 157 [1980]). The trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4- 1 (Jones, Genetics 85: 12 [1977]). The presence of the trpl lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 um circular plasmid pKDl can be used for transformation of Kluyveromyces yeasts. Bianchi et al., Curr. Genet. 12: 185 (1987). More recently, an expression system for large-scale production of recombinant calf chymosin was reported for K. lactis. Van den Berg, Bio/Technology 8: 135 (1990). Stable multi-copy expression vectors for secretion of mature recombinant human serum albumin by industrial strains of Kluyveromyces have also been disclosed. Fleer et al., Bio/Technology 9:968-975 (1991).

Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the heteromultimer nucleic acid. A large number of promoters recognized by a variety of potential host cells are well known. These promoters are operably linked to heteromultimer-encoding DNA by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector.

Promoters suitable for use with prokaryotic hosts include the beta-lactamase and lactose promoter systems (Chang et al., Nature 275:615 [1978]; and Goeddel et al., Nature 281:544

[1979]), alkaline phosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic Acids Res., 8:4057 [1980] and EP 36,776) and hybrid promoters such as the tac promoter (deBoer et al., Proc. Natl. Acad. Sci. USA 80:21-25 [1983]). However, other known bacterial promoters are suitable. Their nucleotide sequences have been published, thereby enabling a skilled worker operably to ligate them to DNA encoding the heteromultimer (Siebenlist et al., Cell 20:269

[1980]) using linkers or adaptors to supply any required restriction sites. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the heteromultimer.

Promoter sequences are known for eukaryotes. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CXCAAT region where X may be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.

Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073 [1980]) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7: 149 [1968]; and Holland, Biochemistry 17:4900 [1978]), such as enolase, glyceraldehyde- 3 -phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3- phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol

dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in Hitzeman et al., EP 73, 657 A. Yeast enhancers also are advantageously used with yeast promoters.

Heteromultimer transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter or from heat- shock promoters.

The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. Fiers et al., Nature 273: 113 (1978); Mulligan and Berg, Science 209: 1422-1427 (1980); Pavlakis et al., Proc. Natl. Acad. Sci. USA 78:7398-7402 (1981). The immediate early promoter of the human

cytomegalovirus is conveniently obtained as a Hindlll E restriction fragment. Greenaway et al., Gene 18:355-360 (1982). A system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. A modification of this system is described in U.S. Pat. No. 4,601,978. See also Gray et al., Nature 295:503-508 (1982) on expressing cDNA encoding immune interferon in monkey cells; Reyes et al., Nature 297:598- 601 (1982) on expression of human beta-interferon cDNA in mouse cells under the control of a thymidine kinase promoter from herpes simplex virus; Canaani and Berg, Proc. Natl. Acad. Sci. USA 79:5166-5170 (1982) on expression of the human interferon betal gene in cultured mouse and rabbit cells; and Gorman et al., Proc. Natl. Acad. Sci. USA 79:6777-6781 (1982) on expression of bacterial CAT sequences in CV-1 monkey kidney cells, chicken embryo fibroblasts, Chinese hamster ovary cells, HeLa cells, and mouse NIH-3T3 cells using the Rous sarcoma virus long terminal repeat as a promoter. Transcription of DNA encoding the heteromultimer by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Enhancers are relatively orientation and position independent, having been found 5' (Laimins et al., Proc. Natl. Acad. Sci. USA 78:993 [1981]) and 3' (Lusky et al., Mol. Cell Bio. 3: 1108 [1983]) to the transcription unit, within an intron (Banerji et al., Cell 33:729 [1983]), as well as within the coding sequence itself (Osborne et al., Mol. Cell Bio. 4: 1293 [1984]). Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297: 17-18 (1982) on enhancing elements for activation of eukaryotic promoters. The enhancer may be spliced into the vector at a position 5' or 3' to the

heteromultimer-encoding sequence, but is preferably located at a site 5' from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the heteromultimer.

Construction of suitable vectors containing one or more of the above listed components employs standard ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to generate the plasmids required.

For analysis to confirm correct sequences in plasmids constructed, the ligation mixtures are used to transform E. coli K12 strain 294 (ATCC 31,446) and successful transformants selected by ampicillin or tetracycline resistance where appropriate. Plasmids from the

transformants are prepared, analyzed by restriction endonuclease digestion, and/or sequenced by the method of Messing et al., Nucleic Acids Res. 9:309 (1981) or by the method of Maxam et al, Methods in Enzymology 65:499 (1980).

Particularly useful in the practice of this invention are expression vectors that provide for the transient expression in mammalian cells of DNA encoding heteromultimer. In general, transient expression involves the use of an expression vector that is able to replicate efficiently in a host cell, such that the host cell accumulates many copies of the expression vector and, in turn, synthesizes high levels of a desired polypeptide encoded by the expression vector. Sambrook et al., supra, pp. 16.17-16.22. Transient expression systems, comprising a suitable expression vector and a host cell, allow for the convenient positive identification of polypeptides encoded by cloned DNAs, as well as for the rapid screening of heteromultimers having desired binding specificities/affinities .

Other methods, vectors, and host cells suitable for adaptation to the synthesis of the heteromultimer in recombinant vertebrate cell culture are described in Gething et al., Nature 293:620-625 (1981); Mantei et al., Nature 281:40-46 (1979); Levinson et al.; EP 117,060; and EP 117,058. A particularly useful plasmid for mammalian cell culture expression of the heteromultimer is pRK5 (EP 307,247) or pSVKB (PCT pub. no. WO 91/08291 published 13 Jun. 1991).

The choice of host cell line for the expression of heteromultimer depends mainly on the expression vector. Another consideration is the amount of protein that is required. Milligram quantities often can be produced by transient transfections. For example, the adenovirus EIA- transformed 293 human embryonic kidney cell line can be transfected transiently with pRK5- based vectors by a modification of the calcium phosphate method to allow efficient

heteromultimer expression. CDM8-based vectors can be used to transfect COS cells by the DEAE-dextran method (Aruffo et al., Cell 61: 1303-1313 [1990]; and Zettmeissl et al., DNA Cell Biol. (US) 9:347-353 [1990]). If larger amounts of protein are desired, the immunoadhesin can be expressed after stable transfection of a host cell line. For example, a pRK5-based vector can be introduced into Chinese hamster ovary (CHO) cells in the presence of an additional plasmid encoding dihydrofolate reductase (DHFR) and conferring resistance to G418. Clones resistant to G418 can be selected in culture. These clones are grown in the presence of increasing levels of DHFR inhibitor methotrexate and clones are selected in which the number of gene copies encoding the DHFR and heteromultimer sequences is co-amplified. If the immunoadhesin contains a hydrophobic leader sequence at its N-terminus, it is likely to be processed and secreted by the transfected cells. The expression of immunoadhesins with more complex structures may require uniquely suited host cells. For example, components such as light chain or J chain may be provided by certain myeloma or hybridoma host cells (Gascoigne et al., supra; and Martin et al., J. Virol. 67:3561-3568 [1993]). Other suitable host cells for cloning or expressing the vectors herein are prokaryote, yeast, or other higher eukaryote cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example,

Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting. Strain W3110 is a particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations. Preferably, the host cell should secrete minimal amounts of proteolytic enzymes. For example, strain W3110 may be modified to effect a genetic mutation in the genes encoding proteins, with examples of such hosts including E. coli W3110 strain 27C7. The complete genotype of 27C7 is tonAAptr3 phoAAE15 Δ (argF-lac)169 ompTAdegP41kan-r Strain 27C7 was deposited on 30 Oct. 1991 in the American Type Culture Collection as ATCC No. 55,244. Alternatively, the strain of E. coli having mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783 issued 7 Aug. 1990 may be employed. Alternatively, methods of cloning, e.g., PCR or other nucleic acid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for heteromultimer-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe (Beach and Nurse, Nature 290: 140 [1981]; EP 139,383 published May 2, 1985); Kluyveromyces hosts (U.S. Pat. No.

4,943,529; Peer et al., supra) such as, e.g., K. lactis [MW98-8C, CBS683, CBS4574;

Louvencourt et al., J. Bacterid., 737 (1983)], K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al., supra), K. thermotolerans, and K. marxianus; yarrowia [EP

402,226]; Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol. 28:265-278

[1988]); Candida; Trichoderma reesia [EP 244,234]; Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA 76:5259-5263 [1979]); Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published 31 Oct. 1990); and filamentous fungi such as, e.g., Neurospora,

Penicillium, Tolypocladium (WO 91/00357 published 10 Jan. 1991), and Aspergillus hosts such as A. nidulans (Ballance et al., Biochem. Biophys. Res. Commun. 112:284-289 [1983]; Tilburn et al., Gene 26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA 81: 1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J. 4:475-479 [1985]).

Suitable host cells for the expression of glycosylated heteromultimer are derived from multicellular organisms. Such host cells are capable of complex processing and glycosylation activities. In principle, any higher eukaryotic cell culture is workable, whether from vertebrate or invertebrate culture. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified. See, e.g., Luckow et al., Bio/Technology 6:47-55 (1988); Miller et al., in Genetic Engineering, Setlow et al., eds., Vol. 8 (Plenum Publishing, 1986), pp. 277-279; and Maeda et al., Nature 315:592-594 (1985). A variety of viral strains for transfection are publicly available, e.g., the L-l variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can be utilized as hosts. Typically, plant cells are transfected by incubation with certain strains of the bacterium Agrobacterium tumefaciens, which has been previously manipulated to contain the heteromultimer DNA. During incubation of the plant cell culture with A. tumefaciens, the DNA encoding the heteromultimer is transferred to the plant cell host such that it is transfected, and will, under appropriate conditions, express the heteromultimer DNA. In addition, regulatory and signal sequences compatible with plant cells are available, such as the nopaline synthase promoter and polyadenylation signal sequences. Depicker et al., J. Mol. Appl. Gen. 1:561 (1982). In addition, DNA segments isolated from the upstream region of the T-DNA 780 gene are capable of activating or increasing transcription levels of plant-expressible genes in recombinant DNA-containing plant tissue. EP 321,196 published 21 Jun. 1989. The preferred hosts are vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years (Tissue Culture, Academic Press, Kruse and Patterson, editors [1973]). Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 [1977]); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/- DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216 [1980]); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 [1980]); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 [1982]); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

Host cells are transfected with the above-described expression or cloning vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Depending on the host cell used, transfection is done using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described in section 1.82 of Sambrook et al., supra, or electroporation is generally used for prokaryotes or other cells that contain substantial cell-wall barriers. Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al., Gene 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. In addition, plants may be transfected using ultrasound treatment as described in WO 91/00358 published 10 Jan. 1991.

For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology 52:456-457 (1978) is preferred. General aspects of mammalian cell host system transformations have been described by Axel in U.S. Pat. No. 4,399,216 issued 16 Aug. 1983. Transformations into yeast are typically carried out according to the method of Van Solingen et al., J. Bact. 130:946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA) 76:3829 (1979). However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene, polyornithine, etc., may also be used. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzymology (1989), Keown et al., Methods in Enzymology 185:527-537 (1990), and Mansour et al., Nature 336:348-352 (1988).

Prokaryotic cells used to produce the heteromultimer polypeptide of this invention are cultured in suitable media as described generally in Sambrook et al., supra.

The mammalian host cells used to produce the heteromultimer of this invention may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ([MEM], Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ([DMEM], Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham and Wallace, Meth. Enz. 58:44 (1979), Barnes and Sato, Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; or 4,560,655; WO 90/03430; WO 87/00195; U.S. Pat. Re. No. 30,985; or U.S. Pat. No. 5,122,469, the disclosures of all of which are incorporated herein by reference, may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as gentamycin drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

In general, principles, protocols, and practical techniques for maximizing the productivity of mammalian cell cultures can be found in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed., IRL Press, 1991.

The host cells referred to in this disclosure encompass cells in culture as well as cells that are within a host animal.

4. Recovery of the Heteromultimer

The heteromultimer preferably is generally recovered from the culture medium as a secreted polypeptide, although it also may be recovered from host cell lysate when directly produced without a secretory signal. If the heteromultimer is membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g. TRITON-X 100)

When the heteromultimer is produced in a recombinant cell other than one of human origin, it is completely free of proteins or polypeptides of human origin. However, it is necessary to purify the heteromultimer from recombinant cell proteins or polypeptides to obtain

preparations that are substantially homogeneous as to heteromultimer. As a first step, the culture medium or lysate is normally centrifuged to remove particulate cell debris.

Heterodimers having antibody constant domains can be conveniently purified by hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography, with affinity chromatography being the preferred purification technique. Where the heteromultimer comprises a CH3 domain, the Bakerbond ABX resin (J. T. Baker, PhiUipsburg, N.J.) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin Sepharose, chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the polypeptide to be recovered. The suitability of protein A as an affinity ligand depends on the species and isotype of the immunoglobulin Fc domain that is used in the chimera. Protein A can be used to purify immunoadhesins that are based on human gammal, gamma2, or gamma4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13

[1983]). Protein G is recommended for all mouse isotypes and for human gamma3 (Guss et al., EMBO J. 5: 15671575 [1986]). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. The conditions for binding an immunoadhesin to the protein A or G affinity column are dictated entirely by the characteristics of the Fc domain; that is, its species and isotype. Generally, when the proper ligand is chosen, efficient binding occurs directly from unconditioned culture fluid. One distinguishing feature of immunoadhesins is that, for human gammal molecules, the binding capacity for protein A is somewhat diminished relative to an antibody of the same Fc type. Bound immunoadhesin can be efficiently eluted either at acidic pH (at or above 3.0), or in a neutral pH buffer containing a mildly chaotropic salt. This affinity chromatography step can result in a heterodimer preparation that is >95% pure. 5. Uses for the Heteromultimer

Many therapeutic applications for the heteromultimer are contemplated. For example, the heteromultimer can be used for redirected cytotoxicity (e.g. to kill tumor cells), as a vaccine adjuvant, for delivering thrombolytic agents to clots, for delivering immunotoxins to tumor cells, for converting enzyme activated prodrugs at a target site (e.g. a tumor), for treating infectious diseases or targeting immune complexes to cell surface receptors.

Therapeutic formulations of the heteromultimer are prepared for storage by mixing the heteromultimer having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A., Ed., [1980]), in the form of lyophilized cake or aqueous solutions. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG).

The heteromultimer also may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly-[methylmethacylate] microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano- particles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.

The heteromultimer to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following

lyophilization and reconstitution. The heteromultimer ordinarily will be stored in lyophilized form or in solution. Therapeutic heteromultimer compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

The route of heteromultimer administration is in accord with known methods, e.g., injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, or intralesional routes, or by sustained release systems as noted below. The heteromultimer is administered continuously by infusion or by bolus injection.

Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the protein, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels [e.g., poly(2-hydroxyethyl-methacrylate) as described by Langer et al., J. Biomed. Mater. Res. 15: 167-277 (1981) and Langer, Chem. Tech. 12:98-105 (1982) or

poly(vinylalcohol)], polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L- glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 [1983]), non-degradable ethylene- vinyl acetate (Langer et al., supra), degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT (injectable microspheres composed of lactic acid- glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid (EP 133,988).

While polymers such as ethylene- vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated proteins remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37 °C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for protein stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S— S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

Sustained-release heteromultimer compositions also include liposomally entrapped heteromultimer. Liposomes containing heteromultimer are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688-3692 (1985), Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP

143,949; EP 142,641; Japanese patent application 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily the liposomes are of the small (about 200-800

Angstroms) unilamellar type in which the lipid content is greater than about 30 mol %

cholesterol, the selected proportion being adjusted for the optimal heteromultimer therapy.

An effective amount of heteromultimer to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the patient. Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. A typical daily dosage might range from about 1 ug/kg to up to 10 mg/kg or more, depending on the factors mentioned above. Typically, the clinician will administer heteromultimer until a dosage is reached that achieves the desired effect. The progress of this therapy is easily monitored by conventional assays.

The heteromultimers described herein can also be used in enzyme immunoassays. To achieve this, one arm of the heteromultimer can be designed to bind to a specific epitope on the enzyme so that binding does not cause enzyme inhibition, the other arm of the heteromultimer can be designed to bind to the immobilizing matrix ensuring a high enzyme density at the desired site. Examples of such diagnostic heteromultimers include those having specificity for IgG as well as ferritin, and those having binding specificities for horse radish peroxidase (HRP) as well as a hormone, for example.

The heteromultimers can be designed for use in two-site immunoassays. For example, two bispecific heteromultimers are produced binding to two separate epitopes on the analyte protein— one heteromultimer binds the complex to an insoluble matrix, the other binds an indicator enzyme.

Heteromultimers can also be used for in vitro or in vivo immunodiagnosis of various diseases such as cancer. To facilitate this diagnostic use, one arm of the heteromultimer can be designed to bind a tumor associated antigen and the other arm can bind a detectable marker (e.g. a chelator which binds a radionuclide). For example, a heteromultimer having specificities for the tumor associated antigen CEA as well as a bivalent hapten can be used for imaging of colorectal and thyroid carcinomas. Other non-therapeutic, diagnostic uses for the heteromultimer will be apparent to the skilled practitioner. For diagnostic applications, at least one arm of the heteromultimer typically will be labeled directly or indirectly with a detectable moiety. The detectable moiety can be any one which is capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as H-3, .C-14, P-32, S-35, or 1-125; a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase (HRP).

Any method known in the art for separately conjugating the heteromultimer to the detectable moiety may be employed, including those methods described by Hunter et al., Nature 144:945 (1962); David et al., Biochemistry 13: 1014 (1974); Pain et al., J. Immunol. Meth.

40:219 (1981); and Nygren, J. Histochem. and Cytochem. 30:407 (1982).

The heteromultimers of the present invention may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and

immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147- 158 (CRC Press, Inc., 1987).

Competitive binding assays rely on the ability of a labeled standard to compete with the test sample analyte for binding with a limited amount of heteromultimer. The amount of analyte in the test sample is inversely proportional to the amount of standard that becomes bound to the heteromultimer. To facilitate determining the amount of standard that becomes bound, the heteromultimers generally are insolubilized before or after the competition, so that the standard and analyte that are bound to the heteromultimers may conveniently be separated from the standard and analyte which remain unbound.

The heteromultimers are particularly useful for sandwich assays which involve the use of two molecules, each capable of binding to a different immunogenic portion, or epitope, of the sample to be detected. In a sandwich assay, the test sample analyte is bound by a first arm of the heteromultimer which is immobilized on a solid support, and thereafter a second arm of the heteromultimer binds to the analyte, thus forming an insoluble three-part complex. See, e.g., U.S. Pat. No. 4,376,110. The second arm of the heteromultimer may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti-immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme. Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

EXAMPLES

Example 1. Selection and characterization of improved heterodimerization domains

A library of randomized IgGl CH3 domains were created and selected for improved heterodimerization by phage display. The identified clones were screened by clonal phage ELISA, and selected clones were further characterized by expression in mammalian cells followed by non-reducing SDS-PAGE and quantitation of heteromeric species (Table 3).

Table 3

TPPVLDSDGSFFLY (SEQ ID NO:27)

FCH QVYTLPP IRECMTDNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO : 28 )

R6-A02 FCL RVRTVPPSREEMTKNQVSLWCHVWGFYPSDIAVEWESNGQPENNYKT 0.808 ND

TPPVLDSDGSFFLY (SEQ ID NO : 29 )

FCH QVYTLPPWREMMTRNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO: 30)

R6-B05 FCL AVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT 0.805 ND

TPPVLDSDGSFFLY (SEQ ID NO: 31)

FCH QVYTLPPVREMMTENQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO: 32)

R6-B07 FCL LVFTMPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT 0.7 ND

TPPVLDSDGSFFLY (SEQ ID NO: 33)

FCH QVYTLPPVREEMTGNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO: 34)

R6-B06 FCL GWTMPPSREEMTKNQVSLWCGVGGFYPSDIAVEWESNGQPENNYKT 0.657 ND

TPPVLDSDGSFFLY (SEQ ID NO: 35)

FCH QVYTLPPVREWMTGNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO : 36 )

R6-B09 FCL GWTAPPSREEMTKNQVSLWCCVGGFYPSDIAVEWESNGQPENNYKT 0.644 ND

TPPVLDSDGSFFLY (SEQ ID NO: 37)

FCH QVYTLPPFREDMTYNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO: 38)

R6-8C FCL WITLPPSREEMTKNQVSLWCMVTGFYPSDIAVEWESNGQPENNYKT 0.593 60

TPPVLDSDGSFFLY (SEQ ID NO : 39 )

FCH QVYTLPP IRELMTSNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO : 40 )

R6-A10 FCL GVYTWPPSREEMTKNQVSLWCLVTGFYPSDIAVEWESNGQPENNYKT 0.558 44

TPPVLDSDGSFFLY (SEQ ID NO : 41 )

FCH QVYTLPPLREGMTGNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO: 42)

R6-C08 FCL VWTLPPSREEMTKNQVSLWCKVKGFYPSDIAVEWESNGQPENNYKT 0.703 ND

TPPVLDSDGSFFLY (SEQ ID NO: 43)

FCH QVYTLPPSREDMTANQWLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO: 44)

R6-C04 FCL LVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT 1.694 ND

TPPVLDSDGSFFLY (SEQ ID NO: 45)

FCH QVYTLPPSREVMTENQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO : 46 )

R6-C03 FCL QVWTLPPSREEMTKNQVSLWCWGGFYPSDIAVEWESNGQPENNYKT 0.75 ND

TPPVLDSDGSFFLY (SEQ ID NO: 47)

FCH QVYTLPPSREEMTRNQWLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO : 48 )

R6-C11 FCL WRTLPPSREEMTKNQVSLWCWGGFYPSDIAVEWESNGQPENNYKT 0.747 ND

TPPVLDSDGSFFLY (SEQ ID NO : 49 )

FCH QVYTLPPSREDMTRNQVWLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO: 50)

R6-C09 FCL WYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT 0.707 ND

TPPVLDSDGSFFLY (SEQ ID NO: 51)

FCH QVYTLPPSREQMTMNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO: 52)

R5-F11 FCL QVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT 0.686 ND

TPPVLCVDGSFGLMSK (SEQ ID NO: 53)

FCH QVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLVSL (SEQ ID NO: 54) R6-E11 FCL QVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT 0.703 N D TPPVLCVDGSFGLMSK (SEQ ID NO: 55)

FCH QVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLVSL (SEQ ID NO : 56 )

R6-F04 FCL QVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT 1.694 N D

FPPTLDSDGSFGLMSK (SEQ ID NO: 57)

FCH QVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT IPPVLDSDGSFMLVSR (SEQ ID NO: 58)

R6-H10 FCL QVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT 0.75 N D

TPPVLDLDGSFDLESKLT (SEQ ID NO : 59 )

FCH QVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYRT TPPVLDSDGSFFLVSFLH (SEQ ID NO: 60)

Al FCL HVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLY (SEQ ID NO: 61)

FCH QVYTLPPFREAMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO: 62)

A2 FCL NVYTLPPSREEMTKNQVSLWCMVKGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLY (SEQ ID NO: 63)

FCH QVYTLPPFREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO: 64)

A3 FCL EVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLY (SEQ ID NO: 65)

FCH QVYTLPP IREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO : 66 )

A4 FCL HVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLY (SEQ ID NO: 67)

FCH QVYTLPP IREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO: 68)

A5 FCL RVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLY (SEQ ID NO : 69 )

FCH QVYTLPP IREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO : 70 )

A6 FCL EVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D 50

TPPVLDSDGSFFLY (SEQ ID NO : 71 )

FCH QVYTLPP IREQMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO: 72)

A7 FCL KVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLY (SEQ ID NO: 73)

FCH QVYTLPP IREGMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO: 74)

A8 FCL KVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLY (SEQ ID NO: 75)

FCH QVYTLPP IREMMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO : 76 )

A9 FCL LVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLY (SEQ ID NO: 77)

FCH QVYTLPP IREEMTTNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO : 78 )

A10 FCL HVYTLPPSREEMTKNQVSLWCLVNGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLY (SEQ ID NO : 79 )

FCH QVYTLPP IREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO : 80 )

All FCL FVYTLPPSREEMTKNQVSLWCMVKGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLY (SEQ ID NO : 81 )

FCH QVYTLPP IREVMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO: 82 )

A12 FCL QVFTLPPSREEMTKNQVSLWCRVEGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLY (SEQ ID NO: 83 )

FCH QVYTLPP IREQMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO: 84 )

Bl FCL QVYTLPPSREEMTKNQVSLWCLVNGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLY (SEQ ID NO: 85 )

FCH QVYTLPPLREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO : 8 6 )

B2 FCL QVFTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLY (SEQ ID NO: 87 )

FCH QVYTLPPLREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO : 88 )

B3 FCL QVYTWPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLY (SEQ ID NO : 8 9 )

FCH QVYTLPPVREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO: 90 )

B4 FCL QVFTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLY (SEQ ID NO: 91 )

FCH QVYTLPPVREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO: 92 )

B5 FCL EVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLY (SEQ ID NO: 93 )

FCH QVYTLPPVREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO: 94 )

B6 FCL HVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLY (SEQ ID NO: 95 )

FCH QVYTLPPVREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO : 96 )

B7 FCL KVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLY (SEQ ID NO: 97 )

FCH QVYTLPPVREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO: 98 )

B8 FCL LVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLY (SEQ ID NO : 99 )

FCH QVYTLPPVREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO : 100 )

B9 FCL MVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLY (SEQ ID NO : 101 )

FCH QVYTLPPVREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO: 102 )

BIO FCL QVFTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D TPPVLDSDGSFFLY (SEQ ID NO: 103 )

FCH QVYTLPPVREGMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO: 104 )

Bll FCL MVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLY (SEQ ID NO: 105 )

FCH QVYTLPPVREGMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO : 10 6 )

B12 FCL YVYTLPPSREEMTKNQVSLWCWKGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLY (SEQ ID NO: 107 )

FCH QVYTLPPVREEMTNNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO : 108 )

CI FCL MVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D TPPVLDSDGSFFLY (SEQ ID NO : 10 9 ) FCH QVYTLPPWREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT

TPPVLDSDGSFFLV (SEQ ID NO : 110 )

C2 FCL RVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLY (SEQ ID NO : 111 )

FCH QVYTLPPWREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO: 112)

C3 FCL SVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLY (SEQ ID NO: 113)

FCH QVYTLPPWREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO: 114)

C4 FCL RVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLY (SEQ ID NO: 115)

FCH QVYTLPPSREGMTKNQVWLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO : 116 )

C5 FCL RVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLY (SEQ ID NO: 117)

FCH QVYTLPPSREEMTGNQVWLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO : 118 )

C6 FCL MVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLY (SEQ ID NO : 119 )

FCH QVYTLPPSREEMTQNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO: 120)

C7 FCL HVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D 48

TPPVLDSDGSFFLY (SEQ ID NO: 121)

FCH QVYTLPPSREEMTNNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO: 122)

C8 FCL QVFTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLY (SEQ ID NO: 123)

FCH QVYTLPPSREEMTENQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLV (SEQ ID NO: 124)

C9 FCL QVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLSSK (SEQ ID NO: 125)

FCH QVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT FPPVLDSDGSFSLVSK (SEQ ID NO: 126)

CIO FCL QVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D TPPVLDSDGSFFLSSK (SEQ ID NO: 127)

FCH QVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT FPPVLDSDGSFLLVSK (SEQ ID NO: 128)

Cll FCL QVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D

SPPFLDSDGSFFLYSK (SEQ ID NO: 129)

FCH QVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT FPPVLDSDGSFFLVSY (SEQ ID NO: 130)

C12 FCL QVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLFSK (SEQ ID NO: 131)

FCH QVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT IPPVLDSDGSFFLVSQ (SEQ ID NO: 132)

Dl FCL QVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D

SPPLLDSDGSFYLYSK (SEQ ID NO: 133)

FCH QVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT IPPVLDSDGSFLLVSK (SEQ ID NO: 134)

D2 FCL QVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D

TPPVLDSDGSFFLYSK (SEQ ID NO: 135)

FCH QVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLVST (SEQ ID NO: 136)

D3 FCL QVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT N D N D SPPVLDSDGSFFLYSK (SEQ ID NO: 137)

FCH QVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT WPPVLDSDGSFGLVSN (SEQ ID NO: 138)

Fc variants were subcloned into a pCEP4 mammalian expression vector (Invitrogen) and transiently expressed using the EXPI293 expression system (Invitrogen). Cells were grown using EXPI293 media in 5% CO2 to a density of 2.5 million cells/mL in a 24-well plate and then transfected with 1 μg of DNA/mL of cells. After six days, the soluble scFvs were harvested by centrifuging the cells at 4000 g. The heteromeric Fes were purified using a 96 well Protein A plate (GE Healthcare Life Sciences). Each well of the plate was washed with IX PBS, and 1.8 mL of supernatant was loaded into each well. The proteins were purified using a vacuum manifold (Whatman) with 10 mbar of pressure. After the supernatant was loaded, the wells were washed with 600 μΐ_^ of IX PBS. To elute the proteins, 200 μΐ_^ of 0.1M acetic acid was added to each well and incubated at room temperature for several minutes. The plate was centrifuged at 100 g for 2 minutes, and 20 μΐ_^ of 1M Tris, pH 8.0 was added to neutralize the proteins.

For non-reducing gel characterization, 10 μΐ of purified protein, 5 μΐ of water, and 5 μΐ of NUPAGE LDS Sample Buffer (Life Technologies) were run on a 4-12% SDS-PAGE gel for 35 minutes at 200 mV. For reducing gels, the same protocol was followed, except that 1.4 M of 2- Mercaptoethanol was added to the sample buffer. The gels were rinsed and stained/destained using the ESTAIN 2.0 Protein Staining System (GenScript). After destaining in water overnight, photos of the gels were taken and quantitated using ImageJ (NIH). Example 2. Further selection and characterization of improved heterodimerization domains A second selection was performed using libraries with randomization of either the A (FCH) or B (FCL) chain of clones 8C and A6. As described in Example 1 above, the identified clones were screened by clonal phage ELISA, and selected clones were further characterized by expression in mammalian cells followed by non-reducing SDS-PAGE and quantitation of heteromeric species (Tables 4-7).

Table 4. wt8c-FCH (SEQ ID NO:40) with 8C-FCL library

(FCL11)

Rdll-A10 140 WTTLPPSREEMTKNQVSLWCIVTGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS 60

FFLY

Rdll-B05 141 W TLPPSREEMTKNQVSLWCIVIGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS 26

FFLR

Rdll-B09 142 WITLPPSREEMTKNQVSLWCMVTGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS ND

FFLS

Rdll-C05 143 WTTLPPSREEMTKNQVSLWCMVSGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS 17

FFLY

Rdll-Cll 144 WSTLPPSREEMTKNQVSLWCMVAGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS ND

FFLY

Rdll-C12 145 VWTLPPSREEMTKNQVSLWCLVSGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS ND

FFLS

Rdll-D06 146 WFTLPPSREEMTKNQVSLWCLVTGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS ND

FFLY

Rdll-D07 147 IVMTLPPSREEMTKNQVSLWCMVTGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS ND

FFLY

Rdll-D09 148 IVITLPPSREEMTKNQVSLWCMVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS ND

FFLY

Rdll-DIO 149 MWTLPPSREEMTKNQVSLWCIVTGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS NE

FFLL

Rdll-D12 150 EVITLPPSREEMTKNQVSLWCTVIGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS ND

FFLH

Rdlll-A02 151 LVITLPPSREEMTKNQVSLWCMVAGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS ND

FFLY

Rdlll-A03 152 EVITLPPSREEMTKNQVSLWCMVQGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS ND

FFLL

Rdlll-A09 153 AVTTLPPSREEMTKNQVSLWCWTGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS ND

FFLS

Rdlll-B02 154 KVITLPPSREEMTKNQVSLWCMVRGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS ND

FFLY

Rdlll-B05 155 QVITLPPSREEMTKNQVSLWCMVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS ND

FFLY

Rdlll-B08 156 WITLPPSREEMTKNQVSLWCMVTGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS 42

FFLW

Rdlll-Cll 157 WLTLPPSREEMTKNQVSLWCLVMGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS ND

FFLS

Rdlll-DOl 158 MVLTLPPSREEMTKNQVSLWCMVTGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS ND

FFLQ

Rdlll-D06 159 FVHTLPPSREEMTKNQVSLWCFVTGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS 51

FFLY

Rdlll-D07 160 VWSVPPSREEMTKNQVSLWCLVTGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS 52

FFLY

Rdlll-D08 161 LVGTLPPSREEMTKNQVSLWCWTGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS ND

FFLY

Rdlll-DIO 162 LVITLPPSREEMTKNQVSLWCRVTGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS ND

FFLY

RdlV-A04 163 WITLPPSREEMTKNQVSLWCMVTGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS ND

FFLF

RdlV-A05 164 WISLPPSREEMTKNQVSLWCMVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS 52

FFLY

RdlV-BOl 165 WIILPPSREEMTKNQVSLWCIVTGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS 60 (FCL-69) FFLY

RdlV-B03 166 LVITLPPSREEMTKNQVSLWCLVIGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS 62

FFLY RdlV-B06 167 WITLPPSREEMTKNQVSLWCLVMGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS N D FFLW

RdlV-D05 168 EVITLPPSREEMTKNQVSLWCWPGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS 40

FFLL

RdlV-D12 169 EVITLPPSREEMTKNQVSLWCMVTGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS N D

FFLL

FCL-1 170 GVSNVPPSREEMTKNQVSLWCAVAGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS 82

FFLY

FCL-7 171 LVMALPPSREEMTKNQVSLWCIVMGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS N D

FFLY

FCL-11 139 LVIALPPSREEMTKNQVSLWCMVMGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS 64

FFLY

FCL-27 172 WIKQPPSREEMTKNQVSLWCIVTGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS 79

FFLY

FCL-67 173 AVI SLPPSREEMTKNQVSLWCLVTGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS N D

FFLY

FCL-69 165 WIILPPSREEMTKNQVSLWCIVTGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS N D

FFLY

FCL-76 174 LV SLPPSREEMTKNQVSLWCMVSGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS 52

FFLD

FCL-79 175 MVFDLPPSREEMTKNQVSLWCMVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS 64

FFLY

FCL-82 176 W APPPSREEMTKNQVSLWCMVIGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS 57

FFLD

FCL-87 177 GV GLPPSREEMTKNQVSLWCTVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS N D

FFLY

Table 5. 8cFCH library with wt8c-FCL (SEQ ID NO:39)

FFLV

RdlV-H09 190 QVYTLPP IREVMTSNQVRLLCTVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS N E

FFLV

RdlV-H05 191 QVYTLPPTRELMTRNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS N E

FFLV

Table 6. A6 FCL library with A6wtFCH (SEQ ID NO:72)

Clone SEQ FCL sequence %

I D het

Rdl l-C07 192 DVYILPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS N D

FFLY

Rdl ll-A02 193 DVYTLPPSREEMTKNQVSLWCSVRGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS N D

FFLY

Rdl l-A02 194 EVYTLPPSREEMTKNQVSLWCQVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS N D

FFLY

Rdl l-B05 195 EVYGLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS N D

FFLY

Rd-CIO 196 EVYLLPPSREEMTKNQVSLWCLVIGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS N D

FFLY

Rdl l-Cll 197 EVYSLPPSREEMTKNQVSLWCLVIGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS N D

FFLY

Rdl l-C12 198 EVYYLPPSREEMTKNQVSLWCGVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS N D

FFLY

Rdl ll-B09 199 EVYSLPPSREEMTKNQVSLWCWIGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS N D

FFLY

Rdl ll-C05 200 EVYILPPSREEMTKNQVSLWCLVYGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS N D

FFLY

Rd-D09 201 EVYSLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS N D

FFLY

RdlV-D02 202 EVYVLPPSREEMTKNQVSLWCWKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS N D

FFLY

RdlV-AlO 203 KVYTLPPSREEMTKNQVSLWCLVEGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS N D

FFLY

Rdl l-D06 204 LVYVLPPSREEMTKNQVSLWCLVNGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS N D

FFLY

Rdl ll-Cll 205 LVYILPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS N D

FFLY

Rdl l-A06 206 RVYALPPSREEMTKNQVSLWCLVTGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS N D

FFLY

Rdl l-BlO 207 WYTLPPSREEMTKNQVSLWCWIGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS N D

FFLY

RdlV-D06 208 YVYTLPPSREEMTKNQVSLWCPVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS N D

FFLY

Table 7. A6 FCH library with A6 wt-FCL (SEQ ID NO:71)

Clone SEQ FCH sequence %

I D het

Rdl ll-G02 209 QVYTLPP IREPMTMNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS N D

FFLV Following the sequencing of the ELISA-confirmed clones, 93 FCL domains were transferred from phagemids to a pFUSEss vector (Invivogen) engineered to contain a partial hlgGl hinge sequence, CH2 domain, and the selected CH3 domains. FCL clones with partial hinges were expressed in 293T mammalian cells and FCLs were detected by SDS-PAGE.

Surprisingly, chains FCL1 and FCL27 appeared to be unstable when expressed without the cognate 8C FCH chain.

To identify clones with enhanced heterodimerization, the FCL domains were co- expressed with 8C FCH and assayed by SDS-PAGE. FCL clones 7, 11, and 27 demonstrated heterodimerization comparable to 8C FCH/FCL with reduced homodimerization. A total of 10 FCL domains were identified that showed little-to-no homodimerization, and like FCL27, some appeared to be unstable when expressed without 8C FCH. Five clones, FCL1, FCL27, FCL76, FCL79, and FCL82 were selected for conversion into full heavy chains. Additional clones were selected for conversion into heavy chains comprising an scFv. The heavy chain converted clones were transiently expressed with varying ratios (1:2, 1:4, 1:9) of transfected FCH and FCL vectors to determine the effect of FCH/FCL ratio for heterodimer formation. Most clones tested had the greatest average percentage of heterodimer formation at a 1:9 ratio of FCH/FCL. FCL1 and FCL27 had the highest percentage of heterodimer formation at all ratios tested.

FCH and FCL genes were subcloned into a double gene vector (DGV; Lonza) the control of a mCMV promoter. The plasmid was stably transfected into CHOKISVGS-KO cells (Lonza). Expression for purification was performed in CM76 media (Lonza). After two weeks of growth, cells were harvested at 5000 g for thirty minutes and filtered using a 0.2 μιη filter. To purify, the supernatant was loaded onto a MABSELECT (GE Healthcare) protein A column at a density of 20 mg antibody/mL of resin. After loading, the protein was washed with IX PBS for four column volumes, and then eluted with 0.1 M acetic acid (2 column volumes). The protein was pooled and neutralized with 1M Tris. The protein was then dialyzed against IX PBS overnight and sterilized with a 0.2 μιη filter.

Example 3. Fc receptor binding of improved heterodimerization domains

Binding of antibodies to Fc gamma receptors was determined by functional ELISAs. Plates were either coated with 1 ug/ml of RIIIA or RIIIB overnight at room temperature. Wells were then blocked with assay buffer (PBS + 1% BSA) at 4 °C overnight. Antibodies diluted to 100 ug/ml, and then three-fold serial dilutions were made and added to the plate in duplicate. Plates were incubated with antibody for 2 hours at room temperature and then washed with assay buffer four times. Biotinylated rabbit anti-human IgG H&L antibody (Abeam, Ab6758) was diluted to 2 ug/ml in assay buffer and 100 ul was added to each well of the plate. The plate was incubated at room temperature for one hour, and washed with IX PBS-T (IX PBS + 0.05% TWEEN-20). Streptavidin-HRP (R&D systems, DY998) was diluted 1:200 in assay buffer and 100 ul/well was added to the assay plate. The plates were incubated at room temperature for 30 minutes in the dark. The plate was then washed four times with IX PBS-T. 100 ul of TMB solution (Cell Signaling Technology, Cat 7004) was added to each well, and after the desired signal was achieved, 100 ul of STOP solution (Cell Signaling Technology, Cat 7002) was added. The plates were read at 450 nM absorbance, and Kds calculated using PRISM. Figures 1 and 2 show the results of these ELISAs. Figure 1 shows the binding to Fc-gamma RIIIA compared to human Fc (hFc) as the control. This shows that the binding of these Fc variants to Fc-gamma RIIIA is very similar to hFc. Figure 2 the results of Binding to Fc-gamma IIIB, as compared to hFc. FCL27 has binding very similar to hFC, however, FCL1 has better affinity to FC gamma IIIB.

Example 4. Exemplary constant regions

Human IgGl constant region (SEQ ID NO:3)

ASTKGPS VFPLAPS S KSTS GGT AALGCLVKD YFPEPVTVS WNS GALTS GVHTFPA

VLQS S GLYS LS SWT VPS S SLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LT VDKS RWQQGN VFS CS VMHE ALHNH YTQKS LS LS PGK

Human IgG2 constant region (SEQ ID NO:4)

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQS S GLYS LS S VVT VPS SNFGTQTYTCNVDHKPSNTKVDKT VERKCC VECPPCPAPPV AGPS VFLFPPKPKDTLMIS RTPE VTC V V VD VS HEDPE VQFNW Y VD G VE VHN AKTKPREE QFNS TFR V VS VLT V VHQD WLNGKE YKC KVS NKGLP APIEKTIS KTKGQPREPQ V YTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTV DKS RWQQGN VFS C S VMHE ALHNH YTQKS LS LS PGK

Human IgG3 constant region (SEQ ID NO:5)

ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQS S GLYS LS SWT VPS S SLGTQT YTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCP EPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVL T VLHQD WLNGKE YKC KVS NKALP APIEKTIS KTKGQPREPQ V YTLPPS REEMTKNQ VS L TCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFS CSVMHEALHNRFTQKSLSLSPGK

Human IgG4 constant region (SEQ ID NO:6)

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQS S GLYS LS SWT VPS S SLGTKT YTCNVDHKPSNTKVDKRVES KYGPPCPSCPAPEFL GGPS VFLFPPKPKDTLMIS RTPE VTC V V VD VS QEDPE VQFNW Y VD G VE VHN AKTKPREE QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTV DKS RWQEGN VFS CS VMHE ALHNH YTQKS LS LS LGK

Human IgA constant region (SEQ ID NO:7)

ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPS QDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPP TPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPE RDLC GC YS VS S VLPGC AEPWNHGKTFTCT A A YPES KTPLT ATLS KS GNTFRPE VHLLPPP SEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVT SILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY

Human IgD constant region (SEQ ID NO:8)

APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFP EIQRRDS Y YMTS S QLS TPLQQWRQGE YKC V VQHT AS KS KKEIFRWPES PKAQ AS S VPT A QPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLL TPA VQDLWLRDKATFTCFVVGSDLKD AHLTWE VAGKVPTGGVEEGLLERHS NGS QS Q HS RLTLPRS LWN AGTS VTCTLNHPS LPPQRLM ALREP A AQ AP VKLS LNLLAS S DPPE A AS WLLCE VS GFS PPNILLM WLEDQRE VNTS GF AP ARPPPQPGS TTFW A WS VLR VP APPS PQP AT YTC V VS HEDS RTLLN AS RS LE VS Y VTDHGPMK

Human IgE constant region (SEQ ID NO:9)

AS TQS PS VFPLTRCC KNIPS NATS VTLGCLATG YFPEP VM VT WDTGS LNGTTMTL P ATTLTLS GH Y ATIS LLT VS G A W AKQMFTCR V AHTPS S TD W VDNKTFS VC S RDFTPPT V KILQSSCDGGGHFPPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELAST QS ELTLS QKHWLS DRT YTC Q VT YQGHTFEDS TKKC ADS NPRG VS A YLS RPS PFDLFIRKS PTITCLVVDLAPSKGTVNLTWSRASGKPVNHSTRKEEKQRNGTLTVTSTLPVGTRDWIE GETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMP EDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHEA ASPS QT VQR A VS VNPGK

Human IgM constant region (SEQ ID NO: 10)

GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGF PSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVS VFVPPRDGFFGNPRKS KLICQATGFSPRQIQVS WLREGKQVGS GVTTDQVQ AE AKES GP TT YKVTS TLTIKES D WLGQS MFTCRVDHRGLTFQQN AS S MC VPDQDT AIR VFAIPPS FAS IFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDD WNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVT GFS P AD VF VQWMQRGQPLS PEKY VTS APMPEPQ APGR YFAHS ILT VS EEE WNTGET YTC VAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

Human IgGl CH3 (SEQ ID NO: 11)

AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Human IgG2 CH3 (SEQ ID NO: 12) TKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Human IgG3 CH3 (SEQ ID NO: 13)

TKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYN TTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

Human IgG4 CH3 (SEQ ID NO: 14)

AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Claims

Claims We claim:

1. A protein comprising a first CH3 domain paired with a non-identical second CH3 domain, wherein a) the first CH3 domain comprises an amino acid at EU position 354 or 364 that creates a protuberance, and an amino acid at EU position 407 that creates a cavity, and the second CH3 domain comprises at least one amino acid mutation at EU position 347, 349, 350, 351, 366, 368, 370, or 407; or b) the first CH3 domain comprises an amino acid at EU position 349 that creates a cavity and an amino acid at EU position 366 that creates a protuberance, and the second CH3 domain comprises at least one amino acid mutation at EU position 351, 354, 357, 360, 364, 366, 368, or 407.

2. The protein of claim 1, wherein the amino acid at EU position 354 of the first CH3 domain of a) is isoleucine (I) or leucine (L), and the amino acid at EU position 349 of the second CH3 domain of a) is selected from the group consisting of cysteine (C), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), arginine (R), serine (S), threonine (T), valine (V), and tryptophan (W); and the amino acid at EU position 407 of the first CH3 domain of a) is a valine (V), and the amino acid at EU position 366 of the second CH3 domain of a) is selected from the group consisting of alanine (A), cysteine (C), glycine (G), lysine (K), leucine (L), methionine (M), arginine (R), serine (S), tryptophan (W), and tyrosine (Y).

3. The protein of claim 2, wherein the amino acid at EU position 357 of the first CH3 domain of a) is leucine (L), and the amino acid at EU position 370 of the second CH3 domain of a) is selected from the group consisting of alanine (A), isoleucine (I), lysine (K), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), and tryptophan (W); and the amino acid at EU position 360 of the first CH3 domain of a) is serine (S), the amino acid at EU position 347 of the second CH3 domain of a) is selected from the group consisting of alanine (A), glutamic acid (E), phenylalanine (F), glycine (G), isoleucine (I), lysine (K), leucine (L), methionine (M), glutamine (Q), serine (S), and valine (V).

4. The protein of any one of claims 1-3, wherein the amino acid at EU position 350 of the second CH3 domain of a) is selected from the group consisting of alanine (A), aspartic acid (D), glycine (G), isoleucine (I), lysine (K), methionine (M), asparagine (N), proline (P), arginine (R), serine (S), threonine (T), and tyrosine

(Y); the amino acid at EU position 351 of the second CH3 domain of a) is selected from the group consisting of phenylalanine (F), histidine (H), lysine (K), leucine (L), methionine (M), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), and tyrosine (Y); the amino acid at EU position 368 of the second CH3 domain of a) is selected from the group consisting of alanine (A), phenylalanine (F), isoleucine (I), leucine (L), methionine (M), asparagine (N), arginine (R), threonine (T), and valine (V); and the amino acid at EU position 407 of the second CH3 domain of a) is selected from the group consisting of alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), histidine (H), isoleucine (I), leucine (L), asparagine (N), glutamine (Q), arginine (R), serine (S), valine (V), tryptophan (W), and tyrosine (Y).

5. The protein of claim 1, wherein the amino acid at EU position 349 of the first CH3 domain of b) is an isoleucine (I), and the amino acid at EU position 354 of the second CH3 domain of b) is selected from the group consisting of cysteine (C), phenylalanine (F), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), arginine (R), serine (S), threonine (T), and valine (V); and the amino acid at EU position 366 the first CH3 domain of b) is a trypophan (W), and the amino acid at EU position 407 of the second CH3 domain of b) is selected from the group consisting of alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), leucine (L), asparagine (N), proline (P), arginine (R), serine (S), valine (V), and tyrosine (Y).

6. The protein of claim 5, wherein the amino acid at EU position 347 of the first CH3 domain of b) is a valine (V), and the amino acid at EU position 360 of the second CH3 domain of b) is selected from the group consisting of cysteine (C), phenylalanine (F), glycine (G), isoleucine (I), lysine (K), leucine (L), asparagine (N), proline (P), arginine (R), serine (S), threonine (T), valine (V), and tyrosine (Y); the amino acid at EU position 368 of the first CH3 domain of b) is a methionine (M), and the amino acid at EU position 364 of the second CH3 domain of b) is selected from the group consisting of alanine (A), cysteine (C), phenylalanine (F), leucine (L), proline (P), arginine (R), serine (S), threonine (T), tryptophan (W), and tyrosine (Y); and the amino acid at EU position 370 position of the first CH3 domain of b) is a threonine

(T), and the amino acid at EU position 357 of the second CH3 domain of b) is selected from the group consisting of cysteine (C), phenylalanine (F), glycine (G), lysine (K), leucine (L), methionine (M), glutamine (Q), serine (S), threonine (T), valine (V), and tryptophan (W).]

7. The protein of claim 5 or 6, wherein, the amino acid at EU position 351 of the second CH3 domain of b) is selected from the group consisting of alanine (A), phenylalanine (F), isoleucine (I), leucine (L), proline (P), glutamine (Q), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y); the amino acid at EU position 366 of the second CH3 domain of b) is selected from the group consisting of alanine (A), aspartic acid (D), lysine (K), leucine (L), methionine (M), proline (P), arginine (R), serine (S), threonine (T), tryptophan (W), and tyrosine (Y); and the amino acid at EU position 368 of the second CH3 domain of b) is selected from the group consisting of alanine (A), aspartic acid (D), glutamic acid (E), glycine (G), isoleucine (I), lysine (K), leucine (L), proline (P), serine (S), threonine (T), and valine (V).

8. A protein comprising a first CH3 domain paired with a non-identical second CH3 domain, wherein a) the first CH3 domain comprises an amino acid at EU position 351 selected from the group consisting of leucine (L), phenylalanine (F), isoleucine (I), and valine (V); an isoleucine (I) at EU position 354; an amino acid at EU position 364 selected from the group consisting of serine (S) and leucine (L); a serine (S) at EU position 366; an alanine (A) at EU position 368; and a valine (V) at EU position 407; and b) the second CH3 domain comprises an amino acid at EU position 347 selected from the group consisting of glycine (G), arginine (R), and glutamine (Q); an amino acid at EU position 350 selected from the group consisting of asparagine (N) and threonine (T); and a tryptophan (W) at EU position 366.

9. The protein of claim 8, wherein the second CH3 domain comprises an amino acid at EU position 347 selected from the group consisting of glycine (G), arginine (R), and glutamine (Q); an amino acid at EU position 349 selected from the group consisting of serine (S), glycine (G), valine (V), alanine (A), arginine (R), methionine, (M), and tryptophan (W); an amino acid at EU position 350 selected from the group consisting of asparagine (N) and threonine (T); an amino acid at EU position 351 selected from the group consisting of valine (V), glycine (G), methionine (M), alanine (A), tryptophan (W), and leucine (L); a tryptophan (W) at EU position 366; an amino acid at EU position 368 selected from the group consisting of alanine (A), glycine (G), cysteine (C), valine (V), histidine (H), and leucine (L); and an amino acid at EU position 370 selected from the group consisting of alanine (A), glycine (G), arginine (R), tryptophan (W), and valine (V).

10. The protein of claim 8, wherein the second CH3 domain comprises an amino acid at EU position 347 selected from the group consisting of glycine (G) and arginine (R); an amino acid at EU position 349 selected from the group consisting of serine (S), glycine (G), valine (V), alanine (A), and arginine (R); an amino acid at EU position 350 selected from the group consisting of asparagine (N) and threonine (T); an amino acid at EU position 351 selected from the group consisting of valine (V), glycine (G), methionine (M), alanine (A), and tryptophan (W); a tryptophan (W) at EU position 366; an amino acid at EU position 368 selected from the group consisting of alanine (A), glycine (G), cysteine (C), valine (V), and histidine (H); and an amino acid at EU position 370 selected from the group consisting of alanine (A), glycine (G), arginine (R), and tryptophan (W).

11. The protein of claim 8, wherein the second CH3 domain comprises a glycine (G) at EU position 347; a serine (S) at EU position 349; an asparagine (N) at EU position 350; a valine (V) at EU position 351; a tryptophan (W) at EU position 366; an alanine (A) at EU position 368; and an alanine (A) at EU position 370.

12. The protein of claim 8, wherein the second CH3 domain comprises a sequence selected from the group consisting of SEQ ID NOs: 15, 17, 21, 25, 27, 29, 35, 37, and 170.

13. A protein comprising a first CH3 domain paired with a non-identical second CH3 domain, wherein a) the first CH3 domain comprises an amino acid at EU position 351 selected from the group consisting of leucine (L), phenylalanine (F), isoleucine (I), and valine (V); an isoleucine (I) at EU position 354; an amino acid at EU position 364 selected from the group consisting of serine (S) and leucine (L); a serine (S) at EU position 366; an alanine (A) at EU position 368; and a valine (V) at EU position 407; and b) the second CH3 domain comprises an amino acid at EU position 347 selected from the group consisting of valine (V), leucine (L), and alanine (A); an amino acid at EU position 349 selected from the group consisting of isoleucine (I), threonine (T), and glycine (G); an amino acid at EU position 350 selected from the group consisting of lysine (K), isoleucine (I), and threonine (T); an amino acid at EU position 351 selected from the group consisting of glutamine (Q) and leucine (L); a tryptophan (W) at EU position 366; an amino acid at EU position 368 selected from the group consisting of isoleucine (I), methionine (M), arginine (R), and valine (V); and an amino acid at EU position 407 selected from the group consisting of tyrosine (Y), serine (S), tryptophan (W), and phenylalanine (F).

14. The protein of claim 13, wherein the second CH3 domain comprises a valine (V) at EU position 347; an amino acid at EU position 349 selected from the group consisting of isoleucine (I) and threonine (T); an amino acid at EU position 350 selected from the group consisting of lysine (K), isoleucine (I), and threonine (T); an amino acid at EU position 351 selected from the group consisting of glutamine (Q) and leucine (L); a tryptophan (W) at EU position 366; an amino acid at EU position 368 selected from the group consisting of isoleucine (I) and methionine (M); and an amino acid at EU position 407 selected from the group consisting of tyrosine (Y), serine (S), tryptophan (W), and phenylalanine (F).

15. The protein of claim 13, wherein the second CH3 domain comprises a valine (V) at EU position 347; an isoleucine (I) at EU position 349; a lysine (K) at EU position 350; a glutamine (Q) at EU position 351; a tryptophan (W) at EU position 366; an isoleucine (I) at EU position 368; and a tyrosine (Y) at EU position 407.

16. The protein of claim 13, wherein the second CH3 domain comprises a sequence selected from the group consisting of SEQ ID NOs: 39, 140, 142, 153, 156, 161, 162, 163, 165, and 172.

17. The protein of any one of claims 8-16, wherein the first CH3 domain comprises an amino acid at EU position 351 selected from the group consisting of leucine (L), phenylalanine (F), isoleucine (I), and valine (V); an isoleucine (I) at EU position 354; an amino acid at EU position 357 selected from the group consisting of leucine (L), methionine (M), serine (S), proline (P), tryptophan (W), glutamic acid (E), glutamine (Q), and glycine (G); an amino acid at EU position 360 selected from the group consisting of serine (S), isoleucine (I), asparagine (N), glycine (G), tyrosine (Y), methionine (M), lysine (K), and arginine (R); an amino acid at EU position 364 selected from the group consisting of serine (S) and leucine (L); a serine (S) at EU position 366; an alanine (A) at EU position 368; and a valine (V) at EU position 407.

18. The protein of any one of claims 8-16, wherein the first CH3 domain comprises a leucine (L) at EU position 351; an isoleucine (I) at EU position 354; an amino acid at EU position 357 selected from the group consisting of glutamine (Q), methionine (M), and glycine (G); a lysine (K) at EU position 360; a leucine (L) at EU position 364; a serine (S) at EU position 366; an alanine (A) at EU position 368; and a valine (V) at EU position 407.

19. The protein of any one of claims 8-14, wherein the first CH3 domain comprises an amino acid at EU position 351 selected from the group consisting of leucine (L), phenylalanine (F), isoleucine (I), and valine (V); an isoleucine (I) at EU position 354; an amino acid at EU position 357 selected from the group consisting of leucine (L), methionine (M), serine (S), proline (P), tryptophan (W), and glutamic acid (E); an amino acid at EU position 360 selected from the group consisting of serine (S), isoleucine (I), asparagine (N), glycine (G), tyrosine (Y), methionine (M), lysine (K), and arginine (R); an amino acid at EU position 364 selected from the group consisting of serine (S) and leucine (L); a serine (S) at EU position 366; an alanine (A) at EU position 368; and a valine (V) at EU position 407.

20. The protein of any one of claims 8-16, wherein the first CH3 domain comprises an amino acid at EU position 351 selected from the group consisting of leucine (L), phenylalanine (F), isoleucine (I), and valine (V); an isoleucine (I) at EU position 354; an amino acid at EU position 357 selected from the group consisting of leucine (L), methionine (M), serine (S), proline (P), and tryptophan (W); an amino acid at EU position 360 selected from the group consisting of serine (S), isoleucine (I), asparagine (N), glycine (G), tyrosine (Y), and methionine (M); an amino acid at EU position 364 selected from the group consisting of serine (S) and leucine (L); a serine (S) at EU position 366; an alanine (A) at EU position 368; and a valine (V) at EU position 407.

21. The protein of any one of claims 8-16, wherein the first CH3 domain comprises an amino acid at EU position 351 selected from the group consisting of leucine (L), phenylalanine (F), isoleucine (I), and valine (V); an isoleucine (I) at EU position 354; an amino acid at EU position 357 selected from the group consisting of leucine (L), methionine (M), and serine (S); an amino acid at EU position 360 selected from the group consisting of serine (S), isoleucine (I), glycine (G), and tyrosine (Y); an amino acid at EU position 364 selected from the group consisting of serine (S) and leucine (L); a serine (S) at EU position 366; an alanine (A) at EU position 368; and a valine (V) at EU position 407.

22. The protein of any one of claims 8-16, wherein the first CH3 domain comprises an isoleucine (I) at EU position 354; a leucine (L) at EU position 357; a serine (S)at EU position 360; a serine (S) at EU position 366; an alanine (A) at EU position 368; and a valine (V) at EU position 407.

23. The protein of any one of claims 8-16, wherein the first CH3 domain comprises an isoleucine (I) at EU position 354; an amino acid at EU position 357 selected from the group consisting of glutamine (Q), methionine (M), and glycine (G); a serine (S) at EU position 366; an alanine (A) at EU position 368; and a valine (V) at EU position 407.

24. The protein of any one of claims 8-16, wherein the first CH3 domain comprises an isoleucine (I) at EU position 354; a glutamine (Q) at EU position 357; a serine (S) at EU position 366; an alanine (A) at EU position 368; and a valine (V) at EU position 407.

25. The protein of any one of claims 8-16, wherein the first CH3 domain comprises a sequence selected from the group consisting of SEQ ID NOs: 40, 72, 74, 76, 78, 82, 84, 178, 179, 180, 181, 182, 183, 184, 185, 189, and 209.

26. A protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-209

27. The protein of any one of claims 1-26, wherein the first CH3 domain and second CH3 domain are each selected from the group consisting of a human IgGl CH3 domain, a human IgG2 CH3 domain, a human IgG3 CH3 domain, a human IgG4 CH3 domain, a human IgA CH3 domain, a human IgD CH3 domain, a human IgE CH3 domain, a human IgM CH3 domain.

28. The protein of any one of claims 1-26, wherein the first CH3 domain and second CH3 domain are present on separate polypeptides.

29. The protein of any one of claims 1-26, wherein the first CH3 domain and second CH3 domain are present on the same polypeptide.

30. The protein of any one of claims 1-29, wherein the protein comprises one or more antibody antigen binding domains, receptor ligand binding domains, or ligand domains.

31. A nucleic acid encoding a protein of any one of claims 1-30 or a polypeptide comprising a CH3 domain of any one of claims 1-30.

32. An expression vector comprising the nucleic acid of claim 31.

33. A cell comprising the nucleic acid of claim 31 or the expression vector of claim 32.

34. A method of producing a polypeptide comprising culturing the cell of claim 33 under conditions wherein the protein or polypeptide is expressed and isolating the protein or polypeptide.