AU2008289225A1 - Formulations of antibodies and Fc-fusion molecules using polycations - Google Patents

Description

WO 2009/026122 PCT/US2008/073250 FORMULATIONS OF ANTIBODIES AND FC-FUSION MOLECULES USING POLYCATIONS [00011 Field of the Invention [00021 The present invention relates, in general, to methods for making antibody and Fe-fusion molecule formulations, comprising an antibody or FEc-fusion molecule and polycations, and compositions comprising the formulations. [0003] Background of the Invention [0004] Proteins having a high pI or distinct polarity may be unstable when put into solution at physiological pH and high concentration of protein in solution. Proteins in solution tend to breakdown over time, and breakdown products aggregate decreasing the biological efficacy of the protein. For example, growth hormone in solution forms insoluble aggregation products resulting in precipitation of the protein and loss of activity (U.S. Patent 4,816,568). In order to prevent protein breakdown in solution, stabilizers (e.g., glycine, polyethylene glycol) are often added to the concentrated protein formulation in an attempt to reduce protein aggregation. U.S. patent 4,816,568 describes a method of stabilizing growth hormones in solution using polyol compounds, single or polymeric amino acids with charged side chains, or choline derivatives. Such degredation and aggregation problems add to the complexity of formulating biologics as pharmaceutical drugs. [0005] Today, antibodies are the fastest growing class of biologics in the pharmaceutical industry. Antibodies are also known to breakdown over time in solution. Hence, scientists have proposed ways of addressing the problem of antibody aggregation in liquid formulations. [00061 Addition of certain positively charged metal ions such as Calcium (Ca2+) and Magnesium (Mg2+) have been proposed as ways to slow isomerization of antibodies and prevention of formation of succinimide intermediates, in solutions (PCT Patent Application Publication WO 2004/039337). However, addition of certain positively charged metal ions (e.g., ZnCl 2 ) may cause protein precipitation (WO 2004/039337).

WO 2009/026122 PCT/US2008/073250 [0007] Summary of the Invention [0008] The present invention relates to methods for making and compositions comprising liquid or lyophilized pharmaceutical formulations, including therapeutic antibody and Fc-fusion formulations. [0009] In one aspect, the invention provides a method for making a liquid or lyophilized formulation of an antibody or Fc-fusion molecule comprising combining an antibody or Fc-fusion molecule with a polycation, thereby, obtaining a formulation that has reduced levels of antibody or Fc-fusion molecule degradation and aggregation compared to the same formulation without the polycation. In one embodiment, the Fc-fusion molecule is a peptibody. [00101 In one embodiment, the polycation is selected from the group consisting of polylysine, polyarginine, polyornithine, polyhistidine, and cationic polysaccharides, or mixtures thereof. [0011] In a related embodiment, the polycation composition comprises at least two of the polycations selected from the group consisting of lysine, arginine, polylysine, polyarginine, polyornithine, polyhistidine, and cationic polysaccharides In a related embodiment, the polycation is composed of lysine and arginine. [0012] In a further embodiment, the polycations are homopolymers or co-polymers, or mixtures thereof. A polycation homopolymer comprises a single repeating unit of the same cation monomer. A polycation co-polymer comprises different cation monomers or different cationic polymers. In one embociment, a polycation co polymer may comprise a mixture of cation monomers, a mixture of polycation homopolymers or a mixture of polycation co-polymers. [0013] In another embodiment, the polycation is a polymer comprising repeating units of a cationic monomer and a neutral monomer. In one embodiment the polycation has the monomeric formula A-B-X-Y, wherein A and X are cationic monomers and B and Y are neutral monomers. In a related embodiment, A and X are the same cationic monomer. In a further embodiment, A and X are different cationic monomers. In another embodiment, B and Y are the same neutral monomer. In a still further embodiment, B and Y are different neutral monomers. In another embodiment, the cationic monomer is a repeating unit which is used as the base of the polycations set out herein. In a related embodiment, the cationic monomer is a basic amino acid, a repeating sequence of a cationic monomer linked to a neutral monomer, WO 2009/026122 PCT/US2008/073250 or other protein molecules or chemical structures having repeating structural characteristics which can be used to construct a polycation composition. [0014] It is further contemplated that the neutral monomer is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine serine, threonine, and proline. In a still further embodiment, the polycation comprises repeating units of lysine and glycine residues. [0015] In another aspect, the invention provides that the antibody formulation of the invention comprises a therapeutic antibody. In one embodiment, the therapeutic antibody is an IgG antibody selected from the group consisting of an IgGI, IgG2, IgG3 and IgG4 antibody or a fragment thereof. [0016] It is contemplated that the polycation in the polycation composition has a molecular weight of between 0.2 kDa to 70 kDa. It is further contemplated that the antibody in said antibody formulation has a pI of 7.5 or less. In one embodiment, the antibody in said antibody formulation has a calculated pI of about 6.4. In a further embodiment, the polycations are at a concentration of about 0.1% to about 10% w/w polycation in the final formulation. [0017] In a related embodiment, the pH of the polycation composition is between about 4.5 to about 7.5, between about 5.0. to about 6.5, or between about 5.5. to about 6.0. [0018] In one embodiment, the antibody or Fc-fusion molecule formulation has a greater solubility when combined with the polycation composition as compared to the solubility of the antibody formulation in the absence of polycations. In an additional embodiment, the antibody or Fe-fusion molecule formulation has an increased shelf life as compared to an antibody formulation that has not been combined with a polycation composition. In a further embodiment, the antibody or Fe-fusion formulation that has been combined with a polycation composition has at least a 25% greater shelf life than a similar antibody formulation that has not been combined with a polycation composition. [0019] The present invention also contemplates an antibody formulation wherein the presence of polycations in said formulation reduces the formation of antibody multimers or aggregates in the antibody or Fc-fusion molecule formulation as compared to such a therapeutic antibody or Fc-fusion formulation that has not been combined with a polycation composition.

WO 2009/026122 PCT/US2008/073250 [0020] In various aspects, the therapeutic antibody is selected from the group consisting of Enbrel (Eternacept), Humira (adalimumab), Synagis (palivizumab), AMG 714 (anti-IL15 antibody), vectibix (panitumumab), Rituxan (rituximab), zevalin (ibritumomab tiuxetan), anti-CD80 monoclonal antibody (mAb) (galiximab), anti CD23 mAb (lumiliximab), M200 (volociximab), anti-Cripto mAb, anti-BR3 mAb, anti-IGFIR mAb, Tysabri (natalizumab), Daclizumab, humanized anti-CD20 mAb (ocrelizumab), soluble BAFF antagonist (BR3-Fc), anti-CD40L mAb, anti-TWEAK mAb, anti-IL5 Receptor mAb, anti-ganglioside GM2 mAb, anti-FGF8 mAb, anti VEGFR/Flt- 1 mAb, anti-ganglioside GD2 mAb, Actilyse@ (alteplase), Metalyse@ (tenecteplase), CAT-3888 and CAT-8015 (anti-CD22 dsFv-PE38 conjugates), CAT 354 (anti-IL13 mAb), CAT-5001 (anti-mesothelin dsFv-PE38 conjugate), GC-1008 (anti-TGF-p mAb), CAM-3001 (anti-GM-CSF Receptor mAb), ABT-874 (anti-IL12 mAb), Lymphostat B (Belimumab; anti-BlyS mAb), HGS-ETR1 (mapatumumab; human anti-TRAIL Receptor-I mAb), HGS-ETR2 (human anti-TRAIL Receptor-2 mAb), ABthraxTM (human, anti-protective antigen (from B. anthracis) mAb), MYO 029 (human anti-GDF-8 mAb), CAT-213 (anti-eotaxinl mAb), Erbitux (Cetuximab), Epratuzumab, Remicade (infliximab; anti-TNF mAb), Herceptin* (traztusumab), Mylotarg (gemtuzumab ozogamicin), VECTIBIX (panatumamab), ReoPro (abciximab), Actemra (anti-IL6 Receptor mAb), HuMax-CD4 (zanolimumab), HuMax-CD20 (ofatumumab), HuMax-EGFr (zalutumumab), HuMax-Inflam, R1507 (anti-IGF-IR mAb), HuMax HepC, HuMax CD38, HuMax-TAC (anti-IL2Ra or anti CD25 mAb), HuMax-ZP3 (anti-ZP3 mAb), Bexxar (tositumomab), Orthoclone OKT3 (muromonab-CD3), MDX-010 (ipilimumab), anti-CTLA4, CNTO 148 (golimumab; anti-TNFa Inflammation mAb), CNTO 1275 (anti-IL12/IL23 mAb), HuMax-CD4 (zanolimumab), HuMax-CD20 (ofatumumab), HuMax-EGFR (zalutumumab), MDX 066 (CDA-1) and MDX-1388 (anti-C. difficile Toxin A and Toxin B C mAbs), MDX 060 (anti-CD30 mAb), MDX-018, CNTO 95 (anti-integrin receptors mAb), MDX 1307 (anti-Mannose Receptor/hCG3 mAb), MDX- 1100 (anti-IP1O Ulcerative Colitis mAb), MDX-1303 (Valortim

TM

), anti-B. anthracis Anthrax, MEDI-545 (MDX- 1103, anti-IFNa), MDX-1106 (ONO-4538; anti-PD1), NVS Antibody #1, NVS Antibody #2, FG-3019 (anti-CTGF Idiopathic Pulmonary Fibrosis Phase I Fibrogen), LLY Antibody, BMS-66513, NI-0401 (anti-CD3 mAb), IMC-18F1 (VEGFR-1), IMC-3G3 (anti-PDGFRc), MDX-1401 (anti-CD30), MDX-1333 (anti-IFNAR), Synagis (palivizumab; anti-RSV mAb), Campath (alemtuzumab), Velcade (bortezomib), MLN0002 (anti- alpha4beta7 mAb), MLN1202 (anti-CCR2 chemokine receptor WO 2009/026122 PCT/US2008/073250 mAb)., Simulect (basiliximab), prexige (lumiracoxib), Xolair (omalizumab), ETI211 (anti-MRSA mAb), Zenapax (Daclizumab), Avastin (Bevacizumab), MabTheraRA (Rituximab), Tarceva (Erlotinib), Zevalin (ibritumomab tiuxetan), Zetia (ezetimibe), Zyttorin (ezetimibe and simvastatin), NI-0401 (human anti-CD3), Adecatumumab, Golimumab (anti-TNFa mAb), Epratuzumab, gemtuzumab, Raptiva (efalizumab), Cimzia (certolizumab pegol, CDP 870), (Soliris) Eculizumab, Pexelizumab (Anti-C5 Complement), MEDI-524 (Numax), Lucentis (Ranibizumab), 17-1A (Panorex), Trabio (lerdelimumab), TheraCim hR3 (Nimotuzumab), Omnitarg (Pertuzumab), Osidem (IDM-1), OvaRex (B43.13), Nuvion (visilizumab), anti-CD40L mAb (IDEC 131), Xanelim(humanized anti-CD 1 Ia) and Cantuzamab. [0021] In a related aspect, the therapeutic Fc-fusion molecule is selected from the group consisting of IL-I Trap (the Fc portion of human IgG1 and the extracellular domains of both IL-I receptor components (the Type I receptor and receptor accessory protein)), VEGF Trap (Ig domains of VEGFR1 fused to IgGI Fc), Atacicept (TACI-Ig), CTLA4-Ig (abatacept), CD4-Ig fusion protein (Pro-542), TNFR1-IgG, Amevive@ (Alefacept, LFA-3/IgG1), CD30-L-IgG, IL-10-Fc, TNRF Fc, IL-2-Ig, OPG-Fc, and leptin(ObR)-Fc. [0022] The invention also provides compositions comprising a therapeutic antibody or Fc-fusion molecule and a polycation. In one embodiment, the composition is a pharmaceutical composition comprising a therapeutic antibody or Fc-fusion molecule, a polycation and a pharmaceutically acceptable excipient. In one embodiment, the invention provides a pharmaceutical composition comprising a therapeutic antibody having a pI of 7.5 or less and a polycation selected from the group consisting of polylysine and polyarginine. The invention also contemplates a pharmaceutical composition comprising a therapeutic antibody having a CDR domain comprising at least two consecutive acidic amino acid residues and a polycation selected from the group consisting of polylysine and polyarginine. In one embodiment, the acidic amino acids are selected from the group consisting of aspartic acid (D) and glutamic acid (E). [0023] In one aspect, the antibody or Fc-fusion molecule in the composition has an increased shelf-life as compared to a pharmaceutical composition that comprises the antibody in the absence of a polycation. In one embodiment, the antibody has a greater solubility in water than a composition containing the antibody or Fc-fusion molecule in the absence of a polycation.

WO 2009/026122 PCT/US2008/073250 [0024] In a related aspect, the dimerization (or other multimerization) and/or aggregate formation of said antibody or Fc-fusion in the pharmaceutical composition is reduced as compared to a pharmaceutical composition comprising the antibody or Fc-fusion in the absence of a polycation. [0025] In a further embodiment, the pharmaceutical composition comprises a therapeutic antibody selected from the group consisting of Enbrel (Eternacept), Humira (adalimumab), Synagis (palivizumab), AMG 714 (anti-IL15 antibody), vectibix (panitumumab), Rituxan (rituximab), zevalin (ibritumomab tiuxetan), anti CD80 monoclonal antibody (mAb) (galiximab), anti-CD23 mAb (lumiliximab), M200 (volociximab), anti-Cripto mAb, anti-BR3 mAb, anti-IGFIR mAb, Tysabri (natalizumab), Daclizumab, humanized anti-CD20 mAb (ocrelizumab), soluble BAFF antagonist (BR3-Fc), anti-CD40L mAb, anti-TWEAK mAb, anti-IL5 Receptor mAb, anti-ganglioside GM2 mAb, anti-FGF8 mAb, anti-VEGFR/Flt- 1 mAb, anti ganglioside GD2 mAb, Actilyse@ (alteplase), Metalyse@ (tenecteplase), CAT-3888 and CAT-8015 (anti-CD22 dsFv-PE38 conjugates), CAT-354 (anti-IL13 mAb), CAT 5001 (anti-mesothelin dsFv-PE38 conjugate), GC-1008 (anti-TGF-6 mAb), CAM 3001 (anti-GM-CSF Receptor mAb), ABT-874 (anti-IL12 mAb), Lymphostat B (Belimumab; anti-BlyS mAb), HGS-ETR1 (mapatumumab; human anti-TRAIL Receptor-I mAb), HGS-ETR2 (human anti-TRAIL Receptor-2 mAb), ABthraxTM (human, anti-protective antigen (from B. anthracis) mAb), MYO-029 (human anti GDF-8 mAb), CAT-213 (anti-eotaxin1 mAb), Erbitux (Cetuximab), Epratuzumab, Remicade (infliximab; anti-TNF mAb), Herceptin* (traztusumab), Mylotarg (gemtuzumab ozogamicin), VECTIBIX (panatumamab), ReoPro (abciximab), Actemra (anti-IL6 Receptor mAb), HuMax-CD4 (zanolimumab), HuMax-CD20 (ofatumumab), HuMax-EGFr (zalutumumab), HuMax-Inflam, R1507 (anti-IGF-IR mAb), HuMax HepC, HuMax CD38, HuMax-TAC (anti-IL2Ra or anti-CD25 mAb), HuMax-ZP3 (anti-ZP3 mAb), Bexxar (tositumomab), Orthoclone OKT3 (muromonab-CD3), MDX-010 (ipilimumab), anti-CTLA4, CNTO 148 (golimumab; anti-TNFa Inflammation mAb), CNTO 1275 (anti-1L12/1L23 mAb), HuMax-CD4 (zanolimumab), HuMax-CD20 (ofatumumab), HuMax-EGFR (zalutumumab), MDX 066 (CDA-1) and MDX-1388 (anti-C. difficile Toxin A and Toxin B C mAbs), MDX 060 (anti-CD30 mAb), MDX-018, CNTO 95 (anti-integrin receptors mAb), MDX 1307 (anti-Mannose Receptor/hCGO mAb), MDX- 1100 (anti-IP10 Ulcerative Colitis mAb), MDX-1303 (Valortim T M ), anti-B. anthracis Anthrax, MEDI-545 (MDX- 1103, WO 2009/026122 PCT/US2008/073250 anti-IFNa), MDX-1106 (ONO-4538; anti-PD 1), NVS Antibody #1, NVS Antibody #2, FG-3019 (anti-CTGF Idiopathic Pulmonary Fibrosis Phase I Fibrogen), LLY Antibody, BMS-66513, NI-0401 (anti-CD3 mAb), IMC-18F1 (VEGFR-1), IMC-3G3 (anti-PDGFRa), MDX-1401 (anti-CD30), MDX-1333 (anti-IFNAR), Synagis (palivizumab; anti-RSV mAb), Campath (alemtuzumab), Velcade (bortezomib), MLN0002 (anti- alpha4beta7 mAb), MLN1202 (anti-CCR2 chemokine receptor mAb)., Simulect (basiliximab), prexige (lumiracoxib), Xolair (omalizumab), ETI211 (anti-MRSA mAb), Zenapax (Daclizumab), Avastin (Bevacizumab), MabTheraRA (Rituximab), Tarceva (Erlotinib), Zevalin (ibritumomab tiuxetan), Zetia (ezetimibe), Zyttorin (ezetimibe and simvastatin), NI-0401 (human anti-CD3), Adecatumumab, Golimumab (anti-TNFa mAb), Epratuzumab, gemtuzumab, Raptiva (efalizumab), Cimzia (certolizumab pegol, CDP 870), (Soliris) Eculizumab, Pexelizumab (Anti-C5 Complement), MEDI-524 (Numax), Lucentis (Ranibizumab), 17-1A (Panorex), Trabio (lerdelimumab), TheraCim hR3 (Nimotuzumab), Omnitarg (Pertuzumab), Osidem (IDM-1), OvaRex (B43.13), Nuvion (visilizumab), anti-CD40L mAb (IDEC 131), Xanelim(humanized anti-CD I 1a) and Cantuzamab.. In a further embodiment, the pharmaceutical composition comprises a therapeutic Fc-fusion molecule selected from the group consisting of IL-I Trap (the Fc portion of human IgG 1 and the extracellular domains of both IL-I receptor components (the Type I receptor and receptor accessory protein)), VEGF Trap (Ig domains of VEGFR1 fused to IgGI Fc), Atacicept (TACI-Ig), CTLA4-Ig (abatacept), CD4-Ig fusion protein (Pro-542), TNFR I -IgG, Amevive@ (Alefacept, LFA-3/IgG 1), CD30-L-IgG, IL- 10-Fe, TNRF Fe, IL-2-Ig, OPG-Fc, and leptin(ObR)-Fc. [0026] In a related embodiment, the present invention can be used in formulating anti-angiopoietins (e.g., anti-Ang2 and/or anti-Ang I) specific antibodies, peptibodies, and related molecules, and the like, including but not limited to those described in International Publication Number WO 03/057134, U.S. Application Publication Number US2003/0229023, and in Oliner et al (Cancer Cell, 507-516, 2004) and supplemental materials, each of which is incorporated herein by reference in its entirety particularly in parts pertinent to Ang2 specific antibodies and peptibodies and the like, especially those of sequences described therein and including but not limited to: L1(N); LI(N) WT; L1(N) 1K WT; 2xL1(N); 2xL1(N) WT; Con4 (N), Con4 (N) 1K WT, 2xCon4 (N) 1K; LIC; LIC 1K; 2xL1C; Con4C; Con4C 1K; 2xCon4C 1K; Con4-L1 (N); Con4-L1C; TN-12-9 (N); C17 (N); TN8-8(N); TN8-14 (N); Con I (N), WO 2009/026122 PCT/US2008/073250 also including anti-Ang 2 antibodies and formulations such as those described in International Publication Number WO 2003/030833 which is incorporated herein by reference in its entirety as to the same, particularly Ab526; Ab528; Ab53 1; Ab533; Ab535; Ab536; Ab537; Ab540; Ab543; Ab544; Ab545; Ab546; A551; Ab553; Ab555; Ab558; Ab559; Ab565; AbF1AbFD; AbFE; AbFJ; AbFK; AbG1D4; AbGC1E8; AbH1C12; AblA1; AblF; AbIK, AbiP; and AbIP, in their various permutations as described therein, each of which is individually and specifically incorporated by reference herein in its entirety fully as disclosed in the foregoing publication. [0027] It is contemplated that for any of the methods of the invention, any of the polycations set out herein may be used in an antibody or Fc-fusion formulation or a polycation composition of the invention. [0028] Brief Description Of The Drawings [00291 Figure 1 shows the relative abundance of aggregates and dimers (or other multimers) formed in a solution comprising polycations and an antibody with consecutive DDD residues after 6 weeks of storage at 52' C. [0030] Detailed Description [0031] The present invention relates to methods for making and compositions comprising liquid or lyophilized pharmaceutical formulations comprising an antibody or Fc-fusion molecule and a polycation, including therapeutic antibody and Fc-fusion formulations. [0032] The term "antibody formulation" as used herein refers to a combination of an antibody and a polycation, with one or more other ingredients for one or more particular uses, such as storage, further processing, sale, and/or administration to a subject, such as, for example, administration to a subject of a specific agent in a specific amount, by a specific route, to treat a specific disease. [0033] The term "Fc-fusion formulation" or "Fc-fusion molecule formulation" as used herein refers to a combination of an Fc-fusion molecule and a polycation, with one or more other ingredients for one or more particular uses, such as storage, further processing, sale, and/or administration to a subject, such as, for example, administration to a subject of a specific agent in a specific amount, by a specific route, to treat a specific disease. C0 WO 2009/026122 PCT/US2008/073250 [0034] The term "polycation" as used herein, refers to a molecule comprising one or more of repeating units of one or more cationic monomers. In various aspects, a polycation comprises repeating units of one or more monomers, or repeating subunits variously comprising one or more cationic monomers linked to one or more neutral monomers, wherein the monomers are naturally-occurring and/or synthetic compounds, covalently linked to provide a compound with repeating structural characteristics having an overall positive charge. "Polycation composition" refers to a composition comprising one or more polycations. [0035] As used herein, a "cation" or "cationic monomer" refers to a single or monomeric unit which may be used to generate a polycation set out herein. As used herein, a "neutral" monomer refers to a monomer having no polarity or charge, such as a single amino acid residue having a nonpolar sidechain, which may be used in combination with a cation to form a polycation contemplated by the invention. [0036] It is further contemplated that the polycations are homopolymers or co polymers, or mixtures thereof. A polycation homopolymer comprises a single repeating unit of the same cation monomer. A polycation co-polymer comprises different cation monomers or different cationic polymers. In one embociment, a polycation co-polymer may comprise a mixture of cation monomers, a mixture of polycation homopolymers or a mixture of polycation co-polymers. [0037] As used herein, the term "antibody" refers to fully assembled antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments that can bind antigen ( e.g., Fab', F'(ab) 2 , Fv, single chain antibodies, diabodies), and recombinant peptides comprising the forgoing as long as they exhibit the desired biological activity. Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antibody fragments or antigen-binding portions include, inter alia, Fab, Fab', F(ab') 2 , Fv, domain antibody (dAb), complementarity determining region (CDR) fragments, single-chain antibodies (scFv), single chain antibody fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, minibody, linear antibody; chelating recombinant antibody, a tribody or bibody, an intrabody, a nanobody, a small modular immunopharmaceutical (SMIP), a antigen-binding-domain immunoglobulin fusion protein, a camelized antibody, a VHH containing antibody, or a variant or a derivative thereof, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen WO 2009/026122 PCT/US2008/073250 binding to the polypeptide, such as a CDR sequence, as long as the antibody retains the desired biological activity. [0038] Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes, IgA, IgD, IgE, IgG and IgM, which may be further divided into subclasses or isotypes, e.g. IgGI, IgG2, IgG3, IgG4, IgAl and IgA2. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Different isotypes have different effector functions; for example, IgG1 and IgG3 isotypes have antibody dependent cell cytotoxicity (ADCC) activity. An antibody of the invention, if it comprises a constant domain, may be of any of these subclasses or isotypes. [0039] A "therapeutic antibody" as used herein refers to an antibody that is used for the treatment of a disease or disorder. [0040] An "antibody having a CDR domain comprising at least two consecutive acidic amino acid residues" as used herein refers to an antibody having at least one CDR amino acid sequence having at least two consecutive, sequential acidic amino acid residues, such as aspartic acid (D) or glutamic acid (E). [0041] An "Fc-fusion" or "Fc-fusion molecule" as used herein refers to a molecule comprising proteins or peptide(s) fused either directly or indirectly to other molecules such as an Fc domain of an antibody, where the protein or peptide moiety specifically binds to a desired target. A "peptibody" as used herein refers to a molecule comprising peptide(s) fused either directly or indirectly to other molecules such as an Fc domain of an antibody. The term "peptibody" does not include Fc-fusion proteins (e.g., full length proteins fused to an Fc domain). The invention includes such molecules comprising an Fc domain modified to comprise a peptide as an internal sequence (preferably in a loop region) of the Fc domain. The Fc internal peptide molecules may include more than one peptide sequence in tandem in a particular internal region, and they may include further peptides in other internal regions. [0042] The term "improving the solubility" as used herein refers to a solution, antibody formulation or Fc-fusion formulation comprising a polycation composition, which comprises fewer aggregates, dimers or multimers, or results in less precipitate compared to another solution, antibody or Fc-fusion formulation lacking the polycation composition.

WO 2009/026122 PCT/US2008/073250 [0043] The term "increased shelf-life" as used herein refers to an increase in the length of time that a composition of the invention comprising a polycation can be stored at proper conditions without degradation of the antibody or Fc-fusion molecule into aggregates, dimers or other multimers, or precipitates, and without significant loss in biological activity, as compared to a composition lacking the polycation composition. [0044] Antibodies [0045] Monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., (Nature, 256:495-7, 1975), or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). The monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., (Nature 352:624-628, 1991) and Marks et al., (J. Mol. Biol. 222:581-597, 1991). [0046] It is further contemplated that antibodies useful in the invention may be used as smaller antigen binding fragments of the antibody well-known in the art and described herein. [0047] Antibody fragments comprise a portion of an intact full length antibody, preferably an antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); multispecific antibody fragments such as bispecfic, trispecific, etc. antibodies (e.g., diabodies, triabodies, tetrabodies); minibody; chelating recombinant antibody; tribodies or bibodies; intrabodies; nanobodies; small modular immunopharmaceuticals (SMIP), binding domain immunoglobulin fusion proteins; camelized antibodies; VHH containing antibodies; and other polypeptides formed from antibody fragments. [0048] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, monovalent fragments consisting of the VL, VH, CL and CH domains each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields a F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, that has two "single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide WO 2009/026122 PCT/US2008/073250 further comprises a polypeptide linker between the VH and VL domains that enables the Fv to form the desired structure for antigen binding, resulting in a single-chain antibody (scFv), in which a VL and V 1 region are paired to form a monovalent molecule via a synthetic linker that enables them to be made as a single protein chain (Bird et al., Science 242:423-426, 1988, and Huston et al., Proc. NatL. Acad. Sci. USA 85:5879-5883, 1988). For a review of sFv see Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 1 13, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). An Fd fragment consists of the VH and CHI domains. [0049] A chimeric antibody refers to an antibody containing sequence derived from two different antibodies (see, e.g., U.S. Patent No. 4,816,567) which typically originate from different species. Most typically, chimeric antibodies comprise human and rodent antibody fragments, generally human constant and mouse variable regions. [0050] Additional antibody fragment include a domain antibody (dAb) fragment (Ward et al., Nature 341:544-546, 1989) which consists of a VH domain. Diabodies are bivalent antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., EP 404,097; WO 93/11161; Holliger et al., Proc. Nati. Acad. Sci. USA 90:6444 6448, 1993, and Poijak et al., Structure 2:1121-1123, 1994). Diabodies can be bispecific or monospecific. [00511 Functional heavy-chain antibodies devoid of light chains are naturally occurring in nurse sharks (Greenberg et al., Nature 374:168-73, 1995), wobbegong sharks (Nuttall et al., Mol Immunol. 38:313-26, 2001) and Camelidae (Hamers Casterman et al., Nature 363: 446-8, 1993; Nguyen et al., J. Mol. Biol. 275: 413, 1998), such as camels, dromedaries, alpacas and llamas. The antigen-binding site is reduced to a single domain, the VHH domain, in these animals. Methods for generating antibodies having camelid heavy chains are described in, for example, in U.S. Patent Publication Nos. 2005/0136049 and 2005/0037421. [0052] Because the variable domain of the heavy-chain antibodies is the smallest fully functional antigen-binding fragment with a molecular mass of only 15 kDa, this entity is referred to as a nanobody (Cortez-Retamozo et al., Cancer Research 64:2853-57, 2004). A nanobody library may be generated from an immunized 1 I WO 2009/026122 PCT/US2008/073250 dromedary as described in Conrath et al., (Antimicrob Agents Chemother 45: 2807-12, 2001) or using recombinant methods as described in Holliger et al. (Nat Biotechnol. 23:1126-36, 2005) and Rahbarizadeh et al. (Hybrid Hybridomics. 23:151-9, 2004) [0053] In a further embodiment, the bispecific antibody may be a chelating recombinant antibody (CRAb). A chelating recombinant antibody recognizes adjacent and non-overlapping epitopes of the target antigen, and is flexible enough to bind to both epitopes simultaneously (Neri et al., J Mol Biol. 246:367-73, 1995). [0054] Production of bispecific Fab-scFv ("bibody") and trispecific Fab-(scFv)(2) ("tribody") are described in Schoonjans et al. (J Immunol. 165:7050-57, 2000) and Willems et al. (J Chromatogr B Analyt Technol Bioned Life Sci. 786:161-76, 2003). For bibodies or tribodies, a scFv molecule is fused to one or both of the VL-CL (L) and VH-CHi (Fd) chains, e.g., to produce a tribody two scFvs are fused to C-term of Fab while in a bibody one scFv is fused to C-term of Fab. [0055] A "minibody" consisting of scFv fused to CH3 via a peptide linker (hingeless) or via an IgG hinge has been described in Olafsen, et al., Protein Eng Des Sel. 2004 Apr;17(4):315-23. [00561 Intrabodies are single chain antibodies which demonstrate intracellular expression and can manipulate intracellular protein function (Biocca, et al., EMBO J. 9:101-108, 1990; Colby et al., Proc Natl Acad Sci U S A. 101:17616-21, 2004). Intrabodies, which comprise cell signal sequences which retain the antibody construct in intracellular regions, may be produced as described in Mhashilkar et al (EMBO J 14:1542-51, 1995) and Wheeler et al. (FASEB J. 17:1733-5. 2003). Transbodies are cell-permeable antibodies in which a protein transduction domain (PTD) is fused with single chain variable fragment (scFv) antibodies ( Heng et al., Med Hypotheses. 64:1105-8, 2005). [0057] Further contemplated are antibodies that are SMIPs or binding domain immunoglobulin fusion proteins specific for target protein. These constructs are single-chain polypeptides comprising antigen binding domains fused to immunoglobulin domains necessary to carry out antibody effector functions. See, for example, WO03/041600, U.S. Patent publication 2003/0133939 and U.S. Patent Publication 2003/0118592. [00581 One or more CDRs may be incorporated into a molecule either covalently or noncovalently to make it an immunoadhesin. An immunoadhesin may incorporate the WO 2009/026122 PCT/US2008/073250 CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) noncovalently. The CDRs permit the immunoadhesin to specifically bind to a particular antigen of interest. [00591 In one aspect, the antibody in the antibody formulation is a therapeutic antibody. Exemplary therapeutic antibodies include, but are not limited to, Enbrel (Eternacept), Humira (adalimumab), Synagis (palivizumab),AMG 714 (anti-IL15 antibody), vectibix (panitumumab), Rituxan (rituximab), zevalin (ibritumomab tiuxetan), anti-CD80 monoclonal antibody (mAb) (galiximab), anti-CD23 mAb (lumiliximab), M200 (volociximab), anti-Cripto mAb, anti-BR3 mAb, anti-IGF1R mAb, Tysabri (natalizumab), Daclizumab, humanized anti-CD20 mAb (ocrelizumab), soluble BAFF antagonist (BR3-Fc), anti-CD40L mAb, anti-TWEAK mAb, anti-IL5 Receptor mAb, anti-ganglioside GM2 mAb, anti-FGF8 mAb, anti-VEGFR/Flt- 1 mAb, anti-ganglioside GD2 mAb, Actilyse@ (alteplase), Metalyse@ (tenecteplase), CAT-3888 and CAT-8015 (anti-CD22 dsFv-PE38 conjugates), CAT-354 (anti-IL13 mAb), CAT-5001 (anti-mesothelin dsFv-PE38 conjugate), GC-1008 (anti-TGF-p mAb), CAM-3001 (anti-GM-CSF Receptor mAb), ABT-874 (anti-IL12 mAb), Lymphostat B (Belimumab; anti-BlyS mAb), HGS-ETR1 (mapatumumab; human anti-TRAIL Receptor- 1 mAb), HGS-ETR2 (human anti-TRAIL Receptor-2 mAb), ABthraxTM (human, anti-protective antigen (from B. anthracis) mAb), MYO-029 (human anti-GDF-8 mAb), CAT-213 (anti-eotaxin1 mAb), Erbitux (Cetuximab), Epratuzumab, Remicade (infliximab; anti-TNF mAb), Herceptin* (traztusumab), Mylotarg (gemtuzumab ozogamicin), VECTIBIX (panatumamab), ReoPro (abciximab), Actemra (anti-IL6 Receptor mAb), HuMax-CD4 (zanolimumab), HuMax-CD20 (ofatumumab), HuMax-EGFr (zalutumumab), HuMax-Inflam, R1507 (anti-IGF- I R mAb), HuMax HepC, HuMax CD38, HuMax-TAC (anti-IL2Ra or anti CD25 mAb), HuMax-ZP3 (anti-ZP3 mAb), Bexxar (tositumomab), Orthoclone OKT3 (muromonab-CD3), MDX-010 (ipilimumab), anti-CTLA4, CNTO 148 (golimumab; anti-TNFa Inflammation mAb), CNTO 1275 (anti-IL12/IL23 mAb), HuMax-CD4 (zanolimumab), HuMax-CD20 (ofatumumab), HuMax-EGFR (zalutumumab), MDX 066 (CDA-1) and MDX-1388 (anti-C. difficile Toxin A and Toxin B C mAbs), MDX 060 (anti-CD30 mAb), MDX-018, CNTO 95 (anti-integrin receptors mAb), MDX 1307 (anti-Mannose Receptor/hCGp mAb), MDX- 1100 (anti-IP1O Ulcerative Colitis mAb), MDX-1303 (Valortim

TM

), anti-B. anthracis Anthrax, MEDI-545 (MDX- 1103, anti-IFNa), MDX- 1106 (ONO-4538; anti-PD1), NVS Antibody #1, NVS Antibody WO 2009/026122 PCT/US2008/073250 #2, FG-3019 (anti-CTGF Idiopathic Pulmonary Fibrosis Phase I Fibrogen), LLY Antibody, BMS-66513, NI-0401 (anti-CD3 mAb), IMC-18F1 (VEGFR-1), IMC-3G3 (anti-PDGFRa), MDX-1401 (anti-CD30), MDX-1333 (anti-IFNAR), Synagis (palivizumab; anti-RSV mAb), Campath (alemtuzumab), Velcade (bortezomib), MLN0002 (anti- alpha4beta7 mAb), MLN1202 (anti-CCR2 chemokine receptor mAb)., Simulect (basiliximab), prexige (lumiracoxib), Xolair (omalizumab), ETI211 (anti-MRSA mAb), Zenapax (Daclizumab), Avastin (Bevacizumab), MabTheraRA (Rituximab), Tarceva (Erlotinib), Zevalin (ibritumomab tiuxetan), Zetia (ezetimibe), Zyttorin (ezetimibe and simvastatin), NI-0401 (human anti-CD3), Adecatumumab, Golimumab (anti-TNFa mAb), Epratuzumab, gemtuzumab, Raptiva (efalizumab), Cimzia (certolizumab pegol, CDP 870), (Soliris) Eculizumab, Pexelizumab (Anti-C5 Complement), MEDI-524 (Numax), Lucentis (Ranibizumab), 17-IA (Panorex), Trabio (lerdelimumab), TheraCim hR3 (Nimotuzumab), Omnitarg (Pertuzumab), Osidem (IDM-1), OvaRex (B43.13), Nuvion (visilizumab), anti-CD40L mAb (IDEC 131), Xanelim(humanized anti-CD 11 a) and Cantuzamab. [0060] Fc-Fusion Molecules [0061] Also contemplated for use in theinvention are Fe-fusion molecules comprising proteins or peptide(s) fused either directly or indirectly to other molecules such as an Fe domain of an antibody. Fc-fusion molecules includes Fc-fusion proteins which comprise full length proteins fused to an Fc-domain, and peptibodies, which refers to a molecule comprising peptide(s) fused either directly or indirectly to other molecules such as an Fe domain of an antibody, where the peptide moiety specifically binds to a desired target. [0062] The term "peptibody" does not include Fc-fusion proteins (e.g., full length proteins fused to an Fe domain). As used herein the term "peptide" refers to molecules of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 ,14 ,15, 16 ,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 ,39, 40, 41, 42, 43, 44, 45, 46, 47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69, 70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92, 93, 94, 95, 96, 97, 98, 99, 100 or more amino acids linked by peptide bonds. Exemplary peptides may be generated by any of the methods known in the art, such as carried in a peptide library (e.g., a phage display library), generated by chemical synthesis, derived by digestion of proteins, or generated using recombinant DNA techniques. Peptides include D and L amino acid forms, either purified or in a WO 2009/026122 PCT/US2008/073250 mixture of the two forms. The production of peptibodies is generally described in PCT publication WO 00/24782, U.S. Patent 7,138,370, U.S. patent 6,660,843 B1 and US patent publication US2004/0044188. [00631 The protein or peptide(s) may be fused to either an Fc region or inserted into an Fc-Loop, a modified Fe molecule. Fc-Loops are described in U.S. Patent Application Publication No. US2006/0140934 incorporated herein by reference in its entirety. The invention includes such molecules comprising an Fe domain modified to comprise a peptide as an internal sequence (preferably in a loop region) of the Fe domain. The Fc internal peptide molecules may include more than one peptide sequence in tandem in a particular internal region, and they may include further peptides in other internal regions. [0064] The invention contemplates the presence of at least one Fe domain attached to a protein or peptide fused to the N or C termini of the protein or peptides, or at both the N and C termini. [0065] In various embodiments of the invention, the Fc component is either a native Fe or an Fc variant. "Fe domain" encompasses native Fe and Fc variant molecules and sequences as defined below. [0066] "Native Fc" refers to molecule or sequence comprising the sequence of a non-antigen-binding fragment resulting from digestion of whole antibody, whether in monomeric or multimeric form. Typically, a native Fc comprises a CH2 and CH3 domain. The immunoglobulin source of the native Fe is, in one aspect, of human origin and may, in alternative embodiments, be of any class of immunoglobulin. Native Fe domains are made up of monomeric polypeptides that may be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and/or non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from one to four depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgGI, IgG2, IgG3, IgA1, IgGA2). One example of a native Fe is a disulfide-bonded dimer resulting from papain digestion of an IgG (see Ellison et al. (1982), Nucleic Acids Res. 10: 4071-9). [0067] "Fe variant" refers to a molecule or sequence that is modified from a native Fe, but still comprises a binding site for the salvage receptor, FcRn. International applications WO 97/34631 (published 25 September 1997) and WO 96/32478 describe exemplary Fc variants, as well as interaction with the salvage receptor, and WO 2009/026122 PCT/US2008/073250 are hereby incorporated by reference. In one aspect, the term "Fe variant" comprises a molecule or sequence that is humanized from a non-human native Fe. In another aspect, a native Fe comprises sites that may be removed because they provide structural features or biological activity that are not required for the fusion molecules of the present invention. Thus, the term "Fe variant" comprises a molecule or sequence that lacks one or more native Fc sites or residues that affect or are involved in (1) disulfide bond formation, (2) incompatibility with a selected host cell (3) N terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC). Fe variants are described in further detail hereinafter. [0068] As with Fe variants and native Fes, the term "Fe domain" includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by other means. Fc sequences are known in the art and are contemplated for use in the invention. For example, Fe IgGI (GenBank Accession No. P01857), Fe IgG2 (GenBank Accession No. P01859), Fe IgG3 (GenBank Accession No. P01860), Fe IgG4 (GenBank Accession No. P01861), Fe IgAl (GenBank Accession No. P01876), Fc IgA2 (GenBank Accession No. P01877), Fe IgD (GenBank Accession No. P01880), Fe IgM (GenBank Accession No. P01871), and Fc IgE (GenBank Accession No. P01854) are some additional Fc sequences contemplated for use herein. [0069] It should be noted that Fe monomers will spontaneously dimerize when the appropriate cysteine residues are present, unless particular conditions are present that prevent dimerization through disulfide bond formation. Even if the cysteine residues that normally form disulfide bonds in the Fe dimer are removed or replaced by other residues, the monomeric chains will generally form a dimer through non-covalent interactions. The term "Fc" herein is used to mean any of these forms: the native monomer, the native dimer (disulfide bond linked), modified dimers (disulfide and/or non-covalently linked), and modified monomers (i.e., derivatives). [0070] As noted, Fe variants are suitable vehicles within the scope of this invention. A native Fc may be extensively modified to form an Fe variant, provided binding to the salvage receptor is maintained; see, for example WO 97/34631 and WO 96/32478. In such Fe variants, one may remove one or more sites of a native Fe that provide structural features or functional activity not required by the fusion molecules WO 2009/026122 PCT/US2008/073250 of this invention. One may remove these sites by, for example, substituting or deleting residues, inserting residues into the site, or truncating portions containing the site. The inserted or substituted residues may also be altered amino acids, such as peptidomimetics or D-amino acids. Fc variants may be desirable for a number of reasons, several of which are described herein. Exemplary Fc variants include molecules and sequences in which: [0071] 1. Sites involved in disulfide bond formation are removed. Such removal may avoid reaction with other cysteine-containing proteins present in the host cell used to produce the molecules of the invention. For this purpose, the cysteine-containing segment at the N-terminus may be truncated or cysteine residues may be deleted or substituted with other amino acids (e.g., alanyl, seryl). Even when cysteine residues are removed, the single chain Fc domains can still form a dimeric Fe domain that is held together non-covalently. [00721 2. A native Fc is modified to make it more compatible with a selected host cell. For example, one may remove the PA sequence near the N-terminus of a typical native Fe, which may be recognized by a digestive enzyme in E. coli such as proline iminopeptidase. One may also add an N-terminal methionine residue, especially when the molecule is expressed recombinantly in a bacterial cell such as E. coli. [00731 3. A portion of the N-terminus of a native Fe is removed to prevent N terminal heterogeneity when expressed in a selected host cell. For this purpose, one may delete any of the first 20 amino acid residues at the N-terminus, particularly those at positions 1, 2, 3, 4 and 5. [0074] 4. One or more glycosylation sites are removed. Residues that are typically glycosylated (e.g., asparagine) may confer cytolytic response. Such residues may be deleted or substituted with unglycosylated residues (e.g., alanine). [00751 5. Sites involved in interaction with complement, such as the Clq binding site, are removed. For example, one may delete or substitute the EKK sequence of human IgG1. Complement recruitment may not be advantageous for the molecules of this invention and so may be avoided with such an Fe variant. [00761 6. Sites are removed that affect binding to Fc receptors other than a salvage receptor. A native Fc may have sites for interaction with certain white blood WO 2009/026122 PCT/US2008/073250 cells that are not required for the fusion molecules of the present invention and so may be removed. [00771 7. The ADCC site is removed. ADCC sites are known in the art; see, for example, Molec. Immunol. 29 (5): 633-9 (1992) with regard to ADCC sites in IgG 1. These sites, as well, are not required for the fusion molecules of the present invention and so may be removed. [00781 8. When the native Fc is derived from a non-human antibody, the native Fc may be humanized. Typically, to humanize a native Fc, one will substitute selected residues in the non-human native Fc with residues that are normally found in human native Fe. Techniques for antibody humanization are well known in the art. [0079] Variants, analogs or derivatives of the Fe portion may be constructed by, for example, making various substitutions of residues or sequences. Variant (or analog) polypeptides include insertion variants, wherein one or more amino acid residues supplement an Fc amino acid sequence. Insertions may be located at either or both termini of the protein, or may be positioned within internal regions of the Fc amino acid sequence. Insertion variants, with additional residues at either or both termini, can include for example, fusion proteins and proteins including amino acid tags or labels. For example, the Fc molecule may optionally contain an N-terminal Met, especially when the molecule is expressed recombinantly in a bacterial cell such as E. coli. [0080] In Fc deletion variants, one or more amino acid residues in an Fc polypeptide are removed. Deletions can be effected at one or both termini of the Fc polypeptide, or with removal of one or more residues within the Fc amino acid sequence. Deletion variants, therefore, include all fragments of an Fe polypeptide sequence. [0081] In Fe substitution variants, one or more amino acid residues of an Fc polypeptide are removed and replaced with alternative residues. In one aspect, the substitutions are conservative in nature and conservative substitutions of this type are well known in the art. Alternatively, the invention embraces substitutions that are also non-conservative. Exemplary conservative substitutions are described in Lehninger, [Biochemistry, 2nd Edition; Worth Publishers, Inc.New York (1975), pp.71-771 and set out immediately below. [00821 Conservative Substitutions I WO 2009/026122 PCT/US2008/073250 SIDE CHAIN AMINO ACID CHARACTERISTIC Non-polar (hydrophobic): A. Aliphatic ALIVP B. Aromatic F W C. Sulfur-containing M D. Borderline G Uncharged-polar: A. Hydroxyl S T Y B. Amides NQ C. Sulfhydryl C D. Borderline G Positively charged (basic) K R H Negatively charged (acidic) D E [0083] Alternative, exemplary conservative substitutions are set out immediately below. [0084] Conservative Substitutions II ORIGINAL RESIDUE EXEMPLARY SUBSTITUTION Ala (A) Val, Leu, Ile Arg (R) Lys, Gin, Asn Asn (N) Gin, His, Lys, Arg Asp (D) Giu Cys (C) Ser Gln (Q) Asn Glu (E) Asp His (H) Asn, Gin, Lys, Arg WO 2009/026122 PCT/US2008/073250 Ile (I) Leu, Val, Met, Ala, Phe, Leu (L) Ile, Val, Met, Ala, Phe Lys (K) Arg, Gln, Asn Met (M) Leu, Phe, Ile Phe (F) Leu, Val, Ile, Ala Pro (P) Gly Ser(S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp, Phe, Thr, Ser Val (V) Ile, Leu, Met, Phe, Ala [0085] For example, cysteine residues can be deleted or replaced with other amino acids to prevent formation of some or all disulfide crosslinks of the Fc sequences. Each cysteine residue can be removed and/or substituted with other amino acids, such as Ala or Ser. As another example, modifications may also be made to introduce amino acid substitutions to (1) ablate the Fc receptor binding site; (2) ablate the complement (Clq) binding site; and/or to (3) ablate the antibody dependent cell mediated cytotoxicity (ADCC) site. Such sites are known in the art, and any known substitutions are within the scope of Fe as used herein. For example, see Molecular Immunology, Vol. 29, No. 5, 633-639 (1992) with regard to ADCC sites in IgG1. [00861 Likewise, one or more tyrosine residues can be replaced by phenylalanine residues. In addition, other variant amino acid insertions, deletions and/or substitutions are also contemplated and are within the scope of the present invention. Conservative amino acid substitutions will generally be preferred. Furthermore, alterations may be in the form of altered amino acids, such as peptidomimetics or D amino acids. [0087] Fc sequences of the compound may also be derivatized as described herein for peptides, i.e., bearing modifications other than insertion, deletion, or substitution of amino acid residues. Preferably, the modifications are covalent in nature, and include for example, chemical bonding with polymers, lipids, other organic, and WO 2009/026122 PCT/US2008/073250 inorganic moieties. Derivatives of the invention may be prepared to increase circulating half-life, or may be designed to improve targeting capacity for the polypeptide to desired cells, tissues, or organs. [0088] It is also possible to use the salvage receptor binding domain of the intact Fc molecule as the Fc part of a compound of the invention, such as described in WO 96/32478, entitled "Altered Polypeptides with Increased Half-Life." Additional members of the class of molecules designated as Fc herein are those that are described in WO 97/34631, entitled "Immunoglobulin-Like Domains with Increased Half Lives." Both of the published PCT applications cited in this paragraph are hereby incorporated by reference. [00891 In one aspect, the Fc-fusion molecule is in the antibody formulation is a therapeutic FC-molecule. Exemplary therapeutic Fc-fusion molecules include, but are not limited to, IL- 1 Trap (the Fc portion of human IgG 1 and the extracellular domains of both IL-I receptor components (the Type I receptor and receptor accessory protein)), VEGF Trap (Ig domains of VEGFR1 fused to IgG1 Fc), Atacicept (TACI Ig), CTLA4-Ig (abatacept), CD4-Ig fusion protein (Pro-542), TNFRI-IgG, Amevive@ (Alefacept, LFA-3/IgGI), CD30-L-IgG, IL-10-Fe, TNRF-Fc, IL-2-Ig, OPG-Fe, and leptin(ObR)-Fc. [0090] It is further contemplated that the present invention can be used in formulating anti-angiopoietins (e.g., anti-Ang2 and/or anti-Angl) specific antibodies, peptibodies, and related molecules, and the like, including but not limited to those described in International Publication Number WO 03/057134 and U.S. Application Publication Number US2003/0229023, each of which is incorporated herein by reference in its entirety particularly in parts pertinent to Ang2 specific antibodies and peptibodies and the like, especially those of sequences described therein and including but not limited to: L1(N); LI(N) WT; L1(N) 1K WT; 2xL1(N); 2xL1(N) WT; Con4 (N), Con4 (N) 1K WT, 2xCon4 (N) 1K; LIC; LIC 1K; 2xL1C; Con4C; Con4C 1K; 2xCon4C 1K; Con4-L1 (N); Con4-L1C; TN-12-9 (N); C17 (N); TN8-8(N); TN8-14 (N); Con I (N), also including anti-Ang 2 antibodies and formulations such as those described in International Publication Number WO 2003/030833 which is incorporated herein by reference in its entirety as to the same, particularly Ab526; Ab528; Ab531; Ab533; Ab535; Ab536; Ab537; Ab540; Ab543; Ab544; Ab545; Ab546; A551; Ab553; Ab555; Ab558; Ab559; Ab565; AbF1AbFD; AbFE; AbFJ; AbFK; AbGID4; AbGCIE8; AbH1C12; AblA1; AblF; AblK, AblP; and AblP, in WO 2009/026122 PCT/US2008/073250 their various permutations as described therein, each of which is individually and specifically incorporated by reference herein in its entirety fully as disclosed in the foregoing publication. [0091] Polycations [0092] Polycations useful in the present invention include positively charged peptides and proteins, both naturally occurring and synthetic, as well as polyamines, carbohydrates or synthetic polycationic polymers. Exemplary polycations useful in the present invention include, but are not limited to, polylysine, polyarginine, polyornithine, polyhistidine, and cationic polysaccharides. Additional polycations useful in the invention include protamine, POLYBRENE@ (1,5-dimethyl- 1,5 diazaundecamethylene polymethobromide, hexadimethrine bromide), histone, myelin basic protein, polymyxin B sulfate, dodecyltrimethylammonium bromide, bradykinin, spermine, putrescine, cadaverine, octylarginine, cationic dendrimer, and synthetic peptides. For certain applications, polycationic carriers may include cationic lipid as well as peptide moieties. See, for example, WO 96/22765. [0093] In a related embodiment, the polycation may comprise a mixture of positively charged monomers, including, but not limited to, lysine, arginine, ornithine histidine, and other cationic monomers set out herein, or a mixture of charged and non-charged monomers. Exemplary monomers include basic amino acids, repeating sequences of a cationic monomer linked to a neutral monomer, or other protein molecules or chemical structures having repeating structural characteristics. Non charged monomers contemplated for use in the invention include neutral amino acids, such as glycine, alanine, valine, leucine, isoleucine, serine, threonine, or proline. [0094] In an additional embodiment, the polycation may comprise a mixture of monomers of any one of the positively charged monomers selected from the group consisting of lysine, arginine, ornithine, histidine, and cationic polysaccharides. [0095] Some polycations useful in the methods and compositions of the invention are proteins or fragments thereof which comprise highly basic regions or amino acids giving the protein an overall positive charge. It is contemplated that the entire full length basic protein may be used in the polycation compositions, or that a portion of the protein having basic amino acid residues may be used as a cationic monomer in the polycation. For example, human protamine (Genbank Accession No. NP_002753) is an arginine rich protein of 102 amino acids. It is contemplated that a WO 2009/026122 PCT/US2008/073250 highly basic portion of the human protamine protein may be used as a cationic monomer as described herein. The protamine monomer could then be used as repeating units in a polycation composition. [0096] Additionally, myelin basic protein (MBP) is a highly basic protein found in the central nervous system (Eylar et al., J Biol Chem. 18:5770-84, 1971). It is contemplated that the entire full-length basic protein may be used in the polycation compositions, or that a highly basic portion of any of the known isoforms of the human myelin basic protein (Genbank Accession No. P02686, NP_001020252, NP_001020263, NP_001020261, and NP_002376) may be used as a cationic monomer as described herein. The MBP monomer may also be used as a repeating unit in a polycation polymer composition. [0097] The cyclic moiety of Polymyxin B (PMB) (Thr-Dab-cyclo[Dab-Dab-d-Phe Leu-Dab-Dab-Thr], where Dab is 2,4-diaminobutyric acid), may be used in a single cationic monomer in a polycation composition of the invention, or can be linearized and used to generate polymers having multiple monomers of the PMB moiety. Polymyxin B heptapeptides, nonapeptides and octapeptides know in the art (J. Antibiot. (Tokyo) 45:742-749 and Tsubery et al., Antimicrob Agents Chemother 49:3122-3128, 2005), also may be used as monomers or linearized and used as repeating units for generating a polycation composition of the invention. [00981 Bradykinin is a nine amino acid peptide chain having the amino acid sequence RPPGFSPFR. The nine amino acid sequence may be used in a polycation composition of the invention, or as an alternative the 9 amino acid sequence comprises a cationic monomer which is used to form a polymer having repeating units of the bradykinin monomer. [0099] Spermine, putrescine, and cadaverine are cationic derivatives of basic amino acids contemplated in the formulations of the invention. Spermine and putrecine are derivatives of arginine. Putrescine is generated by reaction of arginine with arginine decarboxylase and admatine imino hydroxilase. Alternatively, arginine can first be converted to ornithine which is converted to putrescine. Spermine results from reaction of putrescine with an aminopropylic group from decarboxylated S-adenosyl L-methionine to give spermidine, which then is converted to spermine

[NH

2

(CH

2

)

3

NH(CH

2

)

4

NH(CH

2

)

3

NH

2 ] after addition of another aminopropylic group. Cadaverine is a derivative of lysine, generated after reaction of lysine with lysine WO 2009/026122 PCT/US2008/073250 decarboxylase. It is contemplated that a polycation composition useful in the present invention comprises one or more monomers of spermine, putrescine, or cadaverine, and may further comprise a mixture of these monomers. [00100] Cationic polysaccharides are polysaccharides conjugated or otherwise linked to a cationic moiety, such as a cationic monomer set out herein. Cationic polysaccharides, including but not limited to, chitosan, are contemplated for use in the compositions and methods of the invention. Cationic polysaccharides may be generated using techniques known in the art. See, for example, Constantin et al., Drug Deliv. 10:139-49, 2003; U.S. Patent 6,958,325, and International Patent Application No. WO/2003/092739. [00101] In a further embodiment, any cationic monomer known in the art are contemplated for use in a polycation composition of the invention. [00102] In one aspect, the polycation, in the context of a poly-amino acid compound, such as polylysine, polyarginine, polyhistidine, and polyornithine, consists of from 2 to about 500 residues with molecular weight values from 0.2 kDa to 70 kDa. In a poly-amino acid polycation, the number of individual amino acid monomers in the polycation compound may be determined based on the molecular weight of the polycation compound with knowledge of the molecular weight of amino acid(s) making up the polycation. For example, a polymer comprising 7 lysine residues weighs approximately 1 kDa, a polymer comprising 60 lysine residues weighs approximately 8 kDa, and a 70 kDa polylysine comprises 500 lysine residues. [00103] In one embodiment, the molecular weight of the polycation used in the methods and compositions of the invention may be from about 0.2 kDa to 70 kDa, from about 1 kDa to about 70 kDa, from about 5 kDa to about 50 kDa, or from about 10 kD to about 35 kD. It is contemplated that the molecular weight of the polycation in the composition has a molecule weight of less than I kD, about 1 kD, about 5 kD, about 10 kD, about 15 kD, about 20 kD, about 25 kD, about 30 kD, about 35 kD, about 40 kD, about 45 kD, about 50 kD, about 55 kD, about 60 kD, about 65 kD, or about 70 kD. [00104] Method for Making Antibody and Fc-fusion Molecule Formulations Comprising Polycation Compositions [00105] For improved solubility, it is important to have the pH of solution at least 1, or even 2, pH units lower or higher than the isoelectric point (pI) of the antibody or WO 2009/026122 PCT/US2008/073250 Fc-fusion molecule in the solution. The isoelectric point of an antibody or Fe-fusion molecule is the pH at which the net charge of the antibody or Fc-fusion molecule is zero. An antibody or Fc-fusion molecule with a high p1 (e.g., 8-11) generally does not form aggregates in solution at physiological pH of 7.4. [00106] Antibodies of different isotypes share approximately 70% homology, and the pI of a typical antibody is approximately 8.0-8.5, but may range from approximately p 1 6 to approximately pI 9. To increase the solubility of the antibody and reduce aggregation and dimer formation, antibody solutions are formulated at pH 5-6. At this pH, the antibody molecules are positively charged, repel each other, and do not aggregate. This pH is also useful because of the low rate of deamidation of asparagines, oxidation of methionines, and enzymatic and chemical cleavage. [001071 An antibody or Fc-fusion molecule with a lower pI (e.g., pI 5-7), which has more negative charge, may generate aggregates and precipitate in the formulation solution at pH 5-6 or at the injection site during the transition from the formulation pH 5-6 to physiological pH 7.4, which crosses the pH = pI point. Antibodies with lower p1 values show poor solubility. For example, an IgG2 lambda antibody with a calculated pI of 6.4 (Example 1) has poor solubility in a pH of 5.5-6.0. This is likely caused by the pH of the solution approaching the pl of the antibody. When the pH approaches the pI of the antibody, the antibody molecules become electrically neutral and do not repel each other and tend to aggregate in solution. [001081 Additionally, antibodies may have a pI higher than 7.5 but also exhibit a negatively charged region or domain of the antibody that is amenable to association with polycations. For example, an antibody may have a high pI, but contain consecutive acidic amino acid residues in the CDR domains which render the antibody unstable and prone to aggregation in solution. An antibody comprising a string of consecutive amino acids is an antibody having a CDR amino acid sequence having at least two consecutive, sequential acidic amino acid residues, such as aspartic acid (D) or glutamic acid (E). [001091 Additionally, formulation of Fc-fusion molecules in a polycation solution is adjusted based on the pI of the Fc-fusion molecule, as well as any string of negatively charged amino acid residues within the Fc-fusion sequence. The pI of an Fc-fusion molecule may range from pI 4 to p 1 9. Similar to antibodies, at low pI a Fc fusion molecule will likely have low solubility in solution.

WO 2009/026122 PCT/US2008/073250 [001101 To determine the optimal polycation solution for formulation with the antibody of interest, the pl of the antibody is first determined using standard techniques known in the art based on the overall charge of the antibody (Current Protocols in Protein Science, John Wiley and Sons, New York, NY, 1994). An acidic residue lowers the pI of an antibody by 0.1 unit. For example, if the p1 of a typical antibody is approximately 8, then an antibody having additional acidic residues such as aspartic acid (D) or glutamic acid (E) will have p1 lower than normal by approximately 0.1 p1 unit for each negative amino acid in the antibody. If the pH is above the pI of an antibody and the antibody contains aspartic acid or glutamic acid residues, the negatively charged amino acid is deprotonated leading to the negative charge at these acidic residues. [00111] The pIs for some commonly used polycations have been calculated previously. For example, the pI of polylysine is approximately 11, the pI of polyarginine is approximately 10.9, the pI of protamine is approximately 10, and the pI of MBP is approximately 11. The high pI and positive charge of these and other polycations makes these cationic polymers useful for stabilizing a negatively charged antibody or Fc-fusion molecule in solution. [001121 In one aspect, the concentration of polycation in the antibody or Fc-fusion molecule solution is from about 0.1% to about 10% weight/weight (w/w). In one embodiment, the polycation is from about 1% to about 5% w/w, from about 2% to about 4% w/w, or about 3% w/w. In a related embodiment, the antibody or Fc-fusion molecule in solution may be at a concentration of from about 1 mg/ml to about 150 mg/ml. In another embodiment, the concentration of the antibody or Fc-fusion molecule in the formulation may be about I mg/ml, about 2.5/mg/ml, about 5 mg/ml, about 10 mg/ml, about 20 mg/ml, about 30 mg/ml, about 40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about 110 mg/ml, about 120 mg/ml, about 125 mg/ml, about 130 mg/ml, about 140 mg/ml, or about 150 mg/ml. [00113] In another aspect, the polycation/antibody or Fe-fusion molecule composition may be formulated based on the ratio of the polycation to antibody or Fe fusion molecule in the composition. For example, the formulation may be made at a molar ratio of about 2:1 polycation to antibody or Fc-fusion molecule, about 1:1 polycation to antibody or Fe-fusion molecule, or less than a 1:1 ratio of polycation to antibody or Fc-fusion molecule. The ratio may depend on the number of negatively WO 2009/026122 PCT/US2008/073250 charged amino acid residues in the antibody or Fc-fusion molecule, and/or the size and number of cationic charges of the polycation in the formulation. In one instance, an antibody may exhibit negatively charged amino acid residues in the constant region. Because the antibody is a dimeric molecule, each chain of the antibody expresses the negative residues and as such the number of charged residues is two times the number for a single chain of the antibody. Therefore, it may require a 2:1 ratio of polycation:antibody to neutralize the negative residues in the antibody structure. However, if a larger polycation is used, having multiple sites for binding negatively charged residues or regions, less than a 2:1 ratio, or even less than a 1:1 ratio of polycation:antibody may be used in the formulation. [00114] Formulation of Pharmaceutical Compositions [00115] While it may be possible to administer compounds of the invention alone, in the methods described, the compound administered is generally present as an active ingredient in a desired dosage unit formulation, such as pharmaceutically acceptable composition containing conventional pharmaceutically acceptable excipients. Thus, in another aspect of the invention, there is provided a pharmaceutical composition comprising an antibody composition and polycation composition of this invention, or a Fc-fusion molecule and a polycation composition of this invention, in combination with a pharmaceutically acceptable excipient, diluent or carrier. The phrase "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce allergic, or other adverse reactions when administered using routes well-known in the art, as described below. Acceptable pharmaceutical excipients generally include diluents, carriers, adjuvants and the like as described herein. [00116] In addition, compounds may form solvates with water or common organic solvents. Such solvates are contemplated as well. [00117] A pharmaceutical composition of the invention may comprise an effective amount of an antibody or Fc-fusion molecule formulation of the invention or an effective dosage amount of an antibody or Fc-fusion molecule formulation of the invention. An effective dosage amount of an antibody formulation of the invention includes an amount less than, equal to, or greater than an effective amount of the compound. For example, a pharmaceutical composition in which two or more unit dosages, such as in tablets, capsules and the like, are required to administer an WO 2009/026122 PCT/US2008/073250 effective amount of the antibody or Fe-fusion formulation, or alternatively, a multi dose pharmaceutical composition, such as powders, liquids and the like, in which an effective amount of the compound may be administered by administering a portion of the composition. "Unit dosage" is defined as a discrete amount of a therapeutic composition dispersed in a suitable carrier. Those of ordinary skill in the art will readily optimize effective dosages and administration regimens as determined by good medical practice and the clinical condition of the individual patient. [00118] The pharmaceutical composition of the invention is formulated for any route of administration, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intravenous, intraarterial, intraperitoneal, intramuscular, intradermal or subcutaneous administration. In addition, the composition is suitably administered by pulse infusion, particularly with declining doses of the therapeutic antibody or Fc-fusion molecule. Preferably the dosing is given by injections, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Other administration methods are contemplated, including topical, particularly transdermal, transmucosal, rectal, oral or local administration e.g. through a catheter placed close to the desired site. Injection, especially intravenous, is preferred. [001191 Pharmaceutical compositions of the present invention containing an antibody composition or Fc-fusion molecule of the invention as an active ingredient may contain pharmaceutically acceptable excipients or diluents depending on the route of administration. Examples of such excipients include water, a pharmaceutical acceptable organic solvent, collagen, polyvinyl alcohol, polyvinylpyrrolidone, a carboxyvinyl polymer, carboxymethylcellulose sodium, polyacrylic sodium, sodium alginate, water-soluble dextran, carboxymethyl starch sodium, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum Arabic, casein, gelatin, agar, diglycerin, glycerin, propylene glycol, polyethylene glycol, Vaseline, paraffin, stearyl alcohol, benzyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, a pharmaceutically acceptable surfactant and the like. Excipients and diluents used are chosen from, but not limited to, the above or combinations thereof, as appropriate, depending on the dosage form of the present invention. [00120] Formulation of the pharmaceutical composition will vary according to the route of administration selected (e.g., solution, emulsion). An appropriate WO 2009/026122 PCT/US2008/073250 composition comprising the antibody to be administered can be prepared in a physiologically acceptable vehicle or carrier. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers. [001211 A variety of aqueous carriers, e.g., sterile phosphate buffered saline solutions, bacteriostatic water, water, buffered water, 0.4% saline, 0.3% glycine, and the like, and may include other proteins for enhanced stability, such as albumin, lipoprotein, globulin, etc., subjected to mild chemical modifications or the like. [001221 The pharmaceutical compositions may generally be prepared by mixing one or more antibody/polycation compositions or Fc-fusion molecule/polycation compositions of the invention with one or more pharmaceutically acceptable excipients, carriers, binders, adjuvants, diluents, preservatives, solubilizers, emulsifiers and the like, to form a desired administrable formulation to treat or ameliorate a variety of diseases. (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Such compositions include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween 80 also called, Polysorbate 80, Tween 20 also called Polysorbate 20), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., mannitol, sucrose); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Hyaluronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712 which are herein incorporated by reference. The compositions may be prepared in liquid form, or may be in dried powder, such as lyophilized form. Implantable sustained release formulations are also contemplated, as are transdermal formulations. [00123] Additional buffers contemplated include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; WO 2009/026122 PCT/US2008/073250 preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); 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, glutamic acid, proline, histidine, arginine, or lysine; short peptides such as alanine-leucine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as PLURONICS TM or polyethylene glycol (PEG). [00124] The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. [00125] Aqueous suspensions may contain the active compound in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyl eneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n propyl, p-hydroxybenzoate. [001261 The invention also contemplates lyophilized formulations comprising an antibody, or Fe-fusion molecule, and a polycation. Lyophilization is carried out using techniques common in the art and should be optimized for the composition being developed [Tang et al., Pharm Res. 21:191-200, (2004) and Chang et al., Pharm Res.

WO 2009/026122 PCT/US2008/073250 13:243-9 (1996)1. A lyophilized formulation is usually comprised of a buffer, a bulking agent, and a stabilizer. Methods of lyophilizing proteins are described in the art. See for example, US Patent 6,020,469 and US Patent Publication No. 20070053871. [00127] A lyophilization cycle is, in one aspect, composed of three steps: freezing, primary drying, and secondary drying [A.P. Mackenzie, Phil Trans R Soc London, Ser B, Biol 278:167 (1977)]. In the freezing step, the solution is cooled to initiate ice formation. Furthermore, this step induces the crystallization of the bulking agent. The ice sublimes in the primary drying stage, which is conducted by reducing chamber pressure below the vapor pressure of the ice, using a vacuum and introducing heat to promote sublimation. Finally, adsorbed or bound water is removed at the secondary drying stage under reduced chamber pressure and at an elevated shelf temperature. The process produces a material known as a lyophilized cake. Thereafter the cake can be reconstituted with either sterile water or suitable diluent for injection [00128] The lyophilization cycle not only determines the final physical state of the excipients but also affects other parameters such as reconstitution time, appearance, stability and final moisture content. The composition structure in the frozen state proceeds through several transitions (e.g., glass transitions and crystallizations) that occur at specific temperatures and can be used to understand and optimize the lyophilization process. The glass transition temperature (Tg) can provide information about the physical state of a solute and can be determined by differential scanning calorimetry (DSC). This is an important parameter that must be taken into account when designing the lyophilization cycle. Furthermore, in the dried state, the glass transition temperature provides information on the storage temperature of the final product. [00129] In a particular embodiment of the present compositions, a stabilizer is added to the lyophilization formulation to prevent or reduce lyophilization induced or storage induced aggregation and chemical degradation. A hazy or turbid solution upon reconstitution indicates that the protein has precipitated. The term "stabilizer" means an excipient capable of preventing aggregation or other physical degradation, as well as chemical degradation (for example, autolysis, deamidation, oxidation, etc.) in an aqueous and solid state. Stabilizers include a class of compounds that can serve as cryoprotectants, lyoprotectants, and glass forming agents. Cryoprotectants act to WO 2009/026122 PCT/US2008/073250 stabilize proteins during freezing or in the frozen state at low temperatures (P. Cameron, ed., Good Pharmaceutical Freeze-Drying Practice, Interpharm Press, Inc., Buffalo Grove, IL, (1997)). Lyoprotectants stabilize proteins in the freeze-dried solid dosage form by preserving the native-like conformational properties of the protein during dehydration stages of freeze-drying. Glassy state properties have been classified as "strong" or "fragile" depending on their relaxation properties as a function of temperature. It is important that cryoprotectants, lyoprotectants, and glass forming agents remain in the same phase with the protein in order to impart stability. Sugars, polymers, and polyols fall into this category and can sometimes serve all three roles. [00130] Stabilizers that are conventionally employed in pharmaceutical compositions, including, but not limited to, sucrose, trehalose or glycine, may be used [Carpenter et al., Develop. Biol. Standard 74:225, (1991)]. Surfactant stabilizers, such as polysorbate 20 (Tween 20) or polysorbate 80 (Tween 80), may also be added in appropriate amounts to prevent surface related aggregation phenomenon during freezing and drying [Chang, B, J. Pharm. Sci. 85:1325, (1996)]. If desired, the lyophilized compositions also include appropriate amounts of bulking and osmolarity regulating agents suitable for forming a lyophilized "cake". Bulking agents may be either crystalline (for example, mannitol, glycine) or amorphous (for example, sucrose, polymers such as dextran, polyvinylpyrolidone, carboxymethylcellulose. In one embodiment, the bulking agent is mannitol. In a further embodiment, mannitol is incorporated in a concentration of about 2% to about 5% w/v, and in a yet further embodiment in a concentration of about 3% to 4.5% w/v, to produce a mechanically and pharmaceutically stable and elegant cake. In another embodiment, the mannitol concentration is 2% w/v. [001311 The choice of a pharmaceutically-acceptable buffer and pH has also been found to affect the stability of the present compositions. The buffer system present in the compositions is selected to be physiologically compatible and to maintain a desired pH in the reconstituted solution as well as in the solution before lyophilization. In one aspect, the buffers have a pH buffering capacity in the range of from about pH 6.0 to about pH 8.0. A series of screening studies incorporating the above mentioned parameters are typically performed to select the most stable formulation condition.

WO 2009/026122 PCT/US2008/073250 [00132] Lyophilization methods described herein may also comprise one or more of the following steps: adding a stabilizing agent to the antibody or Fe-fusion molecule/polycation mixture prior to lyophilizing, adding at least one agent selected from a bulking agent and an osmolarity regulating agent, and a surfactant to said antibody or Fc-fusion molecule/polycation mixture prior to lyophilization. The bulking agent may be any bulking agent set forth above. In one emboidment, the bulking agent is mannitol. The sugar may be any stabilizing sugar set out above. In one embodiment, the stabilizing agent is sucrose. The surfactant may be any surfactant set out above. In one embodiment, the surfactant is polysorbate 20. [00133] The standard reconstitution practice for lyophilized material is to add back a volume of pure water or sterile water for injection (WFI) (typically equivalent to the volume removed during lyophilization), although dilute solutions of antibacterial agents are sometimes used in the production of pharmaceuticals for parenteral administration [Chen, Drug Development and Industrial Pharmacy, 18:1311-1354 (1992)]. [00134] A variety of aqueous carriers, e.g., sterile water for injection, water with preservatives for multi dose use, or water with appropriate amounts of surfactants (for example, polysorbate 20), 0.4% saline, 0.3% glycine, or aqueous suspensions may contain the active compound in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyl eneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n propyl, p-hydroxybenzoate. [00135] The invention further provides antibody or Fc-fusion molecule formulations which are self-buffering formulations, comprising an antibody or an Fc- WO 2009/026122 PCT/US2008/073250 fusion molecule and one or more polycations. Buffering refers to the resistance to change in pH of a composition upon addition of acid or base. Buffer capacity thus often is defined as the ability of a composition to resist pH change. Self-buffering formulations refers to formulations comprising an antibody or Fc-fusion molecule, which have enough buffer capacity to maintain a formulation within a desired pH range, without additional buffering agents. International Patent Publication W02006/138181 (herein incorporated by reference) describes methods of making self-buffering formulations. [00136] Kits [00137] As an additional aspect, the invention includes kits which comprise one or more antibody or Fc-fusion molecule formulations or compositions packaged in a manner which facilitates their use. In one embodiment, such a kit includes an antibody or Fc-fusion molecule formulation or composition described herein (e.g., a composition comprising an antibody or Fe-fusion molecule, and a polycation), packaged in a container such as a sealed bottle or vessel (e.g., a vial or i.v. bag), with a label affixed to the container or included in the package that describes use of the compound or composition. In an embodiment, an antibody or Fe-fusion molecule formulation or composition is packaged in a unit dosage form. The kit may further include a device suitable for administering the composition according to a specific route of administration. In certain embodiments, the kit contains a label that describes use and administration of an antibody or Fc-fusion molecule formulation or a composition of the invention. [00138] Additional aspects and details of the invention will be apparent from the following examples, which are intended to be illustrative rather than limiting. [00139] Example 1 [00140] Antibody solutions formulated for storage often degrade after long periods of time leading to aggregation and additional smaller degradation products. Antibody constant region amino acid sequences typically have a polar charge, especially those which contain a stretch of aspartic acid residues. In order to further stabilize the solution and prevent antibody aggregation during storage, different stabilizing solutions were added to the antibody formulation and the extent of aggregation and breakdown assessed.

WO 2009/026122 PCT/US2008/073250 [00141] Different formulations of a human monoclonal recombinant IgG2a lambda antibody specific for the CD30 molecule, with a calculated pI of 6.4, were made. The polycations used in the experiment were polyarginine, with average molecular weight (MW) of 35 kDa, polylysine including five lysine residues, and polyarginine with MW greater than 70kDa. The formulation solutions were prepared by diluting a stock solution of 70 mg/mI IgG stock solution in K7.6RT buffer, including 10 mM K 2 P0 4 , 161 mM arginine, 0.004%Tween, pH 7.6. The stock solution was also diluted 7-fold in A5.2Su buffer (acetate buffer at pH 5.2 plus sucrose) to 10 mg/ml IgG solution containing 10 mM sodium acetate, 9% sucrose, 3% polycation, pH 5.2. The final pH values of the polycation solution were approximately the same at pH 5.5-6.0. Results are shown in Table 1. Table 1 Antibody Polycation Measured pH concentration (in A5.2Su buffer, unless indicated otherwise) Buffer alone 5.0 IgG, 10 mg/ml 3% polyR, 35 kDa 5.5-6.0 IgG, 10 mg/mI 3% polyK, 5 lysine 5.5-6.0 IgG, 10 mg/mI Buffer alone 5.5-6.0 IgG, 10 mg/ml 3% polyR, > 70 kDa 5.5-6.0 IgG, 70 mg/ml K7.6RT buffer 7.6 [001421 While the antibody formulations containing acetate and sucrose (at pH 5.5 6.0) were cloudy and viscous, addition of polycations at a concentration of 3% w/w prevented the formation of the cloudy, viscous state and the antibody solutions remained clear. Reverse-phase chromatography of the antibody solution, which tests the stability of the antibody in the solution, shows that the antibody stays in solution in the presence of the polycations.

WO 2009/026122 PCT/US2008/073250 [00143] Example 2 [00144] Many antibody constant regions contain acidic amino acid residues which can confer either an overall or localized negative charge on the antibody in solution. In order to minimize the effect of this negative charge, the effects of polycation solutions were assessed on a negatively charged antibody. [00145] A recombinant human monoclonal IgGI kappa antibody specific for amyloid protein, having a calculated pI of 8.9, and containing three adjacent aspartic acid residues (DDD) in the variable regions, was formulated in different polycation solutions. Methods of making the anti-amyloid antibody, and the structure has been described in International Publication No. WO 2006/081171, (SEQ ID NO: 11 in WO 2006/081171). [00146] Antibody was diluted in control solution (containing 100 mg/mI in 20 mM sodium phosphate, pH 6.9) or polycation solution (containing 100 mg/ml antibody in 20 mM sodium phosphate, pH 5.0, 0.005% Tween, and 10 mg/ml 22K polylysine or 10 mg/ml 83K polylysine) and stored at 520 C for 6 weeks. Relative abundance of aggregates and dimers were measured by size-exclusion chromatography. [00147] The antibody generated dimers and aggregates after long-term storage in a formulation at pH 6.9. Size-exclusion chromatography shows that a solution containing only single lysine residues (10 mg/ml) is unable to reduce the antibody aggregation and actually increases the amount of aggregate formed relative to the control solution. The dimers and aggregates, however, formed under these conditions were significantly reduced in formulations containing 22K or 83K polylysine (Figure 1). [00148] The above experiments show that the addition of polycations to formulations of monoclonal antibodies improve the solubility and/or stability of the formulated antibody. The polycation formulation of the present invention was found effective in increasing the solubility and/or stability of antibodies with a pI below 7.4 and for antibodies with closely located acidic amino acid residues (aspartic acid and glutamic acid). [00149] Example 3 [00150] To determine if the anti-CD30 IgG2a antibody described in Example 1 may be soluble in different concentrations of polycation compositions, the antibody was analyzed for its ability to solubilize over a range of polycation concentrations.

WO 2009/026122 PCT/US2008/073250 [00151] The sample antibody was formulated at 72 mg/mL in 10mM potassium phosphate, 161 mM arginine, 0.004% Tween-20, pH 7.6, was diluted to 10.3 mg/mL in 30 mM sodium acetate, 5% Sorbitol, pH 5.0, containing increasing amounts of poly-lysine of a molecular weight distribution of 15,000 to 30,000 Da. Solutions of the antibody at pH 5 without any polylysine added were turbid. Formulations having polycation concentrations as low as 0.08% polylysine showed a decrease in turbidity of the antibody formulation. The turbidity of the solutions decreased with increasing concentrations of polylysine. Antibody solutions containing 0.5% polylysine and higher were completely clear. [001521 Formulations were also made using a pentalysine (containing five lysine residues) polycation. Results showed that using pentalysine (0.65 KDa) required a higher poly-lysine concentration to be effective. Solutions with 1.28% polylysine were turbid while 2.57% polylysine (pentalysine) solutions were completely clear. [00153] The effects of polycations on peptibody stability were also analyzed. A peptibody (2XCon4C, an anti-angiopoietins 2/1 peptibody with the amino acid sequence: MDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGKGGGGGAQQEECEWDPWTCEHMGSGSATGGSGS TASSGSGSATHQEECEWDPWTCEHMLE) (Oliner et al., Cancer Cell, 507-516, 2004), having a pI of 5.4, was formulated at 27 mg/mL in 10 mM potassium phosphate, 161 mM arginine, 0.004% Tween-20, pH 7.6, and diluted to 3.86 mg/mL in 30 mM sodium acetate 5% sorbitol, pH 5.0, containing increasing amounts of poly lysine of a molecular weight distribution of 15,000 to 30,000 Da. Solutions of the peptibody at pH 5, without any poly-lysine added, were turbid. Peptibody solutions containing 0.08% poly-lysine and higher were completely clear. [00154] Similar results were obtained when the peptibody was formulated with pentalysine; solutions with 0.08% pentalysine were completely clear. [00155] The monomeric amino acid lysine showed an effect in solubilizing the IgG2 antibody with a low pI, but required 2% lysine to eliminate trubidity from solution. Also as shown in Example 2 and which has been previously reported, WO 2009/026122 PCT/US2008/073250 compositions comprising only the single amino acid lysine or arginine are destabilizing to antibodies and cause accelerated aggregation, while polylysine and polyarginine decrease antibody aggregation. [00156] While not wishing to be bound by the following proposed mechanism of action, a possible explanation for the effects of polycations seen in these experiments might be that they bind to negatively charged pockets of an antibody, thus increasing the net positive charge of the antibody, and increasing the effective pI of the antibody, thereby reducing intermolecular interactions. These effects could lead to an increase in antibody solubility at a given pH and decrease the rate of aggregation under certain conditions. The binding of the polycations to these regions, may also reduce the flexibility of the antibody structure in this region and this induced rigidity may further stabilize against degradations such as aggregation, aspartate isomerization, oxidation, and deamidation. While these effects may be more pronounced in the case of low p1 antibodies and antibodies containing sequential aspartate (DD) or glutamate (EE) residues, the potential for polycations to stabilize antibodies outside these characteristics is possible. [001571 Numerous modifications and variations in the invention as set forth in the above illustrative examples are expected to occur to those skilled in the art. Consequently only such limitations as appear in the appended claims should be placed on the invention

Claims (42)

1. A method of making an antibody formulation comprising combining an antibody with a polycation.

2. A method of making an Fc-fusion molecule formulation comprising combining an Fc-fusion molecule with a polycation.

3. A method of making a peptibody formulation comprising combining a peptibody with a polycation.

4. The method of any one of claims 1 to 3 , wherein the polycation is selected from the group consisting of polylysine, polyarginine, polyornithine, polyhistidine, and cationic polysaccharides; or mixtures thereof.

5 The method of claim 4, wherein the polycation is composed of at least two of the polycations selected from the group consisting of polylysine, polyarginine, polyomithine, polyhistidine, and cationic polysaccharides.

6. The method of any one of claims 1 to 3, wherein the polycation is composed of polylysine, or polyarginine; or copolymers thereof.

7. The method of any one of claims I to 3, wherein the polycation is a polymer comprising repeating units of a cationic monomer and a neutral monomer.

8. The method of claim 7, wherein the polycation has a monomeric formula A-B X-Y, wherein A and X are cationic monomers and B and Y are neutral monomers.

9. The method of claim 8, wherein A and X are the same cationic monomer.

10. The method of claim 8, wherein A and X are different cationic monomers.

11. The method of claim 8, wherein B and Y are the same neutral monomer.

12. The method of claim 8, wherein B and Y are different neutral monomers. An~ WO 2009/026122 PCT/US2008/073250

13. The method of any one of claims 9 through 12, wherein one or more of the monomers is an amino acid residue.

14. The method of claim 8, wherein the neutral monomer is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, serine, threonine and proline.

15. The method of claim 7, wherein the polycation comprises repeating units of lysine-glycine residues.

16. The method of claim 1, wherein the antibody formulation comprises a therapeutic antibody.

17. The method of claim 2 wherein the Fc-fusion molecule formulation comprises a therapeutic Fc-fusion molecule.

18. The method of claim 16, wherein said therapeutic antibody is an IgG antibody selected from the group consisting of an IgG1, IgG2, IgG3 and IgG4 antibody.

19. The method of any one of claims 1 to 3, wherein the polycation in said composition has a molecular weight of between about 0.2 kDa to about 70 kDa.

20. The method of claim 1, wherein the antibody in said antibody formulation has a p1 of 7.5 or less.

21. The method of claim 20, wherein the antibody in said antibody formulation is an IgG2 antibody.

22. The method of claim 21, wherein the antibody in said antibody formulation has a calculated pI of about 6.4.

23. The method of any one of claims 1 to 3, wherein the polycations are added to a final concentration of 0.1% to 10% w/w polycation in the final formulation. A I WO 2009/026122 PCT/US2008/073250

24. The method of any one of claims I to 3, wherein the formulation has a greater solubility when combined with said polycation composition as compared to the solubility of said antibody formulation in the absence of polycations.

25. The method of any one of claims I to 3, wherein the formulation has an increased shelf-life as compared to such an antibody formulation that has not been combined with a polycation composition.

26. The method of claim 25, wherein said formulation has at least a 25% greater shelf life than a similar formulation that has not been combined with a polycation composition.

27. The method of any one of claims 1 to 3, wherein the presence of said polycations in said formulation reduces the formation of dimers or aggregates in said formulation as compared to a formulation that has not been combined with a polycation composition.

28. The method of claim 1, wherein said method produces a therapeutic antibody formulation composition that comprises between about 0.1% to about 10 % w/w polycation.

29. The method of any of claims 1 through 28, wherein the pH of said polycation composition is between about 4.5 to about 7.5.

30. The method of any of claims 1 through 28, wherein the pH of said polycation composition is between about 5.0. to about 6.5.

31. The method of any of claims 1 through 28, wherein the pH of said polycation composition is between about 5.5. to about 6.0.

32. A pharmaceutical composition comprising a therapeutic antibody, a polycation and a pharmaceutically acceptable excipient. WO 2009/026122 PCT/US2008/073250

33. A pharmaceutical composition comprising a therapeutic antibody having a pl of 7.5 or less and a polycation selected from the group consisting of polylysine and polyarginine.

34. A pharmaceutical composition comprising a therapeutic antibody having a CDR domain comprising at least two consecutive acidic amino acid residues and a polycation selected from the group consisting of polylysine and polyarginine.

35. The pharmaceutical composition of claim 34 wherein the acidic amino acids are selected from the group consisting of aspartic acid (D) and glutamic acid (E).

36. A pharmaceutical composition comprising an Fc-fusion molecule, a polycation and a pharmaceutically acceptable excipient

37. A pharmaceutical composition comprising a peptibody, a polycation and a pharmaceutically acceptable excipient

38. A pharmaceutical composition comprising a peptidbody that binds Ang2, a polycation and a pharmaceutically acceptable excipient.

39. The composition of any one of claims 36 to 38 wherein the polycation is selected from the group consisting of polylysine and polyarginine.

40. The pharmaceutical composition of any one of claims 32 through 39, wherein said composition has an increased shelf-life as compared to a pharmaceutical composition in the absence of a polycation.

41. The pharmaceutical composition of any one of claims 32 through 39, wherein composition has a greater solubility in water than a composition in the absence of a polycation.

42. The pharmaceutical composition of any one of claims 32 through 40, wherein the dimerization and/or aggregate formation of said pharmaceutical composition is reduced as compared to a pharmaceutical composition in the absence of a polycation. A I