Stable formulations for CTLA4-IG fusion proteins and uses thereof

Abstract

CTLA4-fusion proteins and variants thereof are described, as well as stable formulations comprising such proteins. Such CTLA4-Ig fusion proteins are useful for the treatment and diagnosis of various autoimmune diseases.

Description

[0001] Attorney Docket No. ZEN-016WO1

[0002] STABLE FORMULATIONS FOR CTLA4-IG FUSION PROTEINS AND USES THEREOF

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004]

[0001] This application claims priority to, and the benefit of, U.S. provisional application No. 63 / 696,730, filed on September 19, 2024; and, U.S. provisional application No. 63 / 719,889, filed on November 13, 2024, the content of which is hereby incorporated by reference in its entirety.

[0005] REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0006]

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on September 8, 2025, is named “ZEN-016WO1_SL” and is 11,531 bytes.

[0007] BACKGROUND

[0008]

[0003] Cytotoxic T lymphocyte-associated antigen (CTLA)4, a CD28 homologue expressed on activated T cells and competitively binds to the CD28 ligands, B7-1 (CD80) and B7-2 (CD86). CLTA4-immunoglobulin (CTLA4-Ig) are fusion proteins designed to modulate the T cell co-stimulatory signal mediated through the CD28-CD80 / 86 pathway. Several clinical trials have indicated that this compound can be used in the treatment of several autoimmune diseases and conditions. It has a higher binding affinity to the B7 molecule than CD28 and acts as a negative regulator of CD28-mediated T cell activation.

[0009]

[0004] The CD28-CD80 / 86 pathway regulates interleukin-2 production and the expression of anti-apoptotic molecules such as Bcl-xL. Both CD80 (B7-1) and CD86 (B7- 2) are present on antigen-presenting cells, including dendritic cells, B cells, macrophages and are expressed on activated T cells and are present on T cells obtained from various autoimmune diseases. Engagement with these ligands provides the second signal required for maximal T cell activation, and the absence of a co-stimulatory signal may result in anergy and apoptotic cell death. CTLA4 (CD 152), which is upregulated on T cells following their activation, also interacts with CD80 and CD86, providing an important mechanism for regulating T cell function. Not only does CTLA4 permit interruption of Attorney Docket No. ZEN-016WO1 the activating CD28 pathway but it may also provide important negative signals that permit long-term tolerance. CD28 / B7 interactions are critical for the generation of CD4+, CD25+, CTLA4+ T regulatory cells, and signaling through CTLA4 may promote the release of immunoregulatory cytokines such as TGFp. Individuals with a mutation in a single allele of CTLA4 are highly susceptible to autoimmunity.

[0010]

[0005] CTLA4-Ig, a soluble recombinant form of CTLA4, is a potent inhibitor of

[0011] B7 / CD28 interactions and inhibits in vivo antigen-specific responses. In various animal models, CTLA4-Ig has been shown to inhibit T-cell-dependent antibody responses, significantly prolong transplanted organ survival, induce long-term donor-specific tolerance in some models, slow progression of autoimmune disease and to have immunomodulatory function in several other immunological disease models. It was observed for example that CTLA-4-Ig suppressed the expression of Staphylococcus aureus (SAC)-induced CD80, CD86, TNF-a, and IL6 in human B cells at the transcriptional level. Furthermore, CTLA-4-Ig concomitantly decreased SAC -induced CD80 / CD86 surface expression and TNF-a and IL-6 secretion from B cells. CTLA4-Ig has emerged as a promising new therapeutic agent for induction of donor-specific immunological tolerance, the ultimate goal of clinical immunosuppression.

[0012]

[0006] Developments in biotechnology have made it possible to produce a large variety of biologies for pharmaceutical applications. CTLA4-Ig fusion proteins are larger and more complex than traditional organic and inorganic drugs and therefore formulating these fusion proteins for therapeutic administration poses special problems. One of the problems is maintaining stability of the protein, which is a critical concern in ensuring product quality. Another problem when formulating biologies for therapeutic use is viscosity, particularly when formulating proteins at high concentrations. The unique amino acid sequences and third dimensional structures of proteins causes to differences in physicochemical properties like isoelectric point, hydrophobicity, and surface charge distributions which can be both difficult to predict and greatly impact the behavior of the formulated therapeutic. The optimal pH, ionic strength, buffer type and excipients required for stabilization vary for each protein based on its specific structural characteristics, and a “one-size-fits-all” formulation is not feasible. Attorney Docket No. ZEN-016WO1

[0013] SUMMARY OF THE INVENTION

[0014]

[0007] The present invention provides, among other things, a stable formulation for CTLA4-Ig fusion proteins and methods of treating autoimmune diseases using the same that require less frequent dosing. As described herein, the present disclosure is based, in part, on the identification of stable formulations comprising a CTLA4-Ig fusion protein which maintain low percentage of high molecular weight species when stored in a liquid state for an extended duration. The improved half-life of the CTLA4-Ig fusion proteins disclosed herein furthermore enables the efficacious treatment of autoimmune diseases using a less frequent dosing interval as compared to existing treatment regimens.

[0015]

[0008] In one aspect, the present invention provides a stable formulation comprising a CTLA4-Ig fusion protein, wherein the stable formulation does not comprise acetate, wherein the CTLA4-Ig comprises a first domain comprising a variant CTLA4 and a second domain comprising an IgG Fc region, wherein the variant CTLA4 comprises A29H, T51N, L61E, and K93Q substitutions, and wherein the IgG Fc region comprises M428L and N434S substitutions, wherein the CTLA-4-Ig fusion protein is present at a concentration of greater than 30 mg / ml.

[0016]

[0009] In one aspect, the present invention provides a stable formulation comprising a CTLA-4-Ig fusion protein, wherein the stable formulation comprises a buffer and a pH of 5.5 to 7.5, wherein the CTLA4-Ig comprises a first domain comprising a variant CTLA4 and a second domain comprising an IgG Fc region, wherein the variant CTLA4 comprises A29H, T51N, L61E, and K93Q substitutions, and wherein the IgG Fc region comprises M428L and N434S substitutions, wherein the CTLA-4Ig fusion protein is present at a concentration of greater than 30 mg / ml.

[0017]

[0010] In one aspect, the present invention provides a stable formulation comprising a CTLA4-Ig fusion protein, wherein the stable formulation has an osmolality of greater than 700 mOsmol / kg, and wherein the CTLA4-Ig comprises a first domain comprising a variant CTLA4 and a second domain comprising an IgG Fc region, wherein the variant CTLA4 comprises A29H, T51N, L61E, and K93Q substitutions, and wherein the IgG Fc region comprises M428L and N434S substitutions, and wherein the CTLA4-Ig fusion protein is present at a concentration of greater than 30 mg / ml. Attorney Docket No. ZEN-016WO1

[0018] [Oil] In one aspect, the present invention provides a stable formulation comprising a CTLA4-Ig fusion protein, wherein the stable formulation has a combined concentration of excipients of greater than 350 mM, and wherein the CTLA4-Ig comprises a first domain comprising a variant CTLA4 and a second domain comprising an IgG Fc region, wherein the variant CTLA4 comprises A29H, T51N, L61E, and K93Q substitutions, and wherein the IgG Fc region comprises M428L and N434S substitutions, and wherein the CTLA4-Ig fusion protein is present at a concentration of greater than 30 mg / ml.

[0019]

[0012] In one aspect, the present invention provides a stable formulation comprising 50 mg / mL of a CTLA4-Ig fusion protein, wherein the stable formulation comprises a buffer at a concentration of between 10-75 mM, wherein the CTLA-4Ig comprises a first domain comprising a variant CTLA4 and a second domain comprising an IgG Fc region, wherein the variant CTLA4 comprises A29H, T51N, L61E, and K93Q substitutions, and wherein the IgG Fc region comprises M428L and N434S substitutions, and wherein the amount of HMW species in the formulation is less than 10% upon storage at 5° C for 9 weeks.

[0020]

[0013] In one aspect, the present invention provides a stable formulation comprising a CTLA4-Ig fusion protein, wherein the stable formulation comprises the CTLA4 Ig fusion protein at a concentration of greater than 30 mg / ml and at least 300 mM sucrose, wherein the CTLA4-Ig comprises a first domain comprising a variant CTLA4 and a second domain comprising an IgG Fc region, wherein the variant CTLA4 comprises A29H, T51N, L61E, and K93Q substitutions, and wherein the IgG Fc region comprises M428L and N434S substitutions.

[0021]

[0014] In one aspect, the present invention provides a stable formulation comprising a CTLA4-Ig fusion protein, wherein the stable formulation comprises the CTLA4-Ig fusion protein at a concentration of greater than 30 mg / ml, and an amount of sucrose proportional to the concentration of CTLA4-Ig fusion protein, wherein 5 to 10 mM of sucrose is present for every mg / ml of CTLA4-Ig fusion protein, wherein the CTLA4-Ig comprises a first domain comprising a variant CTLA4 and a second domain comprising an IgG Fc region, wherein the variant CTLA4 comprises A29H, T51N, L61E, Attorney Docket No. ZEN-016WO1 and K93Q substitutions, and wherein the IgG Fc region comprises M428L and N434S substitutions.

[0022]

[0015] In some embodiments, the variant CTLA4 domain comprises an amino acid sequence of SEQ ID NO: 3.

[0023]

[0016] In some embodiments, the IgG Fc region comprises an amino acid sequence of SEQ ID NO: 4.

[0024]

[0017] In some embodiments, the CTLA4-Ig comprises an amino acid sequence of

[0025] SEQ ID NO: 6.

[0026]

[0018] In some embodiments, the stable formulation further comprises an amino acid. In some embodiments, the amino acid is present at a concentration of 50-300 mM. In some embodiments, the amino acid comprises arginine, proline, and / or glycine. In some embodiments, the amino acid is arginine. In some embodiments, the stable formulation comprises arginine at a concentration of 100 mM.

[0027]

[0019] In some embodiments, the stable formulation further comprises a sugar. In some embodiments, the sugar is present at a concentration of 200-600 mM. In some embodiments, the sugar is present at a concentration of 5-15% (w / v). In some embodiments, the sugar comprises sucrose and / or trehalose. In some embodiments, sucrose is present at a concentration of 400 mM.

[0028]

[0020] In some embodiments, the stable formulation further comprises a surfactant. In some embodiments, the surfactant is poloxamer 188. In some embodiments, the concentration of poloxamer 188 is less than 1% (w / v). In some embodiments, the concentration of poloxamer 188 is 0.8% (w / v). In some embodiments, the surfactant is polysorbate 80. In some embodiments, the concentration of polysorbate 80 is less than 0.1%. In some embodiments, the concentration of polysorbate 80 is 0.02%.

[0029]

[0021] In some embodiments, the stable formulation comprises sodium phosphate.

[0030] In some embodiments, sodium phosphate is present at a concentration of 10-70 mM. In some embodiments, the concentration of sodium phosphate is 40 mM.

[0031]

[0022] In some embodiments, the stable formulation comprises succinate. In some embodiments, succinate is present at a concentration of 10-70 mM. In some embodiments, the concentration of succinate buffer is 20 mM. Attorney Docket No. ZEN-016WO1

[0032]

[0023] In some embodiments, the stable formulation has a pH of 5.5 to 7.5. In some embodiments, the stable formulation has a pH of 7 to 7.5. In some embodiments, the stable formulation has a pH of 7.2. In some embodiments, the stable formulation has a pH of 6 to 7. In some embodiments, the stable formulation has a pH of 6.5.

[0033]

[0024] In one aspect, the present invention provides a stable formulation comprising: (i) 30-75 mg / ml CTLA4-Ig fusion protein, (ii) 30-50 mM sodium phosphate buffer, (iii) 350-450 mM sucrose, (iv) 50-150 mM arginine, (v) 0.6%-l% (w / v) poloxamer 188, at pH 6.7-7.7, wherein the CTLA4-Ig fusion protein comprises an amino acid sequence of SEQ ID NO: 6. In some embodiments, the stable formulation comprises: (i) 50 mg / ml CTLA4-Ig fusion protein, (ii) 40mM sodium phosphate, (iii) 400mM sucrose, (iv) lOOmM arginine, (v) 0.8% (w / v) poloxamer 188, at pH of 7.2, wherein the CTLA4-Ig fusion protein comprises an amino acid sequence of SEQ ID NO: 6.

[0034]

[0025] In some embodiments, the viscosity of the formulation is no greater than

[0035] 3.1 mPa s.

[0036]

[0026] In some embodiments, the formulation comprises 85% CTLA4-Ig monomer as measured by size exclusion chromatography. In some embodiments, the formulation comprises 90% CTLA4-Ig monomer as measured by size exclusion chromatography. In some embodiments, the formulation comprises less than 15% high molecular weight species as measured by size exclusion chromatography. In some embodiments, the formulation comprises less than 10% high molecular weight species as measured by size exclusion chromatography.

[0037]

[0027] In some embodiments, the formulation comprises less than 25 pg / mg residual host cell DNA. In some embodiments, the formulation comprises less than 20 pg / mg residual host cell DNA. In some embodiments, the formulation comprises less than 150 ng / mg residual host cell protein. In some embodiments, the formulation comprises less than 100 ng / mg residual host cell protein.

[0038]

[0028] In some embodiments, the formulation comprises less than 100 ng / mg residual protein A. In some embodiments, the formulation comprises less than 50 ng / mg residual protein A. Attorney Docket No. ZEN-016WO1

[0039]

[0029] In some embodiments, the formulation comprises less than 0.4 EU / mg endotoxin.

[0040]

[0030] In some embodiments, the formulation is suitable for subcutaneous administration.

[0041]

[0031] In some embodiments, the formulation is suitable for intravenous administration.

[0042]

[0032] In some embodiments, the present invention provides a method of treating an autoimmune disease in subject comprising administering to the subject in need of treatment a stable formulation.

[0043]

[0033] In one aspect, the present invention provides a method of treating an autoimmune disease in a subject comprising administering to the subject in need of treatment a CTLA4-Ig fusion protein at a therapeutically effective dose no more frequently than once every other week, wherein the CTLA-4-Ig comprises a first domain comprising a variant CTLA-4 and a second domain comprising an IgG Fc region, wherein the variant CTLA-4 comprises A29H, T51N, L61E, and K93Q substitutions, and wherein the IgG Fc region comprises M428L and N434S substitutions.

[0044]

[0034] In some embodiments, the variant CTLA4 domain comprises an amino acid sequence of SEQ ID NO: 3.

[0045]

[0035] In some embodiments, the IgG Fc region comprises an amino acid sequence of SEQ ID NO: 4.

[0046]

[0036] In some embodiments, the CTLA4-Ig fusion protein comprises an amino acid sequence of SEQ ID NO: 6.

[0047]

[0037] In some embodiments, the therapeutically effective dose is 3 mg to 200 mg.

[0048]

[0038] In some embodiments, the therapeutically effective dose is 3 mg. In some embodiments, the therapeutically effective dose is 5 mg. In some embodiments, the therapeutically effective dose is 12.5 mg. In some embodiments, the therapeutically effective dose is 50 mg. In some embodiments, the therapeutically effective dose is 125 mg. In some embodiments, the therapeutically effective dose is 200 mg. Attorney Docket No. ZEN-016WO1

[0049]

[0039] In some embodiments, CTLA4-Ig fusion protein is administered no more frequently than once every week. In some embodiments, CTLA4-Ig fusion protein is administered no more frequently than once every two weeks. In some embodiments, CTLA4-Ig fusion protein is administered no more frequently than once every three weeks. In some embodiments, CTLA4-Ig fusion protein is administered no more frequently than once every four weeks. In some embodiments, CTLA4-Ig fusion protein is administered no more frequently than once every five weeks. In some embodiments, CTLA4-Ig fusion protein is administered no more frequently than once every six weeks. In some embodiments, CTLA4-Ig fusion protein is administered no more frequently than once every seven weeks. In some embodiments, CTLA4-Ig fusion protein is administered no more frequently than once every eight weeks. In some embodiments, CTLA4-Ig fusion protein is administered no more frequently than once every month. In some embodiments, CTLA4-Ig fusion protein is administered no more frequently than once every two months. In some embodiments, CTLA4-Ig fusion protein is administered no more frequently than once every three months. In some embodiments, CTLA4-Ig fusion protein is administered no more frequently than once every four months. In some embodiments, CTLA4-Ig fusion protein is administered no more frequently than once every five months. In some embodiments, CTLA4-Ig fusion protein is administered no more frequently than once every six months.

[0050] BRIEF DESCRIPTION OF THE DRAWINGS

[0051]

[0040] FIG. 1 is an exemplary graph showing CD86 receptor occupancy (RO) in

[0052] CD 14+ monocytes, demonstrating an apparent dose-driven response with the maximum ex vivo stimulated IL-2 inhibition observed approximately 2-6 days after ZB004 administration in each cohort.

[0053] DEFINITIONS

[0054]

[0041] In order for the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. The publications and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference. Attorney Docket No. ZEN-016WO1

[0055]

[0042] Any numerical values used in this application are meant to cover any variations within the standard deviation or normal fluctuations appreciated by one of ordinary skill in the relevant art.

[0056]

[0043] Acidic Species'. As used herein, the term “acidic species” refers to the variants of a protein, e.g., a fusion protein or binding fragment thereof, which are characterized by an overall acidic charge relative to the main species. Acidic species can be detected by charge-based separation techniques such as, for example and without limitation, isoelectric focusing (IEF) gel electrophoresis, capillary isoelectric focusing (cIEF) gel electrophoresis, cation exchange chromatography (CEX), and anion exchange chromatography (AEX). In some embodiments, the level of acidic species is determined by imaging capillary isoelectric focusing (iCIEF).

[0057]

[0044] Acidic species are variants with lower apparent pl relative to the main species when proteins are analyzed using IEF based methods. When analyzed by chromatography -based methods, acidic species are defined based on their retention times relative to the main peak. Acidic species are the variants that elute earlier than the main peak from AEX.

[0058]

[0045] Acidic species of a protein may include charge variants, structure variants, and / or fragmentation variants. Exemplary charge variants include, but are not limited to, deamidation variants, afucosylation variants, methylglyoxal variants, glycation variants, and citric acid variants. Exemplary structure variants include, but are not limited to, glycosylation variants and acetonation variants. Exemplary fragmentation variants include, but are not limited to, any truncated protein species from the protein of interest due to dissociation of peptide chain, enzymatic and / or chemical modifications, including, but not limited to, Fc and Fab fragments, fragments missing a Fab, fragments missing a heavy chain variable domain, C-terminal truncation variants, variants with the excision of N-terminal Asp in the light chain, and variants having N-terminal truncation of the light chain. Other acidic species variants also include, but are not limited to, unpaired disulfides, host cell proteins, and host nucleic acids, chromatographic materials, and media components. The acidic species may be the result of product preparation, storage, and / or purification. Attorney Docket No. ZEN-016WO1

[0059]

[0046] Amino acid. As used herein, the term “amino acid,” in its broadest sense, refers to any compound and / or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure H2N-C(H)(R)- COHO. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a d-amino acid; in some embodiments, an amino acid is an 1-amino acid. In some embodiments, an amino acid is a standard amino acid, which refers to any of the twenty standard 1-amino acids commonly found in naturally occurring peptides. In some embodiments, an amino acid is a nonstandard amino acid, which refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid is a synthetic amino acid, which encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and / or substitutions. Amino acids, including carboxyl- and / or amino-terminal amino acids in peptides, can be modified by methylation, amidation, acetylation, protecting groups, and / or substitution with other chemical groups that can change the peptide’s circulating half-life without adversely affecting their activity. Amino acids may participate in a disulfide bond. Amino acids may comprise one or posttranslational modifications, such as association with one or more chemical entities (e.g., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl moieties, isoprenoid groups, sulfate groups, polyethylene glycol moieties, lipid moi eties, carbohydrate moieties, biotin moieties, etc.). The term “amino acid” is used interchangeably with “amino acid residue,” and may refer to a free amino acid and / or to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide.

[0060]

[0047] Diluent: As used herein, the term "diluent" refers to a pharmaceutically acceptable (e.g., safe and non-toxic for administration to a human) diluting substance useful for the preparation of a reconstituted formulation. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution.

[0061]

[0048] Delivery: As used herein, the term “delivery” encompasses both local, subcutaneous and systemic delivery. Attorney Docket No. ZEN-016WO1

[0062]

[0049] Half-life'. As used herein, the term “half-life” is the time required for a quantity such as nucleic acid or protein concentration or activity to fall to half of its value as measured at the beginning of a time period.

[0063]

[0050] Stability: As used herein, the term "stable" refers to the ability of the therapeutic agent (e.g., a fusion protein) to maintain its therapeutic efficacy (e.g., all or the majority of its intended biological activity and / or physiochemical integrity) over extended periods of time. The stability of a therapeutic agent, and the capability of the pharmaceutical composition to maintain stability of such therapeutic agent, may be assessed over extended periods of time (e.g., for at least 1, 3, 6, 12, 18, 24, 30, 36 months or more). In general, pharmaceutical compositions described herein have been formulated such that they are capable of stabilizing, or alternatively slowing or preventing the degradation, of one or more therapeutic agents formulated therewith (e.g., CTLA4-Ig fusion proteins). In the context of a formulation a stable formulation is one in which the therapeutic agent therein essentially retains its physical and / or chemical integrity and biological activity upon storage and during processes (such as freeze / thaw, mechanical mixing and lyophilization). For protein stability, it can be measure by formation of high molecular weight (HMW) aggregates, loss of enzyme activity, generation of peptide fragments and shift of charge profiles. For the purpose of the present application, the term high molecular weight species (HMW) of the product and “aggregates” are used interchangeably.

[0064]

[0051] Suitable for subcutaneous delivery: As used herein, the phrase "suitable for subcutaneous delivery" or "formulation for subcutaneous delivery" as it relates to the pharmaceutical compositions of the present invention generally refers to the stability, viscosity, and solubility properties of such compositions, as well as the ability of such compositions to deliver an effective amount of protein contained therein to the targeted site of delivery.

[0065]

[0052] Patient: As used herein, the term “patient” or “subject” refers to any organism to which a provided composition may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, a patient is a human. A human includes pre- and post-natal forms. Attorney Docket No. ZEN-016WO1

[0066]

[0053] Pharmaceutically acceptable '. The term “pharmaceutically acceptable” as used herein, refers to substances that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0067]

[0054] Subject'. As used herein, the term “subject” refers to a human or any nonhuman animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre- and post-natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient.” A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.

[0068]

[0055] Substantially. As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0069]

[0056] Treating'. As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and / or reduce incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and / or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease. Attorney Docket No. ZEN-016WO1

[0070] DETAILED DESCRIPTION

[0071] CTLA-4-Ig Fusion Proteins

[0072]

[0057] In some embodiments, inventive compositions and methods provided by the present invention are used to deliver a CTLA-4-Ig fusion protein to a subject in need.

[0073]

[0058] In some embodiments, a human CTLA4 domain comprises an amino acid sequence of SEQ ID NO: 1.

[0074] MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIAS FVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSG NQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLW ILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPI N (SEQ ID NO: 1)

[0075]

[0059] In some embodiments, an extracellular domain of human CTLA4 comprises an amino acid sequence of SEQ ID NO: 2.

[0076] MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYM MGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGN GTQIYVIDPEPCPDS (SEQ ID NO: 2)

[0077]

[0060] In some embodiments, a CTLA4-Ig fusion protein comprises a first domain comprising a CTLA4 domain and a second domain comprising an IgG Fc region. In some embodiments, a CTLA4-Ig fusion protein comprises a CTLA4 domain comprising an amino acid sequence of SEQ ID NO: 3.

[0078] MHVAQPAVVLASSRGIASFVCEYASPGKHTEVRVTVLRQADSQVTEVCAANYM MGNELTFEDDSICTGTSSGNQVNLTIQGLRAMDTGLYICQVELMYPPPYYLGIGN GTQIYVIDPEPCPDSD (SEQ ID NO: 3)

[0079]

[0061] In some embodiments, a CTLA4-Ig fusion protein comprises an IgG Fc region, wherein the IgG Fc region comprises M428L and N434S substitutions. In some embodiments, a CTLA4-Ig fusion protein comprises an Fc region domain comprising an amino acid sequence of SEQ ID NO: 4.

[0080] PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKT Attorney Docket No. ZEN-016WO1

[0081] KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK (SEQ ID NO: 4)

[0082]

[0062] In some embodiments, a CTLA4-Ig fusion protein is ZB004. In some embodiments, a CTLA4-Ig fusion protein comprises a first domain comprising a CTLA4 region comprising an amino acid sequence of SEQ ID NO: 3 and a second domain comprising an IgG Fc region comprising an amino acid sequence of SEQ ID NO: 4. In some embodiments, a CTLA4-Ig fusion protein comprises a first CTLA4 domain and a second IgG Fc region domain, wherein the first domain and second domain are linked via a linker. In some embodiments, a linker comprises an amino acid sequence of SEQ ID NO: 5.

[0083] QEPKSSDKTHTSPPS (SEQ ID NO: 5)

[0084]

[0063] In some embodiments, a CTLA4-Ig fusion protein comprises a first domain comprising a CTLA4 region comprising an amino acid sequence of SEQ ID NO: 3 and a second domain comprising an IgG Fc region comprising an amino acid sequence of SEQ ID NO: 4, wherein the first domain and second domain are linked via a linker comprising an amino acid sequence of SEQ ID NO: 5. In some embodiments, the amino acid sequence of ZB004 is as provided in SEQ ID NO: 6.

[0085] MHVAQPAVVLASSRGIASFVCEYASPGKHTEVRVTVLRQADSQVTEVCAANYM MGNELTFEDDSICTGTSSGNQVNLTIQGLRAMDTGLYICQVELMYPPPYYLGIGN GTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH QDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVLHEALHSHYTQKSLSLSPGK (SEQ ID NO: 6)

[0086]

[0064] The present invention is drawn to CTLA4-Ig fusion proteins with improved

[0087] B7 affinities and enhanced T-cell inhibitory activities. Such fusion proteins are of benefit in a variety of applications as discussed in detail below.

[0088]

[0065] CTLA4 (Cytotoxic T-Lymphocyte Antigen 4), also referred to as CTLA-4 and also known as CD 152 (Cluster of differentiation 152), is a protein that plays an Attorney Docket No. ZEN-016WO1 important regulatory role in the immune system. In humans, the CTLA4 protein is encoded by the CTLA4 gene, the translated amino acid sequence of which is provided in SEQ ID NO: 1. CTLA4 is a member of the immunoglobulin superfamily, and is structurally homologous to CD28, a T cell costimulatory protein. CTLA4 binds to costimulatory ligands B7-1 and B7-2 on the surface of antigen presenting cells (APCs). CLTA4 inhibits immune response in two principal ways - it competes with CD28 for binding to B7-1 and B7-2 and thereby blocks costimulation, and it negatively signals to inhibit T cell activation. A particularly important region of the CTLA4 protein for the present invention is the extracellular domain (ECD) (SEQ ID NO:2), which mediates binding to B7-1 and B7-2 ligands on APCs.

[0089]

[0066] As a result of the critical role of the B7 co-stimulatory pathway in promoting and maintaining immune response, it is desirable to provide therapeutic agents designed to antagonize this pathway. Accordingly, the present invention provides CTLA4-Ig fusion protein.

[0090]

[0067] The invention provides CTLA4 Ig fusion proteins, which may also be referred to as immunoadhesins. “Immunoadhesin” refers to a protein wherein one or more polypeptides is operably linked to an Fc region. Immunoadhesin as used herein is a fusion protein synonymous with the terms “Fc fusion”, “Ig fusion”, “receptor Fc fusion”, “Ig chimera”, and “receptor globulin” (sometimes with dashes), and “TRAPs” as used in the prior art (Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9: 195-200, both entirely incorporated by reference). An immunoadhesin combines the Fc region of an immunoglobulin with a fusion partner, which in general can be any protein or small molecule that has specificity for a target protein. Thus, immunoadhesins have two principal portions - a target binding portion and an Fc portion. The target antigen binding portion may have specificity for virtually any target or target antigen. The Fc portion may bind to one or more Fc receptors or Fc ligands. Fusion partners may be linked to any region of an Fc region, including at the N- or C- termini, or at some residue in-between the termini. Fusion partners can be linked at the N- or C- terminus of the Fc region. While virtually any protein or small molecule may be linked to Fc to generate an Fc fusion, and thus target virtually any target, immunonoadhesins of the present invention comprise a CTLA4 or a variant of CTLA4 as a fusion partner. The Attorney Docket No. ZEN-016WO1 fusion of CTLA4 with an Ig Fc region is referred to herein as a CTLA4-Ig or CTLA4-Ig protein.

[0091] Fc Domains

[0092]

[0068] In some embodiments, a CTLA4-Ig fusion comprises an IgG Fc region or a variant thereof. In some embodiments, a CTLA4-Ig fusion protein comprises an IgGl, an IgG2, an IgG3, or an IgG4 Fc region or a variant thereof. In some embodiments, a CTLA4-Ig fusion protein comprises an IgGl Fc region or a variant thereof. In some embodiments, a CTLA4-Ig fusion protein comprises an IgG2 Fc region or a variant thereof. In some embodiments, a CTLA4-Ig fusion protein comprises an IgG3 Fc region or a variant thereof. In some embodiments, a CTLA4-Ig fusion protein comprises an IgG4 Fc region or a variant thereof. In some embodiments, a CTLA4-Ig fusion protein comprises a hybrid IgGl / IgG2 isotype Fc region or a variant thereof.

[0093]

[0069] In some embodiments, an IgGl Fc region comprises an amino acid sequence of: SEQ ID NO: 7.

[0094] PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7)

[0095]

[0070] In some embodiments, an IgG2 Fc region comprises an amino acid sequence of SEQ ID NO: 8.

[0096] PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 8)

[0097]

[0071] In some embodiments, an IgG3 Fc region comprises an amino acid sequence of SEQ ID NO: 9.

[0098] PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEV HNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKT Attorney Docket No. ZEN-016WO1

[0099] KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTT PPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 9)

[0100]

[0072] In some embodiments, an IgG4 Fc region comprises an amino acid sequence of SEQ ID NO: 10.

[0101] PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 10)

[0102]

[0073] In some embodiments, a CTLA4-Ig fusion protein comprises an IgG Fc region as described in as disclosed in WO2011103584A2 (hereby incorporated by reference in its entirety), or a variant thereof.

[0103] CTLA4 Variants

[0104]

[0074] The target binding portion or fusion partner of the fusion proteins of the invention are comprised of a CTLA4 protein. Thus, the fusion proteins of the invention are directed to bind B7-1, B7-2, and any other ligands or receptors, known or unknown, that are bound by CTLA4. The target binding portion of the invention may comprise an amino acid sequence that is made up of all, any, or part of the human CTLA4 protein (SEQ ID NO: 1). Preferably the fusion protein binding portion comprises all or part of the ECD of CTLA4 (SEQ ID NO:2). As described in the examples, the immunoadhesin binding is preferably a variant of CTLA4 that improves binding to B7-1, B7-2, or both B7-1 and B7-2 and in some cases shows preferential binding to one or the other.

[0105]

[0075] The CTLA4 variants comprise at least one amino acid modification in a native CTLA4 protein, generally the human CTLA4 protein depicted in SEQ ID NO: 1, and in most cases, the variants are within the extracellular domain as the entire CTLA4 protein is generally not used in the fusion proteins of the invention. In this embodiment, one or more modifications are made at one or more of the following positions (numbering as in SEQ ID NO:2): 29, 30, 31, 33, 35, 49, 51, 53, 59, 61, 63, 64, 93, 95, 97, 98, 102, 103, 104, 105 or 106. In some embodiments, the modification is one or more of the following Attorney Docket No. ZEN-016WO1 substitutions: A29E, A29F, A29H, A29K, A29N, A29Q, A29R, T30E, T30H, T30R, T30V, E31D, E31I, E31M, E31T, E31V, R33E, R33F, R33I, R33L, R33M, R33Q, R33T, R33W, R33Y, T35D, T35E, T35F, T35M, T35V, T35Y, A49D, A49E, A49F, A49T, A49W, A49Y, T51D, T51E, T51H, T51L, T51N, T51Q, T51R, T51S, T51V, M53E, M53F, M53H, M53Q, M53W, M53Y, T59H, T59I, T59L, T59N, T59Q, T59V, T59Y, L61A, L61D, L61E, L61F, L61G, L61H, L61I, L61K, L61M, L61N, L61P, L61Q, L61R, L61S, L61T, L61V, L61W, L61Y, D63E, S64K, S64R, S64Y, K93D, K93E, K93F, K93H, K93N, K93Q, K93R, K93S, K93T, K93V, K93W, K93Y, E95D, E95H, E95L, E95Q, E95Y, M97D, M97F, M97I, M97N, M97V, Y98F, Y98W, Y102F, Y102W, Y103D, Y103E, Y103F, Y103H, Y103N, Y103Q, Y103W, L104F, L104H, L104M, L104V, L104Y, G105D, G105E, I106E, and I106Y. Of particular use in some embodiments are CTLA4 variants that have one or more substitutions selected from A29H, T51N, M53Y, L61E, and K93Q, with combinations of particular use including A29H / K93Q, A29H / M53Y, A29H / T51N, T51N / K93Q, T51N / M53Y, A29H / L61E / K93Q, A29H / M53Y / K93Q, A29H / M53Y / L61E, A29H / T51N / L61E, M53Y / L61E / K93Q, T51N / L61E / K93Q, T51N / M53Y / L61E, A29H / M53Y / L61E / K93Q, A29H / T51N / L61E / K93Q, A29H / T51N / M53Y / K93Q, A29H / T51N / M53Y / L61E, T51N / M53Y / L61E / K93Q, and A29H / T51N / M53Y / L61E / K93Q.

[0106]

[0076] In some embodiments, a fusion protein of the present invention comprises a first CTLA4 domain as disclosed in WO2011103584A2 or a variant thereof. In some embodiments, a fusion protein of the present invention refers to a CTLA4-Ig immunoadhension as disclosed in WO2011103584A2, or a variant thereof.

[0107]

[0077] As for all the lists of positions and substitutions herein, it should be understood that combinations of individual substitutions can be made, of any and all possible combinations, and that any individual position or substitution can be independently included or excluded from the list of possibilities. In general, as compared to the wild-type or parent CTLA4 (or Fc region), generally the variants of the invention have 1, 2, 3, 4, or 5 amino acid substitutions in the CTLA4 region, although in some cases more substitutions can be used, as long as the desired function is preserved. Similarly, as described below, the Fc domain may have substitutions in this manner as well. Attorney Docket No. ZEN-016WO1

[0108]

[0078] As described elsewhere, the CTLA4 variants generally preserve or enhance binding to one or more of the CTLA4 ligands, such as enhanced binding to B7-1 and / or B7-2.

[0109] Amino Acid Modifications of CTLA4-Ig Fusion Proteins

[0110]

[0079] CTLA4-Ig fusion proteins disclosed herein may comprise a variant

[0111] CTLA4, a variant Fc region, or both a variant CTLA4 and a variant Fc region. A variant comprises one or more amino acid modifications relative to a parent CTLA4-Ig protein, wherein the amino acid modification(s) provide one or more optimized properties. By “modification” herein is meant an alteration in the physical, chemical, or sequence properties of a protein, polypeptide, antibody, or CTLA4-Ig immunoadhesin. By “amino acid” and “amino acid identity” as used herein is meant one of the 20 naturally occurring amino acids or any non-natural analogues that may be present at a specific, defined position. Thus "amino acid" as used herein means both naturally occurring and synthetic amino acids. For example, homophenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention. "Amino acid" also includes imino acid residues such as proline and hydroxyproline. The side chain may be in either the (R) or the (S) configuration. In some embodiments, the amino acids are in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradation. An amino acid modification can be an amino acid substitution, insertion, and / or deletion in a polypeptide sequence. By “amino acid substitution” or “substitution” herein is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid. By “amino acid insertion” or “insertion” as used herein is meant the addition of an amino acid at a particular position in a parent polypeptide sequence. By “amino acid deletion” or “deletion” as used herein is meant the removal of an amino acid at a particular position in a parent polypeptide sequence.

[0112]

[0080] A variant disclosed herein differs in amino acid sequence from its parent by virtue of at least one amino acid modification. By “parent polypeptide”, “parent protein”, “precursor polypeptide”, or “precursor protein” as used herein is meant an unmodified polypeptide that is subsequently modified to generate a variant. Said parent polypeptide may be a naturally occurring polypeptide, i.e. a WT or native protein, or a variant or Attorney Docket No. ZEN-016WO1 engineered version of a naturally occurring polypeptide. Parent polypeptide may refer to the polypeptide itself, compositions that comprise the parent polypeptide, or the amino acid sequence that encodes it. By “wild type”, “WT”, or “native” herein is meant an amino acid sequence or a nucleotide sequence that is found in nature, including allelic variations. A WT protein, including for example a WT CTLA4 or WT Fc region protein, has an amino acid sequence or a nucleotide sequence that has not been intentionally modified. CTLA4-Ig fusion proteins as disclosed herein may have more than one amino acid modification as compared to the parent, for example from about one to fifty amino acid modifications, e.g., from about one to ten amino acid modifications, from about one to about five amino acid modifications, etc. compared to the parent. Thus, the sequences of the variants and those of the parent polypeptide are substantially homologous. For example, the variant sequences herein will possess about 80% homology with the parent sequence, e.g., at least about 90% homology, at least about 95% homology, at least about 98% homology, at least about 99% homology, etc. Modifications disclosed herein also include glycoform modifications. Modifications may be made genetically using molecular biology, or may be made enzymatically or chemically.

[0113]

[0081] Variants disclosed herein are defined according to the amino acid modifications that compose them. Thus, for example, the substitution T51N in CTLA4 refers a CTLA4 variant in which the threonine at position 51 is replaced with asparagine. As another example, the substitution N434S in the Fc region refers to an Fc variant in which the asparagine at position 434 is replaced with serine. Likewise, M428L / N434S defines an Fc variant with the substitutions M428L and N434S relative to the parent Fc polypeptide. The identity of the WT amino acid may be unspecified, in which case the aforementioned variant is referred to as 428L / 434S. It is noted that the order in which substitutions are provided is arbitrary, that is to say that, for example, 428L / 434S is the same Fc variant as 434S / 428L.

[0114]

[0082] For modifications in CTLA4, numbering of positions herein is according to the sequential numbering of the extracellular region of CTLA4 provided in SEQ ID NO:2. Antibody constant region and Fc region positions discussed herein are numbered according to the EU index or EU numbering scheme (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda, hereby entirely incorporated by reference). The EU index Attorney Docket No. ZEN-016WO1 or EU index as in Kabat or EU numbering scheme refers to the numbering of the EU antibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, hereby entirely incorporated by reference).

[0115]

[0083] The goal of the variants herein is to provide one or more optimized properties, typically by altering affinity for a target ligand or Fc receptor. Affinity may be enhanced or reduced relative to a parent protein. By “greater affinity” or “improved affinity” or “enhanced affinity” or "better affinity" than a parent Fc polypeptide, as used herein is meant that a variant binds to a ligand or receptor with a significantly higher equilibrium constant of association (KA or Ka) or lower equilibrium constant of dissociation (KD or Kd) than the parent polypeptide done under the same conditions, for example, when the amounts of variant and parent polypeptide in the binding assay are essentially the same.

[0116]

[0084] For example, a CTLA4 variant with improved B7-2 binding affinity may display from about 1.2, 1.5, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, fold to about 100 fold or more , e.g. from about 2 fold to about 20 fold improvement in B7-2 binding affinity compared to the parent CTLA4 polypeptide, where B7-2 binding affinity is determined, for example, by the binding methods disclosed herein, including but not limited to Biacore™, by one skilled in the art.

[0117]

[0085] Accordingly, by “reduced affinity” as compared to a parent polypeptide as used herein is meant that a variant binds a ligand or receptor with significantly lower KA or higher KD than the parent polypeptide. Greater or reduced affinity can also be defined relative to an absolute level of affinity.

[0118]

[0086] The fusion proteins herein preferably comprise a variant CTLA4. CTLA4 variants may improve binding to B7-1, B7-2, or both B7-1 and B7-2. CTLA4 variants may improve binding selectively to B7-2 relative to B7-1. That is, variants may enhance affinity of CTLA4 for B7-2, but either reduce affinity for B7-1, not affect affinity for B7- 1, or improve affinity for B7-1 less than the affinity improvement to B7-2. Alternatively, variants may improve binding selectively to B7-1 relative to B7-2.

[0119]

[0087] The fusion proteins herein preferably comprise an Fc variant. The Fc variants disclosed herein may be optimized for improved or reduced binding to Fc receptors or Fc ligands. By “Fc receptor" or "Fc ligand” as used herein is meant a Attorney Docket No. ZEN-016WO1 molecule, preferably a polypeptide, from any organism that binds to the Fc region of an antibody to form an Fc-ligand complex. Fc ligands include but are not limited to FcyRs, FcyRs, FcyRs, FcRn, Clq, C3, mannan binding lectin, mannose receptor, staphylococcal protein A, streptococcal protein G, and viral FcyR. Fc ligands also include Fc receptor homologs (FcRH), which are a family of Fc receptors that are homologous to the FcyRs. Fc ligands may include undiscovered molecules that bind Fc.

[0120]

[0088] In some embodiments, CTLA4-Ig fusion proteins disclosed herein incorporate Fc variants that improve FcRn binding. Such variants may enhance the in vivo pharmacokinetic properties of the CTLA4-Ig fusion proteins. Variants that increase binding to FcRn and / or improve pharmacokinetic properties include but are not limited to substitutions at positions 259, 308, 428, and 434, including but not limited to for example 2591, 308F, 428L, 428M, 434S, 434H, 434F, 434Y, and 434M (US SN 12 / 341,769, filed Dec. 22, 2008, entitled “Fc Variants with Altered Binding to FcRn”, entirely incorporated by reference). Other variants that increase Fc binding to FcRn include but are not limited to: 250E, 250Q, 428L, 428F, 250Q / 428L (Hinton et al., 2004, J. Biol. Chem. 279(8): 6213-6216, Hinton et al. 2006 Journal of Immunology 176:346-356), 256A, 272A, 286A, 305A, 307A, 307Q, 311A, 312A, 376A, 378Q, 380A, 382A, 434A (Shields et al, Journal of Biological Chemistry, 2001, 276(9):6591-6604, entirely incorporated by reference), 252F, 252T, 252Y, 252W, 254T, 256S, 256R, 256Q, 256E, 256D, 256T, 309P, 31 IS, 433R, 433 S, 4331, 433P, 433Q, 434H, 434F, 434Y, 252Y / 254T / 256E, 433K / 434F / 436H, 308T / 309P / 311S (Dall Acqua et al. Journal of Immunology, 2002, 169:5171-5180, Dall'Acqua et al., 2006, Journal of Biological Chemistry 281 :23514-23524, entirely incorporated by reference). Other modifications for modulating FcRn binding are described in Yeung et al., 2010, J Immunol, 182:7663-7671.

[0121]

[0089] Other Fc modifications for use in the present invention include variants that reduce or ablate binding to FcyRs and / or complement proteins, thereby reducing or ablating Fc-mediated effector functions such as ADCC, ADCP, and CDC. Such variants are also referred to herein as “knockout variants” or “KO variants”. Variants that reduce binding to FcyRs and complement are useful for reducing unwanted interactions mediated by the Fc region and for tuning the selectivity of the CTLA4-Ig fusion proteins. Knockout variants are described in USSN11 / 981,606, filed 10 / 31 / 2007, entitled “Fc Variants with Attorney Docket No. ZEN-016WO1

[0122] Optimized Properties, herein expressly incorporated by reference herein. Modifications include but are not limited substitutions, insertions, and deletions at positions 234, 235, 236, 237, 267, 269, 325, and 328, wherein numbering is according to the EU index. Substitutions include but are not limited to 234G, 235G, 236R, 237K, 267R, 269R, 325L, and 328R, wherein numbering is according to the EU index. Variants may be used in the context of any IgG isotype or IgG isotype Fc region, including but not limited to human IgGl, IgG2, IgG3, and / or IgG4. IgG Fc regions for reducing FcyR and complement binding and reducing Fc-mediated effector functions are IgG2 and IgG4 Fc regions. Hybrid isotypes may also be useful, for example hybrid IgGl / IgG2 isotypes as described in USSN11 / 256,060. Other modifications for reducing FcyR and complement interactions include but are not limited to substitutions 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 33 IS, 220S, 226S, 229S, 238S, 233P, and 234V, as well as removal of the glycosylation at position 297 by mutational or enzymatic means or by production in organisms such as bacteria that do not glycosylate proteins. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691, incorporated by reference in its entirety.

[0123]

[0090] Fc modifications that improve binding to FcyRs and / or complement may also find use in the CTLA4-Ig fusion proteins herein. Such Fc variants may enhance Fc- mediated effector functions such as ADCC, ADCP, and / or CDC. Modifications for improving FcyR and complement binding are described in USSN11 / 124,620 and USSN11 / 396,495, expressly incorporated herein by reference. Fc modifications comprise a substitution at a position selected from the group consisting of 236, 239, 268, 324, and 332, wherein numbering is according to the EU index. Substitutions include but are not limited to 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E. Variants include but are not limited to 239D / 332E, 236A / 332E, 236A / 239D / 332E, 268F / 324T, 267E / 268F, 267E / 324T, and 267E / 268F / 324T. Other modifications for enhancing FcyR and complement interactions include but are not limited to substitutions 298A, 333A, 334A, 326A, 2471, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 3051, and 396L. These and other modifications are reviewed in Strohl, 2009, ibid.

[0124]

[0091] In one embodiment, the CTLA4-Ig fusion proteins disclosed herein may incorporate Fc variants that enhance affinity for an inhibitory receptor FcyRIIb. Such Attorney Docket No. ZEN-016WO1 variants may provide the CTLA4-Ig immunoadhesins herein with immunomodulatory activities related to FcyRIIb cells, including for example B cells and monocytes. In one embodiment, the Fc variants provide selectively enhanced affinity to FcyRIIb relative to one or more activating receptors. Modifications for altering binding to FcyRIIb are described in USSN 12 / 156,183, filed May 30, 2008, entitled “Methods and Compositions for Inhibiting CD32b Expressing Cells”, herein expressly incorporated by reference. In particular, Fc variants that improve binding to FcyRIIb may include one or more modifications at a position selected from the group consisting of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328, and 332, according to the EU index. Preferable substitutions for enhancing FcyRIIb affinity include but are not limited to 234D, 234E, 234W, 235D, 235F, 235R, 235Y, 236D, 236N, 237D, 237N, 239D, 239E, 266M, 267D, 267E, 268D, 268E, 327D, 327E, 328F, 328W, 328Y, and 332E. More preferably, substitutions include but are not limited to 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F, 328W, and 328Y. Fc variants for enhancing binding to FcyRIIb include but are not limited to 235Y / 267E, 236D / 267E, 239D / 268D, 239D / 267E, 267E / 268D, 267E / 268E, and 267E / 328F.

[0125]

[0092] CTLA4-Ig fusion proteins described herein can incorporate Fc modifications in the context of any IgG isotype or IgG isotype Fc region, including but not limited to human IgGl, IgG2, IgG3, and / or IgG4. The IgG isotype may be selected such as to alter FcyR- and / or complement- mediated effector function(s). Hybrid IgG isotypes may also be useful. For example, USSN11 / 256,060 describes a number of hybrid IgGl / IgG2 constant regions that may find use in the particular invention. In some embodiments of the invention, CTLA4-Ig fusion proteins may comprise means for isotypic modifications, that is, modifications in a parent IgG to the amino acid type in an alternate IgG. For example, an IgGl / IgG3 hybrid variant may be constructed by a substitutional means for substituting IgGl positions in the CH2 and / or CH3 region with the amino acids from IgG3 at positions where the two isotypes differ. Thus, a hybrid variant IgG antibody may be constructed that comprises one or more substitutional means, e.g., 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R, and 436F. In other embodiments of the invention, an IgGl / IgG2 hybrid variant may be constructed by a substitutional means for substituting IgG2 positions in the CH2 and / or CH3 region Attorney Docket No. ZEN-016WO1 with amino acids from IgGl at positions where the two isotypes differ. Thus, a hybrid variant IgG antibody may be constructed that comprises one or more substitutional means, e.g., one or more of the following amino acid substations: 233E, 234L, 235L, -236G (referring to an insertion of a glycine at position 236), and 327A.

[0126]

[0093] As will be appreciated by those in the art, the disclosure of individual and combination variants in the Fc region can be independently and optionally combined with any of the CTLA4 variants disclosed herein. That is, as described herein, CTLA4 variants are individually and optionally selected and / or combined within the set of disclosed variants, in any combination. Similarly, the lists above of suitable Fc domain variants can be individually and optionally combined in any way, not only within the Fc region but with any CTLA4 variants. That is, a CTLA4 variant may be selected that comprises a number of variants, for example A29H / T51N / L61E / K93Q, and these variants can be combined with Fc domain variants, such as 239D / 332E, and / or 428L / 434S. Thus, the disclosure of a “list” of possible individual variants is meant to include any and all possible combinations within the list as well as with other lists of variants for the same or other purposes.

[0127] Stable Formulations

[0128]

[0094] Formulating proteins can present significant challenges due to various factors, including stability and viscosity. These challenges are particularly prevalent in high concentration formulations with a protein concentration of >50 mg / mL, requiring robust processes for formulation, stabilization, development, and manufacturing. Moreover, high concentration formulations can undergo hydrolytically driven chemical degradations like deamidation, isomerization, and cleavage of peptide bonds, leading to irreversible covalent aggregates that may affect safety and efficacy. To mitigate these risks, formulating high concentration protein products requires addressing challenges related to physical stability, solubility, viscosity, aggregation, and immunogenicity.

[0129]

[0095] In some embodiments, a stable formulation of the present invention comprises one or more excipients. One skilled in the part will appreciate that the term "excipient” encompasses various substances, including but not limited to, buffers, surfactants, antioxidants, etc. In some embodiments, the stable formulation has a Attorney Docket No. ZEN-016WO1 combined concentration of excipients greater than 300 mM. In some embodiments, the stable formulation has a combined concentration of excipients greater than 325 mM. In some embodiments, the stable formulation has a combined concentration of excipients greater than 350 mM. In some embodiments, the stable formulation has a combined concentration of excipients greater than 375 mM. In some embodiments, the stable formulation has a combined concentration of excipients greater than 400 mM.

[0130] Buffers

[0131]

[0096] In some embodiments, a stable formulation as described herein comprises a buffer to control pH. Various buffers can be present in the stable formulation of the present disclosure. Suitable buffers generally include, for example, acetate, citrate, histidine, phosphate, succinate, tri s(hydroxymethyl)aminom ethane ("Tris") and other organic acids. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable buffer, wherein the pharmaceutically acceptable buffer is phosphoric acid buffer, citric acid buffer, acetic acid buffer, citrate buffer, ascorbic acid buffer, glutamic acid buffer, lactic acid buffer, maleic acid buffer, trometamol buffer, and gluconic acid buffer, acetate buffer, succinate buffer, phosphate buffer, histidine buffer or any combination thereof. Unless stated otherwise, a buffer comprises both acid and its conjugate base sufficient to adjust and maintain the pH. One of ordinary skill in the art will be able to ascertain an acid and its conjugate base used in the specific buffer.

[0132]

[0097] In some embodiments, a stable formulation comprises a buffer at a concentration of 5 mM to 80 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of 5 mM to 60 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of 5 mM to 40 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of 5 mM to 30 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of 5 mM to 20 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of 5 mM to 10 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of 10 mM to 50 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of 20 mM to 40 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of less than 80 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of Attorney Docket No. ZEN-016WO1 less than 70 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of less than 60 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of less than 55 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of less than 50 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of less than 45 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of less than 40 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of less than 35 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of less than 30 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of less than 25 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of 5 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of 10 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of 15 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of 20 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of 25 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of 30 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of 35 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of 40 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of 45 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of 50 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of 55 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of 60 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of 70 mM. In some embodiments, a stable formulation comprises a buffer at a concentration of 80 mM.

[0133]

[0098] In some embodiments, a stable formulation comprises sodium phosphate.

[0134] In some embodiments, a stable formulation comprises sodium phosphate at a concentration of 5 mM to 80 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of 5 mM to 60 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of 5 mM to 40 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of 5 Attorney Docket No. ZEN-016WO1 mM to 30 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of 5 mM to 20 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of 5 mM to 10 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of 10 mM to 50 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of 20 mM to 40 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of less than 80 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of less than 70 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of less than 60 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of less than 55 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of less than 50 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of less than 45 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of less than 40 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of less than 35 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of less than 30 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of less than 25 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of 5 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of 10 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of 15 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of 20 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of 25 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of 30 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of 35 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of 40 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of 45 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of 50 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of 55 Attorney Docket No. ZEN-016WO1 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of 60 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of 70 mM. In some embodiments, a stable formulation comprises sodium phosphate at a concentration of 80 mM.

[0135]

[0099] In some embodiments, a stable formulation comprises succinate. In some embodiments, a stable formulation comprises succinate at a concentration of 5 mM to 80 mM. In some embodiments, a stable formulation comprises succinate at a concentration of 5 mM to 60 mM. In some embodiments, a stable formulation comprises succinate at a concentration of 5 mM to 40 mM. In some embodiments, a stable formulation comprises succinate at a concentration of 5 mM to 30 mM. In some embodiments, a stable formulation comprises succinate at a concentration of 5 mM to 20 mM. In some embodiments, a stable formulation comprises succinate at a concentration of 5 mM to 10 mM. In some embodiments, a stable formulation comprises succinate at a concentration of 10 mM to 50 mM. In some embodiments, a stable formulation comprises succinate at a concentration of 20 mM to 40 mM. In some embodiments, a stable formulation comprises succinate at a concentration of less than 80 mM. In some embodiments, a stable formulation comprises succinate at a concentration of less than 70 mM. In some embodiments, a stable formulation comprises succinate at a concentration of less than 60 mM. In some embodiments, a stable formulation comprises succinate at a concentration of less than 55 mM. In some embodiments, a stable formulation comprises succinate at a concentration of less than 50 mM. In some embodiments, a stable formulation comprises succinate at a concentration of less than 45 mM. In some embodiments, a stable formulation comprises succinate at a concentration of less than 40 mM. In some embodiments, a stable formulation comprises succinate at a concentration of less than 35 mM. In some embodiments, a stable formulation comprises succinate at a concentration of less than 30 mM. In some embodiments, a stable formulation comprises succinate at a concentration of less than 25 mM. In some embodiments, a stable formulation comprises succinate at a concentration of 5 mM. In some embodiments, a stable formulation comprises succinate at a concentration of 10 mM. In some embodiments, a stable formulation comprises succinate at a concentration of 15 mM. In some embodiments, a stable formulation comprises succinate at a concentration of 20 mM. In some embodiments, a stable formulation comprises succinate at a concentration of 25 mM. In Attorney Docket No. ZEN-016WO1 some embodiments, a stable formulation comprises succinate at a concentration of 30 mM. In some embodiments, a stable formulation comprises succinate at a concentration of 35 mM. In some embodiments, a stable formulation comprises succinate at a concentration of 40 mM. In some embodiments, a stable formulation comprises succinate at a concentration of 45 mM. In some embodiments, a stable formulation comprises succinate at a concentration of 50 mM. In some embodiments, a stable formulation comprises succinate at a concentration of 55 mM. In some embodiments, a stable formulation comprises succinate at a concentration of 60 mM. In some embodiments, a stable formulation comprises succinate at a concentration of 70 mM. In some embodiments, a stable formulation comprises succinate at a concentration of 80 mM.

[0136]

[0100] In some embodiments, a stable formulation comprises acetate. In some embodiments, a stable formulation comprises acetate at a concentration of 5 mM to 80 mM. In some embodiments, a stable formulation comprises acetate at a concentration of 5 mM to 60 mM. In some embodiments, a stable formulation comprises acetate at a concentration of 5 mM to 40 mM. In some embodiments, a stable formulation comprises acetate at a concentration of 5 mM to 30 mM. In some embodiments, a stable formulation comprises acetate at a concentration of 5 mM to 20 mM. In some embodiments, a stable formulation comprises acetate at a concentration of 5 mM to 10 mM. In some embodiments, a stable formulation comprises acetate at a concentration of 10 mM to 50 mM. In some embodiments, a stable formulation comprises acetate at a concentration of 20 mM to 40 mM. In some embodiments, a stable formulation comprises acetate at a concentration of less than 80 mM. In some embodiments, a stable formulation comprises acetate at a concentration of less than 70 mM. In some embodiments, a stable formulation comprises acetate at a concentration of less than 60 mM. In some embodiments, a stable formulation comprises acetate at a concentration of less than 55 mM. In some embodiments, a stable formulation comprises acetate at a concentration of less than 50 mM. In some embodiments, a stable formulation comprises acetate at a concentration of less than 45 mM. In some embodiments, a stable formulation comprises acetate at a concentration of less than 40 mM. In some embodiments, a stable formulation comprises acetate at a concentration of less than 35 mM. In some embodiments, a stable formulation comprises acetate at a concentration of less than 30 mM. In some embodiments, a stable formulation comprises acetate at a concentration of less than 25 mM. In some Attorney Docket No. ZEN-016WO1 embodiments, a stable formulation comprises acetate at a concentration of 5 mM. In some embodiments, a stable formulation comprises acetate at a concentration of 10 mM. In some embodiments, a stable formulation comprises acetate at a concentration of 15 mM. In some embodiments, a stable formulation comprises acetate at a concentration of 20 mM. In some embodiments, a stable formulation comprises acetate at a concentration of 25 mM. In some embodiments, a stable formulation comprises acetate at a concentration of 30 mM. In some embodiments, a stable formulation comprises acetate at a concentration of 35 mM. In some embodiments, a stable formulation comprises acetate at a concentration of 40 mM. In some embodiments, a stable formulation comprises acetate at a concentration of 45 mM. In some embodiments, a stable formulation comprises acetate at a concentration of 50 mM. In some embodiments, a stable formulation comprises acetate at a concentration of 55 mM. In some embodiments, a stable formulation comprises acetate at a concentration of 60 mM. In some embodiments, a stable formulation comprises acetate at a concentration of 70 mM. In some embodiments, a stable formulation comprises acetate at a concentration of 80 mM.

[0137]

[0101] In some embodiments, a stable formulation comprises histidine. In some embodiments, a stable formulation comprises histidine at a concentration of 5 mM to 80 mM. In some embodiments, a stable formulation comprises histidine at a concentration of 5 mM to 60 mM. In some embodiments, a stable formulation comprises histidine at a concentration of 5 mM to 40 mM. In some embodiments, a stable formulation comprises histidine at a concentration of 5 mM to 30 mM. In some embodiments, a stable formulation comprises histidine at a concentration of 5 mM to 20 mM. In some embodiments, a stable formulation comprises histidine at a concentration of 5 mM to 10 mM. In some embodiments, a stable formulation comprises histidine at a concentration of 10 mM to 50 mM. In some embodiments, a stable formulation comprises histidine at a concentration of 20 mM to 40 mM. In some embodiments, a stable formulation comprises histidine at a concentration of less than 80 mM. In some embodiments, a stable formulation comprises histidine at a concentration of less than 70 mM. In some embodiments, a stable formulation comprises histidine at a concentration of less than 60 mM. In some embodiments, a stable formulation comprises histidine at a concentration of less than 55 mM. In some embodiments, a stable formulation comprises histidine at a concentration of less than 50 mM. In some embodiments, a stable formulation comprises Attorney Docket No. ZEN-016WO1 histidine at a concentration of less than 45 mM. In some embodiments, a stable formulation comprises histidine at a concentration of less than 40 mM. In some embodiments, a stable formulation comprises histidine at a concentration of less than 35 mM. In some embodiments, a stable formulation comprises histidine at a concentration of less than 30 mM. In some embodiments, a stable formulation comprises histidine at a concentration of less than 25 mM. In some embodiments, a stable formulation comprises histidine at a concentration of 5 mM. In some embodiments, a stable formulation comprises histidine at a concentration of 10 mM. In some embodiments, a stable formulation comprises histidine at a concentration of 15 mM. In some embodiments, a stable formulation comprises histidine at a concentration of 20 mM. In some embodiments, a stable formulation comprises histidine at a concentration of 25 mM. In some embodiments, a stable formulation comprises histidine at a concentration of 30 mM. In some embodiments, a stable formulation comprises histidine at a concentration of 35 mM. In some embodiments, a stable formulation comprises histidine at a concentration of 40 mM. In some embodiments, a stable formulation comprises histidine at a concentration of 45 mM. In some embodiments, a stable formulation comprises histidine at a concentration of 50 mM. In some embodiments, a stable formulation comprises histidine at a concentration of 55 mM. In some embodiments, a stable formulation comprises histidine at a concentration of 60 mM. In some embodiments, a stable formulation comprises histidine at a concentration of 70 mM. In some embodiments, a stable formulation comprises histidine at a concentration of 80 mM.

[0138]

[0102] In some embodiments, a stable formulation comprises citrate. In some embodiments, a stable formulation comprises citrate at a concentration of 5 mM to 80 mM. In some embodiments, a stable formulation comprises citrate at a concentration of 5 mM to 60 mM. In some embodiments, a stable formulation comprises citrate at a concentration of 5 mM to 40 mM. In some embodiments, a stable formulation comprises citrate at a concentration of 5 mM to 30 mM. In some embodiments, a stable formulation comprises citrate at a concentration of 5 mM to 20 mM. In some embodiments, a stable formulation comprises citrate at a concentration of 5 mM to 10 mM. In some embodiments, a stable formulation comprises citrate at a concentration of 10 mM to 50 mM. In some embodiments, a stable formulation comprises citrate at a concentration of 20 mM to 40 mM. In some embodiments, a stable formulation comprises citrate at a Attorney Docket No. ZEN-016WO1 concentration of less than 80 mM. In some embodiments, a stable formulation comprises citrate at a concentration of less than 70 mM. In some embodiments, a stable formulation comprises citrate at a concentration of less than 60 mM. In some embodiments, a stable formulation comprises citrate at a concentration of less than 55 mM. In some embodiments, a stable formulation comprises citrate at a concentration of less than 50 mM. In some embodiments, a stable formulation comprises citrate at a concentration of less than 45 mM. In some embodiments, a stable formulation comprises citrate at a concentration of less than 40 mM. In some embodiments, a stable formulation comprises citrate at a concentration of less than 35 mM. In some embodiments, a stable formulation comprises citrate at a concentration of less than 30 mM. In some embodiments, a stable formulation comprises citrate at a concentration of less than 25 mM. In some embodiments, a stable formulation comprises citrate at a concentration of 5 mM. In some embodiments, a stable formulation comprises citrate at a concentration of 10 mM. In some embodiments, a stable formulation comprises citrate at a concentration of 15 mM. In some embodiments, a stable formulation comprises citrate at a concentration of 20 mM. In some embodiments, a stable formulation comprises citrate at a concentration of 25 mM. In some embodiments, a stable formulation comprises citrate at a concentration of 30 mM. In some embodiments, a stable formulation comprises citrate at a concentration of 35 mM. In some embodiments, a stable formulation comprises citrate at a concentration of 40 mM. In some embodiments, a stable formulation comprises citrate at a concentration of 45 mM. In some embodiments, a stable formulation comprises citrate at a concentration of 50 mM. In some embodiments, a stable formulation comprises citrate at a concentration of 55 mM. In some embodiments, a stable formulation comprises citrate at a concentration of 60 mM. In some embodiments, a stable formulation comprises citrate at a concentration of 70 mM. In some embodiments, a stable formulation comprises citrate at a concentration of 80 mM.

[0139]

[0103] In some embodiments, a stable formulation comprises tri s(hydroxymethyl)aminom ethane (Tris). In some embodiments, a stable formulation comprises Tris at a concentration of 5 mM to 80 mM. In some embodiments, a stable formulation comprises Tris at a concentration of 5 mM to 60 mM. In some embodiments, a stable formulation comprises Tris at a concentration of 5 mM to 40 mM. In some embodiments, a stable formulation comprises Tris at a concentration of 5 mM to 30 mM. In some embodiments, a stable formulation comprises Tris at a concentration of 5 mM to Attorney Docket No. ZEN-016WO1

[0140] 20 mM. In some embodiments, a stable formulation comprises Tris at a concentration of 5 mM to 10 mM. In some embodiments, a stable formulation comprises Tris at a concentration of 10 mM to 50 mM. In some embodiments, a stable formulation comprises Tris at a concentration of 20 mM to 40 mM. In some embodiments, a stable formulation comprises Tris at a concentration of less than 80 mM. In some embodiments, a stable formulation comprises Tris at a concentration of less than 70 mM. In some embodiments, a stable formulation comprises Tris at a concentration of less than 60 mM. In some embodiments, a stable formulation comprises Tris at a concentration of less than 55 mM. In some embodiments, a stable formulation comprises Tris at a concentration of less than 50 mM. In some embodiments, a stable formulation comprises Tris at a concentration of less than 45 mM. In some embodiments, a stable formulation comprises Tris at a concentration of less than 40 mM. In some embodiments, a stable formulation comprises Tris at a concentration of less than 35 mM. In some embodiments, a stable formulation comprises Tris at a concentration of less than 30 mM. In some embodiments, a stable formulation comprises Tris at a concentration of less than 25 mM. In some embodiments, a stable formulation comprises Tris at a concentration of 5 mM. In some embodiments, a stable formulation comprises Tris at a concentration of 10 mM. In some embodiments, a stable formulation comprises Tris at a concentration of 15 mM. In some embodiments, a stable formulation comprises Tris at a concentration of 20 mM. In some embodiments, a stable formulation comprises Tris at a concentration of 25 mM. In some embodiments, a stable formulation comprises Tris at a concentration of 30 mM. In some embodiments, a stable formulation comprises Tris at a concentration of 35 mM. In some embodiments, a stable formulation comprises Tris at a concentration of 40 mM. In some embodiments, a stable formulation comprises Tris at a concentration of 45 mM. In some embodiments, a stable formulation comprises Tris at a concentration of 50 mM. In some embodiments, a stable formulation comprises Tris at a concentration of 55 mM. In some embodiments, a stable formulation comprises Tris at a concentration of 60 mM. In some embodiments, a stable formulation comprises Tris at a concentration of 70 mM. In some embodiments, a stable formulation comprises Tris at a concentration of 80 mM. pH

[0141]

[0104] The pH of a stable formulation of a biologic protein is crucial for ensuring the stability and efficacy of the therapeutic. Low pH can cause proteins to unfold and Attorney Docket No. ZEN-016WO1 aggregate, especially during processes like freeze-thaw cycles. Maintaining an optimal pH is essential to minimize aggregation, which can cause unwanted immunogenic responses in patients. Therefore, selecting the appropriate pH and buffer system is vital in developing a formulation for proteins, and is specific to each protein.

[0142]

[0105] In some embodiments, a stable formulation as described herein comprises a particular pH. One skilled in the art will appreciate that pH can influence the properties of a formulation such as viscosity and stability (e.g., monomer purity, increase in high molecular weight (HMW) species, loss of main charge variant and charge distribution). In some embodiments, a stable formulation has a pH of 6.0 to 8.0. In some embodiments, a stable formulation has a pH of 6.7 to 7.7. In some embodiments, a stable formulation has a pH of 6.0 to 7.0. In some embodiments, a stable formulation has a pH of 6.2 to 7.8. In some embodiments, a stable formulation has a pH of 6.4 to 7.6. In some embodiments, a stable formulation has a pH of 6.6 to 7.4. In some embodiments, a stable formulation has a pH of 6.8 to 7.2. In some embodiments, a stable formulation has a pH of 7.0 to 7.5.

[0143]

[0106] In some embodiments, a stable formulation comprises a pH of less than 8.0. In some embodiments, a stable formulation comprises a pH of less than 7.0. In some embodiments, a stable formulation comprises a pH of less than 6.0. In some embodiments, the stable formulation comprises a pH of 8.0. In some embodiments, the stable formulation comprises a pH of 7.9. In some embodiments, the stable formulation comprises a pH of 7.8. In some embodiments, the stable formulation comprises a pH of

[0144] 7.7. In some embodiments, the stable formulation comprises a pH of 7.6. In some embodiments, the stable formulation comprises a pH of 7.5. In some embodiments, the stable formulation comprises a pH of 7.4. In some embodiments, the stable formulation comprises a pH of 7.3. In some embodiments, the stable formulation comprises a pH of 7.2. In some embodiments, the stable formulation comprises a pH of 7.1. In some embodiments, the stable formulation comprises a pH of 7.0. In some embodiments, the stable formulation comprises a pH of 6.9. In some embodiments, the stable formulation comprises a pH of 6.8. In some embodiments, the stable formulation comprises a pH of

[0145] 6.7. In some embodiments, the stable formulation comprises a pH of 6.6. In some embodiments, the stable formulation comprises a pH of 6.5. In some embodiments, the stable formulation comprises a pH of 6.4. In some embodiments, the stable formulation comprises a pH of 6.3. In some embodiments, the stable formulation comprises a pH of Attorney Docket No. ZEN-016WO1

[0146] 6.2. In some embodiments, the stable formulation comprises a pH of 6.1. In some embodiments, the stable formulation comprises a pH of 6.0. In some embodiments, the stable formulation comprises a pH of 5.9. In some embodiments, the stable formulation comprises a pH of 5.9. In some embodiments, the stable formulation comprises a pH of 5.8. In some embodiments, the stable formulation comprises a pH of 5.7. In some embodiments, the stable formulation comprises a pH of 5.6. In some embodiments, the stable formulation comprises a pH of 5.5.

[0147] Protein Concentration

[0148]

[0107] In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 10 mg / mL to 200 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 20 mg / mL to 160 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration from 30 mg / mL to 120 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration from 40 mg / mL to 80 mg / mL. In some embodiments, the stable formulation comprises a CTLA-4-Ig fusion protein at a concentration from 50 mg / mL to 60 mg / mL.

[0149]

[0108] In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 200 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 195 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 190 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 185 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 180 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 175 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 170 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 165 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 160 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 155 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion Attorney Docket No. ZEN-016WO1 protein at a concentration of 150 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 145 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 140 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 135 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 130 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 125 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 120 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 115 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 110 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 105 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 100 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 95 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 90 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 85 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 80 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 75 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 70 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 65 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 60 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 55 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 50 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 45 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 40 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 35 mg / mL. Attorney Docket No. ZEN-016WO1

[0150] In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 30 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 25 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 20 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 15 mg / mL. In some embodiments, a stable formulation comprises a CTLA-4-Ig fusion protein at a concentration of 10 mg / mL.

[0151] Sugar

[0152]

[0109] In some embodiments, a stable formulation further comprises a sugar.

[0153] Suitable sugars include monosaccharides, disaccharides, and / or polysaccharides. In some embodiments, the sugar comprises sucrose, trehalose, fructose, maltose, galactose, glucose, D-mmanose, sorbose, lactose, cellobiose, raffinose, meleitose, maltodextrins, dextrans, or starches. In some embodiments, the sugar comprises sucrose. In some embodiments, the sugar comprises trehalose.

[0154] [HO] In some embodiments, a stable formulation comprises a sugar at a concentration of 0.1% (w / v) to 15% (w / v). In some embodiments, a sugar is present at a concentration of 1% (w / v) to 10% (w / v). In some embodiments, a sugar is present at a concentration of 4% (w / v) to 8% (w / v). In some embodiments, a sugar is present at a concentration of 1% (w / v). In some embodiments, a sugar is present at a concentration of 1.5% (w / v). In some embodiments, a sugar is present at a concentration of 2% (w / v). In some embodiments, a sugar is present at a concentration of 2.5% (w / v). In some embodiments, a sugar is present at a concentration of 3% (w / v). In some embodiments, a sugar is present at a concentration of 3.5% (w / v). In some embodiments, a sugar is present at a concentration of 4% (w / v). In some embodiments, a sugar is present at a concentration of 4.5% (w / v). In some embodiments, a sugar is present at a concentration of 5% (w / v). In some embodiments, a sugar is present at a concentration of 5.5% (w / v). In some embodiments, a sugar is present at a concentration of 6% (w / v). In some embodiments, a sugar is present at a concentration of 6.5% (w / v). In some embodiments, a sugar is present at a concentration of 7% (w / v). In some embodiments, a sugar is present at a concentration of 7.5% (w / v). In some embodiments, a sugar is present at a concentration of 8% (w / v). In some embodiments, a sugar is present at a concentration of Attorney Docket No. ZEN-016WO1

[0155] 8.5% (w / v). In some embodiments, a sugar is present at a concentration of 9% (w / v). In some embodiments, a sugar is present at a concentration of 9.5% (w / v). In some embodiments, a sugar is present at a concentration of 10% (w / v). In some embodiments, a sugar is present at a concentration of 10.5% (w / v). In some embodiments, a sugar is present at a concentration of 11% (w / v). In some embodiments, a sugar is present at a concentration of 11.5% (w / v). In some embodiments, a sugar is present at a concentration of 12% (w / v). In some embodiments, a sugar is present at a concentration of 12.5% (w / v). In some embodiments, a sugar is present at a concentration of 13% (w / v). In some embodiments, a sugar is present at a concentration of 13.5% (w / v). In some embodiments, a sugar is present at a concentration of 14% (w / v). In some embodiments, a sugar is present at a concentration of 14.5% (w / v). In some embodiments, a sugar is present at a concentration of 15% (w / v).

[0156] [Hl] In some embodiments, a sugar comprises sucrose. In some embodiments, sucrose is present at a concentration of 0.1% (w / v) to 15% (w / v). In some embodiments, sucrose is present at a concentration of 1% (w / v) tol0% (w / v). In some embodiments, sucrose is present at a concentration of 4% (w / v) to 8% (w / v). In some embodiments, sucrose is present at a concentration of 1% (w / v). In some embodiments, sucrose is present at a concentration of 1.5% (w / v). In some embodiments, sucrose is present at a concentration of 2% (w / v). In some embodiments, sucrose is present at a concentration of 2.5% (w / v). In some embodiments, sucrose is present at a concentration of 3% (w / v). In some embodiments, sucrose is present at a concentration of 3.5% (w / v). In some embodiments, sucrose is present at a concentration of 4% (w / v). In some embodiments, sucrose is present at a concentration of 4.5% (w / v). In some embodiments, sucrose is present at a concentration of 5% (w / v). In some embodiments, sucrose is present at a concentration of 5.5% (w / v). In some embodiments, sucrose is present at a concentration of 6% (w / v). In some embodiments, sucrose is present at a concentration of 6.5% (w / v). In some embodiments, sucrose is present at a concentration of 7% (w / v). In some embodiments, sucrose is present at a concentration of 7.5% (w / v). In some embodiments, sucrose is present at a concentration of 8% (w / v). In some embodiments, sucrose is present at a concentration of 8.5% (w / v). In some embodiments, sucrose is present at a concentration of 9% (w / v). In some embodiments, sucrose is present at a concentration of 9.5% (w / v). In some embodiments, sucrose is present at a concentration of 10% (w / v). In Attorney Docket No. ZEN-016WO1 some embodiments, sucrose is present at a concentration of 10.5% (w / v). In some embodiments, sucrose is present at a concentration of 11% (w / v). In some embodiments, sucrose is present at a concentration of 11.5% (w / v). In some embodiments, sucrose is present at a concentration of 12% (w / v). In some embodiments, sucrose is present at a concentration of 12.5% (w / v). In some embodiments, sucrose is present at a concentration of 13% (w / v). In some embodiments, sucrose is present at a concentration of 13.5% (w / v). In some embodiments, sucrose is present at a concentration of 14% (w / v). In some embodiments, sucrose is present at a concentration of 14.5% (w / v). In some embodiments, sucrose is present at a concentration of 15% (w / v).

[0157]

[0112] In some embodiments, sucrose is present at a concentration of 10 mM to 800 mM. In some embodiments, sucrose is present at a concentration of 50 mM to 700 mM. In some embodiments, sucrose is present at a concentration of 100 mM to 600 mM. In some embodiments, sucrose is present at a concentration of 200 mM to 600 mM. In some embodiments, sucrose is present at a concentration of 300 mM to 500 mM. In some embodiments, sucrose is present at a concentration of 200 mM. In some embodiments, sucrose is present at a concentration of 250 mM. In some embodiments, sucrose is present at a concentration of 300 mM. In some embodiments, sucrose is present at a concentration of 350 mM. In some embodiments, sucrose is present at a concentration of 400 mM. In some embodiments, sucrose is present at a concentration of 450 mM. In some embodiments, sucrose is present at a concentration of 500 mM. In some embodiments, sucrose is present at a concentration of 550 mM. In some embodiments, sucrose is present at a concentration of 600 mM. In some embodiments, sucrose is present at a concentration of 650 mM. In some embodiments, sucrose is present at a concentration of 700 mM.

[0158]

[0113] In some embodiments, a sugar present in a stable formulation at a concentration proportional to the amount of CTLA4-Ig fusion protein. One skilled in the part would thus appreciate that the suitable concentration of sugar in a stable formulation would vary with the concentration of a CTLA4-Ig protein. In some embodiments, a sugar is present at a concentration of 1 to 10 mM for every mg / ml of CTLA4-Ig fusion protein. In some embodiments, a sugar is present at a concentration of 1 mM for every mg / ml of CTLA4-Ig fusion protein. In some embodiments, a sugar is present at a concentration of 2 mM for every mg / ml of CTLA4-Ig fusion protein. In some embodiments, a sugar is present at a concentration of 3 mM for every mg / ml of CTLA4-Ig fusion protein. In some Attorney Docket No. ZEN-016WO1 embodiments, a sugar is present at a concentration of 4 mM for every mg / ml of CTLA4-Ig fusion protein. In some embodiments, a sugar is present at a concentration of 5 mM for every mg / ml of CTLA4-Ig fusion protein. In some embodiments, a sugar is present at a concentration of 6 mM for every mg / ml of CTLA4-Ig fusion protein. In some embodiments, a sugar is present at a concentration of 7 mM for every mg / ml of CTLA4-Ig fusion protein. In some embodiments, a sugar is present at a concentration of 8 mM for every mg / ml of CTLA4-Ig fusion protein. In some embodiments, a sugar is present at a concentration of 9 mM for every mg / ml of CTLA4-Ig fusion protein. In some embodiments, a sugar is present at a concentration of 10 mM for every mg / ml of CTLA4- Ig fusion protein.

[0159]

[0114] In some embodiments, sucrose present in a stable formulation at a concentration proportional to the amount of CTLA4-Ig fusion protein. One skilled in the part would thus appreciate that the suitable concentration of sucrose in a stable formulation would vary with the concentration of a CTLA4-Ig protein. In some embodiments, sucrose is present at a concentration of 1 to 10 mM for every mg / ml of CTLA4-Ig fusion protein. In some embodiments, sucrose is present at a concentration of 1 mM for every mg / ml of CTLA4-Ig fusion protein. In some embodiments, sucrose is present at a concentration of 2 mM for every mg / ml of CTLA4-Ig fusion protein. In some embodiments, sucrose is present at a concentration of 3 mM for every mg / ml of CTLA4-Ig fusion protein. In some embodiments, sucrose is present at a concentration of 4 mM for every mg / ml of CTLA4-Ig fusion protein. In some embodiments, sucrose is present at a concentration of 5 mM for every mg / ml of CTLA4-Ig fusion protein. In some embodiments, sucrose is present at a concentration of 6 mM for every mg / ml of CTLA4-Ig fusion protein. In some embodiments, sucrose is present at a concentration of 7 mM for every mg / ml of CTLA4-Ig fusion protein. In some embodiments, sucrose is present at a concentration of 8 mM for every mg / ml of CTLA4-Ig fusion protein. In some embodiments, sucrose is present at a concentration of 9 mM for every mg / ml of CTLA4-Ig fusion protein. In some embodiments, sucrose is present at a concentration of 10 mM for every mg / ml of CTLA4-Ig fusion protein. Attorney Docket No. ZEN-016WO1

[0160] Amino Acids

[0161]

[0115] In some embodiments, a stable formulation of the CTLA4-Ig fusion protein further comprises one or more amino acids. In some embodiments, amino acids can be used at to maintain stability and prevent aggregation of the CTLA4-Ig fusion protein. In some embodiments, one or more amino acids are present at a concentration between 10 mM and 250 mM. In some embodiments, one or more amino acids are present at a concentration between 10 mM and 225 mM. In some embodiments, one or more amino acids are present at a concentration between 10 mM and 200 mM. In some embodiments, one or more amino acids are present at a concentration between 10 mM and 175 mM. In some embodiments, one or more amino acids are present at a concentration between 10 mM and 150 mM. In some embodiments, one or more amino acids are present at a concentration between 10 mM and 125 mM. In some embodiments, one or more amino acids are present at a concentration between 10 mM and 100 mM. In some embodiments, one or more amino acids are present at a concentration between 10 mM and 75 mM. In some embodiments, one or more amino acids are present at a concentration between 10 mM and 50 mM. In some embodiments, one or more amino acids are present at a concentration between 10 mM and 25 mM. In some embodiments, one or more amino acids are present at a concentration between 20 mM and 250 mM. In some embodiments, one or more amino acids are present at a concentration between 25 mM and 250 mM. In some embodiments, one or more amino acids are present at a concentration between 50 mM and 250 mM. In some embodiments, one or more amino acids are present at a concentration between 75 mM and 250 mM. In some embodiments, one or more amino acids are present at a concentration between 100 mM and 250 mM. In some embodiments, one or more amino acids are present at a concentration between 125 mM and 250 mM. In some embodiments, one or more amino acids are present at a concentration between 150 mM and 250 mM. In some embodiments, one or more amino acids are present at a concentration between 175 mM and 250 mM. In some embodiments, one or more amino acids are present at a concentration between 200 mM and 250 mM. In some embodiments, one or more amino acids are present at a concentration between 5 mM and 35 mM , between 10 mM and 35 mM, or between 15 mM and 30 mM. Attorney Docket No. ZEN-016WO1

[0162]

[0116] In some embodiments, a stable formulation comprises one or more amino acids selected from arginine, glutamic acid, glycine, histidine, proline, and combinations thereof. In some embodiments, a stable formulation comprises arginine. In some embodiments, a stable formulation comprises glutamic acid. In some embodiments, a stable formulation comprises arginine and glutamic acid. In some embodiments, a stable formulation comprises histidine. In some embodiments, the stable formulation comprises glycine. In some embodiments, a stable formulation comprises proline. In some embodiments, a stable formulation comprises histidine.

[0163]

[0117] Arginine, as used herein, includes the free base form of arginine, as well as any and all salts thereof. In some embodiments, arginine includes a pharmaceutically acceptable salt thereof, e.g., arginine hydrochloride. Arginine as used herein also includes all enantiomers (e.g., L-arginine and S-arginine), and any combination of enantiomers (e.g., 50% L-arginine and 50% S-arginine). In some embodiments, the stable formulation comprising arginine comprises L-arginine hydrochloride. In some embodiments, the stable formulation comprises arginine at a concentration ranging from 10 mM to 250 mM. In some embodiments, the stable formulation comprises arginine at a concentration ranging from 20 mM to 225 mM. In some embodiments, the stable formulation comprises arginine at a concentration ranging from 30 mM to 200 mM. In some embodiments, the stable formulation comprises arginine at a concentration ranging from 40 mM to 175 mM. In some embodiments, the stable formulation comprises arginine at a concentration ranging from 50 mM to 150 mM. In some embodiments, the stable formulation comprises arginine at a concentration ranging from 60 mM to 125 mM. In some embodiments, the stable formulation comprises arginine at a concentration ranging from 70 mM to 100 mM. In some embodiments, the stable formulation comprises arginine at a concentration of 25 mM. In some embodiments, the stable formulation comprises arginine at a concentration of 50 mM. In some embodiments, the stable formulation comprises arginine at a concentration of 75 mM. In some embodiments, the stable formulation comprises arginine at a concentration of 100 mM. In some embodiments, the stable formulation comprises arginine at a concentration of 125 mM. In some embodiments, the stable formulation comprises arginine at a concentration of 150 mM. Attorney Docket No. ZEN-016WO1

[0164] Salts

[0165]

[0118] In some embodiments, a stable formulation further comprises a salt. In some embodiments, a stable formulation comprises less than 100 mM of a salt. In some embodiments, a stable formulation comprises less than 90 mM of a salt, less than 80 mM of a salt, less than 70 mM of a salt, less than 60 mM of a salt, less than 50 mM of a salt, less than 40 mM of a salt, less than 30 mM of a salt, less than 20 mM of a salt, less than 10 mM of a salt or less than 5 mM of a salt. In some embodiments, a stable formulation comprises only residual amounts of salts. In some embodiments, a stable formulation is substantially free of salt.

[0166]

[0119] In other embodiments, a stable formulation comprises a salt. In some embodiments, a salt is present at a concentration of between 25 mM and 250 mM. In some embodiments, a salt is present at a concentration of between 35 mM and 100 mM. In some embodiments, a salt is present at a concentration of 25 mM, 30 mM, 35 mM, 40 mM, 45, mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 165 mM, 170 mM, 175 mM, 180 mM, 185 mM, 190 mM, 195 mM, 200 mM, 205 mM, 210 mM, 215 mM, 220 mM, 225 mM, 230 mM, 235 mM, 240 mM, 245 mM, or 250 mM.

[0167]

[0120] In some embodiments, a salt comprises a halide. In some embodiments, a salt comprises NaCl. In some embodiments, NaCl is present at a concentration of between 25 mM and 250 mM. In some embodiments, NaCl is present at a concentration of between 35 mM and 100 mM. In some embodiments, NaCl is present at a concentration of 25 mM, 30 mM, 35 mM, 40 mM, 45, mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 165 mM, 170 mM, 175 mM, 180 mM, 185 mM, 190 mM, 195 mM, 200 mM, 205 mM, 210 mM, 215 mM, 220 mM, 225 mM, 230 mM, 235 mM, 240 mM, 245 mM, or 250 mM. In some embodiments, NaCl is present at a concentration of 25 to 75 mM. In some embodiments, NaCl is present at a concentration of 50 mM. Attorney Docket No. ZEN-016WO1

[0168] Surfactants

[0169]

[0121] In some embodiments of the invention, a stable formulation further comprises a surfactant. Exemplary surfactants include nonionic surfactants such as polysorbates (e.g., polysorbate 20, polysorbate 40, or polysorbate 80); poloxamers (e.g., pol oxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl ofeyl-taurate; and the MONAQUAT™ series (Mona Industries, Inc., Paterson, N.J.), polyethylene glycol (PEG), polypropylene glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronics, PF68, etc). Typically, the amount of surfactant added is such that it reduces aggregation of the protein and minimizes the formation of particulates.

[0170]

[0122] In some embodiments, a stable formulation comprises a surfactant at a concentration of 0.001 to 0.5% (w / v) (e.g., 0.075%). In some embodiments, a surfactant is present at a concentration of 0.005% (w / v), 0.01 % (w / v), 0.02% (w / v), 0.03% (w / v), 0.04% (w / v), 0.05% (w / v), 0.06% (w / v), 0.07% (w / v), 0.08% (w / v), 0.09% (w / v), or 0.1 % (w / v). In some embodiments, a surfactant is present at a concentration of less than 2% (w / v). In some embodiments, a surfactant is present at a concentration of less than 1% (w / v). In some embodiments, a surfactant is present at a concentration of 0.2% (w / v). In some embodiments, a surfactant is present at a concentration of 0.4% (w / v). In some embodiments, a surfactant is present at a concentration of 0.6% (w / v). In some embodiments, a surfactant is present at a concentration of 0.8% (w / v). In some embodiments, a surfactant is present at a concentration of 1.0% (w / v). In some embodiments, a surfactant is present at a concentration of 1.2% (w / v). In some embodiments, a surfactant is present at a concentration of 1.4% (w / v). In some embodiments, a surfactant is present at a concentration of 1.6% (w / v). In some embodiments, a surfactant is present at a concentration of 1.8% (w / v). Attorney Docket No. ZEN-016WO1

[0171]

[0123] In some embodiments, a surfactant is polysorbate 80 (PS 80). In some embodiments, polysorbate 80 (PS80) is present at a concentration of less than 1% (w / v). In some embodiments, polysorbate 80 (PS80) is present at a concentration of less than 0.1% (w / v). In some embodiments, polysorbate 80 (PS80) is present a concentration of 0.001% (w / v / ) to 0.1% (w / v). In some embodiments, polysorbate 80 (PS80) is present at a concentration of 0.01% (w / v) to 0.6% (w / v). In some embodiments, polysorbate 80 (PS80) is present at a concentration of 0.01% (w / v), 0.02% (w / v), 0.03% (WINI), 0.04% (w / v), 0.05% (w / v), 0.06% (w / v), 0.07% (w / v), 0.08% (w / v), 0.09% (w / v), or 0.1% (w / v). In some embodiments, polysorbate 80 (PS80) is present at a concentration of 0.01% (w / v).

[0172] In some embodiments, polysorbate 80 (PS80) is present at a concentration of 0.02% (w / v).

[0173] In some embodiments, polysorbate 80 (PS80) is present at a concentration of 0.03% (w / v).

[0174] In some embodiments, polysorbate 80 (PS80) is present at a concentration of 0.04% (w / v).

[0175] In some embodiments, polysorbate 80 (PS80) is present at a concentration of 0.05% (w / v).

[0176] In some embodiments, polysorbate 80 (PS80) is present at a concentration of 0.06% (w / v).

[0177] In some embodiments, polysorbate 80 (PS80) is present at a concentration of 0.07% (w / v).

[0178] In some embodiments, polysorbate 80 (PS80) is present at a concentration of 0.08% (w / v).

[0179] In some embodiments, polysorbate 80 (PS80) is present at a concentration of 0.09% (w / v).

[0180] In some embodiments, polysorbate 80 (PS80) is present at a concentration of 0.1% (w / v). Alternatively, or in addition, the surfactant may be added to a lyophilized formulation, pre-lyophilized formulation and / or a reconstituted formulation.

[0181]

[0124] In some embodiments, a surfactant comprises pol oxamer 188 (Pl 88). In some embodiments, pol oxamer 188 (Pl 88) is present at a concentration of less than 2% (w / v). In some embodiments, pol oxamer 188 (Pl 88) is present at a concentration of less than 1% (w / v). In some embodiments, poloxamer 188 (P188) is present a concentration of 0.01% (w / v / ) to 0.1% (w / v). In some embodiments, poloxamer 188 (P188) is present at a concentration of 0.1% (w / v) to 1.0% (w / v). In some embodiments, poloxamer 188 (P188) is present at a concentration of 1.0% (w / v) to 2.0% (w / v).

[0182]

[0125] In some embodiments, poloxamer 188 (Pl 88) is present at a concentration of 0.1% (w / v). In some embodiments, polysorbate 80 (P188) is present at a concentration of 0.2% (w / v). In some embodiments, poloxamer 188 (Pl 88) is present at a concentration of 0.3% (w / v). In some embodiments, poloxamer 188 (P188) is present at a concentration of 0.4% (w / v). In some embodiments, poloxamer 188 (Pl 88) is present at a concentration Attorney Docket No. ZEN-016WO1 of 0.5% (w / v). In some embodiments, pol oxamer 188 (Pl 88) is present at a concentration of 0.6% (w / v). In some embodiments, poloxamer 188 (P188) is present at a concentration of 0.7% (w / v). In some embodiments, poloxamer 188 (P188) is present at a concentration of 0.8% (w / v). In some embodiments, poloxamer 188 (Pl 88) is present at a concentration of 0.9% (w / v). In some embodiments, poloxamer 188 (P188) is present at a concentration of 1.0% (w / v). In some embodiments, poloxamer 188 (P188) is present at a concentration of 1.2% (w / v). In some embodiments, poloxamer 188 (Pl 88) is present at a concentration of 1.4% (w / v). In some embodiments, poloxamer 188 (Pl 88) is present at a concentration of 1.6% (w / v). In some embodiments, poloxamer 188 (P188) is present at a concentration of 1.8% (w / v). Alternatively, or in addition, the surfactant may be added to a lyophilized formulation, pre-lyophilized formulation and / or a reconstituted formulation.

[0183] Osmolality

[0184]

[0126] In some embodiments, a stable formulation comprises an osmolality of greater than 550 mOsmol / kg. In some embodiments, a stable formulation comprises an osmolality of greater than 600 mOsmol / kg. In some embodiments, a stable formulation comprises an osmolality of greater than 650 mOsmol / kg. In some embodiments, a stable formulation comprises an osmolality of greater than 700 mOsmol / kg. In some embodiments, a stable formulation comprises an osmolality of greater than 750 mOsmol / kg. In some embodiments, a stable formulation comprises an osmolality of greater than 800 mOsmol / kg. In some embodiments, a stable formulation comprises an osmolality of greater than 850 mOsmol / kg.

[0185]

[0127] In some embodiments, a stable formulation comprises an osmolality of between 250 and 450 mOsmol / kg. In some embodiments, the stable formulation comprises an osmolality of between 275 and 410 mOsmol / kg. In some embodiments the stable formulation comprises an osmolality of 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 420, 440 or 450 mOsmol / kg. In some embodiments, the stable formulation comprises an osmolality of between 275 and 375 mOsmol / kg. In some embodiments, the stable formulation comprises an osmolality of between 310 and 410 mOsmol / kg.

[0186] Viscosity Attorney Docket No. ZEN-016WO1

[0187]

[0128] In some embodiments of the invention, a stable formulation is a liquid. In some embodiments of the invention, stable formulations were optimized in order to reduce viscosity, while maintaining high protein concentration. Using a constant rate of 0.1 mL / minute, the force required to either aspirate (syringeability) or expel (injectability) the material from / into the syringe can be recorded. In some embodiments, lower viscosity allows for increased injectability of a formulation or effective sample transfer and preparation during manufacturing. In some embodiments, the stable formulation has a viscosity, as measured by microfluidic rheometer. In some embodiments, a stable formulation has a viscosity no greater than 5 mPa s. In some embodiments, a stable formulation has a viscosity no greater than 4 mPa s. In some embodiments, a stable formulation has a viscosity no greater than 3.5 mPa s. In some embodiments, a stable formulation has a viscosity no greater than 3.4 mPa s. In some embodiments, a stable formulation has a viscosity no greater than 3.3 mPa s. In some embodiments, a stable formulation has a viscosity no greater than 3.2 mPa s. In some embodiments, a stable formulation has a viscosity no greater than 3.1 mPa s.

[0188] Exemplary CTLA-4-Ig Fusion Protein Formulations

[0189]

[0129] In some embodiments, a stable formulation comprises: (i) 30-75 mg / ml CTLA4-Ig fusion protein, (ii) 30-50 mM sodium phosphate buffer, (iii) 350-450 mM sucrose, (iv) 50-150 mM arginine, (v) 0.6%-l% (w / v) poloxamer 188, at pH 6.7-7.7.

[0190]

[0130] In some embodiments, a stable formulation comprises: (i) 30-75 mg / ml CTLA4-Ig fusion protein, (ii) 30-50 mM sodium phosphate buffer, (iii) 350-450 mM sucrose, (iv) 50-150 mM arginine, (v) 0.6%-l% (w / v) poloxamer 188, at pH 6.7-7.7, wherein the CTLA-4-Ig fusion protein comprises a first domain comprising a variant CTLA-4 and a second domain comprising an IgG Fc region, wherein the variant CTLA-4 comprises A29H, T51N, L61E, and K93Q substitutions, and wherein the IgG Fc region comprises M428L and N434S substitutions.

[0191]

[0131] In some embodiments, a stable formulation comprises: (i) 30-75 mg / ml CTLA4-Ig fusion protein, (ii) 30-50 mM sodium phosphate buffer, (iii) 350-450 mM sucrose, (iv) 50-150 mM arginine, (v) 0.6%-l% (w / v) poloxamer 188, at pH 6.7-7.7, wherein the CTLA4-Ig fusion protein comprises an amino acid sequence of SEQ ID NO: 6. Attorney Docket No. ZEN-016WO1

[0192]

[0132] In some embodiments, a stable formulation comprises: (i) 50 mg / ml CTLA4-Ig fusion protein, (ii) 40mM sodium phosphate, (iii) 400mM sucrose, (iv) lOOmM arginine, (v) 0.8% (w / v) poloxamer 188, at pH of 7.2.

[0193]

[0133] In some embodiments, a stable formulation comprises: (i) 50 mg / ml CTLA4-Ig fusion protein, (ii) 40mM sodium phosphate, (iii) 400mM sucrose, (iv) lOOmM arginine, (v) 0.8% (w / v) poloxamer 188, at pH of 7.2, wherein the CTLA-4-Ig fusion protein comprises a first domain comprising a variant CTLA-4 and a second domain comprising an IgG Fc region, wherein the variant CTLA-4 comprises A29H, T51N, L61E, and K93Q substitutions, and wherein the IgG Fc region comprises M428L and N434S substitutions.

[0194]

[0134] In some embodiments, a stable formulation comprises: (i) 50 mg / ml CTLA4-Ig fusion protein, (ii) 40mM sodium phosphate, (iii) 400mM sucrose, (iv) lOOmM arginine, (v) 0.8% (w / v) poloxamer 188, at pH of 7.2, wherein the CTLA4-Ig fusion protein comprises an amino acid sequence of SEQ ID NO: 6.

[0195] Stability and Properties of Stable Formulations

[0196]

[0135] In some embodiments, a stable formulation of the present invention is substantially free of impurities. In some embodiments, a stable formulation comprises less than 25 pg / mg residual host cell protein. In some embodiments, a stable formulation comprises less than 20 pg / mg residual host cell protein. In some embodiments, a stable formulation comprises less than 150 ng / mg residual host cell protein. In some embodiments, a stable formulation comprises less than 100 ng / mg residual host cell protein. In some embodiments, a stable formulation comprises less than 50 ng / mg residual protein A. In some embodiments, a stable formulation comprises less than 0.4 EU / mg endotoxin.

[0197]

[0136] Various stability assays are available to the skilled practitioner for confirming the stability of a stable formulation described herein. For example, a stable formulation may be one which is found to be stable upon storage: at about 40° C for at least 4 weeks; at about 5° C or about 15° C for at least 3 months or at least 1 year; and / or about -20° C for at least 3 months. In some embodiments, the formulation is stable at - 20° C for 3 weeks. In some embodiments, a stable formulation is stable at -20° C for 4 Attorney Docket No. ZEN-016WO1 weeks. In some embodiments, a stable formulation is stable at -20° C for 5 weeks. In some embodiments, a stable formulation is stable at -20° C for 1 month. In some embodiments, a stable formulation is stable at -20° C for 2 months. In some embodiments, a stable formulation is stable at -20° C for 3 months. In some embodiments, a stable formulation is stable at -20° C for 4 months. In some embodiments, a stable formulation is stable at -20° C for 5 months. In some embodiments, a stable formulation is stable at -20° C for 6 months. In some embodiments, a stable formulation is stable at -20° C for 7 months. In some embodiments, a stable formulation is stable at -20° C for 8 months. In some embodiments, a stable formulation is stable at -20° C for 9 months. In some embodiments, a stable formulation is stable at -20° C for 10 months . In some embodiments, a stable formulation is stable at -20° C for 11 months . In some embodiments, a stable formulation is stable at -20° C for 12 months . In some embodiments, a stable formulation is stable at -20° C for 18 months . In some embodiments, a stable formulation is stable at -20° C for 12 months . In some embodiments, a stable formulation is stable at -20° C for 24 months . In some embodiments, a stable formulation is stable at -20° C for more than 1 year. In some embodiments, a stable formulation is stable at -20° C for more than 2 years. In some embodiments, a stable formulation is stable at -20° C for more than 3 years. In some embodiments, a stable formulation is stable at -20° C for more than 4 years. In some embodiments, a stable formulation is stable at -20° C for more than 5 years. In some embodiments, a stable formulation is stable at -20° C for more than 6 years. In some embodiments, a stable formulation is stable at more than 2 - 8° C for 1 week. In some embodiments, a stable formulation is stable at more than 2 - 8° C for 2 weeks. In some embodiments, a stable formulation is stable at more than 2 - 8° C for 3 weeks. In some embodiments, a stable formulation is stable at more than 2 - 8° C for 4 weeks. In some embodiments, a stable formulation is stable at more than 2 - 8° C for 5 weeks. In some embodiments, a stable formulation is stable at more than 2 - 8° C for 1 month. In some embodiments, a stable formulation is stable at more than 2 - 8° C for 2 months. In some embodiments, a stable formulation is stable at more than 2 - 8° C for 3 months. In some embodiments, a stable formulation is stable at more than 2 - 8° C for 4 months. In some embodiments, a stable formulation is stable at more than 2 - 8° C for 5 months. In some Attorney Docket No. ZEN-016WO1 embodiments, a stable formulation is stable at more than 2 - 8 C for 6 months. In some embodiments, a stable formulation is stable at more than 2 - 8 C for 7 months. In some embodiments, a stable formulation is stable at more than 2 - 8 C for 8 months. In some embodiments, a stable formulation is stable at more than 2 - 8 C for 9 months. In some embodiments, a stable formulation is stable at more than 2 - 8 C for 10 months. In some embodiments, a stable formulation is stable at more than 2 - 8 C for 11 months. In some embodiments, a stable formulation is stable at more than 2 - 8 C for 12 months. In some embodiments, a stable formulation is stable at more than 2 - 8 C for 18 months. In some embodiments, a stable formulation is stable at more than 2 - 8 C for 20 months. In some embodiments, a stable formulation is stable at more than 2 - 8 C for 24 months. In some embodiments, a stable formulation is stable at more than 2 - 8 C for more than 1 year. In some embodiments, a stable formulation is stable at more than 2 - 8° C for more than 2 years. In some embodiments, a stable formulation is stable at more than 2 - 8° C for more than 3 years. In some embodiments, a stable formulation is stable at more than 2 - 8° C for more than 4 years. In some embodiments, a stable formulation is stable at more than 2 - 8° C for more than 5 years. In some embodiments, a stable formulation is stable at more than 2 - 8° C for more than 6 years.

[0198]

[0137] Stability can be tested by evaluating physical stability, chemical stability, and / or biological activity of the protein in the formulation around the time of formulation as well as following storage at the noted temperatures. Physical and / or stability can be evaluated qualitatively and / or quantitatively in a variety of different ways, including evaluation of aggregate formation (for example using size exclusion chromatography, by measuring turbidity, and / or by visual inspection); by assessing charge heterogeneity using cation exchange chromatography or capillary zone electrophoresis; amino-terminal or carboxy-terminal sequence analysis; mass spectrometric analysis; SDS-PAGE analysis to compare reduced and intact protein; peptide map (for example tryptic or LYS-C) analysis; evaluating biological activity or antigen binding function of the protein; etc. Instability may result in aggregation, deamidation (e.g. Asn deamidation), oxidation (e.g. Met oxidation), isomerization (e.g. Asp isomeriation), clipping / hydrolysis / fragmentation (e.g. hinge region fragmentation), succinimide formation, unpaired cysteine(s), N-terminal extension, C-terminal processing, glycosylation differences, etc. Biological activity or Attorney Docket No. ZEN-016WO1 antigen binding function can be evaluated using various techniques available to the skilled practitioner.

[0199] Monomer, High Molecular Weight (HMW) Species, Low Molecular Weight (LMW) Species

[0200]

[0138] In some embodiments, a stable formulation is characterized by the amount of high molecular weight (HMW) aggregates. In some embodiments, the amount of high molecular weight (HMW) species, monomer, and low molecular weight (LMW) species are measured by SEC or SEC-UPLC. In some embodiments, the amount of high molecular weight species (HMWS) in the stable formulation is less 10% (e.g., less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4% less than 3%, less than 2% or less than 1%) as measured by SEC. In some embodiments, the amount of high molecular weight species (HMWS) in the stable formulation is less than 5% as measured by SEC. In some embodiments, the stability based on the HMW or HMWS is based on the composition that is stored at 40 C for a period of time, such as 4 weeks.

[0201]

[0139] In some embodiments, a stable formulation comprises at least 85% CTLA4-Ig monomer as measured by size exclusion chromatography. In some embodiments, a stable formulation comprises at least 90% CTLA4-Ig monomer as measured by size exclusion chromatography. In some embodiments, a stable formulation comprises at least 95% CTLA4-Ig monomer as measured by size exclusion chromatography. In some embodiments, a stable formulation comprises at least 99% CTLA4-Ig monomer as measured by size exclusion chromatography. In some embodiments, a stable formulation comprises less than 15% high molecular weight species as measured by size exclusion chromatography. In some embodiments, a stable formulation comprises less than 10% high molecular weight species as measured by size exclusion chromatography. In some embodiments, a stable formulation comprises less than 5% high molecular weight species as measured by size exclusion chromatography. In some embodiments, a stable formulation comprises less than 1% high molecular weight species as measured by size exclusion chromatography.

[0202]

[0140] In some embodiments, a stable formulation comprises at least 90% monomer as measured by size exclusion chromatography following storage at 2-8 °C for at least 6 months. In some embodiments, a stable formulation comprises at least 95% Attorney Docket No. ZEN-016WO1 monomer as measured by size exclusion chromatography following storage at 2-8 °C for at least 6 months. In some embodiments, a stable formulation comprises less than 10% high molecular weight species as measured by size exclusion chromatography following storage at 2-8 °C for at least 6 months. In some embodiments, a stable formulation comprises less than 5% high molecular weight species as measured by size exclusion chromatography following storage at 2-8 °C for at least 6 months.

[0203]

[0141] In some embodiments, a stable formulation comprises at least 90% monomer as measured by size exclusion chromatography following storage at 25 °C for at least 6 months. In some embodiments, a stable formulation comprises at least 95% monomer as measured by size exclusion chromatography following storage at 25 °C for at least 6 months. In some embodiments, a stable formulation comprises less than 10% high molecular weight species as measured by size exclusion chromatography following storage at 25 °C for at least 6 months. In some embodiments, a stable formulation comprises less than 5% high molecular weight species as measured by size exclusion chromatography following storage at 25 °C for at least 6 months.

[0204] Imaged Capillary Isoelectric Focusing (iCIEF)

[0205]

[0142] Imaged Capillary Isoelectric Focusing (iCIEF) is used to monitor the distribution of the charge variants. The isoelectric point (pl), being an intrinsic property of a specific protein, is the pH at which the protein molecule does not carry net electrical charge. Under an external electric field, the charge variants move along a continuous pH gradient formed by carrier ampholytes and stop at where the pH equals its pl. This technique used to characterize and quantify protein charge variants.

[0206]

[0143] In some embodiments, the shift in charge profiles may be measured through iCIEF the relative percentages of main peak, acid peak and / or basic peak. For example, a stable formulation can be one in which the change in measured acidic peak increases by less than 40% (e.g., less than 38%, 36%, 34%, 32%, 30%, 28%, 26%, 24%, 22% or less than 20%) relative to an initial value following storage at 25 °C for 6 months as measured by iCIEF and quantified using chromatographic software. In some embodiments, a stable formulation is one in which the main peak percentage decreases by less than 35% (e.g., less than 34%, less than 33%, less than 32%, less than 31%, less than 30%, less than 29%, less than 28%, less than 27%, less than 26%, less than 25%, than less Attorney Docket No. ZEN-016WO1 than 24%, less than 23%, less than 22%, less than 21% or less than 20%) relative to an initial value following storage at 25 °C for 6 months as measured by iCIEF and quantified using chromatographic software. In some embodiments, the stable formulation basic peak, measured by iCIEF, decreases in area percent by less than 6.5% (e.g., less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%,) after 6 months at a temperature of 25 °C, relative to the initial value.

[0207]

[0144] In some embodiments, the acidic peak increases by less than 50% upon storage at 40°C for at least four weeks as measured by iCIEF. In some embodiments, the acidic peak increases by less than 45% upon storage at 25°C for at least 6 months as measured by iCIEF. In some embodiments, the acidic peak increases by less than 40% upon storage at 25 °C for at least 6 months as measured by iCIEF. In some embodiments, the acidic peak increases by less than 35% upon storage at 25 °C for at least 6 months as measured by iCIEF. In some embodiments, the acidic peak increases by less than 30% upon storage at 25 °C for at least 6 months as measured by iCIEF.

[0208]

[0145] In some embodiments, the main peak decreases by less than 50% upon storage at 25 °C for at least 6 months as measured by iCIEF. In some embodiments, the main peak decreases by less than 45% upon storage at 25°C for at least 6 months as measured by iCIEF. In some embodiments, the main peak decreases by less than 40% upon storage at 25 °C for at least 6 months as measured by iCIEF. In some embodiments, the main peak decreases by less than 35% upon storage at 25 °C for at least 6 months as measured by iCIEF. In some embodiments, the main peak decreases by less than 30% upon storage at 25 °C for at least 6 months as measured by iCIEF.

[0209]

[0146] In some embodiments, the basic peak decreases by less than 10% upon storage at 25 °C for at least 6 months as measured by iCIEF. In some embodiments, the basic peak decreases by less than 8% upon storage at 25°C for at least 6 months as measured by iCIEF. In some embodiments, the basic peak decreases by less than 7% upon storage at 25 °C for at least 6 months as measured by iCIEF. In some embodiments, the basic peak decreases by less than 6.5% upon storage at 25°C for at least 6 months as measured by iCIEF. In some embodiments, the basic peak decreases by less than 6% upon storage at 25 °C for at least 6 months as measured by iCIEF. In some embodiments, the basic peak decreases by less than 5.5% upon storage at 25 °C for at least 6 months as Attorney Docket No. ZEN-016WO1 measured by iCIEF. In some embodiments, the basic peak decreases by less than 5% upon storage at 25 °C for at least 6 months as measured by iCIEF.

[0210] Appearance / Aggregation

[0211]

[0147] Appearance is a visual confirmation of a sample compared to a standard. The appearance of all samples is assessed for visible particles, clarity and color. This is routinely examined against black and white backgrounds using a Clarity Detector to shine light directly onto the samples. This technique measures stability. In some embodiments, the stable formulation is free of particles upon storage at 5°C for at least 6 months. In some embodiments, the stable formulation is free of particles upon storage at 25°C for at least 6 months.

[0212] Methods of Administration

[0213]

[0148] In one aspect, the present invention provides a method of treating autoimmune diseases in a patient in need thereof using a CTLA4-Ig fusion protein at a therapeutically effective dose. In some embodiments, a therapeutically effective dose is 1 mg to 300 mg. In some embodiments, a therapeutically effective dose is 3 mg to 200 mg. In some embodiments, a therapeutically effective dose is 12.5 mg to 200 mg. In some embodiments, a therapeutically effective dose is 50 mg to 200 mg.

[0214]

[0149] In some embodiments, a therapeutically effective dose is 1 mg. In some embodiments, a therapeutically effective dose is 3 mg. In some embodiments, the therapeutically effective dose is 5 mg. In some embodiments, the therapeutically effective dose is 7.5 mg. In some embodiments, the therapeutically effective dose is 8 mg. In some embodiments, the therapeutically effective dose is 10 mg. In some embodiments, a therapeutically effective dose is 12.5 mg. In some embodiments, the therapeutically effective dose is 15 mg. In some embodiments, the therapeutically effective dose is 17.5 mg. In some embodiments, the therapeutically effective dose is 20 mg. In some embodiments, the therapeutically effective dose is 25 mg. In some embodiments, the therapeutically effective dose is 30 mg. In some embodiments, the therapeutically effective dose is 40 mg. In some embodiments, a therapeutically effective dose is 50 mg. In some embodiments, the therapeutically effective dose is 60 mg. In some embodiments, Attorney Docket No. ZEN-016WO1 the therapeutically effective dose is 70 mg. In some embodiments, the therapeutically effective dose is 80 mg. In some embodiments, the therapeutically effective dose is 90 mg. In some embodiments, a therapeutically effective dose is 100 mg. In some embodiments, the therapeutically effective dose is 120 mg. In some embodiments, a therapeutically effective dose is 125 mg. In some embodiments, the therapeutically effective dose is 130 mg. In some embodiments, the therapeutically effective dose is 140 mg. In some embodiments, the therapeutically effective dose is 150 mg. In some embodiments, the therapeutically effective dose is 160 mg. In some embodiments, the therapeutically effective dose is 170 mg. In some embodiments, the therapeutically effective dose is 180 mg. In some embodiments, a therapeutically effective dose is 200 mg. In some embodiments, the therapeutically effective dose is 250 mg. In some embodiments, the therapeutically effective dose is 300 mg.

[0215]

[0150] In some embodiments, a CTLA4-Ig fusion protein is administered at a therapeutically effective dose less frequently than once every week. In some embodiments, a CTLA4-Ig fusion protein is administered at a therapeutically effective dose no more frequently than once every other week. In some embodiments, a CTLA4-Ig fusion protein is administered at a therapeutically effective dose no more frequently than once every three weeks. In some embodiments, a CTLA4-Ig fusion protein is administered at a therapeutically effective dose no more frequently than once every four weeks. In some embodiments, a CTLA4-Ig fusion protein is administered at a therapeutically effective dose no more frequently than once every month weeks.

[0216]

[0151] In some embodiments, a CTLA4-Ig fusion protein is administered once every 5 days. In some embodiments, a CTLA4-Ig fusion protein is administered once every week. In some embodiments, a CTLA4-Ig fusion protein is administered once every two weeks. In some embodiments, a CTLA4-Ig fusion protein is administered once every three weeks. In some embodiments, a CTLA4-Ig fusion protein is administered once every four weeks. In some embodiments, a CTLA4-Ig fusion protein is administered once every five weeks. In some embodiments, a CTLA4-Ig fusion protein is administered once every six weeks. In some embodiments, a CTLA4-Ig fusion protein is administered once every seven weeks. In some embodiments, a CTLA4-Ig fusion protein is administered once every eight weeks. In some embodiments, a CTLA4-Ig fusion protein is administered once every nine weeks. In some embodiments, a CTLA4-Ig fusion protein is Attorney Docket No. ZEN-016WO1 administered once every ten weeks. In some embodiments, a CTLA4-Ig fusion protein is administered once every month. In some embodiments, a CTLA4-Ig fusion protein is administered once every two months. In some embodiments, a CTLA4-Ig fusion protein is administered once every three months. In some embodiments, a CTLA4-Ig fusion protein is administered once every four months. In some embodiments, a CTLA4-Ig fusion protein is administered once every five months. In some embodiments, a CTLA4- Ig fusion protein is administered once every six months. In some embodiments, a CTLA4-Ig fusion protein is administered once every seven months. In some embodiments, a CTLA4-Ig fusion protein is administered once every eight months. In some embodiments, a CTLA4-Ig fusion protein is administered once every nine months. In some embodiments, a CTLA4-Ig fusion protein is administered once every ten months.

[0217] Delivery

[0218]

[0152] In some embodiments, a CTLA4-Ig fusion protein is administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and / or mode of administration will vary depending upon the desired results. Routes of administration for the fusion protein of the invention include subcutaneous, intravenous, intramuscular, intradermal, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, a CTLA4-Ig fusion protein can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically. In some embodiments, a CTLA4-Ig fusion protein is administered subcutaneously. In some embodiments, a CTLA4-Ig fusion protein is administered intravenously. Attorney Docket No. ZEN-016WO1

[0219]

[0153] The CTLA4-Ig fusion protein and other therapeutically active agents may also be entrapped in microcapsules prepared by methods including but not limited to coacervation techniques, interfacial polymerization (for example using hydroxymethylcellulose or gelatin-microcapsules, or poly-(methylmethacylate) microcapsules), colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), and macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980, incorporated entirely by reference. Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymer, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides, copolymers of L-glutamic acid and gamma ethyl -L-glutamate, non- degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the Lupron Depot® (which are injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), poly-D-(-)-3 -hydroxybutyric acid, and ProLease® (commercially available from Alkermes), which is a microsphere-based delivery system composed of the desired bioactive molecule incorporated into a matrix of poly-DL-lactide- co-glycolide (PLG).

[0220]

[0154] Therapeutic compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Patent Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules useful in the present invention include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering medicants through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,439, 196, which discloses an osmotic drug delivery system having multichamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug Attorney Docket No. ZEN-016WO1 delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[0221] Kits

[0222]

[0155] Treatment of individuals may comprise the administration of a pharmaceutical composition described herein. The pharmaceutical compositions can be provided in a kit, such as those provided herein. The pharmaceutical compositions can be used or administered alone or in admixture with another therapeutic, analgesic, or diagnostic agent, such as provided for herein. In providing a patient with a pharmaceutical composition comprising a fusion protein, or fragment thereof, capable of binding to CTLA4, or a fusion protein capable of protecting against CTLA4 pathology in a recipient patient, the dosage of administered agent will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition, previous medical history, etc.

[0223] Attorney Docket No. ZEN-016WO1

[0224] EXAMPLES

[0225]

[0156] Examples are provided below to illustrate the present invention. These examples are not meant to constrain the present invention to any particular application or theory of operation.

[0226] Example 1. Formulation Development of 125mg / ml ZB004

[0227]

[0157] This example illustrates that formulations of ZB004 at 125 mg / ml concentration are stable upon storage at 25°C for at least 5 days.

[0228] Table 1. Protein Stability.

[0229]

[0158] All formulations except F4 and F8 had low HMW% upon storage at 25 °C for at least 5 days. Attorney Docket No. ZEN-016WO1

[0230] Table 2. Osmolality, A2, and Viscosity.

[0231]

[0159] Table 2 shows the viscosity was raised along with the increase of sucrose strength. The pH and protein concentration were close to the target value. The osmolality range was 537-877 mOsmol / kg.

[0232] Table 3. Formulation optimization study 1.

[0233]

[0160] The formulation optimization study is aimed to evaluate the protein stability with different pH / buffer systems, different types of excipients, and different surfactants. The protein concentration was 100 mg / mL and all formulations were stored at 25°C for 10 days. Attorney Docket No. ZEN-016WO1

[0234] Table 4. Stabilizer, Amino Acid, and Salt Optimization.

[0235]

[0161] Table 4 shows the data summary of SEC-UPLC results in the formulation optimization study. Attorney Docket No. ZEN-016WO1

[0236] Table 5. Appearance.

[0237]

[0162] Table 5 provides the data of the appearance testing for the formulation optimization study. All formulations were slightly yellow, slightly opalescent and free of visible particles during the study. The pH and protein concentrations were close to the target value. The osmolality was about 491-671 mOsmol / kg at TO.

[0238] Example 2. Evaluating protein concentration and surfactant concentration

[0239]

[0163] The purpose of the formulation optimization study was to evaluate the protein stability with different buffer systems, protein concentrations, excipients, and Attorney Docket No. ZEN-016WO1 strengths of surfactant comprehensively. The stability was assessed upon storage at 5 °C and 25°C for up to 2 weeks.

[0240] Table 6. Protein Concentration and Surfactant Optimization. Attorney Docket No. ZEN-016WO1

[0241]

[0164] Table 6 shows the stability of formulations as assessed by SEC-UPLC storage at 25 °C for 4 weeks.

[0242] Example 3. Optimization of Formulation with 40 mM Phosphate at pH 7.2 at 75 mg / ml

[0243]

[0165] According to previous studies, 40 mM phosphate buffer at pH 7.2 was selected for the formulation optimization study 4th and the protein concentration was 75 mg / mL. Different strengths of excipients and surfactants (sucrose, proline, L-Arg-HCl, Pl 88) were conducted in the formulation optimization study. In this study, thirteen formulations were generated, three were designed similarly to the formulation that had better performance in the 3rd study, and one contained only buffer without excipients or surfactants. A total of seventeen formulations were conducted under 5°C and 25°C for up to 12 months.

[0244] Table 7. Total Excipient Comparison. Attorney Docket No. ZEN-016WO1 Attorney Docket No. ZEN-016WO1

[0245]

[0166] Table 7 shows SEC results indicating that the formulations with sucrose performed better than others after storage at 5°C and 25°C for 8 weeks. Overall, the higher concentration of excipients has obvious advantages on protein stability, and L-Arg- HC1 also has significant effects on protein stability. Therefore, sucrose and arginine were selected as excipients for further study.

[0246] Example 4. Formulations with pH of 7.2

[0247]

[0167] Based on previous studies, different strengths of sucrose (400 mM, 450 mM, and 500 mM) and L-Arg-HCl (25 mM and 50 mM) were evaluated with 0.8% (w / v) Pl 88 in the formulation optimization study. Phosphate buffer at pH 7.2 was selected as the formulation buffer system, and two buffer strengths (10 mM and 40 mM) with two protein concentrations (60 mg / mL and 75 mg / mL) were evaluated simultaneously. A total of seven formulations were conducted under 5°C for up to 6 months.

[0248] Table 8. Formulations with pH of 7.2. Attorney Docket No. ZEN-016WO1

[0249] Example 5. Long-term optimization of phosphate buffer at 7.2pH

[0250]

[0168] This example illustrates that the formulations of the present invention are stable upon long-term storage (e.g., > 6 months) at various temperatures.

[0251] Table 9. Long-term stability optimization.

[0252]

[0169] Table 9 shows the formulation optimization of protein from 50 mg / mL to

[0253] 75 mg / mL while comparing sucrose strength with 400 mM and 500 mM, and buffer strength with 40 mM and 20 mM over 6 months.

[0254] Attorney Docket No. ZEN-016WO1

[0255] Example 6. PK / PD Study of ZB004

[0256]

[0170] ZB004 was assess in both rats and cynomolgus monkeys in single-dose PK studies at doses ranging from 10 to 100 mg / kg via intravenous or subcutaneous administration. ZB004 exhibited a dose-dependent increase in exposure as measured by maximum observed serum concentration (Cmax) and area under the concentration time curve (AUC), low plasma clearance (IV administration), and a low volume of distribution (IV administration). The elimination terminal half-life ranged from 4 to 9 days in rats and 5 to 9 days in cynomolgus monkeys. When the terminal half-life parameter of ZB004 and abatacept was compared in a single-dose IV infusion PK study in female monkeys at 6 mg / kg, the 11 / 2 value of ZB004 was at least 1.39-fold longer than that of abatacept. The SC bioavailability of ZB004 was 45.4% to 72.5% for rats and 73.4% to.79.9% for cynomolgus monkeys, depending upon the doses.

[0257]

[0171] The toxicokinetics of ZB004 following repeat-dose SC administration in rats and cynomolgus monkeys for 13 weeks were assessed at doses ranging from 20 to 200 mg / kg. Results from both studies demonstrated approximately dose-proportional increases in systemic exposure (Cmax and AUC), no sex differences in exposure, and no accumulation in rats. Minimal accumulation (approximately 2-fold) of ZB004 was observed in monkeys. Immune responses to the humanized fusion protein resulted in antidrug antibody (ADA) development in very few animals, implying a low immunogenicity potential for ZB004.

[0258]

[0172] Cynomolgus monkeys and rats were selected as the relevant toxicology species based on binding affinity with targets of CD80 and CD86, as well as similar tissue cross-reactivity of ZB004 in normal human, cynomolgus monkey, and rat tissue panels. Additionally, ZB004 is expected to be pharmacologically active in both cynomolgus monkeys and rats. ZB004 was well tolerated following a single-dose SC administration in monkeys and rats. The maximum tolerated dose was determined to be >300 mg / kg, the highest tested doses in both species. Rats were found to be the more sensitive species as compared to monkey, and a 13-week repeat-dose toxicity study in rats established a noobserved adverse-effect-level with once weekly (Q1W) subcutaneous administration of 60 mg / kg. Attorney Docket No. ZEN-016WO1

[0259]

[0173] Briefly, in the 13-week repeat-dose toxicity study in monkeys, SC injection of ZB004 at doses of 20, 60, or 200 mg / kg, once weekly for 13 weeks (13 injections), resulted in no adverse ZB004-related changes at any dose level. Consequently, the no- observed-adverse-effect level (NOAEL) was considered to be 200 mg / kg. On Day 85, the ZB004 Cmax and AUC0-7d at this dose level were 2,210 pg / mL and 248,000 h*pg / mL (= 10333 pg*day / mL), respectively, in males; and 2,140 pg / mL and 204,000 h*pg / mL (= 8500 pg*day / mL), respectively, in females.

[0260]

[0174] In the 13-week repeat-dose toxicity study in rats, SC administration of ZB 004 at dosages of 20, 60, or 200 mg / kg, once weekly for 13 weeks (14 doses in total), followed by a 13-week recovery period was tolerated. ZB004-related macroscopic findings consisted of an increased incidence of focal or multifocal, red, SC discoloration at the injection site in males administered >20 mg / kg, which were not adverse due to the magnitude of the changes and the lack of evidence of tissue injury. ZB004-related histopathological findings were limited to minimal primary inflammation associated with secondary hepatocyte necrosis at all doses. There was no dose relationship or correlates with any serum chemistry parameters indictive of liver injury. These findings only correlated with non-adverse increases in white blood cell mass. The magnitude of white blood cell mass changes was within the laboratory’s historical reference range and lacked a dose relationship. However, ZB004-related liver findings were deemed adverse during the histological examination because they were considered evidence of cellular injury.

[0261]

[0175] The liver is a large organ with a high regenerative capacity. The in-life and clinical pathology data support that the ZB004-related liver findings were not adverse as they were minimal in severity and incidence, and fully reversible at the end of the recovery period. Additionally, there were no correlates during the in-life evaluation, the macroscopic examination, or during the evaluation of a full panel of serum chemistry, coagulation and urinalysis. The ZB004-related finding can likely be attributed to altered immune responses to normal physiologic insults (such as bacteria or bacterial products) to the liver as a result of the expected immunomodulatory effects of ZB004. The finding of basal cell carcinoma at the injection site of one 200 mg / kg female was considered to be ZB004-related and adverse. Attorney Docket No. ZEN-016WO1

[0262]

[0176] The NOAEL in the 13 -week rat toxicity study was considered to be 60 mg / kg under the conditions of the study. Systemic exposures (Cmax and AUC0-7d) of ZB004 at 60 mg / kg on Day 92 were 154 pg / mL and 22,300 pg*h / mL (= 929 pg*day / mL), respectively, in males and 20,7000 ng / mL and 28,100 pg*h / mL(= 1171 pg*day / mL), respectively, in females.

[0263]

[0177] The local tolerance of ZB004 at the injection site was evaluated macroscopically and microscopically, and no compound-related signs of irritation at the injection site were noted up to the highest concentration tested of 40 mg / mL in rats and 66.7 mg / mL in monkeys.

[0264]

[0178] Accounting for an additional 10-fold safety margin, the maximum recommended starting dose for first-in-human was set to 0.96 mg / kg Q1W (approximately 58 mg Q1W for subjects weighing 60 kg).

[0265] Example 7. Administration of CTLA4-Ig to human patients

[0266]

[0179] The objectives of this study were to evaluate ZB004 safety and tolerability, pharmacokinetics (PK), immunogenicity, and pharmacodynamics (PD) in healthy volunteers (HV).

[0267]

[0180] This study was a double blind, randomized, placebo-controlled, single ascending dose (SAD) study. Male and female HVs ages 18-55 years were enrolled at a single center (New Zealand Clinical Research). Eight HVs were enrolled into each dosing cohort and randomized in a 3 : 1 ratio to receive subcutaneous ZB004 or placebo. Doses tested were 3 mg, 12.5 mg, 50 mg, 125 mg, and 200 mg. ZB004 PK and anti-drug antibodies (ADA) were analysed, CD86 receptor occupancy (RO) levels were measured in the whole blood for evaluation of ZB004 target engagement, and PD activity was assessed using ex vivo stimulated whole blood.

[0268]

[0181] No safety signals were identified, with adverse events consistent with expectations in a HV population, and no serious adverse events observed. PK results showed a ZB004 half-life of between 8-18 days (Table 10). The presence of target mediated drug distribution (TMDD) effect is likely, as evidenced by the nonlinear increase in PK parameters with increase in dose level. PD analysis in whole blood samples Attorney Docket No. ZEN-016WO1 revealed clear target engagement with dose dependent response in %CD86 RO levels, and inhibition of ex vivo stimulated IL-2 in all dose cohorts (See FIG. 1). Maximum ex vivo stimulated IL-2 inhibition was observed approximately 2-6 days after ZB004 administration in each cohort (Table 11). The duration of this PD response appears to be dose dependent.

[0269] Table 10. Summary PK Parameters.

[0270] Table 11. Percentage Change from Baseline in Ex Vivo Stimulated IL-2. Attorney Docket No. ZEN-016WO1

[0271]

[0182] Immunogenicity evaluation showed a lowering of both incidence and titer value with increase in dose level, consistent with the immunosuppressive effect of ZB004. The participants who received ZB004 had their serum analyzed for presence of ADA, and 86.7% of participants (26 / 30) had at least 1 positive ADA sample at any time during the study. The presence of AD As did not have an apparent impact on the serum ZB004 exposure levels. 83.3% of participants (25 / 30) had treatment-induced ADA and 3.3% of participant (1 / 30) had treatment-boosted ADA. A lowering of both incidence and ADA titer value with increase in dose level indicated the higher immunosuppressive effect of ZB004. Due to the minimal hypersensitivity TEAEs, ADA had no apparent association with clinical safety at all doses studied.

[0272]

[0183] This study demonstrated that ZB004 was safe and well tolerated at all doses studied. Observed ZB004 PK and target engagement warrants continued clinical development.

Claims

Attorney Docket No. ZEN-016WO1CLAIMS1. A stable formulation comprising a CTLA4-Ig fusion protein, wherein the stable formulation does not comprise acetate, wherein the CTLA4-Ig comprises a first domain comprising a variant CTLA4 and a second domain comprising an IgG Fc region, wherein the variant CTLA4 comprises A29H, T51N, L61E, and K93Q substitutions, and wherein the IgG Fc region comprises M428L and N434S substitutions, and wherein the CTLA-4-Ig fusion protein is present at a concentration of greater than 30 mg / ml.

2. A stable formulation comprising a CTLA-4-Ig fusion protein, wherein the stable formulation comprises a buffer and a pH of 5.5 to 7.5, wherein the CTLA4-Ig comprises a first domain comprising a variant CTLA4 and a second domain comprising an IgG Fc region, wherein the variant CTLA4 comprises A29H, T51N, L61E, and K93Q substitutions, and wherein the IgG Fc region comprises M428L and N434S substitutions, and wherein the CTLA-4Ig fusion protein is present at a concentration of greater than 30 mg / ml.

3. A stable formulation comprising a CTLA4-Ig fusion protein, wherein the stable formulation has an osmolality of greater than 700 mOsmol / kg, wherein the CTLA4-Ig comprises a first domain comprising a variant CTLA4 and a second domain comprising an IgG Fc region, wherein the variant CTLA4 comprises A29H, T51N, L61E, and K93Q substitutions, and wherein the IgG Fc region comprises M428L and N434S substitutions, and wherein the CTLA4-Ig fusion protein is present at a concentration of greater than 30 mg / ml.Attorney Docket No. ZEN-016WO14. A stable formulation comprising a CTLA4-Ig fusion protein, wherein the stable formulation has a combined concentration of excipients of greater than 350 mM, wherein the CTLA4-Ig comprises a first domain comprising a variant CTLA4 and a second domain comprising an IgG Fc region, wherein the variant CTLA4 comprises A29H, T51N, L61E, and K93Q substitutions, and wherein the IgG Fc region comprises M428L and N434S substitutions, and wherein the CTLA4-Ig fusion protein is present at a concentration of greater than 30 mg / ml.

5. A stable formulation comprising 50 mg / mL of a CTLA4-Ig fusion protein, wherein the stable formulation comprises a buffer at a concentration of between 10-75 mM, wherein the CTLA-4Ig comprises a first domain comprising a variant CTLA4 and a second domain comprising an IgG Fc region, wherein the variant CTLA4 comprises A29H, T51N, L61E, and K93Q substitutions, and wherein the IgG Fc region comprises M428L and N434S substitutions, and wherein the amount of HMW species in the formulation is less than 10% upon storage at 5° C. for 9 weeks.

6. A stable formulation comprising a CTLA4-Ig fusion protein, wherein the stable formulation comprises the CTLA4 Ig fusion protein at a concentration of greater than 30 mg / ml and at least 300 mM sucrose, and wherein the CTLA4-Ig comprises a first domain comprising a variant CTLA4 and a second domain comprising an IgG Fc region, wherein the variant CTLA4 comprises A29H, T51N, L61E, and K93Q substitutions, and wherein the IgG Fc region comprises M428L and N434S substitutions.Attorney Docket No. ZEN-016WO17. A stable formulation comprising a CTLA4-Ig fusion protein, wherein the stable formulation comprises the CTLA4-Ig fusion protein at a concentration of greater than 30 mg / ml, and an amount of sucrose proportional to the concentration of CTLA4-Ig fusion protein, wherein 5 to 10 mM of sucrose is present for every mg / ml of CTLA4-Ig fusion protein, and wherein the CTLA4-Ig comprises a first domain comprising a variant CTLA4 and a second domain comprising an IgG Fc region, wherein the variant CTLA4 comprises A29H, T51N, L61E, and K93Q substitutions, and wherein the IgG Fc region comprises M428L and N434S substitutions.

8. The stable formulation of any one of the preceding claims, wherein the variant CTLA4 domain comprises an amino acid sequence of SEQ ID NO: 3.

9. The stable formulation of any one of the preceding claims, wherein the IgG Fc region comprises an amino acid sequence of SEQ ID NO: 4.

10. The stable formulation of any one of the preceding claims, wherein the CTLA4-Ig comprises an amino acid sequence of SEQ ID NO: 6.

11. The stable formulation of any one of the preceding claims, wherein the stable formulation further comprises an amino acid.

12. The stable formulation of claim 11, wherein the amino acid is present at a concentration of 50-300 mM.

13. The stable formulation of claim 11 or 12, wherein the amino acid comprises arginine, proline, and / or glycine.

14. The stable formulation of any one of claims 11-13, wherein the amino acid is arginine.Attorney Docket No. ZEN-016WO115. The stable formulation of any one of the preceding claims, wherein the stable formulation comprises arginine at a concentration of 100 mM.

16. The stable formulation of any one of claims 1-5 and 8-15, wherein the stable formulation further comprises a sugar.

17. The stable formulation of claim 16, wherein the sugar is present at a concentration of 200-600 mM.

18. The stable formulation of claim 16, wherein the sugar is present at a concentration of 5-15% (w / v).

19. The stable formulation of any one of claims 16-18, wherein the sugar comprises sucrose and / or trehalose.

20. The stable formulation of claim 17, wherein sucrose is present at a concentration of 400 mM.

21. The stable formulation of any one of the preceding claims, wherein the stable formulation further comprises a surfactant.

22. The stable formulation of claim 21, wherein the surfactant is poloxamer 188.

23. The stable formulation of claim 22, wherein the concentration of poloxamer 188 is less than 1% (w / v).

24. The stable formulation of claim 22 or 23, wherein the concentration of poloxamer 188 is 0.8% (w / v).

25. The stable formulation of claim 21, wherein the surfactant is polysorbate 80.Attorney Docket No. ZEN-016WO126. The stable formulation of claim 25, wherein the concentration of polysorbate 80 is less than 0.1%.

27. The stable formulation of claim 25 or 26, wherein the concentration of polysorbate 80 is 0.02%.

28. The stable formulation of any one of the preceding claims, wherein the stable formulation comprises sodium phosphate.

29. The stable formulation of claim 28, wherein sodium phosphate is present at a concentration of 10-70 mM.

30. The stable formulation of claim 28 or 29, wherein the concentration of sodium phosphate is 40 mM.

31. The stable formulation of any one of the preceding claims, wherein the stable formulation comprises succinate.

32. The stable formulation of claim 31, wherein succinate is present at a concentration of 10-70 mM.

33. The stable formulation of claim 31 or 32, wherein the concentration of succinate buffer is 20 mM.

34. The stable formulation of any one of claims 1 and 3-33, wherein the stable formulation has a pH of 5.5 to 7.5.

35. The stable formulation of claim 34, wherein the stable formulation has a pH of 7 to 7.5.

36. The stable formulation of claim 35, wherein the stable formulation has a pH of 7.2.Attorney Docket No. ZEN-016WO137. The stable formulation of claim 34, wherein the stable formulation has a pH of 6 to 7.

38. The stable formulation of claim 37, wherein the stable formulation has a pH of 6.5.

39. A stable formulation comprising:30-75 mg / ml CTLA4-Ig fusion protein,30-50 mM sodium phosphate buffer,350-450 mM sucrose,50-150 mM arginine0.6%-l% (w / v) poloxamer 188, at pH 6.7-7.7, and wherein the CTLA4-Ig fusion protein comprises an amino acid sequence of SEQ ID NO: 6.

40. The stable formulation of claim 39, comprising:50 mg / ml CTLA4-Ig fusion protein,40mM sodium phosphate,400mM sucrose, lOOmM arginine0.8% (w / v) poloxamer 188, at pH of 7.2, and wherein the CTLA4-Ig fusion protein comprises an amino acid sequence of SEQ ID NO: 6.

41. The stable formulation of any one of the preceding claims, wherein the viscosity of the formulation is no greater than 3.1 mPa s.

42. The stable formulation of any one of the preceding claims, wherein the formulation comprises 85% CTLA4-Ig monomer as measured by size exclusion chromatography.

43. The stable formulation of any one of the preceding claims, wherein the formulation comprises 90% CTLA4-Ig monomer as measured by size exclusion chromatography.Attorney Docket No. ZEN-016WO144. The stable formulation of any one of the preceding claims, wherein the formulation comprises less than 15% high molecular weight species as measured by size exclusion chromatography.

45. The stable formulation of any one of the preceding claims, wherein the formulation comprises less than 10% high molecular weight species as measured by size exclusion chromatography.

46. The stable formulation of any one of the preceding claims, wherein the formulation comprises less than 25 pg / mg residual host cell DNA.

47. The stable formulation of any one of the preceding claims, wherein the formulation comprises less than 20 pg / mg residual host cell DNA.

48. The stable formulation of any one of the preceding claims, wherein the formulation comprises less than 150 ng / mg residual host cell protein.

49. The stable formulation of claim 48, wherein the formulation comprises less than 100 ng / mg residual host cell protein.

50. The stable formulation of any one of preceding claims, wherein the formulation comprises less than 100 ng / mg residual protein A.

51. The stable formulation of claim 50, wherein the formulation comprises less than 50 ng / mg residual protein A.

52. The stable formulation of any one of the preceding claims, wherein the formulation comprises less than 0.4 EU / mg endotoxin.

53. The stable formulation of any one of the preceding claims, wherein the formulation is suitable for subcutaneous administration.Attorney Docket No. ZEN-016WO154. The stable formulation of any one of the preceding claims, wherein the formulation is suitable for intravenous administration.

55. A method of treating an autoimmune disease in a subject comprising administering to the subject in need of treatment the stable formulation of any one of the preceding claims.

56. A method of treating an autoimmune disease in a subject comprising administering to the subject in need of treatment a CTLA4-Ig fusion protein at a therapeutically effective dose no more frequently than once every other week, wherein the CTLA-4-Ig comprises a first domain comprising a variant CTLA-4 and a second domain comprising an IgG Fc region, wherein the variant CTLA-4 comprises A29H, T51N, L61E, and K93Q substitutions, and wherein the IgG Fc region comprises M428L and N434S substitutions.

57. The method of claim 56, wherein the variant CTLA4 domain comprises an amino acid sequence of SEQ ID NO: 3.

58. The method of claim 56 or 57, wherein the IgG Fc region comprises an amino acid sequence of SEQ ID NO: 4.

59. The method of any one of claims 56-58, wherein the CTLA4-Ig fusion protein comprises an amino acid sequence of SEQ ID NO: 6.

60. The method of any one of claims 56-59, wherein the therapeutically effective dose is 3 mg to 200 mg.

61. The method of any one of claims 56-60, wherein the therapeutically effective dose is 3 mg, 12.5 mg, 50 mg, 100 mg, 125 mg, or 200 mg.

62. The method of any one of claims 56-61, wherein the CTLA4-Ig fusion protein is administered no more frequently than once every week, once every two weeks, once every four weeks, or once every month.

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