Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2896—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2875—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39591—Stabilisation, fragmentation
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/08—Solutions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

• CD73 cluster of differentiation 73
• CD73 is an enzyme serves to convert AMP to adenosine.
• CD73 catalyzes the formation of extracellular adenosine which contributes to the immunosuppressive tumor environment.
• CD73 is over-expressed in stromal cells and multiple types of tumor cells, as well as in Tregs, M2 and myeloid derived suppressor cells (MDSCs) .
• CD73 inhibition prevented adenosine-mediated lymphocyte suppression, increased the activity of CD8+ effector cells, and reduced both MDSCs and Tregs.
• CD73 antibodies being developed as potential anticancer agents, but none have been approved for clinical use.
• Delivery by injection is generally the delivery route of choice for cancer treatments with antibodies or antigen binding fragments.
• biological, chemical, and physical barriers such as poor long-term storage, osmolality, solubility, and stability make delivery of biologically active agents by injection to mammals problematic. Therefore, there exists a need for improved injectable preparations of antibodies, which are stable and soluble.
• the present disclosure provides a composition comprising an anti-CD73 antibody, 5-50 mM histidine, 2%-20% (w/v) trehalose, and 0.015%-0.05% (w/v) polysorbate 80 (PS80) , at pH 5.6-6.4, wherein the antibody comprises a heavy chain variable region (VH) comprising a CDR1, a CDR2 and a CDR3, and a light chain variable region (VL) comprising a CDR1, a CDR2 and a CDR3, and wherein the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NO: 1-6, respectively.
• VH heavy chain variable region
• VL light chain variable region
• VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NO: 1-6, respectively.
• the composition comprises 10-30 mM histidine, 5%-15% (w/v) trehalose, and 0.015%-0.035% (w/v) PS80, at pH 5.8-6.2. In some embodiments, the composition comprises 15-25 mM histidine, 6%-10% (w/v) trehalose, 0.015%-0.025% (w/v) PS80, at pH 5.9-6.1. In some embodiments, the composition comprises 5-150 mg/mL of the antibody.
• the VH comprises the amino acid sequence of SEQ ID NO: 7
• the VL comprises the amino acid sequence of SEQ ID NO: 8.
• the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a light chain comprising the amino acid sequence of SEQ ID NO: 10.
• a lyophilized composition obtainable by freeze-drying the composition of the disclosure.
• a solution e.g., water
• dissolving the lyophilized composition e.g., water
• compositions for treating cancer such as bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer.
• cancer such as bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer.
• composition is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant.
• a “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
• formulation F5 with 20 nM histidine at pH 6.0 appeared to be able to support the stability of the protein.
• this buffer condition also allowed for high solubility of the protein, for at least 80 mg/mL and 150 mg/mL (Table 7) .
• Example 3 A number of candidate excipients were tested in Example 3, including sucrose, trehalose, sorbitol, mannitol, and arginine. Surprisingly, visible particles were detected in the formulation that contained mannitol, one of the most commonly used excipients for protein formulation, following freeze-thaw testing, even in the presence of PS80 (polysorbate 80) , an excipient identified as important for stabilizing the protein during freeze-thaw cycles (Tables 11-13) . Also, the formulation with sucrose suffered the most fragmentation (Table 17) . Use of another excipient, arginine, also led to relatively large (1%) decrease of protein purity (Table 18) . Yet another excipient, sorbitol, failed to sufficiently support protein stability during 10 cycles of freeze-thaw (Table 23) .
• Example 5 the thermodynamic stability of the formulations was tested and it was found that at least 0.02%PS80 was required to keep the protein stable during the testing procedure. Therefore, through trial and error, the instant inventors were able to identify a suitable formulation for the anti-CD73 antibody that includes histidine, trehalose, and PS80 at about pH 6.0.
• composition that includes anti-CD73 antibody or antigen-binding fragment of the present disclosure, histidine, trehalose, and polysorbate 80.
• the composition is an aqueous solution and the pH of the solution is 5.5 to 6.5.
• histidine e.g., histidine HCl
• histidine’s concentration is at least about 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, or 45 mM.
• histidine’s concentration is not higher than about 100 mM, 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 45 mM, 40 mM, 35 mM, 30 mM, 29 mM, 28 mM, 27 mM, 26 mM, 25 mM, 24 mM, 23 mM, 22 mM, 21 mM, 20 mM, 19 mM, 18 mM, 17 mM, 16 mM, or 15 mM.
• histidine’s concentration is about 5-40 mM, 5-35 mM, 5-30 mM, 5-25 mM, 5-20 mM, 10-50 mM, 10-45 mM, 10-40 mM, 10-35 mM, 10-30 mM, 10-25 mM, 10-20 mM, 15-50 mM, 15-45 mM, 15-40 mM, 15-35 mM, 15-30 mM, 15-25 mM, 15-20 mM, 20-50 mM, 20-45 mM, 20-40 mM, 20-35 mM, 20-30 mM, or 20-25 mM.
• trehalose e.g., trehalose dihydrate
• trehalose is present at a concentration of about 2%-20% (w/v) .
• trehalose’s concentration is at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 15%, 17%or 18% (w/v) .
• trehalose’s concentration is not higher than about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, or 3% (w/v) .
• trehalose’s concentration is about 2%-20%, 2%-19%, 2%-18%, 2%-17%, 2%-16%, 2%-15%, 2%-14%, 2%-13%, 2%-12%, 2%-11%, 2%-10%, 2%-9%, 2%-8%, 3%-20%, 3%-19%, 3%-18%, 3%-17%, 3%-16%, 3%-15%, 3%-14%, 3%-13%, 3%-13%, 3%-11%, 3%-10%, 3%-9%, 3%-8%, 4%-20%, 4%-19%, 4%-18%, 4%-17%, 4%-16%, 4%-15%, 4%-14%, 4%-13%, 4%-14%, 4%-13%, 4%-14%, 4%-11%, 4%-10%, 4%-9%, 4%-8%, 5%-20%, 5%-19%, 5%-18%, 5%-17%, 5%-16%, 5%-15%, 5%-14%, 5%-13%,
• polysorbate 80 is present at a concentration of about 0.015%-0.05% (w/v) . In some embodiments, PS80 is present at a concentration of at least about 0.015%, 0.016%, 0.017%, 0.018%, 0.019%, 0.02%, 0.021%, 0.022%, 0.023%, 0.024%, 0.025%, 0.026%, 0.027%, 0.028%, 0.029%, or 0.03% (w/v) .
• PS80 is present at a concentration of not higher than 0.05%, 0.049%, 0.048%, 0.047%, 0.046%, 0.045%, 0.044%, 0.043%, 0.042%, 0.041%, 0.04%, 0.039%, 0.038%, 0.037%, 0.036%, 0.035%, 0.034%, 0.033%, 0.032%, 0.031%, 0.03%, 0.029%, 0.028%, 0.027%, 0.026%, 0.025%, 0.024%, 0.023%, 0.022%, 0.021%, or 0.02% (w/v) .
• the composition has a pH of 5.6-6.4.
• the pH is not lower than 5.6, 5.7, 5.8, 5.85, 5.9, 5.95, 6, 6.05, 6.1, 6.15, or 6.2.
• the pH is not higher than 6.4, 6.3, 6.2, 6.15, 6.1, 6.05, 6, 5.95, 5.9, 5.85, or 5.8.
• the pH is about 5.7-6.3, 5.8-6.2, 5.85-6.15, 5.9-6.1, 5.95-6.05, or at about 5.9, 5.95, 6, 6.05, or 6.1.
• the composition further includes one or more bulking agents.
• the term “bulking agent” refers to an ingredient that provides bulk to a lyophilized formulation.
• bulking agents include, without limitation, mannitol, trehalose, lactose, sucrose, polyvinyl pyrrolidone, sucrose, glucose, glycine, cyclodextrins, dextran, solid PEGs and derivatives and mixtures thereof.
• a formulation of the present disclosure optionally includes a bulking agent.
• the composition further includes one or more tonicity agents.
• tonicity agent as used herein denotes pharmaceutically acceptable agents used to modulate the tonicity of the formulation. Isotonicity generally relates to the osmotic pressure relative to a solution, usually relative to that of human blood serum.
• a formulation can be hypotonic, isotonic or hypertonic.
• the formulation is isotonic.
• An isotonic formulation is liquid or liquid reconstituted from a solid form, e.g. from a lyophilized form and denotes a solution having the same tonicity as some other solution with which it is compared, such as physiologic salt solution and the blood serum.
• Suitable isotonicity agents include but are not limited to sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars, as defined herein as well as combinations thereof.
• the composition further includes one or more surfactants.
• surfactant refers to a pharmaceutically acceptable organic substance having amphipathic structures; namely, it is composed of groups of opposing solubility tendencies, typically an oil-soluble hydrocarbon chain and a water-soluble ionic group.
• surfactants can be classified, depending on the charge of the surface-active moiety, into anionic, cationic, and nonionic surfactants.
• Surfactants are often used as wetting, emulsifying, solubilizing, and dispersing agents for various pharmaceutical compositions and preparations of biological materials.
• the amount of surfactant is described as a percentage expressed in weight/volume percent (w/v %) .
• Suitable pharmaceutically acceptable surfactants include but are not limited to the group of polyoxyethylensorbitan fatty acid esters (Tween) , polyoxyethylene alkyl ethers (Brij) , alkylphenylpolyoxyethylene ethers (Triton-X) , polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic) , or sodium dodecyl sulphate (SDS) .
• Polyoxyethylenesorbitan-fatty acid esters include polysorbate 20, (sold under the trademark Tween 20 TM ) and polysorbate 80 (sold under the trademark Tween 80 TM ) .
• Polyethylene-polypropylene copolymers include those sold under the names F68 or Poloxamer 188 TM .
• Polyoxyethylene alkyl ethers include those sold under the trademark Brij TM .
• Alkylphenolpolyoxyethylene ethers include those sold under the tradename Triton-X.
• the composition further includes one or more lyoprotectants.
• a “lyoprotectant” refers to a pharmaceutically acceptable substance that stabilizes a protein during lyophilization (the process of rapid freezing and drying in a high vacuum) .
• lyoprotectants include, without limitation, sucrose, trehalose or mannitol.
• the composition further includes one or more antioxidants.
• An “antioxidant” refers to a molecule capable of slowing or preventing the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation reactions can produce free radicals, which start chain reactions that destabilize the protein therapeutics and ultimately affect the product activity. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions by being oxidized themselves. As a result, antioxidants are often reducing agents, chelating agent and oxygen scavengers such as citrate, EDTA, DPTA, thiols, ascorbic acid or polyphenols.
• Non-limiting examples of antioxidants include ascorbic acid (AA, E300) , thiosulfate, methionine, tocopherols (E306) , propyl gallate (PG, E310) , tertiary butylhydroquinone (TBHQ) , butylated hydroxyanisole (BHA, E320) and butylated hydroxytoluene (BHT, E321) .
• the composition further includes one or more preservatives.
• a “preservative” is a natural or synthetic chemical that is added to products such as foods, pharmaceuticals, paints, biological samples, wood, etc. to prevent decomposition by microbial growth or by undesirable chemical changes.
• Preservative additives can be used alone or in conjunction with other methods of preservation.
• Preservatives may be antimicrobial preservatives, which inhibit the growth of bacteria and fungi, or antioxidants such as oxygen absorbers, which inhibit the oxidation of constituents.
• Common antimicrobial preservatives include, benzalkonium chloride, benzoic acid, cholorohexidine, glycerin, phenol, potassium sorbate, thimerosal, sulfites (sulfur dioxide, sodium bisulfite, potassium hydrogen sulfite, etc. ) and disodium EDTA.
• Other preservatives include those commonly used in patenteral proteins such as benzyl alcohol, phenol, m-cresol, chlorobutanol or methylparaben.
• the anti-CD73 antibodies as disclosed herein, have a heavy chain variable (VH) region and a light chain variable (VL) region.
• the VH includes three complementarity determining regions (CDR) , CDR1, CDR2 and CDR3 and the VL also includes three CDRs, CDR1, CDR2 and CDR3.
• the VH CDR1, CDR2 and CDR3 and VL CDR1, CDR2 and CDR3 include the amino acid sequences SEQ ID NO: 1-6, respectively.
• Examples VH and VL sequences include SEQ ID NO: 7 and 8, respectively.
• the antibody can also include heavy chain and light chain constant regions.
• Example heavy chain and light chain sequences include SEQ ID NO: 9 and 10, respectively.
• the composition includes 5-150 mg/mL of the anti-CD73 antibody or antigen-binding fragment of the disclosure, 5-50 mM histidine, 2%-20% (w/v) trehalose, 0.015%-0.05% (w/v) PS80, at pH 5.6-6.4.
• the composition includes 10-120 mg/mL of the anti-CD73 antibody or antigen-binding fragment of the disclosure, 10-30 mM histidine, 5%-15% (w/v) trehalose, 0.015%-0.035% (w/v) PS80, at pH 5.8-6.2.
• the composition includes 10-120 mg/mL of the anti-CD73 antibody or antigen-binding fragment of the disclosure, 15-25 mM histidine, 6%-10% (w/v) trehalose, 0.015%-0.025% (w/v) PS80, at pH 5.9-6.1.
• a lyophilized composition that can be prepared by freeze-drying the aqueous solution as disclosed herein.
• a solution that can be prepared by dissolving the lyophilized composition in a solvent such as water.
• compositions of the present disclosure may be used in certain treatment and diagnostic methods.
• the present disclosure is further directed to antibody-based therapies which involve administering the composition of the disclosure to a patient such as an animal, a mammal, and a human for treating one or more of the disorders or conditions described herein.
• compositions of the disclosure can also be used to treat or inhibit cancer.
• CD73 can be overexpressed in tumor cells.
• Tumor-derived CD73 can function as an ecto-enzyme to produce extracellular adenosine, which promotes tumor growth by limiting antitumor T-cell immunity via adenosine receptor signaling.
• Results with small molecule inhibitors, or monoclonal antibodies targeting CD73 in murine tumor models, indicate that targeted CD73 therapy is an important alternative and realistic approach to effective control of tumor growth. In particular, it helps T-cell-based therapy by enhancing the adaptive immune response machinery, which may increase the function of tumor-infiltrating T lymphocytes, and lead to improved survival in cancer patients.
• the method in one embodiment, entails administering to the patient an effective amount of a composition of the present disclosure.
• at least one of the cancer cells (e.g., stromal cells) in the patient over-expresses CD73.
• Non-limiting examples of cancers include bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer.
• Additional diseases or conditions associated with increased cell survival include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia) ) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia) ) , polycythemia vera, lymphomas (e.g., Hodgkin′sdisease and non-Hodgkin′sdisease) , multiple myeloma, Waldenstrom′smacroglobulinemia, heavy chain disease, and solid tumors including, but not
• a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the particular antibodies, variant or derivative thereof used, the patient′sage, body weight, general health, sex, and diet, and the time of administration, rate of excretion, drug combination, and the severity of the particular disease being treated. Judgment of such factors by medical caregivers is within the ordinary skill in the art.
• the amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.
• compositions include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes.
• the compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc. ) and may be administered together with other biologically active agents.
• pharmaceutical compositions containing the antigen-binding polypeptides of the disclosure may be administered parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch) , bucally, or as an oral or nasal spray.
• parenteral refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intra-articular injection and infusion.
• Administration can be systemic or local.
• Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
• the amount of the antibodies of the disclosure which will be effective in the treatment, inhibition and prevention of an inflammatory, immune or malignant disease, disorder or condition can be determined by standard clinical techniques.
• in vitro assays may optionally be employed to help identify optimal dosage ranges.
• the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease, disorder or condition, and should be decided according to the judgment of the practitioner and each patient′scircumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
• the dosage administered to a patient of the antigen-binding polypeptides of the present disclosure is typically 0.1 mg/kg to 100 mg/kg of the patient′sbody weight, between 0.1 mg/kg and 20 mg/kg of the patient′sbody weight, or 1 mg/kg to 10 mg/kg of the patient′sbody weight.
• human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible.
• the dosage and frequency of administration of antibodies of the disclosure may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.
• the methods for treating an infectious or malignant disease, condition or disorder comprising administration of a composition of the disclosure are typically tested in vitro, and then in vivo in an acceptable animal model, for the desired therapeutic or prophylactic activity, prior to use in humans.
• Suitable animal models, including transgenic animals are well known to those of ordinary skill in the art.
• in vitro assays to demonstrate the therapeutic utility of antigen-binding polypeptide described herein include the effect of an antigen-binding polypeptide on a cell line or a patient tissue sample.
• the effect of the antigen-binding polypeptide on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art, such as the assays disclosed elsewhere herein.
• in vitro assays which can be used to determine whether administration of a specific antigen-binding polypeptide is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.
• compositions of the disclosure are administered in combination with an antineoplastic agent, an antiviral agent, antibacterial or antibiotic agent or antifungal agents. Any of these agents known in the art may be administered in the compositions of the current disclosure.
• compositions of the disclosure are administered in combination with a chemotherapeutic agent.
• Chemotherapeutic agents that may be administered with the compositions of the disclosure include, but are not limited to, antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin) ; antiestrogens (e.g., tamoxifen) ; antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine) ; cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cis-platin, and vincristine sulfate)
• compositions of the disclosure are administered in combination with cytokines.
• Cytokines that may be administered with the compositions of the disclosure include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF- ⁇ .
• compositions of the disclosure are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.
• a humanized anti-CD73 antibody (IM005) was developed that exhibited strong activities in binding to and inhibiting the enzymatic activity of the human CD73 protein.
• the antibody effectively induces the internalization of cell surface CD73.
• the antibody can also completely reverse AMP and tumor cell-mediated suppression of CD4 + and CD8 + T cell responses, and is efficacious in the suppression of tumor-derived CD73 activity, leading to inhibition of tumor growth.
• IM005 binds to the C-terminal domains of the CD73 protein which differs from known anti-CD-73 antibodies that bind to the N-terminal domains.
• the unique binding property of IM005 contributes to its superior CD73 inhibition profile as compared to known antibodies.
• MEDI-9447 Oleclumab
• MEDI-9447 is a human anti-CD73 monoclonal antibody currently under clinical development for the treatment of pancreatic and colorectal and other cancers. Inhibition of CD73 by MEDI-9447 requires that both binding sites on a full MEDI-9447 antibody bind to CD73, while a monovalent binding by Hu101-28 is sufficient.
• MEDI-9447 is incapable of inhibiting soluble CD73, or on cells hat express relatively low level of CD73.
• IM005 can achieve complete inhibition of CD73 activity on cells which express different levels of CD73 on the surface, and soluble CD73.
• IM005 has the following VH/VL and CDR sequences.
• VH and VK genes were produced synthetically and then respectively cloned into vectors containing the human gamma 1 and human kappa constant domains.
• Protein concentration was determined by a Thermo UV spectrophotometer.
• the extinction coefficient used in pH and buffer evaluation study was 1.660 AU*mL*mg -1 *cm -1 , which was applied in all formulation development studies. All measurements were repeated twice with 2.5 ⁇ L sample each time and an average was taken.
• Size exclusion chromatography was performed using an Agilent HPLC system with a TSKGel G3000SWXL column (300 ⁇ 7.8 mm, 5 ⁇ m) .
• the mobile phase was 50mM PB, 300mM NaCl, pH 6.8 ⁇ 0.2 and the flow rate was set as 1.0 mL/min. 100 ⁇ g protein for each sample was injected and the wavelength of detector was set at 280 nm.
• cIEF was performed on a ProteinSimple iCE analyzer equipped with FC-coated cIEF cartridge.
• the cIEF method was used to analyze isoelectric point (pI) and charge variants of IM005 samples.
• the loading mixture contained 20 ⁇ L sample at a concentration of 1.0 mg/mL, 35 ⁇ L 1%Methyl cellulose, 1 ⁇ L Pharmalyte 3-10 and 3 ⁇ L Pharmalyte 8-10.5 carrier ampholyte, 0.5 ⁇ L low pI Marker 6.61 or 6.14, 0.5 ⁇ L high pI Marker 9.46 or 9.77, 25 ⁇ L 8 mol/L urea, and 15 ⁇ L purified water to make the loading sample volume to 100 ⁇ L finally.
• Temperature of auto-sampler was 5°C, sample injection duration was 90 seconds. Focusing was performed in two stages: prefocusing for 1 min under 1500 V, followed by focusing at 3000 V for 8 min. Signals were detected at 280 nm with whole column imaging detection technology. Data were analyzed using Chromperfect Analysis Software.
• Turbidity was performed by a spectrophotometer (Spectra Max) . 150 ⁇ L samples were added into the wells of a 96-well plate, 150 ⁇ L of respective buffers were also added into the corresponding wells as the reference. Then the absorption of the buffers and samples were tested at 350 nm. The UV350 value of the protein was obtained by subtracting the corresponding buffer.
• Caliper-SDS was performed by Caliper-a microchip-based assay.
• the denaturing solution was prepared by mixing sample buffer (from kit) , 10%SDS and 100 mM N-Ethylmaleimide at 20 ⁇ 1 ⁇ 0.7 volume ratio. Two microliters of samples and 7 ⁇ L denaturing solution were mixed well, incubated at 70°C for 10 mins and spun down. H 2 O (35 ⁇ L) was added to the sample, then 42 ⁇ L of the mixture was transferred into 96-well plate and centrifuged at 4000 rpm for 20 mins to remove air bubbles. After the plate was loaded onto the instrument’s plate holder, samples were sipped, stained, separated and detected in the Microchip which was filled with destaining-gel, fluorescent dye and marker. Data was analyzed with LabChip GX Reviewer.
• the denaturing solution was prepared by mixing sample buffer (from kit) , 10%SDS and 1 M Dithiothreitol at 20 ⁇ 1 ⁇ 0.7 volume ratio, while the reference standard or samples were diluted to 1 mg/mL with MQ H 2 O. Two microliters of samples and 7 ⁇ L denaturing solution were mixed well, incubated at 70°C for 10 mins and spun down.
• H 2 O 35 ⁇ L was added to the sample, then 42 ⁇ L of the mixture was transferred into 96-well plate and centrifuged at 4000 rpm for 20 mins to remove air bubbles. After the plate was loaded onto the instrument’s plate holder, samples were sipped, stained, separated and detected in the Microchip which was filled with de-staining-gel, fluorescent dye and marker. Data was analyzed with LabChip GX Reviewer.
• DSC analysis was performed with a GE DSC System. Samples were diluted to 1mg/mL with reference buffer. 400 ⁇ L of respective reference buffers were added into the odd-numbered wells of a 96-well plate and 400 ⁇ L of samples were added into the even-numbered wells of the same plate. Experimental parameters were set such that the scan temperature ramped from 10-110°C with a rate of 200 °C/h. Analysis of thermograms was performed with MicroCal VP-Capillary DSC software.
• Viscosity was assessed using a BROOKFIELD viscometer and the cone type was CPA-40Z.
• the test temperature was 25°C and 0.5 mL sample was required.
• IM005 (20.4 mg/mL) was first exchanged into 9 different buffers as shown in Table 1. The protein concentration was adjusted to 10 mg/mL.
• thermodynamic stability of each formulation was examined using DSC.
• Tm melting temperature
• the onset temperatures of transition ranged from 46.18°C (F1: Acetate pH4.5) to 57.25°C (F7: PB pH6.0) .
• the Tm1 ranged from 54.53°C (F1 Acetate pH4.5) to 76.57°C (F3: Acetate pH5.5)
• Tm2 range from 77.27 °C (F5: Histidine pH6.0) to 86.54°C (F9: PB pH7.0) (except F3: Acetate pH5.5, F6: Histidine pH6.5 and F7: PB pH6.0) .
• the different temperature of transitions were observed among the profiles of 9 formulations.
• Protein concentrations of all formulations were measured at all sampling points, using nanodrop2000, a UV280 method. As the result shows, about 10%decrease in protein concentration was observed in F9 (PB pH7.0) and no substantial change among F1-F8 in protein concentration was seen after incubation at 40°C condition.
• F2 to F6 can be selected as formulation candidates.
• Caliper-SDS is a CE based high-throughput analysis, which is capable of detection of fragmentation of monoclonal antibodies with high sensitivity.
• F1 Acetate pH4.5
• F7 PB pH6.0
• F8 PB pH6.5
• F9 PB pH7.0
• % (LC+HC) reduction were more rapid in F1, F7, F8, and F9 samples.
• Caliper-SDS result shown the similar trend as other assay results that purity decreased more in low pH (pH4.5) and high pH (pH7.0) conditions.
• This example evaluated the solubility of IM005 in the selected buffering system.
• IM005 was exchanged into 20 mM histidine, pH 6.0 buffer and the concentration was adjusted to 80 mg/mL and 150 mg/mL.
• the samples were aseptically filtered with 0.22 ⁇ m PVDF membrane filter.
• 3 2-mL glass vials were filled with 1 mL of DS in a bio-safety hood. The vials were stoppered and crimped immediately after filling.
• a total of 6 vials were placed into different incubators (2-8°C and 25°C) , and 1 vial was removed from each condition for analysis.
• IM005 was successfully concentrated to the target concentrations of 80 mg/mL and 150 mg/mL in 20 mM histidine, pH6.0 buffer.
• the short-term stability results showed that no substantial change was observed after storage at 2 ⁇ 8°C and 25°C for 48 hours. It indicated that IM005 was stable after short term storage at 80 mg/mL or 150 mg/mL in 20 mM histidine, pH 6.0 buffer.
• the pH/buffer screening study investigated 9 pH/buffer types under stress condition (40°C) , and the result showed stability of IM005 is closely related with both pH and buffer type. Poor protein stability was observed under low pH, high pH conditions, and phosphate buffers.
• the selected buffer was 20 mM histidine, pH6.0 buffer (F5) .
• This example examined candidates excipients that could stabilize the IM005 protein in a 20 mM histidine, pH 6.0 buffer system.
• IM005 antibody stock solutions were exchanged into 20 mM histidine pH 6.0 buffer system. Then, the excipients listed in Table 8 were added to this solution, respectively. The protein concentration was adjusted to 50 mg/mL.
• Formulation Composition Osmolality (mOsmol/kg) F1 20 mM Histidine, 8%Sucrose, 0.02%PS80, pH 6.0 334 F2 20 mM Histidine, 8%Trehalose dihydrate, 0.02%PS80, pH 6.0 327 F 20 mM Histidine, 4%Sorbitol, 0.02%PS80, pH 6.0 327 F4 20 mM Histidine, 4%Mannitol , 0.02 %PS80, pH 6.0 303 F5 20 mM Histidine, 140 mM Arginine ⁇ HCl, 0: 02%PS80, pH 6.0 344 F6 20 mM Histidine, 8%sucrose, pH 6.0 332
• thermodynamic stability of each formulation was examined using DSC.
• the melting temperature (Tm) induces the temperature of start of unfolding of protein is considered as an indicator of its conformational stability.
• the onset temperatures of transition ranged from 50.9°C (F5: Arginine ⁇ HCl, pH 6.0) to 58.6°C (F2: Trehalose, pH 6.0) .
• the Tm1 ranged from 59.3°C (F5: Arginine ⁇ HCl, pH 6.0) to 65.2°C (F1: Sucrose, pH 6.0)
• Tm2 range from 76.7°C (F5: Arginine ⁇ HCl, pH 6.0) to 78.5°C (F1: Sucrose, pH 6.0) .
• the maximum difference values of Tm onset , Tm1 and Tm2 among 6 formulations are 7.7°C, 5.8°C and 1.9°C, respectively.
• Protein concentrations of 6 formulations were measured at all sampling points as shown in Tables 11-13, using nanodrop2000, a UV280 method. As the results showed, there was no substantial change in protein concentration after incubation t 25°C, 40°C for 4 weeks or repeated freeze-thaw for 5 cycles /10 cycles.
• Caliper-SDS is a CE-based high-throughput analysis method, which is used to detect fragmentation of monoclonal antibodies with high sensitivity. Caliper-SDS (nr) results at 40°C are shown in Table 17. %Purity reductions were observed in all 6 formulations. From F1 to F6, %purity was reduced by 5.8%, 3.7%, 4.4%, 4.9%, 4.8%and 4.6%, respectively. Among them, F2 (trehalose dihydrate, pH 6.0) showed the least %purity decline.
• the cIEF results at 40°C are shown in Table 20.
• the increase in acidic peak and decrease in main peak were observed in all 6 formulations. From F1 to F6, %Main peak declined by 23.2%, 22.2%, 19.4%, 21.6%, 16.0%and 19.8%, respectively. And %Acidic peak increased by 24.8%, 21.6%, 18.6%, 21.0%, 18.0%and 18.0%, respectively. It’s worth noting that there was no obvious change in %Basic peak.
• %Acidic peaks of 6 formulations were increased with the decrease of %Main peak. From F1 to F6, %Main peak declined by 5.8%, 5.4%, 7.5%, 5.4%, 5.2%and 4.3%, respectively; while %Acidic peaks increased by 6.8%, 4.2%, 7.3%, 4.5%, 5.8%and 2.9%, respectively.
• Table 22 shows the cIEF results of the freeze-thaw experiments. The results indicated that the charge of 6 formulations did not change significantly after 10 cycles of freeze thaw.
• Binding antigen method is an ELISA-based method used to detect the monoclonal antibody binding activity. The results are shown in Table 24. After 4 weeks of incubation at 25°C and 40°C, there was no obvious decrease in the binding antigen activity of F2 (trehalose dihydrate, pH 6.0)
• This example screened for optimal concentrations of PS80 to stabilize IM005 protein.
• the IM005 antibody (20.4 mg/mL) was exchanged into 20 mM histidine, pH 6.0 buffer. The concentration of trehalose dihydrate was adjusted to 8%with its stock solution. Then, 10%stock solution of PS80 was added to make the final PS80 concentration to 0, 0.01%, 0.02%and 0.03%, respectively, as described in Table 25. The concentration of protein was adjusted to 50 mg/mL.
• the prepared formulations were aseptically filtered with 0.22 ⁇ m PVDF membrane filter.
• 1 mL of sample was filled into 2 mL of glass vial.
• the vials were stoppered and crimped immediately after filling. All procedures were carried out in a bio-safety hood except for the crimping operation.
• Vials were then placed in agitation condition (300 rmp and 25°C) .
• the samples were analyzed at the specified time points.
• the osmolality of F1-F4 at T0 was determined using an osmometer (Advanced 2020 Multi-Sample) . Osmolality data are shown in Table 26.
• thermodynamic stability of each formulations (F1-F4) at T0 was examined using DSC. As shown in Table 27, the onset transition temperatures of all formulations ranged from 56.30°C to 57.39°C. The Tm1 value ranged from 64.39°C to 65.23°C and Tm2 value was in the range of 78.60°C to 78.90°C. There was no significant difference in the thermodynamic transition profiles among the four formulations.
• the protein concentrations of F1-F4 were measured using nanodrop2000 (UV280 method) at all sampling points. As shown in Table 28, no substantial change in protein concentration was detected after agitation for 3 days at 25°C.
• Caliper-SDS is a microchip and CE based high-throughput analysis method, which is capable of detecting monoclonal antibodies fragments with high sensitivity.
• the results of Caliper-SDS (nr) are shown in Table 30.
• For all formulations (F1-F4) no substantial %purity reduction was observed after agitation for 3 days at 25°C.
• Sub-visible particle detection was perfo1med using MicroFlow Image (MFI5200) .
• MFI data are shown in Table 29.
• T0 the number of the particles with the particle size ⁇ 25 ⁇ m was at the same level for F1-F4.
• the number of particles with the particle size ⁇ 10 ⁇ m and ⁇ 25 ⁇ m in F1 (without PS80) was much higher than other formulations (F2, F3 and F4) .
• Excipient screening study was conducted by investigating five kinds of excipients under 40°C, 25°C and freeze-thaw conditions, respectively. Fo1mulation with trehalose dihydrate performed the best compared with other four excipients, thus, was selected for the subsequent surfactant screening study.
• the lead formulation included 20 mM Histidine buffer, 8% (w/v) Trehalose Dihydrate, 0.02% (w/v) PS80, at pH6.0.
• IM005 sample (51.7 mg/mL) was filtered with 0.22- ⁇ m filter, aseptically filled into 8 mL glass vials with 3.3 mL IM005 per vial, stoppered and sealed in a bio-safety hood.
• CE-SDS reduced and non-reduced results were summarized in Tables 38-39.
• For CE-SDS reduced results there was no obvious decrease of purity of 2-8°q samples.
• purity of 25°C-8W decreased by 0.6%compared to T0.
• purity of 40°C-4W decreased by 2.3%compared to T0.
• For CE-SDS non-reduced results there was no obvious decrease of purity of 2-8°C samples.
• purity of 25°C-8W decreased by 2.3%compared to T0.
• purity of 40°C-4W decreased by 5.6%compared to T0. This trend is consist with results of previous formulation screening study.

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