Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic 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
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/55—Fab or Fab'
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"

Definitions

• the present application relates to novel molecules comprising binding domains to both VEGF and Ang2.
• Angiogenesis is implicated in the pathogenesis of a variety of disorders including solid tumors, intraocular neovascular syndromes such as proliferative retinopathies or age- related macular degeneration (AMD), rheumatoid arthritis, and psoriasis (Folkman, J., et al., J. Biol. Chem.267 (1992) 10931-10934; Klagsbrun, M., et al, Annu. Rev. Physiol.53 (1991) 217- 239; and Garner, A., Vascular Diseases, in: Pathobiology of Ocular Disease, A Dynamic Approach, Garner, A., and Klintworth, G. K.
• VEGF/VEGF-A Human vascular endothelial growth factor
• VEGF expression contributes to the development of solid tumors by promoting tumor angiogenesis and to the etiology of several additional diseases that are characterized by abnormal angiogenesis (Kim, K. J., et al., 1993. Nature (London) 362, 841– 844; Millauer, B., et al., 1994. Nature (London) 367, 576–579). Consequently, inhibition of VEGF signaling abrogates the development of a wide variety of tumors.
• retinopathies in which partial or general ischemia of the retina is accompanied by overexpression of VEGF and hyperproliferation of blood vessels, blindness can result (Aiello, L. P et al., 1994. N. Engl. J. Med.331, 1480–1487; Adamis, A. P., et al., , Am. J. Ophthalmol. 118, 445–450). Inhibition of VEGF expression in such disease states can treat or prevent resulting blindness.
• Ang2 Human angiopoietin-2 (ANG-2 or Ang-2 or Ang2) (alternatively abbreviated with ANGPT2 or ANG2) is described in Maisonpierre, P.C., et al, Science 277 (1997) 55-60 and Cheung,A.H., et al, Genomics 48 (1998) 389-91.
• Ang2 plays an important role in angiogenesis and its expression levels have been correlated with cancer and eye diseases (Gerald D et al., Cancer Res.2013, 73(6):1649-57; Watanabe et al., Am J Ophthalmol.2005, 139(3):476-81).
• Dual antagonist RG7716 demonstrated superior efficacy than VEGF antagonist ranibizumab in a recent clinical trial.
• the reported dosage for RG7716 at 6 mg per dose was rather high considering the volume of administration to eye is typically low, e.g. 50 micoL). This could require a concentration of 120 mg/ml, a significant challenge for formulation development.
• a dual antagonist with stronger binding affinities to VEGF and/or Ang2 is needed.
• the present invention includes bi-specific molecules with enhanced binding ability and which result in a reduction in the severity of a disease in a patient treated with a molecule disclosed herein.
• the present disclosure relates to novel bispecific chimeric molecules comprising binding domains to both VEGF and Ang-2. Further disclosed are methods of using said chimeric molecules to treat a patient of cancer, proliferative retinopathy, neovascular glaucoma, macular edema, wet age-related macular degeneration (wAMD), macular edema following retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR).
• proliferative retinopathy neovascular glaucoma
• macular edema macular edema
• wAMD wet age-related macular degeneration
• RVO macular edema following retinal vein occlusion
• DME diabetic macular edema
• DR diabetic retinopathy
• said chimeric molecule comprises one or two VEGF-binding moieties and one or two Ang-2 antagonist peptides, wherein:
• said Ang-2 antagonist peptide comprises an amino acid sequence selected from SEQ ID NO: 8-14;
• said VEGF-binding moiety is an antibody, an Fab or an scFv; and wherein said antibody, Fab or scFv comprises light chain CDRs as derived from a light chain with an amino acid sequence as shown in SEQ ID NO: 4, or derived from a scFv with an amino acid sequence as shown in SEQ ID NO: 6, and heavy chain CDRs as derived from a heavy chain with an amino acid sequence as shown in SEQ ID NO: 5, or derived from a scFv with an amino acid sequence as shown in SEQ ID NO: 6.
• said VEGF binding moiety comprises an antibody with a light chain amino acid sequence that is at least 95% identical to that of SEQ ID NO: 4, and heavy chain amino acid sequence that is at least 99% identical to that of SEQ ID NO: 7.
• said Ang-2 antagonist peptide is fused to one or both of the N- terminals of the heavy chains (HC) of the said antibody optionally through a peptide linker.
• the peptide-HC fusion polypeptide comprises an amino acid sequence that is at least 99% identical as one selected from SEQ ID NOS:29, 30, and SEQ ID NO:33.
• said Ang-2 antagonist peptide is fused to one or both of the C- terminal of the heavy chain of the said antibody optionally through a peptide linker.
• the Ang-2 antagonist peptide-heavy chain fusion polypeptide comprises an amino acid sequence that is at least 99% identical or 100% identical as one selected from SEQ ID NOS: 31, 32, and 34.
• said Ang-2 antagonist polypeptide is fused to the N-terminals or the C-terminals of the heavy chain of said antibody through a peptide linker; and wherein the Ang-2 antagonist peptide-heavy chain fusion polypeptide comprises an amino acid sequence at least 99% identical or 100% identical as one selected from SEQ ID NO: 37, 39, 41, 43, 45, 47, 49, 51, and 53.
• said VEGF binding moiety is an Fab with a light chain amino acid sequence of at least 95% identity to SEQ ID NO: 4, and a heavy chain amino acid sequence of at least 95% identity to SEQ ID NO: 5.
• th-e Ang2 antagonist peptide is fused to the N-terminal of the heavy chain of said Fab molecule through a peptide linker.
• the Ang2 antagonist peptide-heavy chain fusion polypeptide has an amino acid sequence at least 99% identical as that of SEQ ID NO:19 or SEQ ID NO:20.
• the Ang-2 antagonist peptide is fused to the C-terminal of the heavy chain of said Fab molecule through a peptide linker.
• the peptide- heavy chain fusion polypeptide has an amino acid sequence at least 99% identical as that of SEQ ID NO: 25 or SEQ ID NO:26.
• said VEGF binding moiety is an scFv with an amino acid sequence that has at least 95% identity to SEQ ID NO: 6.
• the Ang-2 antagonist peptide is fused to the N-terminal of the scFv; and wherein the peptide-scFv fusion has an amino acid sequence selected from SEQ ID NOS:21 and 22.
• the Ang-2 antagonist peptide is fused to the C-terminal of the scFv; and wherein the peptide-scFv fusion polypeptide has an amino acid sequence selected from SEQ ID NO:27 and SEQ ID NO:28.
• said chimeric molecule comprises a fusion protein that has one or more VEGF-binding moieties and one or two Ang-2 antagonist peptides, wherein said VEGF binding moiety is a VEGF trap with an amino acid sequence having at least 95% identity to SEQ ID NO: 3; wherein said chimeric molecule comprises two identical polypeptide chains, which have an amino acid sequence at least 99% identical as one selected from SEQ ID NOS:15-17, 23 and 24.
• polynucleotide or polynucleotides encoding any one of the above said chimeric molecules.
• said polynucleotide comprises a DNA sequence as one selected from SEQ ID NO: 35, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54 and 56.
• an expression vector or vectors comprising the above said polynucleotide or polynucleotides.
• composition comprising the chimeric molecule of any one of the above said chimeric molecule and a pharmaceutically acceptable excipient.
• a method of treating a patient with cancer, proliferative retinopathy, wet age-related macular degeneration (wAMD), macular edema following retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) comprising administering to a subject of above said pharmaceutical composition.
• wAMD wet age-related macular degeneration
• RVO retinal vein occlusion
• DME diabetic macular edema
• DR diabetic retinopathy
• FIG. 1 Protein A Affinity Chromatography. Approximately 150 ml of the clarified HEK 293 cell culture medium of the transient expression of AMD-B was loaded to a Protein A column (1 x 17 cm (Diameter x Height) of Captiv A Protein A resin) at 3 ml/min. The protein A column was equilibrated with an equilibration buffer (25 mM Tris Buffer, 100 mM NaCl, PH approximately 7.2). The column was washed with the Equilibration buffer and eluted with 2 M ariginine solution, PH 4.
• an equilibration buffer 25 mM Tris Buffer, 100 mM NaCl, PH approximately 7.2
• FIG. 4A Blocking of Binding of Ang-1 and Ang-2 to Tie-2 by AMD-A and AMD- B
• fusion proteins and chimeric molecules which comprise two components: an Ang-2 antagonist peptide operationally linked to a VEGF binding domain, which is selected from an anti-VEGF antibody, an anti-VEGF Fab, an anti-VEGF scFv, or a VEGF receptor extracellular domain-Fc fusion protein (or VEGF Trap).
• the Ang-2 antagonist peptide and VEGF binding domain are each defined below with reference to percent identity to a reference sequence.
• chimeric molecules to treat a patient of cancer, proliferative retinopathy, neovascular glaucoma, macular edema, wet age- related macular degeneration (wAMD), macular edema following retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR).
• proliferative retinopathy neovascular glaucoma
• macular edema wet age- related macular degeneration
• wAMD macular edema following retinal vein occlusion
• DME diabetic macular edema
• DR diabetic retinopathy
• Reference to“about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se.
• description referring to“about X” includes description of“X.”
• use of“about” preceding any series of numbers includes“about” each of the recited numbers in that series.
• description referring to “about X, Y, or Z” is intended to describe“about X, about Y, or about Z.”
• the term“antigen-binding moiety” refers to a polypeptide or a set of interacting polypeptides that specifically bind to an antigen, and includes, but is not limited to, an antibody or antibody fragment, such as a monoclonal antibody, polyclonal, a chimeric antibody, a CDR- grafted antibody, a humanized antibody, a Fab, a Fab', a F(ab')2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody (dAb), a diabody, a multispecific antibody, a dual specific antibody, an anti-idiotypic antibody, a bispecific antibody, a functionally active epitope-binding fragment thereof, bifunctional hybrid antibodies, a single chain antibody, and a Fc-containing polypeptide, such as an immunoadhesion.
• an antibody or antibody fragment such as a monoclonal antibody, polyclonal, a chimeric antibody, a CDR
• the antibody may be of any heavy chain isotype (e.g., IgG, IgA, IgM, IgE, or IgD). In some embodiments, the antibody may be of any light chain isotype (e.g., kappa or gamma). The antibody may be non-human (e.g.,
• the antibody is a derivatized antibody.
• an effective amount refers to an amount of a compound or composition sufficient to treat a specified disorder, condition, or disease, such as ameliorate, palliate, lessen, and/or delay one or more of its symptoms.
• an effective amount comprises an amount sufficient to cause a tumor to shrink and/or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation in the cancer.
• the effective amount is an amount sufficient to delay development of a cancer.
• the effective amount is an amount sufficient to prevent or delay recurrence.
• An effective amount can be administered in one or more administrations.
• the effective amount of the drug or composition may: (i) reduce the number of epithelioid cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferably stop the cancer cells infiltration into peripheral organs; (iv) inhibit (e.g., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with the cancer.
• the term“fused” or“fusion” in reference to two or more polypeptide sequences refers to joining of the polypeptide sequences through a backbone peptide bond.
• pharmaceutically acceptable when used to refer to a compound or composition means that the compound or composition is suitable for administration to a subject, including a human subject, to achieve the treatments described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.
• subject refers to a mammal and includes, but is not limited to, human, bovine, horse, feline, canine, rodent, or primate.
• treatment or“treating” is an approach for obtaining beneficial or desired results including clinical results.
• beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or
• treatment is a reduction of a pathological consequence of a disease (such as cancer).
• the methods of the invention contemplate any one or more of these aspects of treatment.
• the fusion protein or chimeric molecule comprises an Ang-2 antagonist peptide component, which binds to Angiopoietin 2 (Ang-2) and inhibits the binding of Ang-2 to its receptor.
• Ang-2 antagonist peptide component which binds to Angiopoietin 2 (Ang-2) and inhibits the binding of Ang-2 to its receptor.
• Ang-2 Angiopoietin 2
• One example of the peptide is called 2xCon4(C), as described in WO2004/092215A2 or WO03/05134A2.
• 2xCon4(C) has an amino acid sequence as shown in SEQ ID NO:1. Additional examples of Ang-2 binding peptides include but are not limited to: L-1-21, L1-7, L1- 10, and L1-15, as described in WO2004/092215A2. Examples of Ang-2 antagonist peptides are shown in SEQ ID NO: 8-14.
• the chimeric molecule further comprises a VEGF-binding moiety.
• said VEGF-binding moiety is an anti-VEGF antibody, an anti-VEGF Fab, or an anti-VEGF scFv that inhibits the binding of VEGF to its receptors.
• VEGF antibody is bevacizumab, which has two heavy chains with amino acid sequence as shown as SEQ ID NO:1, and two light chains with amino acid sequence as shown as SEQ ID NO:2.
• Another example is ranibizumab, an anti-VEGF Fab.
• a third example is Brolucizumab (RTH258), which is a humanized single-chain antibody fragment (scFv) against VEGF.
• said VEGF binding domain is a VEGF receptor-Fc fusion protein which“traps” VEGF (herein, referred to as a“VEGF trap”) and competes with the naturally occurring VEGF cellular receptor to inhibit VEGF.
• a“VEGF trap” VEGF receptor-Fc fusion protein which“traps” VEGF
• afilbercept which has an amino acid sequence as shown in SEQ ID NO:3.
• the VEGF-binding moiety comprises the six complementarity determining regions (CDRs) of Brolucizumab (RTH258), Ranibizumab or Bevacizumab.
• CDRs complementarity determining regions
• RTH258 Brolucizumab
• Ranibizumab Ranibizumab
• Bevacizumab A number of CDR delineations are known in the art and are encompassed herein. A person of skill in the art can readily determine a CDR for a given delineation based on the sequence of the heavy or light chain variable region.
• The“Kabat” Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.
• “Chothia” CDRs refer to the location of the structural loops (Chothia & Lesk, Canonical structures for the hypervariable regions of immunoglobulins, J. Mol. Biol., vol. 196, pp.901-917 (1987)).
• The“AbM” CDRs represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software.
• The“Contact” CDRs are based on an analysis of the available complex crystal structures. The residues from each of these CDRs are noted below in Table 1, in reference to common antibody numbering schemes.
• amino acid number of antibodies refers to the Kabat numbering scheme as described in Kabat et al., supra, including when CDR delineations are made in reference to Kabat, Chothia, AbM, or Contact schemes.
• the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a framework region (FR) or CDR of the variable domain.
• a heavy-chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy-chain FR residue 82.
• the Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a“standard” Kabat numbered sequence.
• the CDRs are“extended CDRs,” and encompass a region that begins or terminates according to a different scheme.
• an extended CDR can be as follows: L24—L36, L26—L34, or L26—L36 (VL-CDR1); L46—L52, L46—L56, or L50—L55 (VL-CDR2); L91—L97 (VL-CDR3); H47—H55, H47—H65, H50—H55, H53—H58, or H53—H65 (VH-CDR2); and/or H93—H102 (VH-CDR3).
• the Ang-2 peptide can be linked or fused to either the C- or N- terminus of the VEGF antibody (e.g., either the heavy or the light chains) or the VEGF receptor-Fc fusion protein.
• the Fc portion of the VEGF receptor-Fc fusion protein may be located at either the C- or N-terminus of the VEGF receptor protein. The Fc portion is further defined herein.
• the present compositions include“Fc fragments” or“Fc regions.”
• the Fc region excludes the variable regions of the heavy and light chains, the heavy-chain constant region 1 (CH1) and the light-chain constant region 1 (CL1) of the immunoglobulin.
• the Fc region may further include a hinge region at the heavy- chain constant region.
• the immunoglobulin Fc region disclosed herein may contain a part
• the immunoglobulin Fc region may be a fragment having a deletion in a relatively long portion of the amino acid sequence of CH2 and/or CH3.
• the immunoglobulin Fc region disclosed herein may comprise 1) a CH1 domain, a CH2 domain, a CH3 domain and a CH4 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, 5) a combination of one or more domains and an immunoglobulin hinge region (or a portion of the hinge region), and 6) a dimer of each domain of the heavy-chain constant regions and the light-chain constant region.
• the immunoglobulin Fc region disclosed herein includes a native amino acid sequence, or a sequence analogue thereof.
• An amino acid sequence analogue is a sequence that is different from the native amino acid sequence due to a deletion, an insertion, a non- conservative or conservative substitution or combinations thereof of one or more amino acid residues.
• analogues are possible, including one in which a region capable of forming a disulfide bond is deleted, or certain amino acid residues are eliminated at the N- terminal end of a native Fc form or a methionine residue is added thereto. Further, to remove effector functions, a deletion may occur in a complement-binding site, such as a C1q-binding site and an ADCC (antibody dependent cell mediated cytotoxicity) site.
• a complement-binding site such as a C1q-binding site and an ADCC (antibody dependent cell mediated cytotoxicity) site.
• the aforementioned Fc analogues are analogues that have a biological activity identical to the Fc region disclosed herein or improved structural stability, for example, against heat, pH, or the like.
• these Fc regions may be obtained from native forms isolated from humans and other animals including cows, goats, pigs, mice, rabbits, hamsters, rats and guinea pigs, or may be recombinants or analogues thereof, obtained from transformed animal cells or microorganisms.
• they may be obtained from a native immunoglobulin by isolating whole immunoglobulins from human or animal organisms and treating them with a proteolytic enzyme.
• a human-derived Fc region is a recombinant immunoglobulin Fc region that is obtained from a microorganism.
• the Fc region may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like.
• the immunoglobulin Fc region disclosed herein may be in the form of having native sugar chains, increased sugar chains compared to a native form or decreased sugar chains compared to the native form, or may be in a deglycosylated form. The increase, decrease or removal of the immunoglobulin Fc sugar chains may be achieved by methods common in the art, such as a chemical method, an enzymatic method and a genetic engineering method using a microorganism.
• an immunoglobulin Fc region in a deglycosylated or aglycosylated form may be more suitable as a drug carrier.
• deglycosylation refers to enzymatically removing sugar moieties from an Fc region
• amino acid sequence means that an Fc region is produced in an unglycosylated form by a prokaryote, preferably E. coli.
• the immunoglobulin Fc region may be an Fc region that is derived from IgG, IgA, IgD, IgE and IgM, or that is made by combinations thereof or hybrids thereof. In an embodiment, it is derived from IgG or IgM, which are among the most abundant proteins in human blood, and further, wherein an IgG, which is known to enhance the half-lives of ligand-binding proteins is an IgG1, IgG2a, IgG2b and/or IgG3.
• polypeptide peptide
• protein protein
• amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
• Methods for obtaining e.g., producing, isolating, purifying,
• compositions of the chimeric molecules are prepared by mixing the antibody fusion molecules or the antibody fusion molecule drug conjugate having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (see Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
• Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arg
• Buffers are used to control the pH in a range which optimizes the therapeutic effectiveness, especially if stability is pH dependent. Buffers are preferably present at concentrations ranging from about 50 mM to about 250 mM.
• Suitable buffering agents for use with the present invention include both organic and inorganic acids and salts thereof, such as citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. Additionally, buffers may comprise histidine and trimethylamine salts such as Tris.
• Preservatives are added to retard microbial growth, and are typically present in a range from 0.2% - 1.0% (w/v).
• Suitable preservatives for use with the present invention include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl or benzyl
• alkyl parabens such as methyl or propyl paraben
• catechol resorcinol
• cyclohexanol 3- pentanol
• m-cresol
• Tonicity agents sometimes known as“stabilizers” are present to adjust or maintain the tonicity of liquid in a composition. When used with large, charged biomolecules such as proteins and antibodies, they are often termed“stabilizers” because they can interact with the charged groups of the amino acid side chains, thereby lessening the potential for inter- and intra- molecular interactions. Tonicity agents can be present in any amount between 0.1% to 25% by weight, or more preferably between 1% to 5% by weight, taking into account the relative amounts of the other ingredients.
• Preferred tonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.
• Non-ionic surfactants or detergents are present to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation- induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody.
• Non-ionic surfactants are present in a range of about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07 mg/ml to about 0.2 mg/ml.
• Suitable non-ionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), polyoxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose.
• Anionic detergents that can be used include sodium lauryl sulfate, dioctyle sodium sulfosuccinate and dioctyl sodium sulfonate.
• Cationic detergents include benzalkonium chloride or benzethonium chloride.
• compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s) or solubilizing agent(s).
• compositions useful in the formulation may be different composition/formulation requirements dependent on the different delivery systems.
• pharmaceutical compositions useful in the formulation may be different composition/formulation requirements dependent on the different delivery systems.
• pharmaceutical compositions useful in the formulation may be different composition/formulation requirements dependent on the different delivery systems.
• present invention may be formulated to be administered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestible solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route.
• the formulation may be designed to be administered by a number of routes.
• said formulation is administrated directly in a tumor or tumors.
• a host cell is a cell that is transfected with an expression vector containing a nucleotide or polynucleotide sequence that encodes one or more protein sequences that can be expressed in a cell.
• a cell, including a host cell is a mammalian cell, a yeast cell, an insect cell, or a bacteria.
• a mammalian cell used as a host cell can be a Chinese hamster ovary (“CHO”) cell, a HeLa cell, an HEK cell, including an HEK-293 cell.
• a yeast cell used as a host cell can be S. cerevisiae or Pichia pastoris.
• an insect cell used as a host cell can be Sf9, Sf21, Hi-5, Schneider 2 cells, Schneider 3 cells or High Five.
• a bacterial cell used as a host cell can be E. coli, Corynebacterium or C. glutamicum.
• an antibody or protein formulation is a lyophilized formulation. In another embodiment, an antibody or protein formulation is an aqueous formulation.
• a fusion protein or chimeric molecule disclosed herein reduces the severity of a disease by, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%.
• a fusion protein or chimeric molecule disclosed herein reduces the severity of a disease from, e.g., about 5% to about 100%, about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about
• a fusion protein or chimeric molecule disclosed herein may comprise a therapeutic compound in an amount sufficient to allow customary administration to an individual and with other excipients may constitute a pharmaceutical composition.
• a therapeutic compound disclosed herein may be, e.g., at least 5 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, or at least 100 mg of a therapeutic compound.
• a therapeutic compound disclosed herein may be, e.g., at least 5 mg, at least 10 mg, at least 20 mg, at least 25 mg, at least 50 mg, at least 75 mg, at least 100 mg, at least 200 mg, at least 300 mg, at least 400 mg, at least 500 mg, at least 600 mg, at least 700 mg, at least 800 mg, at least 900 mg, at least 1,000 mg, at least 1,100 mg, at least 1,200 mg, at least 1,300 mg, at least 1,400 mg, or at least 1,500 mg of a therapeutic compound.
• a therapeutic compound disclosed herein may be in the range of, e.g., about 5 mg to about 100 mg, about 10 mg to about 100 mg, about 50 mg to about 150 mg, about 100 mg to about 250 mg, about 150 mg to about 350 mg, about 250 mg to about 500 mg, about 350 mg to about 600 mg, about 500 mg to about 750 mg, about 600 mg to about 900 mg, about 750 mg to about 1,000 mg, about 850 mg to about 1,200 mg, or about 1,000 mg to about 1,500 mg.
• a therapeutic compound disclosed herein may be in the range of, e.g., about 10 mg to about 250 mg, about 10 mg to about 500 mg, about 10 mg to about 750 mg, about 10 mg to about 1,000 mg, about 10 mg to about 1,500 mg, about 50 mg to about 250 mg, about 50 mg to about 500 mg, about 50 mg to about 750 mg, about 50 mg to about 1,000 mg, about 50 mg to about 1,500 mg, about 100 mg to about 250 mg, about 100 mg to about 500 mg, about 100 mg to about 750 mg, about 100 mg to about 1,000 mg, about 100 mg to about 1,500 mg, about 200 mg to about 500 mg, about 200 mg to about 750 mg, about 200 mg to about 1,000 mg, about 200 mg to about 1,500 mg, about 5 mg to about 1,500 mg, about 5 mg to about 1,000 mg, or about 5 mg to about 250 mg.
• a therapeutic compound disclosed herein may comprise a solvent, emulsion or other diluent in an amount sufficient to dissolve a therapeutic compound disclosed herein.
• a therapeutic compound disclosed herein may comprise a solvent, emulsion or a diluent in an amount of, e.g., less than about 90% (v/v), less than about 80% (v/v),
• a therapeutic compound disclosed herein may comprise a solvent, emulstion or other diluent in an amount in a range of, e.g., about 1% (v/v) to 90% (v/v), about 1% (v/v) to 70% (v/v), about 1% (v/v) to 60% (v/v), about 1% (v/v) to 50% (v/v), about 1% (v/v) to 40% (v/v), about 1% (v/v) to 30% (v/v), about 1% (v/v) to 20% (v/v), about 1% (v/v) to 10% (v/v), about 2% (v/v) to 50% (v/v), about 2% (v/v) to 40% (v/v), about 2% (v/v) to 30% (v/v), about 2% (v/v) to 20% (v/v), about 2% (v/v) to 10% (v/v), about 4% (v/v) to 50% (v/v/v), about
• the final concentration of a therapeutic compound disclosed herein in a pharmaceutical composition disclosed herein may be of any concentration desired.
• the final concentration of a therapeutic compound in a pharmaceutical composition may be a therapeutically effective amount.
• the final concentration of a therapeutic compound in a pharmaceutical composition may be, e.g., at least 0.00001 mg/mL, at least 0.0001 mg/mL, at least 0.001 mg/mL, at least 0.01 mg/mL, at least 0.1 mg/mL, at least 1 mg/mL, at least 10 mg/mL, at least 25 mg/mL, at least 50 mg/mL, at least 100 mg/mL, at least 200 mg/mL, at least 500 mg/mL, at least 700 mg/mL, at least 1,000 mg/mL, or at least 1,200 mg/mL.
• the concentration of a therapeutic compound disclosed herein in the solution may be, e.g., at most 1,000 mg/mL, at most 1,100 mg/mL, at most 1,200 mg/mL, at most 1,300 mg/mL, at most 1,400 mg/mL, at most 1,500 mg/mL, at most 2,000 mg/mL, at most 2,000 mg/mL, or at most 3,000 mg/mL.
• the final concentration of a therapeutic compound in a pharmaceutical composition may be in a range of, e.g., about 0.00001 mg/mL to about 3,000 mg/mL, about 0.0001 mg/mL to about 3,000 mg/mL, about 0.01 mg/mL to about 3,000 mg/mL, about 0.1 mg/mL to about 3,000 mg/mL, about 1 mg/mL to about 3,000 mg/mL, about 250 mg/mL to about 3,000 mg/mL, about 500 mg/mL to about 3,000 mg/mL, about 750 mg/mL to about 3,000 mg/mL, about 1,000 mg/mL to about 3,000 mg/mL, about 100 mg/mL to about 2,000 mg/mL, about 250 mg/mL to about 2,000 mg/mL, about 500 mg/mL to about 2,000
• aspects of the present specification disclose, in part, treating an individual suffering from a disease, including a cancer.
• treating refers to reducing or eliminating in an individual a clinical symptom of cancer; or delaying or preventing in an individual the onset of a clinical symptom of a disease, including a cancer.
• the term "treating" can mean reducing a symptom of a condition characterized by a cancer, including, but not limited to, tumor size, by, e.g., at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, or at least 100%.
• the actual symptoms associated with cancer are well known and can be determined by a person of ordinary skill in the art by taking into account factors, including, without limitation, the location of the disease, including a cancer, the cause of the disease, including a cancer, the severity of the disease, including a cancer, and/or the tissue or organ affected by the disease, including a cancer.
• factors including, without limitation, the location of the disease, including a cancer, the cause of the disease, including a cancer, the severity of the disease, including a cancer, and/or the tissue or organ affected by the disease, including a cancer.
• Those of skill in the art will know the appropriate symptoms or indicators associated with a specific type of disease, including a cancer and will know how to determine if an individual is a candidate for treatment as disclosed herein.
• a therapeutically effective amount of a therapeutic compound disclosed herein reduces a symptom associated with a disease, including a cancer by, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%,
• a therapeutically effective amount of a therapeutic compound disclosed herein reduces a symptom associated with a disease, including a cancer by, e.g., at most 10%, at most 15%, at most 20%, at most 25%, at most 30%, at most 35%, at most 40%, at most 45%, at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 95% or at most 100%.
• a therapeutically effective amount of a therapeutic compound disclosed herein reduces a symptom associated with a disease, including a cancer by, e.g., about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 20%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.
• a therapeutically effective amount of a therapeutic compound disclosed herein generally is in the range of about 0.001 mg/kg/day to about 100 mg/kg/day.
• an effective amount of a therapeutic compound disclosed herein may be, e.g., at least 0.001 mg/kg/day, at least 0.01 mg/kg/day, at least 0.1 mg/kg/day, at least 1.0 mg/kg/day, at least 5.0 mg/kg/day, at least 10 mg/kg/day, at least 15 mg/kg/day, at least 20 mg/kg/day, at least 25 mg/kg/day, at least 30 mg/kg/day, at least 35 mg/kg/day, at least 40 mg/kg/day, at least 45 mg/kg/day, or at least 50 mg/kg/day.
• an effective amount of a therapeutic compound disclosed herein may be in the range of, e.g., about 0.001 mg/kg/day to about 10 mg/kg/day, about 0.001 mg/kg/day to about 15 mg/kg/day, about 0.001 mg/kg/day to about 20 mg/kg/day, about 0.001 mg/kg/day to about 25 mg/kg/day, about 0.001 mg/kg/day to about 30 mg/kg/day, about 0.001 mg/kg/day to about 35 mg/kg/day, about 0.001 mg/kg/day to about 40 mg/kg/day, about 0.001 mg/kg/day to about 45 mg/kg/day, about 0.001 mg/kg/day to about 50 mg/kg/day, about 0.001 mg/kg/day to about 75 mg/kg/day, or about 0.001 mg/kg/day to about 100 mg/kg/day.
• an effective amount of a therapeutic compound disclosed herein may be in the range of, e.g., about 0.01 mg/kg/day to about 10 mg/kg/day, about 0.01 mg/kg/day to about 15 mg/kg/day, about 0.01 mg/kg/day to about 20 mg/kg/day, about 0.01 mg/kg/day to about 25 mg/kg/day, about 0.01 mg/kg/day to about 30 mg/kg/day, about 0.01 mg/kg/day to about 35 mg/kg/day, about 0.01 mg/kg/day to about 40 mg/kg/day, about 0.01 mg/kg/day to about 45 mg/kg/day, about 0.01 mg/kg/day to about 50 mg/kg/day, about 0.01
• an effective amount of a therapeutic compound disclosed herein may be in the range of, e.g., about 0.1 mg/kg/day to about 10 mg/kg/day, about 0.1 mg/kg/day to about 15 mg/kg/day, about 0.1 mg/kg/day to about 20 mg/kg/day, about 0.1 mg/kg/day to about 25 mg/kg/day, about 0.1 mg/kg/day to about 30 mg/kg/day, about 0.1 mg/kg/day to about 35 mg/kg/day, about 0.1 mg/kg/day to about 40 mg/kg/day, about 0.1 mg/kg/day to about 45 mg/kg/day, about 0.1 mg/kg/day to about 50 mg/kg/day, about 0.1 mg/kg/day to about 75 mg/kg/day, or about 0.1 mg/kg/day to about 100 mg/kg/day.
• an effective amount of a therapeutic compound disclosed herein may be in the range of, e.g., about 1 mg/kg/day to about 10 mg/kg/day, about 1 mg/kg/day to about 15 mg/kg/day, about 1 mg/kg/day to about 20 mg/kg/day, about 1 mg/kg/day to about 25 mg/kg/day, about 1 mg/kg/day to about 30 mg/kg/day, about 1 mg/kg/day to about 35 mg/kg/day, about 1 mg/kg/day to about 40 mg/kg/day, about 1 mg/kg/day to about 45 mg/kg/day, about 1 mg/kg/day to about 50 mg/kg/day, about 1 mg/kg/day to about 75 mg/kg/day, or about 1 mg/kg/day to about 100 mg/kg/day.
• an effective amount of a therapeutic compound disclosed herein may be in the range of, e.g., about 5 mg/kg/day to about 10 mg/kg/day, about 5 mg/kg/day to about 15 mg/kg/day, about 5 mg/kg/day to about 20 mg/kg/day, about 5 mg/kg/day to about 25 mg/kg/day, about 5 mg/kg/day to about 30 mg/kg/day, about 5 mg/kg/day to about 35 mg/kg/day, about 5 mg/kg/day to about 40 mg/kg/day, about 5 mg/kg/day to about 45 mg/kg/day, about 5 mg/kg/day to about 50 mg/kg/day, about 5 mg/kg/day to about 75 mg/kg/day, or about 5 mg/kg/day to about 100 mg/kg/day.
• a concentration of a therapeutic compound disclosed herein typically may be between about 50 mg/mL to about 1,000 mg/mL.
• a therapeutically effective amount of a therapeutic compound disclosed herein may be from, e.g., about 50 mg/mL to about 100 mg/mL, about 50 mg/mL to about 200 mg/mL, about 50 mg/mL to about 300 mg/mL, about 50 mg/mL to about 400 mg/mL, about 50 mg/mL to about 500 mg/mL, about 50 mg/mL to about 600 mg/mL, about 50 mg/mL to about 700 mg/mL, about 50 mg/mL to about 800 mg/mL, about 50 mg/mL to about 900 mg/mL, about 50 mg/mL to about 1,000 mg/mL, about 100 mg/mL to about 200 mg/mL, about 100 mg/mL to about 300 mg/mL, about 100 mg/mL to about 400 mg/mL,
• Dosing can be single dosage or cumulative (serial dosing), and can be readily determined by one skilled in the art.
• treatment of a disease, including acancer may comprise a one-time administration of an effective dose of a therapeutic compound or a pharmaceutical composition disclosed herein.
• treatment of a disease, including a cancer may comprise multiple administrations of an effective dose of a pharmaceutical composition carried out over a range of time periods, such as, e.g., once daily, twice daily, trice daily, once every few days, or once weekly.
• the timing of administration can vary from individual to individual, depending upon such factors as the severity of an individual's symptoms.
• an effective dose of a therapeutic compound or pharmaceutical composition disclosed herein can be administered to an individual once daily for an indefinite period of time, or until the individual no longer requires therapy.
• a person of ordinary skill in the art will recognize that the condition of the individual can be monitored throughout the course of treatment and that the effective amount of a therapeutic compound or pharmaceutical composition disclosed herein that is administered can be adjusted accordingly.
• a therapeutic compound disclosed herein is capable of reducing the number of cancer cells or tumor size in an individual suffering from a cancer by, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% as compared to a patient not receiving the
• a therapeutic compound is capable of reducing the number of cancer cells or tumor size in an individual suffering from a cancer by, e.g., about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70% as compared to a patient not receiving the same treatment.
• a therapeutic compound and its derivatives have half-lives of 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, one month, two months, three months, four months or more.
• the period of administration of a therapeutic compound is for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more.
• a period of during which administration is stopped is for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more.
• a therapeutically effective amount of a therapeutic compound disclosed herein reduces or maintains a disease, including a cancer cell population and/or tumor cell size in an individual by, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 100%.
• a therapeutically effective amount of a therapeutic compound disclosed herein reduces or maintains a disease or a cancer cell population and/or tumor cell size in an individual by, e.g., at most 10%, at most 15%, at most
• a therapeutically effective amount of a therapeutic compound disclosed herein reduces or maintains a disease, including a cancer cell population and/or tumor cell size in an individual by, e.g., about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 20%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.
• a pharmaceutical composition or therapeutic compound is administered to an individual.
• An individual is typically a human being, but can be an animal, including, but not limited to, dogs, cats, birds, cattle, horses, sheep, goats, reptiles and other animals, whether domesticated or not.
• any individual who is a candidate for treatment is a candidate with some form of disease, including a cancer, whether the cancer is benign or malignant, a tumor, solid or otherwise, a cancer cell not located in a tumor or some other form of cancer.
• cancer include, but are not limited to, bladder cancer, breast cancer, colon and rectal cancer, endometrial cancer, kidney cancer, renal cancer, leukemia, lung cancer, melanoma, non-Hodgkins lymphoma, pancreatic cancer, prostate cancer, stomach cancer and thyroid cancer.
• Pre-operative evaluation typically includes routine history and physical examination in addition to thorough informed consent disclosing all relevant risks and benefits of the procedure.
• Example 1 Production of the Chimeric Molecule Comprising VEGF Antibody and Ang-2 Binding Peptide in HEK293 Cells
• Chimeric molecules named AMD A, B, C, D and E were expressed through transient expression by HEK-293 cells. Briefly, DNAs (SEQ ID NOs: 58, 59, 60 and 63) for the fusion proteins comprising VEGF antibody light chain with or without Ang2 binding peptides and DNAs (SEQ ID NOs: 57, 61 and 62) for the fusion proteins comprising VEGF antibody heavy chain with Ang2 binding peptides were synthesized and cloned into expression vectors. The complete expression constructs comprising the genes were confirmed by DNA sequencing. DNA constructs were transformed into E. coli DH5alfa competent cells
• the cell culture media were harvested by clarifying centrifugation at 9000 rpm for 30-60 minutes followed by filtration through 0.22 micrometer filters.
• the clarified supernants were loaded to a Protein A affinity column and the chimeric molecules (AMD-A, B, C, D and E) were purified.
• the chimeric molecules were eluted using 2 M arginine solution, pH 4 from the protein A column.
• Figure 1 shows a representative chromatograph of the Protein A column step.
• Table 3 summarizes the results from the purification of the chimeric molecules.
• chimeric molecules containing a total of 2 copies L1-15 peptides (AMD-B and AMD-D), both fused to the N-terminals of the heavy chain, had significantly higher expression levels comparing to the ones with a total of four copies of L1-15 peptides (AMD-A and AMD-C), wherein there is one each of L1-15 peptide fused to the N-terminals of both the light chains and the heavy chains of the antibody.
• ASD-A and AMD-C L1-15 peptide fused to the N-terminals of both the light chains and the heavy chains of the antibody.
• AMD-E has one Peptide 2xCon4(C) fused to each of the C-terminus of the heavy chains of Bevacizumab.
• the purity of the products were analyzed using SDS electrophoresis and/or HPLC methods.
• DNA for the chimeric molecule comprising the VEGF Receptor-Fc fusion protein (VEGF Trap) and the Ang-2 binding peptide (SEQ ID NO: 64, named as ASKB-E06) is synthesized and cloned into an expression vector.
• the complete expression construct comprising the DNA gene is confirmed by DNA sequencing.
• the expression construct is amplified by transforming into DH10B E. coli and culturing the cells overnight.
• DNA for the expression construct was prepared and purified by endo-free plasmid kit (from QIAGEN ® ).
• Cell lines stably expressing ASKB-E06 is obtained by transfecting the expression construct into GS -/- Chinese hamster ovarian cells (CHO) by electroporation and screening for transfected CHO cells using a selective culture medium without glutamine (EX-CELL ® CD CHO Fusion Growth Medium). In this manner 32 or more stable minipools are established and the leading mini-pool is selected based on expression level in batch and fed-batch cultures. The expression levels are detected by ELISA titer assay. Single cloning is performed by limited dilution and using clone media, two leading single clones out of more than100 positive clones are selected based on productivity and cell growth in batch and fed-batch culture.
• the lead clones are expanded and seeded at 0.5x10 6 cells/mL, total 300 mL in 2L shake flasks, and the cells are cultured at 37 o C, 5% CO 2 , 70% HMR conditions and shaking at 120 rpm.
• the cultures are fed by using 5% Acti CHO ® Feed A + 0.5% Feed B (from GE Health) on Day 3, 6, 7, 8 and 9.
• the cell viability, viable cell density are monitored every other day, the cultures are harvested on Day 11-13.
• the cell culture medium is harvested by clarifying approximately 600 mL of the cultured cell medium through centrifugation at 2000 rpm for 10 minutes followed by filtration.
• the clarified supernant is loaded to a Protein A affinity column and the chimeric molecule is purified.
• the protein is further purified using ion exchange chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, and/or mixed mode chromatography.
• the product is further concentrated and buffer exchanged using UFDF and further formulated.
• the purity of the product is analyzed using CE-SDS and HPLC methods.
• Molecular assays (Octet Binding Affinity, Affinity ELISA, and Blocking ELISA) were developed to assess direct binding of the chimeric molecules to ANG-1, Ang-2 and/or VEGF, and the effect of the chimeric molecules on the Ang1:Tie-2 interaction, Ang-2:Tie-2 interaction and/or VEGF:VEGF receptor interaction. These in vitro assays are described as the following: Octet Affinity
• VEGF protein Purified recombinant human VEGF protein was ordered from Life-Technologies (Cat.# PHC9391 ). Human Ang1 or Ang2 protein were ordered from R&D System. Analysis was carried out using Octet Red96 from Pall ForteBio. Using anti-human IgG Fc sensors, a sample of chimeric molecule AMD-B, AMD-D, AMD-E or the control antibody Bevacizumab was loaded for 300 seconds at 3ug/mL in the kinetics buffer. Ligands ANG1, ANG2, or VEGF samples were associated for 300 seconds using a dilution series starting at 5 or 10 ug/mL and sequentially diluting 2-fold for 7 wells. Dissociation was run for 600 seconds.
• Affinity ELISA Purified recombinant human VEGF protein was ordered from Life- Technologies (Cat.# PHC9391 ). VEGF is reconstituted in BSA solution at 0.1 mg/mL as recommended by the manufacturer. Aliquots the samples were made and stored at -20 o C.
• VEGF vascular endothelial growth factor
• PBS phosphate buffered saline
• Tween-20 four times.
• the wells are then blocked using about 250 microliters per well of about 5 percent BSA in PBS, and the plates were incubated at room temperature for about 2 hours. After incubation, excess blocking solution is discarded, and about 100 microliters of AMD-A, B, C, D or E was added to a well in a dilution series starting at a concentration of about 40 nanomolar and then serially diluting 4- fold in PBS containing about 1 percent BSA.
• Figure 3 shows the ELISA results of binding of VEGF to AMD-A, B, C, D, and E.
• the results showed that all the molecules AMD-A, B, C, D and E retained abilities to bind to VEGF.
• the EC-50 results are summarized in Table 5.
• the results showed that the AMD-B and AMD-D had VEGF binding affinity close to ASKB1202.
• AMD- B and AMD-D had stronger VEGF binding affinity than AMD-A and AMD-C.
• chimeric molecules were assessed in their abilities in blocking the binding of Ang1 and Ang2 to their receptor Tie-2.
• 96 well microtiter plate (Nunk) was coated with 100 uL final concentration 100 ng/mL of human Tie2-Fc (R&D System, 313-T1) diluted in 0.1 M carbonate (pH9.3) at 4 o C overnight. The plate was then blocked for 2 hours with 5% BSA in PBST (0.05% Tween 20). Purified chimeric molecule, at starting concentration of 1000 ng/mL, was serially diluted with dilution factor of three in PBS with 1% BSA.
• Human Ang1 or Ang2 protein (R&D System) was added to final concentration of 50 ng/mL and incubated at room temperature for 1 hour. The Chimeric molecule-Ang1 or Chimeric molecule-Ang2 mixture was then added into microtiter plate coated with human Tie2-Fc and incubate for another 1 hour at room temperature. 100 uL anti-Ang1 or anti-Ang2 monoclonal antibody (R&D System) was added into each well at final concentration of 1 ug/mL and incubated for 1 hour at room temperature. Horseradish-peroxidase (HRP) conjugated anti-mouse IgG secondary antibody was added at 1:5000 dilution and incubated for 1 hour at room temperature. Standard colorimetric response was developed by using TMB (Pierce). Absorbance was read at OD450 by spectrophotometer. Between each step, the plate was washed 5 time with 100 uL PBS.
• HRP horseradish-peroxidase conjugated anti-mouse
• Figure 5 shows the inhibition of the binding of Ang-2 to Tie-2 by chimeric molecules 712-O and 712-O2.
• the chimeric molecule 712-O comprises two heavy chain polypeptide chains with an amino acid sequence as shown in SEQ ID NO: 29 and two light chains with an amino acid sequence as shown in SEQ ID NO: 4.
• the chimeric molecule 712-O2 comprises
• the Ang-2 antagonist peptide L1-15 is fused to the N-terminals of the heavy chains of a VEGF-binding antibody in the case of 712-O.
• L1-15 is fused to the C-terminals of the heavy chains.
• the IC-50’s for the Ang-2 blocking assay were approximately 33 pM for 712-O and approximately 78 pM for 712-O2. Since L1-15, together with other peptides including L1-7, L1-10 and L1-21, was considered an N-terminal fusion peptide and was only tested to be active when it is fused to the N-terminal of the Fc as described in WO2004/092215A2. It was surprised that the chimeric molecule 712-O2 was significantly potent with an IC-50 of approximately 78 pM.
• Blocking ELISA Results Inhibition of Binding of Ang-1 or Ang-2 to Tie-2.
• Example 4 Cell-based activity assay: In vitro Human Umbilical Vein Endothelial Cells
• EBM-2 medium having VEGF-A (50 ng/ml) is added thereto, or EBM-2 medium including VEGF- A (50 ng/ml) and 712-O sample at different concentration is added thereto in each well of a 96- well plate, followed by incubation under 5% CO 2 , at 37° C. for 72 hours. Then, 10 ml of WST-1 solution was added thereto, followed by incubation at 37° C. for 4 hours. Absorbance is measured at 410 nm with a reference of 610 nm. The results are shown in Table 7, which indicated that 712-O had similar or higher potency than Lucentis®. It was more potent than ASKB1202 (a biosimilar of bevacizumab).
• a lower chamber is filled with 600 ml of EBM- 2 medium (Lonza), EBM-2 with VEGF-A (50 ng/ml), or EBM-2 with VEGF-A (50 ng/ml) and 712- O sample at different concentration.
• An upper chamber is provided with 100 ml of EBM-2 medium containing 1 ⁇ 10 5 HUVEC.
• a filter is detached from the Transwell and cells are fixed with methanol for 1 minute and stained with Hematoxylin/Eosin. Cells which do not migrate but are left on an upper surface of the transwell are completely removed with a cotton swab. Five random fields among the cells migrated through the filter are arbitrarily chosen under an optical microscope ( ⁇ 100) and the number thereof is counted.
• tube formation assay is performed. More specifically, after a 96-well plate is coated with Growth Factor Reduced Matrigel (BD Biosciences, US), 15,000 HUVEC in 100 ml of EBM-2 medium, EBM-2 medium with VEGF-A (50 ng/ml), or EBM-2 medium with VEGF-A (50 ng/ml) and an antibody sample are added to each well, followed by incubation in 37° C. cell incubator for 6 hours. Then, tube formation is observed by using an inverted microscope.
• Growth Factor Reduced Matrigel BD Biosciences, US
• Example 5 In vivo anti-tumor activity study: Therapeutic Efficacy Studies With Systemically
• the chimeric molecule ASKB712-B is administered subcutaneously to A431 tumor- bearing mice at a once-per-day schedule 72 hours after tumor challenge.
• the doses used are
• a chimeric molecule which comprises one or two VEGF-binding moieties and one or two Ang-2 antagonist peptides, wherein:
• said Ang-2 antagonist peptide comprises an amino acid sequence selected from SEQ ID NO: 8-14;
• said VEGF-binding moiety is an antibody, an Fab or an scFv; and wherein said antibody, Fab or scFv comprises light chain CDRs as derived from a light chain with an amino acid sequence as shown in SEQ ID NO: 4, or derived from a scFv with an amino acid sequence as shown in SEQ ID NO: 6, and heavy chain CDRs as derived from a heavy chain with an amino acid sequence as shown in SEQ ID NO: 5, or derived from a scFv with an amino acid sequence as shown in SEQ ID NO: 6.
• VEGF binding moiety comprises an antibody with a light chain amino acid sequence that is at least 95% identical to that of SEQ ID NO: 4, and heavy chain amino acid sequence that is at least 99% identical to that of SEQ ID NO: 7.
• Ang-2 antagonist peptide-HC fusion polypeptide comprises an amino acid sequence that has at least 99% identity to one of SEQ ID NOS:29, 30, and SEQ ID NO:33.
• Ang-2 antagonist peptide-heavy chain fusion polypeptide comprises an amino acid sequence at least 99% identical or 100% identical as one selected from SEQ ID NOS: 31, 32, and 34.
• Ang-2 antagonist polypeptide is fused to the N-terminals or the C-terminals of the heavy chain of said antibody through a peptide linker; and wherein the Ang-2 antagonist peptide-heavy chain fusion polypeptide comprises an amino acid sequence at least 99% identical or 100% identical as one selected from SEQ ID NO: 37, 39, 41, 43, 45, 47, 49, 51, and 53..
• VEGF binding moiety is an Fab with a light chain amino acid sequence of at least 95% identity to SEQ ID NO: 4, and a heavy chain amino acid sequence of at least 95% identity to SEQ ID NO: 5.
• Ang-2 antagonist peptide-heavy chain fusion polypeptide has an amino acid sequence at least 99% identical as that of SEQ ID NO:19 or SEQ ID NO:20.
• VEGF binding moiety is an scFv with an amino acid sequence having at least 95% identity to SEQ ID NO: 6.
• Ang2 antagonist peptide is fused to the C-terminal of the scFv optionally; and wherein the peptide-scFv fusion has an amino acid sequence selected from SEQ ID NO:27 and SEQ ID NO:28.
• a chimeric molecule comprising a fusion protein that has one or more VEGF-binding moieties and one or two Ang-2 antagonist peptides, wherein said VEGF binding moiety is a VEGF trap with an amino acid sequence having at least 95% identity to SEQ ID NO: 3; wherein the chimeric molecule comprises two identical polypeptide chains, each having an amino acid sequence at least 99% identical to one of SEQ ID NOS:15-17, 23 and 24.
• a method of making the chimeric molecule of any one of claims 1-16 comprising culturing a host cell transfected with one or more expression vectors containing a polynucleotide that encodes a chimeric molecule of one of claims 1 -16 under conditions that allow expression of the chimeric molecule, and isolating the chimeric molecule.
• a pharmaceutical composition comprising the chimeric molecule of any one of claims 1-16 and a pharmaceutically acceptable excipient.
• composition of claim 21, wherein the pharmaceutical composition is in the form of a lyophilized formulation or an aqueous solution.
• a method of treating a patient with cancer, proliferative retinopathy, wet age-related macular degeneration (wAMD), macular edema following retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) comprising administering to a subject a pharmaceutical composition of claim 21.
• wAMD wet age-related macular degeneration
• RVO retinal vein occlusion
• DME diabetic macular edema
• DR diabetic retinopathy
• the open-ended transitional term“comprising” (and equivalent open-ended transitional phrases thereof like including, containing and having) encompasses all the expressly recited elements, limitations, steps and/or features alone or in combination with unrecited subject matter; the named elements, limitations and/or features are essential, but other unnamed elements, limitations and/or features may be added and still form a construct within the scope of the claim.
• the meaning of the open-ended transitional phrase“comprising” is being defined as encompassing all the specifically recited elements, limitations, steps and/or features as well as any optional, additional unspecified ones.
• the meaning of the closed-ended transitional phrase“consisting of” is being defined as only including those elements, limitations, steps and/or features specifically recited in the claim whereas the meaning of the closed-ended transitional phrase“consisting essentially of” is being defined as only including those elements, limitations, steps and/or features specifically recited in the claim and those elements, limitations, steps and/or features that do not materially affect the basic and novel characteristic(s) of the claimed subject matter.
• the open-ended transitional phrase“comprising” (and equivalent open-ended transitional phrases thereof) includes within its meaning, as a limiting case, claimed subject matter specified by the closed- ended transitional phrases“consisting of” or“consisting essentially of.” As such embodiments described herein or so claimed with the phrase“comprising” are expressly or inherently
• SEQ ID NO: 1 Bevacizumab Heavy Chain:
• YTQKSLSLSP G(K) [0144] SEQ ID NO: 4, Protein Sequence for Light Chain, Ranibizumab (VEGF Fab) DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
• SEQ ID NO: 5 Protein Sequence for Heavy Chain Ranibizumab (VEGF Fab) EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGE PTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHL
• SEQ ID NO:6 Protein Sequence for a VEGF ScFv EIVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGVPSRF SGSGAEFTLTISSLQPDDFATYYCQNVYLASTNGANFGQGTKLTVLGGGGGSGGGG SGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGL EWVGFIDPDDDPYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGDHNSG WGLDIWGQGTLVTVSS
• SEQ ID NO:7 Protein Sequence for a heavy chain of a VEGF antibody
• VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K
• SEQ ID NO 16 Protein Sequence for AMD-I (L1-15 fused to VEGF Trap)
• SEQ ID NO 19 Protein Sequence for AMD-K Heavy Chain (L1-15 fused to VEGF Fab)
• SEQ ID NO 20 Protein Sequence for AMD-L Heavy Chain (L1-7 fused to VEGF Fab)
• SEQ ID NO 21 Protein Sequence for AMD-N (L1-15 fused to VEGF ScFv)
• SEQ ID NO 23 Protein Sequence for AMD-I-C terminal (L1-15 fused to C- terminal of VEGF Trap)
• SEQ ID NO 24 Protein Sequence for AMD-J-C terminal (L1-7 fused to C- terminal of VEGF Trap)
• SEQ ID NO 25 Protein Sequence for AMD-K-C terminal Heavy Chain (L1-15 fused to C-terminal VEGF Fab)
• SEQ ID NO 26 Protein Sequence for AMD-L-C terminal Heavy Chain (L1-7 fused to VEGF Fab)
• SEQ ID NO 27 Protein Sequence for AMD-N-C terminal (L1-15 fused to C- terminal VEGF ScFv)
• SEQ ID NO 28 Protein Sequence for AMD-Q-C terminal (L1-7 fused to VEGF ScFv)
• SEQ ID NO: 30 Protein Sequence for ASKB712-O3 (L1-15 fused to the N- terminal of an VEGF-binding antibody)
• Xaa25 is L or deleted
• Xaa26 is E is deleted
• n 0, 1, 2, 3, 4, or 5
• the C- terminal amino acid K may be deleted.
• SEQ ID NO:31 Protein Sequence for ASKB712-O2 (L1-15 fused to the C- terminal of an VEGF-binding antibody)
• SEQ ID NO: 32 Protein Sequence for ASKB712-O4 (L1-15 fused to the C- terminal of an VEGF-binding antibody)
• SEQ ID NO: 33 Protein Sequence for ASKB712-P (L1-7 fused to the N- terminal of an VEGF-binding antibody)
• SEQ ID NO: 34 Protein Sequence for ASKB712-P2 (L1-7 fused to the C- terminal of an VEGF-binding antibody)
• Xaa1 is A, G, or deleted;
• Xaa2 is Q or A or deleted;
• Xaa26 is L or deleted;
• Xaa27 is E is deleted;
• SEQ ID NO 57 DNA sequence (DHAMDH02083016) for 2xCon4(C) fused to the C-terminus of the Heavy Chain of Bevacizumab, with linker peptide
• SEQ ID NO 58 DNA sequence (DHAMDL083016), for the light chain of
• SEQ ID NO 59 DNA sequence (LY2.55.1), for peptide L1-15 (no LE) fused to the N-terminus of the light chain of Bevacizumab
• SEQ ID NO 60 DNA sequence (LY2.55.2), for peptide L1-15 (with LE) fused to the N-terminus of the light chain of Bevacizumab
• SEQ ID NO 61 DNA sequence (LY2.55.3), for peptide L1-15 (no LE) fused to the N-terminus of the heavy chain of Bevacizumab
• SEQ ID NO 62 DNA sequence (LY2.55.4), for peptide L1-15 (with LE) fused to the N-terminus of the heavy chain of Bevacizumab
• SEQ ID NO 63 DNA sequence (LY2.55.5), for the light chain of Bevacizumab ATGGCCTGGATGATGTTGCTTCTCGGACTTCTCGCGTATGGATCAGGGGTGGACTCCG ACATTCAAATGACTCAGTCGCCATCGTCCCTCTCGGCATCCGTGGGAGACAGAGTGA CCATCACTTGTTCCGCCTCGCAAGACATCTCCAACTACCTGAACTGGTACCAGCAGA AGCCCGGGAAGGCCCCCAAAGTGCTCATCTACTTTACTTCCTCACTGCACTCCGGG GTGCCAAGCCGCTTTAGCGGCTCCGGTTCTGGAACCGATTTCACCCTGACCATTAGC TCACTCCAGCCGGAAGATTTCGCTACGTACTACTGCCAGCAGTATTCGACCGTGCCG TGGACTTTCGGACAGGGTACCAAAGTCGAGATCAAGCGGACCGTGGCCCGAG CGTGTTCATTTTCCCGCCTTCCGACGAGCAACTCAAGTCCGTCAGTCGGACAGGGTACCAAAGTCGAAAGCGGACCGTGGCC

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