Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • 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
  • C07K16/2851—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 lectin superfamily, e.g. CD23, CD72
• • 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
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• • 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
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/77—Internalization into the cell
• • 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

• aspects of the present disclosure relate generally to antibodies or binding fragments thereof that block or disrupt the interaction between Galectin-3 (Gal3) and insulin receptor (INSR) or a glucose transporter, such as glucose transporter 1 (GLUT1) or glucose transporter 4 (GLUT4).
• Galectin-3 Galectin-3
• INSR insulin receptor
• a glucose transporter such as glucose transporter 1 (GLUT1) or glucose transporter 4 (GLUT4).
• aspects of the present disclosure relate generally to antibodies or binding fragments thereof that block or disrupt the interaction between Galectin-3 (Gal3) and insulin receptor (INSR) or a glucose transporter, such as glucose transporter 1 (GLUT1) or glucose transporter 4 (GLUT4).
• Galectin-3 Galectin-3
• INSR insulin receptor
• a glucose transporter such as glucose transporter 1 (GLUT1) or glucose transporter 4 (GLUT4).
• Galectin-3 has been implicated to have immunomodulatory activity.
• An example of this is the interaction between Gal3 and T-cell immunoglobulin and mucin- domain containing-3 (TIM-3), which causes suppression of immune responses such as T cell activation and may enable cancer cells to evade immune clearance.
• This phenomenon and methods to inhibit the same are exemplified in WO 2019/023247 and WO 2020/160156, each of which is hereby expressly incorporated by reference in its entirety.
• nucleic acids comprising a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the nucleic acid sequences of SEQ ID NOs: 866-925, 1011-1066, 1239-1410, 1413-1414, or 1515-1614.
• GLUT translocation in the cell is enhanced by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, or 200% after contacting with the anti-Gal3 antibody or binding fragment thereof relative to a cell that is not contacted with the anti-Gal3 antibody or binding fragment thereof.
• the methods may comprise contacting the cell with a preincubated complex of Gal3 and an anti-Gal3 antibody or binding fragment thereof.
• the glucose transporter is glucose transporter 1 (GLUT1) and/or glucose transporter 4 (GLUT4).
• the method is performed in vitro or in vivo.
• GLUT translocation in the cell is enhanced by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, or 200% after contacting with the preincubated complex of Gal3 and the anti-Gal3 antibody or binding fragment thereof relative to a cell that is not contacted with the preincubated complex of Gal3 and the anti-Gal3 antibody or binding fragment thereof.
• the anti-Gal3 antibody or binding fragment is any one or more of the anti-Gal3 antibodies or binding fragments thereof, or any portion or component of any one or more of the anti-Gal3 antibodies or binding fragments thereof disclosed herein, including but not limited to 1, 2, 3, 4, 5, or 6 CDRs, heavy chain variable regions, light chain variable regions, heavy chains, or light chains.
• the methods may comprise administering to the subject a preincubated complex of Gal3 and an anti-Gal3 antibody or binding fragment thereof, thereby improving insulin sensitivity in the subject.
• the GLUT is glucose transporter 1 (GLUT1) and/or glucose transporter 4 (GLUT4).
• the methods further comprise identifying the subject as needing improvement in insulin sensitivity prior to the administering step.
• the methods further comprise detecting an improvement in insulin sensitivity in the subject following the administering step.
• the anti-Gal3 antibody or binding fragment is any one or more of the anti-Gal3 antibodies or binding fragments thereof, or any portion or component of any one or more of the anti-Gal3 antibodies or binding fragments thereof disclosed herein, including but not limited to 1, 2, 3, 4, 5, or 6 CDRs, heavy chain variable regions, light chain variable regions, heavy chains, or light chains.
• the insulin sensitivity in the subject is improved by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, or 200% relative to the insulin sensitivity of the subject prior to the administering step.
• the insulin sensitivity in the subject is improved by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, or 200% relative to the insulin sensitivity of the subject prior to the administering step.
• FIG. 1 demonstrates the ability of galectin-3 targeted antibodies to block the binding of Gal3 and Insulin Receptor (INSR) as measured by enzyme-linked immunosorbent assay (ELISA) at 10, 3, and 1 pg/mL. Bars represent mean +/- standard deviation.
• FIG. 3 A depicts a summary of properties for exemplary anti-Gal3 antibodies.
• FIG. 10 depicts a Kaplan-Meier curve of healthy C57BL6/J mice and Db/Db mice treated with the anti-Gal3 antibody mTBOOl, a PBS negative control, or a semaglutide positive control.
• FIG. 16 depicts the quantification of circulating IFN-g in DSS-induced IBD mice treated with mTBOOl (lOmg/kg and lmg/kg) or PBS compared to normal mice.
• FIG. 19A depicts exemplary variable heavy chain complementarity determining region (CDR) 1 for anti-Gal3 antibodies disclosed herein.
• CDR complementarity determining region
• any of the compositions or methods provided herein can include one or more of the variable heavy chain CDR1 provided herein.
• FIG. 20B depicts exemplary variable light chain CDR2 for anti-Gal3 antibodies disclosed herein.
• any of the compositions or methods provided herein can include one or more of the variable light chain CDR2 provided herein.
• FIG. 21 depicts exemplary heavy chain variable region (VH) sequences for anti-Gal3 antibodies disclosed herein.
• VH heavy chain variable region
• FIG. 31A-B depict graphical representations of relative GLUT4 translocation determined as a fold change between insulin- stimulated and insulin-unstimulated (basal) cells ⁇ Gal3 when contacted with variants of 20H5 (FIG. 31A) or 2D10-VH0-VL0 (2D 10) (FIG. 31B) (or no antibody control) as measured by immunocytochemistry based assay.
• FIG. 43A-C depict the quantification of affinity (KD) of TB006 (FIG. 43A), TB001 (FIG. 43B), and a control antibody Synagis (FIG. 43C) to rat Gal3.
• FIG. 47 depicts a hydrogen-deuterium mass spectrometric heat map of hGal3 complexed with GLUT4, showing the putative regions of Gal3 responsible for binding to GLUT4. On the heat map, darker regions have less deuterium uptake after binding.
• Galectin-3 (Gal3, GAL3) is known to play an important role in cell proliferation, adhesion, differentiation, angiogenesis, and apoptosis. This activity is, at least in part, due to immunomodulatory properties and binding affinity towards other immune regulatory proteins, signaling proteins, and other cell surface markers.
• Gal3 functions by distinct N-terminal and C-terminal domains.
• the N-terminal domain (isoform 1: amino acids 1-111, isoform 3: amino acids 1-125) comprise a tandem repeat domain (TRD, isoform 1: amino acids 36-109, isoform 3: amino acids 50-123) and is largely responsible for oligomerization of Gal3.
• the C-terminal domain (isoform 1: amino acids 112-250, isoform 3: amino acids 126-264) comprise a carbohydrate-recognition-binding domain (CRD), which binds to b-galactosides.
• CCD carbohydrate-recognition-binding domain
• An exemplary sequence for isoform 1 of human Gal3 (NCBI Reference No. NP_002297.2) is shown in SEQ ID NO: 1.
• An exemplary sequence for isoform 3 of human Gal3 (NCBI Reference No. NP_001344607.1) is shown in SEQ ID NO: 2.
• Gal3 has been shown to be elevated in obese humans and is believed to cause insulin resistance and glucose intolerance in these subjects. Gal3 has been shown to bind directly to insulin receptor and to inhibit downstream signaling. Thus, Gal3 may contribute to obesity-induced insulin resistance and chronic tissue inflammation.
• anti-Gal3 antibodies or binding fragments thereof or compositions comprising anti-Gal3 antibodies or binding fragments thereof are provided. In some embodiments, the anti-Gal3 antibodies or binding fragments thereof bind to the N- terminal domain, the N-terminus and/or the TRD of Gal3. In some embodiments, the anti-Gal3 antibodies or binding fragments thereof bind to the C-terminal domain, the C-terminus and/or the CRD of Gal3.
• antibody denotes the meaning ascribed to it by one of skill in the art, and further it is intended to include any polypeptide chain-containing molecular structure with a specific shape that fits to and recognizes an epitope, where one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope.
• Antibodies may be polyclonal antibodies, although monoclonal antibodies may be preferred because they may be reproduced by cell culture or recombinantly and can be modified to reduce their antigenicity.
• amino acids that are the same between two or more sequences relative to the length of the sequence.
• the % similarity will be respective that length.
• deletions and/or insertions may be introduced to obtain the best alignment.
• the similarity of two amino acids may dictate whether a certain substitution is conservative or non-conservative. Methods of determining the conservativeness of an amino acid substitution are generally known in the art and may involve substitution matrices.
• consensus sequence refers to the generalized sequence representing all of the different combinations of permissible amino acids at each location of a group of sequences.
• a consensus sequence may provide insight into the conserved regions of related sequences where the unit (e.g. amino acid or nucleotide) is the same in most or all of the sequences, and regions that exhibit divergence between sequences.
• the consensus sequence of a CDR may indicate amino acids that are important or dispensable for antigen binding. It is envisioned that consensus sequences may be prepared with any of the sequences provided herein, and the resultant various sequences derived from the consensus sequence can be validated to have similar effects as the template sequences.
• a pharmaceutically acceptable diluent, excipient, and/or carrier is a diluent, excipient, and/or carrier approved by a regulatory agency of a Federal, a state government, or other regulatory agency, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans as well as non-human mammals, such as cats and dogs.
• the term diluent, excipient, and/or carrier can refer to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical formulation is administered.
• Such pharmaceutical diluent, excipient, and/or carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin.
• Water, saline solutions and aqueous dextrose and glycerol solutions can be employed as liquid diluents, excipients, and/or carriers, particularly for injectable solutions.
• suitable pharmaceutical diluents and/or excipients include sugars, starch, glucose, fructose, lactose, sucrose, maltose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, salts, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
• a non-limiting example of a physiologically acceptable carrier is an aqueous pH buffered solution.
• a “carrier” refers to a compound, particle, solid, semi-solid, liquid, or diluent that facilitates the passage, delivery and/or incorporation of a compound to cells, tissues and/or bodily organs.
• a lipid nanoparticle is a type of carrier that can encapsulate an oligonucleotide to thereby protect the oligonucleotide from degradation during passage through the bloodstream and/or to facilitate delivery to a desired organ, such as to the lungs.
• the actin-related p41ARC subunit contributes to p21-activated kinase-1 (PAKl)-mediated glucose uptake into skeletal muscle cells”. J Biol Chem. 2017 Sep 25. pii: jbc.Ml 17.801340, each of which is hereby expressly incorporated by reference in its entirety. Improvement in insulin sensitivity, for example with an anti-Gal3 antibody or binding fragment thereof, can be compared to other treatment regimens, such as dietary changes, weight loss, exercise, metformin, and thiazolidinediones, indicated for insulin resistant patients.
• the V H -CDR3 comprises an amino acid sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,
• the antibodies comprise one or more sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to a VL sequence, a VH sequence, a VL/VH pairing, and/or V H -CDR1, V H -CDR2, V H -CDR3, V L -CDR1, V L -CDR2, V L -CDR3 (including 1, 2, 3, 4, or 5 amino acid substitutions of any one or more of these CDRs) set from the heavy chain and light chain sequences as depicted in FIG. 25.
• the antibody or binding fragment thereof comprises a combination of a V H -CDR1, a V H -CDR2, a V H -CDR3, V L -CDR1, a V L -CDR2, and a V L - CDR3, where one or more of these CDRs is defined by a consensus sequence.
• the consensus sequences provided herein have been derived from the alignments of CDRs depicted in FIG. 37A-B. However, it is envisioned that alternative alignments may be done (e.g. using global or local alignment, or with different algorithms, such as Hidden Markov Models, seeded guide trees, Needleman-Wunsch algorithm, or Smith-Waterman algorithm) and as such, alternative consensus sequences can be derived.
• the V H -CDR2 is defined by the formula X1X2X3X4X5X6X7X8X9X10, where Xi is no amino acid, I, or F; X 2 is no amino acid or R; X3 is no amino acid, F, I, F, or V; X 4 is A, D, F, H, K, F, N, S, W, or Y; X 5 is A, D, P, S, T, W, or Y; X 6 is D, E, G, H, K, N, S, V, or Y; X 7 is D, E, G, N, S, or T; X 8 is D, G, I, K, N, Q, R, S, V, or Y; X9 is A, D, E, G, I, K, N, P, S, T, V, or Y; X10 is no amino acid, I, P, S, or T.
• the V H -CDR3 is defined by the formula X 1X2X3X4X5X0X7X8X9X10X11X12X13X14X15X10X17X18X19X20X21X22X23X24X25, where Xi is no amino acid or A; X2 is no amino acid, A, R, or Y; X3 is no amino acid, A, F, H, K, F, R, S, or V; X4 is no amino acid, A, D, K, N, R, S, or T; X5 is no amino acid, A, D, G, H, I, L, N, P, R, S, T, V, or Y; Xe is no amino acid, A, D, G, H, K, N, P, Q, R, S, or Y; X7 is no amino acid, D, F, G, H, P, R, S, W, or Y; Xs is no amino acid, A, A,
• the antibody or binding fragment thereof comprises a light chain, wherein the light chain comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence selected from SEQ ID NOs: 495-538, 805, 851-865, 997-1010, 1196-1238, 1412, 1490-1514.
• the antibodies or binding fragments thereof are anti-Gal3 antibodies or binding fragments thereof.
• A3_hVH5 VL 1 -hlgG 1 (LALAPGv2), 20H5.A3_hVH5VLl-hIgGl (REM), 20H5.
• A3_hVH6VL 1 -hlgG 1 KEMv2
• 20H5.A3_hVH6VLl-hIgGl LALAPGv2
• the antibody or binding fragment thereof comprises a sequence (e.g. CDR, VL, VH, LC, HC) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence similarity to a sequence of TB001, TB006, 12G5.D7, 13A12.2E5, 14H10.2C9, 15F10.2D6, 19B5.2E6, 20D11.2C6, 20H5.A3, 23H9.2E4, 2D10.2B2, 3B11.2G2, 7D8.2D8, mIMTOOl, 4A11.2B5, 4A11.H1L1, 4A11.H4L2, 4G2.2G6, 6B3.2D3, 6H6.2D6, 9H2.2H10, 13G4.2F8, 13H12.2F8, 15G7.2A7, 19D9.2E
• a sequence e.
• the V H -CDR2 comprises an amino acid sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence similarity to SEQ ID NO: 801, 951, 952.
• the V L -CDR2 comprises an amino acid sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence similarity to SEQ ID NO: 222.
• the VH- CDR2 comprises an amino acid sequence having at least 0, 1, 2, 3, 4, 5, or 6 substitutions relative to SEQ ID NO: 801, 951, 952.
• the VH-CDR3 comprises an amino acid sequence having at least 0, 1, 2, 3, 4, 5, or 6 substitutions relative to SEQ ID NO: 802, 953, 954.
• the VL-CDR1 comprises an amino acid sequence having at least 0, 1, 2, 3, 4, 5, or 6 substitutions relative to SEQ ID NO: 171.
• the VL-CDR2 comprises an amino acid sequence having at least 0, 1, 2, 3, 4, 5, or 6 substitutions relative to SEQ ID NO: 222.
• the VL-CDR3 comprises an amino acid sequence having at least 0, 1, 2, 3, 4, 5, or 6 substitutions relative to SEQ ID NO: 257.
• the heavy chain variable region of the anti-Gal3 antibody or binding fragment thereof comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the sequences of SEQ ID NOs: 806-813, 955-968.
• the V H -CDR1 comprises an amino acid sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,
• antibodies or binding fragments thereof are provided.
• the antibodies or binding fragments thereof are anti-Gal3 antibodies or binding fragments thereof.
• the anti-Gal3 antibodies or binding fragments thereof comprises a heavy chain variable region comprising a V H -CDR1, a V H - CDR2, and a V H -CDR3.
• the anti-Gal3 antibodies or binding fragments thereof comprise a light chain variable region comprising a V L -CDR1, a V L -CDR2, and a V L - CDR3.
• the V L -CDR1 comprises an amino acid sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,
• the heavy chain variable region of the anti-Gal3 antibody or binding fragment thereof comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the sequences of SEQ ID NOs: 814-820, 1067-1109.
• the light chain variable region of the anti-Gal3 antibody or binding fragment thereof comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the sequences of SEQ ID NOs: 829-835, 1110-1152.
• the anti-Gal3 antibody or binding fragment thereof comprises a light chain, wherein the light chain comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity to any one of the sequences of SEQ ID NOs: 859-865, 1196-1238.
• the anti-Gal3 antibody or binding fragment thereof comprises a heavy chain, wherein the heavy chain comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the sequences of SEQ ID NOs: 1465-1489.
• the anti-Gal3 antibody or binding fragment thereof binds to Gal3 with a KD of less than 20 nM. In some embodiments, the anti- Gal3 antibody or binding fragment thereof binds to Gal3 with a KD of less than 25 nM. In some embodiments, the anti-Gal3 antibody or binding fragment thereof binds to Gal3 with a KD of less than 30 nM.
• the anti-Gal3 antibody comprises a V H -CDR1 sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence similarity to any one of SEQ ID NOs: 27-70.
• the anti-Gal3 antibody or binding fragment thereof comprises a V L -CDRI sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence similarity to any one of SEQ ID NOs: 170-220.
• the anti-Gal3 antibody or binding fragment thereof comprises a light chain variable region (VL) sequence selected from FIG. 22 (SEQ ID NOs: 374-447, 821-835, 941-943, 969-982, 1110-1152, 1440-1464).
• VL light chain variable region
• A3_hVH6VL 1 -hlgG 1 (REM), 21H6-H0L0, 21H6-H1L1, 21H6-H1L2, 21H6-H1L3, 21H6-H1L4, 21H6-H2L1, 21H6-H2L2, 21H6-H2L3, 21H6-H2L4, 21H6-H3L1, 21H6-H3L2, 21H6-H3L3, 21H6-H3L4, 21H6-H4L1, 21H6-H4L2, 21H6-H4L3, 21H6-H4L4, 21H6-H5L1, 21H6-H5L2, 21H6-H5L3, 21H6-H5L4, 21H6-H6L1, 21H6-H6L2, 21H6-H6L3, 21H6-H6L4, or a binding fragment thereof.
• the anti-Gal3 antibody or binding fragment thereof comprises a heavy chain and/or light chain depicted in FIG. 25.
• the anti-Gal3 antibody or binding fragment thereof can comprise or include any one or more of the sequences provided in any one or more of FIG. 19A-C, 20A- C, 21, 22, 23, 24, 25, or any one or more of a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identical thereto.
• the anti-Gal3 antibody or binding fragment thereof can comprise or include any one or more of the sequences provided in any one or more of FIG. 19A-C, 20A-C, 21, 22, 23, 24, 25, or any one or more of a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater similar thereto.
• the heavy chain variable region comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the amino acid sequences of SEQ ID NOs: 806-820, 955-968, 1067-1109, 1415-1439.
• the anti-Gal3 antibody or binding fragment thereof comprises a heavy chain, wherein the heavy chain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the amino acid sequences of SEQ ID NOs: 836-850, 983-996, 1153-1195, 1411, 1465-1489.
• the anti-Gal3 antibody or binding fragment thereof comprises a light chain, wherein the light chain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the amino acid sequences of SEQ ID NOs: 851-865, 997-1010, 1196-1238, 1412, 1490-1514.
• A3_hVH6VL 1 -hlgG 1 KEMv2
• 20H5.A3_hVH6VLl-hIgGl LALAPGv2
• 20H5.A3_hVH6VLl-hIgGl REM
• the anti-Gal3 antibody or binding fragment thereof comprises a monovalent Fab’, a divalent Fab2, a single-chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb), or a camelid antibody or binding fragment thereof.
• the anti-Gal3 antibody or binding fragment thereof can comprise an IgM, IgG (e.g., IgGl, IgG2, IgG3, or IgG4), IgA, or IgE framework.
• the IgG framework can be IgGl, IgG2, IgG3 or IgG4.
• the anti-Gal3 antibody or binding fragment thereof comprises an IgGl framework.
• the anti- Gal3 antibody or binding fragment thereof comprises an IgG2 framework.
• the anti-Gal3 antibody or binding fragment thereof comprises an IgG4 framework.
• the anti-Gal3 antibody or binding fragment thereof can further comprise a Fc mutation.
• the Fc region comprises one or more mutations that modulate Fc receptor interactions, e.g., to enhance effector functions such as ADCC and/or CDC.
• exemplary residues when mutated modulate effector functions include S239, K326, A330, 1332, or E333, in which the residue position correspond to IgGl and the residue numbering is in accordance to Rabat numbering (EU index of Rabat et al 1991 Sequences of Proteins of Immunological Interest).
• the one or more mutations comprise S239D, R326W, A330L, I332E, E333A, E333S, or a combination thereof.
• an anti-Gal3 antibody or binding fragment thereof comprises a humanization score of above 70, above 80, above 81, above 82, above 83, above 84, above 85, above 86, above 87, above 88, above 89, above 90, or above 95.
• the anti-Gal3 antibody or binding fragment thereof comprises a humanization score of above 80.
• the anti-Gal3 antibody or binding fragment thereof comprises a humanization score of above 83.
• the anti-Gal3 antibody or binding fragment thereof comprises a humanization score of above 85.
• the anti-Gal3 antibody or binding fragment thereof comprises a humanization score of above 87.
• the proteins comprise a sequence having at least 0, 1, 2, 3, 4, 5, or 6 substitutions relative to any one or more sequences of SEQ ID NOs: 27-538, 801- 865, 955-1010, 1067-1238, 1415-1514.
• the proteins comprise six sequences selected from each of SEQ ID NOs: 27-70; SEQ ID NOs: 71-111, 801, 951, 952; SEQ ID NOs: 112-169, 802, 953, 954; SEQ ID NOs: 170-220; SEQ ID NOs: 221-247; SEQ ID NOs: 248-296.
• the protein comprises a sequence defined by the formula X1X2X3X4X5X6X7X8X9X10, where Xi is no amino acid, A, E, F, H, L, M, Q, S, V, or W; X 2 is A, H, or Q; X 3 is D, F, G, H, L, M, N, Q, S, T, W, or Y; X 4 is no amino acid or W; X 5 is A, D, I, K, L, N, Q, R, S, T, V, or Y; X 6 is D, E, H, I, K, L, N, Q, S, or T; X 7 is D, F, K, L, N, P, S, T, V, W, or Y; X 8 is H, P, or S; X9 is F, F, P, Q, R, R, T, W, or Y; X10 is no amino acid, T, or V.
• the protein comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to this consensus sequence. In some embodiments, the protein comprises a sequence having 0, 1, 2, 3, 4, 5, or 6 substitutions from this consensus sequence.
• a pharmaceutical formulation for treating a disease as described herein can comprise an anti-Gal3 antibody or binding fragment thereof described supra.
• the anti-Gal3 antibody or binding fragment thereof can be formulated for systemic administration ⁇
• the anti-Gal3 antibody or binding fragment thereof can be formulated for parenteral administration.
• the pharmaceutical compositions further include diluent which are used to stabilize compounds because they can provide a more stable environment.
• Salts dissolved in buffered solutions are utilized as diluents in the art, including, but not limited to a phosphate buffered saline solution.
• diluents increase bulk of the composition to facilitate compression or create sufficient bulk for homogenous blend for capsule filling.
• the pharmaceutical formulation can further comprise an additional therapeutic agent.
• additional therapeutic agents include alpha- glucosidase inhibitors, including acarbose (Precose ® ) and miglitol (Glyset ® ); biguanides, including metformin-alogliptin (Kazano ® ), metformin-canagliflozin (Invokamet ® ), metformin- dapagliflozin (Xigduo ® XR), metformin-empagliflozin (Synjardy ® ), metformin-glipizide, metformin-glyburide (Glucovance ® ), metformin-linagliptin (Jentadueto ® ), metformin- pioglitazone (Actoplus ® ), metformin-repaglinide (PrandiMet ® ), metformin-rosiglitazone (Avan
• GLUT glucose transporter
• the methods comprise contacting the cell with an anti-Gal3 antibody or binding fragment thereof.
• binding of the anti-Gal3 antibody or binding fragment thereof to Gal3 in the cell inhibits Gal3 -mediated blocking of GLUT translocation.
• the glucose transporter is glucose transporter 1 (GLUT1) and/or glucose transporter 4 (GLUT4).
• the method is performed in vitro or in vivo.
• the V H -CDR1 comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the amino acid sequences of SEQ ID NOs: 27-70.
• the V L -CDR1 comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the amino acid sequences of SEQ ID NOs: 170-220.
• the V L -CDR2 comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the amino acid sequences of SEQ ID NOs: 221-247.
• the anti-Gal3 antibody or binding fragment thereof comprises a heavy chain, wherein the heavy chain comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence selected from SEQ ID NOs: 448-494, 804, 836-850, 983-996, 1153-1195, 1411, 1465-1489.
• the anti-Gal3 antibody or binding fragment thereof is selected from the group consisting of: 20H5.A3, 20H5.A3-VH3VL1, 20H5.A3- VH3VL3, 20H5.A3-VH4VL1, 20H5.A3-VH5VL1, 20H5.A3-VH5VL3, 20H5.A3-VH6VL1, 20H5.A3-VH6VL3, or binding fragment thereof.
• the glucose transporter is glucose transporter 1 (GLUT1) and/or glucose transporter 4 (GLUT4).
• the method is performed in vitro or in vivo.
• GLUT translocation in the cell is enhanced by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, or 200% after contacting with the preincubated complex of Gal3 and the anti-Gal3 antibody or binding fragment thereof relative to a cell that is not contacted with the preincubated complex of Gal3 and the anti-Gal3 antibody or binding fragment thereof.
• the VH-CDR2 comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the amino acid sequences of SEQ ID NOs: 71-111, 801, 951, 952.
• the VL-CDR1 comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the amino acid sequences of SEQ ID NOs: 170-220.
• the method involves administering any antibody or variant thereof as provided herein (including any with one or more of the 6 CDRs provided in the present disclosure), in a therapeutically effective amount, sufficient to interfere with the interaction between GAL3 and GLUT (e.g., GLUT1 and/or GLUT4), so as to treat (either in response to a subject having and/or to reduce the risk of) one or more of: diabetes mellitus, insulin resistance, chronic hyperinsulinemia, dysmetabolic syndrome, type A insulin resistance syndrome, type B insulin resistance syndrome, gestational diabetes, acanthosis nigricans, polycystic ovary syndrome (PCOS), obesity, muscle wasting, cardiovascular diseases, cardiac hypertrophy, myocardial ischemia, hypertension, pancreatic cancer associated diabetes (PCDM), or cancers.
• PCOS polycystic ovary syndrome
• the VH-CDR3 comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the amino acid sequences of SEQ ID NOs: 112-169, 802, 953, 954.
• the VL-CDR3 comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the amino acid sequences of SEQ ID NOs: 248-296.
• the anti-Gal3 antibody or binding fragment thereof comprises a combination of the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1 , VL-CDR2, and VL-CDR3 as illustrated in FIG.23.
• the light chain variable region comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence selected from SEQ ID NOs: 374-447, 821-835, 969-982, 1110-1152, 1440-1464.
• the anti-Gal3 antibody or binding fragment thereof comprises a heavy chain, wherein the heavy chain comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence selected from SEQ ID NOs: 448-494, 804, 836-850, 983-996, 1153-1195, 1411, 1465-1489.
• the anti-Gal3 antibody or binding fragment thereof comprises a light chain, wherein the light chain comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence selected from SEQ ID NOs: 495-538, 805, 851-865, 997-1010, 1196-1238, 1412, 1490-1514.
• the anti-Gal3 antibody or binding fragment thereof is selected from the group consisting of: 20H5.A3, 20H5.A3-VH3VL1, 20H5.A3- VH3VL3, 20H5.A3-VH4VL1, 20H5.A3-VH5VL1, 20H5.A3-VH5VL3, 20H5.A3-VH6VL1, 20H5.A3-VH6VL3, or binding fragment thereof.
• the methods further comprise identifying the subject as needing improvement in insulin sensitivity prior to the administering step.
• the methods further comprise detecting an improvement in insulin sensitivity in the subject following the administering step.
• detecting the improvement in insulin sensitivity in the subject is done by measuring blood sugar levels, measuring blood insulin levels, glucose tolerance testing, or hyperinsulinemic euglycemic clamp.
• the insulin sensitivity in the subject is improved by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%,
• the anti-Gal3 antibody or binding fragment thereof comprises (1) a heavy chain variable region comprising a V H -CDR1, a V H - CDR2, and a V H -CDR3; and (2) a light chain variable region comprising a V L -CDR1, a V L - CDR2, and a V L -CDR3.
• the V H -CDR1 comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the amino acid sequences of SEQ ID NOs: 27-70.
• the V H -CDR2 comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the amino acid sequences of SEQ ID NOs: 71-111, 801, 951, 952.
• the V L -CDR1 comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the amino acid sequences of SEQ ID NOs: 170- 220.
• the heavy chain variable region comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence selected from SEQ ID NOs: 297-373, 803, 806-820, 955-968, 1067-1109, 1415-1439.
• the light chain variable region comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence selected from SEQ ID NOs: 374-447, 821-835, 969- 982, 1110-1152, 1440-1464.
• the anti-Gal3 antibody or binding fragment thereof comprises a light chain, wherein the light chain comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence selected from SEQ ID NOs: 495-538, 805, 851-865, 997-1010, 1196-1238, 1412, 1490-1514.
• the anti-Gal3 antibody or binding fragment thereof is selected from the group consisting of: 2D10-VH0-VL0, 2D 10- hVH4-HVLl, 2D 10-hVH4-HVL2, 2D10-hVH4-HVL3, 2D10-hVH4-HVL4, 2D10-hVH3- HVL1, 2D 10-hVH3 -H VL2, 2D10-hVH3-HVL3, 2D10-hVH3-HVL4, or binding fragment thereof.
• the insulin sensitivity in the subject is improved by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, or 200%, or any percentage within a range defined by any two of the aforementioned percentages, relative to the insulin sensitivity of the subject prior to the administering step.
• the anti-Gal3 antibody or binding fragment thereof selectively binds to an N- terminal domain of Gal3.
• the anti-Gal3 antibody or binding fragment thereof binds to Peptide 1 (SEQ ID NO: 3), Peptide 6 (SEQ ID NO: 8), or Peptide 7 (SEQ ID NO:9), or any combination thereof.
• the anti-Gal3 antibody or binding fragment thereof comprises (1) a heavy chain variable region comprising a V H -CDR1, a V H - CDR2, and a V H -CDR3; and (2) a light chain variable region comprising a V L -CDR1, a V L - CDR2, and a V L -CDR3.
• the V H -CDR3 comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the amino acid sequences of SEQ ID NOs: 112-169, 802, 953, 954.
• the V L -CDR1 comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
• the anti-Gal3 antibody or binding fragment thereof is selected from the group consisting of: TB001, TB006, 12G5.D7, 13A12.2E5, 14H10.2C9, 15F10.2D6, 19B5.2E6, 20D11.2C6, 20H5.A3, 23H9.2E4, 2D10.2B2, 3B11.2G2, 7D8.2D8, mIMTOOl, 4A11.2B5, 4A11.H1L1, 4A11.H4L2, 4G2.2G6, 6B3.2D3, 6H6.2D6, 9H2.2H10, 13G4.2F8, 13H12.2F8, 15G7.2A7, 19D9.2E5, 23B10.2B12, 24D12.2H9, F846C.1B2, F846C.1F5, F846C.1H12, F846C.1H5, F846C.2H3, F846TC.14A2, F846TC.14E4, F846TC.16B5, F846TC.7
• the preincubated complex of Gal3 and the anti-Gal3 antibody or binding fragment thereof is prepared with Gal3 and the anti-Gal3 antibody or binding fragment thereof at a mass ratio of or of about 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, or 1:2.5, or any mass ratio within a range defined by any two of the aforementioned mass ratios.
• the V H -CDR2 comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the amino acid sequences of SEQ ID NOs: 71-111, 801, 951, 952.
• the V H -CDR3 comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the amino acid sequences of SEQ ID NOs: 112-169, 802, 953, 954.
• the heavy chain variable region comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence selected from SEQ ID NOs: 297-373, 803, 806-820, 955-968, 1067-1109, 1415- 1439.
• A3_hVH6VL 1 -hlgG 1 KEMv2
• 20H5.A3_hVH6VLl-hIgGl LALAPGv2
• 20H5.A3_hVH6VLl-hIgGl REM
• 21H6-H0L0 21H6-H1L1, 21H6-H1L2, 21H6-H1L3, 21H6-H1L4, 21H6-H2L1, 21H6-H2L2, 21H6-H2L3, 21H6-H2L4, 21H6-H3L1, 21H6-H3L2, 21H6-H3L3, 21H6-H3L4, 21H6-H4L1, 21H6-H4L2, 21H6-H4L3, 21H6-H4L4, 21H6-H5L1, 21H6-H5L2, 21H6-H5L3, 21H6-H5L4, 21H6-H6L1, 21H6-H6
• the anti-Gal3 antibody or binding fragment thereof binds to an epitope of Gal3 that includes a motif of GxYPG, where x is the amino acids alanine (A), glycine (G), or valine (V).
• an anti-Gal3 antibody as described herein binds to an epitope of Gal3 that includes two GxYPG motifs separated by three amino acids, where x is A, G, or V.
• any of the methods disclosed herein involving an anti-Gal3 antibody or binding fragment can be performed with an antigen binding molecule that binds to Gal3.
• exemplary VH-CDR1 sequences are depicted in FIG. 19A.
• exemplary VH-CDR2 sequences are depicted in FIG. 19B.
• exemplary VH-CDR3 sequences are depicted in FIG. 19C.
• exemplary VL-CDR1 sequences are depicted in FIG. 20A.
• exemplary VL-CDR2 sequences are depicted in FIG. 20B.
• exemplary VL-CDR3 sequences are depicted in FIG. 20C.
• the anti-Gal3 antibody or binding fragment thereof is selected from the group consisting of TB001, TB006, 12G5.D7, 13A12.2E5, 14H10.2C9, 15F10.2D6, 19B5.2E6, 20D11.2C6, 20H5.A3, 23H9.2E4, 2D10.2B2, 3B11.2G2, 7D8.2D8, mIMTOOl, 4A11.2B5, 4A11.H1L1, 4A11.H4L2, 4G2.2G6, 6B3.2D3, 6H6.2D6, 9H2.2H10, 13G4.2F8, 13H12.2F8, 15G7.2A7, 19D9.2E5, 23B10.2B12, 24D12.2H9, F846C.1B2, F846C.1F5, F846C.1H12, F846C.1H5, F846C.2H3, F846TC.14A2, F846TC.14
• A3_hVH6VL 1 -hlgG 1 (REM), 21H6-H0L0, 21H6-H1L1, 21H6-H1L2, 21H6-H1L3, 21H6-H1L4, 21H6-H2L1, 21H6-H2L2, 21H6-H2L3, 21H6-H2L4, 21H6-H3L1, 21H6-H3L2, 21H6-H3L3, 21H6-H3L4, 21H6-H4L1, 21H6-H4L2, 21H6-H4L3, 21H6-H4L4, 21H6-H5L1, 21H6-H5L2, 21H6-H5L3, 21H6-H5L4, 21H6-H6L1, 21H6-H6L2, 21H6-H6L3, 21H6-H6L4, or binding fragment thereof.
• combinations of CDRs are excluded from the sequences associated with the subset of named anti-Gal3 antibodies or binding fragments thereof depicted in any one of FIGS. 38A-D, or excludes any one or more of the CDRs, VH, VL, HC, and/or LC thereof.
• the anti-Gal3 antibody or binding fragment thereof excludes those selected from the group of named anti-Gal3 antibody or binding fragment thereof depicted in FIG. 38A, or excludes any one or more of the CDRs, VH, VL, HC, and/or LC thereof.
• the anti-Gal3 antibody or binding fragment thereof excludes those selected from the group of named anti-Gal3 antibody or binding fragment thereof depicted in FIG. 38B, or excludes any one or more of the CDRs, VH, VL, HC, and/or LC thereof. In some embodiments, the anti-Gal3 antibody or binding fragment thereof excludes those selected from the group of named anti-Gal3 antibody or binding fragment thereof depicted in FIG. 38C, or excludes any one or more of the CDRs, VH, VL, HC, and/or LC thereof. In some embodiments, the anti- Gal3 antibody or binding fragment thereof excludes those selected from the group of named anti-Gal3 antibody or binding fragment thereof depicted in FIG.
• the anti-Gal3 antibody or binding fragment thereof blocks the interaction between Gal3 and GLUT (e.g., GLUT1 and/or GLUT4) and excludes those selected from the group of named anti-Gal3 antibody or binding fragment thereof depicted in FIG. 38B, or excludes any one or more of the CDRs, VH, VL, HC, and/or LC thereof.
• the anti-Gal3 antibody or binding fragment thereof blocks the interaction between Gal3 and GLUT (e.g., GLUT1 and/or GLUT4) and excludes those selected from the group of named anti-Gal3 antibody or binding fragment thereof depicted in FIG.
• combinations of CDRs are selected from the sequences associated with the subset of named anti-Gal3 antibodies or binding fragments thereof depicted in any one of FIGS.39A-E.
• the anti-Gal3 antibody or binding fragment thereof is selected from the group of named anti-Gal3 antibody or binding fragment thereof depicted in FIG. 39A, or comprises any one or more of the CDRs, VH, VL, HC, and/or LC thereof.
• the anti-Gal3 antibody or binding fragment thereof is selected from the group of named anti-Gal3 antibody or binding fragment thereof depicted in FIG.39B, or comprises any one or more of the CDRs, VH, VL, HC, and/or LC thereof. In some embodiments, the anti-Gal3 antibody or binding fragment thereof is selected from the group of named anti-Gal3 antibody or binding fragment thereof depicted in FIG. 39C, or comprises any one or more of the CDRs, VH, VL, HC, and/or LC thereof. In some embodiments, the anti- Gal3 antibody or binding fragment thereof is selected from the group of named anti-Gal3 antibody or binding fragment thereof depicted in FIG.
• the anti-Gal3 antibody or binding fragment thereof is selected from the group of named anti-Gal3 antibody or binding fragment thereof depicted in FIG. 39E, or comprises any one or more of the CDRs, VH, VL, HC, and/or LC thereof.
• the anti-Gal3 antibody or binding fragments inhibits, blocks, or disrupts the interaction between Gal3 and the glucose transporter (e.g., GLUT1 and/or GLUT4) with an IC50 of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 pg/mL, or no more than 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 pg/mL, or any IC50 concentration within a range defined by any two of the aforementioned concentrations.
• the anti-Gal3 antibody or binding fragment thereof is administered enterally, orally, intranasally, parenterally, intracranially, subcutaneously, intramuscularly, intradermally, or intravenously, or any combination thereof.
• the anti-Gal3 antibody or binding fragment thereof is formulated for systemic administration ⁇ In some embodiments, the anti-Gal3 antibody or binding fragment thereof is formulated for parenteral administration. In some embodiments, more than one anti-Gal3 antibody or binding fragment is administered. In some embodiments, when more than one anti- Gal3 antibody or binding fragment is administered, the more than one anti-Gal3 antibodies or binding fragments thereof may be selected from the anti-Gal3 antibodies or binding fragments thereof disclosed herein. In some embodiments, any of the methods disclosed herein involving an anti-Gal3 antibody or binding fragment can be performed with an antigen binding molecule that binds to Gal3.
• the anti-Gal3 antibodies or binding fragments thereof disclosed herein are administered for therapeutic applications.
• the anti- Gal3 antibody or binding fragment thereof is administered once per day, twice per day, three times per day or more.
• the anti-Gal3 antibody or binding fragment thereof is administered daily, every day, every alternate day, five days a week, once a week, every other week, two weeks per month, three weeks per month, once a month, twice a month, three times per month, or more.
• the dose reduction during a drug holiday is from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
• a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, can be reduced, as a function of the symptoms, to a level at which the treated disease, disorder, or condition is retained.
• toxicity and therapeutic efficacy of such therapeutic regimens are determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
• the dose ratio between the toxic and therapeutic effects is the therapeutic index and it is expressed as the ratio between LD50 and ED50.
• Compounds exhibiting high therapeutic indices are preferred.
• the data obtained from cell culture assays and animal studies are used in formulating a range of dosage for use in human.
• the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. The dosage varies within this range depending upon the dosage form employed and the route of administration utilized.
• the present disclosure provides isolated nucleic acids encoding any of the anti-Gal3 antibodies or binding fragments thereof disclosed herein.
• the present disclosure provides vectors comprising a nucleic acid sequence encoding any anti-Gal3 antibody or binding fragment thereof disclosed herein.
• this disclosure provides isolated nucleic acids that encode heavy chain variable regions, light chain variable regions, heavy chains, or light chains of an anti-Gal3 antibody or binding fragment thereof disclosed herein.
• nucleic acid sequences encoding for heavy chain variable regions are depicted in FIG. 33 (SEQ ID NOs: 539-620, 797, 866-880, 1011-1024, 1239-1281, 1515-1539).
• nucleic acid sequences encoding for light chain variable regions are depicted in FIG. 34 (SEQ ID NOs: 621-702, 798, 881-895, 1025-1038, 1282-1324, 1540-1564).
• nucleic acid sequences encoding for heavy chains are depicted in FIG. 35 (SEQ ID NO: 703-749, 799, 896-910, 1039-1052, 1325-1367, 1565-1589).
• nucleic acid sequences encoding for light chains are depicted in FIG. 36 (SEQ ID NO: 750-796, 800, 911-925, 1053-1066, 1368-1410, 1590- 1614).
• any one of the anti-Gal3 antibodies or binding fragments thereof described herein can be prepared by recombinant DNA technology, synthetic chemistry techniques, or a combination thereof.
• sequences encoding the desired components of the anti-Gal3 antibodies, including light chain CDRs and heavy chain CDRs are typically assembled cloned into an expression vector using standard molecular techniques know in the art. These sequences may be assembled from other vectors encoding the desired protein sequence, from PCR- generated fragments using respective template nucleic acids, or by assembly of synthetic oligonucleotides encoding the desired sequences.
• Expression systems can be created by transfecting a suitable cell with an expressing vector which comprises an anti-Gal3 antibody of interest or binding fragment thereof.
• anti-Gal3 antibodies or binding fragments thereof can be produced from phage libraries containing human variable regions. See U.S. Pat. No. 6,174,708, incorporated fully herein by reference in its entirety.
• Humanized antibodies can be engineered to contain human-like immunoglobulin domains and incorporate only the complementarity-determining regions of the animal-derived antibody. This can be accomplished by carefully examining the sequence of the hyper-variable loops of the variable regions of a monoclonal antigen binding unit or monoclonal antibody and fitting them to the structure of a human antigen binding unit or human antibody chains. See, e.g., U.S. Pat. No. 6,187,287, incorporated fully herein by reference.
• a process for humanization of subject antigen binding units can be as follows.
• the best-fit germline acceptor heavy and light chain variable regions are selected based on homology, canonical structure and physical properties of the human antibody germlines for grafting.
• Computer modeling of mVH/VL versus grafted hVH/VL is performed and prototype humanized antibody sequence is generated. If modeling indicated a need for framework back- mutations, second variant with indicated FW changes is generated.
• DNA fragments encoding the selected germline frameworks and murine CDRs are synthesized. The synthesized DNA fragments are subcloned into IgG expression vectors and sequences are confirmed by DNA sequencing.
• the humanized antibodies are expressed in cells, such as 293F and the proteins are tested, for example in MDM phagocytosis assays and antigen binding assays.
• the humanized antigen binding units are compared with parental antigen binding units in antigen binding affinity, for example, by FACS on cells expressing the target antigen. If the affinity is greater than 2-fold lower than parental antigen binding unit, a second round of humanized variants can be generated and tested as described above.
• Multivalent anti-Gal3 antibodies or binding fragments thereof can be further classified on the basis of their binding specificities.
• a “monospecific” anti-Gal3 antibody or binding fragment thereof is a molecule capable of binding to one or more antigens of the same kind.
• a “multispecific” anti-Gal3 antibody or binding fragment thereof is a molecule having binding specificities for at least two different antigens. While such molecules normally will only bind two distinct antigens (i.e. bispecific anti-Gal3 antibodies), antibodies with additional specificities such as trispecific antibodies are encompassed by this expression when used herein.
• This disclosure further provides multispecific anti-Gal3 antibodies.
• Multispecific anti- Gal3 antibodies or binding fragments thereof are multivalent molecules capable of binding to at least two distinct antigens, e.g., bispecific and trispecific molecules exhibiting binding specificities to two and three distinct antigens, respectively.
• any of the above-mentioned methods is suitable for vector delivery.
• Preferred animal cells are vertebrate cells, preferably mammalian cells, capable of expressing exogenously introduced gene products in large quantity, e.g. at the milligram level.
• Non-limiting examples of preferred cells are NIH3T3 cells, COS, HeLa, and CHO cells.
• expression of the anti-Gal3 antibodies or binding fragments thereof can be determined using any nucleic acid or protein assay known in the art.
• the presence of transcribed mRNA of light chain CDRs or heavy chain CDRs, or the anti-Gal3 antibody or binding fragment thereof can be detected and/or quantified by conventional hybridization assays (e.g. Northern blot analysis), amplification procedures (e.g. RT-PCR), SAGE (U.S. Pat. No. 5,695,937), and array-based technologies (see e.g. U.S. Pat. Nos. 5,405,783, 5,412,087 and 5,445,934), using probes complementary to any region of a polynucleotide that encodes the anti-Gal3 antibody or binding fragment thereof.
• the payload comprises a microtubule disrupting agent.
• microtubule disrupting agents include, but are not limited to, 2-methoxyestradiol, auristatin, chalcones, colchicine, combretastatin, cryptophycin, dictyostatin, discodermolide, dolastain, eleutherobin, epothilone, halichondrin, laulimalide, maytansine, noscapinoid, paclitaxel, peloruside, phomopsin, podophyllotoxin, rhizoxin, spongistatin, taxane, tubulysin, vinca alkaloid, vinorelbine, or derivatives or analogs thereof.
• the auristatin derivative is monomethyl auristatin F (MMAF).
• MMAF monomethyl auristatin F
• the auristatin is an auristatin derivative or analog such as described in U.S. Patent No. 6884869, 7659241, 7498298, 7964566, 7750116, 8288352, 8703714, and 8871720.
• the payload comprises a DNA modifying agent.
• the DNA modifying agent comprises DNA cleavers, DNA intercalators, DNA transcription inhibitors, or DNA cross-linkers.
• the DNA cleaver comprises bleomycine A2, calicheamicin, or derivatives or analogs thereof.
• the DNA intercalator comprises doxorubicin, epirubicin, PNU- 159682, duocarmycin, pyrrolobenzodiazepine, oligomycin C, daunorubicin, valrubicin, topotecan, or derivatives or analogs thereof.
• the DNA transcription inhibitor comprises dactinomycin.
• the DNA cross-linker comprises mitomycin C.
• the DNA modifying agent comprises amsacrine, anthracycline, camptothecin, doxorubicin, duocarmycin, enediyne, etoposide, indolinobenzodiazepine, netropsin, teniposide, or derivatives or analogs thereof.
• the analog of camptothecin is topotecan, irinotecan, silatecan, cositecan, exatecan, lurtotecan, gimatecan, belotecan, rubitecan, or SN-38.
• the duocarmycin is duocarmycin A, duocarmycin Bl, duocarmycin B2, duocarmycin Cl, duocarmycin C2, duocarmycin D, duocarmycin SA, or CC- 1065.
• the enediyne is a calicheamicin, esperamicin, or dynemicin A.
• the pyrrolobenzodiazepine is anthramycin, abbeymycin, chicamycin, DC-81, mazethramycin, neothramycins A, neothramycin B, porothramycin, prothracarcin, sibanomicin (DC- 102), sibiromycin, or tomaymycin.
• the pyrrolobenzodiazepine is a tomaymycin derivative, such as described in U.S. Patent Nos. 8404678 and 8163736.
• the pyrrolobenzodiazepine is such as described in U.S. Patent Nos.
• the payload comprises an Akt inhibitor.
• the Akt inhibitor comprises ipatasertib (GDC-0068) or derivatives thereof.
• the payload comprises a polymerase inhibitor, including, but not limited to polymerase II inhibitors such as a-amanitin, and poly(ADP-ribose) polymerase (PARP) inhibitors.
• PARP inhibitors include, but are not limited to Iniparib (BSI 201), Talazoparib (BMN-673), Olaparib (AZD-2281), Olaparib, Rucaparib (AGO 14699, PF-01367338), Veliparib (ABT-888), CEP 9722, MK 4827, BGB-290, or 3- aminobenz amide.
• the payload comprises a detectable moiety.
• a “detectable moiety” may comprise an atom, molecule, or compound that is useful in diagnosing, detecting or visualizing a location and/or quantity of a target molecule, cell, tissue, organ, and the like.
• Detectable moieties that can be used in accordance with the embodiments herein include, but are not limited to, radioactive substances (e.g. radioisotopes, radionuclides, radiolabels or radiotracers), dyes, contrast agents, fluorescent compounds or molecules, bioluminescent compounds or molecules, enzyme and enhancing agents (e.g. paramagnetic ions), or specific binding moieties such as streptavidin, avidin, or biotin.
• some nanoparticles for example quantum dots or metal nanoparticles can be suitable for use as a detectable moiety.
• radioactive substances that can be used as detectable moieties in accordance with the embodiments herein include, but are not limited to, 18 F, 18 F-FAC, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, ⁇ Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y, 90 Y, 89 Sr, 89 Zr, 94 Tc, 94 Tc, "mTc, "Mo, 105 Pd, 105 Rh, in Ag, in In, 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 153 Sm, 154 158 Gd, 161 Tb, 166 Dy, 166 HO, 169 Er, 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At, 211 Pb, 21 3 ⁇ 4i, 212 Pb
• Exemplary paramagnetic ions substances that can be used as detectable markers include, but are not limited to ions of transition and lanthanide metals (e.g. metals having atomic numbers of 6 to 9, 21-29, 42, 43, 44, or 57-71). These metals include ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu.
• transition and lanthanide metals e.g. metals having atomic numbers of 6 to 9, 21-29, 42, 43, 44, or 57-71).
• metals include ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu.
• chelating groups examples include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTP A), DOTA, NOTA, NOGADA, NETA, deferoxamine (DfO), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and like groups.
• EDTA ethylenediaminetetraacetic acid
• DTP A diethylenetriaminepentaacetic acid
• DOTA DOTA
• NOTA NOGADA
• NETA deferoxamine
• porphyrins porphyrins
• polyamines crown ethers
• bis-thiosemicarbazones polyoximes, and like groups.
• chelates when complexed with non-radioactive metals, such as manganese, iron and gadolinium are useful for MRI, when used along with the antigen binding constructs and carriers described herein.
• Macrocyclic chelates such as NOTA, NOGADA, DOTA, and TETA are of use with a variety of metals and radiometals including, but not limited to, radionuclides of gallium, yttrium and copper, respectively.
• Other ring-type chelates such as macrocyclic polyethers, which are of interest for stably binding radionuclides, such as Radium- 223 for RAIT may be used.
• chelating moieties may be used to attach a PET imaging agent, such as an Aluminum- 18 F complex, to a targeting molecule for use in PET analysis.
• Bioluminescent and fluorescent compounds or molecules and dyes that can be used as detectable moieties in accordance with the embodiments of the disclosure include, but are not limited to, fluorescein, fluorescein isothiocyanate (FITC), OREGON GREENTM, rhodamine, Texas red, tetrarhodimine isothiocynate (TRITC), Cy3, Cy5, and the like), fluorescent markers (e.g., green fluorescent protein (GFP), phycoerythrin, and the like), autoquenched fluorescent compounds that are activated by tumor-associated proteases, enzymes (e.g., lucif erase, horseradish peroxidase, alkaline phosphatase, and the like), nanoparticles, biotin, digoxigenin or combinations thereof.
• fluorescent markers e.g., green fluorescent protein (GFP), phycoerythrin, and the like
• enzymes e.g., lucif erase, horseradish peroxidas
• Enzymes that can be used as detectable moieties in accordance with the embodiments of the disclosure include, but are not limited to, horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, b-galactosidase, b-glucoronidase or b- lactamase. Such enzymes may be used in combination with a chromogen, a fluorogenic compound or a luminogenic compound to generate a detectable signal.
• Nanospheres, nanorods, and nanocups are just a few of the shapes that have been grown.
• Semiconductor quantum dots and nanocrystals are examples of additional types of nanoparticles.
• Such nanoscale particles can be used as payloads to be conjugated to any one of the anti-Gal3 antibodies disclosed herein.
• anti-hormones include anti-estrogens including, for example, tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4- hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapnstone, and toremifene; and anti androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and anti adrenal agents.
• anti-estrogens including, for example, tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4- hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapnstone, and toremifene
• anti androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin
• the payload comprises an immunotoxin.
• Immunotoxins include, but are not limited to, ricin, radionuclides, pokeweed antiviral protein, Pseudomonas exotoxin A, diphtheria toxin, ricin A chain, fungal toxins such as restrictocin and phospholipase enzymes. See, generally, “Chimeric Toxins,” Olsnes and Pihl, Pharmac. Ther. 15:355-381 (1981); and “Monoclonal Antibodies for Cancer Detection and Therapy,” eds. Baldwin and Byers, pp. 159-179, 224-266, Academic Press (1985).
• the payload comprises a nucleic acid polymer.
• the nucleic acid polymer comprises short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), an antisense oligonucleotide.
• the nucleic acid polymer comprises an mRNA, encoding, e.g., a cytotoxic protein or peptide or an apoptotic triggering protein or peptide.
• Exemplary cytotoxic proteins or peptides include a bacterial cytotoxin such as an alpha-pore forming toxin (e.g., cytolysin A from E. coli), a beta-pore- forming toxin (e.g., a-Hemolysin, PVL — panton Valentine leukocidin, aerolysin, clostridial Epsilon-toxin, Clostridium perfringens enterotoxin), binary toxins (anthrax toxin, edema toxin, C. botulinum C2 toxin, C spirofome toxin, C. perfringens iota toxin, C.
• a bacterial cytotoxin such as an alpha-pore forming toxin (e.g., cytolysin A from E. coli), a beta-pore- forming toxin (e.g., a-Hemolysin, PVL —
• cyto- lethal toxins A and B
• prion parasporin, a cholesterol-dependent cytolysins (e.g., pneumolysin), a small pore-forming toxin (e.g., Gramicidin A), a cyanotoxin (e.g., microcystins, nodularins), a hemotoxin, a neurotoxin (e.g., botulinum neurotoxin), a cytotoxin, cholera toxin, diphtheria toxin, Pseudomonas exotoxin A, tetanus toxin, or an immunotoxin (idarubicin, ricin A, CRM9, Pokeweed antiviral protein, DT).
• a cholesterol-dependent cytolysins e.g., pneumolysin
• small pore-forming toxin e.g., Gramicidin A
• cyanotoxin e.
• Exemplary apoptotic triggering proteins or peptides include apoptotic protease activating factor- 1 (Apaf-1), cytochrome-c, caspase initiator proteins (CASP2, CASP8, CASP9, CASP10), apoptosis inducing factor (AIF), p53, p73, p63, Bcl-2, Bax, granzyme B, poly-ADP ribose polymerase (PARP), and P 21-activated kinase 2 (PAK2).
• the nucleic acid polymer comprises a nucleic acid decoy.
• the nucleic acid decoy is a mimic of protein-binding nucleic acids such as RNA-based protein-binding mimics.
• exemplary nucleic acid decoys include transactivating region (TAR) decoy and Rev response element (RRE) decoy.
• the payload is an aptamer.
• Aptamers are small oligonucleotide or peptide molecules that bind to specific target molecules.
• Exemplary nucleic acid aptamers include DNA aptamers, RNA aptamers, or XNA aptamers which are RNA and/or DNA aptamers comprising one or more unnatural nucleotides.
• Exemplary nucleic acid aptamers include ARC 19499 (Archemix Corp.), REG1 (Regado Biosciences), and ARC 1905 (Ophthotech).
• Nucleic acids in accordance with the embodiments described herein optionally include naturally occurring nucleic acids, or one or more nucleotide analogs or have a structure that otherwise differs from that of a naturally occurring nucleic acid.
• 2 ’-modifications include halo, alkoxy, and allyloxy groups.
• the 2 ’-OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NEE, NHR, NR2 or CN, wherein R is C1-C6 alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I.
• modified linkages include phosphorothioate and 5’-N-phosphoramidite linkages.
• nucleic acids having a variety of different nucleotide analogs, modified backbones, or non-naturally occurring internucleoside linkages are utilized in accordance with the embodiments described herein.
• nucleic acids include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxy thymidine, deoxyguanosine, and deoxycytidine) or modified nucleosides.
• modified nucleotides include base modified nucleoside (e.g., aracytidine, inosine, isoguanosine, nebularine, pseudouridine, 2,6-diaminopurine, 2-aminopurine, 2-thiothymidine, 3-deaza-5- azacytidine, 2'-deoxyuridine, 3-nitorpyrrole, 4-methylindole, 4-thiouridine, 4-thiothymidine, 2-aminoadenosine, 2-thiothymidine, 2-thiouridine, 5-bromocytidine, 5-iodouridine, inosine, 6- azauridine, 6-chloropurine, 7-deazaadenosine, 7-deazaguanosine, 8-azaadenosine, 8- azidoadenosine, benzimidazole, Ml-methyladenosine, pyrrolo-pyrimidine, 2-amino-6- chloropurine,
• nucleic acids Natural and modified nucleotide monomers for the chemical synthesis of nucleic acids are readily available.
• nucleic acids comprising such modifications display enhanced properties relative to nucleic acids consisting only of naturally occurring nucleotides.
• nucleic acid modifications described herein are utilized to reduce and/or prevent digestion by nucleases (e.g. exonucleases, endonucleases, etc.).
• nucleases e.g. exonucleases, endonucleases, etc.
• the structure of a nucleic acid may be stabilized by including nucleotide analogs at the 3' end of one or both strands order to reduce digestion.
• nucleotide modifications and/or backbone structures may exist at various positions in the nucleic acid.
• modifications include morpholinos, peptide nucleic acids (PNAs), methylphosphonate nucleotides, thiolphosphonate nucleotides, 2’-fluoro N3- P5’-phosphoramidites, 1’, 5’- anhydrohexitol nucleic acids (HNAs), or a combination thereof.
• PNAs peptide nucleic acids
• HNAs anhydrohexitol nucleic acids
• the payload is conjugated to an anti-Gal3 antibody described herein by a site-directed method utilizing an unnatural amino acid incorporated into the binding moiety.
• the unnatural amino acid comprises p- acetylphenylalanine (pAcPhe).
• pAcPhe p- acetylphenylalanine
• the keto group of pAcPhe is selectively coupled to an alkoxy- amine derivatived conjugating moiety to form an oxime bond (see Axup et al., “Synthesis of site-specific antibody-drug conjugates using unnatural amino acids,” PNAS 109(40): 16101-16106 (2012)).
• the payload is conjugated to an anti-Gal3 antibody described herein by a site-directed method utilizing an enzyme-catalyzed process.
• the site-directed method utilizes SMARTagTM technology (Redwood).
• the SMARTagTM technology comprises generation of a formylglycine (FGly) residue from cysteine by formylglycine-generating enzyme (FGE) through an oxidation process under the presence of an aldehyde tag and the subsequent conjugation of FGly to an alkylhydraine-functionalized polynucleic acid molecule via hydrazino-Pictet-Spengler (HIPS) ligation
• FGE formylglycine-generating enzyme
• HIPS hydrazino-Pictet-Spengler
• the enzyme-catalyzed process comprises microbial transglutaminase (mTG).
• the payload is conjugated to the anti-Gal3 antibody utilizing a microbial transglutaminze catalyzed process.
• mTG catalyzes the formation of a covalent bond between the amide side chain of a glutamine within the recognition sequence and a primary amine of a functionalized polynucleic acid molecule.
• mTG is produced from Streptomyces mobarensis. ( see Strop et al, “Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates,” Chemistry and Biology 20(2) 161-167 (2013)).
• a linker described herein comprises a natural or synthetic polymer, consisting of long chains of branched or unbranched monomers, and/or cross-linked network of monomers in two or three dimensions.
• the linker includes a polysaccharide, lignin, rubber, or polyalkylen oxide (e.g., polyethylene glycol).
• the linker includes, but is not limited to, alpha-, omega- dihydroxylpolyethyleneglycol, biodegradable lactone -based polymer, e.g. polyacrylic acid, polylactide acid (PLA), poly(glycolic acid) (PGA), polypropylene, polystyrene, polyolefin, polyamide, polycyanoacrylate, polyimide, polyethylenterephthalat (PET, PETG), polyethylene terephthalate (PETE), polytetramethylene glycol (PTG), or polyurethane as well as mixtures thereof.
• a mixture refers to the use of different polymers within the same compound as well as in reference to block copolymers.
• block copolymers are polymers wherein at least one section of a polymer is built up from monomers of another polymer.
• the linker comprises polyalkylene oxide.
• the linker comprises PEG.
• the linker comprises polyethylene imide (PEI) or hydroxy ethyl starch (HES).
• the polyalkylene oxide (e.g., PEG) is a polydispers or monodispers compound.
• polydispers material comprises disperse distribution of different molecular weight of the material, characterized by mean weight (weight average) size and dispersity.
• the monodisperse PEG comprises one size of molecules.
• the linker is poly- or monodispersed polyalkylene oxide (e.g., PEG) and the indicated molecular weight represents an average of the molecular weight of the polyalkylene oxide, e.g., PEG, molecules.
• the linker comprises a polyalkylene oxide (e.g., PEG) and the molecular weight of the polyalkylene oxide (e.g., PEG) is about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da.
• PEG polyalkylene oxide
• the polyalkylene oxide is a discrete PEG, in which the discrete PEG is a polymeric PEG comprising more than one repeating ethylene oxide units.
• a discrete PEG comprises from 2 to 60, from 2 to 50, or from 2 to 48 repeating ethylene oxide units.
• a dPEG comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 35, 40, 42, 48, 50 or more repeating ethylene oxide units.
• a dPEG comprises about 2 or more repeating ethylene oxide units.
• a dPEG is synthesized as a single molecular weight compound from pure (e.g., about 95%, 98%, 99%, or 99.5%) staring material in a step wise fashion.
• a dPEG has a specific molecular weight, rather than an average molecular weight.
• the linker is a discrete PEG, optionally comprising from 2 to 60, from 2 to 50, or from 2 to 48 repeating ethylene oxide units.
• the linker comprises a dPEG comprising about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 35, 40, 42, 48, 50 or more repeating ethylene oxide units.
• the linker is a polypeptide linker.
• the polypeptide linker comprises at least 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more amino acid residues.
• the polypeptide linker comprises at least 2, 3, 4, 5, 6, 7, 8, or more amino acid residues.
• the polypeptide linker comprises at most 2, 3, 4, 5, 6, 7, 8, or less amino acid residues.
• the polypeptide linker is a cleavable polypeptide linker (e.g., either enzymatically or chemically). In some cases, the polypeptide linker is a non-cleavable polypeptide linker.
• the linker comprises a homobifuctional linker.
• exemplary homobifuctional linkers include, but are not limited to, Lomant's reagent dithiobis (succinimidylpropionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl proprionate (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo DST), ethylene glycobis(succinimidylsuccinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl- 3, 3
• DTSSP
• the linker comprises a heterobifunctional linker.
• exemplary heterobifunctional linker include, but are not limited to, amine-reactive and sulfhydryl cross-linkers such as N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long- chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble-long-chain N- succinimidyl 3-(2-pyridyldithio) propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-a- methyl-a-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[a-methyl-a-(2- pyridyldithio)toluamido]hexanoate (sulfo-
• the maleimide group is maleimidocaproyl (me).
• the peptide group is val-cit.
• the benzoic acid group is PABA.
• the linker comprises a mc-val-cit group.
• the linker comprises a val- cit-PABA group.
• the linker comprises a mc-val-cit-PABA group.
• the linker is a self-immolative linker or a self elimination linker. In some cases, the linker is a self-immolative linker. In other cases, the linker is a self-elimination linker (e.g., a cyclization self-elimination linker). In some instances, the linker comprises a linker described in U.S. Patent No. 9,089,614 or PCT Publication No. WO2015038426.
• the linker is a dendritic type linker.
• the dendritic type linker comprises a branching, multifunctional linker moiety.
• the dendritic type linker comprises PAMAM dendrimers.
• the linker is a traceless linker or a linker in which after cleavage does not leave behind a linker moiety (e.g., an atom or a linker group) to the antibody or payload.
• a linker moiety e.g., an atom or a linker group
• Exemplary traceless linkers include, but are not limited to, germanium linkers, silicium linkers, sulfur linkers, selenium linkers, nitrogen linkers, phosphorus linkers, boron linkers, chromium linkers, or phenylhydrazide linker.
• the linker is a traceless aryl-triazene linker as described in Hejesen, et al., “A traceless aryl-triazene linker for DNA- directed chemistry,” Org Biomol Chem 11(15): 2493-2497 (2013).
• the linker is a traceless linker described in Blaney, et al., “Traceless solid-phase organic synthesis,” Chem. Rev. 102: 2607-2024 (2002).
• a linker is a traceless linker as described in U.S. Patent No. 6,821,783.
• kits and articles of manufacture for use with one or more of the compositions and methods described herein.
• Such kits include a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein.
• Suitable containers include, for example, bottles, vials, syringes, and test tubes.
• the containers are formed from a variety of materials such as glass or plastic.
• the articles of manufacture provided herein contain packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.
• the container(s) include an anti-Gal3 antibody as disclosed herein, host cells for producing one or more antibodies described herein, and/or vectors comprising nucleic acid molecules that encode the antibodies described herein.
• kits optionally include an identifying description or label or instructions relating to its use in the methods described herein.
• a kit typically includes labels listing contents and/or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included.
• a label is on or associated with the container.
• a label is on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself; a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert.
• a label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates directions for use of the contents, such as in the methods described herein.
• the pharmaceutical compositions are presented in a pack or dispenser device which contains one or more unit dosage forms containing a compound provided herein.
• the pack for example, contains metal or plastic foil, such as a blister pack.
• the pack or dispenser device is accompanied by instructions for administration.
• the pack or dispenser is also accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration.
• a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration.
• Such notice for example, is the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert.
• compositions containing a compound provided herein formulated in a compatible pharmaceutical carrier are also prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
• the anti-Gal3 antibody or binding fragment thereof of any one of arrangements 1-3 wherein the anti-Gal3 antibody or binding fragment thereof comprises a heavy chain, wherein the heavy chain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the amino acid sequences of SEQ ID NOs: 836-850, 983-996, 1411, 1153-1195, or 1465-1489.
• the anti-Gal3 antibody or binding fragment thereof comprises a light chain, wherein the light chain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the amino acid sequences of SEQ ID NOs: 851-865, 997-1010, 1196-1238, 1412 or 1490-1514.
• a nucleic acid comprising a sequence having at least 80%, 81%, 82%, 83%,
• glucose transporter is glucose transporter 1 (GLUT1) and/or glucose transporter 4 (GLUT4).
• the anti-Gal3 antibody or binding fragment thereof comprises (1) a heavy chain variable region comprising a V H -CDR1, a V H -CDR2, and a V H -CDR3; and (2) a light chain variable region comprising a V L -CDR1, a V L -CDR2, and a V L -CDR3, wherein the V H -CDR1 comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the amino acid sequences of SEQ ID NOs: 27-70; the V H -CDR2 comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
• the heavy chain variable region comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence selected from SEQ ID NOs: 297-373, 803, 806-820, 955-968, 1067-1109, 1415-1439.
• the light chain variable region comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence selected from SEQ ID NOs: 374-447, 821-835, 969-982, 1110-1152, 1440-1464.
• the anti-Gal3 antibody or binding fragment thereof comprises a light chain
• the light chain comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence selected from SEQ ID NOs: 495-538, 805, 851-865, 997-1010, 1196-1238, 1412, 1490-1514.
• A3_hVH3 VL 1 -hlgG 1 (LALAPGv2), 20H5.A3_hVH3VLl-hIgGl (REM), 20H5. A3_hVH5 VL 1 -hlgG 1 (KEMv2), 20H5.A3_hVH5VLl-hIgGl (LALAPGv2), 20H5. A3_hVH5 VL 1 -hlgG 1 (REM), 20H5.A3_hVH6VLl-hIgGl (KEMv2),
• anti-Gal3 antibody or binding fragment thereof is selected from the group consisting of: 20H5.A3, 20H5.A3-VH3VL1, 20H5.A3-VH3VL3, 20H5.A3-VH4VL1, 20H5.A3-VH5VL1, 20H5.A3- VH5VL3, 20H5.A3-VH6VL1, 20H5.A3-VH6VL3, or binding fragment thereof.
• anti-Gal3 antibody or binding fragment thereof is selected from the group consisting of: 2D10-VH0-VL0, 2D 10-hVH4-HVL 1 , 2D10-hVH4-HVL2, 2D10-hVH4-HVL3, 2D10-hVH4-HVL4, 2D10- hVH3-HVLl, 2D10-hVH3-HVL2, 2D10-hVH3-HVL3, 2D10-hVH3-HVL4, or binding fragment thereof.
• a method of improving insulin sensitivity in a subject in need thereof comprising: administering to the subject an anti-Gal3 antibody or binding fragment thereof, wherein binding of the anti-Gal3 antibody or binding fragment thereof to Gal3 in the subject inhibits Gal3-mediated blocking of glucose transporter (GLUT) translocation in the subject, thereby improving insulin sensitivity in the subject.
• GLUT glucose transporter
• [0388] 34 The method of arrangement 32 or 33, wherein the insulin sensitivity in the subject is improved by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%,
• the anti-Gal3 antibody or binding fragment thereof comprises (1) a heavy chain variable region comprising a V H -CDR1, a V H -CDR2, and a V H -CDR3; and (2) a light chain variable region comprising a V L -CDR1, a V L -CDR2, and a V L -CDR3, wherein the V H -CDR1 comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the amino acid sequences of SEQ ID NOs: 27-70; the V H -CDR2 comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
• the heavy chain variable region comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence selected from SEQ ID NOs: 297-373, 803, 806-820, 955-968, 1067-1109, 1415-1439.
• the anti-Gal3 antibody or binding fragment thereof comprises a heavy chain
• the heavy chain comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence selected from SEQ ID NOs: 448-494, 804, 836-850, 983-996, 1153-1195, 1411, 1465-1489.
• the anti-Gal3 antibody or binding fragment thereof comprises a light chain
• the light chain comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence selected from SEQ ID NOs: 495-538, 805, 851-865, 997-1010, 1196-1238, 1412, 1490-1514.
• A3_hVH3 VL 1 -hlgG 1 (LALAPGv2), 20H5.A3_hVH3VLl-hIgGl (REM), 20H5.A3_hVH5VLl-hIgGl (KEMv2), 20H5.A3_hVH5VLl-hIgGl (LALAPGv2), 20H5. A3_hVH5 VL 1 -hlgG 1 (REM), 20H5.A3_hVH6VLl-hIgGl (KEMv2),
• 20H5.A3_hVH6VLl-hIgGl (LALAPGv2), 20H5.A3_hVH6VLl-hIgGl (REM), 21H6-H0L0, 21H6-H1L1, 21H6-H1L2, 21H6-H1L3, 21H6-H1L4, 21H6-H2L1, 21H6-H2L2, 21H6-H2L3, 21H6-H2L4, 21H6-H3L1, 21H6-H3L2, 21H6-H3L3, 21H6-H3L4, 21H6-H4L1, 21H6-H4L2, 21H6-H4L3, 21H6-H4L4, 21H6-H5L1, 21H6-H5L2, 21H6-H5L3, 21H6-H5L4, 21H6-H6L1, 21H6-H6L2, 21H6-H6L3, 21H6-H6L4, or binding fragment thereof.
• a method of treating a disease associated with insulin resistance in a subject in need thereof comprising: administering to the subject an anti-Gal3 antibody or binding fragment thereof, thereby treating the disease associated with insulin resistance in the subject.
• a method of treating a disease associated with insulin resistance in a subject in need thereof comprising: administering to a subject a preincubated complex of Gal3 and an anti-Gal3 antibody or binding fragment thereof, thereby treating the disease associated with insulin resistance in the subject.
• [0410] 56 The method of arrangement 54 or 55, wherein the insulin sensitivity in the subject is improved by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, or 200% relative to the insulin sensitivity of the subject prior to the administering step.
• the anti-Gal3 antibody or binding fragment thereof comprises (1) a heavy chain variable region comprising a V H -CDR1, a V H -CDR2, and a V H -CDR3; and (2) a light chain variable region comprising a V L -CDR1, a V L -CDR2, and a V L -CDR3, wherein the V H -CDR1 comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the amino acid sequences of SEQ ID NOs: 27-70; the V H -CDR2 comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%
• the heavy chain variable region comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence selected from SEQ ID NOs: 297-373, 803, 806-820, 955-968, 1067-1109, 1415-1439.
• the light chain variable region comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence selected from SEQ ID NOs: 374-447, 821-835, 969-982, 1110-1152, 1440-1464.
• the anti-Gal3 antibody or binding fragment thereof comprises a heavy chain
• the heavy chain comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence selected from SEQ ID NOs: 448-494, 804, 836-850, 983-996, 1153-1195, 1411, 1465-1489.
• the anti-Gal3 antibody or binding fragment thereof comprises a light chain
• the light chain comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence selected from SEQ ID NOs: 495-538, 805, 851-865, 997-1010, 1196-1238, 1412, 1490-1514.
• A3_hVH3 VL 1 -hlgG 1 (LALAPGv2), 20H5.A3_hVH3VLl-hIgGl (REM), 20H5. A3_hVH5 VL 1 -hlgG 1 (KEMv2), 20H5.A3_hVH5VLl-hIgGl (LALAPGv2), 20H5. A3_hVH5 VL 1 -hlgG 1 (REM), 20H5.A3_hVH6VLl-hIgGl (KEMv2),
• any one of arrangements 47-65 wherein the anti-Gal3 antibody or binding fragment thereof is selected from the group consisting of: 2D10-VH0-VL0, 2D 10-hVH4-HVL 1 , 2D10-hVH4-HVL2, 2D10-hVH4-HVL3, 2D10-hVH4-HVL4, 2D10- hVH3-HVLl, 2D10-hVH3-HVL2, 2D10-hVH3-HVL3, 2D10-hVH3-HVL4, or binding fragment thereof.
• lymph nodes axillary, accessory axillary, mediastinal, superficial inguinal, iliac, sacral and popliteal
• LN immunized lymph node
• spleen and bone marrow cells were obtained using 2 sterile frosted glass slides in a tissue culture petri dish with 15 mL DMEM. Bone marrow was extracted from femurs via end-cap flushing with a 5 mL syringe fitted with an 18-gauge needle.
• Cells from 3 animals were pelleted with 5 minutes of centrifugation at 1200 RPM, resuspended in 10 mL of DMEM (GIBCO 10564-011) and nucleated cells were enumerated by hemocytometer count. Cells were pelleted at 1200 RPM and were resuspended in SC-Buffer (PBS, 2% FBS and 1 mM EDTA), and plasma cells were isolated with an Easy SepTM Mouse CD 138 Positive Selection Kit (StemCell Technologies) with the manufacturer recommended protocol.
• DMEM fetal bovine serum
• CD138-positive cells were pelleted with 5 minutes of centrifugation at 1200 RPM, resuspended in 50 mL electrofusion buffer (Eppendorf 940-00-220-6) and were enumerated.
• SP2/0-mIL6 myeloma cells ATCC CRL2016
• Myeloma cells and CD 138- positive plasma cells were combined at a 1:1 ratio, volume was expanded to 50 mL with electrofusion buffer, cells were pelleted with 5 minutes of centrifugation at 1200 RPM and supernatant was discarded.
• cells were resuspended in electrofusion buffer to a concentration of 10c10 L 6 cells/mL, up to 9 mL of cell suspension was added to a BTX electrofusion chamber, and cells were fused with an 800V electrofusion protocol.
• Fused cells were rested for 5 minutes, transferred to a tissue culture dish containing 40 mL medium MM (DMEM, 15% FBS, 1% glutamax and 1% Pen/Strep), incubated for 1 hour at 37°C, 8% CO2, resuspended with a pipette, pelleted with 5 minutes of centrifugation at 1200 RPM, resuspended in ClonaCell HY Liquid HAT Selection Medium (StemCell Technologies), and plated in 96-well tissue culture flat bottomed plates. After 10 days, supernatants were sampled and evaluated for binding to isolated Gal3 by ELISA.
• DMEM fetal bovine serum
• Gal3-targeted antibodies with the ability to block the interaction of Gal3 and INSR
• purified Gal3 and INSR proteins were incubated in the presence of Gal3- immunization hybridoma supernatants described above, or without antibody, and protein interaction was evaluated by ELISA.
• Human Galectin-3 protein (Aero Biosystems, GA3- H5129) was diluted in PBS (Coming, 21-030-CM) to a concentration of 3 mg/ml and added to the wells of a 96-well ELISA plate (Thermo Fisher, 44-2404-21).
• PBST PBS with 0.05% Tween 20 [VWR, 0777]
• the plate was then blocked for an hour with 2% BSA (EMD Millipore, 126609) in PBST at room temperature with gentle rocking. Thereafter, the 2% BSA in PBST was discarded and antibody or inhibitor (3-fold dilutions beginning at 20 pg/ml, 60 pg/ml, or 180 mM) in 2% BSA in PBST was added to the wells.
• the plate was incubated at room temperature for an hour with gentle rocking and then washed three times with PBST.
• TMB substrate (Thermo Scientific, 34029) was then added to each well.
• the reaction was stopped with 1M HC1 (JT Baker, 5620-02) and read using a plate reader (Molecular Devices) at absorbance of 450 nm.
• monoclonal antibodies with reduced capacity to inhibit Gal3-INSR binding at 3 pg/mL to 15- 25% of levels of an unblocked sample were identified, including 7D8.2D8 and 15F10.2D6.
• monoclonal antibodies with the capacity to minimally impact Gal3-INSR binding by 30% or less at 3 pg/ml were identified, including 9H2.2H1, 12G5.D7, 13G4.2F8, and 24D12.2H9.
• the complex was allowed to associate and dissociate for 240 and 300 seconds, respectively.
• the surfaces were regenerated with a 30 second injection of 10 mM Glycine pH 1.7 (flow rate 30 pL/min).
• the data were fit to a simple 1:1 interaction model using the global data analysis option available within BiacoreT200 Evaluation software V2.0.
• Gal3 monoclonal antibodies were confirmed to be greater than 30 nM for all antibodies studied (FIG. 3A).
• 15G7 an antibody which reduced assembly of Gal3 and INSR by greater than 99% at 3 pg/mL exhibited an affinity of 27.8 nM for Gal3, indicating that the ability to block the assembly of Gal3 and INSR is possible with antibodies with affinity at or below this level.
• Gal3-targeted antibodies which poorly block Gal3-INSR assembly 13G4.2F8, 9H2.2H1, 24D12.2H9, and 12G5.D7, exhibited affinities of 18.4, 2.53, 4.13, and 1.9 nM, respectively.
• antibody affinity to Gal3 of less than 10 nM is not sufficient to predict the ability to block assembly of Gal3 and INSR.
• At least 2 pg/ml of hGal3 peptide in 50 pi of PBS or 0.1 pg/ml of full-length human Gal3 protein (GenScript) and human Galectin-3 protein (Aero Biosystems, GA3-H5129) were diluted in PBS (Corning, 21-030-CM) to concentrations of at least 2 pg/ml or 0.1 pg/ml, respectively, and added to the wells of a 96- well ELISA plate (Thermo Fisher, 44-2404-21). After incubating the plate at 4°C overnight, the plate was washed three times with PBST (PBS with 0.05% Tween 20 [VWR, 0777]).
• PBST PBS with 0.05% Tween 20 [VWR, 0777]
• the plate was then blocked for an hour with 2% BSA (EMD Millipore, 126609) in PBST at room temperature with gentle rocking. Thereafter, the 2% BSA in PBST was discarded and human Galectin-3 hybridoma supernatants or antibodies were diluted in 2% BSA in PBST to concentrations of at least 0.1 pg/ml and added to the wells.
• the plate was incubated for an hour at room temperature with gentle rocking and then washed three times with PBST.
• Goat Anti-Mouse IgG-HRP Jackson ImmunoResearch, 115-036-1461
• Goat Anti-Rat IgG HRP Abeam, ab205720
• 2% BSA in PBST 1:4000
• TMB substrate Thermo Scientific, 34029
• the reaction was stopped with 1M HC1 (JT Baker, 5620-02) and read using a plate reader (Molecular Devices) at absorbance of 450 nm.
• Gal3-binding antibodies with strong Gal3-INSR blocking activity 4G2.2G6 and 3B11.2G2 bound peptide 4 of Gal3, corresponding to amino acids 31-50 of Gal3, GAGGYPGASYPGAYPGQAPP (SEQ ID NO: 6).
• Gal3-targeted antibodies with poor Gal3-INSR blocking activity were observed to bind peptide 4.
• Gal3-binding antibodies with Gal3-INSR blocking activity (6H6.2D6, 20H5.A3, 20D11.2C6, 13H12.2F8, 19B5.2E6, 23H9.2E4, 15G7.2A7, 19D9.2E5, 14H10.2C9, 7D8.2D8, and 15F10.2D6) all bound peptide 7 of Gal3, corresponding to amino acids 61-80 of Gal3, AYPGAPGAYPGAPAPGVYPG (SEQ ID NO: 9).
• no Gal3-targeted antibodies with poor Gal3-INSR blocking activity were observed to bind peptide 7.
• binding to Gal3 peptide 7 is predictive of the ability to block the interaction of Gal3 with INSR.
• these data indicate the utility of anti-Gal3 antibodies to Gal3 peptides 1, 4, 6, and 7 as predictive of the ability to block the interaction of Gal3 and INSR.
• peptides 4, 6, and 7 share repeated amino acid sequences comprised of proline-glycine (PG) and tyrosine-proline-glycine (YPG), suggesting a common feature that may explain the ability of Gal3 -targeted antibodies to bind to multiple Gal3 peptides.
• PG proline-glycine
• YPG tyrosine-proline-glycine
• GxYPG amino acid sequence glycine-x-tyrosine-proline-glycine
• x may be the amino acids alanine (A), glycine (G), or valine (V)
• Gal3-binding antibodies with Gal3-INSR blocking activity bind to the same or overlapping regions of the Gal3 molecule
• antibody binning assays were performed to assess the ability of antibodies to simultaneously bind Gal3.
• Amine-reactive probes were loaded onto a Gator biosensor (Probe Life, Palo Alto, CA), equilibrated in dH20 for 60 seconds, dipped into 100 pi EDC 0.2M /NHS 0.05M activation buffer for 30 seconds, then dipped into a solution of 20 pg/pl human Gal3-His in 10 mM NaOAc buffer, pH 5 until binding was saturated, and quenched in 1 M ethanolamine pH 8.5 for 300 seconds.
• tips were dipped in 20 pg/mL saturating antibody, then successively dipped into 5 pg/mL competing antibody.
• FIG. 3A-B antibodies with competitive binding profiles were assigned bins and associations to blocking activity were made.
• Gal3-INSR blocking antibody TB001 reduces weight gain, insulin resistance, glucose insensitivity, liver steatosis, and liver dysfunction in high-fat diet fed diabetic mice.
• HFD high fat diet
• mice were housed in standard facilities and disposable standard cages with filter tops at room temperature with a 06:00-18:00 day-night cycle. Mice were fed with standard chow diet or HFD ad libitum except when fasted as indicated in the experiments.
• Glucose tolerance test (GTT) and insulin tolerance test (ITT) were done at 15 weeks and 16 weeks old (8 weeks and 9 weeks after HFD), respectively, to confirm the insulin resistant and glucose intolerant phenotype.
• GTT Glucose tolerance test
• ITT insulin tolerance test
• mice were fasted for 6 hours by transferring mice to clean cage with no food or faeces but with drinking water and then injected with 2 g/kg glucose (Sigma) and blood was drawn to measure glucose levels at 0, 15, 30, 60, 90, and 120 minutes after glucose injection.
• mice were fasted for 6 hours, and then injected with 0.75 IU/kg body weight of Humalin-R (Eli Lilly) intraperitoneally. Blood from the tail was measured for glucose content using HemoCue Glucose 201 Analyzer at 0, 15, 30, 45, 60 minutes. Based on area under the curve (AUC) from ITT, HFD fed mice were randomized into two groups: human IgG4 isotype group and IMT001-4 treated group. At 17 weeks of age, mice were given antibody treatments by intraperitoneal injection twice a week for 5 doses before ITT and GTT were done at 19 weeks old and 20 weeks old.
• AUC area under the curve
• mice Human IgG4 isotype and IMT001-4 were dosed at 10 mg/kg with 100 pl/mouse and BM was dosed at 10 mg/ml with 100 pl/mouse. Treatments continued until the mice were sacked at 21 weeks old. The mice body weight was monitored one week after the mice arrived at the facility. Body weight was measured once a week using a balance. Mouse blood was collected using cardiac puncture. Liver, gastrocnemius muscle, and posterior subcutaneous white adipose fat were collected and weighed.
• liver sections from animals treated as in FIGs. 3-5 were examined for signs of fat accumulation, e.g., steatosis. Briefly, liver specimens were fixed in 4% paraformaldehyde for 24 hours, placed in 70% EtOH for 3 days, embedded into paraffin and 10 pm sections were cut and mounted to glass slides and stained with hematoxylin and eosin and visualized on a Revolve microscope at 40X magnification. Images were evaluated by ImageJ to quantitate the extent of steatosis.
• liver disease serum markers of liver dysfunction were evaluated in animals treated as above by ELISA. Briefly, mouse plasma samples were obtained from whole blood sample in lithium heparin collection tubes. 100 pi of serum was dispensed into the rotor of a VetScan VS2 Blood Chemistry Analyzer (Abaxis) through the sample port. ALT levels were determined as per the manufacturer’s specification.
• isotype-control HFD-fed diabetic mice exhibited nearly a 3 -fold increase in ALT relative to normal-diet fed non-diabetic mice (FIG. 8). Strikingly, ALT levels were significantly decreased in IMT001-4 treated HFD-fed mice relative to HFD-fed isotype-control treated mice, to levels on-par with normal-diet fed non-diabetic mice. Accordingly, the Gal3- targeted Gal3-INSR blocking antibody, IMT001-4, has utility in reducing serum liver enzymes.
• Example 5 Anti-GAL3 antibodies have therapeutic use in a model of advanced Type II diabetes [0439]
• HFD High Fat Diet
• Db/Db mice were fed with 60% kcal HFD (Research Diet, 12492i) for eight weeks.
• Control animals male C57BL6/J (000662), Jackson lab) were fed normal chow diet.
• Db/Db animals had increased levels circulating Gal-3 (FIG. 9). Animals were randomized based on the body composition measured by employing EchoMRI-500TM (EchoMRI).
• Example 6 Anti-GAL3 antibodies have therapeutic use in a mouse model of Type I diabetes [0440] To assess the therapeutic potential of anti-GAL3 antibody in Type I diabetes, a mouse model of diabetes type I was used. Briefly, seven- weeks-old female NOD/ShiLtJ mice (Jackson lab (001976)) and control group animals NOR/LtJ (Jackson Lab (002050)) were housed in standard disposable cages with filter tops at room temperature with a 6:00-18:00 day-night cycle. Mice were allowed to rest for a week before initiating glucose monitoring. The antibody treatment was initiated when glucose levels reach 250-400 (mg/dL) for two consecutive days.
• NOD/ShiLtJ mice Jackson lab (001976)
• NOR/LtJ Jackson Lab (002050)
• the blood samples were collected from tail vein of the experimental animals following housing in clean cages with water and no food for 4-6 hours.
• the collected samples were analyzed by HemoCue Glucose 201 Analyzer and cuvettes (Mercedes Scientific 110706).
• the animals were dosed twice a week (lOmg/kg) with anti- GAL3 antibodies (mTBOOl) or PBS control.
• the animals were sacrificed when the glucose levels reached >1000 (mg/dL) or until they exhibited morbidity symptoms (i.e. hunched posture, hypothermia, and hypoactivity).
• the plasma samples were collected from symptomatic animals.
• C-peptide levels were determined by Alpco Mouse C-peptide ELISA kit (Alpco, 80-CPTMS-E01). As depicted in FIG. 12, anti-GAL3 treatment prolonged survival of NOD/ShiLtJ animals and restored beta-cell function by stabilizing the levels of blood glucose (FIG. 13A) and restoring C-peptide levels (FIG. 13B). Therefore, anti-Gal3 treatment decreases the symptoms of both Type II and Type I diabetes.
• Anti-GAL3 antibodies have therapeutic use in a mouse model of chronic inflammatory bowel disease (IBP)
• Plasma Inflammatory cytokines were measured using Multi-spot Assay Systems (MSD) Proinflammatory Panel 1 (mouse) kit (MSD, K15048D).
• RT-qPCR was performed on frozen colon samples. Briefly, the colon tissue was disrupted using Qiazol lysis buffer (Qiagen, 79306) and the Qiagen Tissue Lyser II (Qiagen, 85300). RNA was extracted using RNeasy mini kit (Qiagen, 74104). cDNA was synthesized with iScript Reverse Transcription Supermix (BioRad, 1708841) at BioRad C1000 touch thermal cycler (BioRad, 1851138).
• RT-qPCR was performed using SsoAdvanced Universal SYBR green supermix (BioRad, 1725272) at CFX384 Touch Real-Time PCR detection system (BioRad, 1955485) using the primers in FIG. 14 (SEQ ID NO: 926-936).
• Anti-galectin-3 treatment restored DSS-induced reduction in the colon length (FIG. 15) and reduced DSS-induced inflammation determined by the circulating levels of IFN- gamma (FIG. 16).
• mRNA levels of several pro-inflammatory genes e.g. IFN- gamma, IF17a, IF-lbeta, IF-21, IF-22 were reduced in response to TB001 treatment.
• Example 8 Binning and peptide binding assay of additional exemplary anti-Ga!3 antibodies
• a large-scale antibody binning assay was performed on exemplary anti-Gal3 antibodies.
• the epitope binning assay was done in a sandwich format on the high- throughput SPR-based Carterra ESA unit (CarterraBio, Salt Lake City, UT).
• the purified antibodies were diluted to 10 mg/ml concentration in 10 mM NaOAc (pH 5.0) and then were covalently coupled via amine group to HC200M chip activated by EDC and S-NHS to immobilize antibodies to different positions of a 384-spot array.
• One hundred thirty-eight binning cycles were ran on the array of immobilized antibodies.
• Clones IMT001 (TB001) and F847C.21H6 exhibited mutual competitive binding for Gal3, but did not prevent binding of the rest of the clones, thus defining bin 1.
• Clones IMT006 (TB006), 19B5.2E6, 20H5.A3, 23H9.2E4, 2D10.2B2 exhibited mutual competitive binding for Gal3, but did not prevent binding of the rest of the clones, thus defining bin 3.
• Clones 20D11.2C6 and 15G7.2A7 exhibited mutual competitive binding for Gal3, but did not prevent binding of the rest of the clones, thus defining bin 5.
• Clones 3B11.2G2, 13A12.2E5 exhibited mutual competitive binding for Gal3, but did not prevent binding of the rest of the clones, thus defining bin 7.
• Clones 24D12.2H9, 6B3.2D3, 849.1D2 exhibited mutual competitive binding for Gal3, but did not prevent binding of the rest of the clones, thus defining bin 11.
• Clones 13G4.2F8 and 9H2.2H10 exhibited mutual competitive binding for Gal3, but did not prevent binding of the rest of the clones, thus defining bin 12.
• Clones F846C.1B2, F846C.1F5, F846C.1H12, F846C.2H3, F846TC.16B5 exhibited mutual competitive binding for Gal3, but did not prevent binding of the rest of the clones, thus defining bin 17.
• clone 849.5H1 did not compete for binding to Gal3 with other antibodies tested, therefore defining bin 21.
• Antibodies 847.12C4, 847.15D12, 847.15H11, 847.20H7, and 847.27B9 exhibited mutual competitive binding and competed with binding with the commercially available anti-Gal3 antibody B2C10 for hGAL3, but did not prevent binding of the rest of the clones, thus defining bin “B2C10”.
• B2C10 has been epitope mapped to bind to the first 18 amino acids of Gal3.
• Antibodies 847.10C9, 847.11D6, 847.13E2-mH0mLl, 847.13E2- mH0mL2, 847.16D10, 847.23F11, 847.28D1, and 847.3B3 exhibited mutual competitive binding to the C-terminal carbohydrate-recognition domain (CRD), but did not prevent binding of the rest of the clones, thus defining bin “CRD”.
• Gal3 peptide binding activity of additional exemplary anti-Gal3 antibodies was assessed with the same peptides as described in FIG. 18 (SEQ ID NOs: 3-26).
• Human Gal3 peptides LifeTein, custom order
• human Gal3 proteins R&D Systems, 8259-GA; TrueBinding, QCB200349
• PBS PBS
• concentrations of at least 100 pg/mL (peptides) or 1 pg/mL (proteins) were added to the wells of several 96-well ELISA plates (Thermo Fisher, 44-2404-21).
• PBST PBS with 0.05% Tween 20 [VWR, 0777]
• the plates were then blocked for an hour with 2% BSA (EMD Millipore, 126609) in PBST at room temperature with gentle rocking. Thereafter, the 2% BSA in PBST was discarded and Gal3- binding antibodies (reformatted hIgG4 [S228P]) were diluted in 2% BSA in PBST to concentrations of 5 pg/mL and added to the wells.
• the plates were incubated for an hour at room temperature with gentle rocking and then washed three times with PBST.
• FIG. 26 Peptide binding results are depicted in FIG. 26. Peptide numbering is based on FIG. 18. Binding of Gal3-binding antibodies to the peptide array was observed at multiple locations, with the majority of binding observed in peptides 1-8 and some binding to peptides 13 and 17. [0451] 13 separate Gal3-binding antibodies (19B5.2E6, 20D11.2C6, 20H5.A3,
• 847.12C4, 847.10C9) bound peptide 3 of Gal3, corresponding to amino acids WPG AW GN QPAG AGG YPG AS Y (SEQ ID NO: 5) of Gal3.
• F846C.1H12, F847C.21H6) bound peptide 5 of Gal3, corresponding to amino acids PGAYPGQAPPGAYPGQAPPG (SEQ ID NO: 7) of Gal3.
• Gal3 corresponding to amino acids PPSGPGAYPSSGQPSATGAY (SEQ ID NO: 11) of Gal3.
• Gal3-binding antibodies 15G7.2A7, 847.12C4, 847.10C9 all bound peptide 10 of Gal3, corresponding to amino acids SGQPSATGAYPATGPYGAPA (SEQ ID NO: 12) of Gal3.
• 3 separate Gal3-binding antibodies (847.13E2-mH0mLl, 847.13E2- mH0mL2, 847.23F11) all bound peptide 13 of Gal3, corresponding to amino acids LPGGVVPRMLITILGTVKPN (SEQ ID NO: 15) of Gal3.
• peptides 4, 5, 6, and 7 share repeated amino acid sequences comprised of proline-glycine (PG) and tyrosine-proline- glycine (YPG), indicating a common feature that may explain the ability of Gal3 -targeted antibodies to bind to multiple Gal3 peptides.
• PG proline-glycine
• YPG tyrosine-proline- glycine
• GxYPG amino acid sequence glycine-x-tyrosine-proline-glycine
• x may be the amino acids alanine (A), glycine (G), or valine (V)
• the presence of two GxYPG sequences in close apposition is likely predictive of the ability to bind Gal3-targeted antibodies.
• the Grantham distance of alanine, glycine, and valine is Ala-Val: 64, Ala-Gly: 60, Val-Gly: 109, thereby predicting that amino acids with similarly low Grantham distances may similarly be able to substitute at the variable region, including proline and threonine.
• Example 9 Humanized anti-Ga!3 antibodies have high affinity for Gal3 of different species [0465] Cross reactivity of exemplary anti-Gal3 antibodies TB001 and TB006 to human or mouse Gal3 were tested. Kinetics experiments were performed on a Carterra LSA at 25°C. An HC30M chip was immobilized with recombinant Protein A/G. TB001 (IMT001; IMT001-4), TB006 (IMT006; IMT006-5), and a negative control antibody Synagis (10 pg/mL each diluted in HBSTE buffer (HEPES based saline with Tween-20 and EDTA) with 0.5 mg/mL BSA) were captured onto different spots in the 384-spot array.
• HBSTE buffer HEPES based saline with Tween-20 and EDTA
• a CM5 chip coated with anti-mouse Fc/anti-human Fc mixture was used to load purified antibody TB001 or TB006 at 10 pg/mL in HBS-EP+ buffer (HEPES based saline with polysorbate 20 and EDTA) with 0.5 mg/mL BSA for 180 seconds, and then a dilution series of His-enterokinase cleavage site (EK)-cynoGal3 (TrueBinding in-house antigen) in HBS-EP+ with 0.5 mg/mL BSA starting from 100 nM, 1:2 dilution for 7 points for 200 seconds, followed by dissociation for 300 seconds.
• HBS-EP+ buffer HBS based saline with polysorbate 20 and EDTA
• EK His-enterokinase cleavage site
• the affinity of Gal3 binding to antibody was determined by acquiring real time Ligand: Analyte binding kinetics data and fitting the data with a 1:1 monovalent binding model.
• the kinetic evaluation procedure determines association and dissociation constants by fitting the experimental data to a 1 : 1 interaction model between analyte A and ligand B :
• K a is the association rate constant (M V 1 )
• K d is the dissociation rate constant (s 1 ).
• IMT001 also has high affinity for mouse Gal3 (IMT001: 2.3 nM, IMT006a: 40000 nM) and rat Gal3 (IMT001: 14 nM, IMT006a: undetected).
• Insulin leads to glucose uptake by stimulating the translocation of the glucose transporter GLUT4 from intracellular stores to the plasma membrane. It was tested if Gal3 can interfere with GLUT4 translocation and if anti-Gal3 antibodies can block this effect.
• Rat L6 GLUT4myc myoblasts express a Myc-tagged GLUT4 that can be detected with an anti-Myc antibody. These cells were grown in a 48-well plate in MEM a growth media (Gibco, #12-571-063) + 10% fetal bovine serum (FBS; Coming #35-016-CV) until 90-95 % confluence was reached. Cells were then washed in PBS and serum starved for 2 hours in MEM a growth media without FBS.
• MEM a growth media Gibco, #12-571-063
• FBS fetal bovine serum
• the cells were washed three times with PBS, then blocked with 0.5mL 5% goat serum (Vector Laboratories #101098-382) in PBS for 15 minutes. After the blocking agent was aspirated from the cells, 1 pg/ml anti-Myc polyclonal antibody (Sigma-Aldrich #C-3956) in 5% goat serum was added for 1 hour. One well did not receive the primary antibody and was used later for background subtraction. Primary antibody was aspirated away, and then cells washed 5 times with PBS. Next, cells (including background controls) were incubated with 1:1000 goat anti rabbit antibody secondary antibody (Abeam #Ab97051) in 5% Goat serum for 30 minutes at room temperature.
• 1:1000 goat anti rabbit antibody secondary antibody Abeam #Ab97051
• FIG. 30 shows that insulin stimulation elevates the surface level of Myc- tagged GLUT4 that can be detected and that Gal3 co-treatment blocks that increase.
• Antibodies that bind the N-terminal domain (NTD) of Gal3 e.g. TB001, TB006, 2D10-VH0-VL0, 20H5.A3 reduce the ability of Gal3 to block GLUT4 translocation.
• Antibodies that bind to the carbohydrate recognition binding domain (CRD) of Gal3 e.g. 13E2 enhance the ability of Gal3 to block GLUT4 translocation.
• FIG. 31A shows that variants of 20H5 (20H5.A3-VH3VL1, 20H5.A3- VH3VL3, 20H5.A3-VH4VL1, 20H5.A3-VH5VL1, 20H5.A3-VH5VL3, 20H5.A3-VH6VL1, 20H5.A3-VH6VL3) can reduce the ability of Gal3 to block insulin-dependent GLUT4 translocation.
• 31B shows that variants of 2D10-VH0-VL0 (2D10-VH0-VL0, 2D10- hVH4-HVLl, 2D 10-hVH4-HVL2, 2D10-hVH4-HVL3, 2D10-hVH4-HVL4, 2D10-hVH3- HVL1, 2D10-hVH3-HVL2, 2D10-hVH3-HVL3, 2D10-hVH3-HVL4) can reduce the ability of Gal3 to block GLUT4 translocation. It is believed that the variation in blocking between the antibodies is due to variations in the antibodies’ aggregation status, rather than the effectiveness of the specific CDRs for the antibodies.
• the INS-1 832/13 cell line (Sigma-Aldrich #SCC207) is a rat beta cell line that produces insulin. These cells will be grown in a 48-well plate in RPMI-1640 with glucose (Gibco #11875-093) + 10% FBS (Coming #35-016-CV ) until 90-95 % confluence is reached. Cells will then be washed in PBS and serum starved for 2 hours in RPMI without glucose (Gibco #11879-020) and without FBS added.
• Example 12 Anti-Gal3 antibodies for use in the treatment of diabetes
• One or more anti-Gal3 antibodies or binding fragments thereof disclosed herein are administered to the patients enterally, orally, intranasally, parenterally, intracranially, subcutaneously, intramuscularly, intradermally, or intravenously.
• the anti-Gal3 antibodies or binding fragments thereof are administered as doses in at an amount of 1 ng (or in the alternative: 0.1, 10, 100, 1000 ng, or 1, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 pg, or 1, 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mg, or any amount within a range defined by any two of the aforementioned amounts, or any other amount appropriate for optimal efficacy in humans).
• the doses are administered every 1 day (or in the alternative: every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, or 48 days or any time within a range defined by any two of the aforementioned times).
• a treatment of the diabetes or symptoms associated with diabetes is observed in the patients following administration of the anti-Gal3 antibodies or binding fragments thereof.
• Administration of the anti-Gal3 antibodies or binding fragments may be performed in conjunction with another therapy for diabetes, for example, insulin, an insulin derivative or mimetic thereof, insulin aspart, insulin glulisine, insulin lispro, insulin isophane, insulin degludec, insulin detemir, insulin zinc, insulin glargine, vanadium, biguanides, metformin, phenformin, buformin, thiazolidinediones, rosiglitazone, pioglitazone, troglitazone, tolimidone, sulfonylureas, tolbutamide, acetohexamide, tolazamide, chlorpropamide, glipizide, glibenclamide, glimepiride, gliclazide, glyclopyramide, gliquidone, meglitinides, repag
• Example 13 Anti-Gal3 antibodies for use in the treatment of insulin resistance
• the anti-Gal3 antibodies or binding fragments thereof are administered as doses in an amount of 1 ng (or in the alternative: 0.1, 10, 100, 1000 ng, or 1, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 pg, or 1, 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mg, or any amount within a range defined by any two of the aforementioned amounts, or any other amount appropriate for optimal efficacy in humans).
• the doses are administered every 1 day (or in the alternative: every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, or 48 days or any time within a range defined by any two of the aforementioned times).
• a treatment of the insulin resistance or symptoms associated with insulin resistance is observed in the patients following administration of the anti-Gal3 antibodies or binding fragments thereof.
• Administration of the anti-Gal3 antibodies or binding fragments may be performed in conjunction with another therapy for insulin resistance, for example, insulin, an insulin derivative or mimetic thereof, insulin aspart, insulin glulisine, insulin lispro, insulin isophane, insulin degludec, insulin detemir, insulin zinc, insulin glargine, vanadium, biguanides, metformin, phenformin, buformin, thiazolidinediones, rosiglitazone, pioglitazone, troglitazone, tolimidone, sulfonylureas, tolbutamide, acetohexamide, tolazamide, chlorpropamide, glipizide, glibenclamide, glimepiride, gliclazide, glyclopyramide, gliquidone, meglitinides,
• Example 14 Quantification of affinity for exemplary anti-Ga!3 antibodies to Gal3 of different species.
• TB001 showed binding to human Gal3 at an average affinity of 9.7 nM (FIG.
• TB001 showed binding to mouse Gal3 at an average affinity of 4.2 nM (FIG.
• TB001 showed binding to His-EK-cynoGal3 at an affinity of 34 nM (FIG.
• TB001 showed binding to His-EK-ratGal3 at an average affinity of 14 nM
• Example 15 Screening for additional anti-Ga!3 antibodies that induce insulin -in dependent GLUT4 translocation
• Additional anti-Gal3 antibodies were screened for ability to induce insulin- independent GLUT4 translocation in L6 GLUT4myc myoblasts according to the protocol provided in Example 10, but modified such that the cells were not treated with insulin.
• 30 pg/mL (approximately 1 mM) of human Gal3 was incubated with 60 pg/mL (approximately 0.4 pM) of candidate antibodies or MOPC21 control antibody, and cells were treated without insulin.
• the preincubated TB006 and Gal3 complex increased surface GLUT4 levels independent of insulin in tested L6 GLUT4myc cells (FIG. 44A).
• TB006 alone did not result in comparable induction of GLUT4 translocation, suggesting that the antibody- Gal3 complex exhibits an insulin-independent effect relative to antibody alone.
• the 2D10.2B2 (2D 10) antibody and variants thereof disclosed herein were screened for the ability to induce insulin-independent GLUT4 translocation according to the same protocol provided in Example 10, but modified such that the cells were not treated with insulin.
• the 2D 10 variants have the same heavy chain and light chain CDRs and generally vary in framework sequences. Exemplary variants tested (and their corresponding VH and VL) are shown in Table 1.
• Each of the 2D10 variants increased surface GLUT4 levels independent of insulin in tested L6 GLUT4myc cells (FIG. 44B).
• Variants of the 20H5.A3 (20H5) antibody disclosed herein were screened for the ability to induce insulin-independent GLUT4 translocation according to the same protocol provided in Example 10, but modified such that the cells were not treated with insulin.
• the 20H5 variants have the same heavy chain and light chain CDRs and generally vary in the framework sequences. Exemplary variants tested (and their corresponding VH and VL) are shown in Table 2.
• the 20H5 variants exhibited variability in increasing surface GLUT4 levels independent of insulin, with some variants reducing GLUT4 levels, in tested L6 GLUT4myc cells (FIG. 44C).
• Variants of the F847C.21H6 (21H6) antibody disclosed herein were screened for the ability to induce insulin-independent GLUT4 translocation according to the same protocol provided in Example 10, but modified such that the cells were not treated with insulin.
• the 21H6 variants have the same heavy chain and light chain CDRs and generally vary in the framework sequences. Exemplary variants tested (and their corresponding VH and VL) are shown in Table 3.
• Example 16 The Gal3 N-terminal and C-terminal domain are important for inducing GLUT4 translocation
• Gal3 R186S mutant that blocks glycan binding
• amino acids 65-250 of hGal3, hGal3 P64H mutants that makes Gal3 susceptible to cleavage by MMP
• amino acids 2-112 of hGal3 N-terminal domain
• amino acids 111-250 of hGal3 C-terminal carbohydrate recognition domain
• FIG. 45 shows the ability of TB006 complexed with the different Gal3 mutants to induce insulin-independent GLUT4 translocation.
• the 2-112 amino acid N-terminal truncation and the 111-250 amino acid C-terminal truncation both exhibit significantly reduced GLUT4 translocation, suggesting that both domains of the Gal3 protein are important for insulin-independent GLUT4 translocation activity. Similar reduction of ability was observed for the Gal3 R186S mutant, suggesting that glycan binding by Gal3 is also involved in GLUT4 translocation.
• Example 17 Gal3 binds to the glucose transporters GLUT1 and GLUT4 and anti-Ga!3 antibodies block this binding
• Human Gal3 protein was diluted in PBS to a concentration of 4 pg/mL and coated on a 96-well ELISA plate by adding 80 pL per well. Additional wells were coated with a two-fold serial dilution of Gal3 in PBS (2 pg/mL and 1 pg/mL) or no Gal3 coat control. After incubating the plate at 4°C overnight, the plate was washed with 300 pL PBST three times, followed by a blocking step with 150 pL of 2% BSA in PBST per well and incubated for an hour at room temperature (RT) with gentle rocking. The existing blocking solution was then discarded from the plate.
• RT room temperature
• Binding solutions were prepared by diluting GLUT1 and GLUT4 proteins tagged with FLAG in separate buffers of 2% BSA in PBST to a concentration of 4 pg/mL.
• the GLUT1 and GLUT4 dilutions were then applied to the plate by adding 60 pL per well column-wise for each Gal3 coat concentration condition, and then two-fold serially diluted (2 pg/mL and 1 pg/mL) in 2% BSA in PBST.
• the plate was incubated for an hour at RT with gentle rocking, and then washed with 300 pL PBST three times.
• HRP-tagged anti- FLAG antibodies were diluted to 1:4000 in 2% BSA in PBST, and 25 pL was added to all of the wells.
• the plate was incubated for an hour at RT with gentle rocking, and then washed with 300 pL PBST three times.
• 50 pL of ABTS substrate was added to each well and incubated until a sufficiently high signal was achieved.
• the plate was read in a plate reader at an absorbance of 405 nm. Data was graphed using GraphPad Prism 8.0 software (GraphPad Software, Inc.).
• ELISA was also used to quantify IC50s of exemplary anti-Gal3 antibodies TB006 and 2D10-VH0-VL0 for disrupting the interaction between Gal3 and GLUT1 or GLUT4.
• Human Gal3 protein was diluted in PBS to a concentration of 2 pg/mL and used to coat a 96-well ELISA plate with 40 pL per well. After incubating the plate at 4°C overnight, the plate was washed three times with 300 pL PBST. A blocking step was performed with 150 pL of 2% BSA in PBST per well and incubated for an hour at RT with gentle rocking. The existing blocking solution was then discarded form the plate.
• Antibodies TB006, 2D10-VH0- VL0 (2D10), or MOPC21 gG4 isotype control were diluted to 180 pg/mL in 2% BSA in PBST, then applied to the plate by adding 45 pL to each well in the leftmost column and serially diluted 3-fold rightwards across the plate. 30 pL of 3 pg/mL of FLAG-tagged GLUT1 or GLUT4 protein diluted in 2% BSA in PBST was then added to the appropriate wells containing antibody or control solution. The plate was incubated for an hour at RT with gentle rocking and then washed three times with 300 pL PBST.
• HRP-tagged anti-FLAG antibody was diluted to 1:4000 in 2% BSA in PBST, and 25 pL was added to all of the wells.
• the plate was incubated for an hour at RT with gentle rocking, and then washed with 300 pL PBST three times.
• 50 pL of ABTS substrate was added to each well and incubated until a sufficiently high signal was achieved.
• the plate was read with a plate reader at an absorbance of 405 nm. Data was graphed using GraphPad Prism 8.0 software and IC50 values were generated using manufacturer instructions.

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