IGFBP3 ANTIBODIES AND THERAPEUTIC USES THEREOF
20230039165 · 2023-02-09
Inventors
Cpc classification
C07K2317/76
CHEMISTRY; METALLURGY
C07K16/22
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
A61P7/00
HUMAN NECESSITIES
C12N15/66
CHEMISTRY; METALLURGY
International classification
C07K16/22
CHEMISTRY; METALLURGY
A61P7/00
HUMAN NECESSITIES
Abstract
The present invention relates to antibodies or antigen binding fragment thereof that binds specifically to IGFBP3. The antibody inhibits or reduces the binding of IGFBP3 to the TMEM219 receptor. The invention also relates to methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.
Claims
1. An isolated antibody or antigen binding fragment thereof that binds to human IGFBP3 with an affinity constant lower than or equal to 1.1×10.sup.−9 M and which inhibits or reduces the binding of IGFBP3 to TMEM219.
2. The isolated antibody or antigen binding fragment thereof according to claim 1 that inhibits, reduces, or neutralizes the activation of the TMEM219 receptor induced by IGFBP3.
3. The isolated antibody or antigen binding fragment thereof according to claim 1 that is effective in controlling blood glucose levels in an in vivo model.
4. The isolated antibody or antigen binding fragment thereof according to claim 1 that has at least one activity selected from: (a) increase in IGFBP3 treated healthy subject mini-gut growth; (b) increase in IBD-patient mini-gut growth; (c) increase in diabetic enteropathy serum treated healthy subject mini-gut growth; (d) increase in expression of EphB2 and/or LGR5 in IGFBP3 treated healthy subject mini gut; (e) decrease in caspase 8 expression in IGFBP3 treated healthy subject mini-gut; (f) decrease in β-cell loss in IGFBP3 treated β-cell; (g) increase in expression of insulin in IGFBP3 treated β-cell; (h) decrease in apoptosis of β-cell in IGFBP3 treated β-cell; (i) decrease in caspase 8 expression in IGFBP3 treated β-cell; (j) decrease in insulitis score in an animal model of diabetes; and (k) decrease in diabetes onset in an animal model of diabetes.
5. The isolated antibody or antigen binding fragment thereof according to claim 4 wherein the increase in (a), (b), and (c) and is by at least 20%; the increase in (d) and (e) is by at least 50%; the decrease in (f) and the increase in (g) is by at least 10%.
6. The isolated antibody or antigen binding fragment thereof according to claim 1 comprising: (a) a heavy chain variable domain (VH) comprising: (i) a CDR1 sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO: 1, 4, 7 or 9; (ii) a CDR2 sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO: 2, 5, 8 or 10; and (iii) a CDR3 sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO: 3, 6 or 11; and/or (b) a light chain variable domain (VL) comprising: (i) a CDR1 sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO: 12, 15, 17, 20, 23, 25 or 27; (ii) a CDR2 sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO: 13, 18 or 21; and (iii) a CDR3 sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO: 14, 16, 19, 22, 24 or 26.
7. The isolated antibody or antigen binding fragment thereof according to claim 6 comprising the CDRs as indicated in Table 2 and/or in Table 3, including Table 3.1.
8. The isolated antibody or antigen binding fragment thereof according to claim 1 comprising: (a) SEQ ID NO: 9 and SEQ ID NO: 10 and SEQ ID NO: 11 and SEQ ID NO: 27 and SEQ ID NO: 18 and SEQ ID NO: 26 or Kabat, IMGT, Chothia, AbM, or Contact CDRs of M1; or (b) SEQ ID NO: 4 and SEQ ID NO: 5 and SEQ ID NO: 6 and SEQ ID NO: 12 and SEQ ID NO: 13 and SEQ ID NO: 14 or Kabat, IMGT, Chothia, AbM, or Contact CDRs of E08; or (c) SEQ ID NO: 4 and SEQ ID NO: 5 and SEQ ID NO: 6 and SEQ ID NO: 23 and SEQ ID NO: 18 and SEQ ID NO: 24 or Kabat, IMGT, Chothia, AbM, or Contact CDRs of E20.
9. The isolated antibody or antigen binding fragment thereof according to claim 1 comprising: (a) a heavy chain variable domain (VH) comprising: (i) a CDR1 sequence of the amino acid sequence selected from the group consisting of a sequence as defined using abysis tool analysis (abysis.org); (ii) a CDR2 sequence of the amino acid sequence selected from the group consisting of a sequence as defined using abysis tool analysis (abysis.org); and (iii) a CDR3 sequence of as defined using abysis tool analysis (abysis.org); and/or (b) a light chain variable domain (VL) comprising: (ii) CDR1 sequence of the amino acid sequence selected from the group consisting of a sequence as defined using abysis tool analysis (abysis.org); (ii) a CDR2 sequence of the amino acid sequence selected from the group consisting of a sequence as defined using abysis tool analysis (abysis.org); and (iii) a CDR3 sequence of the amino acid sequence selected from the group consisting of a sequence as defined using abysis tool analysis (abysis.org).
10. The isolated antibody or antigen binding fragment thereof according to claim 1 comprising: (a) a heavy chain variable domain sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO:28 to SEQ ID NO:36; or (b) a light chain variable domain sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO: 37 to SEQ ID NO:45; or (c) the light chain variable domain of (a) and the heavy chain variable domain of (b).
11. The isolated antibody or antigen binding fragment thereof according to claim 1, wherein the antibody or antigen binding fragment is selected from the group consisting of antibody E01, E02, E08, E14, E19, E20, E23, E24 M1, or an antigen binding fragment thereof.
12. An isolated antibody or antigen binding fragment thereof that: (a) binds specifically to an epitope on IGFBP3, that is the same or similar epitope as the epitope recognized by the monoclonal antibody E01, E02, E08, E14, E19, E20, E23, E24, or M1 comprising the sequences as defined in Tables 2-7; or (b) cross-competes for binding with the monoclonal antibody E01, E02, E08, E14, E19, E20, E23, E24, or M1 comprising the sequences as defined in Tables 2-7; or (c) shows the same or similar binding affinity or specificity, or both, as any of antibody E01, E02, E08, E14, E19, E20, E23, E24, or M1 comprising the sequences as defined in Tables 2-7; or (d) has one or more biological properties of an antibody chosen from any of E01, E02, E08, E14, E19, E20, E23, E24, or M1 comprising the sequences as defined in Tables 2-7; or (e) has one or more pharmacokinetic properties of an antibody molecule described herein, wherein the antibody is any of E01, E02, E08, E14, E19, E20, E23, E24 or M1 comprising the sequences as defined in Tables 2-7.
13. The isolated antibody or antigen binding fragment thereof according to claim 1, wherein the antibody or antigen binding fragment thereof is a human or a humanized antibody.
14. The isolated antibody or antigen binding fragment thereof according to claim 1, wherein the antibody or antigen binding fragment thereof is an IgG2 or IgG4 antibody.
15. An isolated polynucleotide comprising at least one sequence that encodes the antibody or antigen binding fragment thereof according to claim 1.
16. A vector comprising the polynucleotide according to claim 15, optionally wherein the vector is selected from the group consisting of a plasmid, a viral vector, a non-episomal mammalian vector, an expression vector, and a recombinant expression vector.
17. An isolated cell comprising the polynucleotide according to claim 15, optionally wherein the isolated cell is a hybridoma or a Chinese Hamster Ovary (CHO) cell or a Human Embryonic Kidney (HEK293) cell.
18. A method of treating a disorder, comprising: administering a therapeutically effective amount of the antibody or antigen binding fragment thereof according to claim 1 optionally wherein the disorder is selected from: diabetes, intestinal and/or bowel disorder, malabsorption syndrome, cachexia or diabetic enteropathy.
19. A pharmaceutical composition comprising the isolated antibody or antigen binding fragment thereof according to claim 1, and a pharmaceutically acceptable carrier, optionally wherein for use in the treatment of: diabetes, intestinal and/or bowel disorder, malabsorption syndrome, cachexia or diabetic enteropathy, optionally wherein the intestinal and/or bowel disorder is inflammatory bowel disease, celiac disease, ulcerative colitis, Crohn's disease or intestinal obstruction.
20. A method of inhibiting the binding of IGFBP3 to TMEM219 receptor, comprising contacting IGFBP3 with the antibody or the composition according to claim 1.
21. The isolated antibody or antigen binding fragment thereof according to claim 14, wherein the antibody or antigen binding fragment thereof is a human IgG2 or human IgG4.
22. The isolated polynucleotide according to claim 15, wherein said polynucleotide is a cDNA.
23. An isolated cell comprising the vector according to claim 16, wherein the isolated cell is a hybridoma or a Chinese Hamster Ovary (CHO) cell or a Human Embryonic Kidney cells (HEK293).
24. The isolated antibody or antigen binding fragment thereof according to claim 14, wherein the antibody or antigen binding fragment thereof is selected from the group consisting of an IgG2 kappa antibody, an IgG2 lambda antibody, an IgG4 kappa antibody and an IgG4 lambda antibody.
25. The vector according to claim 16, wherein the vector is a plasmid.
26. The vector according to claim 16, wherein the vector is a viral vector.
27. The vector according to claim 16, wherein the vector is a non-episomal mammalian vector.
28. The vector according to claim 16, wherein the vector is an expression vector.
29. The vector according to claim 16, wherein the vector is a recombinant expression vector.
30. The isolated cell according to claim 17, wherein the isolated cell is hybridoma.
31. The isolated cell according to claim 17, wherein the isolated cell is Chinese Hamster Ovary (CHO) cell.
32. The isolated cell according to claim 17, wherein the isolated cell is Human Embryonic Kidney (HEK293) cell.
33. A method of treating a disorder, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising the isolated antibody or antigen binding fragment thereof according to claim 1, and a pharmaceutically acceptable carrier, optionally wherein for the use in the treatment of: diabetes, intestinal and/or bowel disorder, malabsorption syndrome, cachexia or diabetic enteropathy, optionally wherein the intestinal and/or bowel disorder is inflammatory bowel disease, celiac disease, ulcerative colitis, Crohn's disease or intestinal obstruction.
34. The method of treating a disorder of claim 18, the diabetes disorder, wherein it is Type I or Type II.
35. The method of treating a disorder of claim 18, the intestinal and/or bowel disorder, wherein it is inflammatory bowel disease, celiac disease, ulcerative colitis, Crohn's disease or intestinal obstruction.
36. The isolated antibody or antigen binding fragment thereof according to claim 1 wherein the heavy chain constant region is a human IgG4 including a Ser to Pro substitution at position 228 and a Leu to Glu substitution at position 235.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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[0194] Blood glucose >250 mg/dl for three consecutive measurements defined diabetes onset. Diabetes-free mice do not have Blood glucose >250 mg/dl for three consecutive measurements.
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DETAILED DESCRIPTION OF THE INVENTION
[0196] The antibodies of the invention specifically bind human IGFBP3. As discussed herein, the antibodies of the invention are collectively referred to as “anti-IGFBP3 antibodies”. All such antibodies are encompassed by the discussion herein. The respective antibodies can be used alone or in combination in the methods of the invention.
[0197] By “antibodies that specifically bind” IGFBP3 is intended that the antibodies will not substantially cross react with another, non-homologous, human polypeptide. By “not substantially cross react” is intended that the antibody or fragment has a binding affinity for a non-homologous protein which is less than 10%, more preferably less than 5%, and even more preferably less than 1%, of the binding affinity for IGFBP3.
[0198] In various embodiments, an antibody that “specifically binds” IGFBP3, as used herein, includes antibodies that bind human IGFBP3 with a KD of less than about 1000 nM, less than about 500 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM or about 0.5 nM, as measured with an Octet biolayer interferometry device or in a surface plasmon resonance assay, for example using the BIAcore™ system (Biacore Life Sciences division of GE Healthcare, Piscataway, N.J.) or kinetic exclusion assays or any known method in the art.
[0199] The term “antibody” herein is used in the broadest sense understood in the art, including all polypeptides described as antibodies in (25), incorporated herein by reference.
[0200] For example, the term “antibody”, as used herein encompasses monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as the fragment exhibits the desired antigen-binding activity (antigen-binding fragments). The term has its broadest art-recognized meaning and includes all known formats, including, without limitation: bivalent monospecific monoclonal antibodies, bivalent bispecific antibodies, trivalent trispecific antibodies, F(ab) fragments, F(ab)′2 fragments, scFv fragments, diabodies, single domain antibodies, including camelid VHH single domain antibodies, tandabs, and flexibodies.
[0201] The terms “antigen-binding fragment” of an antibody or equivalently “antigen-binding portion” of an antibody and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that comprises a portion of an antibody and that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.
[0202] As with full antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen.
[0203] In particular embodiments, an antigen-binding fragment of an antibody comprises at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may in various embodiments consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody may in various embodiments comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).
[0204] The term “antigen-binding fragment” of an antibody further includes single domain antibodies.
[0205] A single-domain antibody is an antibody fragment consisting of a single monomeric variable antibody domain. In some embodiments, the single-domain antibody is derived from the variable domain of the antibody heavy chain from camelids (also termed nanobodies, or VHH fragments). In some embodiments, the single-domain antibody is an autonomous human heavy chain variable domain (aVH) or VNAR fragments derived from sharks.
[0206] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, and bivalent nanobodies), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigen-binding fragment,” as used herein.
[0207] An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.
[0208] The antibody or binding molecule of the invention can further be linked to an active substance, preferably a nanoparticle or a radionucleotide.
[0209] As used herein, the term “antigen binding molecule” refers in its broadest sense to a molecule that specifically binds an antigenic determinant. Examples of antigen binding molecules are antibodies, including antigen-binding antibody fragments, and scaffold antigen binding proteins.
[0210] The term “antigen binding moiety” refers to the portion of an antigen binding molecule that specifically binds to an antigenic determinant. Antigen binding moieties include antibodies and antigen-binding fragments thereof, such as scFv, that are capable of specific binding to an antigen on a target cell. In a particular aspect, the antigen binding moiety is able to direct the entity to which it is attached, such as a cell, to a target site. In addition, antigen binding moieties capable of specific binding to a target cell antigen include scaffold antigen binding proteins as defined herein below, e.g. binding domains which are based on designed repeat proteins or designed repeat domains such as designed ankyrin repeat proteins (DARPins) (see e.g. WO 2002/020565) or Lipocalins (Anticalin).
[0211] Designed Ankyrin Repeat Proteins (DARPins), which are derived from Ankyrin, which is a family of proteins that mediate attachment of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two alpha-helices and a beta-turn. They can be engineered to bind different target antigens by randomizing residues in the first alpha-helix and a beta-turn of each repeat. Their binding interface can be increased by increasing the number of modules (a method of affinity maturation). For further details see J. Mol. Biol. 332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003) and J. Mol. Biol. 369, 1015-1028 (2007) and US20040132028.
[0212] In certain embodiments, antibodies and antigen binding molecules provided herein are altered to increase or decrease the extent to which the antigen binding moiety is glycosylated. Glycosylation variants of the molecules may be conveniently obtained by altering the amino acid sequence such that one or more glycosylation sites is created or removed. Where the antigen binding molecule comprises an Fc region, the carbohydrate attached thereto may be altered. In one aspect, variants of antigen binding molecules are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. Such fucosylation variants may have improved ADCC function, see e.g. US Patent Publication Nos. US 2003/0157108 (Presta, L.) or US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Further variants of antigen binding molecules of the invention include those with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region is bisected by GIcNAc. Such variants may have reduced fucosylation and/or improved ADCC function, see for example WO 2003/011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.). Variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function and are described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.).
[0213] In certain embodiments, it may be desirable to create cysteine engineered variants of the antibody or antigen binding molecule of the invention, e.g., “thioMAbs,” in which one or more residues of the molecule are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the molecule. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine engineered antigen binding molecules may be generated as described, e.g., in U.S. Pat. No. 7,521,541.
[0214] In certain aspects, the antibody or antigen binding molecules provided herein may be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody or antigen binding molecule include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
[0215] Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.
[0216] In another aspect, conjugates of an antibody and non-proteinaceous moiety that may be selectively heated by exposure to radiation are provided. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam, N. W. et al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605). The radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the non-proteinaceous moiety to a temperature at which cells proximal to the antibody-non-proteinaceous moiety are killed. In another aspect, immunoconjugates of the antigen binding molecules provided herein may be obtained. An “immunoconjugate” is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.
[0217] The constant region of an antibody is important in the ability of an antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the isotype of an antibody may be selected on the basis of whether it is desirable for the antibody to mediate cytotoxicity. In certain embodiments, the constant region is an IgG1, IgG2, IgG3, IgG4 constant region.
[0218] The invention encompasses in various embodiments antibodies having one or more mutations in the hinge, CH2 or CH3 region which may be desirable, for example, in production, to improve the yield of the desired antibody form. In some embodiments, for example, the antibodies described herein comprise a human IgG4 constant region. In particular embodiments, the IgG4 constant region has a single amino acid substitution in the hinge region of the human IgG4 hinge which reduced Fab arm exchange (Angal et al. (1993) Molecular Immunology 30:105) to levels typically observed using a human IgG1 hinge.
[0219] In certain embodiments, the antibody comprises one or more mutations in the constant region that increase serum half-life, including those described in U.S. Pat. Nos. 7,083,784, 8,323,962 and Dall'Aqua et al., J. Biol. Chem. 281(33):23514-23524 (2006); Hinton et al., J. Immunology 176:346-356 (2006); Yeung et al., J. Immunology 182:7663-7671 (2009); and Petkova et al., Intn'l Immunology, 18: 1759-1769 (2006), incorporated herein by reference in their entireties.
[0220] The term “human antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies featured in the invention may in various embodiments nonetheless include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in in some embodiments CDR3. However, the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences are derived from the germline of another mammalian species, such as a mouse, which have been grafted onto human framework sequences.
[0221] The term “recombinant human antibody”, as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295, incorporated herein by reference in its entirety) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
[0222] An “isolated antibody,” as used herein, means an antibody that has been identified and separated and/or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally exists or is naturally produced, is an “isolated antibody.” In various embodiments, the isolated antibody also includes an antibody in situ within a recombinant cell. In other embodiments, isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. In various embodiments, an isolated antibody may be substantially free of other cellular material and/or chemicals.
[0223] The term “epitope” refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. In certain circumstance, an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.
[0224] The anti-IGFBP3 antibodies described herein and useful for the methods featured herein may in various embodiments include one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases.
[0225] The present invention includes in various embodiments antibodies and methods involving the use of antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as “germline mutations”).
[0226] Numerous antibodies and antigen-binding fragments may be constructed which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and/or CDR residues within the VH and/or VL domains are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived).
[0227] Furthermore, the antibodies may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a certain germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antibodies and antigen-binding fragments that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. The use of antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present invention.
[0228] The present invention also includes anti-IGFBP3 antibodies and methods involving the use of anti-IGFBP3 antibodies comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention includes the use of anti-IL-6R antibodies having HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein. The term “bioequivalent” as used herein, refers to a molecule having similar bioavailability (rate and extent of availability) after administration at the same molar dose and under similar conditions (e.g., same route of administration), such that the effect, with respect to both efficacy and safety, can be expected to be essentially same as the comparator molecule. Two pharmaceutical compositions comprising an anti-IGFBP3 antibody are bioequivalent if they are pharmaceutically equivalent, meaning they contain the same amount of active ingredient (e.g., IGFBP3 antibody), in the same dosage form, for the same route of administration and meeting the same or comparable standards. Bioequivalence can be determined, for example, by an in vivo study comparing a pharmacokinetic parameter for the two compositions. Parameters commonly used in bioequivalence studies include peak plasma concentration (Cmax) and area under the plasma drug concentration time curve (AUC).
[0229] The invention in certain embodiments relates to antibodies and methods comprising administering to the subject an antibody which comprises the heavy chain variable region comprising a sequence chosen from the group of: SEQ ID NO:28 to SEQ ID NO:36 and the light chain variable region comprising a sequence chosen from the group of: SEQ ID NO:37 to SEQ ID NO:45. The disclosure provides pharmaceutical compositions comprising such antibody, and methods of using these compositions.
[0230] The antibody is administered to the subject in various embodiments in a formulation comprising suitable carriers, excipients, and other agents to provide improved transfer, delivery, tolerance, and the like, and suitable for an intravenous or subcutaneous injection.
[0231] The injectable preparations may be prepared by methods publicly known. For example, injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 20 or 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injectable preparation thus prepared can be filled in an appropriate ampoule.
[0232] The antibody according to the invention can be administered to the subject using any acceptable device or mechanism. For example, the administration can be accomplished using a syringe and needle or with a reusable pen and/or autoinjector delivery device. The methods of the present invention include the use of numerous reusable pen and/or autoinjector delivery devices to administer an antibody (or pharmaceutical formulation comprising the antibody). Examples of such devices include, but are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen and/or autoinjector delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but are not limited to, the SOLOSTAR™ pen (Sanofi-Aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), the PENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L. P.), the HUMIRA™ Pen (Abbott Labs, Abbott Park, Ill.), the DAI® Auto Injector (SHL Group) and any auto-injector featuring the PUSHCLICK™ technology (SHL Group), to name only a few.
[0233] In one embodiment, the antibody is administered with a prefilled syringe. In another embodiment, the antibody is administered with a prefilled syringe containing a safety system. For example, the safety system prevents an accidental needlestick injury. In various embodiments, the antibody is administered with a prefilled syringe containing an ÈRIS™ safety system (West Pharmaceutical Services Inc.). See also U.S. Pat. Nos. 5,215,534 and 9,248,242, incorporated herein by reference in their entireties. In another embodiment, the antibody is administered with an auto-injector. In various embodiments, the antibody is administered with an auto-injector featuring the PUSHCLICK™ technology (SHL Group). In various embodiments, the auto-injector is a device comprising a syringe that allows for administration of a dose of the composition and/or antibody to a subject. See also U.S. Pat. Nos. 9,427,531 and 9,566,395, incorporated herein by reference in their entireties.
[0234] According to the invention, “subject” means a human subject or human patient.
EXAMPLES
[0235] Methods
[0236] Patients and Study Design
[0237] Healthy control subjects were individuals lacking a diagnosis of inflammatory bowel disease (IBD) (CTRL) and were enrolled from patients undergoing colonoscopy or intestinal surgery for diverticulosis, colon cancer, irritable bowel syndrome.
[0238] CD individuals had a long history of Crohn's disease and were enrolled at the moment of surgery procedure for disease complications (strictures, fistulas) or during an endoscopy routine examination before undergoing surgery. All subjects provided informed consent before study enrollment.
[0239] Animal Studies
[0240] C57BL/6J (B6) mice were obtained from Charles River Italian Laboratories (Calco, Italy) and were cared for and used in accordance with the Italian law on animal care N° 116/1992 and the European Communities Council Directive EEC/609/86.
[0241] Recombinant Proteins and Interventional Studies
[0242] Recombinant human IGFBP3 was obtained from Life Technologies (IGFBP3, Life Technologies, 10430H07H5). Ecto-TMEM219, which is the extracellular domain of the TMEM219 receptor was used as a positive control. Ecto-TMEM219 has been shown to successfully prevent IGFBP3-mediated injury in vitro and vivo, in relevant disease models. See WO 2016/193496 and WO 2016/193497. Ecto-TMEM was obtained through Genescript's customized protein service. The protein, produced in E. coli, has the following amino acid sequence:
TABLE-US-00001 Human Ecto-TMEM amino acid sequence: (SEQ ID No. 69) THRTGLRSPDIPQDWVSFLRSFGQLTLCPRNGTVTGKWRGSHVVGLLTTL NFGDGPDRNKTRTFQATVLGSQMGLKGSSAGQLVLITARVTTERTAGTCL YFSAVPGILPSSQPPISCSEEGAGNATLSPRMGEECVSVWSHEGLVLTKL LTSEELALCGSR Murine Ecto-TMEM amino acid sequence: (SEQ ID No. 70) THTTGLRSPDIPQDWVSFLRSFGQLSLCPMNETVTGTWQGPHVVGLLTTL NFGDGPDRNKTQTFQAKIHGSQIGLTGSSAGESVLVTARVASGRTPGTCL YFSGVPKVLPSSQPPISCSEEGVGNATLSPVMGEECVRVWSHERLVLTEL LTSEELALCGS
[0243] IGFBP3 at 50 ng/ml and ecto-TMEM219 at 130 ng/ml were added to culture medium at day+1 from mini-guts culture (see below).
[0244] Newly generated anti-IGFBP3 monoclonal antibodies were added at 1:1 molecular ratio as compared to IGFBP3 at 10 ug/ml final concentration.
[0245] Crypts Isolation and Mini-Guts Development
[0246] Humans
[0247] Crypts were extracted from mucosa and sub-mucosa of intestinal samples of healthy subjects (healthy controls) or obtained from patients with established Crohn's disease undergoing surgery for disease complications (strictures, fistulae). Mucosa was incubated with a mixture of antibiotics Normocin, [Invivogen, San Diego, Calif. 92121, USA; catalog code ant-nr], Gentamycin [Invitrogen, Carlsbad, Calif., USA catalog code ant-gn] and Fungizone [Invitrogen 15290018]) for 15 minutes at room temperature, and then tissue was minced into small pieces and incubated with 10 mM Dithiothreitol (DTT) (Sigma) in PBS 2-3 times for several minutes. Samples were then transferred to 8 mM EDTA in PBS and incubated for 30 minutes at 37° C. After this step, vigorous shaking of the sample yielded supernatants enriched in colonic crypts. Fetal bovine serum (FBS, Sigma 12103C-500ML) was added to a final concentration of 5%, and single cells were removed by centrifugation 40×g for 2 minutes. Crypts were mixed with 50 μl of Matrigel (BD Biosciences 354234) and plated on pre-warmed culture dishes. After solidification, crypts were overlaid with complete crypt culture medium: Wnt3a-conditioned medium and Advanced DMEM/F12 (Life Technologies 1263010) 50:50, supplemented with Glutamax, 10 mM (Life Technologies 35050038) HEPES (Life Technologies 15630080), N-2 [1×] (Life Technologies 17502048), B−27 without retinoic acid [1×](Life Technologies 12587010), 10 mM Nicotinamide (Sigma N0636), 1 mM N-Acetyl-L-cysteine (Sigma A965), 50 ng/ml human EGF (Life Technologies PHG0311), 1 μg/ml RSPO1 (Sino Biological 11083-H08H), 100 ng/ml human Noggin (Peprotech 12010C), 1 μg/ml Gastrin (Sigma-Aldrich SCP0152), 500 nM LY2157299 (Axon MedChem 1491), 10 μM SB202190 (Sigma S7067) and 0.01 μM PGE2 (Sigma P6532). Medium was replaced every 3 days. Purified crypts have been cultured for 8 days with/without recombinant proteins/Antibodies as described in the Recombinant proteins and interventional studies section. After 8 days, crypts were collected, and the morphology, mini-gut growth, expression of intestinal signature markers (EphB2, LGR5, h-TERT), and Caspase 8 (Life Technologies) were examined using RT-PCR.
[0248] Percentage of developed mini-guts with at least one crypt domain was assessed as already described (4,18).
[0249] Murine
[0250] Crypts were obtained from C57BL/6J mice. Briefly the colon was cut into 2-4 mm pieces with scissors and fragments were washed in 30 ml of ice-cold PBS and then incubated with 20 mM EDTA-PBS at 37° C. Finally, fragments were treated trypsin/DNAse solution to obtain crypts. After this step, vigorous shaking of the sample yielded supernatants enriched in colonic crypts. Crypts were mixed with Matrigel and plated on pre-warmed culture dishes. After solidification of matrigel (10-15 min at 37° C.), crypts were overlaid with culture medium (ADF, 10 mM HEPES, N-2, B27 without retinoic acid, 10 μM Y−27632, 1 μM JAG1 peptide (Anaspec, Fremont, Calif., USA), 1 μg/ml R-Spondin 1, 50 ng/ml EGF (Invitrogen), and 100 ng/ml Noggin (Peprotech, Rocky Hill, N.J., USA), and medium was changed every other day until day 8. After 8 days, percentage of developed mini-guts was assessed.
[0251] qRT-PCR Analysis
[0252] RNA from purified intestinal crypts was extracted using Trizol Reagent (Invitrogen), and qRT-PCR analysis was performed using TaqMan assays (Life Technologies, Grand Island, N.Y.) according to the manufacturer's instructions. The normalized expression values were determined using the ΔΔCt or the ΔCt method. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) data were normalized for the expression of ACTB. Statistical analysis compared gene expression across all cell populations for each patient via one-way ANOVA followed by Bonferroni post-test for multiple comparisons between the population of interest and all other populations. Analysis was performed in technical and biological triplicates.
[0253] The list of genes which expression has been quantified by qRT-PCR is reported below.
TABLE-US-00002 Gene Band Size Reference Symbol UniGene # Refseq Accession # (bp) Position LGR5 Hs.658889 NM_003667 91 1665 EPHB2 Hs.523329 NM_004442 68 2908 TERT Hs.492203 NM_198253 106 1072 ACTB Hs.520640 NM_001101 174 730 Caspase 8 Hs.599762 NM_001080124.1 124 648
[0254] Competitive ELISA Binding Assay
[0255] The following reagents were used to screen the newly generated anti-IGFBP3 antibodies: Recombinant Human IGFBP3 (0,223 mg/ml R&D System 8874-B3-025), Ecto-TMEM219 (0.5 mg/ml GenScript), newly generated anti-IGFBP3 mAbs (Trianni), anti-human IgG HRP (Life Technologies A24470), bovine serum albumin (BSA), Tween 20 (TW), ELISA colorimetricTMB reagent (HRP substrate, Item H Sigma, RABTMB3), ELISA STOP solution (Item I, Sigma, RABSTOP3). We also employed a blocking reagent solution (3% BSA in PBS) and a diluent solution (0.5% BSA, 0.05% Tw in PBS).
[0256] Microplate (Thermofisher, Electron Corporation, 2801) was coated with 50 μl/well of 4 μg/ml rhIGFBP3 dissolved in PBS or PBS alone (no coating). Plate was incubated 90 minutes at 37° C. and washed with PBS (300 μl/well) and incubated with the blocking reagent (200 μl/well) 2 hours at room temperature. Samples were then diluted in the diluent solution (50 μl/well) and added to the plate as following: diluent solution (none), ecto-TMEM219 10 μg/ml, ecto-TMEM219 10 μg/ml+ anti-IGFBP3 mAbs 10 μg/ml, anti-IGFBP3 mAbs 10 μg/ml alone. After washing steps, plate was then incubated at room temperature for 1 hour with anti 6× His tag HRP diluted 1:2000 in Diluent solution (50 μl/well). ELISA plate was then read after adding visualization solution at ELISAreader and adsorbance was measured.
[0257] Beta-Cells
[0258] Betalox-5 cells, a human beta cell line (36) were grown in culture flasks containing DMEM (glucose 1 g/L), BSA fraction V (0.02% wt/vol), Non-essential amino acids (1×) penicillin (100 units/mL), and streptomycin (100 μg/mL). The cells were cultured at 37° C. in a humidified incubator in 5% CO2. The cells were passaged once every second week. Beta cells were cultured with or without IGFBP3, with or without ecto-TMEM219, with or without newly generated monoclonal antibodies (see Recombinant proteins and interventional studies) and cells were collected for immunofluorescence studies, RNA extraction, apoptosis detection, and protein analysis. Supernatants were collected for assessment of insulin. Insulin levels were assayed with a microparticle enzyme immunoassay (Mercodia Iso-Insulin ELISA, 10-1113-01).
[0259] Statistical Analysis
[0260] Data are presented as mean and standard error of the mean (SEM) and were tested for normal distribution with the Kolmogorov-Smirnov test and for homogeneity of variances with Levene's test. The statistical significance of differences was tested with two-tailed t-test. Significance between the two groups was determined by two-tailed unpaired Student's t test. For multiple comparisons, the ANOVA test with Bonferroni correction was employed. Graphs and data were generated using GraphPad Prism version 6.0 (GraphPad Software, La Jolla, Calif.). All statistical tests were performed at the 5% significance level.
[0261] Anti-IGFBP3 mAbs Efficacy in T1D Mouse Model Following Intraperitoneal (IP) Administration
[0262] Animals
[0263] Female non-obese diabetic (NOD) mice (10 weeks old) were obtained from the Charles River Laboratories, Calco, Varese, Italy (stock #613). All mice were cared for and used in accordance with Italian law on animal care N° 116/1992 and the European Communities Council Directive EEC/609/86.
[0264] Diabetes Monitoring and Treatment
[0265] Overt diabetes (the most advanced stage, characterized by elevated fasting blood glucose concentration and classical symptoms) was defined as blood glucose levels above 250 mg/dL for three consecutive measurements. Glycemia was monitored twice a week.
[0266] Inventors set up the following treatment groups:
[0267] 1) Untreated
[0268] 2) Ecto-TMEM219 0.1 mg/day (i.p) for 10 days
[0269] 3) Anti-IGFBP3 M1 0.5 mg/day (i.p) for 10 days
[0270] Ecto-TMEM and antibody were dissolved in PBS.
[0271] N=10 mice were included in each group of treatment. Treatment started when mice were weeks old at day 0. Mice were followed up for up to 23 weeks of age. Mice were harvested when diabetes was assessed or at week 23. Plasma samples and pancreas were collected for ex vivo analysis. The experimental timelines are described in
[0272] Insulitis Scoring and Pancreatic Islet Histopathology
[0273] Insulitis scoring was performed on 5-μm-thick formalin-fixed, paraffin-embedded, hematoxylin and eosin (H&E)-stained pancreatic sections as previously described (Vergani A et al. Diabetes 2010; Ben Nasr M et al. Sci Transl Med 2017). Insulitis scoring was performed on hematoxylin and eosin (H&E) and Insulin stained pancreatic sections. A score of 0 to 4 was assigned based on islet infiltration by an experienced pathologist. Insulitis scores were graded as follows: grade 0, normal islets; grade 1, mild mononuclear infiltration (25%) at the periphery; grade 2, 25-50% of the islets infiltrated; grade 3, (50% of the islets infiltrated); grade 4, islets completely infiltrated with no residual parenchyma remaining. At least 30 islets per group were analyzed and pooled from sections obtained from different mice.
[0274] Statistical Analysis
[0275] Data are presented as mean and standard error of the mean (SEM) unless otherwise reported. Diabetes incidence among different groups was analyzed with the log-rank (Mantel-Cox) test. Statistical analysis was conducted using GraphPad Prism version 7.0 (GraphPad Software, La Jolla, Calif.). All statistical tests were performed at the 5% significance level.
Example 1: Monoclonal Antibodies Development
[0276] Monoclonal anti-IGFBP3 antibodies were discovered through the utilization of transgenic mouse, where the relevant human immunoglobulin sequences have been introduced into the genome of the animal by genetic engineering, the Trianni Mouse™ (Trianni). Through use of such technology, chimeric monoclonal antibodies containing the full repertoire of human heavy- and light-chain variable domains and the retention of the mouse constant domains were produced.
[0277] Essentially, two cohorts of Trianni Mouse™ (Cohort 1: ALD/MDP adjuvant and Cohort 2: SAS/Ribi adjuvant) were immunized with a purified preparation of IGFPP3 antigen (lot #AB08BP1210), two injections a week for 4 weeks then 2 weeks extension. Then, lymphatic cells (such as B-cells) were recovered from the mice that express antibodies, such cells were fused with a myeloid-type cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines were screened and selected to identify hybridoma cell lines that produce antibodies specific to human IGFBP3 (lot #AB08BP1210) by ELISA. Hybridoma cell lines that were reactive for the antigen of interest were expanded. Sequencing was accomplished by RNA isolation, followed by cDNA sequencing of the human VH and human VK using Sanger sequencing methods.
[0278] Antibodies can be expressed in cell lines other than hybridoma cell lines. Sequences encoding antibodies can be used for transformation of a suitable mammalian host cell. In fact, the monoclonal antibody deriving from cohort 1, M1 was expressed in a transient gene expression system in mammalian cell.
[0279] Method of Expressing Recombinant Protein in CHO Cells
[0280] The corresponding M1 cDNAs was cloned into evitria's vector system using conventional (non-PCR based) cloning techniques to produce a fully human IgG4 mAb. The evitria vector plasmids were gene synthesized. Plasmid DNA was prepared under low-endotoxin conditions based on anion exchange chromatography. Correctness of the sequences was verified with Sanger sequencing (with up to two sequencing reactions per plasmid depending on the size of the cDNA.)
[0281] Suspension-adapted CHO K1 cells (evitria) was used for production. The seed was grown in eviGrow medium, a chemically defined, animal-component free, serum-free medium. Cells were transfected with eviFect, evitria's custom-made, proprietary transfection reagent, and cells were grown after transfection in eviMake, an animal-component free, serum-free medium, at 37° C. and 5% CO2 for 7 days. Supernatant was harvested by centrifugation and subsequent filtration (0.2 μm filter).
[0282] The antibody was purified using MabSelect™ SuRe™ with Dulbecco's PBS (Lonza BE17-512Q) as wash buffer and 0.1 M Glycine pH 3.5 as elution buffer. Subsequent size exclusion chromatography was performed on a HiLoad Superdex 200 pg column using the final buffer as running buffer.
[0283] Monomericity was determined by analytical size exclusion chromatography with an Agilent AdvanceBio SEC column (300A 2.7 um 7.8×300 mm) and DPBS as running buffer at 0.8 ml/min. Remarkably, the monomericity of M1 was >95%, exhibiting only <5% of aggregated. The high monomericity of the protein is an exceptional property that should aid in its manufacture.
[0284] Affinity Measurement
[0285] Octet BLI-Based Analysis
[0286] Antibodies possessed high affinity to the target. The binding affinity measurements were performed using an Octet instrument (Octet BMIA), which is a Biolayer Interferometry (BLI) platform based on Biomolecular Interaction Analysis. To establish the assay, the target monoclonal antibody (30 μg/ml in PBS) was immobilized via Fc on the via Anti-Mouse IgG Fc Capture (AMC) or Anti-Human IgG Fc Capture (AMC) biosensors and the interaction with the antigen, human IGFBP3 (R&D, cat n° 675 B3) at 150 nM was measured.
[0287] The affinity measurement of the anti-IGFBP3 mAbs for the target human IGFBP3 are reported in Table 1.
TABLE-US-00003 TABLE 1 Affinity measurement of exemplified antibodies Antibody KD (M) E01 1.2E−09 E02 >1.0E−12 E08 6.1E−10 E14 8.5E−10 E19 6.9E−10 E20 8.5E−10 E23 1.1E−09 E24 >1.0E−12 M1 >1.0E−12
[0288] The sequences of the 9 novel anti-IGFBP3 antibodies are reported in Tables 2-5 below.
TABLE-US-00004 TABLE 2 VH CDR Sequences of exemplified antibodies Antibody CDR1 CDR2 CDR3 E01 GFTFSSYG ISYDGSNK ARGGEYFYYYGLDV (SEQ ID No. 1) (SEQ ID No. 2) (SEQ ID No. 3) E02 GYTFSNYG INTYNGNT ARDRGYSSSPYYYYYGMDV (SEQ ID No. 4) (SEQ ID No. 5) (SEQ ID No. 6) E08 GYTFSNYG INTYNGNT ARDRGYSSSPYYYYYGMDV (SEQ ID No. 4) (SEQ ID No. 5) (SEQ ID No. 6) E14 GYTFSNYG INTYNGNT ARDRGYSSSPYYYYYGMDV (SEQ ID No. 4) (SEQ ID No. 5) (SEQ ID No. 6) E19 GFTFSSYG ISYDGSNK ARGGEYFYYYGLDV (SEQ ID No. 1) (SEQ ID No. 2) (SEQ ID No. 3) E20 GYTFSNYG INTYNGNT ARDRGYSSSPYYYYYGMDV (SEQ ID No. 4) (SEQ ID No. 5) (SEQ ID No. 6) E23 GFTFSSYG ISYDGSNK ARGGEYFYYYGLDV (SEQ ID No. 1) (SEQ ID No. 2) (SEQ ID No. 3) E24 GYTFTNYG INAYNGNT ARDRGYSSSPYYYYYGMDV (SEQ ID No. 7) (SEQ ID No. 8) (SEQ ID No. 6) M1 GGSISTYY (SEQ IYYSGST (SEQ ARYDIVTGYPHYYYYVMDV ID No. 9) ID No. 10) (SEQ ID No. 11)
TABLE-US-00005 TABLE 3 VL CDR sequences of exemplified antibodies Antibody CDR1 CDR2 CDR3 E01 QSVSSSS (SEQ ID GAS (SEQ QQDYNLPLT (SEQ ID No. No. 12) ID No. 13) 14) E02 QSVSSSH (SEQ ID GAS (SEQ QQDYNLTIT (SEQ ID No. No. 15) ID No. 13) 16) E08 QSVSSSS (SEQ ID GAS (SEQ QQDYNLPLT (SEQ ID No. No. 12) ID No. 13) 14) E14 QGISNY (SEQ ID AAS (SEQ QQYNSYPFT (SEQ ID No. No. 17) ID No. 18) 19) E19 QGISSA (SEQ ID DAS (SEQ QQFNNYPST (SEQ ID No. 20) ID No. 21) No. 22) E20 QGIRND (SEQ ID AAS (SEQ LQHNSYPYT (SEQ ID No. No. 23) ID No. 18) 24) E23 QGISNY (SEQ ID AAS (SEQ QQYNSYPFT (SEQ ID No. No. 17) ID No. 18) 19) E24 QGIRNA (SEQ ID AAS (SEQ LQDYNYPLT (SEQ ID No. No. 25) ID No. 18) 26) M1 RGIRNA (SEQ ID AAS (SEQ LQDYNYPLT (SEQ ID No. No. 27) ID No. 18) 26)
[0289] CDR definition is provided using annotation tool from http://www.abysis.org/ based on full VH and VL amino acid sequences as defined in Tables 4 and 5.
[0290] For example, the VH amino acid sequence of any antibody disclosed herein is plugged into the annotation tool and Kabat defined CDR sequences, or IMGT, or Chothia, or AbM or Contact defined CDR sequences are provided. Using the “All, side by side” feature, defined CDR sequences are provided. The following example is based on SEQ ID No. 36 and 45.
TABLE-US-00006 TABLE 3.1 All, side by side defined CDR sequences of VH (SEQ ID No. 36) and VL (SEQ ID No. 45): Regions Definition - All, side by side Region Definition Sequence Fragment Residues HFR1 Chothia QVQLQESGPGLVKPSETLSLTCTVS----- (SEQ ID No. 71) 1-25 AbM QVQLQESGPGLVKPSETLSLTCTVS-----(SEQ ID No. 71) 1-25 Kabat QVQLQESGPGLVKPSETLSLTCTVSGGSIS(SEQ ID No. 72) 1-30 Contact QVQLQESGPGLVKPSETLSLTCTVSGGSI-(SEQ ID No. 73) 1-29 IMGT QVQLQESGPGLVKPSETLSLTCTVS-----(SEQ ID No. 71) 1-25 CDR-H1 Chothia GGSISTY---(SEQ ID No. 74) 26-32 AbM GGSISTYYWS (SEQ ID No. 75) 26-35 Kabat -----TYYWS (SEQ ID No. 76) 31-35 Contact ----STYYWS (SEQ ID No. 77) 30-35 IMGT GGSISTYY-(SEQ ID No. 9) 26-33 HFR2 Chothia YWSWIRQPPGKGLEWIGYI (SEQ ID No. 78) 33-51 AbM ---WIRQPPGKGLEWIG-- (SEQ ID No. 79) 36-49 Kabat ---WIRQPPGKGLEWIG-- (SEQ ID No. 79) 36-49 Contact ---WIRQPPGKGLE-----(SEQ ID No. 80) 36-46 IMGT -WSWIRQPPGKGLEWIGY- (SEQ ID No. 81) 34-50 CDR-H2 Chothia -----YYSGS--------- (SEQ ID No. 82) 52-56 AbM ---YIYYSGSTN------ (SEQ ID No. 83) 50-58 Kabat ---YIYYSGSTNYNPSLKS (SEQ ID No. 84) 50-65 Contact WIGYIYYSGSTN-------(SEQ ID No. 85) 47-58 IMGT ----IYYSGST--------(SEQ ID No. 10) 51-57 HFR3 Chothia TNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ 57-97 ID No. 86) AbM --YNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR 59-97 (SEQ ID No. 87) Kabat ---------RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ 66-97 ID No. 88) Contact --YNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYC- 59-95 (SEQ ID No. 89) IMGT -NYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYC- 58-95 (SEQ ID No. 90) CDR-H3 Chothia --YDIVTGYPHYYYYVMDV (SEQ ID No. 91) 98-114 AbM --YDIVTGYPHYYYYVMDV (SEQ ID No. 91) 98-114 Kabat --YDIVTGYPHYYYYVMDV (SEQ ID No. 91) 98-114 Contact ARYDIVTGYPHYYYYVMD- (SEQ ID No. 92) 96-113 IMGT ARYDIVTGYPHYYYYVMDV (SEQ ID No. 11) 96-114 HFR4 Chothia -WGQGTTVTVSS (SEQ ID No. 93) 115-125 AbM -WGQGTTVTVSS (SEQ ID No. 93) 115-125 Kabat -WGQGTTVTVSS (SEQ ID No. 93) 115-125 Contact VWGQGTTVTVSS (SEQ ID No. 94) 114-125 IMGT -WGQGTTVTVSS (SEQ ID No. 93) 115-125 LFR1 Chothia AIQMTQSPSSLSASVGDRVTITC------(SEQ ID No. 95) 1-23 AbM AlOMTOSPSSLSASVGDRVTITC------(SEQ ID No. 95) 1-23 Kabat AIQMTQSPSSLSASVGDRVTITC------(SEQ ID No. 95) 1-23 Contact AIQMTQSPSSLSASVGDRVTITCRASRGI (SEQ ID No. 96) 1-29 IMGT AIQMTQSPSSLSASVGDRVTITCRAS---(SEQ ID No. 97) 1-26 CDR-L1 Chothia RASRGIRNALG--(SEQ ID No. 98) 24-34 AbM RASRGIRNALG--(SEQ ID No. 98) 24-34 Kabat RASRGIRNALG--(SEQ ID No. 98) 24-34 Contact ------RNALGWY (SEQ ID No. 99) 30-36 IMGT ---RGIRNA----(SEQ ID No. 27) 27-32 LFR2 Chothia --WYQQKPGTAPKLLIY (SEQ ID No. 100) 35-49 AbM --WYQQKPGTAPKLLIY (SEQ ID No. 100) 35-49 Kabat --WYQQKPGTAPKLLIY (SEQ ID No. 100) 35-49 Contact ----QQKPGTAPK----(SEQ ID No. 101) 37-45 IMGT LGWYQQKPGTAPKLLIY (SEQ ID No. 102) 33-49 CDR-L2 Chothia ----AASSLQS (SEQ ID No. 103) 50-56 AbM ----AASSLQS (SEQ ID No. 103) 50-56 Kabat ----AASSLQS (SEQ ID No. 103) 50-56 Contact LLIYAASSLQ- (SEQ ID No. 104) 46-55 IMGT ----AA----- 50-51 LFR3 Chothia -----GVPSRFSGSGSGTDFTLTISSLQPEDSATYYC (SEQ ID 57-88 No. 105) AbM -----GVPSRFSGSGSGTDFTLTISSLQPEDSATYYC (SEQ ID 57-88 No. 105) Kabat -----GVPSRFSGSGSGTDFTLTISSLQPEDSATYYC (SEQ ID 57-88 No. 105) Contact ----SGVPSRFSGSGSGTDFTLTISSLQPEDSATYYC (SEQ ID 56-88 No. 106) IMGT SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDSATYYC (SEQ ID 52-88 No. 107) CDR-L3 Chothia LQDYNYPLT (SEQ ID No. 26) 89-97 AbM LQDYNYPLT (SEQ ID No. 26) 89-97 Kabat LQDYNYPLT (SEQ ID No. 26) 89-97 Contact LQDYNYPL-(SEQ ID No. 108) 89-96 IMGT LQDYNYPLT (SEQ ID No. 26) 89-97 LFR4 Chothia -FGGGTKVEIK (SEQ ID No. 109) 98-107 AbM -FGGGTKVEIK (SEQ ID No. 109) 98-107 Kabat -FGGGTKVEIK (SEQ ID No. 109) 98-107 Contact TFGGGTKVEIK (SEQ ID No. 110) 97-107 IMGT -FGGGTKVEIK (SEQ ID No. 109) 98-107
TABLE-US-00007 TABLE 4 VH amino acid sequences of exemplified antibodies Antibody AA of VH E01 QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLE WVAVISYDGSNKNYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY YCARGGEYFYYYGLDVWGQGTTVTVSS (SEQ ID No. 28) E02 QVQLVQSGAEVKKPGASVKVSCKASGYTFSNYGISWVRQAPGQGLE WMGWINTYNGNTNYAQKLQGRVTMTTDTSTSTAYMALRGLRSDDTA VYYCARDRGYSSSPYYYYYGMDVWGQGTTVTVSS (SEQ ID No. 29) E08 QVQLVQSGAEVKKPGASVKVSCKASGYTFSNYGISWVRQAPGQGLE WMGWINTYNGNTNYAQKLQGRVTMTTDTSTSTAYMALRGLRSDDTA VYYCARDRGYSSSPYYYYYGMDVWGQGTTVTVSS (SEQ ID No. 30) E14 QVQLVQSGAEVKKPGASVKVSCKASGYTFSNYGISWVRQAPGQGLE WMGWINTYNGNTNYAQKLQGRVTMTTDTSTSTAYMALRGLRSDDTA VYYCARDRGYSSSPYYYYYGMDVWGQGTTVTVSS (SEQ ID No. 31) E19 QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLE WVAVISYDGSNKNYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY YCARGGEYFYYYGLDVWGQGTTVTVSS (SEQ ID No. 32) E20 QVQLVQSGAEVKKPGASVKVSCKASGYTFSNYGISWVRQAPGQGLE WMGWINTYNGNTNYAQKLQGRVTMTTDTSTSTAYMALRGLRSDDTA VYYCARDRGYSSSPYYYYYGMDVWGQGTTVTVSS (SEQ ID No. 33) E23 QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLE WVAVISYDGSNKNYWDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAV YYCARGGEYFYYYGLDVWGQGTTVTVSS (SEQ ID No. 34) E24 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGISWRQAPGQGLE WMGWINAYNGNTNYAQKLQGRVTMTTVTYTSTAYMELRSLRSDDTA VYYCARDRGYSSSPYYYYYGMDVWGQGTTVTVSS (SEQ ID No. 35) M1 QVQLQESGPGLVKPSETLSLTCTVSGGSISTYYWSWIRQPPGKGLEWI GYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR YDIVTGYPHYYYYVMDVWGQGTTVTVSS (SEQ ID No. 36)
TABLE-US-00008 TABLE 5 VL amino acid sequences of exemplified antibodies Antibody AA OF VK E01 EIVMTQSPATLSLSPGERATLSCRASQSVSSSSLSWYQQKPGQAPR LLIYGASTRATGIPARFSGSGSGTDFTLTISSLQPEDFAVYYCQQDYN LPLTFGGGTKVEIK (SEQ ID No. 37) E02 EIVMTQSPATLSLSPGERATLSCRASQSVSSSHLSWYQQKPGQAPR LLIYGASTRATGIPARFSGSGSGTDFTLTISSLQPEDFAVYYCQQDYN LTITFGQGTRLEIK (SEQ ID No. 38) E08 EIVMTQSPATLSLSPGERATLSCRASQSVSSSSLSWYQQKPGQAPR LLIYGASTRATGIPARFSGSGSGTDFTLTISSLQPEDFAVYYCQQDYN LPLTFGGGTKVEIK (SEQ ID No. 39) E14 DIQMTQSPSSLSASIGDRVTITCRASQGISNYLAWFQQKPGKAPKSLI YAASSLQSGVPSKFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYP FTFGPGTKVDIK (SEQ ID No. 40) E19 AIQLTQSPSSLSASVGDRVTITCRAGQGISSALAWYQQKPGKAPKILIY DASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNNYPS TFGQGTKLEIK (SEQ ID No. 41) E20 DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRL IYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYP YTFGQGTKLEIK (SEQ ID No. 42) E23 DIQMTQSPSSLSASIGDRVTITCRASQGISNYLAWFQQKPGKAPKSLI YAASSLQSGVPSKFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYP FTFGPGTKVDIK (SEQ ID No. 43) E24 AIQMTQSPSSLSASVGDKVTITCRASQGIRNALGWYQQKPGTAPKLLI YAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDSATYYCLQDYNYP LTFGGGTKVEIK (SEQ ID No. 44) M1 AIQMTQSPSSLSASVGDRVTITCRASRGIRNALGWYQQKPGTAPKLLI YAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDSATYYCLQDYNYP LTFGGGTKVEIK (SEQ ID No. 45)
TABLE-US-00009 TABLE 6 VH nucleotide sequences of exemplified antibodies Antibody DNA of VH E01 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGG GAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGT AGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCT GGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAAAACTAT GTAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCA AGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACA CGGCTGTGTATTACTGTGCGAGAGGAGGGGAGTACTTCTACTATTA CGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTC A (SEQ ID No. 46) E02 CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGG GCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTTCCA ATTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTG AGTGGATGGGATGGATCAACACTTACAATGGTAACACAAACTATGC ACAGAAGCTCCAGGGCAGAGTCACCATGACCACTGACACATCCAC GAGCACAGCCTACATGGCGCTGAGGGGCCTGAGATCTGACGACAC GGCCGTGTATTATTGTGCGAGAGATAGGGGGTATAGCAGCAGCCC TTACTACTACTACTACGGAATGGACGTCTGGGGCCAAGGGACCAC GGTCACCGTCTCCTCA (SEQ ID No. 47) E08 CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGG GCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTTCCA ATTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTG AGTGGATGGGATGGATCAACACTTACAATGGTAACACAAACTATGC ACAGAAGCTCCAGGGCAGAGTCACCATGACCACTGACACATCCAC GAGCACAGCCTACATGGCGCTGAGGGGCCTGAGATCTGACGACAC GGCCGTGTATTATTGTGCGAGAGATAGGGGGTATAGCAGCAGCCC TTACTACTACTACTACGGAATGGACGTCTGGGGCCAAGGGACCAC GGTCACCGTCTCCTCA (SEQ ID No. 48) E14 CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGG GCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTTCCA ATTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTG AGTGGATGGGATGGATCAACACTTACAATGGTAACACAAACTATGC ACAGAAGCTCCAGGGCAGAGTCACCATGACCACTGACACATCCAC GAGCACAGCCTACATGGCGCTGAGGGGCCTGAGATCTGACGACAC GGCCGTGTATTATTGTGCGAGAGATAGGGGGTATAGCAGCAGCCC TTACTACTACTACTACGGAATGGACGTCTGGGGCCAAGGGACCAC GGTCACCGTCTCCTCA (SEQ ID No. 49) E19 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGG GAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGT AGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCT GGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAAAACTAT GTAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCA AGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACA CGGCTGTGTATTACTGTGCGAGAGGAGGGGAGTACTTCTACTATTA CGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTC A (SEQ ID No. 50) E20 CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGG GCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTTCCA ATTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTG AGTGGATGGGATGGATCAACACTTACAATGGTAACACAAACTATGC ACAGAAGCTCCAGGGCAGAGTCACCATGACCACTGACACATCCAC GAGCACAGCCTACATGGCGCTGAGGGGCCTGAGATCTGACGACAC GGCCGTGTATTATTGTGCGAGAGATAGGGGGTATAGCAGCAGCCC TTACTACTACTACTACGGAATGGACGTCTGGGGCCAAGGGACCAC GGTCACCGTCTCCTCA (SEQ ID No. 51) E23 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGG GAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGT AGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCT GGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAAAACTAT GTAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCA AGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACA CGGCTGTGTATTACTGTGCGAGAGGAGGGGAGTACTTCTACTATTA CGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTC A (SEQ ID No. 52) E24 CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGA GCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCA ACTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTG AGTGGATGGGATGGATCAACGCTTACAATGGTAACACAAACTATGC ACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGTCACATACAC GAGTACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACAC GGCCGTGTATTACTGTGCGAGAGATAGGGGGTATAGCAGCAGCCC TTATTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACG GTCACCGTCTCCTCA (SEQ ID No. 53) M1 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTC GGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGT ACTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTG GAGTGGATTGGGTATATCTATTACAGTGGGAGCACCAACTACAACC CCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAA CCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCTGCGGACACGGC CGTTTATTACTGTGCGAGGTACGATATTGTGACTGGTTATCCTCACT ACTACTACTACGTTATGGACGTCTGGGGCCAAGGGACCACGGTCA CCGTCTCCTCA (SEQ ID No. 54)
TABLE-US-00010 TABLE 7 VL nucleotide sequences of exemplified antibodies Antibody DNA ofVL E01 GAAATTGTAATGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAG GGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCA GCAGCTCCTTATCCTGGTACCAGCAGAAACCTGGGCAGGCTCCCA GGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGCATCCCAG CCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCAT CAGCAGCCTGCAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAG GATTATAACTTACCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGA TCAAA (SEQ ID No. 55) E02 GAAATTGTAATGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAG GGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCA GCAGCCATTTATCCTGGTACCAGCAGAAACCTGGGCAGGCTCCCA GGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGCATCCCAG CCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCAT CAGCAGCCTGCAGCCTGAAGATTTTGCAGTTTATTATTGTCAGCAG GATTATAATTTAACGATCACCTTCGGCCAAGGGACACGACTGGAGA TTAAA (SEQ ID No. 56) E08 GAAATTGTAATGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAG GGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCA GCAGCTCCTTATCCTGGTACCAGCAGAAACCTGGGCAGGCTCCCA GGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGCATCCCAG CCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCAT CAGCAGCCTGCAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAG GATTATAACTTACCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGA TCAAA (SEQ ID No. 57) E14 GACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTATAG GAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGCATTAGCA ATTATTTAGCCTGGTTTCAGCAGAAACCAGGGAAAGCCCCTAAGTC CCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAAG TTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCA GCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAAT AGTTACCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA (SEQ ID No. 58) E19 GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAG GAGACAGAGTCACCATCACTTGCCGGGCAGGTCAGGGCATTAGCA GTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGAT CCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGG TTCAGCGGCAGTGGATCGGGGACAGATTTCACTCTCACCATCAGCA GCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAAT AATTACCCTAGCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA (SEQ ID No. 59) E20 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAG GAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAA ATGATTTAGGCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCG CCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGG TTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCA GCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCTACAGCATAAT AGTTACCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA (SEQ ID No. 60) E23 GACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTATAG GAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGCATTAGCA ATTATTTAGCCTGGTTTCAGCAGAAACCAGGGAAAGCCCCTAAGTC CCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAAG TTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCA GCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAAT AGTTACCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA (SEQ ID No. 61) E24 GCCATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAG GAGACAAAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAAA TGCTTTAGGCTGGTATCAGCAGAAACCAGGAACAGCCCCTAAACTC CTGATCTATGCTGCATCCAGTTTACAGAGTGGGGTCCCATCAAGGT TCAGCGGCAGTGGATCTGGCACAGATTTCACTCTCACCATCAGCAG CCTGCAGCCTGAAGATTCTGCAACTTATTACTGTCTACAAGATTACA ATTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA (SEQ ID No. 62) M1 GCCATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAG GAGACAGAGTCACCATCACTTGCCGGGCAAGTCGGGGCATTAGAA ATGCTTTAGGCTGGTATCAGCAGAAACCAGGAACAGCCCCTAAACT CCTGATCTATGCTGCATCCAGTTTACAGAGTGGGGTCCCATCAAGG TTCAGCGGCAGTGGATCTGGCACAGATTTCACTCTCACCATCAGCA GCCTGCAGCCTGAAGATTCTGCAACTTATTACTGTCTACAAGATTAC AATTACCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA (SEQ ID No. 63)
TABLE-US-00011 TABLE 8 Constant region amino acid sequences Constant region AA Human IgG4 heavy ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA chain LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPS P01861.1 NTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISR TPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST YRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK (SEQ ID No. 64) Human IgG2 heavy ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA chain P01859 LTSGVHTFPAVLQSSGLYSLSSWTVPSSNFGTQTYTCNVDHKPS NTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRT PEVTCVWDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTF RWSVLTWHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENN YKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK (SEQ ID No. 65) Human light chain, GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKAD lambda 1 GSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQV P0CG04 THEGSTVEKTVAPTECS (SEQ ID No. 66) Human light chain, GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKAD lambda 2 SSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQV P0DOY2 THEGSTVEKTVAPTECS (SEQ ID No. 67) Human light chain, RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDN kappa ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT P01834 HQGLSSPVTKSFNRGEC (SEQ ID No. 68)
Example 2
[0291] Anti-IGFBP3 mAbs Produced by Hybridoma Inhibit IGFBP3-TMEM219 Binding
[0292] The novel anti-IGFBP3 monoclonal antibody generated using hybridoma was screened for its ability to compete with ecto-TMEM219 for the interaction with IGFBP3 using a competitive ELISA binding assay. IGFBP3, ecto-TMEM219 and the available antibodies were all used in a 1:1 ratio. The anti-IGFBP3 mAb was able to inhibit the IGFBP3-Ecto-TMEM219 (
[0293] Newly Generated Monoclonal Anti-IGFBP3 Antibodies Rescue IGFBP3-Damage in the Mini-Guts Assay in Humans.
[0294] The newly generated monoclonal Antibody was also tested in the mini-gut assay. Briefly, mini-guts were generated from crypts obtained from healthy controls (n=3) and cultured for 8 days in presence of IGFBP3 and treated with either ecto-TMEM219 or newly generated anti-IGFBP3 mAb at a ratio of 1:1 (mAbs/ecto-TMEM219: IGFBP3).
[0295] We observed that Anti-IGFBP3 mAb was comparable to ecto-TMEM219 in rescuing the negative effects of IGFBP3 on self-renewal ability (% development) and morphology (absence of crypts domain, generation of small spheroids) of large crypt organoids. (
[0296] Newly Generated Monoclonal Anti-IGFBP3 Antibodies Rescue IGFBP3-Damage on ISCs Markers
[0297] Newly generated anti-IGFBP3 mAbs, which were shown to be effective in promoting mini-guts development upon IGFBP3 exposure were also able to restore the expression of ISCs markers EphB2 and LGR5 (
[0298] The Newly Discovered Anti-IGFBP3 Antibody Rescue Mini-Guts Growth in Disease Models.
[0299] In order to confirm that the newly discovered monoclonal anti-IGFBP3 antibody prevent the detrimental effects of IGFBP3 on TMEM219-expressing intestinal stem cells, the inventors further tested them in vitro in the mini-gut obtained from IBD patients.
[0300] The novel anti-IGFBP3 mAb significantly improved the development of mini-guts from IBD patients of at least 20%, similarly to Ecto-TMEM219 treatment (
[0301] This highlights that anti-IGFBP3 mAbs of the invention, selected for their ability to competitively inhibit ecto-TMEM binding to IGFBP3 are capable of rescuing ISCs function and preserve ISCs pool from IGFBP3-detrimental effects.
[0302] Newly Generated Monoclonal Anti-IGFBP3 Antibodies Rescue IGFBP3-Damage in Murine Mini-Guts
[0303] Crypts Isolation and Murine Mini-Guts Development
[0304] In order to confirm that the present invention antibodies have a similar tissue cross-reactivity profile in murine tissue in respect to the human tissues, the inventors further tested the monoclonal anti-IGFBP3 antibodies of the invention in the in vitro mini-gut assay in murine crypts. Crypts were obtained from control mice (n=3) (632C57BL/6J Charles River Laboratories, Lyon, France).
[0305] Isolated crypts were cultured to generate large crypts organoids namely mini-guts for 8 days in the presence of IGFBP3 with/without ecto-TMEM219. Newly generated anti-IGFBP3 mAbs were added at day 0 at a ratio of 1:1 (mAbs/ecto-TMEM219: IGFBP3).
[0306] Mini-guts development was calculated as a percentage of organoids growth after 8 days as compared to the plated isolated crypts (D′Addio F et al. Cell Stem Cell 2015 Oct. 1; 17(4): 486-498).
[0307] As shown in
[0308] Newly generated monoclonal anti-IGFBP3 antibodies inhibit IGFBP3-mediated overexpression of caspase 8 in human beta cells IGFBP3-detrimental effects on human beta cells are Caspase-8 mediated. Interestingly, newly discovered anti-IGFBP3 mAbs were able to inhibit the caspase-8 upregulation induced by IGFBP3 treatment by at least 50% when compared to samples treated only with IGFBP3 (
[0309] These results suggest that the discovered anti-IGFBP3 mAbs exert a protective effect on human beta-cells by blocking the IGFBP3/TMEM219 Caspase-8-mediated apoptotic injury.
Example 3
[0310] Newly Generated Monoclonal Anti-IGFBP3 Antibodies Rescue IGFBP3-Damage in the Mini-Guts Assay in Humans.
[0311] Anti-IGFBP3 monoclonal antibodies were tested in the mini-gut assay. Briefly, mini-guts were generated from crypts obtained from healthy controls (n=3) and cultured for 8 days upon IGFBP3 exposure and treated with anti-IGFBP3 mAbs at a ratio of 1:1 (mAbs: IGFBP3). Inventors observed that among 8 mAbs, E08 and E20 were comparable to ecto-TMEM219 in rescuing the self-renewal ability of large crypt organoids in the presence of IGFBP3, thus supporting a relevant effect in preventing IGFBP3-mediated damage on local stem cells (
[0312] Newly Generated Monoclonal Anti-IGFBP3 Antibodies Rescue IGFBP3-Damage on ISCs Markers
[0313] Anti-IGFBP3 mAbs, which showed to be effective in promoting mini-guts development, are also able to restore the expression of ISCs markers EphB2 and LGR5 (
[0314] Newly Generated Monoclonal Anti-IGFBP3 Antibodies Rescue IGFBP3-Damage in Murine Mini-Guts
[0315] Crypts Isolation and Murine Mini-Guts Development
[0316] In order to confirm that the present invention antibodies have a similar tissue cross-reactivity profile in murine tissue in respect to the human tissues, the inventors further tested the monoclonal anti-IGFBP3 antibodies of the invention in the in vitro mini-gut assay in murine crypts. Crypts were obtained from control mice (n=3) (632C57BL/6J Charles River Laboratories, Lyon, France).
[0317] Isolated crypts were cultured to generate large crypts organoids namely mini-guts for 8 days in the presence of IGFBP3 with/without ecto-TMEM219. Newly generated anti-IGFBP3 mAbs were added at day 0 at a ratio of 1:1 (mAbs/ecto-TMEM219: IGFBP3). Mini-guts development was calculated as a percentage of organoids growth after 8 days as compared to the plated isolated crypts (D′Addio F et al. Cell Stem Cell 2015 Oct. 1; 17(4): 486-498).
[0318] As shown in
[0319] Anti-IGFBP3 mAbs Protect a Beta Cell Line from Apoptosis In Vitro
[0320] In order to confirm that the anti-IGFBP3 mAbs prevent the pro-apoptotic effects of IGFBP3 on TMEM219-expressing cells within the pancreas, inventors further tested them in vitro in a human beta cell line, Betalox-5. Exposure of beta cells to pooled T1 D serum increased CASP8 expression and anti-IGFBP3 mAb E08 was able to counterbalance this effect, thus supporting the beneficial effects of the newly generated monoclonal anti-TMEM219 antibodies in preventing pancreatic beta cells apoptosis (
Example 3: T1D Mouse Model
[0321] As shown in
[0322] Next, pancreatic tissue sections of NOD mice from untreated mice, M1S and Ecto-TMEM treated groups were analyzed at 24 weeks of age and demonstrated a reduction in islet infiltrate, with a slight increased detection of insulin positive cells as compared to untreated controls (
INCORPORATION BY REFERENCE
[0323] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes.
EQUIVALENTS
[0324] While various specific embodiments have been illustrated and described, the above specification is not restrictive. It will be appreciated that various changes can be made without departing from the spirit and scope of the invention(s). Many variations will become apparent to those skilled in the art upon review of this specification.
REFERENCES
[0325] 1. Baxter R C. J Cell Commun Signal. 2013; 7(3):179-89. [0326] 2. Oh Y, et al., Prog Growth Factor Res. 1995; 6(2-4):503-12. [0327] 3. Ingermann A R, et al. JBC. 2010; 285(39):30233-46. [0328] 4. D′Addio F, et al. Cell stem cell. 2015; 17(4):486-98. [0329] 5. Brennand K, and Melton D. J. of cellular and molecular med. 2009; 13(3):472-87. [0330] 6. Yi P, Park J S, and Melton D A. Cell. 2014; 159(3):467-8. [0331] 7. Ben Nasr M, et al., Pharmacological res.: the official journal of the Italian Pharmacological Society. 2015; 98:31-8. [0332] 8. Keenan H A, et al. Diabetes. 2010; 59(11):2846-53. [0333] 9. Meier J J, et al., Diabetologia. 2005; 48(11):2221-8. [0334] 10. Atkinson M A, et al., Diabetes care. 2015; 38(6):979-88. [0335] 11. Nguyen K H, et al., Endocrinology. 2011; 152(6):2184-96. [0336] 12. Yakar S, et al., FASEB J. 2009; 23(3):709-19. [0337] 13. Drogan D, et al., Am J Epidemiol. 2016; 183(6):553-60. [0338] 14. Peet A, et al., Eur J Endocrinol. 2015; 173(2):129-37. [0339] 15. Kaplan G G. Nat Rev Gastroenterol Hepatol. 2015; 12(12):720-7. [0340] 16. Cui S, and Chang P Y. World J Gastroenterol. 2016; 22(31):7099-110. [0341] 17. Yancu D, et al., J Gastroenterol Hepatol. 2017; 32(1):146-53. [0342] 18. Jung P, et al., Nature Medecine 2011; 17:1225-1227. [0343] 20. George M J, et al., Curr Diab Rep 2013; 13(1):72-80. [0344] 21. Marsha J D. Am Health Drug Benefits 2011; 4(5): 312-322 [0345] 22. Dhingra A K et al., Antiinflamm Antiallergy Agents Med Chem. 2015; 14(2):81-97. [0346] 23. Pithadia A B, SunitaJ. Pharmacoligal rep 2011; 63: 629-642 [0347] 24. Zhe Wang, et al., Expert Opin Drug Deliv. 2010 February; 7(2):159-71 [0348] 25. Sumit G, Wei W, Tsutomu and Satoshi O, Antibodies 2013; 2: 452-500; [0349] 26. Beck A, et al., Nat Rev Imm 2010; 10: 345-352 [0350] 27. Huch M, et al., Nature 2013; 494 (7436): 247-250 [0351] 28. Mahé M M, et al., Curr Protoc Mouse Biol 2013; 3: 217-240 [0352] 29. Shimkets R A, Gene Expression Profiling. Meth. in Mol. Biology; 258. Humana Press [0353] 30. Raghavachari N, et al., Gene Expression Analaysis, Meth. in Mol. Biol; 1783. Humana Press [0354] 31. Kurien B T, et al., Western Blotting, Methods in Molecullar Biology; 1312. Humana Press [0355] 32. Hnasko R, ELISA, Methods in Molecular Blology, 1318. Humana Press [0356] 33. Lipton M S, et al., Mass Spec. of Proteins and Peptides, Meth. in Mol. Biol., 492. Humana Press [0357] 34. Brady H, Apoptosis Meth. and Protocols, Meth. in Mol. Biol., 282. Humana Press [0358] 35. Cheryl L et al., Protein-Protein interactions, Meth. in Mol. Biol., 1278. Humana Press