ANTIBODIES AND USES THEREOF
20210388072 · 2021-12-16
Inventors
Cpc classification
C07K2317/76
CHEMISTRY; METALLURGY
C07K16/22
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
C07K2317/33
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to an antibody or antigen binding fragment thereof that binds specifically to IGFBP3 and does not displace the binding of IGF-I 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 which: does not displace the binding of IGF-I to IGFBP3, and inhibits or reduces the binding of IGFBP3 to the TMEM219 receptor.
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 any one of previous claims that has at least one activity selected from: a—increase in IGFBP3 treated healthy subject minigut growth b—increase in IBD-patient minigut growth; c—increase in diabetic enteropathy serum treated healthy subject minigut growth; d—increase in expression of EphB2 and/or LGR5 in IGFBP3 treated healthy subject minigut; e—decrease in caspase 8 expression in IGFBP3 treated healthy subject minigut; f—decrease in β-cell loss in IGFBP3 treated β-cell; g—increase in expression of insulin in IGFBP3 treated β-cell; and h—decrease in apoptosis of β-cell in IGFBP3 treated β-cell.
4. The isolated antibody or antigen binding fragment thereof according to claim 3 wherein the increase in a), b) and c) 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%.
5. The isolated antibody or antigen binding fragment thereof according any one of previous claims 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 to SEQ ID NO: 7; ii. a CDR2 sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO:8 to SEQ ID NO: 16; and iii. a CDR3 sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO:17 to SEQ ID NO:26; 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:27 to SEQ ID NO:36; ii. a CDR2 sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO:37 to SEQ ID NO:44; and iii. a CDR3 sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO:45 to SEQ ID NO:54.
6. The isolated antibody or antigen binding fragment thereof according to claim 5 comprising the CDRs as indicated in Table 2 and/or in Table 3.
7. The isolated antibody or antigen binding fragment thereof according to any one of previous claim comprising: a. a heavy chain variable domain sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO:55 to SEQ ID NO:65; or b. a light chain variable domain sequence of the amino acid sequence selected from the group consisting of: SEQ ID NO: 66 to SEQ ID NO:76; or c. the light chain variable domain of (a) and the heavy chain variable domain of (b).
8. The isolated antibody or antigen binding fragment thereof according to any one of previous claim being Yu139-A02 having SEQ ID NO:55 and SEQ ID NO:66, Yu139-A03 having SEQ ID NO:56 and SEQ ID NO:67, Yu139-B01 having SEQ ID NO:57 and SEQ ID NO:68, Yu139-C01 having SEQ ID NO:58 and SEQ ID NO:69, Yu139-C02 having SEQ ID NO:59 and SEQ ID NO:70, Yu139-D02 having SEQ ID NO:60 and SEQ ID NO:71, Yu139-D03 having SEQ ID NO:61 and SEQ ID NO:72, Yu139-F02 having SEQ ID NO:62 and SEQ ID NO:73, Yu139-G01 having SEQ ID NO:63 and SEQ ID NO:74, Yu139-G03 having SEQ ID NO:64 and SEQ ID NO:75 or Yu139-H03 having SEQ ID NO:65 and SEQ ID NO:76, preferably Yu139-A03, Yu139-C01, Yu139-G03 or Yu139-H03.
9. The isolated antibody or antigen binding fragment thereof according to any one of previous claim that binds to human IGFBP3 with an affinity constant lower than or equal to 4×10.sup.−6 M.
10. An isolated antibody or antigen binding fragment thereof that: (a) binds specifically to an epitope on IGFBP3, e.g., the same or similar epitope as the epitope recognized by the monoclonal antibody Yu139-A02, A03, B01, C01, C02, D02, D03, F02, G01, G03, H03 as defined in Tables 4 and 5; or (b) cross-competes for binding with the monoclonal antibody Yu139-A02, A03, B01, C01, C02, D02, D03, F02, G01, G03, H03 as defined in Tables 4 and 5; or (c) shows the same or similar binding affinity or specificity, or both, as any of Yu139-A02, A03, B01, C01, C02, D02, D03, F02, G01, G03, H03 as defined in Tables 4 and 5; or (d) has one or more biological properties of an antibody molecule described herein, e.g., an antibody molecule chosen from, e.g., any of Yu139-A02, A03, B01, C01, C02, D02, D03, F02, G01, G03, H03 as defined in Tables 4 and 5; or (e) has one or more pharmacokinetic properties of an antibody molecule described herein, e.g., an antibody molecule chosen from, e.g., any of Yu139-A02, A03, B01, C01, C02, D02, D03, F02, G01, G03, H03 as defined in Table 4 and 5.
11. The isolated antibody or antigen binding fragment thereof according to any one of previous claim being a human or a humanized antibody.
12. The isolated antibody or antigen binding fragment thereof according to any one of previous claim being an IgG2 or IgG4 antibody, preferably an IgG2 kappa antibody, an IgG2 lambda antibody, an IgG4 kappa antibody or an IgG4 lambda antibody.
13. An isolated polynucleotide comprising at least one sequence that encodes the antibody or antigen binding fragment thereof according to any one of previous claim, preferably said polynucleotide is a cDNA.
14. A vector comprising the polynucleotide according to claim 13, preferably said 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.
15. An isolated cell comprising the polynucleotide according to claim 13 or the vector according to claim 14, preferably the isolated cell is a hybridoma or a Chinese Hamster Ovary (CHO) cell or a Human Embryonic Kidney cells (HEK293).
16. The antibody or antigen binding fragment thereof according to any one of claims 1 to 12 or the polynucleotide according to claim 13 or the vector according to claim 14 or the cell according to claim 15 for use as a medicament, preferably for use in the treatment of: diabetes, intestinal and/or bowel disorder, malabsorption syndrome, cachexia or diabetic enteropathy, preferably diabetes is Type I or Type II diabetes, preferably the intestinal and/or bowel disorder is inflammatory bowel disease, celiac disease, ulcerative colitis, Crohn's disease or intestinal obstruction.
17. A pharmaceutical composition comprising the isolated antibody or antigen binding fragment thereof or the isolated polynucleotide or the vector or the isolated cell according to any one of previous claim and pharmaceutically acceptable carrier, preferably for use in the treatment of: diabetes, intestinal and/or bowel disorder, malabsorption syndrome, cachexia or diabetic enteropathy, preferably the intestinal and/or bowel disorder is inflammatory bowel disease, celiac disease, ulcerative colitis, Crohn's disease or intestinal obstruction.
18. A method of inhibiting the binding of IGFBP3 to TMEM219 receptor, comprising contacting IGFBP3 with the antibody or the composition according to any one of claim 1 to 12 or 17.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0166] The antibodies of the invention include antibodies that specifically bind IGFBP3. As discussed herein, these antibodies are collectively referred to as “anti-IGFBP3 antibodies”. Thus, by “anti-IGFBP3 antibodies” is intended antibodies specific for IGFBP3. All of these antibodies are encompassed by the discussion herein. The respective antibodies can be used alone or in combination in the methods of the invention.
[0167] By “antibodies that specifically bind” is intended that the antibodies will not substantially cross react with another 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.
[0168] 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, antibody fragments and scaffold antigen binding proteins.
[0169] The term “antigen binding moiety” refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one aspect, the antigen binding moiety is able to activate signaling through its target cell antigen. In a particular aspect, the antigen binding moiety is able to direct the entity to which it is attached to a target site. Antigen binding moieties include antibodies and fragments thereof capable of specific binding to a target cell antigen. 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).
[0170] The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
[0171] Additional terms are defined below and throughout the application.
[0172] The term “antibody” is intended to refer to immunoglobulin molecules comprising at least one heavy (H) chain or at least one light (L) chain. Said chains may be inter-connected by disulfide bonds. The term “antibody” also comprises multimers of said chains (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region.
[0173] The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of at least one CDR, preferably three CDRs and at least one FR, preferably four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments, the FRs of the antibody (or antigen-binding portion thereof) may be identical to the human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.
[0174] The term “antibody,” as used herein, also includes ADC (antibody drug conjugate), payload fusion antibodies, bispecific, Fab, scFv, diabodies, triabodies, minibodies and single domain antibodies, camelid IgG, IgNAR, as described in (25) incorporated by reference.
[0175] The term “antibody,” as used herein, also includes antigen-binding fragments of full antibody molecules. The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein 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.
[0176] Designed Ankyrin Repeat Proteins (DARPins) 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.
[0177] A single-domain antibody is an antibody fragment consisting of a single monomeric variable antibody domain. The first single domain was derived from the variable domain of the antibody heavy chain from camelids (nanobodies or VHH fragments). Furthermore, the term single-domain antibody includes an autonomous human heavy chain variable domain (aVH) or VNAR fragments derived from sharks.
[0178] 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.
[0179] 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.
[0180] In certain embodiments, an antigen-binding fragment of an antibody may contain 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)).
[0181] In certain embodiments, antigen binding molecules provided herein are altered to increase or decrease the extent to which the antibody 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. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in antigen binding molecules may be made in order to create variants with certain improved properties. 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 GlcNAc. 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.).
[0182] In certain embodiments, it may be desirable to create cysteine engineered variants of the 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.
[0183] In certain aspects, the 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 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. 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. 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.
[0184] 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. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, may in various embodiments be adapted for use in the context of an antigen-binding fragment of an anti-IL-6R antibody using routine techniques available in the art.
[0185] 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.
[0186] 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 derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
[0187] 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.
[0188] Human antibodies can exist in two forms that are associated with hinge heterogeneity. In an embodiment, an immunoglobulin molecule comprises a stable four chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond. In another embodiment, the dimers are not linked via inter-chain disulfide bonds and a molecule of about 75-80 kDa is formed composed of a covalently coupled light and heavy chain (half-antibody). These embodiments/forms have been extremely difficult to separate, even after affinity purification. The frequency of appearance of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular Immunology 30:105) to levels typically observed using a human IgG1 hinge. The instant 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.
[0189] 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.
[0190] The term “specifically binds,” or the like, means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiologic conditions. Methods for determining whether an antibody specifically binds to an antigen are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, an antibody that “specifically binds” IGFBP3, as used herein, includes antibodies that bind IGFBP3or portion thereof 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 in a surface plasmon resonance assay.
[0191] The term “surface plasmon resonance”, as used herein, refers to an optical phenomenon that allows for the analysis of real-time interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore™ system (Biacore Life Sciences division of GE Healthcare, Piscataway, N.J.) or kinetic exclusion assays.
[0192] The term “KD”, as used herein, is intended to refer to the equilibrium dissociation constant of an antibody-antigen interaction.
[0193] 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.
[0194] The anti-IGFBP3 antibodies 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. The present invention includes in various embodiments 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”). 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). 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.
[0195] The present invention also includes 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.
[0196] 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).
[0197] The invention in certain embodiments relates to 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:55 to SEQ ID NO:65 and the light chain variable region comprising a sequence chosen from the group of: SEQ ID NO:66 to SEQ ID NO:76. The disclosure provides pharmaceutical compositions comprising such antibody, and methods of using these compositions.
[0198] 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.
[0199] 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.
[0200] 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.
[0201] 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 ERIS™ 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.
[0202] According to the invention, “subject” means a human subject or human patient.
EXAMPLES
Methods
Patients and Study Design
[0203] Healthy subjects (CTRL) were enrolled (n=10) among patients undergoing to colonoscopy or intestinal surgery for diverticulosis, colon cancer, irritable bowel syndrome. Age: 41.3±2.2 (mean±SEM); Sex (M/F): 7/3.
[0204] IBD individuals had a 5-year 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. Age: 47.1±3.1 (mean±SEM); Sex (M/F): 4/6.
[0205] DE serum was obtained from n=10 individuals with long-standing type 1 diabetes (history of diabetes >20 years), intestinal symptoms and motility dysfunction, with age ranging from 41 to 43 years old, and pooled for in vitro experiments. All subjects provided informed consent before study enrollment.
Recombinant Proteins and Interventional Studies
[0206] Recombinant human IGFBP3 was obtained from Life Technologies (IGFBP3, Life Technologies, 10430H07H5). ecto-TMEM219 was obtained through Genescript's customized protein service. The protein, produced in E. coli, has the following amino acid sequence,
TABLE-US-00001 (SEQ ID NO: 104) THRTGLRSPDIPQDWVSFLRSFGQLTLCPRNGTVTGKWRGSHVVGLLTT LNFGDGPDRNKTRTFQATVLGSQMGLKGSSAGQLVLITARVTTERTAGT CLYFSAVPGILPSSQPPISCSEEGAGNATLSPRMGEECVSVWSHEGLVL TKLLTSEELALCGSR.
[0207] 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).
[0208] Monoclonal and polyclonal anti-IGFBP3 (LSBio LS-045037/107162 mouse IgG1 clone 83.8F9; LSBIO LS-0149975/72664, goat IgG) were added to culture medium at day +1 from mini-guts culture at 1, 10, 50 ug/ml and at 10 ug/ml final concentration respectively.
[0209] Newly generated anti-IGFBP3 monoclonal antibodies were added at 1:1 molecular ratio as compared to IGFBP3 at 10 ug/ml final concentration.
Crypts Isolation and Mini-Guts Development
[0210] 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 ant-nr], Gentamycin [Invitrogen, Carlsbad, Calif., USA 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). Isolated 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. Percentage of developed mini-guts with at least one crypt domain was assessed as already described (4,18).
In Vitro Mini-Gut Generation Study
[0211] Crypts were isolated from healthy subject biopsy samples and cultured as previously described to generate mini-guts. To mimic the pathologic conditions, the crypt culturing medium was modified by adding human serum of individuals with diabetic enteropathy (DE), namely “DE” medium, in place of regular FBS, L-Wnt3 cells were grown in 10% “DE” serum to generate conditioned medium that was further added 50:50 to Advanced DMEM/F12 medium in order to obtain the crypts culture medium as described above (see Crypts isolation and mini-guts development). 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. This has been established as model of intestinal stem cells-damage (4).
qRT-PCR Analysis
[0212] 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, and ΔCt values were calculated. Analysis was performed in technical and biological triplicates.
[0213] The list of genes which expression have been quantified by qRT-PCR is reported below.
TABLE-US-00002 TABLE 1 Gene Refseq Band Size Reference Symbol UniGene # 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 CasDase 8 Hs.599762 NM_001080124.1 124 648
Competitive ELISA Binding Assay
[0214] The following reagents were used to screen the newly generated anti-IGFBP3 antibodies: Recombinant Human IGFBP3 (0,223 mg/ml R&D System 8874-63-025), Ecto-TMEM219 (0.5 mg/ml GenScript), newly generated anti-IGFBP3 mAbs (Yumab, under contract), goat polyclonal anti-IGFBP3 (LS-C149975 LSBio), mouse monoclonal anti-IGFBP3 (LS-C45037 LSBio), mouse 6×His tag HRP (GTX30506 Genetex), bovine serum albumin (BSA A9418 Sigma), Tween 20 (TW P2287 Sigma), ELISA colorimetric TMB reagent (HRP substrate, Item H Sigma, RABTMB3), ELISA STOP solution (Item I, Sigma, RABSTOP3), blocking reagent solution (3% BSA in PBS) and a diluent solution (0.5% BSA, 0.05% Tw in PBS).
[0215] Microplate 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 follows: diluent solution (none), ecto-TMEM 10 μg/ml, ecto-TMEM 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.
Epitope Binning
[0216] Epitope binning is a competitive immunoassay used to characterize and then sort a library of monoclonal antibodies against a target protein (Abdiche, Y. N.; D. S. Malashock; A. Pinkerton; J. Pons (March 2009). “Exploring blocking assays using Octet, ProteOn, and Biacore biosensors”. Analytical Biochemistry. 386 (2): 172-180. doi:10.1016/j.ab.2008.11.038. PMID 19111520). Antibodies against a similar target are tested against all other antibodies in the library in a pairwise fashion to see if antibodies block one another's binding to the epitope of an antigen. After each antibody has a profile created against all of the other antibodies in the library, a competitive blocking profile is created for each antibody relative to the others in the library. Closely related binning profiles indicate that the antibodies have the same or a closely related epitope and are “binned” together. Epitope binning is referenced in the literature under different names such as epitope mapping and epitope characterization (Brooks, B. D. (2014). “The Importance of Epitope Binning in Drug Discovery”. Current Drug Discovery Technology. 11.).
[0217] CVC microplates were coated with: i) 100 μl/well of 2 μg/ml rhIGFBP3 dissolved in PBS or PBS alone (no coating) and incubated 60 minutes at RT; ii) 100 μl/well of 2 μg/ml anti-IGFBP3 (LS-C45037 LSBio) and incubated o.n at 4° C. Plates were washed with H.sub.2O-Tween20 (0.05%) and incubated with the blocking reagent (2% BSA-PBS-T (0.05%)). For condition ii) 100 μl/well of 2 μg/ml rhIGFBP3 was incubated o.n at 4° C. After washing the newly generated antibodies (in 2% BSA-PBS-T(0.05%)) were titrated onto the plate and incubated for 1 hour at RT. After washing steps, detection of bound antibodies was performed using a goat anti-human Fc-HRP antibody (Sigma A0170) in 2% BPS-PBS-T (0.05%) followed by washing and read out with TMB (100 μl/well). ELISA plates were then read measuring the absorbance at 450 nm.
Beta-Cells
[0218] 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).
Statistical Analysis
[0219] Data are presented as mean and standard deviation (SD) 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 and the chi-square (×2) tests. Significance between the two groups was determined by two-tailed unpaired Student's t test. For multiple comparisons, the 1-way ANOVA test with Bonferroni correction was employed. Statistical analysis was conducted using GraphPad Prism version 5.0 (GraphPad Software, La Jolla, Calif.). All statistical tests were performed at the 5% significance level.
Example 1
Monoclonal Antibodies Development
[0220] Monoclonal anti-IGFBP3 antibodies were discovered from naïve human phage-display libraries using a recombinant full length human IGFBP3 (R&D cat n° 8874-B3) as antigen for the screening. Briefly, human IGFBP3 (R&D cat n° 8874-B3) was biotinylated and immobilized via streptavidin onto 96-well ELISA plates at 4° C. After washing and blocking of the wells with BSA, the antibody-phage libraries were added. Tested libraries were first cleared from antibody-phage that bind to Streptavidin (John deKruif and Ton Logtenberg, Phage Display of Antibodies, Encyclopedia of Immunology (Second Edition) 1998, Pages 1931-1934). Those phages that carried an antigen-specific antibody were captured on the plate surface. After removal by washing with PBS-T of unbound/weakly bound phage with, antigen-specific phage were eluted and amplified by phage infection in E. coli. This amplified library subset was again selected for target binding under more stringent conditions. In total, three selection rounds were performed to enrich antigen specific antibody-phage.
[0221] At the end of the discovery process, each selection output was screened for antigen-specific antibodies. For this purpose, 384 clones were chosen from each selection output and used for production of monoclonal scFv antibodies. These were then tested for specific antigen binding by ELISA. 24 target specific hits were identified which were sequenced. All antibodies showing unique CDRs sequences were cloned into a mammalian scFv-Fc expression vector. As a result, the scFv was genetically fused to a Fc Fragment of human IgG4.
[0222] This resulted in 11 scFv-Fc antibodies which were produced by transient transfection of HEK293 cells. Then, the antibodies were purified by affinity chromatography (Protein A) and re-buffered in PBS (no additives). The protein concentration was determined by UV/VIS spectrometry and purity was checked by Coomassie staining.
[0223] The sequences of the 11 novel anti-IGFBP3 antibodies are reported in the Tables below.
TABLE-US-00003 TABLE 2 VH CDR Sequences of exemplified antibodies Antibody VH CDR1 VH CDR2 VH CDR3 YU139- SYAIS GIIPIFGTANYAQKFQG DYYDSSGYYFDAFDI A02 (SEQ ID NO: 1) (SEQ ID NO: 8) (SEQ ID NO: 17) YU139- SYGIH IVSYDGRHKYYADSVKG DSGNSGLEGIHDY A03 (SEQ ID NO: 2) (SEQ ID NO: 9) (SEQ ID NO: 18) YU139- SSNWWS (SEQ ID EVYHSGSTNYNPSLKS NIPLSSSWPNYYYYYGMDV B01 NO: 3) (SEQ ID NO: 10) (SEQ ID NO: 19) YU139- SYGIS WINTYNGNTNYAQKLQG DIGYSGSYYSPYYYYGMDV C01 (SEQ ID NO: 4) (SEQ ID NO: 11) (SEQ ID NO: 20) YU139- SYGIS WISAYNGNTNYAQKLQG VPSEYYDFWSGYYTEENAFDI C02 (SEQ ID NO: 4) (SEQ ID NO: 12) (SEQ ID NO: 21) YU139- SYGMH (SEQ ID VISYDGSNKYYADSVKG DNGDYGYYYYYYMDV D02 NO: 5) (SEQ ID NO: 13) (SEQ ID NO: 22) YU139- DYGIS WISGDNVKTTYAKKFQG GGIVFDY D03 (SEQ ID NO: 6) (SEQ ID NO: 14) (SEQ ID NO: 23) YU139- SYGIS WISAYNGNTNYAQKLQG GGIVFDY F02 (SEQ ID NO: 4) (SEQ ID NO: 12) (SEQ ID NO: 23) YU139- SYGIS WISAYNGNTYYAQKLQG LSYYNYAMDV G01 (SEQ ID NO: 4) (SEQ ID NO: 15) (SEQ ID NO: 24) YU139- TYVTS GIIPMFDTTEYAQKLEG AGGLSHFDY G03 (SEQ ID NO: 7) (SEQ ID NO: 16) (SEQ ID NO: 25) YU139- SYGIS WISAYNGNTNYAQKLQG VKWELGWAFDI H03 (SEQ ID NO: 4) (SEQ ID NO: 12) (SEQ ID NO: 26)
TABLE-US-00004 TABLE 3 VL CDR sequences of exemplified antibodies Antibody VL CDR1 VL CDR2 VL CDR3 YU139- SGSSSNIGTNTVS NNNERPS AAWDDNLTGLV A02 (SEQ ID NO: 27) (SEQ ID NO: 37) (SEQ ID NO: 45) YU139- RASQGISSWLA AASSLQS QQANSFPLT A03 (SEQ ID NO: 28) (SEQ ID NO: 38) (SEQ ID NO: 46) YU139- QGDSLRNYYAS GKNNRPS NSRDSSAKHWV B01 (SEQ ID NO: 29) (SEQ ID NO: 39) (SEQ ID NO: 47) YU139- QGDGLRNYFAS GKNNRPS NSRDGSGKHLV C01 (SEQ ID NO: 30) (SEQ ID NO: 39) (SEQ ID NO: 48) YU139- RASQSISSYLN AASSLQS QQSYSTWT C02 (SEQ ID NO: 31) (SEQ ID NO: 38) (SEQ ID NO: 49) YU139- RASQSISRWLA DASSLES QQSYSTPLT D02 (SEQ ID NO: 32) (SEQ ID NO: 40) (SEQ ID NO: 50) YU139- RASQSVSGNYLA GASSRAT QQYGSSPGT D03 (SEQ ID NO: 33) (SEQ ID NO: 41) (SEQ ID NO: 51) YU139- RASQSISSYLN AASSLQS QQSYSTPLT F02 (SEQ ID NO: 31) (SEQ ID NO: 38) (SEQ ID NO: 50) YU139- TGTSSDVGGYNHVS EVSNRPS SSYTSSNTWV G01 (SEQ ID NO: 34) (SEQ ID NO: 42) (SEQ ID NO: 52) YU139- RSSQSLLHSNGNNYLN LSSRRAS MQGLQTPWT G03 (SEQ ID NO: 35) (SEQ ID NO: 43) (SEQ ID NO: 53) YU139- TGSSSNIGAGYDVH GNSNRPS QSYDSSLSTV H03 (SEQ ID NO: 36) (SEQ ID NO: 44) (SEQ ID NO: 54)
TABLE-US-00005 TABLE 4 VH amino acid sequences of exemplified antibodies Antibody AA of VH YU139- QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQK A02 FQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDYYDSSGYYFDAFDIWGQGTMVTVSS (SEQ ID NO: 55) YU139- QVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGIHWVRQAPGKGLEWVAIVSYDGRHKYYAD A03 SVKGRFTISRDDSKNTIYLQMDSLRAEDTAVYYCAKDSGNSGLEGIHDYWGQGTLVTVSS (SEQ ID NO: 56) YU139- QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEVYHSGSTNYNP B01 SLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARNIPLSSSWPNYYYYYGMDVWGQGTTVTV SS (SEQ ID NO: 57) YU139- QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWINTYNGNTNYA C01 QKLQGRVTMTTDTSTTTAYMELRSLRSDDTAVYYCARDIGYSGSYYSPYYYYGMDVWGQGTTV TVSS (SEQ ID NO: 58) YU139- EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYA C02 QKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVPSEYYDFWSGYYTEENAFDIWGQGT MVTVSS (SEQ ID NO: 59) YU139- EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYAD D02 SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDNGDYGYYYYYYMDVWGKGTTVTVSS (SEQ ID NO: 60) YU139- QVQLVQSGAEVKKPGASVKVSCKASGYTFSDYGISWVRQAPGQGLEWMGWISGDNVKTTYA D03 KKFQGRVTLTTDTSTSTAYMELRSLTSDDTAAYYCARGGIVFDYWGQGTLVTVSS (SEQ ID NO: 61) YU139- QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQATGQGLEWMGWISAYNGNTNYA F02 QKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGGIVFDYWGQGTLVTVSS (SEQ ID NO: 62) YU139- QMQLVQSGAEVKMPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTYY G01 AQKLQGRVTMTADTSTSTAYMDLRSLRSDDTAVYYCARLSYYNYAMDVWGQGTTVTVSS (SEQ ID NO: 63) YU139- QVQLQQSGAEVKKPGSSVKVSCKASGGIFSTYVTSWVRQAPGQGLEWMGGIIPMFDTTEYAQ G03 KLEGRVTITVDESTNTAYMELSSLRFEDTAVYYCARAGGLSHFDYWGQGTLVTVSS (SEQ ID NO: 64) YU139- QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYA H03 QKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVKWELGWAFDIWGQGTMVTVSS (SEQ ID NO: 65)
TABLE-US-00006 TABLE 5 VL amino acid sequences of exemplified antibodies Antibody AA of VL YU139- QAGLTQPPSASGTPGQRVIISCSGSSSNIGTNTVSWYQHLPGTAPKLLIYNNNERPSGVPRRFS A02 GSRSGASASLAISGLQSDDEAHYYCAAWDDNLTGLVFGGGTKLTVL (SEQ ID NO: 66) YU139- DVVMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFS A03 GSGSGTDFTLTISSLQPEDFGTYYCQQANSFPLTFGGGTKVEIK (SEQ ID NO: 67) YU139- SSELTQDPAVSVALGQTVRITCQGDSLRNYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSG B01 SSSGNTASLTINGAQAEDEADYYCNSRDSSAKHWVFGGGTKLTV (SEQ ID NO: 68) YU139- SSELTQDPAVSVALGQTVRITCQGDGLRNYFASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSG C01 SSSGNTASLRIAGAQAEDEADYYCNSRDGSGKHLVFGGGTKLTV (SEQ ID NO: 69) YU139- DIVMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGS C02 GSGTDFTLTISSLQPEDFATYYCQQSYSTWTFGQGTKVEIK (SEQ ID NO: 70) YU139- DIQMTQSPSTLSASVGDRVTITCRASQSISRWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSG D02 SGSGTEFTLTISSLQPDDFATYYCQQSYSTPLTFGPGTKVDIK (SEQ ID NO: 71) YU139- ETTLTQSPGTLSLSPGERATLSCRASQSVSGNYLAWYQQKPGQPPRLLIFGASSRATGIPDRFSG D03 SGSGTDFTLTISRLEPEDFGVYYCQQYGSSPGTFGQGTKVEIK (SEQ ID NO: 72) YU139- DVVMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGS F02 GSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK (SEQ ID NO: 73) YU139- QSALTQPPSASGSPGQSVTISCTGTSSDVGGYNHVSWYQQHPGKAPKLMIYEVSNRPSGVPN G01 RFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSNTWVFGGGTKLTVL (SEQ ID NO: 74) YU139- EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGNNYLNWYLQKPGQSPQLLIYLSSRRASGVPDR G03 FSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQTPWTFGQGTKVETK (SEQ ID NO: 75) YU139- QAVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDR H03 FSGSKSGTSAPLAITGLQAEDEADYYCQSYDSSLSTVFGGGTKLTVL (SEQ ID NO: 76)
TABLE-US-00007 TABLE 6 VH nucleotide sequences of exemplified antibodies Antibody DNA of VH YU139- CAGGTTCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGT A02 CTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGC CCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTA CGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTA CATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGATTA CTATGATAGTAGTGGTTATTACTTTGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTC ACCGTCTCTTCA (SEQ ID NO: 77) YU139- CAGGTCCAGCTGGTACAATCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACT A03 CTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATCCACTGGGTCCGCCAGGCT CCAGGCAAGGGGCTGGAGTGGGTGGCAATTGTATCATATGATGGAAGACATAAATATTAT GCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACGATTCCAAGAACACGATCTAT CTGCAAATGGACAGCCTGAGAGCTGAGGACACGGCTGTATATTACTGTGCGAAAGATAGT GGGAACTCCGGTCTGGAAGGTATCCATGACTACTGGGGCCAGGGAACCCTGGTCACCGTC TCCTCA (SEQ ID NO: 78) YU139- CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGGGACCCTGTCCCTC B01 ACCTGCGCTGTCTCTGGTGGCTCCATCAGCAGTAGTAACTGGTGGAGTTGGGTCCGCCAG CCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAGTCTATCATAGTGGGAGCACCAACTA CAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACAAGTCCAAGAACCAGTTCTCC CTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGAGAAACATC CCCCTTAGCAGCAGCTGGCCTAATTACTACTACTACTACGGTATGGACGTCTGGGGCCAAG GGACCACGGTCACCGTCTCCTCA (SEQ ID NO: 79) YU139- CAGGTCCAGCTGGTGCAATCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGT C01 CTCCTGCAAGGCTTCTGGCTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCC CCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACACTTACAATGGTAACACAAACTAT GCACAGAAACTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGACCACAGCCTAC ATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTATATTACTGTGCGAGAGATATC GGGTATAGTGGGAGCTACTATTCGCCCTACTACTACTACGGTATGGACGTCTGGGGCCAA GGGACCACGGTCACCGTCTCCTCA (SEQ ID NO: 80) YU139- GAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCTTCAGTGAAGGT C02 CTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCC CCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTAT GCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTAC ATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCTGTGTATTACTGTGCGAGAGTTCCC TCAGAGTATTACGATTTTTGGAGTGGTTATTATACCGAAGAGAATGCTTTTGATATCTGGG GCCAAGGGACAATGGTCACCGTCTCTTCA (SEQ ID NO: 81) YU139- GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACT D02 CTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCT CCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTAT GCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTAT CTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAAAGATAAC GGTGACTACGGATACTACTACTACTACTACATGGACGTCTGGGGCAAAGGGACCACGGTC ACCGTCTCCTCA (SEQ ID NO: 82) YU139- CAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGT D03 CTCCTGCAAGGCTTCTGGTTACACCTTTTCCGACTATGGTATCAGCTGGGTGCGACAGGCC CCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGGTGACAATGTTAAGACTACCTAT GCAAAGAAGTTCCAGGGCAGAGTCACCCTGACCACAGACACATCCACGAGCACAGCCTAC ATGGAGCTGAGGAGCCTGACATCTGACGACACGGCCGCATATTACTGTGCGAGAGGGGG GATCGTATTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 83) YU139- CAGGTTCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGT F02 CTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCC ACTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTAT GCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTAC ATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGGGGG GATCGTATTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 84) YU139- CAAATGCAGCTGGTACAGTCTGGGGCTGAGGTGAAGATGCCTGGGGCCTCAGTGAAGGT G01 CTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCC CCTGGACAAGGGCTTGAGTGGATGGGGTGGATCAGCGCTTACAATGGTAACACATACTAT GCACAGAAACTCCAGGGCAGAGTCACCATGACCGCAGACACATCCACGAGCACAGCCTAC ATGGACCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGACTATCA TATTACAACTACGCTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO: 85) YU139- CAGGTACAGCTGCAGCAGTCAGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGT G03 GTCCTGCAAGGCTTCTGGAGGCATCTTCAGCACCTATGTGACCAGCTGGGTGCGACAGGC CCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATGTTTGACACAACAGAGTA CGCACAGAAGCTCGAGGGCAGAGTCACGATCACCGTGGACGAATCCACGAACACAGCCT ACATGGAGCTGAGCAGCCTGAGATTTGAGGACACGGCCGTATATTACTGTGCGAGAGCG GGAGGTTTGAGCCACTTTGACTACTGGGGCCAGGGAACTCTGGTCACCGTCTCGTCA (SEQ ID NO: 86) YU139- CAGGTCCAGCTGGTACAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGT H03 CTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCC CCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTAT GCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACAAGCACAGCCTAC ATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGTGAA GTGGGAGCTAGGGTGGGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTC A (SEQ ID NO: 87)
TABLE-US-00008 TABLE 7 VL nucleotide sequences Antibody DNA of VL YU139- CAGGCTGGGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCATCATC A02 TCTTGTTCTGGAAGCAGCTCCAACATCGGAACTAATACTGTAAGCTGGTATCAACACCTCC CAGGAACGGCCCCCAAGCTCCTCATCTATAACAATAATGAACGGCCCTCAGGGGTCCCTCG CCGATTCTCTGGCTCCAGGTCTGGCGCCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCT GACGATGAGGCTCATTATTATTGTGCAGCCTGGGATGACAACCTGACTGGCTTGGTGTTCG GCGGAGGGACCAAACTGACCGTCCTA (SEQ ID NO: 88) YU139- GATGTTGTGATGACTCAGTCTCCTTCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCA A03 TCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAG GGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAG GTTCAGCGGCAGTGGATCTGGGACAGATTTCACACTCACCATCAGCAGCCTGCAGCCTGA AGATTTTGGAACTTACTATTGTCAACAGGCTAACAGTTTTCCGCTCACTTTCGGCGGAGGG ACCAAGGTGGAGATCAAA (SEQ ID NO: 89) YU139- TCTTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCA B01 CATGCCAAGGAGACAGCCTCAGAAACTATTATGCAAGCTGGTACCAGCAGAAGCCAGGAC AGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATT CTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCAATGGGGCTCAGGCGGAAGA TGAGGCTGACTATTACTGTAACTCCCGGGACAGCAGTGCTAAACATTGGGTGTTCGGCGG AGGGACCAAGCTGACCGTC (SEQ ID NO: 90) YU139- TCTTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCA C01 CGTGCCAAGGAGACGGCCTCAGAAACTATTTTGCAAGTTGGTACCAGCAGAAGCCAGGAC AGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATT CTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGAGAATCGCTGGGGCTCAGGCGGAAGA TGAGGCTGACTATTACTGTAATTCCCGGGACGGCAGTGGTAAGCATCTGGTATTCGGCGG AGGGACCAAGTTGACCGTC (SEQ ID NO: 91) YU139- GACATCGTGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCA C02 TCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAG GGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAG GTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAA GATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCTGGACGTTCGGCCAAGGGACCA AGGTGGAAATCAAA (SEQ ID NO: 92) YU139- GACATCCAGATGACTCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCA D02 TCACTTGCCGGGCCAGTCAGAGTATTAGTAGGTGGTTGGCCTGGTATCAGCAGAAACCAG GGAAAGCCCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAA GGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTG ATGATTTTGCAACTTATTACTGTCAACAGAGTTACAGTACCCCTCTTACTTTCGGCCCTGGG ACCAAAGTGGATATCAAA (SEQ ID NO: 93) YU139- GAAACGACACTCACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACC D03 CTCTCCTGCAGGGCCAGTCAGAGTGTTAGTGGCAACTACTTAGCCTGGTACCAGCAGAAA CCTGGCCAGCCTCCCAGGCTCCTCATCTTTGGTGCATCCAGCAGGGCCACTGGCATCCCAG ACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGC CTGAAGATTTTGGAGTGTATTACTGTCAGCAGTATGGTAGCTCACCAGGGACGTTCGGCC AAGGGACCAAGGTGGAAATCAAA (SEQ ID NO: 94) YU139- GATGTTGTGATGACTCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCA F02 TCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAG GGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAG GTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAA GATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCGCTCACTTTCGGCGGAGGGA CCAAGGTGGAGATCAAA (SEQ ID NO: 95) YU139- CAGTCTGCCCTGACTCAGCCTCCCTCCGCGTCCGGGTCCCCTGGACAGTCAGTCACCATCTC G01 CTGCACTGGAACCAGCAGTGATGTTGGTGGTTATAACCATGTCTCCTGGTACCAGCAACAC CCAGGCAAAGCCCCCAAACTCATGATTTATGAGGTCAGTAATCGGCCCTCAGGAGTTCCTA ATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGC TGAGGACGAGGCTGATTATTACTGCAGCTCATATACAAGCAGCAACACTTGGGTGTTCGG CGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 96) YU139- GAAATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCA G03 TCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGAAACAACTATTTGAATTGGTA CCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGAGTTCTCGTCGGGCCTCC GGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATTAGC AGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTCTACAAACTCCGTGG ACGTTCGGCCAAGGGACCAAGGTGGAAACCAAA (SEQ ID NO: 97) YU139- CAGGCAGTGCTGACTCAGCCACCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATC H03 TCCTGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGTACACTGGTACCAGCAG CTTCCAGGAACAGCCCCCAAACTCCTCATCTATGGTAACAGCAATCGGCCCTCAGGGGTCC CTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCCCCCTGGCCATCACTGGGCTCCA GGCTGAGGACGAGGCTGATTATTACTGCCAGTCTTATGACAGCAGCCTGAGTACGGTATT CGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 98)
TABLE-US-00009 TABLE 8 Constant region amino acid sequences Constant region AA Human IgG4 heavy ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF chain PAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC P01861.1 PSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK (SEQ ID NO: 99) Human IgG2 heavy ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF chain P01859 PAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVE CPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNW YVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKG LPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVE WESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK (SEQ ID NO: 100) Human light chain, GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKA lambda 1 GVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPT P0CG04 ECS (SEQ ID NO: 101) Human light chain, GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAG lambda 2 VETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTE P0DOY2 CS (SEQ ID NO: 102) Human light chain, RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN kappa SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR P01834 GEC (SEQ ID NO: 103)
Example 2
[0224] A Commercially Available Anti-IGFBP3 Monoclonal Antibody does not Affect IGFBP3/TMEM219 Binding
[0225] A commercially available anti-IGFBP3 antibody was tested for its ability to displace the binding of ecto-TMEM219 to IGFBP3. Displacement of binding would indicate both (i) the ability of the antibody to inhibit binding of IGFBP3 to the native TMEM219 receptor, and (ii) the ability of the antibody to mimic the neutralizing activities of the ecto-TMEM219 ligand trap fusion protein.
[0226] As shown in
[0227] As shown in
Commercially Available Anti-IGFBP3 Antibodies do not Rescue IGFBP3-Mediated Damage of Mini-Guts in Different Disease Models.
[0228] In order to assess whether commercially available antibodies, both monoclonal and polyclonal, could mimic the ability of the ecto-TMEM219 ligand trap to rescue mini-gut growth in intestinal stem cell (ISO) injury disease conditions though preventing the binding of IGFBP3 to TMEM219, they were further tested in vitro in the mini-gut assay. Mini-guts were generated from healthy controls as already described (4, 18). As shown in the left-most 3 bars in
[0229] Monoclonal and polyclonal antibodies were tested at three different concentrations in the presence of medium from individuals with diabetic enteropathy (“Medium DE”). Neither commercially available monoclonal nor polyclonal anti-IGFBP3 antibodies significantly improved the development of mini-guts in the presence of medium from individuals with diabetic enteropathy. None of the tested antibodies mimicked the ability of ecto-TMEM219 to rescue mini-gut development, suggesting that commercial anti-IGFBP3 antibodies would not be able to prevent ISO damage in disease conditions such as diabetic enteropathy; consistent with the data from Example 1, we infer that this lack of functional activity can be attributed to their inability to prevent binding of IGFBP3 to TMEM219 binding.
[0230] This highlights the need for other anti-IGFBP3 antibodies that selectively prevent the IGFBP3-TMEM219 interaction.
[0231] Antibodies were also tested in mini-gut assays using crypts obtained from patients with established Crohn's disease (an inflammatory bowel disease, IBD) undergoing surgery for disease complications (strictures, fistulae). This model is highly clinically relevant as it recapitulates in vitro the major features of the disease condition in patients.
[0232] As shown in
[0233] Next, we showed that the expression of the ISC markers EphB2, LGR5, and h-TERT in IBD patients, detected by RT-PCR was not rescued by the addition of commercially available anti-IGFBP3 Abs, in contrast to Ecto-TMEM219 (
[0234] These results confirm the poor effects of the commercial anti-IGFBP3 monoclonal and polyclonal antibodies in targeting ISCs function (mini-guts development) and in rescuing ISC markers expression. This also emphasizes that different disease conditions (e.g. diabetic enteropathy, IBD) in which ISCs are damaged due to the activation of IGFBP3-TMEM219 signaling, may benefit from a strategy that selectively targets it.
Example 3
[0235] Novel Anti-IGFBP3 mAbs Inhibit IGFBP3-TMEM219 Binding
[0236] As discussed in Example 1, a set of anti-IGFBP3 monoclonal antibodies was selected by phage display. These novel antibodies were then screened for their ability to compete with ecto-TMEM for the binding to IGFBP3 using a competitive ELISA binding assay. IGFBP3 (coating), ecto-TMEM219 and the anti-IGFBP3 antibodies selected by phage display were all used in a 1:1 molecular ratio. As shown in
[0237] This demonstrates that the majority of the newly discovered Anti-IGFBP3 mAbs may prevent the IGFBP3-TMEM219 binding to a higher rate as compared to the commercially available anti-IGFBP3 antibody.
Novel Anti-IGFBP3 Antibodies Rescue IGFBP3-Damage in the Mini-Guts Assay
[0238] The 11 newly discovered monoclonal antibodies were also tested in the mini-gut assay. Mini-guts were generated from crypts obtained from healthy controls and cultured for 8 days in presence of IGFBP3 and treated with either ecto-TMEM219 or the newly discovered anti-IGFBP3 mAbs All factors were included after the first day of culture at a 1:1 ratio (mAbs/ecto-TMEM219:IGFBP3).
[0239] As shown in
[0240] Further, antibodies YU139-001, YU139-A03, YU139-G03, and YU139-H03 also rescue the negative effects of IGFBP3 on self-renewal ability (% development) and morphology (absence of crypts domain, generation of small spheroids) of large crypt organoids (
Novel Monoclonal Anti-IGFBP3 Antibodies Rescue IGFBP3-Damage on ISCs Markers
[0241] The newly discovered anti-IGFBP3 mAbs which were shown to be effective in promoting mini-guts development upon IGFBP3 exposure were also able to significantly upregulate the expression of ISCs markers EphB2 and LGR5 (
Novel Monoclonal Anti-IGFBP3 Antibodies Inhibit IGFBP3-Mediated Overexpression of Caspase 8
[0242] IGFBP3-detrimental effects on ISCs 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 (
[0243] These results suggest that the discovered anti-IGFBP3 mAbs exert a protective effect on ISCs pool by blocking the IGFBP3/TMEM219 Caspase-8-mediated apoptotic injury.
The Newly Discovered Anti-IGFBP3 Antibodies Rescue Mini-Guts Growth in Disease Models.
[0244] In order to confirm that the newly discovered monoclonal anti-IGFBP3 antibodies prevent the detrimental effects of IGFBP3 on TMEM219-expressing intestinal stem cells, the inventors further tested them in vitro in the mini-gut assay in an established model of DE.
[0245] The novel anti-IGFBP3 mAbs significantly improved the development of mini-guts in DE of at least 20%, similarly to Ecto-TMEM219 treatment (
[0246] This highlights that anti-IGFBP3 mAbs of the invention, selected both for binding to IGFBP3 and screened 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.
[0247] The inventors also demonstrated that mini-guts growth was significantly recovered, by at least 20%, when treating mini-guts obtained from IBD patients with the newly discovered anti-IGFBP3 mAbs. The effects obtained with the novel anti-IGFBP3 mAbs were similar to that exerted by ecto-TMEM219 (
[0248] These results show a potential benefit in preserving the ISCs pool and function also in this disease model.
The Newly Discovered Human Monoclonal Anti-IGFBP3 Antibodies Bind a Different Region of the Antigen from the Commercial Mouse Monoclonal Anti-IGFBP3
[0249] An epitope binning experiment was performed to check whether commercial antibody LSBIO LS-C45037 (clone 83.8F9) and the newly developed anti-IGFBP3 antibodies bind to the same epitope region of IGFBP3. Briefly, a sandwich ELISA was performed coating the multiwell plate with the prior art antibody. All of the novel anti-IGFBP3 monoclonal antibodies selected by phage display were able to bind to IGFBP3 in the presence of commercial antibody LSBIO LS-C45037 (clone 83.8F9).
[0250] This result suggests that the epitope on IGFBP3 bound by the newly discovered antibodies is different from that of commercially available antibody (
Example 4
Newly Generated Monoclonal Anti-IGFBP3 Antibodies do not Displace IGF-I-IGFBP3 Binding
Competitive ELISA Binding Assay
[0251] Newly generated anti-IGFBP3 mAbs were tested for their ability to displace the IGF-I-IGFBP3 interaction by using a competitive ELISA in vitro. Briefly, plate was coated with IGFBP3 (Life Technologies) 4 μg/ml. IGF-I (R&D, 291-G1-01M) was added in a dose dependent ratio with IGFBP3 (0:1, 0.5:1 and 2:1). The newly generated anti-IGFBP3 mAbs (Yumab) of the invention were added at a concentration of 1:1 as compared to IGFBP3. IGFBP3-IGF-I binding was revealed by using anti-IGF-I HRP conjugated antibody.
[0252] The following reagents were used to screen the anti-IGFBP3 Antibodies: Recombinant Human IGFBP-3 (0,223 mg/ml R&D System, 675-B3-025), IGF-I (R&D, 291-G1-01M), anti-IGFBP3 mAbs (Yumab), mouse monoclonal anti-IGFBP3 (Novus Biological, NBP2-12364), anti-IGF-I HRP (LSBio, C547140), bovine serum albumin (BSA, Sigma A7906), Tween 20 (TW), ELISA colorimetric TMB reagent (HRP substrate, Item H Sigma, RABTMB3), ELISA STOP solution (Item I, Sigma, RABSTOP3), blocking reagent solution (3% BSA in PBS) and a diluent solution (0.5% BSA, 0.05% Tw in PBS).
[0253] 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), IGF-I at different ratios with rhIGFBP3 (0:1, 0.5:1 and 2:1) and anti-IGFBP3 mAbs at 10 μg/ml. After washing steps, plate was then incubated at room temperature for 1 hour with anti-IGF-I HRP diluted in Diluent solution (50 μl/well). ELISA plate was then read after adding visualization solution at ELISA reader.
[0254] As shown in
[0255] In other words, when IGF1 is available, IGFBP3 preferentially binds to IGFF1 and the newly generated mAbs do not interfere with the IGF1-IGFBP3 binding.
Newly Generated Monoclonal Anti-IGFBP3 Antibodies Rescue IGFBP3-Damage in Murine Mini-Guts
Crypts Isolation and Murine Mini-Guts Development
[0256] 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 C57BL/6J mice (632C57BL/6J Charles River Laboratories, Lyon, France). 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. Then, 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.
[0257] 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). Miniguts 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).
[0258] As shown in
Affinity Measurement
Octet BLI-Based Analysis
[0259] To assess the binding affinity in more detail, affinity measurements were performed using Biomolecular Interaction Analysis performed by an Octet instrument (BLItz System), which is a Biolayer Interferometry (BLI) platform. To establish the assay, the target monoclonal antibody (30 μg/ml in 1% BSA-PBST) was immobilized via Fc on the via AHC biosensor and the interaction with the antigen human IGFBP3 (R&D, cat n° 675 B3) and mouse IGFBP3 (Creative Biomart, cat n° igfbp3 829M) at different concentration was measured.
[0260] The affinity measurement of YU139-C01, YU139-H03, YU139-G03 and YU139-A03 for the target human and murine IGFBP3 are reported in the Table 9.
TABLE-US-00010 TABLE 9 Affinity of YU139-C01, YU139-H03, YU139-G03 and YU139-A03 for the target human and murine IGFBP3. Molecule EctoTMEM- YU139- YU139- YU139- id His C01 H03 A03 hIGFBP-3 3.5 × 10.sup.−8 1.69 × 10.sup.−9 M 4.0 × 10.sup.−6 3.28 × 10.sup.−7 KD (M) mIGFBP-3 6.0 × 10.sup.−8 3.42 × 10.sup.−7 >10.sup.−6 7.35 × 10.sup.−7 KD (M)
[0261] Newly generated anti-IGFBP3 mAbs show good human antigen binding affinity with KD below 4×10.sup.−6 M. The antibodies also show murine cross reactivity. This data confirmed that mice can be considered a relevant animal species for testing of the monoclonal antibodies during preclinical development.
Anti-IGFBP3 mAbs Efficacy in IBD Mouse Model Following Intraperitoneal (IP) Administration
[0262] The model of colitis induced by Dextran sulfate sodium (DSS) in C57BL/6J mice is a validated animal model to evaluate and also to confirm the anti-inflammatory properties of drugs in IBD. DSS (oral administration in the drinking water) induces prominent diarrhea followed by inflammation. This model is well characterized, reliable, reproducible and admitted by regulatory authorities in IBD with no mortality. This study was performed in C57BL/6J mice. Indeed, in this particular genetic background, mice develop acute colitis when analyzed 3 days after the last DSS administration or a chronic-like inflammation when analyzed 7 days after the last DSS administration.
Animals
[0263] Male C57BL/6J mice were supplied by Charles River laboratories, l'Arbresle, France. The mice were housed at 20±5° C. and provided with water and food ad libitum. All experimental protocols were performed in accredited facilities at Institut Pasteur from Lille according to governmental guidelines.
Establishment of DSS-Induced Mice Colitis Model and Treatment
[0264] Acute colitis was induced by feeding mice with 2.5% (w/v) DSS (45 kD; TDB Consultancy AB, Uppsala, Sweden, Batch number DB001-41) dissolved in drinking water for 5 days. The mice were randomly divided into eight groups: control group; DSS+vehicle; DSS+Yu139-CO1 0.6 mg/mouse, DSS+Ecto-TMEM 0.1 mg/mouse, DSS+mouse anti-TNFα 0.1 mg/mouse (Ozyme, B270358), DSS+Pentoxifylline (Sigma P1784, BCBQ05 15), DSS+Humira (Abbvie, 1108722) ip, DSS+Humira sc. To assess the preventive therapeutic effects of anti-IGFBP3 mAb on DSS-induced acute colitis in C57BL/6J mice, the mice were treated daily by intraperitoneal administration with indicated dose of Yu139-CO1 starting 3 days before colitis induction and were performed until euthanasia occurring 7 days after the last DSS administration. The experimental timelines of the animal model are described in
[0265] The therapeutic properties of the biologics were compared to those induced by positive control groups, mouse anti-TNFα antibody, Pentoxifylline and Humira (Taghipour N. et al Gastroenterol Hepatol Bed Bench 2016; 9(1):45-52).
[0266] As shown on
[0267] As shown on
[0268]
Clinical Scoring
[0269] In all groups, mice weight, stool consistency and blood in stool were recorded daily. The disease activity index (DAI) scores were based on changes in body weight, consistency of stool, and hemoccult bleeding according to a standard scoring system. These parameters were assessed on a scale as described in the Table 10. The DAI data are presented as an average score of these parameters taken daily. Animals were sacrificed by cervical dislocation under anesthesia. At euthanasia, colons were carefully dissected, and its weight and size were measured. At euthanasia, the presence of Occult Blood (OB) is recorded using the hemoccult method.
TABLE-US-00011 TABLE 10 Parameters assessed for DAI score Parameters Scores Disease Weight loss 0: no; 1: <10%; 2: ≥10% Activity Index Stool consistency 0: regular; 1: soft; 2: diarrhea (DAI) (0-5) Blood occurrence 0: absence; 1: presence
Statistical Analysis
[0270] All comparisons were analysed using the Permutation Test for two independent samples. Statistics have been calculated using the GraphPad Prism version 7.0 (GraphPad Software, San Diego, Calif.). Differences were considered statistically significant if the p value was <0.05.
Anti-IGFBP3 mAbs Efficacy in T1D Mouse Model Following Intraperitoneal (IP) Administration
Animals
[0271] Female non-obese diabetic (NOD) mice (10 weeks old) were obtained from the Jackson Laboratory, Bar Harbor, Me. (JAX stock#001976). All mice were cared for and used in accordance with institutional guidelines approved by the Jackson Laboratory Institutional Animal Care and Use Committee.
Diabetes Monitoring and Treatment
[0272] Overt diabetes (the most advanced stage, characterized by elevated fasting blood glucose concentration and classical symptoms) was defined as blood glucose levels above 300 mg/dL. Blood glucose was measured using the Breeze2 (Bayer S.p.A) blood glucose meter.
[0273] The experiment was randomly divided into three groups: control group; Yu139-CO1 0.25 mg/mouse and Ecto-TMEM 0.1 mg/mouse. To determine whether the anti-IGFBP3 mAb prevents diabetes in NOD mice, animals were treated daily for 10 days by intraperitoneal administration with the indicated dose of Yu139-CO1. The experimental timelines are described in
Statistical Analysis
[0274] Data are presented as mean and standard deviation (SD) 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 and the chi-square (χ2) tests. Significance between the two groups was determined by two-tailed unpaired Student's t test. For multiple comparisons, the 1-way ANOVA test with Bonferroni correction was employed. 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.
[0275] As shown in
INCORPORATION BY REFERENCE
[0276] 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
[0277] 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.
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