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WO2025224297A1 - Antibodies having specificity to tgfbi and uses thereof - Google Patents

Antibodies having specificity to tgfbi and uses thereof

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Publication number
WO2025224297A1
WO2025224297A1 PCT/EP2025/061321 EP2025061321W WO2025224297A1 WO 2025224297 A1 WO2025224297 A1 WO 2025224297A1 EP 2025061321 W EP2025061321 W EP 2025061321W WO 2025224297 A1 WO2025224297 A1 WO 2025224297A1
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WIPO (PCT)
Prior art keywords
amino acid
mab
acid residue
antibody
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/061321
Other languages
French (fr)
Inventor
Andrei Turtoi
Bilguun ERKHEM-OCHIR
Bruno Robert
Pierre Martineau
Claire CRAMPES
Hajar ABEDI-JONI
Marie-Alix Poul
Haruka OKAMI
Takehiko Yokobori
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institut National de la Sante et de la Recherche Medicale INSERM
Universite de Montpellier
Institut Regional du Cancer de Montpellier
Gunma University NUC
Original Assignee
Institut National de la Sante et de la Recherche Medicale INSERM
Universite de Montpellier
Institut Regional du Cancer de Montpellier
Gunma University NUC
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Application filed by Institut National de la Sante et de la Recherche Medicale INSERM, Universite de Montpellier, Institut Regional du Cancer de Montpellier, Gunma University NUC filed Critical Institut National de la Sante et de la Recherche Medicale INSERM
Publication of WO2025224297A1 publication Critical patent/WO2025224297A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to antibodies having specificity to TGFBI and uses thereof.
  • Cancer cachexia is a multifactorial syndrome characterized by a continuous loss of skeletal muscle mass (with or without fat loss) that cannot be completely reversed by conventional nutritional support. Cachexia occurs in 50-80% of advanced cancers (mainly of gastrointestinal and lung type), and as such it represents a true challenge in the clinics.
  • the cachexic syndrome is fueled by continuous metabolic wasting, frequently accompanied by reduced appetite and food intake (Fearon K et al., 2011, Lancet Oncol). Beyond cancer, chronic infections (e.g. HIV infection, tuberculosis) as well as kidney failure can also cause cachexia accompanied with muscle weakness and organ damage.
  • macrophages that inherent with their ability to assume pro- and anti-inflammatory states, can both enhance (Shukla S.K et al., 2020, Cancer Lett) as well as suppress cachexia (Erdem M et al., 2019, J Cachexia Sarcopenia Muscle). Indeed in obesity, a shift of resident macrophages from M2 (antiinflammatory) to Ml (pro-inflammatory) phenotype has been previously observed in adipose tissue (McNelis J.C et al., 2014, Immunity). There, the newly established pro-inflammatory condition favors T-cell infiltration, adipocyte necrosis and insulin resistance. While the link between macrophage polarization states and cachexia is yet to be investigated in detail, it is not unreasonable to assume that factors that determine macrophage polarization may be involved in modulating cachexia.
  • TGFBI loss-of-function in macrophages inhibited their polarization to M2 phenotype (Zhou J et al., 2023, Cancer Lett), while other findings have clearly demonstrated that M2 macrophages in tumors secrete high levels of TGFBI (Zhou J et al., 2023, Cancer Lett Peng P et al., 2022, Theranostics & Lecker L.S.M et al., 2021, Cancer Res).
  • TGFBI has been reported to be also secreted by cancer-associated fibroblast (CAF) as well as cancer cells in limited manner.
  • CAF cancer-associated fibroblast
  • TGFBI tumor associated macrophage
  • the anti-TGFBI antibody is selected among 7 antibodies (called also here A6, C7, C9, D2, D5, E4 and G7).
  • the anti-TGFBI antibody is selected among 2 antibodies (called also here A6 and C9).
  • the invention relates to an anti-TGFBI antibody having a heavy chain comprising i) the H-CDR1 of A6 mab, ii) the H-CDR2 of A6 mab and iii) the H-CDR3 of A6 mab and a light chain comprising i) the L-CDR1 of A6 mab, ii) the L-CDR2 of A6 mab and iii) the L-CDR3 of A6 mab
  • the H-CDR1 of the A6 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO: 1;
  • H-CDR2 of A6 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO: 1;
  • the H-CDR3 of the A6 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO: 1.
  • the L-CDR1 of the A6 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO:2;
  • the L-CDR2 of the A6 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO:2;
  • the L-CDR3 of the A6 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO:2; or an anti-TGFBI antibody having a heavy chain comprising i) the H-CDR1 of C7 mab, ii) the H-CDR2 of C7 mab and iii) the H-CDR3 of C7 mab and a light chain comprising i) the L-CDR1 of C7 mab, ii) the L-CDR2 of C7 mab and iii) the L-CDR3 of C7 mab
  • an anti-TGFBI antibody having a heavy chain comprising i) the H-CDR1 of C7 mab, ii) the H-CDR2 of C7 mab and iii) the H-CDR3 of C7 mab and a light chain comprising i) the L-CDR1 of C7 mab, ii) the L-C
  • the H-CDR1 of the C7 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO:3;
  • H-CDR2 of C7 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO:3;
  • the H-CDR3 of the C7 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO:3.
  • the L-CDR1 of the C7 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO:4;
  • the L-CDR2 of the C7 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO:4;
  • the L-CDR3 of the C7 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO:4; or an antibody having a heavy chain comprising i) the H-CDR1 of C9 mab, ii) the H- CDR2 of C9 mab and iii) the H-CDR3 of C9 mab and a light chain comprising i) the L- CDR1 of C9 mab, ii) the L-CDR2 of C9 mab and iii) the L-CDR3 of C9 mab
  • the H-CDR1 of the C9 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO: 5;
  • H-CDR2 of C9 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO:5;
  • the H-CDR3 of the C9 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO: 5.
  • the L-CDR1 of the C9 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO:6;
  • the L-CDR2 of the C9 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO:6;
  • the L-CDR3 of the C9 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO: 6; or an antibody having a heavy chain comprising i) the H-CDR1 of D2 mab, ii) the H- CDR2 of D2 mab and iii) the H-CDR3 of D2 mab and a light chain comprising i) the L-CDR1 of D2 mab, ii) the L-CDR2 of D2 mab and iii) the L-CDR3 of D2 mab
  • the H-CDR1 of the D2 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO: 7;
  • the H-CDR2 of D2 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO:7;
  • the H-CDR3 of the D2 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO:7.
  • the L-CDR1 of the D2 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO:8;
  • the L-CDR2 of the D2 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO:8;
  • the L-CDR3 of the D2 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO:8; or an antibody having a heavy chain comprising i) the H-CDR1 of D5 mab, ii) the H- CDR2 of D5 mab and iii) the H-CDR3 of D5 mab and a light chain comprising i) the L-CDR1 of D5 mab, ii) the L-CDR2 of D5 mab and iii) the L-CDR3 of D5 mab
  • the H-CDR1 of the D5 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO: 9;
  • the H-CDR2 of D5 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO:9;
  • the L-CDR2 of the D5 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO: 10;
  • the L-CDR3 of the D5 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO: 10; or an antibody having a heavy chain comprising i) the H-CDR1 of E4 mab, ii) the H- CDR2 of E4 mab and iii) the H-CDR3 of E4 mab and a light chain comprising i) the L- CDR1 of E4 mab, ii) the L-CDR2 of E4 mab and iii) the L-CDR3 of E4 mab
  • the H-CDR1 of the E4 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO: 11;
  • H-CDR2 of E4 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO: 11;
  • the H-CDR3 of the E4 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO: 11.
  • the L-CDR3 of the E4 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO: 12; or an antibody having a heavy chain comprising i) the H-CDR1 of G7 mab, ii) the H- CDR2 of G7 mab and iii) the H-CDR3 of G7 mab and a light chain comprising i) the L-CDR1 of G7 mab, ii) the L-CDR2 of G7 mab and iii) the L-CDR3 of G7 mab
  • the H-CDR1 of the G7 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO: 13;
  • the H-CDR3 of the G7 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO: 13.
  • the L-CDR2 of the G7 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO: 14;
  • TGFBI Transforming Growth Factor-Beta-Induced
  • Pig-h3 Transforming Growth Factor-Beta-Induced
  • the Entrez reference number of the human gene coding for TGFBI is 7045 and the Uniprot reference number of TGFBI human protein is QI 5582.
  • TGFBI is involved in endochondrial bone formation in cartilage. TGFBI can activate cancer aggressiveness via the activation of integrin signaling.
  • TGFBI has been reported to be also secreted by cancer-associated fibroblast (CAF) as well as cancer cells in limited manner which express TGFBI especially when metastatic (such as CTCs) (Chiavarina B et al., 2021, Theranostics). TGFBI expression is induced by the activation of transforming growth factor-P (TGF-P) signal (Yokobori T et al., 2017, J Clin Med).
  • CAF cancer-associated fibroblast
  • TGF-P transforming growth factor-P
  • antibody or “immunoglobulin” have the same meaning, and will be used equally in the present invention.
  • the term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen.
  • the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments.
  • two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (1) and kappa (k).
  • the heavy chain includes two domains, a variable domain (VL) and a constant domain (CL).
  • the heavy chain includes four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH).
  • VL variable domain
  • VH variable domain
  • CH constant domain
  • the constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR).
  • the Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain.
  • the specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant.
  • Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FR) can participate to the antibody binding site or influence the overall domain structure and hence the combining site.
  • Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site.
  • the light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L- CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively.
  • An antigen-binding site therefore, typically includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region.
  • Framework Regions refer to amino acid sequences interposed between CDRs.
  • the antibody of the invention is a monoclonal antibody.
  • the amino acid residues of the antibody of the invention are numbered according to the KABAT numbering system.
  • This system is set forth in Kabat et al., 1987, in sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NTH, USA (hereafter “Kabat et al.”). This numbering system is used in the present specification.
  • the Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues in SEQ ID sequences.
  • the actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure.
  • CDR complementarity determining region
  • the correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a “standard” Kabat numbered sequence.
  • the CDRs of the heavy chain variable domain are located at residues 31-35B (H- CDR1), residues 50-65 (H-CDR2) and residues 95-102 (H-CDR3) according to the Kabat numbering system.
  • the CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2) and residues 89-97 (L-CDR3) according to the Kabat numbering system. (http://www.bioinf.org.Uk/abs/#cdrdef).
  • the term “specificity” refers to the ability of an antibody to detectably bind an epitope presented on an antigen, such as TGFBI, while having relatively little detectable reactivity with non-TGFBI proteins or structures (such as other proteins presented on cancerous cell, or on other cell types). Specificity can be relatively determined by binding or competitive binding assays, using, e.g., Biacore instruments, as described elsewhere herein. Specificity can be exhibited by, e.g., an about 10: 1, about 20: 1, about 50: 1, about 100: 1, 10.000: 1 or greater ratio of affinity/avidity in binding to the specific antigen versus nonspecific binding to other irrelevant molecules (in this case the specific antigen is TGFBI).
  • affinity refers to the strength of the binding of an antibody to an epitope.
  • the affinity of an antibody is given by the dissociation constant Kd, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of the unbound antibody and [Ag] is the molar concentration of the unbound antigen.
  • Kd dissociation constant
  • Ka is defined by 1/Kd.
  • monoclonal antibody As used herein, the terms “monoclonal antibody”, “monoclonal Ab”, “monoclonal antibody composition”, “mAb”, or the like, refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • the VH region of the A6 mab consists of the sequence of SEQ ID NO: 1. Accordingly, the H-CDR1 of the A6 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO: 1. Accordingly, the H-CDR2 of A6 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO: 1. Accordingly, the H-CDR3 of the A6 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO: 1.
  • SEQ ID NO:1 VH region of the A6 mab FR1 -CDR 1 -FR2-CDR2-FR3 -CDR3 -FR4
  • the VL region of the A6 mab consists of the sequence of SEQ ID NO:2. Accordingly, the L-CDR1 of the A6 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO:2. Accordingly, the L-CDR2 of the A6 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO:2. Accordingly, the L-CDR3 of the A6 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO:2.
  • SEQ ID NO:2 VL region of the A6 mab FR1 -CDR 1 -FR2-CDR2-FR3 -CDR3 -FR4
  • the VH region of the C7 mab consists of the sequence of SEQ ID NO: 3.
  • the H-CDR1 of the C7 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO:3.
  • the H-CDR2 of C7 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO:3.
  • the H-CDR3 of the C7 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO:3.
  • SEQ ID NO:3 VH region of the C7 mab FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
  • the VL region of the C7 mab consists of the sequence of SEQ ID NO:4. Accordingly, the L-CDR1 of the C7 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO:4. Accordingly, the L-CDR2 of the C7 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO:4. Accordingly, the L-CDR3 of the C7 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO:4.
  • SEQ ID NO:4 VL region of the C7 mab FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
  • the VH region of the C9 mab consists of the sequence of SEQ ID NO:5.
  • the H-CDR1 of the C9 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO:5.
  • the H-CDR2 of C9 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO:5.
  • the H-CDR3 of the C9 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO: 5.
  • SEQ ID NO:5 VH region of the C9 mab FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
  • the VL region of the C9 mab consists of the sequence of SEQ ID NO:6.
  • the L-CDR1 of the C9 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO:6.
  • the L-CDR2 of the C9 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO:6.
  • the L-CDR3 of the C9 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO:6.
  • the VH region of the D2 mab consists of the sequence of SEQ ID NO:7.
  • the H-CDR1 of the D2 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO:7.
  • the H-CDR2 of D2 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO:7.
  • the H-CDR3 of the D2 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO:7.
  • SEQ ID NO:7 VH region of the D2 mab FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
  • the VL region of the D2 mab consists of the sequence of SEQ ID NO: 8. Accordingly, the L-CDR1 of the D2 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO: 8. Accordingly, the L-CDR2 of the D2 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO:8. Accordingly, the L-CDR3 of the D2 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO: 8.
  • SEQ ID NO:8 VL region of the D2 mab FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
  • the VH region of the D5 mab consists of the sequence of SEQ ID NO:9.
  • the H-CDR1 of the D5 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO:9.
  • the H-CDR2 of D5 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO:9.
  • the H-CDR3 of the D5 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO:9.
  • SEQ ID NO:9 VH region of the D5 mab FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
  • the VL region of the D5 mab consists of the sequence of SEQ ID NO: 10. Accordingly, the L-CDR1 of the D5 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO: 10. Accordingly, the L-CDR2 of the D5 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO: 10. Accordingly, the L-CDR3 of the D5 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO: 10.
  • SEQ ID NQ:10 VL region of the D5 mab FR1 -CDR1 -FR2-CDR2-FR3 -CDR3 -FR4
  • the VH region of the E4 mab consists of the sequence of SEQ ID NO: 11. Accordingly, the H-CDR1 of the E4 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO: 11. Accordingly, the H-CDR2 of E4 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO: 11. Accordingly, the H-CDR3 of the E4 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO: 11.
  • SEQ ID NO:11 VH region of the E4 mab FR1 -CDR1 -FR2-CDR2-FR3 -CDR3 -FR4
  • the VL region of the E4 mab consists of the sequence of SEQ ID NO: 12. Accordingly, the L-CDR1 of the E4 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO: 12. Accordingly, the L-CDR2 of the E4 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO: 12. Accordingly, the L-CDR3 of the E4 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO: 12.
  • SEQ ID NO:12 VL region of the E4 mab FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
  • SEQ ID NO:13 VH region of the G7 mab FR1 -CDR1 -FR2-CDR2-FR3 -CDR3 -FR4
  • the VL region of the G7 mab consists of the sequence of SEQ ID NO: 14. Accordingly, the L-CDR1 of the G7 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO: 14. Accordingly, the L-CDR2 of the G7 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO: 14. Accordingly, the L-CDR3 of the G7 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO: 14.
  • SEQ ID NO:14 VL region of the G7 mab FR1 -CDR1 -FR2-CDR2-FR3 -CDR3 -FR4 QSVLTQPASVSGSPGQSITISCAGTSSDVGGSYSVSWYQQHPGKAPKLMIYSDS
  • the antibodies of the present invention are produced by any technique known in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination.
  • any technique known in the art such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination.
  • one skilled in the art can readily produce said antibodies, by standard techniques for production of polypeptides. For instance, they can be synthesized using well-known solid phase method, preferably using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, California) and following the manufacturer’s instructions.
  • antibodies of the present invention can be synthesized by recombinant DNA techniques well-known in the art.
  • antibodies can be obtained as DNA expression products after incorporation of DNA sequences encoding the antibodies into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired antibodies, from which they can be later isolated using well-known techniques.
  • the antibody of the invention is a human antibody.
  • human antibody is intended to include antibodies having variable and constant regions derived from human immunoglobulin sequences.
  • human antibodies of the present invention may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced by random or sitespecific mutagenesis in vitro or by somatic mutation in vivo).
  • 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.
  • Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, cur. Opin. Pharmacol. 5; 368- 74 (2001) and lonberg, cur. Opin. Immunol. 20; 450-459 (2008). Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal’s chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated.
  • Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 13: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51- 63 (Marcel Dekker, Inc., New York, 1987); and Boemer et al., J. Immunol., 147: 86(1991)).
  • Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S.
  • Patent No. 7,189,826 (describing production of monoclonal human igM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas).
  • Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein,, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-91 (2005).
  • Fully human antibodies can also be derived from phage-display libraries (as disclosed in Hoogenboom et al., 1991, J. Mol. Biol.
  • Phage display techniques mimic immune selection through the display of antibody repertoires on the surface of filamentous bacteriophage, and subsequent selection of phage by their binding to an antigen of choice.
  • PCT publication No. WO 99/10494 Human antibodies described herein can also be prepared using SCID mice into which human immune cells have been reconstituted such that a human antibody response can be generated upon immunization. Such mice are described in, for example, U.S. Patent Nos. 5,476,996 and 5,698,767 to Wilson et al.
  • the monoclonal antibody of the invention is a chimeric antibody, particularly a chimeric mouse/human antibody.
  • chimeric antibody refers to an antibody which comprises a VH domain and a VL domain of a non-human antibody, and a CH domain and a CL domain of a human antibody.
  • the human chimeric antibody of the present invention can be produced by obtaining nucleic sequences encoding VL and VH domains as previously described, constructing a human chimeric antibody expression vector by inserting them into an expression vector for animal cell having genes encoding human antibody CH and human antibody CL, and expressing the coding sequence by introducing the expression vector into an animal cell.
  • the CH domain of a human chimeric antibody it may be any region which belongs to human immunoglobulin, but those of IgG class are suitable and any one of subclasses belonging to IgG class, such as IgGl, IgG2, IgG3 and IgG4, can also be used.
  • the CL of a human chimeric antibody may be any region which belongs to Ig, and those of kappa class or lambda class can be used.
  • Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques are well known in the art (See Morrison SL. et al. (1984) and patent documents US5,202,238; and US5,204, 244).
  • the antibody of the invention is an antigen biding fragment selected from the group consisting of a Fab, a F(ab)’2, a single domain antibody, a ScFv, a Sc(Fv)2, a diabody, a triabody, a tetrabody, an unibody, a minibody, a maxibody, a small modular immunopharmaceutical (SMIP), minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody as an isolated complementary determining region (CDR), and fragments which comprise or consist of the VL or VH chains as well as amino acid sequence having at least 70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99 or 100% of identity with SEQ ID NO: 1 to 28.
  • SMIP small modular immunopharmaceutical
  • the antibody of the present invention is an antibody having a heavy chain comprising i) the H-CDR1 of A6, C7, C9, D2, D5, E4 or G7 mabs, ii) the H-CDR2 of A6, C7, C9, D2, D5, E4 or G7 mabs, and iii) the H-CDR3 of A6, C7, C9, D2, D5, E4 or G7 mabs, and a light chain comprising i) the L-CDR1 of A6, C7, C9, D2, D5, E4 or G7 mabs, ii) the L-CDR2 of A6, C7, C9, D2, D5, E4 or G7 mabs, and iii) the L-CDR3 of A6, C7, C9, D2, D5, E4 or G7 mabs.
  • the antibody of the present invention is an antibody having a heavy chain having at least 70 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% of identity with SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13 and a light chain having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% of identity with SEQ ID NO:2, 4, 6, 8, 10, 12 or 14.
  • the antibody has an heavy chain having at least 70% of identity with SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13 and a light chain having at least 70 % of identity with SEQ ID NO:2, 4, 6, 8, 10, 12 or 14.
  • the antibody of the present invention is an antibody having a heavy chain identical to SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13 and a light chain identical to SEQ ID NO:2, 4, 6, 8, 10, 12 or 14.
  • antigen binding fragment of an antibody refers to one or more fragments of an intact antibody that retain the ability to specifically binds to a given antigen (e.g., [TGFBI]).
  • Antigen biding functions of an antibody can be performed by fragments of an intact antibody.
  • biding fragments encompassed within the term antigen biding fragment of an antibody include a Fab fragment, a monovalent fragment consisting of the VL,VH,CL and CHI domains; a Fab’ fragment, a monovalent fragment consisting of the VL,VH,CL,CH1 domains and hinge region; a F(ab’)2 fragment, a bivalent fragment comprising two Fab’ fragments linked by a disulfide bridge at the hinge region; an Fd fragment consisting of VH domains of a single arm of an antibody; a single domain antibody (sdAb) fragment (Ward et al., 1989 Nature 341 :544-546), which consists of a VH domain or a VL domain; and an isolated complementary determining region (CDR).
  • Fab fragment a monovalent fragment consisting of the VL,VH,CL and CHI domains
  • a Fab’ fragment a monovalent fragment consisting of the VL,VH,CL,CH1 domains and hinge region
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by an artificial peptide linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (ScFv); see, e.g., Bird et al., 1989 Science 242:423-426; and Huston et al., 1988 proc. Natl. Acad. Sci. 85:5879-5883).
  • dsFv is a VH::VL heterodimer stabilised by a disulfide bond.
  • Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)2.
  • Such single chain antibodies include one or more antigen biding portions or fragments of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
  • a unibody is another type of antibody fragment lacking the hinge region of IgG4 antibodies. The deletion of the hinge region results in a molecule that is essentially half the size of traditional IgG4 antibodies and has a univalent binding region rather than the bivalent biding region of IgG4 antibodies.
  • Antigen binding fragments can be incorporated into single domain antibodies, SMTP, maxibodies, minibodies, intrabodies, diabodies, triabodies and tetrabodies (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136).
  • the term "diabodies” “tribodies” or “tetrabodies” refers to small antibody fragments with multivalent antigen-binding sites (2, 3 or four), which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL).
  • Antigen biding fragments can be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) Which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al., 1995 Protein Eng. 8(10); 1057-1062 and U.S. Pat. No. 5,641,870).
  • the Fab of the present invention can be obtained by treating an antibody which specifically reacts with TGFBI with a protease, papaine. Also, the Fab can be produced by inserting DNA encoding Fab of the antibody into a vector for prokaryotic expression system, or for eukaryotic expression system, and introducing the vector into a procaryote or eucaryote (as appropriate) to express the Fab.
  • the Fab' of the present invention can be obtained treating F(ab')2 which specifically reacts with TGFBI with a reducing agent, dithiothreitol. Also, the Fab' can be produced by inserting DNA encoding Fab' fragment of the antibody into an expression vector for prokaryote, or an expression vector for eukaryote, and introducing the vector into a prokaryote or eukaryote (as appropriate) to perform its expression.
  • the scFv of the present invention can be produced by obtaining cDNA encoding the VH and VL domains as previously described, constructing DNA encoding scFv, inserting the DNA into an expression vector for prokaryote, or an expression vector for eukaryote, and then introducing the expression vector into a prokaryote or eukaryote (as appropriate) to express the scFv.
  • Domain Antibodies are the smallest functional binding units of antibodies - molecular weight approximately 13 kDa - and correspond to the variable regions of either the heavy (VH) or light (VL) chains of antibodies. Further details on domain antibodies and methods of their production are found in US 6,291,158; 6,582,915; 6,593,081; 6,172,197; and 6,696,245; US 2004/0110941; EP 1433846, 0368684 and 0616640; WO 2005/035572, 2004/101790, 2004/081026, 2004/058821, 2004/003019 and 2003/002609, each of which is herein incorporated by reference in its entirety.
  • UniBodies are another antibody fragment technology, based upon the removal of the hinge region of IgG4 antibodies. The deletion of the hinge region results in a molecule that is essentially half the size of a traditional IgG4 antibody and has a univalent binding region rather than a bivalent binding region. Furthermore, because UniBodies are about smaller, they may show better distribution over larger solid tumors with potentially advantageous efficacy. Further details on UniBodies may be obtained by reference to WO 2007/059782, which is incorporated by reference in its entirety.
  • Another object of the invention relates to a nucleic acid molecule encoding an antibody according to the invention. More particularly the nucleic acid molecule encodes a heavy chain or a light chain of an antibody of the present invention.
  • nucleic acid molecule comprises a nucleic acid sequence having 70% of identity with SEQ ID NO: 15 and 16 or SEQ ID NO: 17 and 18 or SEQ ID NO: 19 and 20 or SEQ ID NO: 21 and 22 or SEQ ID NO: 23 and 24 or SEQ ID NO: 25 and 26 or SEQ ID NO: 27 and 28.
  • said nucleic acid is a DNA or RNA molecule, which may be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector.
  • a vector such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector.
  • vector cloning vector
  • expression vector mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence.
  • a further aspect of the invention relates to a vector comprising a nucleic acid of the invention.
  • Such vectors may comprise regulatory elements, such as a promoter, enhancer, terminator and the like, to cause or direct expression of said antibody upon administration to a subject.
  • promoters and enhancers used in the expression vector for animal cell include early promoter and enhancer of SV40 (Mizukami T. et al. 1987), LTR promoter and enhancer of Moloney mouse leukemia virus (Kuwana Y et al. 1987), promoter (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983) of immunoglobulin H chain and the like. Any expression vector for animal cell can be used, so long as a gene encoding the human antibody C region can be inserted and expressed.
  • Suitable vectors include pAGE107 (Miyaji H et al. 1990), pAGE103 (Mizukami T et al. 1987), pHSG274 (Brady G et al. 1984), pKCR (O'Hare K et al. 1981), pSGl beta d2-4-(Miyaji H et al. 1990) and the like.
  • plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like.
  • viral vector include adenoviral, retroviral, herpes virus and AAV vectors.
  • Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses.
  • Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc.
  • Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO 95/14785, WO 96/22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94/19478.
  • a further aspect of the invention relates to a host cell which has been transfected, infected or transformed by a nucleic acid and/or a vector according to the invention.
  • expression system means a host cell and compatible vector under suitable conditions, e.g. for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell.
  • Common expression systems include E. coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and vectors.
  • Other examples of host cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.).
  • E.coli Escherreocoli
  • Kluyveromyces or Saccharomyces yeasts mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or established mammalian cell cultures (e.g., produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.).
  • mammalian cell lines e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.
  • primary or established mammalian cell cultures e.g., produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.
  • Examples also include mouse SP2/0-Agl4 cell (ATCC CRL1581), mouse P3X63-Ag8.653 cell (ATCC CRL1580), CHO cell in which a dihydrofolate reductase gene (hereinafter referred to as "DHFR gene") is defective (Urlaub G et al; 1980), rat YB2/3HL.P2.G11.16Ag.2O cell (ATCC CRL1662, hereinafter referred to as "YB2/0 cell”), and the like.
  • DHFR gene dihydrofolate reductase gene
  • the present invention also relates to a method of producing a recombinant host cell expressing an antibody according to the invention, said method comprising the steps of: (i) introducing in vitro or ex vivo a recombinant nucleic acid or a vector as described above into a competent host cell, (ii) culturing in vitro or ex vivo the recombinant host cell obtained and (iii), optionally, selecting the cells which express and/or secrete said antibody.
  • recombinant host cells can be used for the production of antibodies of the present invention.
  • Antibodies of the present invention are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A- Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • Another object of the invention relates to antibodies comprising functional variants of the VL region, VH region, or one or more CDRs of the antibodies A6, C7, C9, D2, D5, E4 or G7.
  • a functional variant of a VL or VH used in the context of a monoclonal antibody of the present invention still allows the antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity/avidity and/or the specificity/selectivity of the parent antibody (i.e. A6, C7, C9, D2, D5, E4 or G7 antibody) and in some cases such a monoclonal antibody of the present invention may be associated with greater affinity, selectivity and/or specificity than the parent Ab.
  • Such variants can be obtained by a number of affinity maturation protocols including mutating the CDRs (Yang et al., J. Mol. Biol., 254, 392-403, 1995), chain shuffling (Marks et al., Bio/Technology, 10, 779-783, 1992), use of mutator strains of E. coli (Low et al., J. Mol. Biol., 250, 359-368, 1996), DNA shuffling (Patten et al., Curr. Opin. Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J. Mol.
  • the sequence of CDR variants may differ from the sequence of the CDR of the parent antibody sequences through mostly conservative substitutions; for instance at least about 35%, about 50% or more, about 60% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, (e.g., about 65-95%, such as about 92%, 93% or 94%) of the substitutions in the variant are conservative amino acid residue replacements.
  • sequences of CDR variants may differ from the sequence of the CDRs of the parent antibody sequences through mostly conservative substitutions; for instance at least 10, such as at least 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant are conservative amino acid residue replacements.
  • conservative substitutions may be defined by substitutions within the classes of amino acids reflected as follows:
  • More conservative substitutions groupings include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.
  • Conservation in terms of hydropathic/hydrophilic properties and residue weight/size also is substantially retained in a variant CDR as compared to a CDR of A6, C7, C9, D2, D5, E4 or G7.
  • hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art. It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8) ; phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophane (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (- 3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • Suitable variants typically exhibit at least about 70% of identity to the parent peptide.
  • a first amino acid sequence having at least 70% of identity with a second amino acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; or 100% of identity with the second amino acid sequence.
  • a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; or 100% of identity with the second amino acid sequence.
  • the antibodies described above bind to the same antigen and have the same properties of the antibody of the invention i.e. the antibody with the VH and VL of SEQ ID NO: 1 and 2, 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12 or 13 and 14.
  • Another object of the invention relates to an antibody that competes for binding to TGFBI with the antibodies of the invention.
  • binding in the context of the binding of an antibody to a predetermined antigen or epitope typically is a binding with an affinity corresponding to a KD of about 10' 7 M or less, such as about 10' 8 M or less, such as about 10' 9 M or less, about 10-10 M or less, or about 10' 11 M or even less when determined by for instance surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using a soluble form of the antigen as the ligand and the antibody as the analyte.
  • SPR surface plasmon resonance
  • BIACORE® GE Healthcare, Piscaataway, NJ
  • BIACORE® is one of a variety of surface plasmon resonance assay formats that are routinely used to epitope bin panels of monoclonal antibodies.
  • an antibody binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100-fold lower, for instance at least 1,000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000-fold lower than its KD for binding to a non-specific antigen (e.g., BSA, casein), which is not identical or closely related to the predetermined antigen.
  • a non-specific antigen e.g., BSA, casein
  • An T1 antibody is said to essentially not bind an antigen or epitope if such binding is either not detectable (using, for example, plasmon resonance (SPR) technology in a BIAcore 3000 instrument using a soluble form of the antigen as the ligand and the antibody as the analyte), or is 100 fold, 500 fold, 1000 fold or more than 1000 fold less than the binding detected by that antibody and an antigen or epitope having a different chemical structure or amino acid sequence.
  • SPR plasmon resonance
  • Additional antibodies can be identified based on their ability to cross-compete (e.g., to competitively inhibit the binding of, in a statistically significant manner) with other antibodies of the invention in standard TGFBI binding assays.
  • test antibody to inhibit the binding of antibodies of the present invention to TGFBI demonstrates that the test antibody can compete with that antibody for binding to TGFBI; such an antibody may, according to non-limiting theory, bind to the same or a related (e.g., a structurally similar or spatially proximal) epitope on TGFBI as the antibody with which it competes.
  • another aspect of the invention provides antibodies that bind to the same antigen as, and compete with, the antibodies disclosed herein.
  • an antibody “competes” for binding when the competing antibody inhibits TGFBI binding of an antibody or antigen binding fragment of the invention by more than 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% in the presence of an equimolar concentration of competing antibody.
  • the antibodies or antigen binding fragments of the invention bind to one or more epitopes of TGFBI.
  • the epitopes to which the present antibodies or antigen binding fragments bind are linear epitopes.
  • the antibodies of the invention may be assayed for specific binding by any method known in the art. Many different competitive binding assay format(s) can be used for epitope binding.
  • the immunoassays which can be used include, but are not limited to, competitive assay systems using techniques such western blots, radioimmunoassays, ELISA, "sandwich” immunoassays, immunoprecipitation assays, precipitin assays, gel diffusion precipitin assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and complement-fixation assays.
  • Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, 1994 Current Protocols in Molecular Biology, Vol. 1, John Wiley & sons, Inc., New York).
  • Engineered antibodies of the invention include those in which modifications have been made to framework residues within VH and/or VL, e.g. to improve the properties of the antibody. Typically such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived.
  • the somatic mutations can be "backmutated” to the germline sequence by, for example, site-directed mutagenesis or PCR- mediated mutagenesis.
  • Such “backmutated” antibodies are also intended to be encompassed by the invention.
  • the framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell -epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U. S. Patent Publication No. 20030153043 by Carr et al.
  • the glycosylation of an antibody is modified.
  • Glycosylation can be altered to, for example, increase the affinity of the antibody for the antigen.
  • Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence.
  • one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
  • Such aglycosylation may increase the affinity of the antibody for antigen.
  • some mutations are made to the amino acids localized in aggregation “hotspots” within and near the first CDR (CDR1) to decrease the antibodies susceptibility to aggregation (see Joseph M. Perchiacca et al., Proteins 2011; 79:2637-2647). • Isotype
  • the antibody of the present invention may be of any isotype.
  • the choice of isotype typically will be guided by the desired effector functions.
  • IgGl and IgG3 are isotypes that mediate such effectors functions as ADCC or CDC, when IgG2 and IgG4 don’t or in a lower manner. Either of the human light chain constant regions, kappa or lambda, may be used.
  • the class of a monoclonal antibody of the present invention may be switched by known methods. Typical, class switching techniques may be used to convert one IgG subclass to another, for instance from IgGl to IgG2.
  • the effector function of the monoclonal antibodies of the present invention may be changed by isotype switching to, e.g., an IgGl, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody for various therapeutic uses.
  • the full-length antibody is an IgGl antibody. In some embodiments, the full-length antibody is an IgG3 antibody.
  • the antibody of the present invention is an antibody of a non-IgG2/4 type, e.g. IgGl or IgG3 which has been mutated such that the ability to mediate effector functions, such as ADCC, has been reduced or even eliminated.
  • a non-IgG2/4 type e.g. IgGl or IgG3 which has been mutated such that the ability to mediate effector functions, such as ADCC, has been reduced or even eliminated.
  • Such mutations have e.g. been described in Dall'Acqua WF et al., J Immunol. 177(2): 1129-1138 (2006) and Hezareh M, J Virol. 75(24): 12161-12168 (2001).
  • the hinge region of CHI is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased.
  • This approach is described further in U.S. Patent No. 5,677,425 by Bodmer et al.
  • the number of cysteine residues in the hinge region of CHI is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.
  • the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody.
  • one or more amino acids can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody.
  • the effector ligand to which affinity is altered can be, for example, an Fc receptor or the Cl component of complement.
  • the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fc receptor by modifying one or more amino acids.
  • ADCC antibody dependent cellular cytotoxicity
  • This approach is described further in PCT Publication WO 00/42072 by Presta.
  • the binding sites on human IgGI for FcyRI, FcyRII, FcyRIII and FcRn have been mapped and variants with improved binding have been described (see Shields, R. L. et al., 2001 J. Biol. Chen. 276:6591-6604, W02010106180).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcRs Fc receptors
  • Non-specific cytotoxic cells that mediate ADCC include natural killer (NK) cells, macrophages, monocytes, neutrophils, and eosinophils.
  • the term "Effector functions" refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype.
  • Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.
  • one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC).
  • CDC complement dependent cytotoxicity
  • one or more amino acid residues are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in PCT Publication WO 94/29351 by Bodmer et al.
  • an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated or non-fucosylated antibody having reduced amounts of or no fucosyl residues or an antibody having increased bisecting GlcNac structures.
  • Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies.
  • carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the present invention to thereby produce an antibody with altered glycosylation.
  • the monoclonal antibodies of the present invention may be produced by recombinant expression in a cell line which exhibit hypofucosylation or non-fucosylation pattern, for example, a mammalian cell line with deficient expression of the FUT8 gene encoding fucosyltransferase.
  • PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R.L. et al, 2002 J. Biol. Chem. 277:26733-26740).
  • PCT Publication WO 99/54342 by Umana et al.
  • glycoprotein-modifying glycosyl transferases e.g., beta(l,4)-N acetylglucosaminyltransf erase III (GnTIII)
  • GnTIII glycoprotein-modifying glycosyl transferases
  • Eureka Therapeutics further describes genetically engineered CHO mammalian cells capable of producing antibodies with altered mammalian glycosylation pattern devoid of fucosyl residues (http:/ www.eiirekainc.com &boutus/ 'companyoverview.html).
  • the monoclonal antibodies of the present invention can be produced in yeasts or filamentous fungi engineered for mammalian- like glycosylation pattern and capable of producing antibodies lacking fucose as glycosylation pattern (see for example EP1297172B1).
  • the antibody is modified to increase its biological half-life.
  • the antibody can be altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121 ,022 by Presta et al.
  • Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311,312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434, e.g., substitutions of Fc region residue 434 (US Patent No. 7,371,826).
  • An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody.
  • the antibody, or fragment thereof typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment.
  • PEG polyethylene glycol
  • the pegylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer).
  • polyethylene glycol is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (Cl - CIO) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide.
  • the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the invention. See for example, EP0154316 by Nishimura et al. and EP0401384 by Ishikawa et al.
  • Another modification of the antibodies that is contemplated by the invention is a conjugate or a protein fusion of at least the antigen-binding region of the antibody of the invention to serum protein, such as human serum albumin or a fragment thereof to increase half-life of the resulting molecule.
  • serum protein such as human serum albumin or a fragment thereof to increase half-life of the resulting molecule.
  • Another possibility is a fusion of at least the antigen-binding region of the antibody of the invention to proteins capable of binding to serum proteins, such human serum albumin to increase half-life of the resulting molecule.
  • Such approach is for example described in Nygren et al., EP 0 486 525.
  • PSA polymer polysialic acid
  • PSA is a polymer of sialic acid (a sugar).
  • sialic acid a sugar
  • polysialic acid provides a protective microenvironment on conjugation. This increases the active life of the therapeutic protein in the circulation and prevents it from being recognized by the immune system.
  • the PSA polymer is naturally found in the human body. It was adopted by certain bacteria which evolved over millions of years to coat their walls with it. These naturally polysialylated bacteria were then able, by virtue of molecular mimicry, to foil the body's defense system. PSA, nature's ultimate stealth technology, can be easily produced from such bacteria in large quantities and with predetermined physical characteristics. Bacterial PSA is completely non-immunogenic, even when coupled to proteins, as it is chemically identical to PSA in the human body.
  • HES hydroxy ethyl starch
  • Another technology includes the use of hydroxy ethyl starch (“HES”) derivatives linked to antibodies.
  • HES is a modified natural polymer derived from waxy maize starch and can be metabolized by the body's enzymes.
  • HES solutions are usually administered to substitute deficient blood volume and to improve the rheological properties of the blood. Hesylation of an antibody enables the prolongation of the circulation half-life by increasing the stability of the molecule, as well as by reducing renal clearance, resulting in an increased biological activity.
  • a wide range of HES antibody conjugates can be customized.
  • the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody.
  • one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding.
  • SpA Staphylococcyl protein A
  • antibodies have been engineered to remove sites of deamidation.
  • Deamidation is known to cause structural and functional changes in a peptide or protein. Deamidation can result in decreased bioactivity, as well as alterations in pharmacokinetics and antigenicity of the protein pharmaceutical. (Anal Chem. 2005 Mar 1 ;77(5): 1432-9).
  • the antibodies have been engineered to increase pl and improve their drug-like properties.
  • the pl of a protein is a key determinant of the overall biophysical properties of a molecule.
  • Antibodies that have low pls have been known to be less soluble, less stable, and prone to aggregation. Further, the purification of antibodies with low pl is challenging and can be problematic especially during scale-up for clinical use.
  • Increasing the pl of the anti-TGFBI antibodies of the invention or fragments thereof improved their solubility, enabling the antibodies to be formulated at higher concentrations (>100 mg/ml). Formulation of the antibodies at high concentrations (e.g.
  • the pl is greater than or equal to 8.2.
  • Glycosylation modifications can also induce enhanced anti-inflammatory properties of the antibodies by addition of sialylated glycans.
  • the addition of terminal sialic acid to the Fc glycan reduces FcyR binding and converts IgG antibodies to anti-inflammatory mediators through the acquisition of novel binding activities (see Robert M. Anthony et al., J Clin Immunol (2010) 30 (Suppl 1): S9— S 14; Kai-Ting C et al., Antibodies 2013, 2, 392-414).
  • the heavy and light chains, variable regions domains and CDRs that are disclosed can be used to prepare polypeptides that contain antigen binding region that can specifically bind to TGFBI.
  • the CDRs of the A6, C7, C9, D2, D5, E4 or G7 mabs can be incorporated into a molecule (e.g., a polypeptide) covalently or noncovalently to make an immunoadhesion.
  • a molecule e.g., a polypeptide
  • An immunoadhesion may incorporate the CDRs as part of a larger polypeptide chain, may covalently link the CDRs to another polypeptide chain, or may incorporate the CDRs noncovalently.
  • the CDRs enable the immunoadhesion to bind specifically to a particular antigen of interest (e.g., TGFBI or epitope thereof).
  • polypeptide and protein refer to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well to naturally occurring amino acids polymers and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.
  • the antigen biding fragment of the invention is grafted into nonimmunoglobulin based antibodies also called antibody mimetics selected from the group consisting of an affibody, an affilin, an affitin, an adnectin, an atrimer, an evasin, a DARPin, an anticalin, an avimer, a fynomer, and a versabody.
  • nonimmunoglobulin based antibodies also called antibody mimetics selected from the group consisting of an affibody, an affilin, an affitin, an adnectin, an atrimer, an evasin, a DARPin, an anticalin, an avimer, a fynomer, and a versabody.
  • antibody mimetic refers to molecules capables of mimicking an antibody’s ability to bind an antigen, but which are not limited to native antibody structures.
  • antibody mimetics include, but are not limited to, Adnectins, Affibodies, DARPins, Anticalins, Avimers, and versabodies, all of which employ binding structures that, while they mimic traditional antibody binding, are generated from and function via distinct mechanisms.
  • An affibody is well known in the art and refers to affinity proteins based on a 58 amino acid residue protein domain, derived from one of the IgG binding domain of staphylococcal protein A.
  • DARPins Designed Ankyrin Repeat Proteins
  • DRP designed repeat protein
  • Anticalins are well known in the art and refer to another antibody mimetic technology, wherein the binding specificity is derived from lipocalins. Anticalins may also be formatted as dual targeting protein, called Duocalins.
  • Avimers are well known in the art and refer to another antibody mimetic technology, Avimers are derived from natural A-domain containing protein.
  • Versabodies are well known in the art and refer to another antibody mimetic technology, they are small proteins of 3-5 kDa with >15% cysteines, which form a high disulfide density scaffold, replacing the hydrophobic core the typical proteins have.
  • Such antibody mimetic can be comprised in a scaffold.
  • the invention pertains to generating non-immunoglobulin-based antibodies also called antibody mimetics using non-immunoglobulins scaffolds onto which CDRs of the invention can be grafted.
  • non-immunoglobulin-based antibodies also called antibody mimetics
  • non-immunoglobulins scaffolds onto which CDRs of the invention can be grafted.
  • Known or future non-immunoglobulin frameworks and scaffolds may be employed, as long as they comprise a binding region specific for the target TGFBI protein.
  • Antigen biding fragments of antibodies can be grafted into scaffolds based on polypeptides such as Fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies).
  • the fibronectin scaffolds are based on fibronectin type III domain (e.g., the tenth module of the fibronectin type III (10 Fn3 domain)).
  • the fibronectin type III domain has 7 or 8 beta strands which are distribued between two beta sheets, which themselves pack against each other to form the core of the protein, and further containing loops (analogous to CDRs) which connect the beta strands to each other and are solvent exposed.
  • fibronectin-based scaffolds are not an immunoglobulin, although the overall fold is closely related to that of the smallest functional antibody fragment, the variable region of the heavy chain, which comprise the entire antigen recognition unit in camel and llama IgG. Because of this structure, the non-immunoglobulin antibody mimics antigen binding properties that are similar in nature and affinity to those of antibodies.
  • These scaffolds can be used in a loop randomisation and shuffling strategy in vitro that is similar to the process of affinity maturation of antibodies in vivo.
  • These fibronectin-based molecules can be used as scaffolds where the loop regions of the molecule can be replaced with CDRs of the invention using standard cloning techniques.
  • the Ankyrin technology is based on using proteins with Ankyrin derived repeat modules as scaffolds for bearing variable regions which can be used for binding to different targets.
  • the Ankyrin repeat module is a 33 amino acid polypeptide consisting of two anti-parallel a-helices and a P-tum. Binding of the variable regions is mostly optimized by using ribosome display.
  • Avimers are derived from natural A-domain containing protein such as LRP-1. These domains are used by nature for protein-protein interactions and in human over 250 proteins are structurally based on “A-domains” monomers (2-10) linked via amino acids linkers. Avimers can be created that can bind to the target antigen using the methodology described in, for example, U.S. patent Application publication Nos. 20040175756; 20050053973; 20050048512; and 20060008844.
  • Affibody affinity ligands are small, simple proteins composed of a three-helix bundle based on the scaffold of one of the IgG-binding domains of protein A.
  • protein A is a surface protein form the bacterium Staphylococcus aureus. This scaffold domain consist of 58 amino acids, 13 of which are randomized to generate affibody librairies with a large number of ligand variants (See e.g., US 5,831,012).
  • Affibody molecules mimic antibodies, they have a molecular weight of 6kDa. In spite of its small size, the binding site of affibody molecules is similar to that of an antibody.
  • Anticalins are products developed by the company Pieris ProteoLab AG. They are derived from lipocalins, a widespread group of small and robust proteins that are usually involved in the physiological transport or storage of chemically sensitive or insoluble compounds. Several natural lipocalins occur in human tissues or body liquids. The protein architecture is reminiscent of immunoglobulins, with hypervariable loops on top of a rigid framework. However, in contrast with antibodies or their recombinant fragments, lipocalins are composed of a single polypeptide chain with 160 to 180 amino acids residues, being just marginally bigger than a single immunoglobulin domain. The set of four loops, which makes up the binding pocket, shows pronounced structural plasticity and tolerates a variety of side chains.
  • the binding site can thus be reshaped in a proprietary process in order to recognize prescribed target molecules of different shape with high affinity and specificity.
  • One protein of lipocalin family the bilin-binding protein (BBP) of Pieris Brassicae has been used to develop anticalins by mutagenizing the set of four loops.
  • BBP bilin-binding protein
  • One example of a patent application describing anticalins is in PCT Publication No. WO 199916873.
  • Affilin molecules are small non-immunoglobulin proteins which are designed for specific affinities towards proteins and small molecules.
  • New affilin molecules can be very quickly selected from two libraries, each of which is based on a different human derived scaffold protein. Affilin molecules do not show any structural homology to immunoglobulin proteins.
  • two affilin scaffolds are employed, one of which is gamma crystalline, a human structural eye lens protein and the other is “ubiquitin” superfamily proteins. Both human scaffolds are very small, show high temperature stability and are almost resistant to pH changes and denaturing agents. This high stability is mainly due to the expanded beta sheet structure of the proteins. Examples of “ubiquitin-like” proteins are described in W02004106368.
  • Versabodies are highly soluble and can be formulated to high concentrations. Versabodies are exceptionally heat stable and offer extended shelf-life. Additional information regarding Versabodies can be found in US 2007/0191272, which is hereby incorporated by reference in its entirety.
  • the present invention also provides chimeric antigen receptors (CARs) comprising an antigen binding domain of the antibodies of the present invention.
  • CARs chimeric antigen receptors
  • said chimeric antigen receptor comprises at least one VH and/or VL sequence of the antibodies of the present invention.
  • the chimeric antigen receptor of the present invention also comprises an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain.
  • chimeric antigen receptor has its general meaning in the art and refers to an artificially constructed hybrid protein or polypeptide containing the antigen binding domains of an antibody (e.g., scFv) linked to T- cell signaling domains. Characteristics of CARs include their ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC-restricted manner, exploiting the antigenbinding properties of monoclonal antibodies. The non-MHC-restricted antigen recognition gives T cells expressing CARs the ability to recognize antigen independent of antigen processing, thus bypassing a major mechanism of tumor escape. Moreover, when expressed in T-cells, CARs advantageously do not dimerize with endogenous T cell receptor (TCR) alpha and beta chains.
  • TCR endogenous T cell receptor
  • the invention provides CARs comprising an antigen-binding domain comprising, consisting of, or consisting essentially of, a single chain variable fragment (scFv) of the antibody of the invention.
  • scFv single chain variable fragment
  • the antigen binding domain comprises a linker peptide.
  • the linker peptide may be positioned between the light chain variable region and the heavy chain variable region.
  • the CAR comprises an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain selected from the group consisting of CD28, 4-1BB, and CD3( ⁇ intracellular domains.
  • CD28 is a T cell marker important in T cell co-stimulation.
  • 4- IBB transmits a potent costimulatory signal to T cells, promoting differentiation and enhancing long-term survival of T lymphocytes.
  • CD3( ⁇ associates with TCRs to produce a signal and contains immunoreceptor tyrosine-based activation motifs (ITAMs).
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • the CAR of the present invention can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, e.g., a disulfide bridge, or converted into an acid addition salt and/or optionally dimerized or polymerized.
  • the invention also provides a nucleic acid encoding for a CAR of the present invention.
  • the nucleic acid is incorporated in a vector as such as described above.
  • the present invention also provides a host cell comprising a nucleic acid encoding for a chimeric antigen receptor of the present invention. While the host cell can be of any cell type, can originate from any type of tissue, and can be of any developmental stage; the host cell is a T cell, e.g. isolated from peripheral blood lymphocytes (PBL) or peripheral blood mononuclear cells (PBMC).
  • PBL peripheral blood lymphocytes
  • PBMC peripheral blood mononuclear cells
  • the T cell can be any T cell, such as a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupTl, etc., or a T cell obtained from a mammal. If obtained from a mammal, the T cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, or other tissues or fluids. T cells can also be enriched for or purified.
  • a cultured T cell e.g., a primary T cell
  • a T cell from a cultured T cell line e.g., Jurkat, SupTl, etc.
  • T cell obtained from a mammal e.g., the T cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, or other tissues or fluids. T cells can also be enriched for or purified.
  • the T cell can be any type of T cell and can be of any developmental stage, including but not limited to, CD4+/CD8+ double positive T cells, CD4+ helper T cells, e.g., Th2 cells, CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, and the like.
  • the T cell may be a CD8+ T cell or a CD4+ T cell.
  • T cells prepared as described above can be utilized in methods and compositions for adoptive immunotherapy in accordance with known techniques, or variations thereof that will be apparent to those skilled in the art based on the instant disclosure. See, e.g., US Patent Application Publication No. 2003/0170238 to Gruenberg et al; see also US Patent No. 4,690,915 to Rosenberg.
  • Adoptive immunotherapy of cancer refers to a therapeutic approach in which immune cells with an antitumor reactivity are administered to a tumorbearing host, with the aim that the cells mediate either directly or indirectly, the regression of an established tumor. Transfusion of lymphocytes, particularly T lymphocytes, falls into this category.
  • ALT autolymphocyte therapies
  • These therapies involve processing the patient's own lymphocytes to either enhance the immune cell mediated response or to recognize specific antigens or foreign substances in the body, including the cancer cells.
  • the treatments are accomplished by removing the patient's lymphocytes and exposing these cells in vitro to biologies and drugs to activate the immune function of the cells. Once the autologous cells are activated, these ex vivo activated cells are reinfused into the patient to enhance the immune system to treat cancer.
  • a treatment-effective amount of cells in the composition is dependent on the relative representation of the T cells with the desired specificity, on the age and weight of the recipient, on the severity of the targeted condition and on the immunogenicity of the targeted Ags. These amount of cells can be as low as approximately 103/kg, preferably 5xl03/kg; and as high as 107/kg, preferably 108/kg. The number of cells will depend upon the ultimate use for which the composition is intended, as will the type of cells included therein. For example, if cells that are specific for a particular Ag are desired, then the population will contain greater than 70%, generally greater than 80%, 85% and 90-95% of such cells.
  • the cells are generally in a volume of a liter or less, can be 500 ml or less, even 250 ml or 100 ml or less.
  • the clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed the desired total amount of cells.
  • the cells of the present invention are particularly suitable for the treatment of cancer.
  • the invention provides a multispecific antibody comprising a first antigen binding site from an antibody of the present invention molecule described herein above and at least one second antigen binding site.
  • the second antigen-binding site is used for recruiting a killing mechanism such as, for example, by binding an antigen on a human effector cell as a BiTE (Bispecific T-Cell engager) antibody which is a bispecific scFv2 directed against target antigen and CD3 on T cells described in US7235641, or by binding a cytotoxic agent or a second therapeutic agent.
  • a killing mechanism such as, for example, by binding an antigen on a human effector cell as a BiTE (Bispecific T-Cell engager) antibody which is a bispecific scFv2 directed against target antigen and CD3 on T cells described in US7235641, or by binding a cytotoxic agent or a second therapeutic agent.
  • effector cell refers to an immune cell which is involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response.
  • exemplary immune cells include a cell of a myeloid or lymphoid origin, for instance lymphocytes (such as B cells and T cells including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, mast cells and granulocytes, such as neutrophils, eosinophils and basophils.
  • lymphocytes such as B cells and T cells including cytolytic T cells (CTLs)
  • killer cells such as B cells and T cells including cytolytic T cells (CTLs)
  • killer cells such as B cells and T cells including cytolytic T cells (CTLs)
  • killer cells such as B cells and T cells including cytolytic T cells (CTLs)
  • killer cells such as B cells and T cells including cytolytic T cells (CTLs)
  • killer cells such as B cells and T cells including cytolytic T
  • an effector cell may phagocytose a target antigen or target cell.
  • the expression of a particular FcR on an effector cell may be regulated by humoral factors such as cytokines.
  • An effector cell can phagocytose a target antigen or phagocytose or lyse a target cell.
  • Suitable cytotoxic agents and second therapeutic agents are exemplified below, and include toxins (such as radiolabeled peptides), chemotherapeutic agents and prodrugs
  • the second antigen-binding site binds to an antigen on a human B cell, such as, e.g., CD19, CD20, CD21, CD22, CD23, CD46, CD80, CD138 and HLA-DR.
  • the second antigen-binding site binds a tissue-specific antigen, promoting localization of the bispecific antibody to a specific tissue.
  • the second antigen-binding site binds to an antigen located on the same type of cell as the TGFBI-expressing cell, typically a tumor-associated antigen (TAA), but has a binding specificity different from that of the first antigen-binding site.
  • TAA tumor-associated antigen
  • Such multi- or bispecific antibodies can enhance the specificity of the tumor cell binding and/or engage multiple effector pathways.
  • TAAs include carcinoembryonic antigen (CEA), prostate specific antigen (PSA), RAGE (renal antigen), a-fetoprotein, CAMEL (CTL- recognized antigen on melanoma), CT antigens (such as MAGE-B5, -B6, -C2, -C3, and D; Mage-12; CT10; NY-ESO-1, SSX-2, GAGE, BAGE, MAGE, and SAGE), mucin antigens (e.g., MUC1, mucin-CA125, etc.), ganglioside antigens, tyrosinase, gp75, c-Met, Marti, MelanA, MUM-1, MUM-2, MUM-3, HLA-B7, Ep-CAM or a cancer-associated integrin, such as a5p3 integrin.
  • CEA carcinoembryonic antigen
  • PSA prostate specific antigen
  • RAGE renal antigen
  • CAMEL CTL-
  • the second antigen- binding site binds to a different epitope of TGFBI.
  • the second antigen-binding site may alternatively bind an angiogenic factor or other cancer- associated growth factor, such as a vascular endothelial growth factor, a fibroblast growth factor, epidermal growth factor, angiogenin or a receptor of any of these, particularly receptors associated with cancer progression.
  • the second antigen-binding site is from a second antibody or ADC of the invention, such as the antibody of the present invention.
  • Exemplary formats for the multispecific antibody molecules of the invention include, but are not limited to (i) two antibodies cross-linked by chemical heteroconjugation, one with a specificity to TGFBI and another with a specificity to a second antigen; (ii) a single antibody that comprises two different antigen-binding regions; (iii) a single-chain antibody that comprises two different antigen-binding regions, e.g., two scFvs linked in tandem by an extra peptide linker; (iv) a dual-variable-domain antibody (DVD-Ig), where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-IgTM) Molecule, In : Antibody Engineering, Springer Berlin Heidelberg (2010)); (v) a chemically- linked bispecific (Fab')2 fragment; (vi) a Tandab, which is a fusion of
  • IgG-like molecules with complementary CH3 domains to force heterodimerization is IgG-like molecules with complementary CH3 domains to force heterodimerization.
  • Such molecules can be prepared using known technologies, such as, e.g., those known as Triomab/Quadroma (Trion Pharma/Fresenius Biotech), Knob-into-Hole (Genentech), CrossMAb (Roche) and electrostatically-matched (Amgen), LUZ-Y (Genentech), Strand Exchange Engineered Domain body (SEEDbody)(EMD Serono), Biclonic (Merus) and DuoBody (Genmab A/S) technologies.
  • the bispecific antibody is obtained or obtainable via a controlled Fab-arm exchange, typically using DuoBody technology.
  • controlled Fab-arm exchange In vitro methods for producing bispecific antibodies by controlled Fab-arm exchange have been described in WO2008119353 and WO 2011131746 (both by Genmab A/S).
  • a bispecific antibody is formed by "Fab-arm" or "half- molecule” exchange (swapping of a heavy chain and attached light chain) between two monospecific antibodies, both comprising IgG4-like CH3 regions, upon incubation under reducing conditions.
  • the resulting product is a bispecific antibody having two Fab arms which may comprise different sequences.
  • bispecific antibodies of the present invention are prepared by a method comprising the following steps, wherein at least one of the first and second antibodies is the antibody of the present invention : a) providing a first antibody comprising an Fc region of an immunoglobulin, said Fc region comprising a first CH3 region; b) providing a second antibody comprising an Fc region of an immunoglobulin, said Fc region comprising a second CH3 region; wherein the sequences of said first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions; c) incubating said first antibody together with said second antibody under reducing conditions; and d) obtaining said bispecific antibody, wherein the first antibody is the antibody of the present invention and the second antibody has a different binding specificity, or vice versa.
  • the reducing conditions may, for example, be provided by adding a reducing agent, e.g. selected from 2-mercaptoethylamine, dithiothreitol and tris(2- carboxy ethyl)phosphine.
  • Step d) may further comprise restoring the conditions to become nonreducing or less reducing, for example by removal of a reducing agent, e.g. by desalting.
  • the sequences of the first and second CH3 regions are different, comprising only a few, fairly conservative, asymmetrical mutations, such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions.
  • the first Fc region has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405, 407 and 409
  • the second Fc region has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405, 407 and 409, and wherein the first and second Fc regions are not substituted in the same positions.
  • the first Fc region has an amino acid substitution at position 405, and said second Fc region has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 407 and 409, optionally 409.
  • the first Fc region has an amino acid substitution at position 409
  • said second Fc region has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405, and 407, optionally 405 or 368.
  • both the first and second Fc regions are of the IgGl isotype, with the first Fc region having a Leu at position 405, and the second Fc region having an Arg at position 409.
  • the antibodies of the invention can be conjugated with a detectable label to form an anti-TGFBI immunoconjugate.
  • Suitable detectable labels include, for example, a radioisotope, a fluorescent label, a chemiluminescent label, an enzyme label, a bioluminescent label or colloidal gold. Methods of making and detecting such detectably-labeled immunoconjugates are well-known to those of ordinary skill in the art, and are described in more detail below.
  • the detectable label can be a radioisotope that is detected by autoradiography. Isotopes that are particularly useful for the purpose of the invention are 3H, 1251, 1311, 35S and 14C.
  • Anti-TGFBI immunoconjugates can also be labeled with a fluorescent compound.
  • fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • anti-TGFBI immunoconjugates can be detectably labeled by coupling an antibody to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged immunoconjugate is determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemiluminescent labeling compounds include luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt and an oxalate ester.
  • Bioluminescent compound can be used to label anti-TGFBI immunoconjugates of the invention.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
  • Bioluminescent compounds that are useful for labeling include luciferin, luciferase and aequorin.
  • anti-TGFBI immunoconjugates can be detectably labeled by linking an anti-TGFBI antibody to an enzyme.
  • the enzyme moiety reacts with the substrate to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or visual means.
  • enzymes that can be used to detectably label polyspecific immunoconjugates include P-galactosidase, glucose oxidase, peroxidase and alkaline phosphatase.
  • the convenience and versatility of immunochemical detection can be enhanced by using anti-TGFBI monoclonal antibodies that have been conjugated with avidin, streptavidin, and biotin.
  • anti-TGFBI monoclonal antibodies that have been conjugated with avidin, streptavidin, and biotin.
  • biotin See, e.g., Wilchek et al. (eds.), “Avidin-Biotin Technology,” Methods In Enzymology (Vol. 184) (Academic Press 1990); Bayer et al., “Immunochemical Applications of Avidin-Biotin Technology,” in Methods In Molecular Biology (Vol. 10) 149-162 (Manson, ed., The Humana Press, Inc. 1992).
  • the antibody of the present invention is conjugated to a therapeutic moiety, i.e. a drug.
  • the therapeutic moiety can be, e.g., a cytotoxin, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immune stimulator, a lytic peptide, or a radioisotope.
  • cytotoxin e.g., a cytotoxin, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immune stimulator, a lytic peptide, or a radioisotope.
  • ADCs antibody-drug conjugates
  • the antibody is conjugated to a cytotoxic moiety.
  • the cytotoxic moiety may, for example, be selected from the group consisting of taxol; cytochalasin B; gramicidin D; ethidium bromide; emetine; mitomycin; etoposide; tenoposide; vincristine; vinblastine; colchicin; doxorubicin; daunorubicin; dihydroxy anthracin dione; a tubulin- inhibitor such as maytansine or an analog or derivative thereof; an antimitotic agent such as monomethyl auristatin E or F or an analog or derivative thereof; dolastatin 10 or 15 or an analogue thereof; irinotecan or an analogue thereof; mitoxantrone; mithramycin; actinomycin D; 1 -dehydrotestosterone; a glucocorticoid; procaine; tetracaine; lidocaine; propranolol;
  • the antibody is conjugated to a nucleic acid or nucleic acid- associated molecule.
  • the conjugated nucleic acid is a cytotoxic ribonuclease (RNase) or deoxy-ribonuclease (e.g., DNase I), an antisense nucleic acid, an inhibitory RNA molecule (e.g., a siRNA molecule) or an immunostimulatory nucleic acid (e.g., an immunostimulatory CpG motif-containing DNA molecule).
  • RNase cytotoxic ribonuclease
  • DNase I deoxy-ribonuclease
  • an antisense nucleic acid e.g., an inhibitory RNA molecule
  • an inhibitory RNA molecule e.g., a siRNA molecule
  • an immunostimulatory nucleic acid e.g., an immunostimulatory CpG motif-containing DNA molecule.
  • the antibody is conjugated to an aptamer or a rib
  • the antibody is conjugated, e.g., as a fusion protein, to a lytic peptide such as CLIP, Magainin 2, mellitin, Cecropin and Pl 8.
  • a lytic peptide such as CLIP, Magainin 2, mellitin, Cecropin and Pl 8.
  • the antibody is conjugated to a cytokine, such as, e.g., IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNa, IFN3, IFNy, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and TNFa.
  • a cytokine such as, e.g., IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNa, IFN3, IFNy, GM-CSF, CD40L, Flt3 ligand, stem cell factor,
  • the antibody is conjugated to a radioisotope or to a radioisotopecontaining chelate.
  • the antibody can be conjugated to a chelator linker, e.g. DOTA, DTPA or tiuxetan, which allows for the antibody to be complexed with a radioisotope.
  • the antibody may also or alternatively comprise or be conjugated to one or more radiolabeled amino acids or other radiolabeled moleculesNon-limiting examples of radioisotopes include 3H, 14C, 15N, 35S, 90Y, 99Tc, 1251, 1311, 186Re, 213Bi, 225Ac and 227Th.
  • a radioisotope emitting beta- or alpha-particle radiation can be used, e.g., 1311, 90Y, 211 At, 212Bi, 67Cu, 186Re, 188Re, and 212Pb.
  • an antibody-drug conjugate comprises an anti-tubulin agent.
  • anti-tubulin agents include, for example, taxanes (e.g., Taxol® (paclitaxel), Taxotere® (docetaxel)), T67 (Tularik), vinca alkyloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine) and dolastatins (e.g., auristatin E, AFP, MMAF, MMAE, AEB, AEVB).
  • taxanes e.g., Taxol® (paclitaxel), Taxotere® (docetaxel)
  • T67 Tularik
  • vinca alkyloids e.g., vincristine, vinblastine, vindesine, and vinorelbine
  • dolastatins e.g., auristatin E, AFP, MMAF, MMAE, AEB, AEVB
  • antitubulin agents include, for example, baccatin derivatives, taxane analogs (e.g., epothilone A and B), nocodazole, colchicine and colcimid, estramustine, cryptophysins, cemadotin, maytansinoids, combretastatins, discodermolide, and eleutherobin.
  • the cytotoxic agent is a maytansinoid, another group of anti-tubulin agents.
  • the maytansinoid is maytansine or DM-1 (ImmunoGen, Inc.; see also Chari et al., Cancer Res. 52: 127-131, 1992).
  • the cytotoxic agent is an antimetabolite.
  • the antimetabolite can be, for example, a purine antagonist (e.g., azothioprine or mycophenolate mofetil), a dihydrofolate reductase inhibitor (e.g., methotrexate), acyclovir, gangcyclovir, zidovudine, vidarabine, ribavarin, azidothymidine, cytidine arabinoside, amantadine, dideoxyuridine, iododeoxyuridine, poscarnet, or trifluridine.
  • a purine antagonist e.g., azothioprine or mycophenolate mofetil
  • a dihydrofolate reductase inhibitor e.g., methotrexate
  • acyclovir gangcyclovir
  • zidovudine vidarabine
  • ribavarin azidothymidine
  • an anti-TGFBI antibody is conjugated to a pro-drug converting enzyme.
  • the pro-drug converting enzyme can be recombinantly fused to the antibody or chemically conjugated thereto using known methods.
  • Exemplary pro-drug converting enzymes are carboxypeptidase G2, P-glucuronidase, penicillin- V-amidase, penicillin-G-amidase, P- lactamase, P-glucosidase, nitroreductase and carboxypeptidase A.
  • Other molecule using as therapeutic moiety can be PyrroloBenzoDiazepine dimers (PBD).
  • the antibody is a chimeric antibody having a heavy chain identical to SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13 and a light chain identical to SEQ ID NO:2, 4, 6, 8, 10, 12 or 14 and conjugated to the MMAE.
  • the antibody is a chimeric antibody having a heavy chain identical to SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13 and a light chain identical to SEQ ID NO:2, 4, 6, 8, 10, 12 or 14 and conjugated to PyrroloBenzoDiazepine dimers (PBD).
  • PBD PyrroloBenzoDiazepine dimers
  • the antibody-drug conjugate compounds comprise a linker unit between the drug unit and the antibody unit.
  • the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the drug unit from the antibody in the intracellular environment.
  • the linker unit is not cleavable and the drug is released, for example, by antibody degradation.
  • the linker is cleavable by a cleaving agent that is present in the intracellular environment (e.g., within a lysosome or endosome or caveolea).
  • the linker can be, e.g., a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease.
  • the peptidyl linker is at least two amino acids long or at least three amino acids long.
  • Cleaving agents can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives resulting in the release of active drug inside target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123).
  • peptidyl linkers that are cleavable by enzymes that are present in 191P4D12-expressing cells. Examples of such linkers are described, e.g., in U.S. Pat. No. 6,214,345, incorporated herein by reference in its entirety and for all purposes.
  • the peptidyl linker cleavable by an intracellular protease is a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin with the Val-Cit linker).
  • One advantage of using intracellular proteolytic release of the therapeutic agent is that the agent is typically attenuated when conjugated and the serum stabilities of the conjugates are typically high.
  • the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at certain pH values.
  • the pH-sensitive linker hydrolyzable under acidic conditions.
  • an acid-labile linker that is hydrolyzable in the lysosome e.g., a hydrazone, semi carb azone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like
  • an acid-labile linker that is hydrolyzable in the lysosome e.g., a hydrazone, semi carb azone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like
  • the hydrolyzable linker is a thioether linker (such as, e.g., a thioether attached to the therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929).
  • the linker is cleavable under reducing conditions (e.g., a disulfide linker).
  • a disulfide linker e.g., a disulfide linker.
  • disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2- pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N- succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene), SPDB and SMPT.
  • SATA N-succinimidyl-S-acetylthioacetate
  • SPDP N-succinimidyl-3-
  • the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15: 1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1305- 12).
  • the linker unit is not cleavable and the drug is released by antibody degradation.
  • the linker is not substantially sensitive to the extracellular environment.
  • “not substantially sensitive to the extracellular environment,” in the context of a linker means that no more than about 20 %, typically no more than about 15 %, more typically no more than about 10 %, and even more typically no more than about 5 %, no more than about 3 %, or no more than about 1 % of the linkers, in a sample of antibody-drug conjugate compound, are cleaved when the antibody-drug conjugate compound is present in an extracellular environment (e.g., in plasma).
  • Whether a linker is not substantially sensitive to the extracellular environment can be determined, for example, by incubating with plasma the antibody-drug conjugate compound for a predetermined time period (e.g., 2, 4, 8, 16, or 24 hours) and then quantitating the amount of free drug present in the plasma.
  • a predetermined time period e.g. 2, 4, 8, 16, or 24 hours
  • nucleic acid molecule is covalently attached to lysines or cysteines on the antibody, through N- hydroxysuccinimide ester or maleimide functionality respectively.
  • TDCs cysteine-based site-specific conjugation
  • ADCs cysteine-based site-specific conjugation
  • Conjugation to unnatural amino acids that have been incorporated into the antibody is also being explored for ADCs; however, the generality of this approach is yet to be established (Axup et al., 2012).
  • Fc-containing polypeptide engineered with an acyl donor glutamine-containing tag e.g., Gin-containing peptide tags or Q- tags
  • an endogenous glutamine that are made reactive by polypeptide engineering (e.g., via amino acid deletion, insertion, substitution, or mutation on the polypeptide).
  • a transglutaminase can covalently crosslink with an amine donor agent (e.g., a small molecule comprising or attached to a reactive amine) to form a stable and homogenous population of an engineered Fc-containing polypeptide conjugate with the amine donor agent being site- specifically conjugated to the Fc- containing polypeptide through the acyl donor glutamine- containing tag or the accessible/exposed/reactive endogenous glutamine (WO 2012059882).
  • an amine donor agent e.g., a small molecule comprising or attached to a reactive amine
  • the inventors clearly showed in their results the impact of a TGFBI inhibitor and particularly the anti-TGFBI antibodies on muscle and fat tissue and the macrophage activity in those tissues demonstrating that a TGFBI inhibitor can have an impact on cachexia and cachexia linked to other diseases.
  • another obj ect of the invention relates to a TGFBI inhibitor for use in the treatment of cachexia in a subject in need thereof.
  • the invention relates to a TGFBI inhibitor for use in the treatment of a cachexia linked to a disease selected in the list consisting of cancer, congestive heart failure, chronic obstructive pulmonary disease, chronic kidney disease, and AIDS in a subject in need thereof.
  • the invention relates to a TGFBI inhibitor for use in the treatment of a cancer in a subject in need thereof.
  • the invention relates to a TGFBI inhibitor for use in the treatment of a cancer cachexia in a subject in need thereof.
  • the cancer of the cancer cachexia is a resistant cancer or a resistant cancer cachexia.
  • TGFBI inhibitor refers to any compound natural or not which is capable of reducing orblocking the activity or expression of TGFBI.
  • TGFBI inhibitor encompasses any TGFBI inhibitor that is currently known in the art or that will be identified in the future and includes any chemical entity that, upon administration to a patient, results in inhibition or down-regulation of a biological activity associated with activation of TGFBI. The term also encompasses inhibitor of expression.
  • TGFBI inhibitor is an inhibitor which neutralizes, blocks, inhibits, abrogates, reduces or interferes with the biological activity of TGFBI. In particular it refers to an inhibitor which reduces the weight loss, the muscle wasting and/or the cytokine secretion.
  • the TGFBI inhibitor according to the invention is :
  • biological activity of TGFBI is meant, in the context of the present invention, reducing the weight loss, the muscle wasting and/or the cytokine secretion.
  • Tests for determining the capacity of a compound to be an TGFBI inhibitor are well known to the person skilled in the art.
  • the ability of the inhibitor to inhibits the biological activity of TGFBI is well known to the person skilled in the art.
  • the ability to bind to TGFBI may be determined by assaying cells treated with TGFBI inhibitor (e.g. siRNA, antibodies) with fluorescence ELISA test or surface plasmon resonance (SPR) (see Figures 2A, 2B, 2C, 2D, 2E).
  • SPR surface plasmon resonance
  • the ability to stop weight loss or increase food intake may be determined by assaying the body weight, plasma glucose or albumin level (see Figures 3 A, 3B, 3C, 3D, 3E, 3F).
  • the ability to decrease muscle wasting or lipolysis may be determined by microscopic analysis, magnetic resonance tomography or muscle loss marker expression analysis (Trim63 and Fbox32) (see Figures 4A, 4B, 4C, 4D, 4E, 4F, 4G, 4H, 41).
  • muscle loss marker expression analysis Trim63 and Fbox32
  • Figures 4A, 4B, 4C, 4D, 4E, 4F, 4G, 4H, 41 can be evaluated using established clinical parameters such as handheld dynamometer for humans or wire hanging for rodents.
  • cytokine secretion and inflammation may be determined by ELISA test, Immunohistochemistry in target tissue or FACs analysis of isolated cells from tumors or blood (see Figures 5 A, 5B, 5C, 5D, 5E & 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 61, 6J, 6K).
  • Cancer cachexia refers to a multifactorial syndrome characterized by a continuous loss of skeletal muscle mass (with or without fat loss) provoked by the cancer.
  • the cachexic syndrome is fueled by continuous metabolic wasting, frequently accompanied by reduced appetite and food intake (Fearon K et al., 2011, Lancet Oncol). It results in decreased tolerability of anti-cancer treatments and decreased quality of life, both of which ultimately lead to death in at least 20-30% of patients (Mattox T.W et al., 2017, Nutr Clin Pract & Ferrara M et al., 2022, Front Cell Dev BioT).
  • Cancer refers to liquid or a solid cancer.
  • Cancer may be a cancer selected from the group consisting in adrenal cortical cancer, anal cancer, bile duct cancer (e.g. perihilar cancer, distal bile duct cancer, intrahepatic bile duct cancer), bladder cancer, bone cancer (e.g. osteoblastoma, osteochrondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of the bone, chordoma), brain and central nervous system cancer (e.g.
  • bile duct cancer e.g. perihilar cancer, distal bile duct cancer, intrahepatic bile duct cancer
  • bladder cancer e.g. osteoblastoma, osteochrondroma, hemangioma, chondromyxoid fibroma, osteos
  • meningioma astocytoma, oligodendrogliomas, ependymoma, gliomas, medulloblastoma, ganglioglioma, Schwannoma, germinoma, craniopharyngioma), breast cancer (e.g. ductal carcinoma in situ, infiltrating ductal carcinoma, infiltrating lobular carcinoma, lobular carcinoma in situ, gynecomastia), Castleman disease (e.g. giant lymph node hyperplasia, angiofollicular lymph node hyperplasia), cervical cancer, colorectal cancer, endometrial cancer (e.g.
  • adenocarcinoma endometrial adenocarcinoma, adenocanthoma, papillary serous adenocarcinoma, clear cell
  • esophagus cancer gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g. choriocarcinoma, chorioadenoma destruens), Hodgkin's disease, Kaposi's sarcoma, kidney cancer (e.g. renal cell cancer), laryngeal and hypopharyngeal cancer, liver cancer (e.g.
  • lung cancer e.g. small cell lung cancer, non-small cell lung cancer
  • mesothelioma plasmacytoma, nasal cavity and paranasal sinus cancer (e.g. esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g.
  • rhabdomyosarcoma embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer (e.g. melanoma, nonmelanoma skin cancer), stomach cancer, testicular cancer (e.g. seminoma, nonseminoma germ cell cancer), thymus cancer, thyroid cancer (e.g. follicular carcinoma, anaplastic carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma,), vaginal cancer, vulvar cancer, uterine cancer (e.g.
  • leukaemia like acute myeloid leukaemia, acute lymphoid leukaemia, chronic myelomonocytic leukemia (CMML)
  • CMML chronic myelomonocytic leukemia
  • MDS myelodysplastic syndrome
  • the compounds and methods of the present invention are suitable for treating colorectal cancer (CRC).
  • methods of the present invention are suitable for advanced or resistant CRC.
  • the term “patient” or “subject” or “individual” refers to a subject to be treated by the antibody disclosed herein.
  • the patient suffers from a Cancer.
  • the patient is a mammal.
  • mammals include rodents (e.g., mice and rats), primates (e.g., lemurs, bushbabies, monkeys, apes and humans), rabbits, dogs, horses, cats, livestock (such as pigs, bovines, donkeys, mules, bison, goats, camels, and sheep), and deer.
  • the mammal is a human.
  • the “subject” is a human with a cancer, in particular a cancer cachexia, according to the invention.
  • the TGFBI inhibitor according to the invention may be a low molecular weight compound, e. g. a small organic molecule (natural or not).
  • small organic molecule refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals.
  • Preferred small organic molecules range in size up to about 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to about 1000 Da.
  • the TGFBI inhibitor according to the invention is an anti-TGFBI antibody.
  • Antibodies directed against TGFBI can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from camels, pigs, cows, horses, rabbits, goats, sheep, and mice, among others, or organotypic cultures of primary human cells from tonsils, lymph nodes or peripheral blood.
  • a host animal selected, e.g., from camels, pigs, cows, horses, rabbits, goats, sheep, and mice, among others, or organotypic cultures of primary human cells from tonsils, lymph nodes or peripheral blood.
  • Various adjuvants known in the art can be used to enhance antibody production.
  • antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred.
  • Monoclonal antibodies against TGFBI can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture.
  • Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al., 1983); and the EBV-hybridoma technique (Cole et al. 1985).
  • techniques described for the production of single chain antibodies can be adapted to produce anti- TGFBI single chain antibodies.
  • Compounds useful in practicing the present invention also include anti- TGFBI antibody fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to TGFBI.
  • Humanized or human anti-TGFBI antibodies and antibody fragments therefrom can also be prepared according to known techniques.
  • “Humanized antibodies” are forms of non-human (e.g., rodent) chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the anti-TGFBI antibody according to the invention is one of the antibodies of the invention (A6, C7, C9, D2, D5, E4 or G7, see above).
  • the antibody according to the invention is a single domain antibody against TGFBI.
  • the term “single domain antibody” (sdAb) or “VHH” refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb.
  • VHH refers to the single heavy chain having 3 complementarity determining regions (CDRs): CDR1, CDR2 and CDR3.
  • CDRs complementarity determining region
  • CDR complementarity determining region
  • VHH according to the invention can readily be prepared by an ordinarily skilled artisan using routine experimentation.
  • VHH variants and modified form thereof may be produced under any known technique in the art such as in-vitro maturation.
  • VHHs or sdAbs are usually generated by PCR cloning of the V-domain repertoire from blood, lymph node, or spleen cDNA obtained from immunized animals into a phage display vector, such as pHEN2.
  • Antigen-specific VHHs are commonly selected by panning phage libraries on immobilized antigen, e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells.
  • immobilized antigen e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells.
  • VHHs often show lower affinities for their antigen than VHHs derived from animals that have received several immunizations.
  • VHHs from immune libraries are attributed to the natural selection of variant VHHs during clonal expansion of B-cells in the lymphoid organs of immunized animals.
  • the affinity of VHHs from non-immune libraries can often be improved by mimicking this strategy in vitro, i.e., by site directed mutagenesis of the CDR regions and further rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH, and low antigen concentrations).
  • VHHs derived from camelid are readily expressed in and purified from the E. coli periplasm at much higher levels than the corresponding domains of conventional antibodies.
  • VHHs generally display high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells.
  • the “Hamers patents” describe methods and techniques for generating VHH against any desired target (see for example US 5,800,988; US 5,874, 541 and US 6,015,695).
  • the “Hamers patents” more particularly describe production of VHHs in bacterial hosts such as E. coli (see for example US 6,765,087) and in lower eukaryotic hosts such as moulds (for example Aspergillus or Trichoderma) or in yeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see for example US 6,838,254).
  • the compound according to the invention is an aptamer.
  • Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition.
  • Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
  • Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990.
  • the random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence.
  • Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).
  • the compound according to the invention is a polypeptide.
  • the polypeptide is an antagonist or a reverse agonist of TGFBI and is capable to prevent the function of TGFBI.
  • the polypeptide can be a mutated TGFBI protein or a similar protein without the function of TGFBI.
  • the mutated version of the TGFBI protein is used as a decoy receptor.
  • polypeptide of the invention may be linked to a “cellpenetrating peptide” to allow the penetration of the polypeptide in the cell.
  • cell-penetrating peptides are well known in the art and refers to cell permeable sequence or membranous penetrating sequence such as penetratin, TAT mitochondrial penetrating sequence and compounds (Bechara and Sagan, 2013; Jones and Sayers, 2012; Khafagy el and Morishita, 2012; Malhi and Murthy, 2012).
  • polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art.
  • expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention.
  • the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule.
  • Systems for cloning and expression of a polypeptide in a variety of different host cells are well known.
  • the polypeptide is preferably generated by expression from an encoding nucleic acid in a host cell.
  • Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown. A common, preferred bacterial host is E coli.
  • polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy.
  • modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution.
  • the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution.
  • adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters.
  • a strategy for improving drug viability is the utilization of water-soluble polymers.
  • Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body.
  • water- soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain.
  • PEG Polyethylene glycol
  • Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity.
  • PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule.
  • copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications.
  • PEGylation techniques for the effective modification of drugs.
  • drug delivery polymers that consist of alternating polymers of PEG and tri -functional monomers such as lysine have been used by VectraMed (Plainsboro, N. J.).
  • the PEG chains (typically 2000 Daltons or less) are linked to the a- and e-amino groups of lysine through stable urethane linkages.
  • Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain.
  • the reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules.
  • These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer.
  • the molecular weight of the PEG segments affects the spacing of the drug/linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading).
  • increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory halflife of the conjugate. Nevertheless, the conjugate must either be readily degradable or have a molecular weight below the threshold-limiting glomerular filtration (e.g., less than 60 kDa).
  • linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue.
  • a specific trigger typically enzyme activity in the targeted tissue.
  • tissue activated drug delivery is particularly useful where delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of pathology.
  • Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes. Such linkers may be used in modifying the protein or fragment of the protein described herein for therapeutic delivery.
  • the TGFBI inhibitor according to the invention is an inhibitor of TGFBI gene expression.
  • Small inhibitory RNAs can also function as inhibitors of TGFBI expression for use in the present invention.
  • TGFBI gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that TGFBI gene expression is specifically inhibited (i.e. RNA interference or RNAi).
  • dsRNA small double stranded RNA
  • RNAi RNA interference
  • Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ.
  • siRNAs against TGFBI include, but are not limited to, those described in Chaoyu Ma (2008) Genes & Development 22:308-321.
  • MicroRNA can also function as inhibitors of TGFBI expression for use in the present invention.
  • MicroRNA are small, single-stranded, non-coding RNA molecules containing 21 to 23 nucleotides. Found in plants, animals and some viruses, miRNAs are involved in RNA silencing and post-transcriptional regulation of gene expression. miRNAs base-pair to complementary sequences in mRNA molecules, then silence said mRNA molecules.
  • Ribozymes can also function as inhibitors of TGFBI gene expression for use in the present invention.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of TGFBI mRNA sequences are thereby useful within the scope of the present invention.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.
  • antisense oligonucleotides and ribozymes useful as inhibitors of TGFBI gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramidite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life.
  • Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.
  • Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector.
  • a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing TGFBI.
  • the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences.
  • Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • adenovirus adeno
  • Non-cytopathic viral vectors are based on non-cytopathic eukaryotic viruses in which non- essential genes have been replaced with the gene of interest.
  • Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA.
  • Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle).
  • retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo.
  • adeno-viruses and adeno-associated viruses are double-stranded DNA viruses that have already been approved for human use in gene therapy.
  • the adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions.
  • the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection.
  • wildtype adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event.
  • the adeno-associated virus can also function in an extrachromosomal fashion.
  • Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al., 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigenencoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid.
  • Plasmids may be delivered by a variety of parenteral, mucosal and topical routes.
  • the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally.
  • the plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.
  • the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter.
  • the promoter may be specific for Muller glial cells, microglia cells, endothelial cells, pericyte cells and astrocytes
  • a specific expression in Muller glial cells may be obtained through the promoter of the glutamine synthetase gene is suitable.
  • the promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters.
  • an endonuclease can be used to abolish the expression of the gene, transcript or protein variants of TGFBI.
  • new technologies provide the means to manipulate the genome.
  • natural and engineered nuclease enzymes have attracted considerable attention in the recent years.
  • the mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the error prone non homologous end-joining (NHEJ) and the high-fidelity homology-directed repair (HDR).
  • NHEJ error prone non homologous end-joining
  • HDR high-fidelity homology-directed repair
  • the endonuclease is CRISPR-cas.
  • CRISPR-cas has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences.
  • the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes.
  • the CRISPR/Cas9 system has been described in US 8697359 Bl and US 2014/0068797. Originally an adaptive immune system in prokaryotes (Barrangou and Marraffini, 2014), CRISPR has been recently engineered into a new powerful tool for genome editing. It has already been successfully used to target important genes in many cell lines and organisms, including human (Mali et al., 2013, Science, Vol. 339 : 823-826), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol.
  • the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).
  • the TGFBI inhibitor is an antibody of the invention.
  • the cachexia is a cachexia linked to a cancer and more particularly, the cachexia is a cancer cachexia.
  • composition of the present invention may e.g. be formulated for any mode of administration suitable for the treatment of cancer.
  • the form of the composition, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the subject, etc. Then, the uses are adjusted to provide the optimum desired response (e.g., a therapeutic response).
  • the pharmaceutical compositions may contain vehicles which are pharmaceutically acceptable for a formulation capable of treating cancer.
  • the term “pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administrated to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluents, encapsulating material or formulation auxiliary of any type.
  • the carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • Carriers or “vehicles” include any such material known in the art and may be any liquid, gel, solvent, liquid diluent, solubilizer, or like, which is non-toxic and which does not interect with any components of the composition in a deleterious manner.
  • nutritionally acceptable carriers include, for example, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.
  • the use of liposomes and/or nanoparticles is contemplated for the introduction of the inhibitor or the antibodies of the invention into host cells.
  • Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 pm) are generally designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be are easily made.
  • Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)).
  • MLVs generally have diameters of from 25 nm to 4 pm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core.
  • SAVs small unilamellar vesicles
  • the physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations.
  • the pharmaceutic composition is a therapeutic composition.
  • the inhibitor of the invention (particularly the antibodies of the invention) or the pharmaceutical composition of the invention are administrated in a therapeutically effective amount.
  • Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutic compositions.
  • the term "therapeutically effective amount” or “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.
  • a therapeutically effective amount of the inhibitor or the composition of the present invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the inhibitor or the composition of the present invention to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the inhibitor or the composition are outweighed by the therapeutically beneficial effects.
  • the efficient dosages and dosage regimens for the inhibitor or the composition of the present invention depend on the disease or condition to be treated and may be determined by the persons skilled in the art.
  • a physician having ordinary skill in the art may readily determine and prescribe the effective amount of the inhibitor or the composition of the invention required.
  • the physician could start doses of the inhibitor or the composition of the present invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable dose of the inhibitor or the composition of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen.
  • Such an effective dose will generally depend upon the factors described above.
  • a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of disease.
  • the ability of a compound to inhibit inflammatory disorders and pain disorders may, for example, be evaluated in an animal model system predictive of efficacy in human.
  • this property of a composition may be evaluated by examining the ability of the compound to induce cytotoxicity by in vitro assays known to the skilled practitioner.
  • a therapeutically effective amount of a therapeutic compound may decrease tumor size, or otherwise ameliorate symptoms in a subject.
  • One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.
  • An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg/kg, about 5 mg/kg or about 8 mg/kg.
  • An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is 0.02-100 mg/kg, such as about 0.02-30 mg/kg, such as about 0.05-10 mg/kg or 0.1-3 mg/kg, for example about 0.5-2 mg/kg. Administration may e.g.
  • Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time.
  • the efficacy may be monitored by visualization of the disease area, or by other diagnostic methods described further herein, e.g. by performing one or more PET-CT scans, for example using a labeled antibody of the present invention, fragment or mini-antibody derived from the antibody of the present invention.
  • an effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • the monoclonal antibodies of the present invention are administered by slow continuous infusion over a long period, such as more than 24 hours, in order to minimize any unwanted side effects.
  • An effective dose of an antibody of the present invention may also be administered using a weekly, biweekly or triweekly dosing period.
  • the dosing period may be restricted to, e.g., 8 weeks, 12 weeks or until clinical progression has been established.
  • treatment according to the present invention may be provided as a daily dosage of an antibody of the present invention in an amount of about 0.1-100 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
  • Administration may be topical, oral, intranasal, parenteral, intravenous, intrathecal, epidural, intraocular, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target.
  • Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time.
  • the efficacy may be monitored by visualization of the disease area, or by other diagnostic methods described further herein, e.g. by performing one or more PET-CT scans.
  • an effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • the oligomers of the present invention are administered by slow continuous infusion over a long period, such as more than 24 hours, in order to minimize any unwanted side effects.
  • An effective dose of the agent of the present invention may also be administered using a weekly, biweekly or triweekly dosing period.
  • the dosing period may be restricted to, e.g., 8 weeks, 12 weeks or until clinical progression has been established.
  • treatment according to the present invention may be provided as a daily dosage of the agent of the present invention in an amount of about 0.1- 100 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
  • the quantity of the immune cell or the population of immune cells administered to a subject in need thereof is between 104 to 109 cells per kg.
  • the quantity of cells injected is 106 or 107 cells per kg.
  • the immune cell or the population of immune cells of the invention can be administrated is 1, 2, 3, 4 or 5 times to the subject in need thereof.
  • compositions for example, the form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the subject, etc.
  • compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze- dried compositions.
  • these may be in organic solvent such as DMSO, ethanol which upon addition, depending on the case, of sterilized water or physiological saline permit the constitution of injectable solutions.
  • compositions include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.
  • Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 pm) are generally designed using polymers able to be degraded in vivo. Biodegradable polyalkyl -cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be easily made.
  • Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)).
  • MLVs generally have diameters of from 25 nm to 4 gm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core.
  • SUVs small unilamellar vesicles
  • the physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations.
  • the inhibitor of the invention and particularly the antibodies of the invention may be used alone or in combination with further therapeutic active agent.
  • the inhibitor of the invention or the anti-TGFBI antibodies (or anti-TGFBI antibodies-drug conjugate) are delivered in a manner consistent with conventional methodologies associated with management of the disease or disorder for which treatment is sought (particularly, cachexia and cancer cachexia)
  • Another object of the invention relates to a combination of TGFBI inhibitor and a further therapeutic active agent for use in the treatment of cachexia in a subject in need thereof.
  • the invention relates to a pharmaceutical composition according to the invention and a further therapeutic active agent for use in the treatment of cachexia in a subject in need thereof.
  • the cachexia is a cancer cachexia.
  • therapeutic active agent relates to a substance (particularly a chemical substance) inducing an effect such as a therapeutic or a preventive effect. It may be a bioactive chemical compound from a drug or the drug itself. Active agent can be a single molecule or a mixture of several substances.
  • the therapeutic active agent may be conventional cancer therapies such as, e.g., radiotherapy, chemotherapy (or combinations thereof). Surgery can also be combined with the inhibitor of the invention
  • therapeutic active agent used in combination with the inhibitor of the invention comprising anti-cancer antibodies, cytotoxic agents, chemotherapeutic agents, anti -angiogenic agents, anti-cancer immunogens, cell cycle control/ apoptosis regulating agents, hormonal regulating agents, and other agents described below.
  • the inhibitor of the present invention is used in combination with a chemotherapeutic agent.
  • chemotherapeutic agent refers to chemical compounds that are effective in inhibiting tumor growth.
  • chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaorarnide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a carnptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
  • calicheamicin especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Inti. Ed. Engl. 33: 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino
  • paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • antihormonal agents that act to regulate or inhibit honnone action on tumors
  • anti -estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti -androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • the inhbitor of the present invention is used in combination with a targeted cancer therapy.
  • Targeted cancer therapies are drugs or other substances that block the growth and spread of cancer by interfering with specific molecules ("molecular targets") that are involved in the growth, progression, and spread of cancer.
  • Targeted cancer therapies are sometimes called “molecularly targeted drugs,” “molecularly targeted therapies,” “precision medicines,” or similar names.
  • the targeted therapy consists of administering the subject with a tyrosine kinase inhibitor.
  • tyrosine kinase inhibitor refers to any of a variety of therapeutic agents or drugs that act as selective or non-selective inhibitors of receptor and/or non-receptor tyrosine kinases. Tyrosine kinase inhibitors and related compounds are well known in the art and described in U.S Patent Publication 2007/0254295, which is incorporated by reference herein in its entirety.
  • a compound related to a tyrosine kinase inhibitor will recapitulate the effect of the tyrosine kinase inhibitor, e.g., the related compound will act on a different member of the tyrosine kinase signaling pathway to produce the same effect as would a tyrosine kinase inhibitor of that tyrosine kinase.
  • the tyrosine kinase inhibitor is a small molecule kinase inhibitor that has been orally administered and that has been the subject of at least one Phase I clinical trial, more preferably at least one Phase II clinical, even more preferably at least one Phase III clinical trial, and most preferably approved by the FDA for at least one hematological or oncological indication.
  • inhibitors include, but are not limited to, Gefitinib, Erlotinib, Lapatinib, Canertinib, BMS- 599626 (AC-480), Neratinib, KRN-633, CEP-11981, Imatinib, Nilotinib, Dasatinib, AZM- 475271, CP-724714, TAK-165, Sunitinib, Vatalanib, CP-547632, Vandetanib, Bosutinib, Lestaurtinib, Tandutinib, Midostaurin, Enzastaurin, AEE-788, Pazopanib, Axitinib, Motasenib, OSI-930, Cediranib, KRN-951, Dovitinib, Seliciclib, SNS-032, PD-0332991, MKC-I (Ro- 317453; R-440), Sorafenib, ABT
  • the inhibitor of the present invention is used in combination with an immunotherapeutic agent.
  • immunotherapeutic agent refers to a compound, composition or treatment that indirectly or directly enhances, stimulates or increases the body's immune response against cancer cells and/or that decreases the side effects of other anticancer therapies. Immunotherapy is thus a therapy that directly or indirectly stimulates or enhances the immune system's responses to cancer cells and/or lessens the side effects that may have been caused by other anti-cancer agents. Immunotherapy is also referred to in the art as immunologic therapy, biological therapy biological response modifier therapy and biotherapy.
  • immunotherapeutic agents examples include, but are not limited to, cytokines, cancer vaccines, monoclonal antibodies and non-cytokine adjuvants.
  • the immunotherapeutic treatment may consist of administering the subject with an amount of immune cells (T cells, NK, cells, dendritic cells, B cells).
  • Immunotherapeutic agents can be non-specific, i.e. boost the immune system generally so that the human body becomes more effective in fighting the growth and/or spread of cancer cells, or they can be specific, i.e. targeted to the cancer cells themselves immunotherapy regimens may combine the use of non-specific and specific immunotherapeutic agents.
  • Non-specific immunotherapeutic agents are substances that stimulate or indirectly improve the immune system.
  • Non-specific immunotherapeutic agents have been used alone as a main therapy for the treatment of cancer, as well as in addition to a main therapy, in which case the non-specific immunotherapeutic agent functions as an adjuvant to enhance the effectiveness of other therapies (e.g. cancer vaccines).
  • Non-specific immunotherapeutic agents can also function in this latter context to reduce the side effects of other therapies, for example, bone marrow suppression induced by certain chemotherapeutic agents.
  • Non-specific immunotherapeutic agents can act on key immune system cells and cause secondary responses, such as increased production of cytokines and immunoglobulins. Alternatively, the agents can themselves comprise cytokines.
  • Nonspecific immunotherapeutic agents are generally classified as cytokines or non-cytokine adjuvants.
  • cytokines have found application in the treatment of cancer either as general non-specific immunotherapies designed to boost the immune system, or as adjuvants provided with other therapies. Suitable cytokines include, but are not limited to, interferons, interleukins and colony-stimulating factors. Interferons (IFNs) contemplated by the present invention include the common types of IFNs, IFN-alpha (IFN-a), IFN-beta (IFN-P) and IFN- gamma (IFN-y). IFNs can act directly on cancer cells, for example, by slowing their growth, promoting their development into cells with more normal behavior and/or increasing their production of antigens thus making the cancer cells easier for the immune system to recognise and destroy.
  • IFNs Interferons
  • IFN-a IFN-alpha
  • IFN-P IFN-beta
  • IFN-y IFN-gamma
  • IFNs can act directly on cancer cells, for example, by slowing their growth, promoting
  • IFNs can also act indirectly on cancer cells, for example, by slowing down angiogenesis, boosting the immune system and/or stimulating natural killer (NK) cells, T cells and macrophages.
  • Recombinant IFN-alpha is available commercially as Roferon (Roche Pharmaceuticals) and Intron A (Schering Corporation).
  • Interleukins contemplated by the present invention include IL-2, IL-4, IL-11 and IL-12. Examples of commercially available recombinant interleukins include Proleukin® (IL-2; Chiron Corporation) and Neumega® (IL- 12; Wyeth Pharmaceuticals). Zymogenetics, Inc.
  • Colony-stimulating factors contemplated by the present invention include granulocyte colony stimulating factor (G-CSF or filgrastim), granulocyte-macrophage colony stimulating factor (GM-CSF or sargramostim) and erythropoietin (epoetin alfa, darbepoietin). Treatment with one or more growth factors can help to stimulate the generation of new blood cells in subjects undergoing traditional chemotherapy.
  • such methods may comprise infusion or re-infusion of immune system cells (for instance tumor-infiltrating lymphocytes (TILs), such as CC2+ and/or CD8+ T cells (for instance T cells expanded with tumor-specific antigens and/or genetic enhancements), antibody-expressing B cells or other antibody-producing or -presenting cells, dendritic cells (e.g., dendritic cells cultured with a DC-expanding agent such as GM-CSF and/or Flt3-L, and/or tumor-associated antigen-loaded dendritic cells), anti -tumor NK cells, so- called hybrid cells, or combinations thereof.
  • TILs tumor-infiltrating lymphocytes
  • CC2+ and/or CD8+ T cells for instance T cells expanded with tumor-specific antigens and/or genetic enhancements
  • antibody-expressing B cells or other antibody-producing or -presenting cells for instance dendritic cells cultured with a DC-expanding agent such as GM-CSF and/
  • the inhibitor of the invention may be used in combination with another antibody like the antibody Ha22-2 (Seattle Genetics) described in the patent application WO2012047724.
  • the inhibitor of the present invention is used in combination with an antibody that is specific for a costimulatory molecule.
  • antibodies that are specific for a costimulatory molecule include but are not limited to anti-CTLA4 antibodies (e.g. Ipilimumab), anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-TIMP3 antibodies, anti-LAG3 antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies or anti-B7H6 antibodies.
  • the cachexia is cancer cachexia.
  • the second agent is an agent that induces, via ADCC, the death of a cell expressing an antigen to which the second agent binds.
  • the agent is an antibody (e.g. of IgGl or IgG3 isotype) whose mode of action involves induction of ADCC toward a cell to which the antibody binds.
  • NK cells have an important role in inducing ADCC and increased reactivity of NK cells can be directed to target cells through use of such a second agent.
  • the second agent is an antibody specific for a cell surface antigens, e.g., membrane antigens.
  • the second antibody is specific for a tumor antigen as described above (e.g., molecules specifically expressed by tumor cells), such as CD20, CD52, ErbB2 (or HER2/Neu), CD33, CD22, CD25, MUC-1, CEA, KDR, > VD3, etc., particularly lymphoma antigens (e.g., CD20).
  • a tumor antigen as described above (e.g., molecules specifically expressed by tumor cells), such as CD20, CD52, ErbB2 (or HER2/Neu), CD33, CD22, CD25, MUC-1, CEA, KDR, > VD3, etc., particularly lymphoma antigens (e.g., CD20).
  • a tumor antigen as described above (e.g., molecules specifically expressed by tumor cells), such as CD20, CD52, ErbB2 (or HER2/Neu), CD33, CD22, CD25, MUC-1, CEA, KDR, > VD3, etc., particularly lymphoma antigens (e.g
  • the inhibitor of the present invention is used in combination with an agonist of the ghrelin/growth hormone secretagogue receptor (GHSR).
  • GHSR ghrelin/growth hormone secretagogue receptor
  • the cachexia is cancer cachexia.
  • the agonist of the GHSR can be the anamorelin, the adenosine, the alexamorelin, the capromorelin, the CP -464709, the corti statin- 14, the examorelin (hexarelin), the ghrelin (lenomorelin), the GHRP-1, the GHRP-3, the GHRP-4, the GHRP-5, the GHRP-6, the Ibutamoren (MK-677), the ipamorelin, the L-692,585, the LY-426410, the LY-444711, the macimorelin, the pralmorelin (GHRP-2), the relam orelin, the SM- 130,686, the tabimorelin or the ulimorelin.
  • the invention relates to a combination of TGFBI inhibitor (particularly an antibody of the invention) and anamorelin for use in the treatment of cachexia in a subject in need thereof.
  • Another object of the invention relates to a kit of part comprising the inhibitor of the invention or the pharmaceutical composition of the invention and at least one therapeutic active agent as a combined preparation for simultaneous, separate or sequential use in the treatment of cachexia and particularly cancer cachexia.
  • the term “simultaneous use” denotes the use of the antibody and at least one active agent occurring at the same time.
  • the term “separate use” denotes the use of the antibody and at least one active agent not occurring at the same time.
  • sequential use denotes the use of the antibody and at least one active agent occurring by following an order.
  • FIG. 1 High stromal TGFBI positivity in primary tumors was associated with cachexia in advanced CRC patients.
  • Figure 2 Development of antibodies against Tgfbi.
  • X-axis Human TGFBI schema and target peptide region.
  • D ELISA determined the binding affinity of anti-Tgfbi antibody clones against human recombinant TGFBI protein. The binding affinity ratio was calculated as an absorbance ratio relative to the control (IgG).
  • E Western blot and ELISA of Tgfbi in conditioned medium from murine cell lines. Ponceau red staining was used to evaluate the protein transfer efficiency to the membrane. All data are presented as mean ⁇ SD. *, P value ⁇ 0.05; **, P value ⁇ 0.01; ***, P value ⁇ 0.001; ****, P value ⁇ 0.0001. Ab, antibody.
  • Tgfbi antibodies prevent cancer cachexia and prolong the survival in cachexia model mice.
  • the right lower bar graph shows each group's frequency of invasive and non-invasive tumors. Scale bar 100 pm. Magnification x200. Insets showing low magnification views xlOO.
  • D Plasma glucose and albumin levels of the Colon26 cachexic model mice treated by Isotype IgG or Tgfbi antibodies on day 17. The dashed line indicates the reference ranges of healthy mice.
  • E This figure shows the Kaplan-Meier curve for overall survival in the Colon26 cachexic model mice treated with isotype IgG or Tgfbi antibodies. The foot table shows the P value, hazard ratio (HR), and 95% confidence interval for each group (A6 and C9) compared to the IgG.
  • Tgfbi antibody recovered the muscle wasting and lipolysis in cachexia model mice to the level of control mice without tumor.
  • A Images and weight of quadriceps and gastrocnemius muscles in the control mice and Colon26 cachexic model mice treated by Isotype IgG or Tgfbi antibody (C9) at day 17.
  • B Images and weight of visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) in the control mice and Colon26 cachexic model mice treated by Isotype IgG or Tgfbi antibody at day 17.
  • C Left panel: Representative images of muscle fibers in the control mice and Colon26 cachexic model mice treated by Isotype IgG or Tgfbi antibody at day 17.
  • Right panel The bar graph shows the relation between muscle fiber area and frequency in the model mice. Scale bar 100 pm.
  • Magnification x400 The muscle fiber area was quantified by four independent images using Image J.
  • D The bar graphs show the mRNA expression levels of muscle degradation marker Trim63 (known as Murfl) and Fbxo32 (known as Atrogin) in the control mice and Colon26 cachexic model mice treated by Isotype IgG or Tgfbi antibody at day 17.
  • E Left panel: Representative images of subcutaneous adipose tissues in the control mice and Colon26 cachexic model mice treated by Isotype IgG or Tgfbi antibody at day 17.
  • Right panel The bar graph shows the relation between adipocyte area and frequency in the model mice. Scale bar 100 pm. Magnification x200.
  • the adipocyte cross-sectional area was quantified by eight independent images using Image J.
  • F Left panel: Representative IHC images of Ucpl expression in subcutaneous adipose tissues in the control mice and Colon26 cachexic model mice treated by Isotype IgG or Tgfbi antibody at day 17.
  • Right panel The bar graph shows the quantitative evaluation of Ucpl expression in subcutaneous adipocyte area in the model mice. Scale bar 100 pm. Magnification x200.
  • Ucpl expression was quantified by four independent images using Image J.
  • G Alteration of body weight, wire hanging time, and food intake in the control mice and the Colon26 cachexic model mice treated by Isotype IgG or Tgfbi antibody C9 at indicated days.
  • Tgfbi antibodies inhibit macrophage-derived cytokines.
  • the bar graph shows 11-6, Tnf-a, and Tgfbi levels in plasma samples from the Colon26 cachexic model mice treated with isotype IgG or Tgfbi antibodies (A6 and C9) (day 17), as determined by the ELISA.
  • B Left panel: Representative IHC images show the intratumoral Cd86+ and Cd206+ macrophages in the Colon26 cachexic model mice treated with isotype IgG or Tgfbi antibodies (day 17). Scale bar 100 pm. Magnification x200.
  • Right panel The bar graphs show the Cd86+ and Cd206+ macrophage numbers per field of view (FOV), as quantified by the Hybrid cell count system.
  • FIG. 1 Left panel: Multicolor immunofluorescent images of macrophage markers F4/80 (green) and 11-6 (red) in the tumor samples of the Colon26 cachexic model mice treated with isotype IgG or Tgfbi antibodies (day 17). Scale bar 100 pm. Magnification x400.
  • Tgfbi can induce the macrophage-derived cytokine production via integrins/pFAK signaling.
  • A The anti -Tgfbi antibody treatment repressed secretion of 11-6 and Tnf-a proteins in the culture medium from murine macrophage cell line J774.1 treated with isotype IgG or Tgfbi antibodies (A6 and C9) (10 pg/ml, 48 h). Each dot represents replicates of 4 independent ELISA experiments.
  • B The recombinant Tgfbi (rec. Tgfbi) increased secretion of 11-6 and Tnf-a proteins in the culture medium from murine macrophage cell line J774.1. Each dot represents replicates of 5 independent ELISA experiments.
  • ELISA determined the secreted 11-6 and Tnf-a protein levels in the culture medium from the J774.1 cells treated with or without rec.Tgfbi(100 ng/mL) and cilengitide (10 pM) for 48 h. Each dot shows the replicates from 4 independent experiments.
  • D The integrins siRNAs (siltgav and siltgbS) suppressed macrophage-derived cytokine production induced by rec.Tgfbi treatment.
  • ELISA determined the levels of secreted 11-6 and Tnf-a protein in the culture medium of integrins-suppressed J774.1 cells after 48 h of treatment with or without rec.Tgfbi (100 ng/mL).
  • E FACS histogram of VivoTag® 680 XL labeled antibodies (A6 and C9, 5 ng/mL) binding to the integrins-suppressed J774.1 cells with or without rec.Tgfbi treatment (100 ng/ml, 30 min).
  • Left panel Binding ability of the labeled Tgfbi antibodies to exogenous Tgfbi on J774.1 was depending on the integrins.
  • Right panel The addition of exogenous rec.Tgfbi enhanced the binding ability of the labeled Tgfbi antibodies to the Tgfbi on the cell surface (the siNT control group). However, the binding ability was less enhanced in the integrins siRNA groups.
  • Isotype IgG antibody was used as a control.
  • F The basal protein expression of 11-6 and Tnf-a in culture medium from NIH3T3, Colon26 and J774.1 cells was determined by ELISA.
  • G The anti -Tgfbi antibody treatment repressed the mRNA expression of 11-6 and Tnf-a in J774.1 cells treated with isotype IgG or Tgfbi antibodies (10 pg/ml, 48 h) . Each dot represents replicates of 4 independent experiments.
  • H The recombinant Tgfbi (rec.Tgfbi, 100 ng/ml, 48 h) increased the mRNA expression of 11-6 and Tnf-a in J774.1 cells.
  • Figure 7 Combination treatment of anti-TGFBI (antibody clone C9) with immune-checkpoint therapy anti PD-1.
  • A Kaplan-Meier curve for overall survival in the Colon26 cachexic model mice treated with isotype IgG, Tgfbi antibody (C9), anti-Pdl antibody, and/or combination. Tumors were resected at day 17. The foot table shows the P value, hazard ratio (HR), and 95% confidence interval for each group (A6 and C9) compared to the IgG.
  • B Quantification of tumor volume on the day of tumor excision (day 17).
  • Figure 8 Comparison of anti-TGFBI (antibody clone C9) with state of art cachexia treatment, Ghrelin agonist Anamorelin.
  • E Percentage of white blood cell fractions (lymphocyte, monocyte, and neutrophils) in the peripheral blood of the Colon26 cachexic model mice treated with control, Anamorelin, and Tgfbi antibody (C9) at the time of sacrifice.
  • G Weight of different adipose tissues (SAT, VAT, BAT), muscles (GA, Quad) as well as spleen and liver (left panel) and estimated total weight of adipose and muscle tissues (right panel) in the Colon26 cachexic model mice treated with control, Anamorelin, and Tgfbi antibody (C9) at the time of sacrifice.
  • FIG. 9 Combination treatment of anti-TGFBI (antibody clones A6 and C9) with state of art cancer treatment (5-FU chemotherapy).
  • A Schema of the treatment plan (anti-TGFBI Ab and tumor resection) against the cachexia model mice.
  • Female Balb/c mice were inoculated with the Colon26-luc cells on day 0.
  • the mice were treated with IgG control or anti-TGFBI Abs (A6 and C9 clones) from day 1 (2 times a week, 200 pg/body, intraperitoneal injection), and the tumors were surgically resected on day 17.
  • the animals were recovered from surgery for a week and followed by an antineoplastic agent 5-FU (30mg/kg, intraperitoneal injection on days 23 and 26) in adjuvant chemotherapy setting and followed up to evaluate survival and tumor progression.
  • 5-FU antineoplastic agent
  • the foot table shows the P value, hazard ratio (HR), and 95% confidence interval for each group comparison.
  • Figure 10 Relationship between preoperative serum TGFBI levels and cachexia in 69 pancreatic cancer patients.
  • Figure 11 Validation of the therapeutic effect of TGFBI antibody in a pancreatic cancer patient-derived xenograft (PDX) cachexia model.
  • PDX pancreatic cancer patient-derived xenograft
  • FIG. 1 Schematic representation of the TGFBI antibody (Ab) treatment regimen in the pancreatic cancer PDX (PC-PDX)-induced cachexia model.
  • PC-PDX pancreatic cancer PDX
  • Figure 12 Validation of the cachexia treatment effect of TGFBI Ab using pancreatic cancer patients derived xenograft tumor model.
  • Top panel ELISA quantifications of 11-6, Tnf-a and Tgfbi levels in plasma samples from the no tumor-bearing control mice and PC PDX cachexic model mice treated by isotype IgG or Tgfbi Ab on day 45.
  • Bottom left panel Frequency of the muscle’s cross-sectional area in the model mice.
  • Bottom right panel Western blot analysis of muscle degradation marker MurFl and downstream of 11-6, Stat-3 activation in the TA muscles from PC-PDX animal model mice treated by isotype IgG or Tgfbi Ab on day 45.
  • Figure 13 Comparison of the cachexia treatment effect of TGFBI Ab and TGFBI Ab-LALAPG using colon26 cancer cachexia model.
  • Figure 14 Comparison of the cachexia treatment effect of TGFBI Ab and TGFBI Ab-LALAPG using colon26 cancer cachexia model.
  • Figure 15 Western blot analysis of muscle degradation markers in colon26 cancer cachexia model treated by TGFBI Ab.
  • Example 1 Use of anti- Tgfbi antibodies in the treatment of cachexia and cancer cachexia
  • Phage antibody expression library Husc I (Caucheteur D et al., Methods Mol Biol) was probed with recombinant human TGFBI to isolate polyclonal human antibodies in scFv format. The binding specificty of these antibody fragments was tested against immobilized TGFBI using ELISA. Following the phage-enrichment by four rounds of bio-panning, seven monoclonal antibodies were selected based on their binding to recombinant protein. Complementarity-determining regions (CDR) of the seven anti-TGFBI scFv were sequenced using Next Generation sequencing.
  • CDR complementarity-determining regions
  • 2 anti- TGFBI scFv (hereafter called A6 and C9) were selected based on their diversity and sequence redundance with other scFv clones.
  • the anti-TGFBI scFv were cloned into murine IgG2a framework and outsourced for bioproduction to external company (Evitria AG, Schlieren, Switzerland). All antibody preparations had purity > 90%, were sterile and endotoxin free.
  • the binding of anti-Tgfbi antibodies was evaluated by surface plasmon resonance (SPR) analysis using a BIAcore X-100 apparatus (Cytiva, Marlborough, MA, USA).
  • SPR surface plasmon resonance
  • Human TGFBI and murine Tgfbi (20 pg/ml in 10 mM sodium acetate, pH 4.0) were allowed to react with a flow cell of a CM5 sensor chip previously activated with a mixture of 0.2 M N-ethyl-N’-(3- dimethylaminopropyl)-carbodiimide hydrochloride and 0.05 M N-hydroxysuccinimide (35 pl, flow rate 10 pl/min).
  • the NIH3T3, Colon26-luc, and J774.1 cell lines were from the Japanese Collection of Research Bioresources Cell Bank. All cell lines were cultured in RPML1640 supplemented with 10% fetal bovine serum (FBS) (Gibco, Invitrogen, CA, USA) and 1% penicillin/streptomycin (Wako, Osaka, Japan) at 37 °C in 5% CO2. Cell lines were tested for mycoplasma contamination using Mycostrip (Invivogen, California, USA). For in vitro treatment, J774.1 cells were seeded at 70% confluency and adhered to overnight.
  • FBS fetal bovine serum
  • Pako penicillin/streptomycin
  • J774.1 and Colon26-luc cells were suspended at a density of 1.0 * 10 6 cells in 100 pL of Opti-MEM I Reduced Serum Media (Thermo Fisher Scientific) and then mixed with specific siRNAs (siTgfbi cocktail/siltgav cocktail/siltgb5 cocktail) or non-target control siRNA (siNT) (Ajinomoto Bio-Pharma, Japan, see supplementary table). Transfection was performed using a CUY21 EDIT II electroporator (BEX, Tokyo, Japan) according to the manufacture's protocol. After 48 h incubation, the cells were used for further analysis.
  • J774.1 and Colon26-luc cells were transfected with the siRNAs mentioned above. After 48 h, cells were collected and co-incubated with antibody (5 ng/ml, control IgG and Tgfbi Ab labeled by VivoTag680) at room temperature for 30 min. The cells binding to the labeled antibodies were analyzed using the FACSVerse and FACSSuite software (BD Biosciences, New Jersey, USA). The dissected tumors were minced and incubated with Dri Tumor & Tissue Dissociation Reagent (BD, USA) in RPMI-1640 for 30 min at 37 °C under shaking. Following this, the mixture was filtrated using 100 pm size cell strainers.
  • Mononuclear cells were isolated by gradient centrifugation using Percoll according to the manufacturer’s protocol, and centrifugation was gradually performed at 2200 rpm for 20 min. Following the washing, cell viability and cell numbers were calculated using trypan blue staining and cell counter. The single-cell suspensions were first blocked with murine Fc blocking buffer for 15 min at room temperature, then stained with fluorescently labeled antibodies against CD45, CD3, CD4, CD8, CDllb, F4/80, CD86, and CD206 for 30 min at 4°C prior to flow cytometry analysis (see the supplementary table). Flow-cytometric data were analyzed using the FACSVerse and FACSSuite software (BD Biosciences, New Jersey, USA).
  • Colon26-luc (5x104 cells/lOOul in PBS) were subcutaneously injected into the right flank of the animal under general anesthesia (2.0% Isoflurane given by inhalation).
  • mice were sacrificed at indicated end-points (days 9-17) of the proposed experiments under deep anesthesia (5.0% isoflurane given by inhalation).
  • Internal organs, fat depots (visceral, subcutaneous, and interscapular), muscles (quadriceps and gastrocnemius), and tumors were dissected, weighed, and subjected to further analysis.
  • the blood was collected in a K2EDTA-containing tube (Greiner, Kremsmunster, Austria), and plasma samples were prepared with refrigerated centrifugation at 3000 rpm for 15 min.
  • Plasma (non-fastened) biochemical parameters were analyzed with a MultiRotor I Preventive Care Panel using Vetscan (Zoetis, New Jersey, USA).
  • Plasma (non-fastened) biochemical parameters were analyzed with a MultiRotor I Preventive Care Panel using Vetscan (Zoetis, New Jersey, USA). Magnetic resonance tomography (MRI) was performed to evaluate the fat and muscle composition of the animals.
  • In vivo luciferase assay for detecting the Colon26-luc and biodistributions of the labeled antibodies were performed using the IVIS Lumina Series III system. Detailed methods is described in the supplementary material.
  • Magnetic Resonance Imaging MRI
  • VAT visceral adipose tissue
  • SAT subcutaneous adipose tissue
  • Antibodies were labeled with a VivoTag® 680XL protein labeling kit (Perkin Elmer, Massachusettes, USA) according to the manufacturer’s instructions.
  • mice were fed with iVid-neo (Cat #: 2403000, Oriental East Corp., Tokyo, Japan) alfalfa-free food starting a week before the experiment.
  • Fifty pg VivoTag® 680XL labeled antibodies were intraperitoneally injected.
  • Biodistribution of VivoTag® 680XL labeled antibodies was imaged with IVIS Lumina Series III system fluorescent setting with (Exc: 673nm/ Emm: 691nm) 48h after the antibody injection.
  • Total Radiant Efficiency [p/s]/[pW/cm 2 ] in the tumors was analyzed by Living Image Software (version 4.3.1, Xenogen, Alameda, USA).
  • FFPE paraffin-embedded
  • the sections were prepared, and endogenous peroxidase was blocked, as described above. Nonspecific binding sites were blocked by incubation with Protein Block Serum-Free Reagent for 30 min, and the sections were incubated overnight at 4 °C with the primary antibodies against macrophage marker F4/80 (Cell Signaling Technology, Massachusetts, USA) and murine 11-6 (PeproTech, NJ, USA). Multiplex covalent labeling (F4/40, TSA coumarin; murine 11-6, Opal 690) with tyramide signal amplification (Akoya Biosciences, MA, USA) was performed according to the manufacturer’s protocol.
  • the indirect ELISA was used to quantify Tgfbi, 11-6, and Tnf-a levels in plasma samples and the conditioned medium. Maxisorp 96-well plates (Thermo Fisher, USA) were coated with 100 pL of samples. Serial dilutions of murine recombinant Tgfbi (R&D, USA), 11-6, and Tnf-a (PeproTech, USA) were prepared in PBS as a calibration curve. The coating was performed overnight at 4°C on agitation. The wells were washed three times with PBS/Tween-20 and residual-binding sites were blocked with 4% (w/v) skim milk in PBS (200 pl/well) for 2 h at room temperature.
  • Protein samples of concentrated condition media and cell lysates were suspended in RIPA buffer (Wako, Osaka, Japan). Laemmli buffer (Biorad, California, USA) was added to 10 pl of samples. After boiling for 5 minutes, identical amounts of proteins were loaded on 7.5% polyacrylamide gels. Proteins were separated with electrophoresis and transferred to nitrocellulose membranes at 90V for 90 min. After blocking in 5% skim milk or 5% BSA for 1 h, membranes were incubated (4 °C, overnight) with primary antibodies (see supplementary table). The membranes were washed by TBS-T three times, followed by corresponding secondary antibody incubation for 1 h at room temperature. Then, washing was repeated, and protein detection was visualized using chemiluminescence.
  • Cytokines in the plasma samples (200pl) from 6 mice were analyzed using Proteome Profiler Mouse Cytokine array kits (R&D Systems, cat. #ARY006), according to the manufacturer's instructions. Cytokine abundance was calculated as the integrated average density of the spot replicates using the ImageJ software. Altered cytokines in each group were normalized by the negative control spot intensity.
  • cDNA synthesis was performed using the ReverTra Ace qPCR RT Master Mix (Toyobo, Japan).
  • Quantitative real-time RT-PCR was performed using the KAPA SYBR Fast qPCR Kit (KAPA biosystem, MA, USA) with a total reaction volume of 20 pL. The relative levels of candidate genes were calculated using the 2-ddCT method. All primer sequences used in our study are listed in supplementary table.
  • High stromal TGFBI expression was associated with the cachexic condition in advanced CRC patients.
  • Tgfbi-expressing murine cell lines consisting of 3 colon cancer (Colon26, MC38, and CT26), a fibroblast (NH43T3), and a macrophage cell line J774.1 and found that Colon26 and J774.1 cells were abundantly secreting Tgfbi compared to the other cell lines (Fig. 2E).
  • Fig. 2E Tgfbi-expressing murine cell lines consisting of 3 colon cancer (Colon26, MC38, and CT26), a fibroblast (NH43T3), and a macrophage cell line J774.1 and found that Colon26 and J774.1 cells were abundantly secreting Tgfbi compared to the other cell lines.
  • Fig. 2E Both A6 and C9 antibodies specifically detected murine Tgfbi (Fig.
  • Tgfbi targeting antibody prevents cancer cachexia and prolongs survival in vivo.
  • Colon26-luc tumor-bearing mice with anti-Tgfbi A6 and C9 antibodies twice a week (total 5 times, intraperitoneal injection) (Data not shown). It is well reported that the Colon26 tumorbearing mice show representative cachexic symptoms such as body weight loss, muscle weakening, and food intake reduction (Bonetto A et al., 2016, J Vis Exp & Aulino P et al., 2010, BMC Cancer). Thus, we monitored these parameters during the treatment (Fig. 3 A).
  • mice from the control group had local and distant tumor recurrence, in contrast to 40% of the animals in the treatment arm (Data not shown). Having observed that anti-Tgfbi treatment has an anticachectic effect, we further sought to examine the mechanism behind this observation. Tgfbi antibody inhibits muscle wasting and lipolysis.
  • Placebo-treated tumor-bearing mice had significantly small muscle fiber cross-sections compared to the no tumor and the anti-Tgfbi treated animals.
  • Fig. 4D suggests, muscles recovered from placebo-treated animals featured high expression of both genes.
  • Anti-Tgfbi treatment dampens cytokine levels elevated by tumor-induced cachexia.
  • Tnf-a and 11-6 Treatment with anti-Tgfbi antibodies reduced a number of cytokines, including Tnf-a and 11-6, which were previously established as cachexia relevant (Baazim H et al., 2022, Nat Rev Immunol Kasprzak Aet al., 2Q2 , IntJMol Sci & DzierlegaK et al., 2023, J Immunol). Following this semiquantitative approach, we validated the changes of Tnf-a and 11-6 along with the target protein Tgfbi, in the plasma samples using quantitative ELISA measurements (Fig. 5A).
  • T-cells and macrophages essentially are mainly responsible for the secretion of pro-inflammatory cytokines
  • tumor-infiltrating immune cells in the Colon26 tumors were characterized by IHC analysis.
  • the total number of Cd8 + cytotoxic T-cells was increased in A6 and C9 anti-Tgfbi treated conditions.
  • the total number of Cd86 + macrophages was increased in A6 and C9 anti-Tgfbi treated conditions (Fig. 5B).
  • macrophages are known to reside also in the fat and muscle tissues (Shukla S.K et al., 2020, Cancer Lett Lazaro T et al., 2023, Nature & Jaitin D.A et al., 2019, Cell). Considering that we demonstrated a significant impact of our antibodies on the adipose and muscle tissues, we also examined the status of macrophages in these tissues. IHC analysis showed that the numbers of both Cd86 + and Cd206 + macrophage cells were significantly increased in both tissues (Fig. 5D & Fig. 5E). We next sought to examine if altered cytokine levels (observed in the Fig. 5A) were directly related to the macrophages present in respective tumors.
  • Anti-Tgfbi targeting in macrophages inhibits 11-6 and Tnf-a gene expression and protein secretion.
  • J774.1 cells To test the possible relationship between cytokine expression in macrophages and Tgfbi activity, we selected J774.1 cells to serve as in vitro model. The selection was supported by the data showing their strong Tgfbi expression (Fig. 2E). The J77.1 cells are Cd86 positive and have relatively strong expression and secretion of both 11-6 and Tnf-a compared to the fibroblasts and cancer cells (Fig. 6F & Data not shown). Next, we sought to determine if Tgfbi targeting in the J774.1cells could alter the 11-6 and Tnf-a levels. As shown in the Fig. 6A and Fig. 6G, anti -Tgfbi antibody treatment diminished the secretion and expression of 11-6 and Tnf- a.
  • Tgfbi binds to the cell surface of the macrophage cells to regulate cytokine production and have thus sought to determine the possible receptor.
  • Previous literature evidence supports that extracellular Tgfbi interacts with several integrins on cancer cells, macrophages, and Cd8 + lymphocytes (Peng P et al., 2022, Theranostics Costanza B et al., 2019, Int J Cancer Goehrig D et al., 2019, Gut & Corona A et al., 2021, Cell Signal).
  • Example 2 The combination effect of anti-TGFBI antibody with anti-PD-1 antibody
  • a mouse model of cachexia was created by subcutaneous transplantation of colon26 cells of mouse colon cancer cell line into Balb/c mice.
  • mice were grouped into IgG isotype control group, anti-TGFBI antibody (Ab C9) group (200 ug/body/2 times per week), anti-PD-1 antibody group (200 ug/body/2 times per week) and combination group.
  • Ab C9 anti-TGFBI antibody
  • Anti-PD-1 antibody group 200 ug/body/2 times per week
  • Tumor volume in the combination group was significantly smaller than that of the control and anti-TGFBI antibody alone groups.
  • anti-TGFBI antibody tended to increase the infiltration of cytotoxic T cells into the tumor compared to the single antibody.
  • the appearance suggestive of cachexia was not observed in the anti-TGFBI antibody, anti-PD-1 antibody, and the combination groups, in contrast to the control group, which presented cachexia.
  • the body weight in the anti- TGFBI antibody, anti-PD-1 antibody, and the combination groups was higher than that of the control group.
  • the wire hanging time in the anti-TGFBI antibody group was significantly longer than in the control group, suggesting that the TGFBI blockage can maintain muscle strength in the cachexic condition.
  • Example 3 Comparative study of anti-TGFBI antibody and ghrelin agonist anamorelin in a mouse model of cachexia
  • a mouse model of cachexia was created by subcutaneous transplantation of colon26 cells of mouse colon cancer cell line into Balb/c mice.
  • mice were grouped into a control group (PBS via p.o. every other day), an anti- TGFBI antibody group (200 ug/body/2 times per week), and an anamorelin, a ghrelin agonist group (30 mg/kg p.o. every other day).
  • Both the anti-TGFBI antibody and anamorelin treatment significantly prolonged the survival of the tumor-bearing mouse without any surgical intervention.
  • Median survival days were 26.5, 22, and 18 days, respectively.
  • the anti-TGFBI antibody treatment outperformed the anamorelin treatment in terms of survival and well-being (body weight, maintaining muscle strength, and plasma glucose level) of the tumor-bearing animal.
  • food intake and plasma albumin levels were higher in anamorelin treatment group animals (Fig. 8A and 8B).
  • animals treated with anti-TGFBI antibodies or anamorelin maintained more fat in spite of their body weight loss.
  • Anti-TGFBI antibody treatment significantly elongated the survival in the cachexia model mice compared to the control (PBS) and anamorelin treatment.
  • tumor weight and volume were not suppressed in the anti-TGFBI antibody and anamorelin groups compared to the control group (Fig. 8C).
  • Plasma cytokine levels including 11-6, Tnf-a, and Tgfbi levels were significantly lower in anti-TGFBI antibody-treated animals compared to the control group. More, Tgfbi antibody treated groups tend to have lower levels of cytokines at the endpoints (Fig. 8F and 8G).
  • a mouse model of cachexia was created by subcutaneous transplantation of colon26 cells of mouse colon cancer cell line into Balb/c mice.
  • mice were grouped into IgG isotype control group and anti-TGFBI antibody (Ab C9) group (200 ug/body/2 times per week). Then mouse tumors were surgically resected at day 17 after tumor inoculation. After the tumor resection mice were treated with anti -turn or agent (5FU, 30mg/kg via i.p. injection) at day 23 and day 26, and further followed up to monitor survival until day 60.
  • anti -turn or agent 5FU, 30mg/kg via i.p. injection
  • cancer treatment becomes a chronic systemic therapy with sets of different (and usually cytotoxic) agents.
  • the cancer cells can reprogram the body metabolism, which frequently results in cachexia.
  • Cachexic patients cannot support recommended dose regimens, leading to lowering of the cytotoxic doses and hence ultimately treatment failure and cancer resistance.
  • Our ability to continue the patient treatment therefore critically depends on the auxiliary means of controlling cachexic process and hence widening the window of therapeutic intervention.
  • nutritional supplements are some of limited means to delaying the onset of cachexia; however, the clinical evidence for this remains rather heterogeneous (Van de Worp W et al., 2020, Front Niitr).
  • New drugs are therefore critically needed to curb the cachexic syndrome, especially knowing that in some types of cancer over 3 /4 of patients will experience cachexia (e.g. pancreatic cancer).
  • cachexia e.g. pancreatic cancer
  • the development of anti-cachexic treatments has been a difficult process, largely in part due to the complex etiology of this syndrome.
  • both protein catabolism in the muscle as well as lipolysis in adipose tissue are largely regulated by cytokine messengerging.
  • IL-6 and TNF-a derived from macrophages in the muscle and adipose tissue microenvironment have been reported to cause a typical cachexic phenotypes (muscle atrophy, lipolysis, fat browning) (Baazim H et al., 2022, Nat Rev Immunol). While the systemic surge of cytokines is in part responsible for cachexia, the selective blockade of these cytokines fails to produce tangible effects in the clinics (Prado B.L et al., 2019, Ann Palliat Med). This points out to the fact that cachexia remains a complex metabolic and inflammatory disorder where de-regulated levels of cytokines play importnat part, yet they are not exclusively responsible for the observed muscle and fat tissue wasting.
  • cytokine function is very context and tissue dependent and their systemic targeting may or may not achieve desired effects. Keeping this in mind it may be rather more productive to focus our attention on the cellular “platforms”, primarily immune cells, that are responsible for the cytokine de-regulation in the first place.
  • integrin activation on macrophages significantly enhances the expression of NF-KB-dependent proinflammatory cytokines, such as TNF-a, IL-ip, and IL-6, and that integrin activation on macrophages is responsible for the persistence of chronic inflammation; suggesting the importance of macrophages and integrin signals in cancer cachexia with chronic inflammatory organ damage (Antonov A. S et al . , 2011 , J Cell Physiol). Integrin signaling in the tumor microenvironment is in turn regulated by complex crosstalk with various extracellular factors (e.g., extracellular matrix and stromal proteins) (Desgrosellier J.S et al., 2010, Nat Rev Cancer).
  • extracellular factors e.g., extracellular matrix and stromal proteins
  • TGFBI stromal protein
  • Tgfbi is strongly involved in macrophage biology. Namely, Tgfbi KO mice had more M2 macrophages in the adipose tissue, while the mice had significantly augmented resistance to adipose tissue hypertrophy, liver steatosis, and insulin resistance (Lee S.G et al., 2023, Exp Mol Med).
  • M2 macrophages are themselves characterized by high levels of TGFBI, while the secretion of TGFBI by macrophages is associated with the immune-suppressed tumor microenvironment (Lecker L.S.M et al., 2021, Cancer Res). Recently, Zhou et al. expanded these findings by demonstrating that Tgfbi is not only a marker of but an essential factor for macrophage polarization to immune- suppressive alternatively activated macrophages (Zhou J et al., 2023, Cancer Lett).
  • Goehrig et al. use PMA (phorbol ester) stimulated Raw 264.7 mouse macrophage cell line, which as such is programmed towards a strong pro-inflammatory phenotype (Goehrig D et al., 2019, Guf). They also measure intracellularly the cytokine levels, which is different to our measurement in the extracellular medium. More studies will be necessary to elucidate why these differences are present and if they stem from different cell models or perhaps from inherently different antibodies used in the two studies. Indeed, Goehrig et al.
  • Tgfbi has been shown to interact with integrins heterodimers such as aVp3, aipi and aVp5 (Nam J.O et al., 2003, J Biol Chem ; Ohno S et al., 1999, Biochim Biophys Acta & Ma C et al., 2008, Genes Dev). Goehrig et al.
  • Tgfbi mainly acted through Cd61 (or integrin P3).
  • Cd61 or integrin P3
  • Tgfbi bound to aVp5 we found that Tgfbi bound to aVp5
  • Tnf-a levels in J774.1 macrophages led to an increase in 11-6 and Tnf-a levels in J774.1 macrophages. Additional studies are therefore needed to fully characterize the domains on the Tgfbi protein that are necessary for the respective integrin interactions. This could, in part, explain subtle yet important differences between the two antibodies and hence guide their future clinical application.
  • Tgfbi inhibitory antibody In conclusion, we developed a Tgfbi inhibitory antibody and showed that the antibody administration improved symptoms and prognosis of cachexia by suppressing cachexia- associated cytokine production from macrophages via integrin signals. Furthermore, the Tgfbi antibody could regulate not only cytokine production but also the level of immune cell infiltration in the tumor microenvironment. Our study highlights that a therapeutic strategy targeting Tgfbi protein, a cachexic extracellular factor, may be promising for controlling cachexia symptoms through the regulation of immune cell infiltration and cytokine production in the tumor microenvironment, thus may improve survival and quality of life in many advanced cancer patients suffering from refractory cancer cachexia.
  • PC PDX pancreatic cancer patient-derived xenograft
  • Human pancreatic tumor tissues were cut into small fragments (approximately 5 x 5 x 5 mm) and implanted into the right flank of each mouse under general anesthesia.
  • anti -TGFBI antibody was administered intraperitoneally at a dose of 200 pg per mouse, twice weekly, following the same regimen used in the Colon26 tumor-bearing mouse model. The observation period was set at 45 days ( Figure 11, top panel). Tumor engraftment was confirmed prior to initiating antibody treatment on day 10 post-implantation ( Figure 11 A, top panel).
  • Tumor size (W; width. L; length) was measured with a caliper.
  • Tumor volume (mm 3 ) was calculated as (W 2 xL)/2 (Fig. 1 ID). All animal studies were approved by the Institutional Animal Care and Use Committee of Gunma University.
  • ELISA assay for plasma 11-6, Tnf-a, and Tgfbi in mouse models Indirect ELISA was used to quantify 11-6, Tnf-a, and Tgfbi levels in plasma samples collected from experimental animals. Maxisorp 96-well plates (Thermo Fisher, USA) were coated with 100 pl of plasma or concentrated medium samples. Serial dilutions of murine recombinant Tgfbi (R&D Systems, USA), 11-6 (PeproTech, USA), and Tnf-a (PeproTech, USA) in PBS were prepared to generate standard calibration curves. Coating was performed overnight at 4°C under gentle agitation.
  • Histological evaluation of mouse tissue Histological analysis was performed using FFPE sections prepared from skeletal muscle tissues of experimental animals. Four-micrometer-thick sections were stained with hematoxylin and eosin (HE) to assess the cross-sectional area of muscle fibers. For each sample, at least three independent fields of view (FOVs) were acquired. Muscle fiber area was quantified using an ImageJ plugin designed for cross-sectional area analysis with a defined threshold. The measured areas were classified into three size categories, and the frequency distribution of each category was calculated for every sample.
  • HE hematoxylin and eosin
  • membranes were incubated overnight at 4°C with the following primary antibodies: anti-mouse MuRFl (R&D Systems, 1 :500), anti-mouse phospho-STAT3 (Y705) (Cell Signaling Technology, 1 : 1000), and anti-mouse STAT3 (Cell Signaling Technology, 1 : 1000).
  • Anti-mouse MuRFl R&D Systems, 1 :500
  • anti-mouse phospho-STAT3 Y705
  • anti-mouse STAT3 Cell Signaling Technology, 1 : 1000
  • Membranes were then washed three times with TBS-T and incubated with appropriate HRP -conjugated secondary antibodies for 1 hour at room temperature. After additional washes, protein bands were visualized using the ECL Prime Western Blot Detection System and an ImageQuant LAS 4000 system (GE Healthcare Life Sciences). Ponceau red- stained membranes were imaged to document total protein loading and transfer consistency.
  • Colon26-luc cell line was obtained from the Japanese Collection of Research Bioresources Cell Bank. The cell lines were cultured in RPMI-1640 (Wako, Japan) supplemented with 10% FBS and 1% SM/PC at 37°C in 5%CO2. The cell line was tested for mycoplasma contamination using Mycostrip (Invivogen, California, USA). Colon26-luc (5xl0 4 cells/100 pl in PBS) were subcutaneously injected under general anesthesia into the right flank of female BALB/cAJcl(CLEA, Japan) mice at 8 weeks age. The observation period was set at 17 days ( Figure 13, top left panel).
  • Tumor size (W; width. L; length) was measured with a caliper.
  • Tumor volume (mm 3 ) was calculated as (W 2 xL)/2. All animal studies were approved by the Institutional Animal Care and Use Committee of Gunma University.
  • TGFBI a secreted protein detectable in the bloodstream
  • Fc-mediated effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC)
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • Fc-silent anti-TGFBI antibody carrying the L234A, L235A, and P329G (LALAPG) mutations in the Fc region. These mutations were introduced to abolish binding to Fey receptors and complement component Clq.
  • Cancer cachexia model mice were established using Colon26 cells, and the therapeutic effects of the C9 antibody and the C9- LALAPG antibody were evaluated (Figure 13, top left panel). Both antibodies significantly improved cachexia-related symptoms, including food intake, body weight, and muscle strength, compared to the control IgG group.
  • TGFBI C9 antibody inhibited muscle degradation signaling in colon26 cachexia mice
  • muscle tissues were collected on day 17 from Colon26 cachexia model mice and analyzed by Western blot to assess muscle degradation signaling. Comparisons were made among no tumor-bearing (NTB) mice, control IgG-treated mice, and TGFBI C9 antibody- treated mice. The results showed that cachexia induced by Colon26 tumor implantation activated muscle degradation signaling, which was attenuated by C9 antibody treatment, as confirmed at the protein level by Western blot analysis ( Figure 15). These findings are consistent with the results shown in Figure 4C-4D, where muscle degradation was evaluated by PCR analysis.

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Abstract

The present invention is in the field of medicine. Here, the inventors constructed a novel fully human anti-TGFBI monoclonal antibody using phage display. They explored the new phenotype mediated by Tgfbi-targeting in vivo, showing that Tgfbi can indeed regulate cachexia-associated cytokine production via macrophage integrin signaling. Thus, the present invention relates to an anti-TGFBI antibody and uses thereof.

Description

ANTIBODIES HAVING SPECIFICITY TO TGFBI AND USES THEREOF
FIELD OF THE INVENTION
The present invention relates to antibodies having specificity to TGFBI and uses thereof.
BACKGROUND OF THE INVENTION
Cancer cachexia is a multifactorial syndrome characterized by a continuous loss of skeletal muscle mass (with or without fat loss) that cannot be completely reversed by conventional nutritional support. Cachexia occurs in 50-80% of advanced cancers (mainly of gastrointestinal and lung type), and as such it represents a true challenge in the clinics. The cachexic syndrome is fueled by continuous metabolic wasting, frequently accompanied by reduced appetite and food intake (Fearon K et al., 2011, Lancet Oncol). Beyond cancer, chronic infections (e.g. HIV infection, tuberculosis) as well as kidney failure can also cause cachexia accompanied with muscle weakness and organ damage. Severe emaciation resulting from cachexia results in decreased tolerability of anti -cancer treatments and decreased quality of life, both of which ultimately lead to death in at least 20-30% of patients (Mattox T.W et al., 2017, Nutr Clin Pract & Ferrara M et al., 2022, Front Cell Dev Biol). Despite being a serious clinical challenge, the etiology of cachexia is poorly understood. There is thus an unmet need to develop diagnostic markers that can be used in clinical practice and therapeutic tools that can prolong the survival of patients with cancer cachexia.
Cancer cachexia has been known to be accompanied by increased blood levels of inflammatory cytokines identified as cachexia-associated cytokines, such as tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), transforming growth factor-P (TGF-P), and interleukin- 1 (IL-1) (Baazim H et al., 2022, Nat Rev Immunol). While these cytokines have been molecularly linked to muscle wasting or transformation of white adipose tissue to brown adipose tissue (Baazim H et al., 2022, Nat Rev Immunol), their targeting in clinics has only in part achieved a reversal of cachexic phenotype. Indeed, results from previous clinical trials suggest some benefit from targeting IL-1, IL-6, while no improvement in cachexia symptoms was observable with targeting TNF-a (Prado B.L et al., 2019, Ann Palliat Med). There is no doubt that cytokines are central to the cachexic phenotype (Baracos V.E et al., 2018, Nat Rev Dis Primers), but at present it appears simplistic to single out cytokines as exclusive masterregulators of the cachexic process. Along these lines it may be more relevant to focus on cellular platforms, i.e. immune cells, that by and large regulate inflammatory reactions in physiology and pathophysiology. One of the most versatile cells in this group are macrophages, that inherent with their ability to assume pro- and anti-inflammatory states, can both enhance (Shukla S.K et al., 2020, Cancer Lett) as well as suppress cachexia (Erdem M et al., 2019, J Cachexia Sarcopenia Muscle). Indeed in obesity, a shift of resident macrophages from M2 (antiinflammatory) to Ml (pro-inflammatory) phenotype has been previously observed in adipose tissue (McNelis J.C et al., 2014, Immunity). There, the newly established pro-inflammatory condition favors T-cell infiltration, adipocyte necrosis and insulin resistance. While the link between macrophage polarization states and cachexia is yet to be investigated in detail, it is not unreasonable to assume that factors that determine macrophage polarization may be involved in modulating cachexia.
One of the factors shown to be important for the macrophage polarization is transforming growth factor-beta-induced protein (TGFBI). TGFBI is a 683-amino acid extracellular matrix protein with evolutionarily conserved integrin-binding RGD motifs, that can activate cancer aggressiveness via the activation of integrin signaling. Recent study showed that TGFBI loss-of-function in macrophages inhibited their polarization to M2 phenotype (Zhou J et al., 2023, Cancer Lett), while other findings have clearly demonstrated that M2 macrophages in tumors secrete high levels of TGFBI (Zhou J et al., 2023, Cancer Lett Peng P et al., 2022, Theranostics & Lecker L.S.M et al., 2021, Cancer Res). In more general terms, TGFBI has been reported to be also secreted by cancer-associated fibroblast (CAF) as well as cancer cells in limited manner. The latter do express TGFBI especially when metastatic (such as CTCs) (Chiavarina B et al., 2021, Theranostics). TGFBI expression is induced by the activation of transforming growth factor-P (TGF-P) signal (Yokobori T et al., 2017, J Clin Med). We have reported that high expression of stromal TGFBI in esophageal cancer and gastric cancer was associated with cancer progression and poor prognosis (Suzuki M et al., 2018, J Surg Oncol & Ozawa D et al., 2016, Ann Surg Oncol).
SUMMARY OF THE INVENTION
Given the prominent pro-tumoral role TGFBI assumes in numerous cancers as well as its function in tumor associated macrophage (TAM) and CAF biology, the inventors hypothesized that TGFBI may be involved in modulating cachexia and more particularly cancer cachexia. To test this hypothesis, they first developed means to efficiently target TGFBI in the tumors. To this end, they constructed a novel fully human anti-TGFBI monoclonal antibodies using phage display. They explored the new phenotype mediated by Tgfbi -targeting in vivo, showing that Tgfbi can indeed regulate cachexia-associated cytokine production via macrophage integrin signaling. The present data offer a new rationale for TGFBI targeting in future clinical trials, targeting cachexia and providing therapeutic opportunities for further interventions and treatment combinations.
Thus, the present invention relates to antibodies having specificity to TGFBI and uses thereof. Particularly, the present invention is defined by the claims.
DETAILED DESCRIPTION OF THE INVENTION
In the present study, the inventors constructed novel fully human anti-TGFBI monoclonal antibodies using phage display. They explored the new phenotype mediated by Tgfbi-targeting in vivo, showing that Tgfbi can indeed regulate cachexia-associated cytokine production via macrophage integrin signaling.
A first object of the invention relates to an anti-TGFBI antibody and uses thereof.
In one embodiment, the anti-TGFBI antibody is selected among 7 antibodies (called also here A6, C7, C9, D2, D5, E4 and G7).
In one embodiment, the anti-TGFBI antibody is selected among 2 antibodies (called also here A6 and C9).
Particularly, the invention relates to an anti-TGFBI antibody having a heavy chain comprising i) the H-CDR1 of A6 mab, ii) the H-CDR2 of A6 mab and iii) the H-CDR3 of A6 mab and a light chain comprising i) the L-CDR1 of A6 mab, ii) the L-CDR2 of A6 mab and iii) the L-CDR3 of A6 mab
Wherein
- the H-CDR1 of the A6 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO: 1;
- the H-CDR2 of A6 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO: 1;
- the H-CDR3 of the A6 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO: 1.
- the L-CDR1 of the A6 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO:2;
- the L-CDR2 of the A6 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO:2;
- the L-CDR3 of the A6 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO:2; or an anti-TGFBI antibody having a heavy chain comprising i) the H-CDR1 of C7 mab, ii) the H-CDR2 of C7 mab and iii) the H-CDR3 of C7 mab and a light chain comprising i) the L-CDR1 of C7 mab, ii) the L-CDR2 of C7 mab and iii) the L-CDR3 of C7 mab Wherein
- the H-CDR1 of the C7 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO:3;
- the H-CDR2 of C7 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO:3;
- the H-CDR3 of the C7 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO:3.
- the L-CDR1 of the C7 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO:4;
- the L-CDR2 of the C7 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO:4;
- the L-CDR3 of the C7 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO:4; or an antibody having a heavy chain comprising i) the H-CDR1 of C9 mab, ii) the H- CDR2 of C9 mab and iii) the H-CDR3 of C9 mab and a light chain comprising i) the L- CDR1 of C9 mab, ii) the L-CDR2 of C9 mab and iii) the L-CDR3 of C9 mab
Wherein
- the H-CDR1 of the C9 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO: 5;
- the H-CDR2 of C9 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO:5;
- the H-CDR3 of the C9 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO: 5.
- the L-CDR1 of the C9 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO:6;
- the L-CDR2 of the C9 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO:6;
- the L-CDR3 of the C9 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO: 6; or an antibody having a heavy chain comprising i) the H-CDR1 of D2 mab, ii) the H- CDR2 of D2 mab and iii) the H-CDR3 of D2 mab and a light chain comprising i) the L-CDR1 of D2 mab, ii) the L-CDR2 of D2 mab and iii) the L-CDR3 of D2 mab
Wherein
- the H-CDR1 of the D2 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO: 7;
- the H-CDR2 of D2 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO:7;
- the H-CDR3 of the D2 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO:7.
- the L-CDR1 of the D2 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO:8;
- the L-CDR2 of the D2 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO:8;
- the L-CDR3 of the D2 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO:8; or an antibody having a heavy chain comprising i) the H-CDR1 of D5 mab, ii) the H- CDR2 of D5 mab and iii) the H-CDR3 of D5 mab and a light chain comprising i) the L-CDR1 of D5 mab, ii) the L-CDR2 of D5 mab and iii) the L-CDR3 of D5 mab
Wherein
- the H-CDR1 of the D5 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO: 9;
- the H-CDR2 of D5 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO:9;
- the H-CDR3 of the D5 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO:9.
- the L-CDR1 of the D5 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO: 10;
- the L-CDR2 of the D5 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO: 10;
- the L-CDR3 of the D5 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO: 10; or an antibody having a heavy chain comprising i) the H-CDR1 of E4 mab, ii) the H- CDR2 of E4 mab and iii) the H-CDR3 of E4 mab and a light chain comprising i) the L- CDR1 of E4 mab, ii) the L-CDR2 of E4 mab and iii) the L-CDR3 of E4 mab
Wherein
- the H-CDR1 of the E4 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO: 11;
- the H-CDR2 of E4 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO: 11;
- the H-CDR3 of the E4 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO: 11.
- the L-CDR1 of the E4 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO: 12;
- the L-CDR2 of the E4 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO: 12;
- the L-CDR3 of the E4 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO: 12; or an antibody having a heavy chain comprising i) the H-CDR1 of G7 mab, ii) the H- CDR2 of G7 mab and iii) the H-CDR3 of G7 mab and a light chain comprising i) the L-CDR1 of G7 mab, ii) the L-CDR2 of G7 mab and iii) the L-CDR3 of G7 mab
Wherein
- the H-CDR1 of the G7 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO: 13;
- the H-CDR2 of G7 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO: 13;
- the H-CDR3 of the G7 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO: 13.
- the L-CDR1 of the G7 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO: 14;
- the L-CDR2 of the G7 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO: 14;
- the L-CDR3 of the G7 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO: 14; As used herein, the term “TGFBI” or “Transforming Growth Factor-Beta-Induced” also known as “Pig-h3” is a 683-amino acid extracellular matrix protein with evolutionarily conserved integrin-binding RGD motifs. The Entrez reference number of the human gene coding for TGFBI is 7045 and the Uniprot reference number of TGFBI human protein is QI 5582. TGFBI is involved in endochondrial bone formation in cartilage. TGFBI can activate cancer aggressiveness via the activation of integrin signaling. TGFBI has been reported to be also secreted by cancer-associated fibroblast (CAF) as well as cancer cells in limited manner which express TGFBI especially when metastatic (such as CTCs) (Chiavarina B et al., 2021, Theranostics). TGFBI expression is induced by the activation of transforming growth factor-P (TGF-P) signal (Yokobori T et al., 2017, J Clin Med).
As used herein, the term "antibody" or "immunoglobulin" have the same meaning, and will be used equally in the present invention. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments. In natural antibodies, two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (1) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FR) can participate to the antibody binding site or influence the overall domain structure and hence the combining site. Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L- CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. An antigen-binding site, therefore, typically includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs.
In one embodiment, the antibody of the invention is a monoclonal antibody.
In the context of the invention, the amino acid residues of the antibody of the invention are numbered according to the KABAT numbering system. This system is set forth in Kabat et al., 1987, in sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NTH, USA (hereafter “Kabat et al.”). This numbering system is used in the present specification. The Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues in SEQ ID sequences. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure. The correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a “standard” Kabat numbered sequence. The CDRs of the heavy chain variable domain are located at residues 31-35B (H- CDR1), residues 50-65 (H-CDR2) and residues 95-102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2) and residues 89-97 (L-CDR3) according to the Kabat numbering system. (http://www.bioinf.org.Uk/abs/#cdrdef).
As used herein, the term “specificity” refers to the ability of an antibody to detectably bind an epitope presented on an antigen, such as TGFBI, while having relatively little detectable reactivity with non-TGFBI proteins or structures (such as other proteins presented on cancerous cell, or on other cell types). Specificity can be relatively determined by binding or competitive binding assays, using, e.g., Biacore instruments, as described elsewhere herein. Specificity can be exhibited by, e.g., an about 10: 1, about 20: 1, about 50: 1, about 100: 1, 10.000: 1 or greater ratio of affinity/avidity in binding to the specific antigen versus nonspecific binding to other irrelevant molecules (in this case the specific antigen is TGFBI).
As used herein, the term “affinity” refers to the strength of the binding of an antibody to an epitope. The affinity of an antibody is given by the dissociation constant Kd, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of the unbound antibody and [Ag] is the molar concentration of the unbound antigen. The affinity constant Ka is defined by 1/Kd. Preferred methods for determining the affinity of mAbs can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which references are entirely incorporated herein by reference. One preferred and standard method well known in the art for determining the affinity of mAbs is the use of Biacore instruments.
As used herein, the terms "monoclonal antibody", "monoclonal Ab", "monoclonal antibody composition", "mAb", or the like, refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
According to the present invention, the VH region of the A6 mab consists of the sequence of SEQ ID NO: 1. Accordingly, the H-CDR1 of the A6 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO: 1. Accordingly, the H-CDR2 of A6 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO: 1. Accordingly, the H-CDR3 of the A6 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO: 1.
SEQ ID NO:1: VH region of the A6 mab FR1 -CDR 1 -FR2-CDR2-FR3 -CDR3 -FR4
EVQLVESGGSLVKPGGSLRLSCAASGFTFSNYNMNWVRQAPGKGLEWISGISG
GTLVTVSS
According to the present invention, the VL region of the A6 mab consists of the sequence of SEQ ID NO:2. Accordingly, the L-CDR1 of the A6 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO:2. Accordingly, the L-CDR2 of the A6 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO:2. Accordingly, the L-CDR3 of the A6 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO:2.
SEQ ID NO:2: VL region of the A6 mab FR1 -CDR 1 -FR2-CDR2-FR3 -CDR3 -FR4
QSVLTQPASVSGSPGQSITISCAGTSSDVGGSYSVSWYQQHPGKAPKLMIYSDS YRPSGVSNRFSGSKSGNTASLTISGLOAEDEADYYCSSGTYQSTRVFGGGTKLEIK
According to the present invention, the VH region of the C7 mab consists of the sequence of SEQ ID NO: 3. Accordingly, the H-CDR1 of the C7 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO:3. Accordingly, the H-CDR2 of C7 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO:3. Accordingly, the H-CDR3 of the C7 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO:3.
SEQ ID NO:3: VH region of the C7 mab FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
EVQLVESGGSLVKPGGSLRLSCAASGFTFSNYGMNWVRQAPGKGLEWISGISG SSRYINYADFVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRSYYYGMDVWGR GTLVTVSS
According to the present invention, the VL region of the C7 mab consists of the sequence of SEQ ID NO:4. Accordingly, the L-CDR1 of the C7 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO:4. Accordingly, the L-CDR2 of the C7 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO:4. Accordingly, the L-CDR3 of the C7 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO:4.
SEQ ID NO:4: VL region of the C7 mab FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
QSVLTQPASVSGSPGQSITISCAGTSSDVGGYYSVSWYQQHPGKAPKLMIYSDS YRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSGTQGSTRVFGGGTKLEIK
According to the present invention, the VH region of the C9 mab consists of the sequence of SEQ ID NO:5. Accordingly, the H-CDR1 of the C9 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO:5. Accordingly, the H-CDR2 of C9 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO:5. Accordingly, the H-CDR3 of the C9 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO: 5.
SEQ ID NO:5: VH region of the C9 mab FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
EVQLVESGGSLVKPGGSLRLSCAASGFTFSNYYMNWVRQAPGKGLEWISSISG S S S YIG YADF VKGRFTI SRDN AKNSLYLQMNSLR AEDTAV Y YC VRS S YYNAMD VWG RGTLVTVSS
According to the present invention, the VL region of the C9 mab consists of the sequence of SEQ ID NO:6. Accordingly, the L-CDR1 of the C9 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO:6. Accordingly, the L-CDR2 of the C9 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO:6. Accordingly, the L-CDR3 of the C9 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO:6.
SEQ ID NO:6: VL region of the C9 mab FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
QSVLTQPASVSGSPGQSITISCAGTSSDVGGSGSVSWYQQHPGKAPKLMIYYDS QRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTYYSTRVFGGGTKLEIK
According to the present invention, the VH region of the D2 mab consists of the sequence of SEQ ID NO:7. Accordingly, the H-CDR1 of the D2 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO:7. Accordingly, the H-CDR2 of D2 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO:7. Accordingly, the H-CDR3 of the D2 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO:7.
SEQ ID NO:7: VH region of the D2 mab FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
EVQLVESGGSLVKPGGSLRLSCAASGFTFSNYAMNWVRQAPGKGLEWISSISG SSRSINYADFVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRSYYYGMDVWGR GTLVTVSS
According to the present invention, the VL region of the D2 mab consists of the sequence of SEQ ID NO: 8. Accordingly, the L-CDR1 of the D2 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO: 8. Accordingly, the L-CDR2 of the D2 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO:8. Accordingly, the L-CDR3 of the D2 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO: 8.
SEQ ID NO:8: VL region of the D2 mab FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
QSVLTQPASVSGSPGQSITISCAGTSSDVGGYYSVSWYQQHPGKAPKLMIYSDS YRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSSTDYSTRVFGGGTKLEIK
According to the present invention, the VH region of the D5 mab consists of the sequence of SEQ ID NO:9. Accordingly, the H-CDR1 of the D5 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO:9. Accordingly, the H-CDR2 of D5 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO:9. Accordingly, the H-CDR3 of the D5 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO:9.
SEQ ID NO:9: VH region of the D5 mab FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
EVQLVESGGSLVKPGGSLRLSCAASGFTFSNYGMNWVRQAPGKGLEWISSISG SSRSIGYADFVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRSYYYGMDVWGR GTLVTVSS
According to the present invention, the VL region of the D5 mab consists of the sequence of SEQ ID NO: 10. Accordingly, the L-CDR1 of the D5 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO: 10. Accordingly, the L-CDR2 of the D5 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO: 10. Accordingly, the L-CDR3 of the D5 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO: 10.
SEQ ID NQ:10: VL region of the D5 mab FR1 -CDR1 -FR2-CDR2-FR3 -CDR3 -FR4
QSVLTQPASVSGSPGQSITISCAGTSSDVGGSYSVSWYQQHPGKAPKLMIYSDS YRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSSTOQSTRVFGGGTKLEIK
According to the present invention, the VH region of the E4 mab consists of the sequence of SEQ ID NO: 11. Accordingly, the H-CDR1 of the E4 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO: 11. Accordingly, the H-CDR2 of E4 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO: 11. Accordingly, the H-CDR3 of the E4 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO: 11.
SEQ ID NO:11: VH region of the E4 mab FR1 -CDR1 -FR2-CDR2-FR3 -CDR3 -FR4
EVQLVESGGSLVKPGGSLRLSCAASGFTFSNYAMNWVRQAPGKGLEWISYISG SSRSIGYADFVKGRFTISRDNAkNSLYLQMNSLRAEDTAVYYCVRSYYYGMDVWGRG TLVTVSS
According to the present invention, the VL region of the E4 mab consists of the sequence of SEQ ID NO: 12. Accordingly, the L-CDR1 of the E4 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO: 12. Accordingly, the L-CDR2 of the E4 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO: 12. Accordingly, the L-CDR3 of the E4 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO: 12.
SEQ ID NO:12: VL region of the E4 mab FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
QSVLTQPASVSGSPGQSITISCAGTSSDVGGSYSVSWYQQHPGKAPKLMIYSDS YRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSSTYYSTRVFGGGTKLEIK
According to the present invention, the VH region of the G7 mab consists of the sequence of SEQ ID NO: 13. Accordingly, the H-CDR1 of the G7 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO: 13. Accordingly, the H-CDR2 of G7 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO: 13. Accordingly, the H-CDR3 of the G7 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO:13.
SEQ ID NO:13: VH region of the G7 mab FR1 -CDR1 -FR2-CDR2-FR3 -CDR3 -FR4
EVQLVESGGSLVKPGGSLRLSCAASGFTFSNYGMNWVRQAPGKGLEWISYISG SSRYIGYADFVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRSYYYGMDVWGR GTLVTVSS
According to the present invention, the VL region of the G7 mab consists of the sequence of SEQ ID NO: 14. Accordingly, the L-CDR1 of the G7 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO: 14. Accordingly, the L-CDR2 of the G7 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO: 14. Accordingly, the L-CDR3 of the G7 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO: 14.
SEQ ID NO:14: VL region of the G7 mab FR1 -CDR1 -FR2-CDR2-FR3 -CDR3 -FR4 QSVLTQPASVSGSPGQSITISCAGTSSDVGGSYSVSWYQQHPGKAPKLMIYSDS
The antibodies of the present invention are produced by any technique known in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination. Typically, knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce said antibodies, by standard techniques for production of polypeptides. For instance, they can be synthesized using well-known solid phase method, preferably using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, California) and following the manufacturer’s instructions. Alternatively, antibodies of the present invention can be synthesized by recombinant DNA techniques well-known in the art. For example, antibodies can be obtained as DNA expression products after incorporation of DNA sequences encoding the antibodies into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired antibodies, from which they can be later isolated using well-known techniques.
In one embodiment the antibody of the invention is a human antibody.
As used herein the term "human antibody” is intended to include antibodies having variable and constant regions derived from human immunoglobulin sequences.
The human antibodies of the present invention may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced by random or sitespecific mutagenesis in vitro or by somatic mutation in vivo). 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.
Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, cur. Opin. Pharmacol. 5; 368- 74 (2001) and lonberg, cur. Opin. Immunol. 20; 450-459 (2008). Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal’s chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat.Biotech. 23; 1117-1125 (2005). See also, e.g., U.S Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSETM technology; U.S. Patent No. 5,770,429 describing HUMAB® technology; U.S. Patent No. 7,041,870 describing K-M MOUSE® technology, and U.S. Patent Application publication No. US 2007/0061900, describing VELOCIMOUSE® technology. Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region Human antibodies can also be made by hybridoma-based methods.
Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 13: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51- 63 (Marcel Dekker, Inc., New York, 1987); and Boemer et al., J. Immunol., 147: 86(1991)). Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing production of monoclonal human igM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein,, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-91 (2005). Fully human antibodies can also be derived from phage-display libraries (as disclosed in Hoogenboom et al., 1991, J. Mol. Biol. 227:381; and Marks et al., 1991, J. Mol. Biol. 222:581). Phage display techniques mimic immune selection through the display of antibody repertoires on the surface of filamentous bacteriophage, and subsequent selection of phage by their binding to an antigen of choice. One such technique is described in PCT publication No. WO 99/10494. Human antibodies described herein can also be prepared using SCID mice into which human immune cells have been reconstituted such that a human antibody response can be generated upon immunization. Such mice are described in, for example, U.S. Patent Nos. 5,476,996 and 5,698,767 to Wilson et al. In one embodiment, the monoclonal antibody of the invention is a chimeric antibody, particularly a chimeric mouse/human antibody.
As used herein, the term "chimeric antibody" refers to an antibody which comprises a VH domain and a VL domain of a non-human antibody, and a CH domain and a CL domain of a human antibody.
In one embodiment, the human chimeric antibody of the present invention can be produced by obtaining nucleic sequences encoding VL and VH domains as previously described, constructing a human chimeric antibody expression vector by inserting them into an expression vector for animal cell having genes encoding human antibody CH and human antibody CL, and expressing the coding sequence by introducing the expression vector into an animal cell. As the CH domain of a human chimeric antibody, it may be any region which belongs to human immunoglobulin, but those of IgG class are suitable and any one of subclasses belonging to IgG class, such as IgGl, IgG2, IgG3 and IgG4, can also be used. Also, as the CL of a human chimeric antibody, it may be any region which belongs to Ig, and those of kappa class or lambda class can be used. Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques are well known in the art (See Morrison SL. et al. (1984) and patent documents US5,202,238; and US5,204, 244).
In one embodiment, the antibody of the invention is an antigen biding fragment selected from the group consisting of a Fab, a F(ab)’2, a single domain antibody, a ScFv, a Sc(Fv)2, a diabody, a triabody, a tetrabody, an unibody, a minibody, a maxibody, a small modular immunopharmaceutical (SMIP), minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody as an isolated complementary determining region (CDR), and fragments which comprise or consist of the VL or VH chains as well as amino acid sequence having at least 70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99 or 100% of identity with SEQ ID NO: 1 to 28.
In one embodiment, the antibody of the present invention is an antibody having a heavy chain comprising i) the H-CDR1 of A6, C7, C9, D2, D5, E4 or G7 mabs, ii) the H-CDR2 of A6, C7, C9, D2, D5, E4 or G7 mabs, and iii) the H-CDR3 of A6, C7, C9, D2, D5, E4 or G7 mabs, and a light chain comprising i) the L-CDR1 of A6, C7, C9, D2, D5, E4 or G7 mabs, ii) the L-CDR2 of A6, C7, C9, D2, D5, E4 or G7 mabs, and iii) the L-CDR3 of A6, C7, C9, D2, D5, E4 or G7 mabs. In one embodiment, the antibody of the present invention is an antibody having a heavy chain having at least 70 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% of identity with SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13 and a light chain having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% of identity with SEQ ID NO:2, 4, 6, 8, 10, 12 or 14.
In one embodiment, the antibody has an heavy chain having at least 70% of identity with SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13 and a light chain having at least 70 % of identity with SEQ ID NO:2, 4, 6, 8, 10, 12 or 14.
In one embodiment, the antibody of the present invention is an antibody having a heavy chain identical to SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13 and a light chain identical to SEQ ID NO:2, 4, 6, 8, 10, 12 or 14.
As used herein, the term “antigen binding fragment” of an antibody refers to one or more fragments of an intact antibody that retain the ability to specifically binds to a given antigen (e.g., [TGFBI]). Antigen biding functions of an antibody can be performed by fragments of an intact antibody. Examples of biding fragments encompassed within the term antigen biding fragment of an antibody include a Fab fragment, a monovalent fragment consisting of the VL,VH,CL and CHI domains; a Fab’ fragment, a monovalent fragment consisting of the VL,VH,CL,CH1 domains and hinge region; a F(ab’)2 fragment, a bivalent fragment comprising two Fab’ fragments linked by a disulfide bridge at the hinge region; an Fd fragment consisting of VH domains of a single arm of an antibody; a single domain antibody (sdAb) fragment (Ward et al., 1989 Nature 341 :544-546), which consists of a VH domain or a VL domain; and an isolated complementary determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by an artificial peptide linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (ScFv); see, e.g., Bird et al., 1989 Science 242:423-426; and Huston et al., 1988 proc. Natl. Acad. Sci. 85:5879-5883). "dsFv" is a VH::VL heterodimer stabilised by a disulfide bond. Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)2. Such single chain antibodies include one or more antigen biding portions or fragments of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as are intact antibodies. A unibody is another type of antibody fragment lacking the hinge region of IgG4 antibodies. The deletion of the hinge region results in a molecule that is essentially half the size of traditional IgG4 antibodies and has a univalent binding region rather than the bivalent biding region of IgG4 antibodies. Antigen binding fragments can be incorporated into single domain antibodies, SMTP, maxibodies, minibodies, intrabodies, diabodies, triabodies and tetrabodies (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). The term "diabodies" “tribodies” or “tetrabodies” refers to small antibody fragments with multivalent antigen-binding sites (2, 3 or four), which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
Antigen biding fragments can be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) Which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al., 1995 Protein Eng. 8(10); 1057-1062 and U.S. Pat. No. 5,641,870).
The Fab of the present invention can be obtained by treating an antibody which specifically reacts with TGFBI with a protease, papaine. Also, the Fab can be produced by inserting DNA encoding Fab of the antibody into a vector for prokaryotic expression system, or for eukaryotic expression system, and introducing the vector into a procaryote or eucaryote (as appropriate) to express the Fab.
The F(ab')2 of the present invention can be obtained treating an antibody which specifically reacts with TGFBI with a protease, pepsin. Also, the F(ab')2 can be produced by binding Fab' described below via a thioether bond or a disulfide bond.
The Fab' of the present invention can be obtained treating F(ab')2 which specifically reacts with TGFBI with a reducing agent, dithiothreitol. Also, the Fab' can be produced by inserting DNA encoding Fab' fragment of the antibody into an expression vector for prokaryote, or an expression vector for eukaryote, and introducing the vector into a prokaryote or eukaryote (as appropriate) to perform its expression.
The scFv of the present invention can be produced by obtaining cDNA encoding the VH and VL domains as previously described, constructing DNA encoding scFv, inserting the DNA into an expression vector for prokaryote, or an expression vector for eukaryote, and then introducing the expression vector into a prokaryote or eukaryote (as appropriate) to express the scFv. To generate a humanized scFv fragment, a well-known technology called CDR grafting may be used, which involves selecting the complementary determining regions (CDRs) from a donor scFv fragment, and grafting them onto a human scFv fragment framework of known three dimensional structure (see, e. g., W098/45322; WO 87/02671; US5,859,205; US5,585,089; US4,816,567; EP0173494).v
Domain Antibodies (dAbs) are the smallest functional binding units of antibodies - molecular weight approximately 13 kDa - and correspond to the variable regions of either the heavy (VH) or light (VL) chains of antibodies. Further details on domain antibodies and methods of their production are found in US 6,291,158; 6,582,915; 6,593,081; 6,172,197; and 6,696,245; US 2004/0110941; EP 1433846, 0368684 and 0616640; WO 2005/035572, 2004/101790, 2004/081026, 2004/058821, 2004/003019 and 2003/002609, each of which is herein incorporated by reference in its entirety.
UniBodies are another antibody fragment technology, based upon the removal of the hinge region of IgG4 antibodies. The deletion of the hinge region results in a molecule that is essentially half the size of a traditional IgG4 antibody and has a univalent binding region rather than a bivalent binding region. Furthermore, because UniBodies are about smaller, they may show better distribution over larger solid tumors with potentially advantageous efficacy. Further details on UniBodies may be obtained by reference to WO 2007/059782, which is incorporated by reference in its entirety.
Nucleic acid sequence, vectors and host cells
Another object of the invention relates to a nucleic acid molecule encoding an antibody according to the invention. More particularly the nucleic acid molecule encodes a heavy chain or a light chain of an antibody of the present invention.
More particularly the nucleic acid molecule comprises a nucleic acid sequence having 70% of identity with SEQ ID NO: 15 and 16 or SEQ ID NO: 17 and 18 or SEQ ID NO: 19 and 20 or SEQ ID NO: 21 and 22 or SEQ ID NO: 23 and 24 or SEQ ID NO: 25 and 26 or SEQ ID NO: 27 and 28.
SEQ ID NO: 15 Heavy chain variable region of the A6 mab: acid nucleic sequence - FR1 -CDR1-FR2-CDR2-FR3 -CDR3-FR4 : gaggtgcagctggtggagtctgggggaagcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattc accttcagtaactacaatatgaactgggtccgccaggctccagggaaggggctggagtggatctcagggatttccggtagtagtagaa acataaactacgcagacttcgtgaagggccgattcaccatctccagagacaacgccaagaactcactgtacctgcaaatgaacagcct gagagccgaggacacggctgtttattactgtgtgagatcctactattacggaatggacgtctggggcagaggcaccctggtcaccgtct cctca
SEQ ID NO: 16 Light chain variable region of the A6 mab: acid nucleic sequence - FR1 -CDR1-FR2-CDR2-FR3 -CDR3-FR4 : cagtctgtgctgactcagcctgcctccgtgtctgggagccctggacagtcgatcaccatctcctgcgctggaaccagcagtg acgttggtggttcatactccgtctcctggtaccaacaacacccaggcaaagcccccaaactcatgatttatagcgacagttaccggccct caggggtttctaatcgcttctctggctccaagtctggcaacacggcctccctgacaatctctgggctccaggctgaggacgaggctgatt attactgcagctcaggcacatatcagagcactcgagttttcggcggagggaccaagctggagatcaaa
SEQ ID NO: 17 Heavy chain variable region of the C7 mab: acid nucleic sequence - FR1 -CDR1-FR2-CDR2-FR3 -CDR3-FR4 : gaggtgcagctggtggagtctgggggaagcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattc accttcagtaactacgggatgaactgggtccgccaggctccagggaaggggctggagtggatctcaggtatttccggtagtagtagat atataaattacgcagacttcgtgaagggccgattcaccatctccagagacaacgccaagaactcactgtacctgcaaatgaacagcctg agagccgaggacacggctgtttattactgtgtgagatcctattactacggcatggacgtctggggcagaggcaccctggtcaccgtctc ctca
SEQ ID NO: 18 Light chain variable region of the C7 mab: acid nucleic sequence - FR1 -CDR1-FR2-CDR2-FR3 -CDR3-FR4 : cagtctgtgctgactcagcctgcctccgtgtctgggagccctggacagtcgatcaccatctcctgcgctggaaccagcagtg acgttggtggttattacagcgtctcctggtaccaacaacacccaggcaaagcccccaaactcatgatttattccgacagttatcggccctc aggggtttctaatcgcttctctggctccaagtctggcaacacggcctccctgacaatctctgggctccaggctgaggacgaggctgatta ttactgcagctcaggcacacagggtagcactcgagttttcggcggagggaccaagctggagatcaaa
SEQ ID NO: 19 Heavy chain variable region of the C9 mab: acid nucleic sequence - FR1 -CDR1-FR2-CDR2-FR3 -CDR3-FR4 : gaggtgcagctggtggagtctgggggaagcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattc accttcagtaactactacatgaactgggtccgccaggctccagggaaggggctggagtggatctcatccattagtggtagtagttcctat ataggctacgcagacttcgtgaagggccgattcaccatctccagagacaacgccaagaactcactgtacctgcaaatgaacagcctga gagccgaggacacggctgtttattactgtgtgagatccagctactataacgccatggacgtctggggcagaggcaccctggtcaccgt ctcctca
SEQ ID NO: 20 Light chain variable region of the C9 mab: acid nucleic sequence - FR1 -CDR1-FR2-CDR2-FR3 -CDR3-FR4 : cagtctgtgctgactcagcctgcctccgtgtctgggagccctggacagtcgatcaccatctcctgcgctggaaccagcagtg acgttggtggttccggtagcgtctcctggtaccaacaacacccaggcaaagcccccaaactcatgatttattatgacagtcagcggccct caggggtttctaatcgcttctctggctccaagtctggcaacacggcctccctgacaatctctgggctccaggctgaggacgaggctgatt attactgcagctcatatacatactatagcactcgagttttcggcggagggaccaagctggagatcaaa
SEQ ID NO: 21 Heavy chain variable region of the D2 mab: acid nucleic sequence - FR1 -CDR1-FR2-CDR2-FR3 -CDR3-FR4 : gaggtgcagctggtggagtctgggggaagcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattc accttcagtaactatgcaatgaactgggtccgccaggctccagggaaggggctggagtggatctcatctatttctggtagtagtagatct ataaactacgcagacttcgtgaagggccgattcaccatctccagagacaacgccaagaactcactgtacctgcaaatgaacagcctga gagccgaggacacggctgtttattactgtgtgagatcctactattatggtatggacgtctggggcagaggcaccctggtcaccgtctcct ca
SEQ ID NO: 22 Light chain variable region of the D2 mab: acid nucleic sequence - FR1 -CDR1-FR2-CDR2-FR3 -CDR3-FR4 : cagtctgtgctgactcagcctgcctccgtgtctgggagccctggacagtcgatcaccatctcctgcgctggaaccagcagtg acgttggtggttactactccgtctcctggtaccaacaacacccaggcaaagcccccaaactcatgatttattcagacagttaccggccct caggggtttctaatcgcttctctggctccaagtctggcaacacggcctccctgacaatctctgggctccaggctgaggacgaggctgatt attactgcagctcatccacagattacagcactcgagttttcggcggagggaccaagctggagatcaaa
SEQ ID NO: 23 Heavy chain variable region of the D5 mab: acid nucleic sequence - FR1 -CDR1-FR2-CDR2-FR3 -CDR3-FR4 : gaggtgcagctggtggagtctgggggaagcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattc accttcagtaactacgggatgaactgggtccgccaggctccagggaaggggctggagtggatctcaagcatttcaggtagtagtcgca gtataggttacgcagacttcgtgaagggccgattcaccatctccagagacaacgccaagaactcactgtacctgcaaatgaacagcct gagagccgaggacacggctgtttattactgtgtgagatcctattactatggcatggacgtctggggcagaggcaccctggtcaccgtct cctca
SEQ ID NO: 24 Light chain variable region of the D5 mab: acid nucleic sequence - FR1 -CDR1-FR2-CDR2-FR3 -CDR3-FR4 : cagtctgtgctgactcagcctgcctccgtgtctgggagccctggacagtcgatcaccatctcctgcgctggaaccagcagtg acgttggtggttcctattctgtctcctggtaccaacaacacccaggcaaagcccccaaactcatgatttattcagacagttatcggccctca ggggtttctaatcgcttctctggctccaagtctggcaacacggcctccctgacaatctctgggctccaggctgaggacgaggctgattat tactgcagctcatcaacacaacaaagcactcgagttttcggcggagggaccaagctggagatcaaa
SEQ ID NO: 25 Heavy chain variable region of the E4 mab: acid nucleic sequence - FR1 -CDR1-FR2-CDR2-FR3 -CDR3-FR4 : gaggtgcagctggtggagtctgggggaagcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattc accttcagtaactatgccatgaactgggtccgccaggctccagggaaggggctggagtggatctcatatattagtggtagtagtcgctc aataggttacgcagacttcgtgaagggccgattcaccatctccagagacaacgccaagaactcactgtacctgcaaatgaacagcctg agagccgaggacacggctgtttattactgtgtgagatcctattattacgggatggacgtctggggcagaggcaccctggtcaccgtctc ctca
SEQ ID NO: 26 Light chain variable region of the E4 mab: acid nucleic sequence - FR1 -CDR1-FR2-CDR2-FR3 -CDR3-FR4 : cagtctgtgctgactcagcctgcctccgtgtctgggagccctggacagtcgatcaccatctcctgcgctggaaccagcagtg acgttggtggtagctatagcgtctcctggtaccaacaacacccaggcaaagcccccaaactcatgatttattctgacagttatcggccctc aggggtttctaatcgcttctctggctccaagtctggcaacacggcctccctgacaatctctgggctccaggctgaggacgaggctgatta ttactgcagctcatccacatactatagcactcgagttttcggcggagggaccaagctggagatcaaa
SEQ ID NO: 27 Heavy chain variable region of the G7 mab: acid nucleic sequence - FR1 -CDR1-FR2-CDR2-FR3 -CDR3-FR4 : gaggtgcagctggtggagtctgggggaagcctggtcaagcctggggggtccctgagactctcctgtgcagcctctggattc accttcagtaactacggtatgaactgggtccgccaggctccagggaaggggctggagtggatctcatacatttccggtagtagtcggta cataggctacgcagacttcgtgaagggccgattcaccatctccagagacaacgccaagaactcactgtacctgcaaatgaacagcctg agagccgaggacacggctgtttattactgtgtgagatcctactactacggcatggacgtctggggcagaggcaccctggtcaccgtctc ctca
SEQ ID NO: 28 Light chain variable region of the G7 mab: acid nucleic sequence - FR1 -CDR1-FR2-CDR2-FR3 -CDR3-FR4 : cagtctgtgctgactcagcctgcctccgtgtctgggagccctggacagtcgatcaccatctcctgcgctggaaccagcagtg acgttggtggttcatattccgtctcctggtaccaacaacacccaggcaaagcccccaaactcatgatttatagtgacagttatcggccctc aggggtttctaatcgcttctctggctccaagtctggcaacacggcctccctgacaatctctgggctccaggctgaggacgaggctgatta ttactgcagctcaaacacatatcagagcactcgagttttcggcggagggaccaagctggagatcaaa
Typically, said nucleic acid is a DNA or RNA molecule, which may be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector. As used herein, the terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. A further aspect of the invention relates to a vector comprising a nucleic acid of the invention.
Such vectors may comprise regulatory elements, such as a promoter, enhancer, terminator and the like, to cause or direct expression of said antibody upon administration to a subject. Examples of promoters and enhancers used in the expression vector for animal cell include early promoter and enhancer of SV40 (Mizukami T. et al. 1987), LTR promoter and enhancer of Moloney mouse leukemia virus (Kuwana Y et al. 1987), promoter (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983) of immunoglobulin H chain and the like. Any expression vector for animal cell can be used, so long as a gene encoding the human antibody C region can be inserted and expressed. Examples of suitable vectors include pAGE107 (Miyaji H et al. 1990), pAGE103 (Mizukami T et al. 1987), pHSG274 (Brady G et al. 1984), pKCR (O'Hare K et al. 1981), pSGl beta d2-4-(Miyaji H et al. 1990) and the like. Other examples of plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like. Other examples of viral vector include adenoviral, retroviral, herpes virus and AAV vectors. Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO 95/14785, WO 96/22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94/19478.
A further aspect of the invention relates to a host cell which has been transfected, infected or transformed by a nucleic acid and/or a vector according to the invention.
The term "transformation" means the introduction of a "foreign" (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNAbas been "transformed".
The nucleic acids of the invention may be used to produce an antibody of the present invention in a suitable expression system.
The term "expression system" means a host cell and compatible vector under suitable conditions, e.g. for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell. Common expression systems include E. coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E.coli, Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or established mammalian cell cultures (e.g., produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.). Examples also include mouse SP2/0-Agl4 cell (ATCC CRL1581), mouse P3X63-Ag8.653 cell (ATCC CRL1580), CHO cell in which a dihydrofolate reductase gene (hereinafter referred to as "DHFR gene") is defective (Urlaub G et al; 1980), rat YB2/3HL.P2.G11.16Ag.2O cell (ATCC CRL1662, hereinafter referred to as "YB2/0 cell"), and the like.
The present invention also relates to a method of producing a recombinant host cell expressing an antibody according to the invention, said method comprising the steps of: (i) introducing in vitro or ex vivo a recombinant nucleic acid or a vector as described above into a competent host cell, (ii) culturing in vitro or ex vivo the recombinant host cell obtained and (iii), optionally, selecting the cells which express and/or secrete said antibody. Such recombinant host cells can be used for the production of antibodies of the present invention.
Antibodies of the present invention are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A- Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
Functional variants
Another object of the invention relates to antibodies comprising functional variants of the VL region, VH region, or one or more CDRs of the antibodies A6, C7, C9, D2, D5, E4 or G7.
A functional variant of a VL or VH used in the context of a monoclonal antibody of the present invention still allows the antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity/avidity and/or the specificity/selectivity of the parent antibody (i.e. A6, C7, C9, D2, D5, E4 or G7 antibody) and in some cases such a monoclonal antibody of the present invention may be associated with greater affinity, selectivity and/or specificity than the parent Ab.
Such variants can be obtained by a number of affinity maturation protocols including mutating the CDRs (Yang et al., J. Mol. Biol., 254, 392-403, 1995), chain shuffling (Marks et al., Bio/Technology, 10, 779-783, 1992), use of mutator strains of E. coli (Low et al., J. Mol. Biol., 250, 359-368, 1996), DNA shuffling (Patten et al., Curr. Opin. Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J. Mol. Biol., 256, 77-88, 1996) and sexual PCR (Crameri et al., Nature, 391, 288-291, 1998). Vaughan et al. (supra) discusses these methods of affinity maturation. Such functional variants typically retain significant sequence identity to the parent Ab. The sequence of CDR variants may differ from the sequence of the CDR of the parent antibody sequences through mostly conservative substitutions; for instance at least about 35%, about 50% or more, about 60% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, (e.g., about 65-95%, such as about 92%, 93% or 94%) of the substitutions in the variant are conservative amino acid residue replacements.
The sequences of CDR variants may differ from the sequence of the CDRs of the parent antibody sequences through mostly conservative substitutions; for instance at least 10, such as at least 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant are conservative amino acid residue replacements. In the context of the present invention, conservative substitutions may be defined by substitutions within the classes of amino acids reflected as follows:
Aliphatic residues I, L, V, and M
Cycloalkenyl-associated residues F, H, W, and Y
Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, and Y
Negatively charged residues D and E
Polar residues C, D, E, H, K, N, Q, R, S, and T
Positively charged residues H, K, and R
Small residues A, C, D, G, N, P, S, T, and V
Very small residues A, G, and S
Residues involved in turn A, C, D, E, G, H, K, N, Q, R, S, P, and formation T
Flexible residues Q, T, K, S, G, P, D, E, and R
More conservative substitutions groupings include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Conservation in terms of hydropathic/hydrophilic properties and residue weight/size also is substantially retained in a variant CDR as compared to a CDR of A6, C7, C9, D2, D5, E4 or G7.
The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art. It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8) ; phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophane (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (- 3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The retention of similar residues may also or alternatively be measured by a similarity score, as determined by use of a BLAST program (e.g., BLAST 2.2.8 available through the NCBI using standard settings BLOSUM62, Open Gap= 1 1 and Extended Gap= 1). Suitable variants typically exhibit at least about 70% of identity to the parent peptide.
According to the present invention a first amino acid sequence having at least 70% of identity with a second amino acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; or 100% of identity with the second amino acid sequence.
According to the present invention a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; or 100% of identity with the second amino acid sequence.
In one embodiment, the antibodies described above bind to the same antigen and have the same properties of the antibody of the invention i.e. the antibody with the VH and VL of SEQ ID NO: 1 and 2, 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12 or 13 and 14.
Antibody which compete with the antibody of the invention
Another object of the invention relates to an antibody that competes for binding to TGFBI with the antibodies of the invention.
As used herein, the term "binding" in the context of the binding of an antibody to a predetermined antigen or epitope typically is a binding with an affinity corresponding to a KD of about 10'7 M or less, such as about 10'8 M or less, such as about 10'9 M or less, about 10-10 M or less, or about 10'11 M or even less when determined by for instance surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using a soluble form of the antigen as the ligand and the antibody as the analyte. BIACORE® (GE Healthcare, Piscaataway, NJ) is one of a variety of surface plasmon resonance assay formats that are routinely used to epitope bin panels of monoclonal antibodies. Typically, an antibody binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100-fold lower, for instance at least 1,000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000-fold lower than its KD for binding to a non-specific antigen (e.g., BSA, casein), which is not identical or closely related to the predetermined antigen. When the KD of the antibody is very low (that is, the antibody has a high affinity), then the KD with which it binds the antigen is typically at least 10,000-fold lower than its KD for a non-specific antigen. An T1 antibody is said to essentially not bind an antigen or epitope if such binding is either not detectable (using, for example, plasmon resonance (SPR) technology in a BIAcore 3000 instrument using a soluble form of the antigen as the ligand and the antibody as the analyte), or is 100 fold, 500 fold, 1000 fold or more than 1000 fold less than the binding detected by that antibody and an antigen or epitope having a different chemical structure or amino acid sequence.
Additional antibodies can be identified based on their ability to cross-compete (e.g., to competitively inhibit the binding of, in a statistically significant manner) with other antibodies of the invention in standard TGFBI binding assays.
The ability of a test antibody to inhibit the binding of antibodies of the present invention to TGFBI demonstrates that the test antibody can compete with that antibody for binding to TGFBI; such an antibody may, according to non-limiting theory, bind to the same or a related (e.g., a structurally similar or spatially proximal) epitope on TGFBI as the antibody with which it competes. Thus, another aspect of the invention provides antibodies that bind to the same antigen as, and compete with, the antibodies disclosed herein.
As used herein, an antibody “competes” for binding when the competing antibody inhibits TGFBI binding of an antibody or antigen binding fragment of the invention by more than 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% in the presence of an equimolar concentration of competing antibody.
In one embodiment, the antibodies or antigen binding fragments of the invention bind to one or more epitopes of TGFBI.
In one embodiment, the epitopes to which the present antibodies or antigen binding fragments bind are linear epitopes.
In one embodiment, the epitopes to which the present antibodies or antigen binding fragments bind are non-linear, conformational epitopes.
The antibodies of the invention may be assayed for specific binding by any method known in the art. Many different competitive binding assay format(s) can be used for epitope binding. The immunoassays which can be used include, but are not limited to, competitive assay systems using techniques such western blots, radioimmunoassays, ELISA, "sandwich" immunoassays, immunoprecipitation assays, precipitin assays, gel diffusion precipitin assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and complement-fixation assays. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, 1994 Current Protocols in Molecular Biology, Vol. 1, John Wiley & sons, Inc., New York).
Antibody engineering
• Variable regions
Engineered antibodies of the invention include those in which modifications have been made to framework residues within VH and/or VL, e.g. to improve the properties of the antibody. Typically such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations can be "backmutated" to the germline sequence by, for example, site-directed mutagenesis or PCR- mediated mutagenesis. Such "backmutated" antibodies are also intended to be encompassed by the invention. In one embodiment, the framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell -epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U. S. Patent Publication No. 20030153043 by Carr et al.
In one embodiment, the glycosylation of an antibody is modified. Glycosylation can be altered to, for example, increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.
In one embodiment, some mutations are made to the amino acids localized in aggregation “hotspots” within and near the first CDR (CDR1) to decrease the antibodies susceptibility to aggregation (see Joseph M. Perchiacca et al., Proteins 2011; 79:2637-2647). • Isotype
The antibody of the present invention may be of any isotype. The choice of isotype typically will be guided by the desired effector functions. IgGl and IgG3 are isotypes that mediate such effectors functions as ADCC or CDC, when IgG2 and IgG4 don’t or in a lower manner. Either of the human light chain constant regions, kappa or lambda, may be used. If desired, the class of a monoclonal antibody of the present invention may be switched by known methods. Typical, class switching techniques may be used to convert one IgG subclass to another, for instance from IgGl to IgG2. Thus, the effector function of the monoclonal antibodies of the present invention may be changed by isotype switching to, e.g., an IgGl, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody for various therapeutic uses.
In one embodiment, the antibody of the present invention is a full-length antibody.
In one embodiment, the full-length antibody is an IgGl antibody. In some embodiments, the full-length antibody is an IgG3 antibody.
In one embodiment, the antibody of the present invention is an antibody of a non-IgG2/4 type, e.g. IgGl or IgG3 which has been mutated such that the ability to mediate effector functions, such as ADCC, has been reduced or even eliminated. Such mutations have e.g. been described in Dall'Acqua WF et al., J Immunol. 177(2): 1129-1138 (2006) and Hezareh M, J Virol. 75(24): 12161-12168 (2001).
• Fc region
In one embodiment, the hinge region of CHI is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is described further in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine residues in the hinge region of CHI is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.
In one embodiment, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody. For example, one or more amino acids can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the Cl component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al. In one embodiment, the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fc receptor by modifying one or more amino acids. This approach is described further in PCT Publication WO 00/42072 by Presta. Moreover, the binding sites on human IgGI for FcyRI, FcyRII, FcyRIII and FcRn have been mapped and variants with improved binding have been described (see Shields, R. L. et al., 2001 J. Biol. Chen. 276:6591-6604, W02010106180).
As used herein, the term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a term well understood in the art, and refers to a cell-mediated reaction in which non- specific cytotoxic cells that express Fc receptors (FcRs) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. Non-specific cytotoxic cells that mediate ADCC include natural killer (NK) cells, macrophages, monocytes, neutrophils, and eosinophils.
As used herein, the term "Effector functions" refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.
In one embodiment, one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent Nos. 6,194,551 by Idusogie et al.
In one embodiment, one or more amino acid residues are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in PCT Publication WO 94/29351 by Bodmer et al.
Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated or non-fucosylated antibody having reduced amounts of or no fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the present invention to thereby produce an antibody with altered glycosylation. For example, EPl, 176,195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation or are devoid of fucosyl residues. Therefore, in some embodiments, the monoclonal antibodies of the present invention may be produced by recombinant expression in a cell line which exhibit hypofucosylation or non-fucosylation pattern, for example, a mammalian cell line with deficient expression of the FUT8 gene encoding fucosyltransferase. PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R.L. et al, 2002 J. Biol. Chem. 277:26733-26740). PCT Publication WO 99/54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(l,4)-N acetylglucosaminyltransf erase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al, 1999 Nat. Biotech. 17: 176-180). Eureka Therapeutics further describes genetically engineered CHO mammalian cells capable of producing antibodies with altered mammalian glycosylation pattern devoid of fucosyl residues (http:/ www.eiirekainc.com &boutus/ 'companyoverview.html). Alternatively, the monoclonal antibodies of the present invention can be produced in yeasts or filamentous fungi engineered for mammalian- like glycosylation pattern and capable of producing antibodies lacking fucose as glycosylation pattern (see for example EP1297172B1).
• Half life
In one embodiment, the antibody is modified to increase its biological half-life.
Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent No. 6,277,375 by Ward. Alternatively, to increase the biological half-life, the antibody can be altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121 ,022 by Presta et al. Antibodies with increased half lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the foetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. immunol. 24:249 (1994)), are described in US2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311,312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434, e.g., substitutions of Fc region residue 434 (US Patent No. 7,371,826).
Another modification of the antibodies herein that is contemplated by the invention is pegylation. An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. The pegylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (Cl - CIO) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In one embodiment, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the invention. See for example, EP0154316 by Nishimura et al. and EP0401384 by Ishikawa et al.
Another modification of the antibodies that is contemplated by the invention is a conjugate or a protein fusion of at least the antigen-binding region of the antibody of the invention to serum protein, such as human serum albumin or a fragment thereof to increase half-life of the resulting molecule. Such approach is for example described in Ballance et al. EP0322094. Another possibility is a fusion of at least the antigen-binding region of the antibody of the invention to proteins capable of binding to serum proteins, such human serum albumin to increase half-life of the resulting molecule. Such approach is for example described in Nygren et al., EP 0 486 525.
Polysialytion is another technology, which uses the natural polymer polysialic acid (PSA) to prolong the active life and improve the stability of therapeutic peptides and proteins. PSA is a polymer of sialic acid (a sugar). When used for protein and therapeutic peptide drug delivery, polysialic acid provides a protective microenvironment on conjugation. This increases the active life of the therapeutic protein in the circulation and prevents it from being recognized by the immune system. The PSA polymer is naturally found in the human body. It was adopted by certain bacteria which evolved over millions of years to coat their walls with it. These naturally polysialylated bacteria were then able, by virtue of molecular mimicry, to foil the body's defense system. PSA, nature's ultimate stealth technology, can be easily produced from such bacteria in large quantities and with predetermined physical characteristics. Bacterial PSA is completely non-immunogenic, even when coupled to proteins, as it is chemically identical to PSA in the human body.
Another technology includes the use of hydroxy ethyl starch ("HES") derivatives linked to antibodies. HES is a modified natural polymer derived from waxy maize starch and can be metabolized by the body's enzymes. HES solutions are usually administered to substitute deficient blood volume and to improve the rheological properties of the blood. Hesylation of an antibody enables the prolongation of the circulation half-life by increasing the stability of the molecule, as well as by reducing renal clearance, resulting in an increased biological activity. By varying different parameters, such as the molecular weight of HES, a wide range of HES antibody conjugates can be customized.
In one embodiment, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody.
More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Patent No. 6,165,745 by Ward et al.
• Others modifications
In one embodiment of the invention antibodies have been engineered to remove sites of deamidation. Deamidation is known to cause structural and functional changes in a peptide or protein. Deamidation can result in decreased bioactivity, as well as alterations in pharmacokinetics and antigenicity of the protein pharmaceutical. (Anal Chem. 2005 Mar 1 ;77(5): 1432-9).
In one embodiment of the invention the antibodies have been engineered to increase pl and improve their drug-like properties. The pl of a protein is a key determinant of the overall biophysical properties of a molecule. Antibodies that have low pls have been known to be less soluble, less stable, and prone to aggregation. Further, the purification of antibodies with low pl is challenging and can be problematic especially during scale-up for clinical use. Increasing the pl of the anti-TGFBI antibodies of the invention or fragments thereof improved their solubility, enabling the antibodies to be formulated at higher concentrations (>100 mg/ml). Formulation of the antibodies at high concentrations (e.g. >100mg/ml) offers the advantage of being able to administer higher doses of the antibodies into eyes of patients via intravitreal injections, which in turn may enable reduced dosing frequency, a significant advantage for treatment of chronic diseases including cardiovascular disorders. Higher pls may also increase the FcRn- mediated recycling of the IgG version of the antibody thus enabling the drug to persist in the body for a longer duration, requiring fewer injections. Finally, the overall stability of the antibodies is significantly improved due to the higher pi resulting in longer shelf-life and bioactivity in vivo. Preferably, the pl is greater than or equal to 8.2.
Glycosylation modifications can also induce enhanced anti-inflammatory properties of the antibodies by addition of sialylated glycans. The addition of terminal sialic acid to the Fc glycan reduces FcyR binding and converts IgG antibodies to anti-inflammatory mediators through the acquisition of novel binding activities (see Robert M. Anthony et al., J Clin Immunol (2010) 30 (Suppl 1): S9— S 14; Kai-Ting C et al., Antibodies 2013, 2, 392-414).
• Antiboby mimetics
In one embodiment, the heavy and light chains, variable regions domains and CDRs that are disclosed can be used to prepare polypeptides that contain antigen binding region that can specifically bind to TGFBI.
For example, the CDRs of the A6, C7, C9, D2, D5, E4 or G7 mabs can be incorporated into a molecule (e.g., a polypeptide) covalently or noncovalently to make an immunoadhesion. An immunoadhesion may incorporate the CDRs as part of a larger polypeptide chain, may covalently link the CDRs to another polypeptide chain, or may incorporate the CDRs noncovalently. The CDRs enable the immunoadhesion to bind specifically to a particular antigen of interest (e.g., TGFBI or epitope thereof).
As used herein, the terms “polypeptide” and “protein” refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well to naturally occurring amino acids polymers and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.
In one embodiment, the antigen biding fragment of the invention is grafted into nonimmunoglobulin based antibodies also called antibody mimetics selected from the group consisting of an affibody, an affilin, an affitin, an adnectin, an atrimer, an evasin, a DARPin, an anticalin, an avimer, a fynomer, and a versabody.
As used herein, the term “antibody mimetic” refers to molecules capables of mimicking an antibody’s ability to bind an antigen, but which are not limited to native antibody structures. Examples of such antibody mimetics include, but are not limited to, Adnectins, Affibodies, DARPins, Anticalins, Avimers, and versabodies, all of which employ binding structures that, while they mimic traditional antibody binding, are generated from and function via distinct mechanisms.
An affibody is well known in the art and refers to affinity proteins based on a 58 amino acid residue protein domain, derived from one of the IgG binding domain of staphylococcal protein A.
DARPins (Designed Ankyrin Repeat Proteins) are well known in the art and refer to an antibody mimetic DRP (designed repeat protein) technology developed to exploit the binding abilities of non-antibody proteins.
Anticalins are well known in the art and refer to another antibody mimetic technology, wherein the binding specificity is derived from lipocalins. Anticalins may also be formatted as dual targeting protein, called Duocalins.
Avimers are well known in the art and refer to another antibody mimetic technology, Avimers are derived from natural A-domain containing protein.
Versabodies are well known in the art and refer to another antibody mimetic technology, they are small proteins of 3-5 kDa with >15% cysteines, which form a high disulfide density scaffold, replacing the hydrophobic core the typical proteins have. Such antibody mimetic can be comprised in a scaffold.
The term “scaffold” refers to a polypeptide platform for the engineering of new products with tailored functions and characteristics.
In one aspect, the invention pertains to generating non-immunoglobulin-based antibodies also called antibody mimetics using non-immunoglobulins scaffolds onto which CDRs of the invention can be grafted. Known or future non-immunoglobulin frameworks and scaffolds may be employed, as long as they comprise a binding region specific for the target TGFBI protein.
Antigen biding fragments of antibodies can be grafted into scaffolds based on polypeptides such as Fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies). The fibronectin scaffolds are based on fibronectin type III domain (e.g., the tenth module of the fibronectin type III (10 Fn3 domain)). The fibronectin type III domain has 7 or 8 beta strands which are distribued between two beta sheets, which themselves pack against each other to form the core of the protein, and further containing loops (analogous to CDRs) which connect the beta strands to each other and are solvent exposed. There are at least three such loops at each edge of the beta sheet sandwich, where the edge is the boundary of the protein perpendicular to the direction of the beta strands (see US 6,818,418). These fibronectin-based scaffolds are not an immunoglobulin, although the overall fold is closely related to that of the smallest functional antibody fragment, the variable region of the heavy chain, which comprise the entire antigen recognition unit in camel and llama IgG. Because of this structure, the non-immunoglobulin antibody mimics antigen binding properties that are similar in nature and affinity to those of antibodies. These scaffolds can be used in a loop randomisation and shuffling strategy in vitro that is similar to the process of affinity maturation of antibodies in vivo. These fibronectin-based molecules can be used as scaffolds where the loop regions of the molecule can be replaced with CDRs of the invention using standard cloning techniques.
The Ankyrin technology is based on using proteins with Ankyrin derived repeat modules as scaffolds for bearing variable regions which can be used for binding to different targets. The Ankyrin repeat module is a 33 amino acid polypeptide consisting of two anti-parallel a-helices and a P-tum. Binding of the variable regions is mostly optimized by using ribosome display.
Avimers are derived from natural A-domain containing protein such as LRP-1. These domains are used by nature for protein-protein interactions and in human over 250 proteins are structurally based on “A-domains” monomers (2-10) linked via amino acids linkers. Avimers can be created that can bind to the target antigen using the methodology described in, for example, U.S. patent Application publication Nos. 20040175756; 20050053973; 20050048512; and 20060008844.
Affibody affinity ligands are small, simple proteins composed of a three-helix bundle based on the scaffold of one of the IgG-binding domains of protein A. protein A is a surface protein form the bacterium Staphylococcus aureus. This scaffold domain consist of 58 amino acids, 13 of which are randomized to generate affibody librairies with a large number of ligand variants (See e.g., US 5,831,012). Affibody molecules mimic antibodies, they have a molecular weight of 6kDa. In spite of its small size, the binding site of affibody molecules is similar to that of an antibody.
Anticalins are products developed by the company Pieris ProteoLab AG. They are derived from lipocalins, a widespread group of small and robust proteins that are usually involved in the physiological transport or storage of chemically sensitive or insoluble compounds. Several natural lipocalins occur in human tissues or body liquids. The protein architecture is reminiscent of immunoglobulins, with hypervariable loops on top of a rigid framework. However, in contrast with antibodies or their recombinant fragments, lipocalins are composed of a single polypeptide chain with 160 to 180 amino acids residues, being just marginally bigger than a single immunoglobulin domain. The set of four loops, which makes up the binding pocket, shows pronounced structural plasticity and tolerates a variety of side chains. The binding site can can thus be reshaped in a proprietary process in order to recognize prescribed target molecules of different shape with high affinity and specificity. One protein of lipocalin family, the bilin-binding protein (BBP) of Pieris Brassicae has been used to develop anticalins by mutagenizing the set of four loops. One example of a patent application describing anticalins is in PCT Publication No. WO 199916873.
Affilin molecules are small non-immunoglobulin proteins which are designed for specific affinities towards proteins and small molecules. New affilin molecules can be very quickly selected from two libraries, each of which is based on a different human derived scaffold protein. Affilin molecules do not show any structural homology to immunoglobulin proteins. Currently, two affilin scaffolds are employed, one of which is gamma crystalline, a human structural eye lens protein and the other is “ubiquitin” superfamily proteins. Both human scaffolds are very small, show high temperature stability and are almost resistant to pH changes and denaturing agents. This high stability is mainly due to the expanded beta sheet structure of the proteins. Examples of “ubiquitin-like” proteins are described in W02004106368.
Versabodies are highly soluble and can be formulated to high concentrations. Versabodies are exceptionally heat stable and offer extended shelf-life. Additional information regarding Versabodies can be found in US 2007/0191272, which is hereby incorporated by reference in its entirety.
The above descriptions of antibody fragment and mimetic technologies is not intended to be comprehensive. A variety of additional technologies including alternative polypeptide- based technologies, such as fusions of complementarity determining regions as outlined in Qui et al., Nature Biotechnology, 25(8) 921-929 (2007), as well as nucleic acid- based technologies, such as the RNA aptamer technologies described in US 5,789,157; 5,864,026; 5,712,375; 5,763,566; 6,013,443; 6,376,474; 6,613,526; 6,114,120; 6,261,774; and 6,387,620; all of which are hereby incorporated by reference, could be used in the context of the instant invention.
• CAR-T cells
The present invention also provides chimeric antigen receptors (CARs) comprising an antigen binding domain of the antibodies of the present invention.
Typically, said chimeric antigen receptor comprises at least one VH and/or VL sequence of the antibodies of the present invention. The chimeric antigen receptor of the present invention also comprises an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain.
As used herein, the term “chimeric antigen receptor” or “CAR” has its general meaning in the art and refers to an artificially constructed hybrid protein or polypeptide containing the antigen binding domains of an antibody (e.g., scFv) linked to T- cell signaling domains. Characteristics of CARs include their ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC-restricted manner, exploiting the antigenbinding properties of monoclonal antibodies. The non-MHC-restricted antigen recognition gives T cells expressing CARs the ability to recognize antigen independent of antigen processing, thus bypassing a major mechanism of tumor escape. Moreover, when expressed in T-cells, CARs advantageously do not dimerize with endogenous T cell receptor (TCR) alpha and beta chains.
In one embodiment, the invention provides CARs comprising an antigen-binding domain comprising, consisting of, or consisting essentially of, a single chain variable fragment (scFv) of the antibody of the invention.
In one embodiment, the antigen binding domain comprises a linker peptide. The linker peptide may be positioned between the light chain variable region and the heavy chain variable region.
In one embodiment, the CAR comprises an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain selected from the group consisting of CD28, 4-1BB, and CD3(^ intracellular domains. CD28 is a T cell marker important in T cell co-stimulation. 4- IBB transmits a potent costimulatory signal to T cells, promoting differentiation and enhancing long-term survival of T lymphocytes. CD3(^ associates with TCRs to produce a signal and contains immunoreceptor tyrosine-based activation motifs (ITAMs).
In one embodiment, the CAR of the present invention can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, e.g., a disulfide bridge, or converted into an acid addition salt and/or optionally dimerized or polymerized.
The invention also provides a nucleic acid encoding for a CAR of the present invention.
In one embodiment, the nucleic acid is incorporated in a vector as such as described above.
The present invention also provides a host cell comprising a nucleic acid encoding for a chimeric antigen receptor of the present invention. While the host cell can be of any cell type, can originate from any type of tissue, and can be of any developmental stage; the host cell is a T cell, e.g. isolated from peripheral blood lymphocytes (PBL) or peripheral blood mononuclear cells (PBMC).
In one embodiment, the T cell can be any T cell, such as a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupTl, etc., or a T cell obtained from a mammal. If obtained from a mammal, the T cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, or other tissues or fluids. T cells can also be enriched for or purified. The T cell can be any type of T cell and can be of any developmental stage, including but not limited to, CD4+/CD8+ double positive T cells, CD4+ helper T cells, e.g., Th2 cells, CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, and the like. The T cell may be a CD8+ T cell or a CD4+ T cell.
The population of those T cells prepared as described above can be utilized in methods and compositions for adoptive immunotherapy in accordance with known techniques, or variations thereof that will be apparent to those skilled in the art based on the instant disclosure. See, e.g., US Patent Application Publication No. 2003/0170238 to Gruenberg et al; see also US Patent No. 4,690,915 to Rosenberg. Adoptive immunotherapy of cancer refers to a therapeutic approach in which immune cells with an antitumor reactivity are administered to a tumorbearing host, with the aim that the cells mediate either directly or indirectly, the regression of an established tumor. Transfusion of lymphocytes, particularly T lymphocytes, falls into this category. Currently, most adoptive immunotherapies are autolymphocyte therapies (ALT) directed to treatments using the patient's own immune cells. These therapies involve processing the patient's own lymphocytes to either enhance the immune cell mediated response or to recognize specific antigens or foreign substances in the body, including the cancer cells. The treatments are accomplished by removing the patient's lymphocytes and exposing these cells in vitro to biologies and drugs to activate the immune function of the cells. Once the autologous cells are activated, these ex vivo activated cells are reinfused into the patient to enhance the immune system to treat cancer. In some embodiments, the cells are formulated by first harvesting them from their culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a "pharmaceutically acceptable" carrier) in a treatment-effective amount. Suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but also 5% dextrose in water or Ringer's lactate can be utilized. The infusion medium can be supplemented with human serum albumin. A treatment-effective amount of cells in the composition is dependent on the relative representation of the T cells with the desired specificity, on the age and weight of the recipient, on the severity of the targeted condition and on the immunogenicity of the targeted Ags. These amount of cells can be as low as approximately 103/kg, preferably 5xl03/kg; and as high as 107/kg, preferably 108/kg. The number of cells will depend upon the ultimate use for which the composition is intended, as will the type of cells included therein. For example, if cells that are specific for a particular Ag are desired, then the population will contain greater than 70%, generally greater than 80%, 85% and 90-95% of such cells. For uses provided herein, the cells are generally in a volume of a liter or less, can be 500 ml or less, even 250 ml or 100 ml or less. The clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed the desired total amount of cells. In particular the cells of the present invention are particularly suitable for the treatment of cancer.
Multispecific antibodies
In one embodiment, the invention provides a multispecific antibody comprising a first antigen binding site from an antibody of the present invention molecule described herein above and at least one second antigen binding site.
In one embodiment, the second antigen-binding site is used for recruiting a killing mechanism such as, for example, by binding an antigen on a human effector cell as a BiTE (Bispecific T-Cell engager) antibody which is a bispecific scFv2 directed against target antigen and CD3 on T cells described in US7235641, or by binding a cytotoxic agent or a second therapeutic agent.
As used herein, the term "effector cell" refers to an immune cell which is involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response. Exemplary immune cells include a cell of a myeloid or lymphoid origin, for instance lymphocytes (such as B cells and T cells including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, mast cells and granulocytes, such as neutrophils, eosinophils and basophils. Some effector cells express specific Fc receptors (FcRs) and carry out specific immune functions. In some embodiments, an effector cell is capable of inducing ADCC, such as a natural killer cell. For example, monocytes, macrophages, which express FcRs, are involved in specific killing of target cells and presenting antigens to other components of the immune system. In some embodiments, an effector cell may phagocytose a target antigen or target cell. The expression of a particular FcR on an effector cell may be regulated by humoral factors such as cytokines. An effector cell can phagocytose a target antigen or phagocytose or lyse a target cell. Suitable cytotoxic agents and second therapeutic agents are exemplified below, and include toxins (such as radiolabeled peptides), chemotherapeutic agents and prodrugs
In one embodiment, the second antigen-binding site binds to an antigen on a human B cell, such as, e.g., CD19, CD20, CD21, CD22, CD23, CD46, CD80, CD138 and HLA-DR.
In one embodiment, the second antigen-binding site binds a tissue- specific antigen, promoting localization of the bispecific antibody to a specific tissue.
In one embodiment, the second antigen-binding site binds to an antigen located on the same type of cell as the TGFBI-expressing cell, typically a tumor-associated antigen (TAA), but has a binding specificity different from that of the first antigen-binding site. Such multi- or bispecific antibodies can enhance the specificity of the tumor cell binding and/or engage multiple effector pathways. Exemplary TAAs include carcinoembryonic antigen (CEA), prostate specific antigen (PSA), RAGE (renal antigen), a-fetoprotein, CAMEL (CTL- recognized antigen on melanoma), CT antigens (such as MAGE-B5, -B6, -C2, -C3, and D; Mage-12; CT10; NY-ESO-1, SSX-2, GAGE, BAGE, MAGE, and SAGE), mucin antigens (e.g., MUC1, mucin-CA125, etc.), ganglioside antigens, tyrosinase, gp75, c-Met, Marti, MelanA, MUM-1, MUM-2, MUM-3, HLA-B7, Ep-CAM or a cancer-associated integrin, such as a5p3 integrin. Alternatively, the second antigen- binding site binds to a different epitope of TGFBI. The second antigen-binding site may alternatively bind an angiogenic factor or other cancer- associated growth factor, such as a vascular endothelial growth factor, a fibroblast growth factor, epidermal growth factor, angiogenin or a receptor of any of these, particularly receptors associated with cancer progression.
In one embodiment, the second antigen-binding site is from a second antibody or ADC of the invention, such as the antibody of the present invention.
Exemplary formats for the multispecific antibody molecules of the invention include, but are not limited to (i) two antibodies cross-linked by chemical heteroconjugation, one with a specificity to TGFBI and another with a specificity to a second antigen; (ii) a single antibody that comprises two different antigen-binding regions; (iii) a single-chain antibody that comprises two different antigen-binding regions, e.g., two scFvs linked in tandem by an extra peptide linker; (iv) a dual-variable-domain antibody (DVD-Ig), where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig™) Molecule, In : Antibody Engineering, Springer Berlin Heidelberg (2010)); (v) a chemically- linked bispecific (Fab')2 fragment; (vi) a Tandab, which is a fusion of two single chain diabodies resulting in a tetravalent bispecific antibody that has two binding sites for each of the target antigens; (vii) a flexibody, which is a combination of scFvs with a diabody resulting in a multivalent molecule; (viii) a so called "dock and lock" molecule, based on the "dimerization and docking domain" in Protein Kinase A, which, when applied to Fabs, can yield a trivalent bispecific binding protein consisting of two identical Fab fragments linked to a different Fab fragment; (ix) a so-called Scorpion molecule, comprising, e.g., two scFvs fused to both termini of a human Fab-arm; and (x) a diabody. Another exemplary format for bispecific antibodies is IgG-like molecules with complementary CH3 domains to force heterodimerization. Such molecules can be prepared using known technologies, such as, e.g., those known as Triomab/Quadroma (Trion Pharma/Fresenius Biotech), Knob-into-Hole (Genentech), CrossMAb (Roche) and electrostatically-matched (Amgen), LUZ-Y (Genentech), Strand Exchange Engineered Domain body (SEEDbody)(EMD Serono), Biclonic (Merus) and DuoBody (Genmab A/S) technologies.
In one embodiment, the bispecific antibody is obtained or obtainable via a controlled Fab-arm exchange, typically using DuoBody technology. In vitro methods for producing bispecific antibodies by controlled Fab-arm exchange have been described in WO2008119353 and WO 2011131746 (both by Genmab A/S). In one exemplary method, described in WO 2008119353, a bispecific antibody is formed by "Fab-arm" or "half- molecule" exchange (swapping of a heavy chain and attached light chain) between two monospecific antibodies, both comprising IgG4-like CH3 regions, upon incubation under reducing conditions. The resulting product is a bispecific antibody having two Fab arms which may comprise different sequences. In another exemplary method, described in WO 2011131746, bispecific antibodies of the present invention are prepared by a method comprising the following steps, wherein at least one of the first and second antibodies is the antibody of the present invention : a) providing a first antibody comprising an Fc region of an immunoglobulin, said Fc region comprising a first CH3 region; b) providing a second antibody comprising an Fc region of an immunoglobulin, said Fc region comprising a second CH3 region; wherein the sequences of said first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions; c) incubating said first antibody together with said second antibody under reducing conditions; and d) obtaining said bispecific antibody, wherein the first antibody is the antibody of the present invention and the second antibody has a different binding specificity, or vice versa. The reducing conditions may, for example, be provided by adding a reducing agent, e.g. selected from 2-mercaptoethylamine, dithiothreitol and tris(2- carboxy ethyl)phosphine. Step d) may further comprise restoring the conditions to become nonreducing or less reducing, for example by removal of a reducing agent, e.g. by desalting. Preferably, the sequences of the first and second CH3 regions are different, comprising only a few, fairly conservative, asymmetrical mutations, such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions. More details on these interactions and how they can be achieved are provided in WO 2011131746, which is hereby incorporated by reference in its entirety. The following are exemplary embodiments of combinations of such assymetrical mutations, optionally wherein one or both Fc-regions are of the IgGl isotype.
In one embodiment, the first Fc region has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405, 407 and 409, and the second Fc region has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405, 407 and 409, and wherein the first and second Fc regions are not substituted in the same positions.
In one embodiment, the first Fc region has an amino acid substitution at position 405, and said second Fc region has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 407 and 409, optionally 409.
In one embodiment, the first Fc region has an amino acid substitution at position 409, and said second Fc region has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405, and 407, optionally 405 or 368.
In one embodiment, both the first and second Fc regions are of the IgGl isotype, with the first Fc region having a Leu at position 405, and the second Fc region having an Arg at position 409.
Immunoconjugates
• Detectable label
In one embodiment, the antibodies of the invention can be conjugated with a detectable label to form an anti-TGFBI immunoconjugate.
Suitable detectable labels include, for example, a radioisotope, a fluorescent label, a chemiluminescent label, an enzyme label, a bioluminescent label or colloidal gold. Methods of making and detecting such detectably-labeled immunoconjugates are well-known to those of ordinary skill in the art, and are described in more detail below. The detectable label can be a radioisotope that is detected by autoradiography. Isotopes that are particularly useful for the purpose of the invention are 3H, 1251, 1311, 35S and 14C. Anti-TGFBI immunoconjugates can also be labeled with a fluorescent compound. The presence of a fluorescently-labeled antibody is determined by exposing the immunoconjugate to light of the proper wavelength and detecting the resultant fluorescence. Fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
Alternatively, anti-TGFBI immunoconjugates can be detectably labeled by coupling an antibody to a chemiluminescent compound. The presence of the chemiluminescent-tagged immunoconjugate is determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of chemiluminescent labeling compounds include luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt and an oxalate ester.
Similarly, a bioluminescent compound can be used to label anti-TGFBI immunoconjugates of the invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Bioluminescent compounds that are useful for labeling include luciferin, luciferase and aequorin.
Alternatively, anti-TGFBI immunoconjugates can be detectably labeled by linking an anti-TGFBI antibody to an enzyme. When the anti-TGFBI-enzyme conjugate is incubated in the presence of the appropriate substrate, the enzyme moiety reacts with the substrate to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or visual means. Examples of enzymes that can be used to detectably label polyspecific immunoconjugates include P-galactosidase, glucose oxidase, peroxidase and alkaline phosphatase.
Those of skill in the art will know of other suitable labels which can be employed in accordance with the invention. The binding of marker moieties to anti-TGFBI monoclonal antibodies can be accomplished using standard techniques known to the art. Typical methodology in this regard is described by Kennedy et al., Clin. Chim. Acta 70: 1, 1976; Schurs et al., Clin. Chim. Acta 81:l, 1977; Shih et al., /«z7J. Cancer 46: 1101, 1990; Stein et al., Cancer Res. 50: 1330, 1990; and Coligan, supra.
Moreover, the convenience and versatility of immunochemical detection can be enhanced by using anti-TGFBI monoclonal antibodies that have been conjugated with avidin, streptavidin, and biotin. (See, e.g., Wilchek et al. (eds.), “Avidin-Biotin Technology,” Methods In Enzymology (Vol. 184) (Academic Press 1990); Bayer et al., “Immunochemical Applications of Avidin-Biotin Technology,” in Methods In Molecular Biology (Vol. 10) 149-162 (Manson, ed., The Humana Press, Inc. 1992).)
Methods for performing immunoassays are well-established. (See, e.g., Cook and Self, “Monoclonal Antibodies in Diagnostic Immunoassays,” in Monoclonal Antibodies: Production, Engineering, and Clinical Application 180-208 (Ritter and Ladyman, eds., Cambridge University Press 1995); Perry, “The Role of Monoclonal Antibodies in the Advancement of Immunoassay Technology,” in Monoclonal Antibodies: Principles and Applications 107-120 (Birch and Lennox, eds., Wiley -Liss, Inc. 1995); Diamandis, Immunoassay (Academic Press, Inc. 1996).)
• Immunoconi ugates
In one embodiment, the antibody of the present invention is conjugated to a therapeutic moiety, i.e. a drug.
The therapeutic moiety can be, e.g., a cytotoxin, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immune stimulator, a lytic peptide, or a radioisotope. Such conjugates are referred to herein as an "antibody-drug conjugates" or "ADCs".
In one embodiment, the antibody is conjugated to a cytotoxic moiety. The cytotoxic moiety may, for example, be selected from the group consisting of taxol; cytochalasin B; gramicidin D; ethidium bromide; emetine; mitomycin; etoposide; tenoposide; vincristine; vinblastine; colchicin; doxorubicin; daunorubicin; dihydroxy anthracin dione; a tubulin- inhibitor such as maytansine or an analog or derivative thereof; an antimitotic agent such as monomethyl auristatin E or F or an analog or derivative thereof; dolastatin 10 or 15 or an analogue thereof; irinotecan or an analogue thereof; mitoxantrone; mithramycin; actinomycin D; 1 -dehydrotestosterone; a glucocorticoid; procaine; tetracaine; lidocaine; propranolol; puromycin; calicheamicin or an analog or derivative thereof; an antimetabolite such as methotrexate, 6 mercaptopurine, 6 thioguanine, cytarabine, fludarabin, 5 fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, or cladribine; an alkylating agent such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C; a platinum derivative such as cisplatin or carboplatin; duocarmycin A, duocarmycin SA, rachelmycin (CC-1065), or an analog or derivative thereof; an antibiotic such as dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)); pyrrolo[2,l-c][l,4]- benzodiazepines (PDB); diphtheria toxin and related molecules such as diphtheria A chain and active fragments thereof and hybrid molecules, ricin toxin such as ricin A or a deglycosylated ricin A chain toxin, cholera toxin, a Shiga-like toxin such as SLT I, SLT II, SLT IIV, LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alorin, saporin, modeccin, gelanin, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins such as PAPI, PAPII, and PAP-S, momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin toxins; ribonuclease (RNase); DNase I, Staphylococcal enterotoxin A; pokeweed antiviral protein; diphtherin toxin; and Pseudomonas endotoxin.
In one embodiment, the antibody is conjugated to a nucleic acid or nucleic acid- associated molecule. In one such embodiment, the conjugated nucleic acid is a cytotoxic ribonuclease (RNase) or deoxy-ribonuclease (e.g., DNase I), an antisense nucleic acid, an inhibitory RNA molecule (e.g., a siRNA molecule) or an immunostimulatory nucleic acid (e.g., an immunostimulatory CpG motif-containing DNA molecule). In some embodiments, the antibody is conjugated to an aptamer or a ribozyme.
In one embodiment, the antibody is conjugated, e.g., as a fusion protein, to a lytic peptide such as CLIP, Magainin 2, mellitin, Cecropin and Pl 8.
In one embodiment, the antibody is conjugated to a cytokine, such as, e.g., IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNa, IFN3, IFNy, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and TNFa.
In one embodiment, the antibody is conjugated to a radioisotope or to a radioisotopecontaining chelate. For example, the antibody can be conjugated to a chelator linker, e.g. DOTA, DTPA or tiuxetan, which allows for the antibody to be complexed with a radioisotope. The antibody may also or alternatively comprise or be conjugated to one or more radiolabeled amino acids or other radiolabeled moleculesNon-limiting examples of radioisotopes include 3H, 14C, 15N, 35S, 90Y, 99Tc, 1251, 1311, 186Re, 213Bi, 225Ac and 227Th. For therapeutic purposes, a radioisotope emitting beta- or alpha-particle radiation can be used, e.g., 1311, 90Y, 211 At, 212Bi, 67Cu, 186Re, 188Re, and 212Pb.
In one embodiment, an antibody-drug conjugate comprises an anti-tubulin agent. Examples of anti-tubulin agents include, for example, taxanes (e.g., Taxol® (paclitaxel), Taxotere® (docetaxel)), T67 (Tularik), vinca alkyloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine) and dolastatins (e.g., auristatin E, AFP, MMAF, MMAE, AEB, AEVB). Other antitubulin agents include, for example, baccatin derivatives, taxane analogs (e.g., epothilone A and B), nocodazole, colchicine and colcimid, estramustine, cryptophysins, cemadotin, maytansinoids, combretastatins, discodermolide, and eleutherobin. In some embodiments, the cytotoxic agent is a maytansinoid, another group of anti-tubulin agents. For example, in specific embodiments, the maytansinoid is maytansine or DM-1 (ImmunoGen, Inc.; see also Chari et al., Cancer Res. 52: 127-131, 1992).
In one embodiment, the cytotoxic agent is an antimetabolite. The antimetabolite can be, for example, a purine antagonist (e.g., azothioprine or mycophenolate mofetil), a dihydrofolate reductase inhibitor (e.g., methotrexate), acyclovir, gangcyclovir, zidovudine, vidarabine, ribavarin, azidothymidine, cytidine arabinoside, amantadine, dideoxyuridine, iododeoxyuridine, poscarnet, or trifluridine.
In one embodiment, an anti-TGFBI antibody is conjugated to a pro-drug converting enzyme. The pro-drug converting enzyme can be recombinantly fused to the antibody or chemically conjugated thereto using known methods. Exemplary pro-drug converting enzymes are carboxypeptidase G2, P-glucuronidase, penicillin- V-amidase, penicillin-G-amidase, P- lactamase, P-glucosidase, nitroreductase and carboxypeptidase A. Other molecule using as therapeutic moiety can be PyrroloBenzoDiazepine dimers (PBD).
In one embodiment, the antibody is a chimeric antibody having a heavy chain identical to SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13 and a light chain identical to SEQ ID NO:2, 4, 6, 8, 10, 12 or 14 and conjugated to the MMAE.
In one embodiment, the antibody is a chimeric antibody having a heavy chain identical to SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13 and a light chain identical to SEQ ID NO:2, 4, 6, 8, 10, 12 or 14 and conjugated to PyrroloBenzoDiazepine dimers (PBD).
• Linkers
Typically, the antibody-drug conjugate compounds comprise a linker unit between the drug unit and the antibody unit.
In one embodiment, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the drug unit from the antibody in the intracellular environment. In yet other embodiments, the linker unit is not cleavable and the drug is released, for example, by antibody degradation.
In one embodiment, the linker is cleavable by a cleaving agent that is present in the intracellular environment (e.g., within a lysosome or endosome or caveolea). The linker can be, e.g., a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long. Cleaving agents can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives resulting in the release of active drug inside target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123).
Most typical are peptidyl linkers that are cleavable by enzymes that are present in 191P4D12-expressing cells. Examples of such linkers are described, e.g., in U.S. Pat. No. 6,214,345, incorporated herein by reference in its entirety and for all purposes. In a specific embodiment, the peptidyl linker cleavable by an intracellular protease is a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin with the Val-Cit linker). One advantage of using intracellular proteolytic release of the therapeutic agent is that the agent is typically attenuated when conjugated and the serum stabilities of the conjugates are typically high.
In one embodiment, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at certain pH values.
Typically, the pH-sensitive linker hydrolyzable under acidic conditions. For example, an acid-labile linker that is hydrolyzable in the lysosome (e.g., a hydrazone, semi carb azone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like) can be used. (See, e.g., U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264: 14653-14661.) Such linkers are relatively stable under neutral pH conditions, such as those in the blood, but are unstable at below pH 5.5 or 5.0, the approximate pH of the lysosome. In certain embodiments, the hydrolyzable linker is a thioether linker (such as, e.g., a thioether attached to the therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929).
In one embodiment, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2- pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N- succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene), SPDB and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935.)
In one embodiment, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15: 1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1305- 12). In one embodiment, the linker unit is not cleavable and the drug is released by antibody degradation.
Typically, the linker is not substantially sensitive to the extracellular environment. As used herein, “not substantially sensitive to the extracellular environment,” in the context of a linker, means that no more than about 20 %, typically no more than about 15 %, more typically no more than about 10 %, and even more typically no more than about 5 %, no more than about 3 %, or no more than about 1 % of the linkers, in a sample of antibody-drug conjugate compound, are cleaved when the antibody-drug conjugate compound is present in an extracellular environment (e.g., in plasma). Whether a linker is not substantially sensitive to the extracellular environment can be determined, for example, by incubating with plasma the antibody-drug conjugate compound for a predetermined time period (e.g., 2, 4, 8, 16, or 24 hours) and then quantitating the amount of free drug present in the plasma.
Techniques for conjugating molecules to antibodies, are well-known in the art (See, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in Monoclonal Antibodies And Cancer Therapy (Reisfeld et al. eds., Alan R. Liss, Inc., 1985); Hellstrom et al., “Antibodies For Drug Delivery,” in Controlled Drug Delivery (Robinson et al. eds., Marcel Deiker, Inc., 2nd ed. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biological And Clinical Applications (Pinchera et al. eds., 1985); “Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy,” in Monoclonal Antibodies For Cancer Detection And Therapy (Baldwin et al. eds., Academic Press, 1985); and Thorpe et al., 1982, Immunol. Rev. 62: 119-58. See also, e.g., PCT publication WO 89/12624.) Typically, the nucleic acid molecule is covalently attached to lysines or cysteines on the antibody, through N- hydroxysuccinimide ester or maleimide functionality respectively.
Methods of conjugation using engineered cysteines or incorporation of unnatural amino acids have been reported to improve the homogeneity of the conjugate (Axup, J.Y., Bajjuri, K.M., Ritland, M., Hutchins, B.M., Kim, C.H., Kazane, S.A., Halder, R., Forsyth, J.S., Santidrian, A.F., Stafin, K., et al. (2012). Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc. Natl. Acad. Sci. USA 109, 16101-16106.; Junutula, J.R., Flagella, K.M., Graham, R.A., Parsons, K.L., Ha, E., Raab, H., Bhakta, S., Nguyen, T., Dugger, D.L., Li, G., et al. (2010). Engineered thio-trastuzumab-DMl conjugate with an improved therapeutic index to target humanepidermal growth factor receptor 2-positive breast cancer. Clin. Cancer Res.16, 4769-4778.). Junutula et al. (2008) developed cysteine-based site-specific conjugation called “THIOMABs” (TDCs) that are claimed to display an improved therapeutic index as compared to conventional conjugation methods. Conjugation to unnatural amino acids that have been incorporated into the antibody is also being explored for ADCs; however, the generality of this approach is yet to be established (Axup et al., 2012). In particular the one skilled in the art can also envisage Fc-containing polypeptide engineered with an acyl donor glutamine-containing tag (e.g., Gin-containing peptide tags or Q- tags) or an endogenous glutamine that are made reactive by polypeptide engineering (e.g., via amino acid deletion, insertion, substitution, or mutation on the polypeptide). Then a transglutaminase, can covalently crosslink with an amine donor agent (e.g., a small molecule comprising or attached to a reactive amine) to form a stable and homogenous population of an engineered Fc-containing polypeptide conjugate with the amine donor agent being site- specifically conjugated to the Fc- containing polypeptide through the acyl donor glutamine- containing tag or the accessible/exposed/reactive endogenous glutamine (WO 2012059882).
Therapeutic uses
The inventors clearly showed in their results the impact of a TGFBI inhibitor and particularly the anti-TGFBI antibodies on muscle and fat tissue and the macrophage activity in those tissues demonstrating that a TGFBI inhibitor can have an impact on cachexia and cachexia linked to other diseases.
Thus, another obj ect of the invention relates to a TGFBI inhibitor for use in the treatment of cachexia in a subject in need thereof.
Particularly, the invention relates to a TGFBI inhibitor for use in the treatment of a cachexia linked to a disease selected in the list consisting of cancer, congestive heart failure, chronic obstructive pulmonary disease, chronic kidney disease, and AIDS in a subject in need thereof.
In a particular embodiment, the invention relates to a TGFBI inhibitor for use in the treatment of a cancer in a subject in need thereof.
In a particular embodiment, the invention relates to a TGFBI inhibitor for use in the treatment of a cancer cachexia in a subject in need thereof.
In a particular embodiment the cancer of the cancer cachexia is a resistant cancer or a resistant cancer cachexia.
Another object of the invention relates to a method for treating cachexia or cancer cachexia in a subject in need thereof comprising administering a therapeutically effective amount of a TGFBI inhibitor according to the invention. As used herein, the term “TGFBI inhibitor” refers to any compound natural or not which is capable of reducing orblocking the activity or expression of TGFBI. The term “TGFBI inhibitor” encompasses any TGFBI inhibitor that is currently known in the art or that will be identified in the future and includes any chemical entity that, upon administration to a patient, results in inhibition or down-regulation of a biological activity associated with activation of TGFBI. The term also encompasses inhibitor of expression.
TGFBI inhibitors are well known in the state of the art and include those described in :
Rosemary Kim H et al., 2009, Dev Dyn, DOI : 10.1002/dvdy.21812
- Taketani Y et al., 2017, Sci Rep, DOI : 10.1038/s41598-017-16308-2 Zhou J et al., 2023, Cancer Lett, DOI : 10.1016/j.canlet.2023.216457 Chiavarina B et al., 2021, Theranostics, DOI : 10.7150/thno.51507
In the context of the present invention, “TGFBI inhibitor” is an inhibitor which neutralizes, blocks, inhibits, abrogates, reduces or interferes with the biological activity of TGFBI. In particular it refers to an inhibitor which reduces the weight loss, the muscle wasting and/or the cytokine secretion.
In one embodiment, the TGFBI inhibitor according to the invention is :
1) an inhibitor of TGFBI activity and/or
2) an inhibitor of TGFBI expression.
By "biological activity" of TGFBI is meant, in the context of the present invention, reducing the weight loss, the muscle wasting and/or the cytokine secretion.
Tests for determining the capacity of a compound to be an TGFBI inhibitor are well known to the person skilled in the art. In a particular embodiment, the ability of the inhibitor to inhibits the biological activity of TGFBI is well known to the person skilled in the art. The ability to bind to TGFBI may be determined by assaying cells treated with TGFBI inhibitor (e.g. siRNA, antibodies) with fluorescence ELISA test or surface plasmon resonance (SPR) (see Figures 2A, 2B, 2C, 2D, 2E). The ability to stop weight loss or increase food intake may be determined by assaying the body weight, plasma glucose or albumin level (see Figures 3 A, 3B, 3C, 3D, 3E, 3F). The ability to decrease muscle wasting or lipolysis may be determined by microscopic analysis, magnetic resonance tomography or muscle loss marker expression analysis (Trim63 and Fbox32) (see Figures 4A, 4B, 4C, 4D, 4E, 4F, 4G, 4H, 41). In particular the ability to maintain muscle strength can be evaluated using established clinical parameters such as handheld dynamometer for humans or wire hanging for rodents. Finally, the ability to decrease cytokine secretion and inflammation may be determined by ELISA test, Immunohistochemistry in target tissue or FACs analysis of isolated cells from tumors or blood (see Figures 5 A, 5B, 5C, 5D, 5E & 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 61, 6J, 6K).
As used herein, the term “Cancer cachexia” refers to a multifactorial syndrome characterized by a continuous loss of skeletal muscle mass (with or without fat loss) provoked by the cancer. The cachexic syndrome is fueled by continuous metabolic wasting, frequently accompanied by reduced appetite and food intake (Fearon K et al., 2011, Lancet Oncol). It results in decreased tolerability of anti-cancer treatments and decreased quality of life, both of which ultimately lead to death in at least 20-30% of patients (Mattox T.W et al., 2017, Nutr Clin Pract & Ferrara M et al., 2022, Front Cell Dev BioT).
As used herein, the term “Cancer” refers to liquid or a solid cancer. Cancer may be a cancer selected from the group consisting in adrenal cortical cancer, anal cancer, bile duct cancer (e.g. perihilar cancer, distal bile duct cancer, intrahepatic bile duct cancer), bladder cancer, bone cancer (e.g. osteoblastoma, osteochrondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of the bone, chordoma), brain and central nervous system cancer (e.g. meningioma, astocytoma, oligodendrogliomas, ependymoma, gliomas, medulloblastoma, ganglioglioma, Schwannoma, germinoma, craniopharyngioma), breast cancer (e.g. ductal carcinoma in situ, infiltrating ductal carcinoma, infiltrating lobular carcinoma, lobular carcinoma in situ, gynecomastia), Castleman disease (e.g. giant lymph node hyperplasia, angiofollicular lymph node hyperplasia), cervical cancer, colorectal cancer, endometrial cancer (e.g. endometrial adenocarcinoma, adenocanthoma, papillary serous adenocarcinoma, clear cell), esophagus cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g. choriocarcinoma, chorioadenoma destruens), Hodgkin's disease, Kaposi's sarcoma, kidney cancer (e.g. renal cell cancer), laryngeal and hypopharyngeal cancer, liver cancer (e.g. hemangioma, hepatic adenoma, focal nodular hyperplasia, hepatocellular carcinoma), lung cancer (e.g. small cell lung cancer, non-small cell lung cancer), mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer (e.g. esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer (e.g. melanoma, nonmelanoma skin cancer), stomach cancer, testicular cancer (e.g. seminoma, nonseminoma germ cell cancer), thymus cancer, thyroid cancer (e.g. follicular carcinoma, anaplastic carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma,), vaginal cancer, vulvar cancer, uterine cancer (e.g. uterine leiomyosarcoma), leukaemia (like acute myeloid leukaemia, acute lymphoid leukaemia, chronic myelomonocytic leukemia (CMML)...), lymphoma and myelodysplastic syndrome (MDS).
In one embodiment, the compounds and methods of the present invention are suitable for treating colorectal cancer (CRC). In particular, methods of the present invention are suitable for advanced or resistant CRC.
As used herein, the term “patient” or “subject” or “individual” refers to a subject to be treated by the antibody disclosed herein. In particular, the patient suffers from a Cancer. In one embodiment, the patient is a mammal. Non-limiting examples of mammals include rodents (e.g., mice and rats), primates (e.g., lemurs, bushbabies, monkeys, apes and humans), rabbits, dogs, horses, cats, livestock (such as pigs, bovines, donkeys, mules, bison, goats, camels, and sheep), and deer. In one embodiment, the mammal is a human.
In a preferred embodiment, the “subject” is a human with a cancer, in particular a cancer cachexia, according to the invention.
In one embodiment, the TGFBI inhibitor according to the invention may be a low molecular weight compound, e. g. a small organic molecule (natural or not).
The term "small organic molecule" refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e. g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to about 1000 Da.
In one embodiment, the TGFBI inhibitor according to the invention is an anti-TGFBI antibody.
Antibodies directed against TGFBI can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from camels, pigs, cows, horses, rabbits, goats, sheep, and mice, among others, or organotypic cultures of primary human cells from tonsils, lymph nodes or peripheral blood. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies against TGFBI can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al., 1983); and the EBV-hybridoma technique (Cole et al. 1985). Alternatively, techniques described for the production of single chain antibodies (see e.g., U.S. Pat. No. 4,946,778) can be adapted to produce anti- TGFBI single chain antibodies. Compounds useful in practicing the present invention also include anti- TGFBI antibody fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to TGFBI.
Humanized or human anti-TGFBI antibodies and antibody fragments therefrom can also be prepared according to known techniques. "Humanized antibodies" are forms of non-human (e.g., rodent) chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Methods for making humanized antibodies are described, for example, by Winter (U.S. Pat. No. 5,225,539) and Boss (Celltech, U.S. Pat. No. 4,816,397). Human antibodies can be generated by organotypic cultures of primary human cells from tonsils, lymph nodes or peripheral blood (see Wagar Lisa E. et al. Nature Medicine volume 27, pagesl25-135 (2021)).
For a more important description of the term “antibody” see the parts above. In a particular embodiment the anti-TGFBI antibody according to the invention is one of the antibodies of the invention (A6, C7, C9, D2, D5, E4 or G7, see above).
In another embodiment, the antibody according to the invention is a single domain antibody against TGFBI. The term “single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb. The term “VHH” refers to the single heavy chain having 3 complementarity determining regions (CDRs): CDR1, CDR2 and CDR3. The term “complementarity determining region” or “CDR” refers to the hypervariable amino acid sequences which define the binding affinity and specificity of the VHH.
The VHH according to the invention can readily be prepared by an ordinarily skilled artisan using routine experimentation. The VHH variants and modified form thereof may be produced under any known technique in the art such as in-vitro maturation.
VHHs or sdAbs are usually generated by PCR cloning of the V-domain repertoire from blood, lymph node, or spleen cDNA obtained from immunized animals into a phage display vector, such as pHEN2. Antigen-specific VHHs are commonly selected by panning phage libraries on immobilized antigen, e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells. However, such VHHs often show lower affinities for their antigen than VHHs derived from animals that have received several immunizations. The high affinity of VHHs from immune libraries is attributed to the natural selection of variant VHHs during clonal expansion of B-cells in the lymphoid organs of immunized animals. The affinity of VHHs from non-immune libraries can often be improved by mimicking this strategy in vitro, i.e., by site directed mutagenesis of the CDR regions and further rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH, and low antigen concentrations). VHHs derived from camelid are readily expressed in and purified from the E. coli periplasm at much higher levels than the corresponding domains of conventional antibodies. VHHs generally display high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells. For example, the “Hamers patents” describe methods and techniques for generating VHH against any desired target (see for example US 5,800,988; US 5,874, 541 and US 6,015,695). The “Hamers patents” more particularly describe production of VHHs in bacterial hosts such as E. coli (see for example US 6,765,087) and in lower eukaryotic hosts such as moulds (for example Aspergillus or Trichoderma) or in yeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see for example US 6,838,254).
In one embodiment, the compound according to the invention is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).
Then, for this invention, neutralizing aptamer of TGFBI is selected.
In one embodiment, the compound according to the invention is a polypeptide.
In a particular embodiment the polypeptide is an antagonist or a reverse agonist of TGFBI and is capable to prevent the function of TGFBI. Particularly, the polypeptide can be a mutated TGFBI protein or a similar protein without the function of TGFBI. In this case, the mutated version of the TGFBI protein is used as a decoy receptor.
In one embodiment, the polypeptide of the invention may be linked to a “cellpenetrating peptide” to allow the penetration of the polypeptide in the cell.
The term “cell-penetrating peptides” are well known in the art and refers to cell permeable sequence or membranous penetrating sequence such as penetratin, TAT mitochondrial penetrating sequence and compounds (Bechara and Sagan, 2013; Jones and Sayers, 2012; Khafagy el and Morishita, 2012; Malhi and Murthy, 2012).
The polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art. In order to produce sufficient amounts of polypeptide or functional equivalents thereof for use in accordance with the present invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention. Preferably, the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. When expressed in recombinant form, the polypeptide is preferably generated by expression from an encoding nucleic acid in a host cell. Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown. A common, preferred bacterial host is E coli.
In specific embodiments, it is contemplated that polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution. In example adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters.
A strategy for improving drug viability is the utilization of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained-release effect, water- soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain.
Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity. PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications.
Those of skill in the art are aware of PEGylation techniques for the effective modification of drugs. For example, drug delivery polymers that consist of alternating polymers of PEG and tri -functional monomers such as lysine have been used by VectraMed (Plainsboro, N. J.). The PEG chains (typically 2000 Daltons or less) are linked to the a- and e-amino groups of lysine through stable urethane linkages. Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain. The reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules. These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer. The molecular weight of the PEG segments affects the spacing of the drug/linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading). In general, increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory halflife of the conjugate. Nevertheless, the conjugate must either be readily degradable or have a molecular weight below the threshold-limiting glomerular filtration (e.g., less than 60 kDa).
In addition, to the polymer backbone being important in maintaining circulatory halflife, and biodistribution, linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue. For example, this type of tissue activated drug delivery is particularly useful where delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of pathology. Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes. Such linkers may be used in modifying the protein or fragment of the protein described herein for therapeutic delivery.
In another embodiment, the TGFBI inhibitor according to the invention is an inhibitor of TGFBI gene expression.
Small inhibitory RNAs (siRNAs) can also function as inhibitors of TGFBI expression for use in the present invention. TGFBI gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that TGFBI gene expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).
Examples of siRNAs against TGFBI include, but are not limited to, those described in Chaoyu Ma (2008) Genes & Development 22:308-321.
MicroRNA (miRNA) can also function as inhibitors of TGFBI expression for use in the present invention. MicroRNA (miRNA) are small, single-stranded, non-coding RNA molecules containing 21 to 23 nucleotides. Found in plants, animals and some viruses, miRNAs are involved in RNA silencing and post-transcriptional regulation of gene expression. miRNAs base-pair to complementary sequences in mRNA molecules, then silence said mRNA molecules.
Ribozymes can also function as inhibitors of TGFBI gene expression for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of TGFBI mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.
Both antisense oligonucleotides and ribozymes useful as inhibitors of TGFBI gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramidite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.
Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing TGFBI. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.
Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non- essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, 1990 and in Murry, 1991).
Preferred viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wildtype adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.
Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al., 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigenencoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.
In a particular embodiment, the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter. The promoter may be specific for Muller glial cells, microglia cells, endothelial cells, pericyte cells and astrocytes For example, a specific expression in Muller glial cells may be obtained through the promoter of the glutamine synthetase gene is suitable. The promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters.
In a particular embodiment, an endonuclease can be used to abolish the expression of the gene, transcript or protein variants of TGFBI. Indeed, as an alternative to more conventional approaches, such as cDNA overexpression or downregulation by RNA interference, new technologies provide the means to manipulate the genome. Indeed, natural and engineered nuclease enzymes have attracted considerable attention in the recent years. The mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the error prone non homologous end-joining (NHEJ) and the high-fidelity homology-directed repair (HDR).
In a particular embodiment, the endonuclease is CRISPR-cas. As used herein, the term “CRISPR-cas” has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences.
In some embodiment, the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes. The CRISPR/Cas9 system has been described in US 8697359 Bl and US 2014/0068797. Originally an adaptive immune system in prokaryotes (Barrangou and Marraffini, 2014), CRISPR has been recently engineered into a new powerful tool for genome editing. It has already been successfully used to target important genes in many cell lines and organisms, including human (Mali et al., 2013, Science, Vol. 339 : 823-826), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), zebrafish (Hwang et al., 2013, PLoS One, Vol. 8:e68708.), C. elegans (Hai et al., 2014 Cell Res. doi: 10.1038/cr.2014.11.), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), plants (Mali et al., 2013, Science, Vol. 339 : 823-826), Xenopus tropicalis (Guo et al., 2014, Development, Vol. 141 : 707-714.), yeast (DiCarlo et al., 2013, Nucleic Acids Res., Vol. 41 : 4336-4343.), Drosophila (Gratz et al., 2014 Genetics, doi: 10.1534/genetics, 113.160713), monkeys (Niu et al., 2014, Cell, Vol. 156 : 836- 843.), rabbits (Yang et al., 2014, J. Mol. Cell Biol., Vol. 6 : 97-99.), pigs (Hai et al., 2014, Cell Res. doi: 10.1038/cr.2014.11.), rats (Ma et al., 2014, Cell Res., Vol. 24 : 122-125.) and mice (Mashiko et al., 2014, Dev. Growth Differ. Vol. 56 : 122-129.). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA. Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations. A recent exciting development is the use of the dCas9 version of the CRISPR/Cas9 system to target protein domains for transcriptional regulation, epigenetic modification, and microscopic visualization of specific genome loci. In some embodiment, the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).
Pharmaceutical composition
Another object of the invention relates to a pharmaceutical composition for use in the treatment of cachexia comprising a therapeutically effective amount of an inhibitor according to the invention.
Particularly, the TGFBI inhibitor is an antibody of the invention.
Particularly the cachexia is a cachexia linked to a cancer and more particularly, the cachexia is a cancer cachexia.
The composition of the present invention may e.g. be formulated for any mode of administration suitable for the treatment of cancer. The form of the composition, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the subject, etc. Then, the uses are adjusted to provide the optimum desired response (e.g., a therapeutic response).
The pharmaceutical compositions may contain vehicles which are pharmaceutically acceptable for a formulation capable of treating cancer.
As used herein, the term “pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administrated to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluents, encapsulating material or formulation auxiliary of any type. The carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
“Carriers” or “vehicles” include any such material known in the art and may be any liquid, gel, solvent, liquid diluent, solubilizer, or like, which is non-toxic and which does not interect with any components of the composition in a deleterious manner. Examples of nutritionally acceptable carriers include, for example, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like. In one embodiment, the use of liposomes and/or nanoparticles is contemplated for the introduction of the inhibitor or the antibodies of the invention into host cells.
The formation and use of liposomes and/or nanoparticles are known to those of skill in the art. Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 pm) are generally designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be are easily made. Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)). MLVs generally have diameters of from 25 nm to 4 pm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations.
In a particular embodiment, the pharmaceutic composition is a therapeutic composition.
According to the invention, the inhibitor of the invention (particularly the antibodies of the invention) or the pharmaceutical composition of the invention are administrated in a therapeutically effective amount.
Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutic compositions.
As used herein, the term "therapeutically effective amount" or “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of the inhibitor or the composition of the present invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the inhibitor or the composition of the present invention to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the inhibitor or the composition are outweighed by the therapeutically beneficial effects. The efficient dosages and dosage regimens for the inhibitor or the composition of the present invention depend on the disease or condition to be treated and may be determined by the persons skilled in the art. A physician having ordinary skill in the art may readily determine and prescribe the effective amount of the inhibitor or the composition of the invention required. For example, the physician could start doses of the inhibitor or the composition of the present invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable dose of the inhibitor or the composition of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen. Such an effective dose will generally depend upon the factors described above. For example, a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of disease. Typically, and for example, the ability of a compound to inhibit inflammatory disorders and pain disorders may, for example, be evaluated in an animal model system predictive of efficacy in human. Alternatively, this property of a composition may be evaluated by examining the ability of the compound to induce cytotoxicity by in vitro assays known to the skilled practitioner. A therapeutically effective amount of a therapeutic compound may decrease tumor size, or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected. An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg/kg, about 5 mg/kg or about 8 mg/kg. An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is 0.02-100 mg/kg, such as about 0.02-30 mg/kg, such as about 0.05-10 mg/kg or 0.1-3 mg/kg, for example about 0.5-2 mg/kg. Administration may e.g. be topical, oral, intranasal, parenteral, intravenous, intraocular, intrathecal, epidural, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target. Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time. In some embodiments, the efficacy may be monitored by visualization of the disease area, or by other diagnostic methods described further herein, e.g. by performing one or more PET-CT scans, for example using a labeled antibody of the present invention, fragment or mini-antibody derived from the antibody of the present invention. If desired, an effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In some embodiments, the monoclonal antibodies of the present invention are administered by slow continuous infusion over a long period, such as more than 24 hours, in order to minimize any unwanted side effects. An effective dose of an antibody of the present invention may also be administered using a weekly, biweekly or triweekly dosing period. The dosing period may be restricted to, e.g., 8 weeks, 12 weeks or until clinical progression has been established. As nonlimiting examples, treatment according to the present invention may be provided as a daily dosage of an antibody of the present invention in an amount of about 0.1-100 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.
Administration may be topical, oral, intranasal, parenteral, intravenous, intrathecal, epidural, intraocular, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target. Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time. In some embodiments, the efficacy may be monitored by visualization of the disease area, or by other diagnostic methods described further herein, e.g. by performing one or more PET-CT scans. If desired, an effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In some embodiments, the oligomers of the present invention are administered by slow continuous infusion over a long period, such as more than 24 hours, in order to minimize any unwanted side effects. An effective dose of the agent of the present invention may also be administered using a weekly, biweekly or triweekly dosing period. The dosing period may be restricted to, e.g., 8 weeks, 12 weeks or until clinical progression has been established. As non-limiting examples, treatment according to the present invention may be provided as a daily dosage of the agent of the present invention in an amount of about 0.1- 100 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.
In other words, the quantity of the immune cell or the population of immune cells administered to a subject in need thereof is between 104 to 109 cells per kg. Particularly, the quantity of cells injected is 106 or 107 cells per kg. The immune cell or the population of immune cells of the invention can be administrated is 1, 2, 3, 4 or 5 times to the subject in need thereof.
The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the subject, etc.
The pharmaceutical compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like.
Particularly, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze- dried compositions. In particular, these may be in organic solvent such as DMSO, ethanol which upon addition, depending on the case, of sterilized water or physiological saline permit the constitution of injectable solutions.
In addition, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.
Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 pm) are generally designed using polymers able to be degraded in vivo. Biodegradable polyalkyl -cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be easily made.
Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)). MLVs generally have diameters of from 25 nm to 4 gm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations.
Combinations and kit of part
The inhibitor of the invention and particularly the antibodies of the invention may be used alone or in combination with further therapeutic active agent. In each of the embodiments of the therapeutical uses described in the present invention, the inhibitor of the invention or the anti-TGFBI antibodies (or anti-TGFBI antibodies-drug conjugate) are delivered in a manner consistent with conventional methodologies associated with management of the disease or disorder for which treatment is sought (particularly, cachexia and cancer cachexia)
Thus, another object of the invention relates to a combination of TGFBI inhibitor and a further therapeutic active agent for use in the treatment of cachexia in a subject in need thereof.
In one embodiment, the invention relates to a pharmaceutical composition according to the invention and a further therapeutic active agent for use in the treatment of cachexia in a subject in need thereof.
In one embodiment, the cachexia is a cancer cachexia.
As used herein, the term “therapeutic active agent”, “therapeutic agent” or “active agent” or “active substance” or “active principle” or “active ingredient” relates to a substance (particularly a chemical substance) inducing an effect such as a therapeutic or a preventive effect. It may be a bioactive chemical compound from a drug or the drug itself. Active agent can be a single molecule or a mixture of several substances.
Particularly, the therapeutic active agent may be conventional cancer therapies such as, e.g., radiotherapy, chemotherapy (or combinations thereof). Surgery can also be combined with the inhibitor of the invention
In one embodiment, therapeutic active agent used in combination with the inhibitor of the invention comprising anti-cancer antibodies, cytotoxic agents, chemotherapeutic agents, anti -angiogenic agents, anti-cancer immunogens, cell cycle control/ apoptosis regulating agents, hormonal regulating agents, and other agents described below. In some embodiments, the inhibitor of the present invention is used in combination with a chemotherapeutic agent. The term "chemotherapeutic agent" refers to chemical compounds that are effective in inhibiting tumor growth. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaorarnide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a carnptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estrarnustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimus tine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Inti. Ed. Engl. 33: 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idanrbicin, marcellomycin, mitomycins, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomgrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pento statin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogennanium; tenuazonic acid; triaziquone; 2, 2', 2"- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridinA and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit honnone action on tumors such as anti -estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti -androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
In some embodiments, the inhbitor of the present invention is used in combination with a targeted cancer therapy. Targeted cancer therapies are drugs or other substances that block the growth and spread of cancer by interfering with specific molecules ("molecular targets") that are involved in the growth, progression, and spread of cancer. Targeted cancer therapies are sometimes called "molecularly targeted drugs," "molecularly targeted therapies," "precision medicines," or similar names. In some embodiments, the targeted therapy consists of administering the subject with a tyrosine kinase inhibitor. The term “tyrosine kinase inhibitor” refers to any of a variety of therapeutic agents or drugs that act as selective or non-selective inhibitors of receptor and/or non-receptor tyrosine kinases. Tyrosine kinase inhibitors and related compounds are well known in the art and described in U.S Patent Publication 2007/0254295, which is incorporated by reference herein in its entirety. It will be appreciated by one of skill in the art that a compound related to a tyrosine kinase inhibitor will recapitulate the effect of the tyrosine kinase inhibitor, e.g., the related compound will act on a different member of the tyrosine kinase signaling pathway to produce the same effect as would a tyrosine kinase inhibitor of that tyrosine kinase. Examples of tyrosine kinase inhibitors and related compounds suitable for use in methods of embodiments of the present invention include, but are not limited to, dasatinib (BMS-354825), PP2, BEZ235, saracatinib, gefitinib (Iressa), sunitinib (Sutent; SU11248), erlotinib (Tarceva; OSI-1774), lapatinib (GW572016; GW2016), canertinib (CI 1033), semaxinib (SU5416), vatalanib (PTK787/ZK222584), sorafenib (BAY 43-9006), imatinib (Gleevec; STI571), leflunomide (SU101), vandetanib (Zactima; ZD6474), MK-2206 (8-[4-aminocyclobutyl)phenyl]-9-phenyl-l,2,4-triazolo[3,4-f][l,6]naphthyridin- 3(2H)-one hydrochloride) derivatives thereof, analogs thereof, and combinations thereof. Additional tyrosine kinase inhibitors and related compounds suitable for use in the present invention are described in, for example, U.S Patent Publication 2007/0254295, U.S. Pat. Nos. 5,618,829, 5,639,757, 5,728,868, 5,804,396, 6,100,254, 6,127,374, 6,245,759, 6,306,874, 6,313,138, 6,316,444, 6,329,380, 6,344,459, 6,420,382, 6,479,512, 6,498,165, 6,544,988, 6,562,818, 6,586,423, 6,586,424, 6,740,665, 6,794,393, 6,875,767, 6,927,293, and 6,958,340, all of which are incorporated by reference herein in their entirety. In some embodiments, the tyrosine kinase inhibitor is a small molecule kinase inhibitor that has been orally administered and that has been the subject of at least one Phase I clinical trial, more preferably at least one Phase II clinical, even more preferably at least one Phase III clinical trial, and most preferably approved by the FDA for at least one hematological or oncological indication. Examples of such inhibitors include, but are not limited to, Gefitinib, Erlotinib, Lapatinib, Canertinib, BMS- 599626 (AC-480), Neratinib, KRN-633, CEP-11981, Imatinib, Nilotinib, Dasatinib, AZM- 475271, CP-724714, TAK-165, Sunitinib, Vatalanib, CP-547632, Vandetanib, Bosutinib, Lestaurtinib, Tandutinib, Midostaurin, Enzastaurin, AEE-788, Pazopanib, Axitinib, Motasenib, OSI-930, Cediranib, KRN-951, Dovitinib, Seliciclib, SNS-032, PD-0332991, MKC-I (Ro- 317453; R-440), Sorafenib, ABT-869, Brivanib (BMS-582664), SU-14813, Telatinib, SU- 6668, (TSU-68), L-21649, MLN-8054, AEW-541, and PD-0325901.
In some embodiments, the inhibitor of the present invention is used in combination with an immunotherapeutic agent. The term "immunotherapeutic agent," as used herein, refers to a compound, composition or treatment that indirectly or directly enhances, stimulates or increases the body's immune response against cancer cells and/or that decreases the side effects of other anticancer therapies. Immunotherapy is thus a therapy that directly or indirectly stimulates or enhances the immune system's responses to cancer cells and/or lessens the side effects that may have been caused by other anti-cancer agents. Immunotherapy is also referred to in the art as immunologic therapy, biological therapy biological response modifier therapy and biotherapy. Examples of common immunotherapeutic agents known in the art include, but are not limited to, cytokines, cancer vaccines, monoclonal antibodies and non-cytokine adjuvants. Alternatively the immunotherapeutic treatment may consist of administering the subject with an amount of immune cells (T cells, NK, cells, dendritic cells, B cells...). Immunotherapeutic agents can be non-specific, i.e. boost the immune system generally so that the human body becomes more effective in fighting the growth and/or spread of cancer cells, or they can be specific, i.e. targeted to the cancer cells themselves immunotherapy regimens may combine the use of non-specific and specific immunotherapeutic agents. Non-specific immunotherapeutic agents are substances that stimulate or indirectly improve the immune system. Non-specific immunotherapeutic agents have been used alone as a main therapy for the treatment of cancer, as well as in addition to a main therapy, in which case the non-specific immunotherapeutic agent functions as an adjuvant to enhance the effectiveness of other therapies (e.g. cancer vaccines). Non-specific immunotherapeutic agents can also function in this latter context to reduce the side effects of other therapies, for example, bone marrow suppression induced by certain chemotherapeutic agents. Non-specific immunotherapeutic agents can act on key immune system cells and cause secondary responses, such as increased production of cytokines and immunoglobulins. Alternatively, the agents can themselves comprise cytokines. Nonspecific immunotherapeutic agents are generally classified as cytokines or non-cytokine adjuvants. A number of cytokines have found application in the treatment of cancer either as general non-specific immunotherapies designed to boost the immune system, or as adjuvants provided with other therapies. Suitable cytokines include, but are not limited to, interferons, interleukins and colony-stimulating factors. Interferons (IFNs) contemplated by the present invention include the common types of IFNs, IFN-alpha (IFN-a), IFN-beta (IFN-P) and IFN- gamma (IFN-y). IFNs can act directly on cancer cells, for example, by slowing their growth, promoting their development into cells with more normal behavior and/or increasing their production of antigens thus making the cancer cells easier for the immune system to recognise and destroy. IFNs can also act indirectly on cancer cells, for example, by slowing down angiogenesis, boosting the immune system and/or stimulating natural killer (NK) cells, T cells and macrophages. Recombinant IFN-alpha is available commercially as Roferon (Roche Pharmaceuticals) and Intron A (Schering Corporation). Interleukins contemplated by the present invention include IL-2, IL-4, IL-11 and IL-12. Examples of commercially available recombinant interleukins include Proleukin® (IL-2; Chiron Corporation) and Neumega® (IL- 12; Wyeth Pharmaceuticals). Zymogenetics, Inc. (Seattle, Wash.) is currently testing a recombinant form of IL-21, which is also contemplated for use in the combinations of the present invention. Colony-stimulating factors (CSFs) contemplated by the present invention include granulocyte colony stimulating factor (G-CSF or filgrastim), granulocyte-macrophage colony stimulating factor (GM-CSF or sargramostim) and erythropoietin (epoetin alfa, darbepoietin). Treatment with one or more growth factors can help to stimulate the generation of new blood cells in subjects undergoing traditional chemotherapy. Accordingly, treatment with CSFs can be helpful in decreasing the side effects associated with chemotherapy and can allow for higher doses of chemotherapeutic agents to be used. Various-recombinant colony stimulating factors are available commercially, for example, Neupogen® (G-CSF; Amgen), Neulasta (pelfilgrastim; Amgen), Leukine (GM-CSF; Berlex), Procrit (erythropoietin; Ortho Biotech), Epogen (erythropoietin; Amgen), Arnesp (erytropoietin). Combination compositions and combination administration methods of the present invention may also involve "whole cell" and "adoptive" immunotherapy methods. For instance, such methods may comprise infusion or re-infusion of immune system cells (for instance tumor-infiltrating lymphocytes (TILs), such as CC2+ and/or CD8+ T cells (for instance T cells expanded with tumor-specific antigens and/or genetic enhancements), antibody-expressing B cells or other antibody-producing or -presenting cells, dendritic cells (e.g., dendritic cells cultured with a DC-expanding agent such as GM-CSF and/or Flt3-L, and/or tumor-associated antigen-loaded dendritic cells), anti -tumor NK cells, so- called hybrid cells, or combinations thereof. Cell lysates may also be useful in such methods and compositions. Cellular "vaccines" in clinical trials that may be useful in such aspects include Canvaxin™, APC-8015 (Dendreon), HSPPC-96 (Antigenics), and Melacine® cell lysates. Antigens shed from cancer cells, and mixtures thereof (see for instance Bystryn et al., Clinical Cancer Research Vol. 7, 1882-1887, July 2001), optionally admixed with adjuvants such as alum, may also be components in such methods and combination compositions.
Particularly, the inhibitor of the invention may be used in combination with another antibody like the antibody Ha22-2 (Seattle Genetics) described in the patent application WO2012047724.
In some embodiments, the inhibitor of the present invention is used in combination with an antibody that is specific for a costimulatory molecule. Examples of antibodies that are specific for a costimulatory molecule include but are not limited to anti-CTLA4 antibodies (e.g. Ipilimumab), anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-TIMP3 antibodies, anti-LAG3 antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies or anti-B7H6 antibodies.
Thus the present invention also relates to a combination of TGFBI inhibitor and an anti- PD-1 antibody for use in the treatment of cachexia in a subject in need thereof.
Particularly, the cachexia is cancer cachexia. In some embodiments, the second agent is an agent that induces, via ADCC, the death of a cell expressing an antigen to which the second agent binds. In some embodiments, the agent is an antibody (e.g. of IgGl or IgG3 isotype) whose mode of action involves induction of ADCC toward a cell to which the antibody binds. NK cells have an important role in inducing ADCC and increased reactivity of NK cells can be directed to target cells through use of such a second agent. In some embodiments, the second agent is an antibody specific for a cell surface antigens, e.g., membrane antigens. In some embodiments, the second antibody is specific for a tumor antigen as described above (e.g., molecules specifically expressed by tumor cells), such as CD20, CD52, ErbB2 (or HER2/Neu), CD33, CD22, CD25, MUC-1, CEA, KDR, > VD3, etc., particularly lymphoma antigens (e.g., CD20). Accordingly, the present invention also provides methods to enhance the anti-tumor effect of monoclonal antibodies directed against tumor antigen(s). In the methods of the invention, ADCC function is specifically augmented, which in turn enhances target cell killing, by sequential administration of an antibody directed against one or more tumor antigens, and an antibody of the present invention.
In some embodiments, the inhibitor of the present invention is used in combination with radiotherapy. Radiotherapy may comprise radiation or associated administration of radiopharmaceuticals to a patient. The source of radiation may be either external or internal to the patient being treated (radiation treatment may, for example, be in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that may be used in practicing such methods include, e.g., radium, cesium-137, iridium-192, americium-241, gold- 198, cobalt-57, copper-67, technetium-99, iodide-123, iodide-131, and indium-ill.
In a particular embodiment, the inhibitor of the present invention is used in combination with an agonist of the ghrelin/growth hormone secretagogue receptor (GHSR).
Thus, the present invention also relates to a combination of a TGFBI inhibitor (particularly an antibody of the invention) and an agonist of the GHSR for use in the treatment of cachexia in a subject in need thereof.
Particularly, the cachexia is cancer cachexia.
As used herein, the term “Growth hormone secretagogue receptor” or “GHS-R” or “GHSR” also known as ghrelin receptor, denotes a G protein-coupled receptor that binds growth hormone secretagogues (GHSs), such as ghrelin, the "hunger hormone". The role of GHS-R is thought to be in regulating energy homeostasis and body weight. In the brain, they are most highly expressed in the hypothalamus, specifically the ventromedial nucleus and arcuate nucleus. GSH-Rs are also expressed in other areas of the brain, including the ventral tegmental area, hippocampus, and substantia nigra. Outside the central nervous system, too, GSH-Rs are also found in the liver, in skeletal muscle, and even in the heart.
Particularly the agonist of the GHSR can be the anamorelin, the adenosine, the alexamorelin, the capromorelin, the CP -464709, the corti statin- 14, the examorelin (hexarelin), the ghrelin (lenomorelin), the GHRP-1, the GHRP-3, the GHRP-4, the GHRP-5, the GHRP-6, the Ibutamoren (MK-677), the ipamorelin, the L-692,585, the LY-426410, the LY-444711, the macimorelin, the pralmorelin (GHRP-2), the relam orelin, the SM- 130,686, the tabimorelin or the ulimorelin.
In a particular embodiment, the invention relates to a combination of TGFBI inhibitor (particularly an antibody of the invention) and anamorelin for use in the treatment of cachexia in a subject in need thereof.
Another object of the invention relates to a kit of part comprising the inhibitor of the invention or the pharmaceutical composition of the invention and at least one therapeutic active agent as a combined preparation for simultaneous, separate or sequential use in the treatment of cachexia and particularly cancer cachexia.
As used herein, the term “simultaneous use” denotes the use of the antibody and at least one active agent occurring at the same time.
As used herein, the term “separate use” denotes the use of the antibody and at least one active agent not occurring at the same time.
As used herein, the term “sequential use” denotes the use of the antibody and at least one active agent occurring by following an order.
The invention will be further illustrated by the following figures and examples. However, the examples and figures should not be interpreted in any way as limiting the scope of the present invention.
FIGURES
Figure 1 : High stromal TGFBI positivity in primary tumors was associated with cachexia in advanced CRC patients.
(A) Relationship of cachexic conditions and TGFBI positivity area in the primary tumor lesion. (B) Relationship of tumor size (small <50 mm; large >50 mm) and TGFBI positivity area in the primary tumor lesion. TGFBI positive area percentage was evaluated on three fields of view (FOV) from each specimen. *, P value <0.05. ns, not significant. Figure 2 : Development of antibodies against Tgfbi.
(A) Anti-Tgfbi antibody clones against murine recombinant Tgfbi protein determined by ELISA. The binding affinity ratio is calculated as an absorbance ratio relative to the control (IgG). (B) Left panel: FACS histogram of VivoTag® 680 XL labeled antibodies (5 ng/mL) binding to murine cell lines J774.1 and Colon26 with Tgfbi expression. Isotype IgG antibody was used as a control. Data are representative of two independent experiments. Right panel: Bar graphs show quantitation of Tgfbi positivie percentage on the cells by the labeled antibodies. (C) Identification of the binding peptides against the Tgfbi antibodies. Y axis: ELISA absorbance. X-axis: Human TGFBI schema and target peptide region. (D) ELISA determined the binding affinity of anti-Tgfbi antibody clones against human recombinant TGFBI protein. The binding affinity ratio was calculated as an absorbance ratio relative to the control (IgG). (E) Western blot and ELISA of Tgfbi in conditioned medium from murine cell lines. Ponceau red staining was used to evaluate the protein transfer efficiency to the membrane. All data are presented as mean ±SD. *, P value <0.05; **, P value <0.01; ***, P value <0.001; ****, P value <0.0001. Ab, antibody.
Figure 3 : Tgfbi antibodies prevent cancer cachexia and prolong the survival in cachexia model mice.
(A) Alteration of body weight, wire hanging time, and food intake in the Colon26 cachexic model mice treated by Isotype IgG or Tgfbi antibodies (A6 and C9) at indicated days. All the data is expressed as mean ± SD. (B) Mouse appearance, tumor volume, and proliferating Ki67 positive tumor cell number of the Colon26 cachexic model mice treated by Isotype IgG or Tgfbi antibodies. (C) Representative HE images of the tumor from Colon26 cachexic model mice treated by Isotype IgG or Tgfbi antibodies on day 17. Two marginal areas in one tumor were evaluated to identify tumor invasion into the surrounding non-cancerous area (Black arrow). The right lower bar graph shows each group's frequency of invasive and non-invasive tumors. Scale bar 100 pm. Magnification x200. Insets showing low magnification views xlOO. (D) Plasma glucose and albumin levels of the Colon26 cachexic model mice treated by Isotype IgG or Tgfbi antibodies on day 17. The dashed line indicates the reference ranges of healthy mice. (E) This figure shows the Kaplan-Meier curve for overall survival in the Colon26 cachexic model mice treated with isotype IgG or Tgfbi antibodies. The foot table shows the P value, hazard ratio (HR), and 95% confidence interval for each group (A6 and C9) compared to the IgG. (F) Plasma biochemical profile of the Colon26 cachexic model mice treated with isotype IgG or Tgfbi antibodies (day 17). All data are presented as mean ±SD. *, P value <0.05; **, P value <0.01; ***, P value <0.001; ****, P value <0.0001. ns, not-significant. Ab, antibody
Figure 4 : Tgfbi antibody recovered the muscle wasting and lipolysis in cachexia model mice to the level of control mice without tumor.
(A) Images and weight of quadriceps and gastrocnemius muscles in the control mice and Colon26 cachexic model mice treated by Isotype IgG or Tgfbi antibody (C9) at day 17. (B) Images and weight of visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) in the control mice and Colon26 cachexic model mice treated by Isotype IgG or Tgfbi antibody at day 17. (C) Left panel: Representative images of muscle fibers in the control mice and Colon26 cachexic model mice treated by Isotype IgG or Tgfbi antibody at day 17. Right panel: The bar graph shows the relation between muscle fiber area and frequency in the model mice. Scale bar 100 pm. Magnification x400. The muscle fiber area was quantified by four independent images using Image J. (D) The bar graphs show the mRNA expression levels of muscle degradation marker Trim63 (known as Murfl) and Fbxo32 (known as Atrogin) in the control mice and Colon26 cachexic model mice treated by Isotype IgG or Tgfbi antibody at day 17. (E) Left panel: Representative images of subcutaneous adipose tissues in the control mice and Colon26 cachexic model mice treated by Isotype IgG or Tgfbi antibody at day 17. Right panel: The bar graph shows the relation between adipocyte area and frequency in the model mice. Scale bar 100 pm. Magnification x200. The adipocyte cross-sectional area was quantified by eight independent images using Image J. (F) Left panel: Representative IHC images of Ucpl expression in subcutaneous adipose tissues in the control mice and Colon26 cachexic model mice treated by Isotype IgG or Tgfbi antibody at day 17. Right panel: The bar graph shows the quantitative evaluation of Ucpl expression in subcutaneous adipocyte area in the model mice. Scale bar 100 pm. Magnification x200. Ucpl expression was quantified by four independent images using Image J. (G) Alteration of body weight, wire hanging time, and food intake in the control mice and the Colon26 cachexic model mice treated by Isotype IgG or Tgfbi antibody C9 at indicated days. (H) Upper panel: Representative images of abdominal MRI in the control mice and Colon26 cachexic model mice treated by Isotype IgG or Tgfbi antibody at day 17. Lower panel: The bar graph shows the fat volume (Total, VAT, and SAT) in each group. (I) This figure shows the microscopic and macroscopic examination of vital organs in the control mice and Colon26 cachexic model mice treated by Isotype IgG or Tgfbi antibody at day 17. Bar graphs show the weight of the organs. Data are presented as mean ±SD. *, P value <0.05; **, P value <0.01; ***, P value <0.001; ****, P value <0.0001. ns, not-significant. Ab, antibody. Visceral adipose tissue, VAT. Subcutaneous adipose tissue, SAT.
Figure 5 : Tgfbi antibodies inhibit macrophage-derived cytokines.
(A) The bar graph shows 11-6, Tnf-a, and Tgfbi levels in plasma samples from the Colon26 cachexic model mice treated with isotype IgG or Tgfbi antibodies (A6 and C9) (day 17), as determined by the ELISA. (B) Left panel: Representative IHC images show the intratumoral Cd86+ and Cd206+ macrophages in the Colon26 cachexic model mice treated with isotype IgG or Tgfbi antibodies (day 17). Scale bar 100 pm. Magnification x200. Right panel: The bar graphs show the Cd86+ and Cd206+ macrophage numbers per field of view (FOV), as quantified by the Hybrid cell count system. (C) Left panel: Multicolor immunofluorescent images of macrophage markers F4/80 (green) and 11-6 (red) in the tumor samples of the Colon26 cachexic model mice treated with isotype IgG or Tgfbi antibodies (day 17). Scale bar 100 pm. Magnification x400. Right panel: The bar graph shows the 11-6 and F4/80 double-positive macrophage percentages among the F4/80+ cell numbers as total macrophages in nine fields of view (FOV). (D & E) Left panel: Representative IHC images show the Cd86+ and Cd206+ macrophages in the subcutaneous adipose tissues (D) and muscle tissues (E) from the control (no tumor) mice and the Colon26 cachexic model mice treated with isotype IgG or Tgfbi antibody C9 (day 17). Scale bar 100 pm. Magnification x200. Right panel: The bar graphs show the Cd86+ and Cd206+ macrophage number per field of view (FOV) in the subcutaneous adipose tissues (D) and muscle tissues (E), as quantified by the Hybrid cell counter system. High-power view images show the infiltrating macrophages. Scale bar 20 pm. Cd86+: pro- inflammatory macrophage subset marker, Cd206+: anti-inflammatory macrophage subset marker.
All data are presented as mean ±SD. *, P value <0.05; **, P value <0.01; ***, P value <0.001; ****, p value <0.0001. ns, not-significant. Ab, antibody.
Figure 6 : Tgfbi can induce the macrophage-derived cytokine production via integrins/pFAK signaling.
(A) The anti -Tgfbi antibody treatment repressed secretion of 11-6 and Tnf-a proteins in the culture medium from murine macrophage cell line J774.1 treated with isotype IgG or Tgfbi antibodies (A6 and C9) (10 pg/ml, 48 h). Each dot represents replicates of 4 independent ELISA experiments. (B) The recombinant Tgfbi (rec. Tgfbi) increased secretion of 11-6 and Tnf-a proteins in the culture medium from murine macrophage cell line J774.1. Each dot represents replicates of 5 independent ELISA experiments. (C) ELISA determined the secreted 11-6 and Tnf-a protein levels in the culture medium from the J774.1 cells treated with or without rec.Tgfbi(100 ng/mL) and cilengitide (10 pM) for 48 h. Each dot shows the replicates from 4 independent experiments. (D) The integrins siRNAs (siltgav and siltgbS) suppressed macrophage-derived cytokine production induced by rec.Tgfbi treatment. ELISA determined the levels of secreted 11-6 and Tnf-a protein in the culture medium of integrins-suppressed J774.1 cells after 48 h of treatment with or without rec.Tgfbi (100 ng/mL). Each dot shows the replicates from two independent experiments. (E) FACS histogram of VivoTag® 680 XL labeled antibodies (A6 and C9, 5 ng/mL) binding to the integrins-suppressed J774.1 cells with or without rec.Tgfbi treatment (100 ng/ml, 30 min). Left panel: Binding ability of the labeled Tgfbi antibodies to exogenous Tgfbi on J774.1 was depending on the integrins. Right panel: The addition of exogenous rec.Tgfbi enhanced the binding ability of the labeled Tgfbi antibodies to the Tgfbi on the cell surface (the siNT control group). However, the binding ability was less enhanced in the integrins siRNA groups. Isotype IgG antibody was used as a control. (F) The basal protein expression of 11-6 and Tnf-a in culture medium from NIH3T3, Colon26 and J774.1 cells was determined by ELISA. (G) The anti -Tgfbi antibody treatment repressed the mRNA expression of 11-6 and Tnf-a in J774.1 cells treated with isotype IgG or Tgfbi antibodies (10 pg/ml, 48 h) . Each dot represents replicates of 4 independent experiments. (H) The recombinant Tgfbi (rec.Tgfbi, 100 ng/ml, 48 h) increased the mRNA expression of 11-6 and Tnf-a in J774.1 cells. Each dot represents replicates of 5 independent experiments. (I) The integrin inhibitor cilengitide inhibited rec.Tgfbi-induced 11-6 and Tnf-a expression in J774.1 cells (rec.Tgfbi, 100 ng/ml, 48 h) (cilengitide, 10 pM, 48h). Each dot shows the replicates from two independent experiments. (J) The basal mRNA expression of Itgav, Itgb3, Itgb5 in NIH3T3, Colon26, and J774.1 cells was determined by qRT-PCR. (K) The suppression of Itgav and Itgb5 mRNA expressions in J774.1 cells treated with specific integrin siRNAs was validated by qRT-PCR.
All data are presented as mean ±SD. *, P value <0.05; **, P value <0.01; ***, P value <0.001; ****, p value <0.0001. ns, not-significant. The expression of murine 18S rRNA was used as internal control for qRT-PCR experiments.
Figure 7: Combination treatment of anti-TGFBI (antibody clone C9) with immune-checkpoint therapy anti PD-1.
(A) Kaplan-Meier curve for overall survival in the Colon26 cachexic model mice treated with isotype IgG, Tgfbi antibody (C9), anti-Pdl antibody, and/or combination. Tumors were resected at day 17. The foot table shows the P value, hazard ratio (HR), and 95% confidence interval for each group (A6 and C9) compared to the IgG. (B) Quantification of tumor volume on the day of tumor excision (day 17).
(C) Quantification of CD8+ T-cell numbers per field of view (FOV) in the tumor at day 17, as quantified by the Hybrid cell count system.
(D) Body weight of the Colon26 cachexic model mice treated by isotype IgG, Tgfbi antibody (C9), anti-Pdl antibody, and/or combination at tumor excision (day 17). All the data is expressed as mean ± SD.
(E) Estimation of muscle strength using wire-hanging test at day 17 (left panel), and wire-hanging time of animals with tumors greater than 75 mm3 (right panel).
Figure 8: Comparison of anti-TGFBI (antibody clone C9) with state of art cachexia treatment, Ghrelin agonist Anamorelin.
(A) Kaplan-Meier curve for overall survival in the Colon26 cachexic model mice treated with control, Anamorelin, and Tgfbi antibody (C9). Survival was observed for 4 weeks after tumor engraftment. The foot table shows the P value, hazard ratio (HR), and 95% confidence interval for each group comparison.
(B) Analysis of tumor volume (left panel) and tumor weight (right panel) at sacrifice.
(C) Alteration of body weight (left panel), muscle strength by wire hanging time (middle panel), and food intake (right panel) in the Colon26 cachexic model mice treated by with control, Anamorelin, and Tgfbi antibody (C9) at indicated days. All the data is expressed as mean ± SD.
(D) Quantification of plasma glucose and albumin levels of the Colon26 cachexic model mice treated with control, Anamorelin and Tgfbi antibody (C9) at the time of sacrifice.
(E) Percentage of white blood cell fractions (lymphocyte, monocyte, and neutrophils) in the peripheral blood of the Colon26 cachexic model mice treated with control, Anamorelin, and Tgfbi antibody (C9) at the time of sacrifice.
(F) Plasma levels of 11-6, Tnf-a, and Tgfbi levels in plasma samples from the Colon26 cachexic model mice treated with control, Anamorelin, and Tgfbi antibody (C9) at the time of sacrifice, as determined by the ELISA.
(G) Weight of different adipose tissues (SAT, VAT, BAT), muscles (GA, Quad) as well as spleen and liver (left panel) and estimated total weight of adipose and muscle tissues (right panel) in the Colon26 cachexic model mice treated with control, Anamorelin, and Tgfbi antibody (C9) at the time of sacrifice.
Figure 9: Combination treatment of anti-TGFBI (antibody clones A6 and C9) with state of art cancer treatment (5-FU chemotherapy). (A) Schema of the treatment plan (anti-TGFBI Ab and tumor resection) against the cachexia model mice. Female Balb/c mice were inoculated with the Colon26-luc cells on day 0. The mice were treated with IgG control or anti-TGFBI Abs (A6 and C9 clones) from day 1 (2 times a week, 200 pg/body, intraperitoneal injection), and the tumors were surgically resected on day 17. After the tumor resection, the animals were recovered from surgery for a week and followed by an antineoplastic agent 5-FU (30mg/kg, intraperitoneal injection on days 23 and 26) in adjuvant chemotherapy setting and followed up to evaluate survival and tumor progression.
(B) Kaplan-Meier curve for overall survival after adjuvant chemotherapy. The foot table shows the P value, hazard ratio (HR), and 95% confidence interval for each group comparison.
Figure 10: Relationship between preoperative serum TGFBI levels and cachexia in 69 pancreatic cancer patients.
High serum TGFBI levels are associated with cachexia in pancreatic cancer patients. ***P < 0.001, Mann-Whitney U test.
Figure 11: Validation of the therapeutic effect of TGFBI antibody in a pancreatic cancer patient-derived xenograft (PDX) cachexia model.
(A) Schematic representation of the TGFBI antibody (Ab) treatment regimen in the pancreatic cancer PDX (PC-PDX)-induced cachexia model. Ten days after PDX engraftment, mice were treated with either isotype control IgG (n = 6) or TGFBI Ab (n = 6) twice per week (200 pg/body, intraperitoneally) for 35 days. Non-tumor-bearing BALB/c-nu/nu mice (n = 3) were used as healthy controls.
(B) Wire hanging time in PC-PDX-induced cachexia model mice treated with isotype IgG or TGFBI Ab, measured at the indicated time points. The dotted line represents the maximum measurement limit (180 seconds).
(C) Body weight and food intake in PC-PDX-induced cachexia model mice treated with isotype IgG or TGFBI Ab at the indicated time points.
(D) Tumor volume and tumor weight in PC-PDX mice after 35 days of treatment with isotype IgG or TGFBI Ab.
All data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant. Ab, antibody; PC-PDX, pancreatic cancer patient-derived xenograft; i.p., intraperitoneal injection.
Figure 12: Validation of the cachexia treatment effect of TGFBI Ab using pancreatic cancer patients derived xenograft tumor model. Top panel: ELISA quantifications of 11-6, Tnf-a and Tgfbi levels in plasma samples from the no tumor-bearing control mice and PC PDX cachexic model mice treated by isotype IgG or Tgfbi Ab on day 45. Bottom left panel: Frequency of the muscle’s cross-sectional area in the model mice. Bottom right panel: Western blot analysis of muscle degradation marker MurFl and downstream of 11-6, Stat-3 activation in the TA muscles from PC-PDX animal model mice treated by isotype IgG or Tgfbi Ab on day 45.
All data are presented as mean ± SD . * , P value <0.05 ; * * , P value <0.01 ; P value <0.001; ****, P value <0.0001; ns, not-significant; Ab, antibody; PC PDX, pancreatic cancer patient- derived xenograft; i.p., intraperitoneal injection.
Figure 13 : Comparison of the cachexia treatment effect of TGFBI Ab and TGFBI Ab-LALAPG using colon26 cancer cachexia model.
Schema of the Tgfbi Ab treatment plan against Colon26 cachexic model mice. Measurement of food intake, body weight changes and wire hanging time in the Colon26 cachexic model mice treated by isotype IgG or Tgfbi Ab at indicated timepoint. Tumor volume and tumor weight of the Colon26 tumors at the end of treatment by isotype IgG or Tgfbi Ab or Tgfbi Ab-LALAPG.
All data are presented as mean ± SD . * , P value <0.05 ; * * , P value <0.01 ; P value <0.001; ****, P value <0.0001; ns, not-significant
Figure 14 : Comparison of the cachexia treatment effect of TGFBI Ab and TGFBI Ab-LALAPG using colon26 cancer cachexia model.
ELISA quantifications of 11-6, Tnf-a and Tgfbi levels in plasma samples from the Colon26 cachexic model mice treated by isotype IgG or Tgfbi Ab or Tgfbi Ab-LALAPG at the end of the experiment. Muscle weight of the Colon26 cachexic model mice treated by isotype IgG or Tgfbi Ab or Tgfbi Ab-LALAPG at the end of the experiment. Western blot analysis of muscle degradation marker MurFl and downstream of 11-6, Stat-3 activation in the TA muscles from PC-PDX animal model mice treated by isotype IgG or Tgfbi Ab or Tgfbi Ab-LALAPG. Representative animals from each group was used in the WB analysis.
All data are presented as mean ± SD . * , P value <0.05 ; * * , P value <0.01 ; P value <0.001; ****, P value <0.0001; ns, not-significant
Figure 15 : Western blot analysis of muscle degradation markers in colon26 cancer cachexia model treated by TGFBI Ab.
Western blot analysis of muscle degradation marker MurFl and downstream of 11-6, Stat-3 activation in the TA muscles from Colon26 cancer cachexic model mice treated by isotype IgG or Tgfbi Ab on day 17. EXAMPLES
Example 1: Use of anti- Tgfbi antibodies in the treatment of cachexia and cancer cachexia
Material & Methods
Patient samples
This study included 76 patients with advanced primary CRC who underwent radical surgery at Gunma University Hospital within a time period from July 2013 to February 2020. Exclusion criteria were patients who received neo-adjuvant chemotherapy or radiation therapy and patients who did not undergo radical resection involving distant metastasis. Clinical data, including patient characteristics, were described in Supplementary Table 1. Patients with body weight loss >5% during the last six months, body weight loss >2% with sarcopenia, and body weight loss >2% with BMI<20 kg/m2 were defined as cachexia conditions (Fearon K et al., 2011, Lancet Oncol). This study conformed to the tenets of the Declaration of Helsinki and was approved by the Institutional Review Board for Clinical Research of Gunma University Hospital (approval number: HS2021-020). Patient consent was obtained using the opt-out method.
Tgfbi anibody selection by phage display
Phage antibody expression library Husc I (Caucheteur D et al., Methods Mol Biol) was probed with recombinant human TGFBI to isolate polyclonal human antibodies in scFv format. The binding specificty of these antibody fragments was tested against immobilized TGFBI using ELISA. Following the phage-enrichment by four rounds of bio-panning, seven monoclonal antibodies were selected based on their binding to recombinant protein. Complementarity-determining regions (CDR) of the seven anti-TGFBI scFv were sequenced using Next Generation sequencing. Following the comparison of all CDR sequences, 2 anti- TGFBI scFv (hereafter called A6 and C9) were selected based on their diversity and sequence redundance with other scFv clones. Next, the anti-TGFBI scFv were cloned into murine IgG2a framework and outsourced for bioproduction to external company (Evitria AG, Schlieren, Switzerland). All antibody preparations had purity > 90%, were sterile and endotoxin free.
Biacore Affinity Measurement
The binding of anti-Tgfbi antibodies was evaluated by surface plasmon resonance (SPR) analysis using a BIAcore X-100 apparatus (Cytiva, Marlborough, MA, USA). Human TGFBI and murine Tgfbi (20 pg/ml in 10 mM sodium acetate, pH 4.0) were allowed to react with a flow cell of a CM5 sensor chip previously activated with a mixture of 0.2 M N-ethyl-N’-(3- dimethylaminopropyl)-carbodiimide hydrochloride and 0.05 M N-hydroxysuccinimide (35 pl, flow rate 10 pl/min). After ligand immobilization, matrix neutralization was performed with 1.0 M ethanolamine (pH 8.5) (35 pl, flow rate 10 pl/min) and activated/deactivated dextran was used as a reference (control) system. Increasing concentrations of A6 and C9 anti-Ttgfbi antibodies (from 18.75 to 600 nM) were injected over the Tgfbi-coated sensor chip and the response was recorded by tracking the SPR intensity change upon binding progression. Injection lasted for 2 min (flow rate 10 pl/min) to allow the association with immobilized Tgfbi and was followed by 10 min of dissociation; each run was performed in HBS-EP buffer (Cytiva) and the sensor chip was regenerated with glycine, pH 2. The equilibrium (plateau) values of the SPR sensorgrams were used to build the binding isotherms after normalization. Binding isotherm points were fitted with the Langmuir equation for monovalent binding to evaluate the mass surface dissociation constant, Kd. Steady-state binding affinity was measured at pH 6.0. The best-fitting procedure was performed with the SigmaPlot 11.0 software package (Systat Software Inc.).
Cell culture
The NIH3T3, Colon26-luc, and J774.1 cell lines were from the Japanese Collection of Research Bioresources Cell Bank. All cell lines were cultured in RPML1640 supplemented with 10% fetal bovine serum (FBS) (Gibco, Invitrogen, CA, USA) and 1% penicillin/streptomycin (Wako, Osaka, Japan) at 37 °C in 5% CO2. Cell lines were tested for mycoplasma contamination using Mycostrip (Invivogen, California, USA). For in vitro treatment, J774.1 cells were seeded at 70% confluency and adhered to overnight. After rinsing two times with PBS, cells were incubated with following conditions as follows; anti-Tgfbi Ab (A6 and C9) at a concentration 10 pg/mL for 48 h; murine recombinant Tgfbi at a concentration 100 ng/mL for 1 h and 48 h; established integrin inhibitor cilengitide at a concentration 10 nM for 30 min prior to the recombinant Tgfbi treatement. Conditioned medium was prepared from indicated cell lines. Briefly, cells were plated at 70% confluency and adhered for one day. After washing twice with PBS, serum-free (NIH3T3 cells 0.1% FBS) medium was added and cultured for 48 h. Following this, the medium was collected and centrifuged for 5 min at xl50 g to remove the cell debris. The conditioned medium was further concentrated 10 times with centrifugation using an Amicon column (10 kDa, Merck Millipore, Germany). siRNA transfection
J774.1 and Colon26-luc cells were suspended at a density of 1.0 * 106 cells in 100 pL of Opti-MEM I Reduced Serum Media (Thermo Fisher Scientific) and then mixed with specific siRNAs (siTgfbi cocktail/siltgav cocktail/siltgb5 cocktail) or non-target control siRNA (siNT) (Ajinomoto Bio-Pharma, Japan, see supplementary table). Transfection was performed using a CUY21 EDIT II electroporator (BEX, Tokyo, Japan) according to the manufacture's protocol. After 48 h incubation, the cells were used for further analysis.
FACS analyses for detecting the labeled Tgfbi antibody and immune cell markers
J774.1 and Colon26-luc cells were transfected with the siRNAs mentioned above. After 48 h, cells were collected and co-incubated with antibody (5 ng/ml, control IgG and Tgfbi Ab labeled by VivoTag680) at room temperature for 30 min. The cells binding to the labeled antibodies were analyzed using the FACSVerse and FACSSuite software (BD Biosciences, New Jersey, USA). The dissected tumors were minced and incubated with Dri Tumor & Tissue Dissociation Reagent (BD, USA) in RPMI-1640 for 30 min at 37 °C under shaking. Following this, the mixture was filtrated using 100 pm size cell strainers. Mononuclear cells were isolated by gradient centrifugation using Percoll according to the manufacturer’s protocol, and centrifugation was gradually performed at 2200 rpm for 20 min. Following the washing, cell viability and cell numbers were calculated using trypan blue staining and cell counter. The single-cell suspensions were first blocked with murine Fc blocking buffer for 15 min at room temperature, then stained with fluorescently labeled antibodies against CD45, CD3, CD4, CD8, CDllb, F4/80, CD86, and CD206 for 30 min at 4°C prior to flow cytometry analysis (see the supplementary table). Flow-cytometric data were analyzed using the FACSVerse and FACSSuite software (BD Biosciences, New Jersey, USA).
Establishment of cachexia model mice
Colon26-luc (5x104 cells/lOOul in PBS) were subcutaneously injected into the right flank of the animal under general anesthesia (2.0% Isoflurane given by inhalation). Anti-Tgfbi antibodies and respective control isotype-IgG2a were administered via intraperitoneal injection at 200 pg/body per injection and given two times a week (total five times treatment). Tumor growth was monitored physically with a caliper. Tumor volume was calculated as Tv=(W2xL)/2 mm3. Tumors were surgically removed on day 17 after tumor inoculation and continued to be monitored for further survival. Tumor growth, recurrence, and metastasis were monitored with an in vivo luciferase assay.
Cachexia evaluation
Animal body weights were measured at the beginning of the experiment (day 0) and monitored two times a week. Wire hanging tests were used to evaluate the animal muscle function (strength/ wasting) according to the previous report (Caucheteur D et al., 2018, Methods Mol Biol). Briefly, mice were hung on 30 cm long and 3 mm diameter metal wire with forelimbs (vertically 30 cm above from bedding). Duration to fall was recorded twice weekly. When mice hung from the wire for 180 seconds, the measurement was stopped and recorded. Food intake was measured twice weekly based on the leftover food amount. Surgical procedures and blood sample collection were performed under general anesthesia. Mice were sacrificed at indicated end-points (days 9-17) of the proposed experiments under deep anesthesia (5.0% isoflurane given by inhalation). Internal organs, fat depots (visceral, subcutaneous, and interscapular), muscles (quadriceps and gastrocnemius), and tumors were dissected, weighed, and subjected to further analysis. The blood was collected in a K2EDTA-containing tube (Greiner, Kremsmunster, Austria), and plasma samples were prepared with refrigerated centrifugation at 3000 rpm for 15 min. Plasma (non-fastened) biochemical parameters were analyzed with a MultiRotor I Preventive Care Panel using Vetscan (Zoetis, New Jersey, USA).
In vivo study of cachexia
Female BALB/cAJcl mice at 8 weeks old were used in all animal experiments (CLEA, Japan). Animal experiments were approved by the Institutional Animal Care and Use Committee of Gunma University (Approval# 23-001). Briefly, Colon26-luc (5x104 cells/lOOul in PBS) were subcutaneously injected into the right flank of the animal under general anesthesia. Anti-Tgfbi antibodies and respective control isotype-IgG2a were administered via intraperitoneal injection at 200 pg/body per injection, and given two times a week (total 5 times treatment). Cachexic status of the experimental animals were evlauted using body weight loss, muscle strength and food intake. Plasma (non-fastened) biochemical parameters were analyzed with a MultiRotor I Preventive Care Panel using Vetscan (Zoetis, New Jersey, USA). Magnetic resonance tomography (MRI) was performed to evaluate the fat and muscle composition of the animals. In vivo luciferase assay for detecting the Colon26-luc and biodistributions of the labeled antibodies were performed using the IVIS Lumina Series III system. Detailed methods is described in the supplementary material.
Magnetic Resonance Imaging (MRI)
MRI scans were performed on a Bruker ICON EOT MRI System. The mice were anesthetized with 2% isoflurane to ensure their well-being throughout the procedure. The parameter used in this study was based on the manufacturer's recommendations, and scans were performed on day 9 and day 17. Nine axial images of spin-echo T1 -weighted (TR/TR 291/6 ms; FOV 35x35 mm; slice thickness 1 mm; inter-slice gap 1.5 mm) were obtained with the abdominal coil starting from the L3 area. The image's resolution was 3,216x2,136 pixels and 16-bit, and the image type was subsequently changed to 8-bit and de-noised by subtracting a value of 20. The volume of visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) were both assessed by the ImageJ software (fat volume = fat image area (mm2) x (1.5 mm (inter-slice gap)+ 1 mm (slice thickness)(mm3)/body weight (g). Biodistributions of the labeled antibodies
Antibodies were labeled with a VivoTag® 680XL protein labeling kit (Perkin Elmer, Massachusettes, USA) according to the manufacturer’s instructions. In order to exclude the autofluorescence, mice were fed with iVid-neo (Cat #: 2403000, Oriental East Corp., Tokyo, Japan) alfalfa-free food starting a week before the experiment. Fifty pg VivoTag® 680XL labeled antibodies were intraperitoneally injected. Biodistribution of VivoTag® 680XL labeled antibodies was imaged with IVIS Lumina Series III system fluorescent setting with (Exc: 673nm/ Emm: 691nm) 48h after the antibody injection. Total Radiant Efficiency [p/s]/[pW/cm2] in the tumors was analyzed by Living Image Software (version 4.3.1, Xenogen, Alameda, USA).
In vivo luciferase assay
In vivo luminescence imaging was performed on an IVIS Lumina Series III (Perkin Elmer, Massachusettes, USA) system to monitor tumor growth. The mice were anesthetized and intraperitoneally injected with D-luciferin (0.5 mg/ml; 300pl) 5 min prior to the imaging (exposure time: 3 min 30 sec). Image display analyses were performed with Living Image Software (version 4.3.1, Xenogen, Alameda, USA).
Immunohistochemistry (IHC)
Human and mouse formalin-fixed paraffin-embedded (FFPE) tissue sections were sliced with 4 pm thickness, deparaffinated three times in Clear-plus (xylene substitute solution, FALMA, Japan) for 5 min, and hydrated in an ethanol gradient. Antigen retrieval was performed using the boiling method with ImmunoSaver solution (1 :200 in DDW, Nisshin EM, Tokyo, Japan) at 98°C-100°C for 45 min. Endogenous peroxidase blocking was carried out for 30 min with 0.3% H2O2 and 100% methanol followed by a gradient ethanol series. Following this, tissues were treated with Protein Block Serum-Free solution (Dako, Glostrup, Denmark) for 30 min at room temperature, preceding the primary antibody incubation. Primary antibodies and dilutions used for IHC are listed in the supplementary table. Sections were incubated with primary antibody overnight at 4°C. Following the antibody incubation, they were washed with PBS and then the tissues were incubated with corresponding secondary antibodies Histofine Simple Stain MAX PO (Multi for human tissues and Rabbit for mouse tissues) (Nichirei Biosciences Inc., Tokyo, Japan). The signal was revealed using 3,3 ’-diaminobenzidine tetrachlorhydrate dihydrate (Dojindo, Tokyo, Japan) in 5% H2O2. The slides were counterstained with hematoxylin, then air-dried and mounted with clear-plus followed by a Limo-mount solution (FALMA, Tokyo, Japan). Images of representative fields were taken using a BZX-700 light microscope (Keyence, Osaka, Japan). The positive staining area and positive cell number were analyzed using a Hybrid cell count system (Keyence, Osaka, Japan).
Multiplexed immunofluorescence
The sections were prepared, and endogenous peroxidase was blocked, as described above. Nonspecific binding sites were blocked by incubation with Protein Block Serum-Free Reagent for 30 min, and the sections were incubated overnight at 4 °C with the primary antibodies against macrophage marker F4/80 (Cell Signaling Technology, Massachusetts, USA) and murine 11-6 (PeproTech, NJ, USA). Multiplex covalent labeling (F4/40, TSA coumarin; murine 11-6, Opal 690) with tyramide signal amplification (Akoya Biosciences, MA, USA) was performed according to the manufacturer’s protocol. All sections were counterstained with hematoxylin and examined under an All-in-One BZ-X710 fluorescence microscope (Keyence, Osaka, Japan). The positive staining area and positive cell number were analyzed using a Hybrid cell count system (Keyence, Osaka, Japan). Twelve fields of view from each group were subjected to the quantification. The number of F4/80 positive macrophages and the number of 11-6 and F4/80 double positive macrophages were analyzed using a Hybrid cell count system (Keyence, Osaka, Japan).
Indirect enzyme-linked immunosorbent assay (ELISA)
The indirect ELISA was used to quantify Tgfbi, 11-6, and Tnf-a levels in plasma samples and the conditioned medium. Maxisorp 96-well plates (Thermo Fisher, USA) were coated with 100 pL of samples. Serial dilutions of murine recombinant Tgfbi (R&D, USA), 11-6, and Tnf-a (PeproTech, USA) were prepared in PBS as a calibration curve. The coating was performed overnight at 4°C on agitation. The wells were washed three times with PBS/Tween-20 and residual-binding sites were blocked with 4% (w/v) skim milk in PBS (200 pl/well) for 2 h at room temperature. After washing three times, 100 pL of primary antibodies were added and incubated for 2 h at room temperature (see supplementary table). The antibodies were detected by incubation with corresponding secondary HRP-conjugated antibody (1 h at RT) followed by incubation with enzymatic chromogenic substrate solution one Step-ULTRA-TMB (Thermo Scientific). Enzymatic reaction was terminated using 100 pl of sulphuric acid and optical density at 450 nm was measured.
Western blot analysis
Protein samples of concentrated condition media and cell lysates were suspended in RIPA buffer (Wako, Osaka, Japan). Laemmli buffer (Biorad, California, USA) was added to 10 pl of samples. After boiling for 5 minutes, identical amounts of proteins were loaded on 7.5% polyacrylamide gels. Proteins were separated with electrophoresis and transferred to nitrocellulose membranes at 90V for 90 min. After blocking in 5% skim milk or 5% BSA for 1 h, membranes were incubated (4 °C, overnight) with primary antibodies (see supplementary table). The membranes were washed by TBS-T three times, followed by corresponding secondary antibody incubation for 1 h at room temperature. Then, washing was repeated, and protein detection was visualized using chemiluminescence.
Cytokine array
Cytokines in the plasma samples (200pl) from 6 mice (No tumor, n=2; IgG, n=2; Tgfbi Ab C9, n=2) were analyzed using Proteome Profiler Mouse Cytokine array kits (R&D Systems, cat. #ARY006), according to the manufacturer's instructions. Cytokine abundance was calculated as the integrated average density of the spot replicates using the ImageJ software. Altered cytokines in each group were normalized by the negative control spot intensity.
Quantitative PCR
Total RNA was extracted from tissues and cell lines using the RNeasy Kit (Qiagen, Hilden, Germany), and the quantity of total RNA was measured using an nanodrop spectrophotometer. cDNA synthesis was performed using the ReverTra Ace qPCR RT Master Mix (Toyobo, Japan). Quantitative real-time RT-PCR was performed using the KAPA SYBR Fast qPCR Kit (KAPA biosystem, MA, USA) with a total reaction volume of 20 pL.The relative levels of candidate genes were calculated using the 2-ddCT method. All primer sequences used in our study are listed in supplementary table. Murine 18S rRNA was used to normalise the RNA input for all RT-PCR analyses.
Statistical analysis
Statistically significant differences were analyzed with the t-test and Wilcoxon test for continuous variables and the chi-squared test for categorical variables. Kaplan-Meier curves were generated for overall survival, and statistical significance was determined using the logrank test. All differences were statistically significant at the level of p < 0.05. All statistical analyses were performed using PrismlO (Graphpad, California, USA).
Results
High stromal TGFBI expression was associated with the cachexic condition in advanced CRC patients.
To clarify the impact of TGFBI expression on cachexia, we performed the IHC staining of TGFBI in the surgically resected tumor tissues from 76 advanced CRC patients. Consistent with the previous reports, TGFBI was mainly expressed in the tumor stroma (Data not shown). According to the cachexia diagnostic criteria, 61.8% (n=47) of the patients had cachexic conditions at the time of the tumor resection. Not surprisingly, patients with cachexia had significantly lower BMI than those of non-cachexic patients (Data not shown). As a result, we found that the percentage of the stromal TGFBI positive area was significantly higher in the cachexic patients than the non-cachexic patients (Fig. 1A). The TGFBI positive area was not associated with the tumor size (Fig. IB). To better understand these clinical observations, we developed an anti-Tgfbi antibody and investigated whether antibody -based targeting of Tgfbi can prevent cachexia in a cachexic CRC model.
Development of anti-Tgfbi monoclonal antibodies cross-react to human TGFBI and mouse Tgfbi.
Using a previously published synthetic phage-display library with different complementary determining regions (Caucheteur D et al., 2018, Methods Mol Biol), we generated fully human anti-Tgfbi antibodies hereafter named A6 and C9. To enable their repeated usage in the murine model, we next generated a chimeric antibody, where the human Fc portion of the antibody was replaced with the murine Fc, yielding a chimeric IgG2a antibody isotype. Next, surface plasmon resonance analysis revealed that both A6 and C9 bind to the human recombinant TGFBI in the range of 15-70 nM approximately (Fig. 2C, 2D & Data not shown), while both clones are cross-reactive to the murine Tgfbi protein in the range of 20-40 nM approximately (Fig. 2A& Data not shown). We next screened Tgfbi-expressing murine cell lines consisting of 3 colon cancer (Colon26, MC38, and CT26), a fibroblast (NH43T3), and a macrophage cell line J774.1 and found that Colon26 and J774.1 cells were abundantly secreting Tgfbi compared to the other cell lines (Fig. 2E). We further validated the binding affinity of these antibodies against endogenous Tgfbi in vitro and in vivo. Both A6 and C9 antibodies specifically detected murine Tgfbi (Fig. 2B) and human TGFBI (Data not shown) since when Tgfbi was suppressed with siRNA, the fluorescence signal was no longer detected (Fig. 2B & Data not shown). Furthermore, we evaluated the biodistribution of our antibody in vivo by labeling our antibodies and corresponding control antibody with VivoTag® 680 fluorophore and injecting them into the Colon26-luc tumor-bearing mice. Fluorescence imaging showed that anti-Tgfbi antibody was more accumulated in the subcutaneous Colon26 tumors compared to the isotype control (Data not shown). These data showed that our antibodies can specifically target the Tgfbi protein in its native condition, both in vitro and in vivo.
Tgfbi targeting antibody prevents cancer cachexia and prolongs survival in vivo. In order to investigate the potential anti-cachectic effects of Tgfbi targeting, we treated the Colon26-luc tumor-bearing mice with anti-Tgfbi A6 and C9 antibodies twice a week (total 5 times, intraperitoneal injection) (Data not shown). It is well reported that the Colon26 tumorbearing mice show representative cachexic symptoms such as body weight loss, muscle weakening, and food intake reduction (Bonetto A et al., 2016, J Vis Exp & Aulino P et al., 2010, BMC Cancer). Thus, we monitored these parameters during the treatment (Fig. 3 A). Two weeks after the tumor inoculation, the Colon26-luc tumor-bearing mice displayed cachexic symptoms in the control group, treated with isotype IgG. In sharp contrast, administration of anti-Tgfbi A6 and C9 antibodies resulted in retained body weight, stronger muscle endurance, and increased food intake (Fig. 3A). Interestingly, targeting Tgfbi had no significant effect on the tumor volume and proliferation as judged by the microscopic evaluation of Ki67 (Fig. 3B & Data not shown). While the tumor proliferation was not affected, further histological analysis confirmed that tumor invasiveness was reduced in the Tgfbi treatment groups compared to the IgG control (Fig. 3C). This observation was consistent with previous reports (Suriben R et al., 2020, Nat Med & Queiroz A.L et al., 2022, Nat Commuri). As shown in the Fig. 3C, tumor borders were well delineated from fat and muscle tissue in the anti-Tgfbi treatment condition, while muscular invasion was readily observed in the control group. To further explore the molecular facets of the anti-cachexic phenotype, we assessed vital biochemical parameters in the plasma of the tumor-bearing mice. As demonstrated in the Fig. 3D, plasma glucose and albumin levels were significantly reduced in cachexic mice, while both parameters were significantly improved in the anti-Tgfbi treatment condition. Assessment of other parameters is shown in the Fig. 3F. Having demonstrated the positive impact of anti-Tgfbi treatment on a series of physical and biochemical parameters, we next sought to examine if this would translate to a better outcome. To this end, we surgically excised the tumors at day 17 and followed the survival for an additional 4.5 weeks. The survival analysis shown in Fig. 3E demonstrates a significantly better survival (on average, a 3.5-fold increase in survival probability) in the anti- Tgfbi treatment arms compared to the control treatment. At the end of the observation, cumulatively, only 18.2% of the animals survived in placebo group, while in the anti-Tgfbi treated arm more than 55% of animals survived (55.5% in the A6 group; 66.6% in the C9 group). Eighty percent of the mice from the control group had local and distant tumor recurrence, in contrast to 40% of the animals in the treatment arm (Data not shown). Having observed that anti-Tgfbi treatment has an anticachectic effect, we further sought to examine the mechanism behind this observation. Tgfbi antibody inhibits muscle wasting and lipolysis.
Muscle and fat are the major tissues affected during cachexia. Thus, we performed an additional animal experiment with autopsy to evaluate whether Tgfbi targeting could prevent these major phenomena (muscle degradation and lipolysis). Considering the similar behavior between the A6 and C9 antibodies, only one antibody was further used for these experiments (reducing the total number of animals). We observed no significant changes on cachexic parameters and gross pathological examniation on day 9 (prechacexic stage) between the three groups (data not shown). However, on day 17, the experiments in Fig. 4G & Data not shown replicated the cachexic phenotype (body weight and food intake) as in Fig. 3. Abdominal MRI analysis of fat and lumbar muscle analysis shows that our antibody prevented the reduction of fat and muscle tissues in cachexic animals (Data not shown). Further, the analysis revealed that the weights of muscles (quadriceps, gastrocnemius, and heart) as well as visceral and subcutaneous fat tissues were significantly reduced in the IgG control mice in comparison to the anti-Tgfbi antibody treatment (Fig. 4A ; Fig. 4B, Fig. 4H, 41 & Data not shown). Next, we performed a histological examination of the excised muscle and fat. The cross-sectional area analysis revealed that the anti-Tgfbi treated animals had muscle fiber cross-sections equivalent to those of non-tumor bearing animals (Fig. 4C). Placebo-treated tumor-bearing mice had significantly small muscle fiber cross-sections compared to the no tumor and the anti-Tgfbi treated animals. We next sought to verify if microscopic findings could be validated at the molecular level. To this end, we analyzed the gene expression of muscle wasting markers, Trim63 and Fbox32, in the muscle samples. As Fig. 4D suggests, muscles recovered from placebo-treated animals featured high expression of both genes. We next examined the adipose tissue, where significant tissue lysis was observable in the placebo treatment cohort in comparison to the anti-Tgfbi treatment group (Fig. 4E). Conversion of white to brown adipose tissue is a known phenomenon in cachexia (Xie H et al., 2022, Proc Natl Acad Sci U.S.A & Han J et al., 2018, Lipids Health Dis). We therefore sought to test the white to brown adipose tissue via the surrogate marker Ucpl. As expected, cachexic phenotype induced strong fat tissue browning, which was significantly rescued, in part, by anti-Tgfbi treatment (Fig. 4F). Exacerbated inflammation and cytokine secretion are known mechanisms of lipolysis and sarcopenia in cachexia. To further clarify the mechanism by which Tgfbi controls the cachexia process, we next aimed to identify the cytokines at play.
Anti-Tgfbi treatment dampens cytokine levels elevated by tumor-induced cachexia. To assess which cytokines are perturbed in our model, we collected the blood from mice on day 17 and have screened for cytokines using a mouse cytokine array. Expectedly, a surge in cytokine levels was observable in cachexic mice compared to the healthy littermates (Data not shown). Treatment with anti-Tgfbi antibodies reduced a number of cytokines, including Tnf-a and 11-6, which were previously established as cachexia relevant (Baazim H et al., 2022, Nat Rev Immunol Kasprzak Aet al., 2Q2 , IntJMol Sci & DzierlegaK et al., 2023, J Immunol). Following this semiquantitative approach, we validated the changes of Tnf-a and 11-6 along with the target protein Tgfbi, in the plasma samples using quantitative ELISA measurements (Fig. 5A). Knowing that immune cells (T-cells and macrophages essentially) are mainly responsible for the secretion of pro-inflammatory cytokines, we next sought to better understand the immune microenvironment of the tumors under anti-Tgfbi treatment. To this end, tumor-infiltrating immune cells in the Colon26 tumors were characterized by IHC analysis. The total number of Cd8+ cytotoxic T-cells was increased in A6 and C9 anti-Tgfbi treated conditions. On the other hand, among two macrophage sub-populations, namely the Cd86+ and Cd206+ cells, the total number of Cd86+ macrophages was increased in A6 and C9 anti-Tgfbi treated conditions (Fig. 5B). Apart from the tumor itself, macrophages are known to reside also in the fat and muscle tissues (Shukla S.K et al., 2020, Cancer Lett Lazaro T et al., 2023, Nature & Jaitin D.A et al., 2019, Cell). Considering that we demonstrated a significant impact of our antibodies on the adipose and muscle tissues, we also examined the status of macrophages in these tissues. IHC analysis showed that the numbers of both Cd86+ and Cd206+ macrophage cells were significantly increased in both tissues (Fig. 5D & Fig. 5E). We next sought to examine if altered cytokine levels (observed in the Fig. 5A) were directly related to the macrophages present in respective tumors. Of the two cytokines (11-6 and Tnf-a), we could establish a multiplexed immunofluorescent staining only with 11-6. As shown in Fig. 5C, macrophages in the anti-Tgfbi treated tumors expressed significantly lower levels of 11-6. These findings suggested that macrophages may have been directly targeted by the anti-Tgfbi antibodies and that Tgfbi itself has a cytokine-regulatory role in these cells. To further test this hypothesis, we next sought to determine if the observations made in vivo can be recapitulated in vitro.
Anti-Tgfbi targeting in macrophages inhibits 11-6 and Tnf-a gene expression and protein secretion.
To test the possible relationship between cytokine expression in macrophages and Tgfbi activity, we selected J774.1 cells to serve as in vitro model. The selection was supported by the data showing their strong Tgfbi expression (Fig. 2E). The J77.1 cells are Cd86 positive and have relatively strong expression and secretion of both 11-6 and Tnf-a compared to the fibroblasts and cancer cells (Fig. 6F & Data not shown). Next, we sought to determine if Tgfbi targeting in the J774.1cells could alter the 11-6 and Tnf-a levels. As shown in the Fig. 6A and Fig. 6G, anti -Tgfbi antibody treatment diminished the secretion and expression of 11-6 and Tnf- a. Consistently, external Tgfbi treatment stimulated the production of these cytokines in J774.1 macrophages (Fig. 6B and Fig. 6H). Based on these findings, we hypothesized that Tgfbi might bind to the cell surface of the macrophage cells to regulate cytokine production and have thus sought to determine the possible receptor. Previous literature evidence supports that extracellular Tgfbi interacts with several integrins on cancer cells, macrophages, and Cd8+ lymphocytes (Peng P et al., 2022, Theranostics Costanza B et al., 2019, Int J Cancer Goehrig D et al., 2019, Gut & Corona A et al., 2021, Cell Signal). Based on these evidences, we next used an integrin inhibitor, cilengitide, and tested if the integrin blockade can revert the Tgfbi- induced cytokine secretion from macrophages. The results revealed that integrin blockade using cilengitide abrogated 11-6 and Tnf-a secretion and expression in J774.1 macrophages (Fig. 6C & Fig. 61). Furthermore, the notion of integrin-dependent Tgfbi signaling was reinforced by the observed decrease of phosphorylated focal-adhesion kinase (pFAK) (Data not shown). Indeed, FAK signaling has been reported as preferential axis of integrin-mediated Tgfbi signaling in different types of cells (Costanza B et al., 2019, Int J Cancer Han B et al., 2015, Mol Cancer & Ahmed A.A et al., 2007, Cancer Cell). To further pinpoint which integrin receptors are relevant for the above-mentioned observations, we screened for the expression of several integrins in J774.1 macrophages. We found that the J774.1 macrophage cells express Itgav and Itgb5 (Fig. 6J). Silencing both Itgav and Itgb5 abrogated Tgfbi-induced FAK activation, 11-6, and Tnf-a secretion in J774.1 macrophages (Fig. 6D & Data not shown) (Fig. 6K), while FACS analysis showed decreased cell surface binding of recombinant Tgfbi when both integrins were solely silenced in J774.1 cells (Fig. 6E).
Example 2: The combination effect of anti-TGFBI antibody with anti-PD-1 antibody
Materials & Methods
A mouse model of cachexia was created by subcutaneous transplantation of colon26 cells of mouse colon cancer cell line into Balb/c mice.
Those mice were grouped into IgG isotype control group, anti-TGFBI antibody (Ab C9) group (200 ug/body/2 times per week), anti-PD-1 antibody group (200 ug/body/2 times per week) and combination group.
The effects of each treatment on tumor-infiltrating CD8-positive cytotoxic T cells in model mice (assessed by subcutaneous tumor sampling on Day 17) and on survival after subcutaneous tumor resection were evaluated.
Results (figure 7A, 7B, 7C, 7D, 7E)
Anti-TGFBI antibody alone, anti-PD-1 antibody alone, and the combination of anti- TGFBI and anti-PD-1 antibodies all showed prolonged survival compared to the control group.
Tumor volume in the combination group was significantly smaller than that of the control and anti-TGFBI antibody alone groups.
In addition, the combination of anti-TGFBI antibody and anti-PD-1 antibody tended to increase the infiltration of cytotoxic T cells into the tumor compared to the single antibody.
Regarding the cachexia symptoms, the appearance suggestive of cachexia was not observed in the anti-TGFBI antibody, anti-PD-1 antibody, and the combination groups, in contrast to the control group, which presented cachexia. Moreover, the body weight in the anti- TGFBI antibody, anti-PD-1 antibody, and the combination groups was higher than that of the control group. The wire hanging time in the anti-TGFBI antibody group was significantly longer than in the control group, suggesting that the TGFBI blockage can maintain muscle strength in the cachexic condition.
Example 3: Comparative study of anti-TGFBI antibody and ghrelin agonist anamorelin in a mouse model of cachexia
Materials & Methods
A mouse model of cachexia was created by subcutaneous transplantation of colon26 cells of mouse colon cancer cell line into Balb/c mice.
Those mice were grouped into a control group (PBS via p.o. every other day), an anti- TGFBI antibody group (200 ug/body/2 times per week), and an anamorelin, a ghrelin agonist group (30 mg/kg p.o. every other day).
The effects of each treatment on preventing cachexia were monitored by body weight loss, muscle strength, and food intake for four weeks. Animals were sacrificed when body weight was measured below 18g, and terminal blood and tissue sampling were subjected to further evaluation. Results (Figure 8)
Both the anti-TGFBI antibody and anamorelin treatment significantly prolonged the survival of the tumor-bearing mouse without any surgical intervention. Median survival days were 26.5, 22, and 18 days, respectively. Notably, the anti-TGFBI antibody treatment outperformed the anamorelin treatment in terms of survival and well-being (body weight, maintaining muscle strength, and plasma glucose level) of the tumor-bearing animal. At each endpoint, food intake and plasma albumin levels were higher in anamorelin treatment group animals (Fig. 8A and 8B).
In addition, animals treated with anti-TGFBI antibodies or anamorelin maintained more fat in spite of their body weight loss.
Anti-TGFBI antibody treatment significantly elongated the survival in the cachexia model mice compared to the control (PBS) and anamorelin treatment. On the other hand, tumor weight and volume were not suppressed in the anti-TGFBI antibody and anamorelin groups compared to the control group (Fig. 8C).
Body weight was maintained with the anti-TGFBI antibody and anamorelin treatments. In addition, muscle strength (wire hanging time) was significantly better in the anti-TGFBI antibody group compared to the anamorelin and control group, particularly on day 17 and after. Food intake was reduced only in control group animals but not in anamorelin and anti-TGFBI antibody treatment groups (Fig. 8D and 8E).
Plasma cytokine levels, including 11-6, Tnf-a, and Tgfbi levels were significantly lower in anti-TGFBI antibody-treated animals compared to the control group. More, Tgfbi antibody treated groups tend to have lower levels of cytokines at the endpoints (Fig. 8F and 8G).
Example 4: Adjuvant chemotherapy effect after anti-Tgfbi antibody treatment
Materials & Methods
A mouse model of cachexia was created by subcutaneous transplantation of colon26 cells of mouse colon cancer cell line into Balb/c mice.
Those mice were grouped into IgG isotype control group and anti-TGFBI antibody (Ab C9) group (200 ug/body/2 times per week). Then mouse tumors were surgically resected at day 17 after tumor inoculation. After the tumor resection mice were treated with anti -turn or agent (5FU, 30mg/kg via i.p. injection) at day 23 and day 26, and further followed up to monitor survival until day 60.
Results (Figure 9) Anti-TGFBI antibody treatment followed by anti-tumor agent 5FU in adjuvant settings significantly prolonged the survival of the animal (P value = 0.0330, HR 7.65) (Fig. 9A, 9B).
Discussion
When efforts focusing on local tumor control fail, cancer treatment becomes a chronic systemic therapy with sets of different (and usually cytotoxic) agents. During this exhausting process, the cancer cells can reprogram the body metabolism, which frequently results in cachexia. Cachexic patients cannot support recommended dose regimens, leading to lowering of the cytotoxic doses and hence ultimately treatment failure and cancer resistance. Our ability to continue the patient treatment therefore critically depends on the auxiliary means of controlling cachexic process and hence widening the window of therapeutic intervention. Today, in the clinical setting, nutritional supplements are some of limited means to delaying the onset of cachexia; however, the clinical evidence for this remains rather heterogeneous (Van de Worp W et al., 2020, Front Niitr). New drugs are therefore critically needed to curb the cachexic syndrome, especially knowing that in some types of cancer over 3/4 of patients will experience cachexia (e.g. pancreatic cancer). Despite this need, the development of anti-cachexic treatments has been a difficult process, largely in part due to the complex etiology of this syndrome. We know however that both protein catabolism in the muscle as well as lipolysis in adipose tissue are largely regulated by cytokine messenging. In particular, IL-6 and TNF-a derived from macrophages in the muscle and adipose tissue microenvironment have been reported to cause a typical cachexic phenotypes (muscle atrophy, lipolysis, fat browning) (Baazim H et al., 2022, Nat Rev Immunol). While the systemic surge of cytokines is in part responsible for cachexia, the selective blockade of these cytokines fails to produce tangible effects in the clinics (Prado B.L et al., 2019, Ann Palliat Med). This points out to the fact that cachexia remains a complex metabolic and inflammatory disorder where de-regulated levels of cytokines play importnat part, yet they are not exclusively responsible for the observed muscle and fat tissue wasting. Indeed cytokines, despite their specificity for a given receptor , have pleiotropic effects in the human body. For example, IL6 has a myriad of functions in both adult and developing brain (Erta M et al., 2012, Int J Biol Sci, DOI : 10.7150/ijbs.4679). Therefore cytokine function is very context and tissue dependent and their systemic targeting may or may not achieve desired effects. Keeping this in mind it may be rather more productive to focus our attention on the cellular “platforms”, primarily immune cells, that are responsible for the cytokine de-regulation in the first place. Indeed, immune cells are known to be both circulating as well as tissue resident, permitting them to delicately regulate cytokine-mediated processes both at distance as well as few cell diameters away. One such key cell type are macrophages that have been reported as the major source of cytokines in the tumor microenvironment, especially via the regulation of the integrin signals (Kasprzak A et al., 2021, Int J Mol Set Liu Z et al., 2018, Int J Mol Med Wang Q et al., 2010, Cell Res Han Z.P et al., 2020, Transl Cancer Res & Hwang M.A et al., 2022, Int J Mol Sei). Interestingly, Antonov et al. reported that integrin activation on macrophages significantly enhances the expression of NF-KB- dependent proinflammatory cytokines, such as TNF-a, IL-ip, and IL-6, and that integrin activation on macrophages is responsible for the persistence of chronic inflammation; suggesting the importance of macrophages and integrin signals in cancer cachexia with chronic inflammatory organ damage (Antonov A. S et al . , 2011 , J Cell Physiol). Integrin signaling in the tumor microenvironment is in turn regulated by complex crosstalk with various extracellular factors (e.g., extracellular matrix and stromal proteins) (Desgrosellier J.S et al., 2010, Nat Rev Cancer). Despite this obvious link between the stromal regulators of integrin signaling and inflammation, cachexia-associated extracellular factors that regulate cytokine production in cancer-associated macrophages remain under investigated. One such stromal protein is TGFBI, which we previously described as a deregulated ECM protein in cancer (Turtoi A et al., 2011, J Proteome Res). Recent studies have now established that Tgfbi is strongly involved in macrophage biology. Namely, Tgfbi KO mice had more M2 macrophages in the adipose tissue, while the mice had significantly augmented resistance to adipose tissue hypertrophy, liver steatosis, and insulin resistance (Lee S.G et al., 2023, Exp Mol Med). M2 macrophages are themselves characterized by high levels of TGFBI, while the secretion of TGFBI by macrophages is associated with the immune-suppressed tumor microenvironment (Lecker L.S.M et al., 2021, Cancer Res). Recently, Zhou et al. expanded these findings by demonstrating that Tgfbi is not only a marker of but an essential factor for macrophage polarization to immune- suppressive alternatively activated macrophages (Zhou J et al., 2023, Cancer Lett).
In the present study, we report for the first time that high stromal Tgfbi is associated with cachexia in patients with advanced colon cancer. Importantly, our first-in-class fully human Tgfbi inhibitory antibodies, in a neo-adjuvant setting, could significantly curb the cachexia symptoms in vivo. Tumor-bearing animals treated with anti-Tgfbi antibody had lower weight loss, muscle atrophy, lipolysis, fat browning, hypoglycemia and hypoalbuminemia. They also experienced higher muscle strength, food intake and prolonged survival post-surgery. Notably, the antibody treatment led to enhanced tumoral infiltration of Cd8+ T-cells as well as of Cd86+ macrophages. This in part confirms previous study by Goehrig et al. which showed higher Cd8+ infiltration in pancreatic cancer when animals were under anti-Tgfbi treatment (Goehrig D et al., 2019, Guf). Same study found however that intratumoral Cd206+ macrophages, that the authors designate as M2, were decreased by anti-Tgfbi treatment. Our data are in discordance with these findings, as we noted an overall increase of Cd86+ macrophages found in Colon-26 tumors, though the Cd206+ macrophages were not changed. At this point we cannot explain these differences other than to hypothesize that the choice of animal model (C57BL/6 mice versus BALB/cAJcl mice) as well as the tumor cell line (KPC versus Colon-26) could have been the reasons. Furthermore, Goehrig et al. noted that macrophages cultured on collagen with recombinant Tgfbi had a reduced production of interferon gamma (Ifn-y) and Tnf-a. Our data are in contrast to these findings, as we shown that recombinant Tgfbi induces Tnf-a expression and secretion (along that of 11-6) in unstimulated J774.1 macrophages. Here as well, the choice of cell model may have had an impact as Goehrig et al. use PMA (phorbol ester) stimulated Raw 264.7 mouse macrophage cell line, which as such is programmed towards a strong pro-inflammatory phenotype (Goehrig D et al., 2019, Guf). They also measure intracellularly the cytokine levels, which is different to our measurement in the extracellular medium. More studies will be necessary to elucidate why these differences are present and if they stem from different cell models or perhaps from inherently different antibodies used in the two studies. Indeed, Goehrig et al. used a fully murine 18B3 anti-Tgfbi antibody developed by Bae and colleagues (Bae J.S et al., 2014, Acta Physiol , whose isotype and subclass remain, to our knowledge, undescribed. The antibody has been developed using a synthesized peptide of human TGFBI (amino acids 498-637). Our antibody is a fully human clone that was selected using the entire TGFBI protein and which, for the purpose of the present work, has been re-formatted to a chimeric format, consisting of human F(ab) and murine Fc domain. The latter is an IgG isotype of the 2a subclass, specifically selected for its ability to induce both opsonization and complement activation. The format differences, as well as the epitopes targeted by the two antibodies, should be, therefore, further examined for their ability to evoke different effects on macrophage polarization. It is indeed conceivable that the interaction of Tgfbi with its receptor may be obstructed differently with the two antibodies. Tgfbi has been shown to interact with integrins heterodimers such as aVp3, aipi and aVp5 (Nam J.O et al., 2003, J Biol Chem ; Ohno S et al., 1999, Biochim Biophys Acta & Ma C et al., 2008, Genes Dev). Goehrig et al.29 reported that in their model, Tgfbi mainly acted through Cd61 (or integrin P3). In our study, we found that Tgfbi bound to aVp5, and this interaction led to an increase in 11-6 and Tnf-a levels in J774.1 macrophages. Additional studies are therefore needed to fully characterize the domains on the Tgfbi protein that are necessary for the respective integrin interactions. This could, in part, explain subtle yet important differences between the two antibodies and hence guide their future clinical application.
In conclusion, we developed a Tgfbi inhibitory antibody and showed that the antibody administration improved symptoms and prognosis of cachexia by suppressing cachexia- associated cytokine production from macrophages via integrin signals. Furthermore, the Tgfbi antibody could regulate not only cytokine production but also the level of immune cell infiltration in the tumor microenvironment. Our study highlights that a therapeutic strategy targeting Tgfbi protein, a cachexic extracellular factor, may be promising for controlling cachexia symptoms through the regulation of immune cell infiltration and cytokine production in the tumor microenvironment, thus may improve survival and quality of life in many advanced cancer patients suffering from refractory cancer cachexia. Moreover, this study showed promising results of combination (anti-Tgfbi + anti-PD-1 or anti-Tgfbi + ghrelin agonist or anti- Tgfbi + chemotherapy) for the treatment of cachexia and particularly cancer cachexia. The advantages of such combination are numerous, especially because immune-checkpoint inhibitors and chemotherapy act on the tumor size. Ghrelin agonist (such as Anamorelin) are effective for increasing appetite, leading especially to the increase in the fat tissue. Anti-Tgfbi treatment primarily acts on inflammatory signals, hence impeding muscle destruction and fat reduction. Therefore, we foresee that the combination of several strategies that act through complementary pathways may represent a powerful treatment combination in the clinics - especially for complex systemic diseases such as cancer cachexia.
Example 5 :
Materials & Methods
Clinical samples and serum TGFBI evaluation by ELISA
Preoperative serum samples were obtained from 69 pancreatic cancer patients who underwent curative surgery at Gunma University Hospital. This study was conducted in accordance with the tenets of the Declaration of Helsinki and was approved by the Institutional Review Board for Clinical Research of Gunma University Hospital. Patient consent was obtained via the opt-out method. Cachexia was diagnosed according to the Asia Working Group of Cachexia definition. Serum concentrations of TGFBI were determined by enzyme-linked immunosorbent assay (ELISA). All samples were collected before surgical tumor resection. For TGFBI, we used a Human beta IG H3 (TGFBI) ELISA Kit (ab 155426) (Abeam). Assays were evaluated as per the manufacturer’s instructions, and all samples were tested in duplicate. Pancreatic cancer patient-derived xenograft (PC-PDX) model and TGFBI antibody treatment.
A pancreatic cancer patient-derived xenograft (PC PDX) tumor model was established using female Balb/cAJcl-nu/nu mice (8 weeks old). Human pancreatic tumor tissues were cut into small fragments (approximately 5 x 5 x 5 mm) and implanted into the right flank of each mouse under general anesthesia. In the PC PDX model, anti -TGFBI antibody was administered intraperitoneally at a dose of 200 pg per mouse, twice weekly, following the same regimen used in the Colon26 tumor-bearing mouse model. The observation period was set at 45 days (Figure 11, top panel). Tumor engraftment was confirmed prior to initiating antibody treatment on day 10 post-implantation (Figure 11 A, top panel). Outcome measures included muscle strength (assessed by the wire hanging test, Fig. 11B), body weight, food intake (Fig. 11C), tumor growth, plasma cytokine levels (11-6 and Tnf-a), plasma Tgfbi concentrations, skeletal muscle mass, and expression of muscle degradation markers (MuRFl and phosphorylated Stat3). Tumor size (W; width. L; length) was measured with a caliper. Tumor volume (mm3) was calculated as (W2xL)/2 (Fig. 1 ID). All animal studies were approved by the Institutional Animal Care and Use Committee of Gunma University.
ELISA assay for plasma 11-6, Tnf-a, and Tgfbi in mouse models. Indirect ELISA was used to quantify 11-6, Tnf-a, and Tgfbi levels in plasma samples collected from experimental animals. Maxisorp 96-well plates (Thermo Fisher, USA) were coated with 100 pl of plasma or concentrated medium samples. Serial dilutions of murine recombinant Tgfbi (R&D Systems, USA), 11-6 (PeproTech, USA), and Tnf-a (PeproTech, USA) in PBS were prepared to generate standard calibration curves. Coating was performed overnight at 4°C under gentle agitation. Wells were then washed three times with PBS containing 0.01% (v/v) Tween- 20 and subsequently with PBS alone. Residual binding sites were blocked with 4% skim milk in PBS for 2 hours at room temperature under agitation. After washing, 100 pl of diluted primary antibodies were added to each well and incubated for 2 hours at room temperature with agitation. The antibodies used were rabbit anti -mouse 11-6 (PeproTech, 1 : 1000), goat antimouse Tnf-a (PeproTech, 1 : 1000), and sheep anti-mouse TGFBI (R&D Systems, 1 :500). After another washing step, HRP -conjugated secondary antibodies were added and incubated for 1 hour at room temperature with agitation. Detection was performed using the One Step-ULTRA- TMB substrate solution (Thermo Scientific), followed by termination of the enzymatic reaction with 100 pl of 0.5 M sulfuric acid. Absorbance was measured at 450 nm.
Histological evaluation of mouse tissue Histological analysis was performed using FFPE sections prepared from skeletal muscle tissues of experimental animals. Four-micrometer-thick sections were stained with hematoxylin and eosin (HE) to assess the cross-sectional area of muscle fibers. For each sample, at least three independent fields of view (FOVs) were acquired. Muscle fiber area was quantified using an ImageJ plugin designed for cross-sectional area analysis with a defined threshold. The measured areas were classified into three size categories, and the frequency distribution of each category was calculated for every sample.
Western blot
Western blot analysis was performed using protein samples prepared from muscle tissue lysates suspended in RIPA buffer (Wako, Osaka, Japan) supplemented with protease and phosphatase inhibitors. Protein concentrations were determined using a Bradford assay kit (BioRad, California, USA). Laemmli sample buffer (4x) (Bio-Rad, California, USA) was added to 10 pl of each sample. After boiling for 5 minutes, equal amounts of samples (10 pl of conditioned medium and 10 pg of cell lysate) were loaded onto 7.5% polyacrylamide gels. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes at 90 V for 90 minutes. After blocking, membranes were incubated overnight at 4°C with the following primary antibodies: anti-mouse MuRFl (R&D Systems, 1 :500), anti-mouse phospho-STAT3 (Y705) (Cell Signaling Technology, 1 : 1000), and anti-mouse STAT3 (Cell Signaling Technology, 1 : 1000). Membranes were then washed three times with TBS-T and incubated with appropriate HRP -conjugated secondary antibodies for 1 hour at room temperature. After additional washes, protein bands were visualized using the ECL Prime Western Blot Detection System and an ImageQuant LAS 4000 system (GE Healthcare Life Sciences). Ponceau red- stained membranes were imaged to document total protein loading and transfer consistency.
Cell lines and mice models
Colon26-luc cell line was obtained from the Japanese Collection of Research Bioresources Cell Bank. The cell lines were cultured in RPMI-1640 (Wako, Japan) supplemented with 10% FBS and 1% SM/PC at 37°C in 5%CO2. The cell line was tested for mycoplasma contamination using Mycostrip (Invivogen, California, USA). Colon26-luc (5xl04 cells/100 pl in PBS) were subcutaneously injected under general anesthesia into the right flank of female BALB/cAJcl(CLEA, Japan) mice at 8 weeks age. The observation period was set at 17 days (Figure 13, top left panel). Outcome measures included muscle strength (assessed by the wire hanging test), body weight, food intake, tumor growth, plasma cytokine levels (IL-6 and TNF-a), plasma Tgfbi concentrations, skeletal muscle weight, and expression of muscle degradation markers (MuRFl and phosphorylated Stat3). Tumor size (W; width. L; length) was measured with a caliper. Tumor volume (mm3) was calculated as (W2xL)/2. All animal studies were approved by the Institutional Animal Care and Use Committee of Gunma University.
Result
Association Between Serum TGFBI Levels and Cancer Cachexia in 69 Pancreatic Cancer Patients
In our investigations, we demonstrated that the high stromal TGFBI in primary tumor tissues was associated with the presence of cancer cachexia. We next examined whether TGFBI, a secreted protein detectable in the bloodstream, is correlated with cachexia-related symptoms in clinical pancreatic cancer patients (n=69). As a result, the preoperative serum TGFBI levels were significantly higher in pancreatic cancer patients with cancer cachexia than in those without cachexia (P < 0.001) (Figure 10).
Therapeutic effect of TGFBI antibody against cachexia symptoms in a pancreatic cancer patient-derived xenograft (PDX) model
To determine whether the promising anti-cachectic effect of TGFBI antibody observed in a cell line-derived tumor-bearing mouse model could be recapitulated in a patient-derived xenograft (PDX) model, we established a pancreatic cancer (PC) PDX model (Figure 11, top panel) and administered TGFBI antibody. Muscle strength, body weight, food intake, and tumor growth were assessed and compared between the control IgG group and the TGFBI antibody C9-treated group. As a result, the wire hanging test demonstrated that muscle strength was significantly preserved in the C9 group compared to the control group (Figure 11, middle panel). In contrast, the C9 antibody did not exert a significant effect on body weight, food intake, or tumor growth in this PDX model (Figure 11, middle panel and bottom panel). In the PC PDX model, plasma levels of cytokines (11-6 and Tnf- a ) and Tgfbi were significantly reduced in the C9 antibody-treated group compared to the IgG control group (Figure 12, top panel). Furthermore, comparison of the distribution of muscle fiber areas among the no tumor control, IgG control, and C9 antibody-treated groups showed that cachexia-induced muscle fiber atrophy was evident in the IgG group, whereas muscle fiber size was preserved in the C9- treated group to levels comparable to those in the no tumor control group (Figure 12, bottom left panel). In addition, Western blot analysis of Murfl, a muscle degradation marker, and phosphorylated STAT3, a downstream effector of 11-6, revealed enhanced muscle degradation in the IgG control group (Figure 12, bottom right panel). The anti-cachectic effect of the TGFBI antibody is independent of Fc-mediated effector functions
To eliminate the potential confounding effects of Fc-mediated effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), we generated an Fc-silent anti-TGFBI antibody carrying the L234A, L235A, and P329G (LALAPG) mutations in the Fc region. These mutations were introduced to abolish binding to Fey receptors and complement component Clq. Cancer cachexia model mice were established using Colon26 cells, and the therapeutic effects of the C9 antibody and the C9- LALAPG antibody were evaluated (Figure 13, top left panel). Both antibodies significantly improved cachexia-related symptoms, including food intake, body weight, and muscle strength, compared to the control IgG group. No significant differences were observed between the C9 and C9-LALAPG groups (Figure 13, top right panel and middle panel). Similarly, there was no significant difference in tumor growth between the two treatment groups (Figure 13, bottom panel). Plasma levels of cytokines (11-6 and Tnf-a) and Tgfbi were significantly reduced in both the C9 and C9-LALAPG antibody groups compared to the control IgG group, with no significant difference observed between the two treatment groups (Figure 14, top panel). In addition, analysis of muscle weight in cachectic mice showed no significant difference between the C9 and C9-LALAPG groups (Figure 14, bottom panel). Western blot analysis of Murfl, a muscle degradation marker, and phosphorylated STAT3, a downstream effector of 11-6, revealed muscle degradation in the IgG control group compared to both C9 and C9-LALAPG groups (Figure 12, bottom right panel). Western blot analysis of the muscle degradation marker MURF1 and the 11-6 downstream effector phosphorylated STAT3 revealed increased muscle degradation in the control IgG group compared to both the C9 and C9-LALAPG groups. However, no clear difference was observed between the C9 and C9-LALAPG groups (Figure 14, bottom right panel). The comparable therapeutic efficacy against cancer cachexia and the similar suppression of plasma cytokines observed between the C9 antibody and the Fc-silent C9-LALAPG antibody indicates that the anti -cachectic activity of the C9 antibody is not dependent on Fc-mediated cytotoxicity toward macrophages producing cachexia-related cytokines. Rather, these findings support the therapeutic concept that the C9 antibody exerts its efficacy by inhibiting cytokine production from macrophages.
TGFBI C9 antibody inhibited muscle degradation signaling in colon26 cachexia mice
For this analysis, muscle tissues were collected on day 17 from Colon26 cachexia model mice and analyzed by Western blot to assess muscle degradation signaling. Comparisons were made among no tumor-bearing (NTB) mice, control IgG-treated mice, and TGFBI C9 antibody- treated mice. The results showed that cachexia induced by Colon26 tumor implantation activated muscle degradation signaling, which was attenuated by C9 antibody treatment, as confirmed at the protein level by Western blot analysis (Figure 15). These findings are consistent with the results shown in Figure 4C-4D, where muscle degradation was evaluated by PCR analysis.
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Claims

CLAIMS :
1. An anti -Transforming growth factor-beta-induced antibody having a heavy chain comprising i) the H-CDR1 of A6 mab, ii) the H-CDR2 of A6 mab and iii) the H-CDR3 of A6 mab and a light chain comprising i) the L-CDR1 of A6 mab, ii) the L-CDR2 of A6 mab and iii) the L-CDR3 of A6 mab
Wherein
- the H-CDR1 of the A6 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO: 1;
- the H-CDR2 of A6 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO: 1;
- the H-CDR3 of the A6 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO: 1.
- the L-CDR1 of the A6 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO:2;
- the L-CDR2 of the A6 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO:2;
- the L-CDR3 of the A6 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO:2; or an antibody having a heavy chain comprising i) the H-CDR1 of C7 mab, ii) the H- CDR2 of C7 mab and iii) the H-CDR3 of C7 mab and a light chain comprising i) the L- CDR1 of C7 mab, ii) the L-CDR2 of C7 mab and iii) the L-CDR3 of C7 mab
Wherein
- the H-CDR1 of the C7 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO:3;
- the H-CDR2 of C7 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO:3;
- the H-CDR3 of the C7 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO:3.
- the L-CDR1 of the C7 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO:4;
- the L-CDR2 of the C7 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO:4; the L-CDR3 of the C7 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO:4; or an antibody having a heavy chain comprising i) the H-CDR1 of C9 mab, ii) the H- CDR2 of C9 mab and iii) the H-CDR3 of C9 mab and a light chain comprising i) the L- CDR1 of C9 mab, ii) the L-CDR2 of C9 mab and iii) the L-CDR3 of C9 mab
Wherein
- the H-CDR1 of the C9 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO: 5;
- the H-CDR2 of C9 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO: 5;
- the H-CDR3 of the C9 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO: 5.
- the L-CDR1 of the C9 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO:6;
- the L-CDR2 of the C9 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO:6;
- the L-CDR3 of the C9 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO: 6; or an antibody having a heavy chain comprising i) the H-CDR1 of D2 mab, ii) the H- CDR2 of D2 mab and iii) the H-CDR3 of D2 mab and a light chain comprising i) the L-CDR1 of D2 mab, ii) the L-CDR2 of D2 mab and iii) the L-CDR3 of D2 mab
Wherein
- the H-CDR1 of the D2 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO: 7;
- the H-CDR2 of D2 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO:7;
- the H-CDR3 of the D2 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO:7.
- the L-CDR1 of the D2 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO:8;
- the L-CDR2 of the D2 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO:8; the L-CDR3 of the D2 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO:8; or an antibody having a heavy chain comprising i) the H-CDR1 of D5 mab, ii) the H- CDR2 of D5 mab and iii) the H-CDR3 of D5 mab and a light chain comprising i) the L-CDR1 of D5 mab, ii) the L-CDR2 of D5 mab and iii) the L-CDR3 of D5 mab
Wherein
- the H-CDR1 of the D5 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO: 9;
- the H-CDR2 of D5 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO:9;
- the H-CDR3 of the D5 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO:9.
- the L-CDR1 of the D5 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO: 10;
- the L-CDR2 of the D5 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO: 10;
- the L-CDR3 of the D5 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO: 10; or an antibody having a heavy chain comprising i) the H-CDR1 of E4 mab, ii) the H- CDR2 of E4 mab and iii) the H-CDR3 of E4 mab and a light chain comprising i) the L- CDR1 of E4 mab, ii) the L-CDR2 of E4 mab and iii) the L-CDR3 of E4 mab
Wherein
- the H-CDR1 of the E4 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO: 11;
- the H-CDR2 of E4 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO: 11;
- the H-CDR3 of the E4 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO: 11.
- the L-CDR1 of the E4 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO: 12;
- the L-CDR2 of the E4 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO: 12; the L-CDR3 of the E4 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO: 12; or an antibody having a heavy chain comprising i) the H-CDR1 of G7 mab, ii) the H- CDR2 of G7 mab and iii) the H-CDR3 of G7 mab and a light chain comprising i) the L-CDR1 of G7 mab, ii) the L-CDR2 of G7 mab and iii) the L-CDR3 of G7 mab
Wherein
- the H-CDR1 of the G7 mab is defined by the sequence ranging from the amino acid residue at position 31 to the amino acid residue at position 35 in SEQ ID NO: 13;
- the H-CDR2 of G7 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 65 in SEQ ID NO: 13;
- the H-CDR3 of the G7 mab is defined by the sequence ranging from the amino acid residue at position 95 to the amino acid residue at position 102 in SEQ ID NO: 13.
- the L-CDR1 of the G7 mab is defined by the sequence ranging from the amino acid residue at position 24 to the amino acid residue at position 34 in SEQ ID NO: 14;
- the L-CDR2 of the G7 mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 56 in SEQ ID NO: 14;
- the L-CDR3 of the G7 mab is defined by the sequence ranging from the amino acid residue at position 89 to the amino acid residue at position 97 in SEQ ID NO: 14;
2. An antibody according to claim 1 having a heavy chain having at least 70% of identity with SEQ ID NO: 1, 3, 5, 7, 9 or 13 and a light chain having at least 70 % of identity with SEQ ID NO:2, 4, 6, 8, 10, 12 or 14.
3. An antibody according to claim 1 having a heavy chain identical to SEQ ID NO: 1, 3, 5, 7, 9 or 13 and a light chain identical to SEQ ID NO:2, 4, 6, 8, 10, 12 or 14.
4. The antibodies according to claim 1 which are human antibodies or chimeric antibodies.
5. The antibodies of claims 1 to 4 which are conjugated to a therapeutic moiety or cytotoxic moiety.
6. The nucleic acid molecules encoding the antibodies of claims 1 to 4.
7. A TGFBI inhibitor for use in the treatment of cachexia in a subject in need thereof. Ill
8. The TGFBI inhibitor for use according to the claim 7 wherein the cachexia is a cancer cachexia.
9. The TGFBI inhibitor for use according to the claims 7 or 8 wherein the inhibitor is anti- TGFBI antibody according to the claim 1.
10. A combination of a TGFBI inhibitor and an agonist of the GHSR for use in the treatment of cachexia in a subject in need thereof.
11. A combination of TGFBI inhibitor and an anti-PD-1 antibody for use in the treatment of cachexia in a subject in need thereof.
12. A pharmaceutical composition for use in the treatment of cachexia comprising a therapeutically effective amount of an inhibitor according to the invention.
13. A method for treating cachexia or cancer cachexia in a subject in need thereof comprising administering a therapeutically effective amount of a TGFBI inhibitor.
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