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WO2025012417A1 - Anti-neurotensin long fragment and anti-neuromedin n long fragment antibodies and uses thereof - Google Patents

Anti-neurotensin long fragment and anti-neuromedin n long fragment antibodies and uses thereof Download PDF

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Publication number
WO2025012417A1
WO2025012417A1 PCT/EP2024/069780 EP2024069780W WO2025012417A1 WO 2025012417 A1 WO2025012417 A1 WO 2025012417A1 EP 2024069780 W EP2024069780 W EP 2024069780W WO 2025012417 A1 WO2025012417 A1 WO 2025012417A1
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Prior art keywords
antibody
seq
malignant
carcinoma
cell
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PCT/EP2024/069780
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French (fr)
Inventor
Patricia Forgez
Marco Alifano
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Institut National de la Santé et de la Recherche Médicale
Université Paris Cité
Sorbonne Université
Assistance Publique-Hôpitaux De Paris (Aphp)
Verticalsentinel,
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Publication of WO2025012417A1 publication Critical patent/WO2025012417A1/en

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    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • 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
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • 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

  • NTS Neurotensin
  • NTS action is mediated by two different G protein coupled receptors, the high and low affinity neurotensin receptors NTSR1 and NTSR2, respectively, and by a nonspecific single transmembranous sorting receptor encoded by the SORT1 gene, NTSR3/sortiline (4).
  • the NTS gene is abnormally expressed or overexpressed, under physiological or pathological contexts (tumors, inflammation, infection, allergies, and chemotherapy treatment, obesity, ...) (8-12) facilitating the production and the release of the proform (PF), the large form of NTS and/or NN.
  • PF proform
  • LF NTS LF NTS
  • LF NN LF NTS
  • LF NN LF NTS
  • NTSR1 high affinity neurotensin receptor 1
  • the PF, the LF NTS and the LF NN represent the active form whereas the N terminal fragment of 140 among acid maned R for remnant is inactive on the neurotensinergic system.
  • the high affinity neurotensin receptor 1 (NTSR1) is abnormally expressed or overexpressed in tumor cells of a large number of cancers, especially from epithelial origin (13, 14).
  • the present invention relates to an antibody, which is capable of binding to the Neuromedin N long fragment, and Neurotensin long fragment with high affinity.
  • the antibody of the present invention neutralises the activity of the Neuromedin N long fragment, and Neurotensin long fragment, in particular their oncogenic activities.
  • the present invention provides a human antibody, particularly in a purified form or in an isolated form.
  • the invention relates to a human antibody comprising:
  • variable domain comprises: a H-CDR1 having a sequence set forth as SEQ ID NO: 2, a H-CDR2 having a sequence set forth as SEQ ID NO:3; a H-CDR3 having a sequence set forth as SEQ ID NO: 4;
  • variable domain comprises : a L-CDR1 having a sequence set forth as SEQ ID NO: 6; a L-CDR2 having a sequence set forth as SEQ ID NO: 7; a L-CDR3 having a sequence set forth as SEQ ID NO: 8
  • the antibody of the present invention comprises a heavy chain wherein the variable domain comprises at least one CDR having a sequence selected from the group consisting of SEQ ID NO:2 for H-CDR1, SEQ ID NO:3 for H-CDR2 and SEQ ID NON for H- CDR3.
  • the antibody of the present invention comprises a light chain wherein the variable domain comprises at least one CDR having a sequence selected from the group consisting of SEQ ID NO:6 for L-CDR1, SEQ ID NO:7 for L-CDR2 and SEQ ID NO:8 for L- CDR3.
  • the antibody of the present invention comprises a heavy chain wherein the variable domain comprises at least one CDR having a sequence selected from the group consisting of SEQ ID NO:2 for H-CDR1, SEQ ID NO:3 for H-CDR2 and SEQ ID NON for H- CDR3 and a light chain wherein the variable domain comprises at least one CDR having a sequence selected from the group consisting of SEQ ID NO:6 for L-CDR1, SEQ ID NO:7 for L-CDR2 and SEQ ID NO: 8 for L-CDR3.
  • the antibody of the present invention comprises a heavy chain variable region comprising SEQ ID NO:2 in the H-CDR1 region, SEQ ID NON in the H-CDR2 region and SEQ ID NON in the H- CDR3 region ; and a light chain variable region comprising SEQ ID NO:6 in the L-CDR1 region, SEQ ID NON in the L-CDR2 region and SEQ ID NO:8 in the L-CDR3 region.
  • the antibody of the present invention comprises a heavy chain variable region having at least 70% of identity with SEQ ID NO:1 and/or a light chain variable region having at least 70% of identity with SEQ ID NO:5.
  • 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.
  • the antibody of the present invention comprises a heavy chain variable region of having the amino acid sequence set forth as SEQ ID NO: 1 and/or a light chain variable region having the amino acid sequence set forth as SEQ ID NO: 5.
  • the invention relates to a human antibody comprising: a heavy chain wherein the variable domain has a sequence set forth as SEQ ID NO: 1 a light chain wherein the variable domain has a sequence set forth as SEQ ID NO: 5
  • the present invention provides a human anti-neurotensin long fragment (LF anti-NTS) antibody or a human anti-neuromedin N long fragment (LF anti-NN) antibody (named NAM02), particularly in a purified form or in an isolated form.
  • LF anti-NTS human anti-neurotensin long fragment
  • LF anti-NN human anti-neuromedin N long fragment
  • the invention relates to a human anti-neurotensin long fragment (LF anti-NTS) antibody or a human anti-neuromedin N long fragment (LF anti-NN) antibody comprising:
  • variable domain comprises: a H-CDR1 having a sequence set forth as SEQ ID NO: 2, a H-CDR2 having a sequence set forth as SEQ ID NO:3; a H-CDR3 having a sequence set forth as SEQ ID NO: 4;
  • variable domain comprises : a L-CDR1 having a sequence set forth as SEQ ID NO: 6; a L-CDR2 having a sequence set forth as SEQ ID NO: 7; a L-CDR3 having a sequence set forth as SEQ ID NO: 8
  • the human anti-neurotensin long fragment (LF anti-NTS) antibody or the human anti-neuromedin N long fragment (LF anti-NN) antibody of the present invention comprises a heavy chain wherein the variable domain comprises at least one CDR having a sequence selected from the group consisting of SEQ ID NO:2 for H-CDR1, SEQ ID NO:3 for H-CDR2 and SEQ ID NON for H-CDR3.
  • the human anti-neurotensin long fragment (LF anti-NTS) antibody or the human anti-neuromedin N long fragment (LF anti-NN) antibody of the present invention comprises a light chain wherein the variable domain comprises at least one CDR having a sequence selected from the group consisting of SEQ ID NO:6 for L-CDR1, SEQ ID NO:7 for L-CDR2 and SEQ ID NO: 8 for L-CDR3.
  • the human anti-neurotensin long fragment (LF anti-NTS) antibody or the human anti-neuromedin N long fragment (LF anti-NN) antibody of the present invention comprises a heavy chain wherein the variable domain comprises at least one CDR having a sequence selected from the group consisting of SEQ ID NO:2 for H-CDR1, SEQ ID NON for H-CDR2 and SEQ ID NON for H-CDR3 and a light chain wherein the variable domain comprises at least one CDR having a sequence selected from the group consisting of SEQ ID NO: 6 for L-CDR1, SEQ ID NON for L-CDR2 and SEQ ID NO: 8 for L-CDR3.
  • the human anti-neurotensin long fragment (LF anti-NTS) antibody or the human anti-neuromedin N long fragment (LF anti-NN) antibody of the present invention comprises a heavy chain variable region comprising SEQ ID NO:2 in the H-CDR1 region, SEQ ID NO:3 in the H-CDR2 region and SEQ ID NO:4 in the H- CDR3 region ; and a light chain variable region comprising SEQ ID NO:6 in the L-CDR1 region, SEQ ID NO:7 in the L-CDR2 region and SEQ ID NO: 8 in the L-CDR3 region.
  • the human anti-neurotensin long fragment (LF anti-NTS) antibody or the human anti-neuromedin N long fragment (LF anti-NN) antibody is having at least 70% of identity with SEQ ID NO: 1 and/or a light chain variable region having at least 70% of identity with SEQ ID NO: 5.
  • 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.
  • the human anti-neurotensin long fragment (LF anti-NTS) antibody or the human anti-neuromedin N long fragment (LF anti-NN) antibody comprises a heavy chain variable region of having the amino acid sequence set forth as SEQ ID NO: 1 and/or a light chain variable region having the amino acid sequence set forth as SEQ ID NO: 5.
  • the invention relates to a human anti-neurotensin long fragment (LF anti- NTS) antibody or a human anti-neuromedin N long fragment (LF anti-NN) antibody comprising an amino acid sequence having 70% (+ until 99%) of identity with SEQ ID NO:1 or SEQ ID NO:5.
  • the invention relates to a human anti-neurotensin long fragment (LF anti-NTS) antibody or a human anti-neuromedin N long fragment (LF anti-NN) antibody comprising: a heavy chain wherein the variable domain has a sequence set forth as SEQ ID NO: 1 a light chain wherein the variable domain has a sequence set forth as SEQ ID NO: 5
  • the VH region of the antibody of the present invention consists of the sequence of SEQ ID NO: 1.
  • the H-CDR1 of the antibody of the present invention 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 El- CDR2 of the antibody of the present invention mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 66 in SEQ ID NO: 1.
  • the H-CDR3 of the antibody of the present invention mab is defined by the sequence ranging from the amino acid residue at position 99 to the amino acid residue at position 112 in SEQ ID NO: 1.
  • the VL region of the antibody of the present invention consists of the sequence of SEQ ID NO:5.
  • the L-CDR1 of mab is defined by the sequence ranging from the amino acid residue at position 23 to the amino acid residue at position 36 in SEQ ID NO:5.
  • the L-CDR2 of mab is defined by the sequence ranging from the amino acid residue at position 52 to the amino acid residue at position 58 in SEQ ID NO: 5.
  • the L-CDR3 of mab is defined by the sequence ranging from the amino acid residue at position 91 to the amino acid residue at position 100 in SEQ ID NO: 5.
  • SEQ ID NO: 5 VL region of antibody of the present invention FR1-CDR1-FR2-CDR2-FR3- CDR3-FR4
  • 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 nonhypervariable 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 residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al.
  • 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, while having relatively little detectable reactivity with nonantigen proteins or structures (such as other proteins 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 Neurotensin long fragment and Neuromedin N long fragment).
  • affinity means 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.
  • 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.
  • Percent (%) amino acid sequence identity with respect to a peptide or polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table A below.
  • the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code shown in Table A below has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU5 10087.
  • the ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, Calif, or may be compiled from the source code provided in FIG. 8 below.
  • the ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • monoclonal antibody refers 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 antibody is isolated.
  • An “isolated” antibody is one, which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials, which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • the invention relates to antibodies that bind to the human neurotensin long fragment (LF NTS) or the human neuromedin N long fragment (LF NN) with a KD of 17.7 nM nM or 2.8 nM respectively.
  • KD is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e. Kd/Ka) and is expressed as a molar concentration (M).
  • KD values for antibodies can be determined using methods well established in the Art. One method for determining the KD of an antibody is by using surface Plasmon resonance, or using a biosensor system such as a Biacore® system.
  • the invention relates to antibodies restoring responses to treatment, ie tumors which are resistant or partially resistant to existing treatments, especially chemotherapeutic agents, targeted therapeutics into tumor fully or partially responsive to existing treatments in patients with cancer.
  • the invention relates to antibodies reestablishing the state of “cold tumor(s)”, ie tumors which have not or poorly been infiltrated with immune cells into “hot tumor(s)”, tumor infiltrated with immune cells rushing to fight the cancerous cells.
  • the invention relates to antibodies that restore the immune cells depletion in the primary and secondary immune organs induced by cancer.
  • Antibodies of the invention restore the depletion of immune cells tumor suppressor.
  • 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 site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • 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.
  • 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 Boerner 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.
  • 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.
  • the antibody of the invention is an antigen binding 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 or SEQ ID NO: 5.
  • 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.
  • 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, SMIP, maxibodies, minibodies, intrabodies, diabodies, triabodies and tetrabodies (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136).
  • 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 present invention further provides fragments said antibodies which include but are not limited to Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2 and diabodies.
  • Fab denotes an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, in which about a half of the N-terminal side of H chain and the entire L chain, among fragments obtained by treating IgG with a protease, papaine, are bound together through a disulfide bond.
  • F(ab')2 refers to an antibody fragment having a molecular weight of about 100,000 and antigen binding activity, which is slightly larger than the Fab bound via a disulfide bond of the hinge region, among fragments obtained by treating IgG with a protease, pepsin.
  • Fab refers to an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, which is obtained by cutting a disulfide bond of the hinge region of the F(ab')2.
  • scFv single chain Fv
  • VH::VL heterodimer which is usually expressed from a gene fusion including VH and VL encoding genes linked by a peptide-encoding linker.
  • dsFv refers to 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.
  • diabodies refers to small antibody fragments with two antigenbinding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light- chain variable domain (VL) in the same polypeptide chain (VH-VL).
  • VH heavy-chain variable domain
  • VL light- chain variable domain
  • the Fab of the present invention can be obtained by treating an antibody which specifically reacts with human neurotensin long fragment (LF NTS) or human neuromedin N long fragment (LF anti-NN) with a protease, papaine.
  • 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 human neurotensin long fragment (LF NTS) or human neuromedin N long fragment (LF anti-NN) 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 human neurotensin long fragment (LF NTS) or human neuromedin N long fragment (LF anti- NN) with a reducing agent, dithiothreitol.
  • 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.
  • CDR grafting 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).
  • 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.
  • 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.
  • single domain antibody has its general meaning in the art and 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 single domain antibody are also called VHH or “nanobody®”.
  • VHH single domain antibody
  • the nanobody has a molecular weight approximately one-tenth that of a human IgG molecule, and the protein has a physical diameter of only a few nanometers.
  • camelid nanobodies are useful as reagents to detect antigens that are otherwise cryptic using classical immunological techniques, and as possible therapeutic agents.
  • nanobody can inhibit as a result of binding to a specific site in a groove or narrow cleft of a target protein, and hence can serve in a capacity that more closely resembles the function of a classical low molecular weight drug than that of a classical antibody.
  • the low molecular weight and compact size further result in nanobodies being extremely thermostable, stable to extreme pH and to proteolytic digestion, and poorly antigenic.
  • nanobodies readily move from the circulatory system into tissues, and even cross the blood-brain barrier and can treat disorders that affect nervous tissue. Nanobodies can further facilitated drug transport across the blood brain barrier. See U.S. patent application 20040161738 published August 19, 2004.
  • the amino acid sequence and structure of a single domain antibody can be considered to be comprised of four framework regions or "FRs” which are referred to in the art and herein as “Framework region 1" or “FR1”; as “Framework region 2" or “FR2”; as “Framework region 3” or “FR3”; and as “Framework region 4" or “FR4” respectively; which framework regions are interrupted by three complementary determining regions or "CDRs”, which are referred to in the art as "Complementarity Determining Region for "CDR1”; as “Complementarity Determining Region 2" or “CDR2” and as “Complementarity Determining Region 3" or “CDR3”, respectively.
  • the single domain antibody can be defined as an amino acid sequence with the general structure : FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4 respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3.
  • the amino acid residues of the single domain antibody are numbered according to the general numbering for VH domains given by the International ImMunoGeneTics information system aminoacid numbering (http://imgt.cines.fr/).
  • hydropathic index of amino acids may be considered.
  • 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).
  • a further object of the present invention also encompasses function-conservative variants of the antibodies of the present invention.
  • “Function-conservative variants” are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like).
  • certain amino acids may be substituted by other amino acids in a protein structure without appreciable loss of activity. Since the interactive capacity and nature of a protein define the protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence, and, of course, in its DNA encoding sequence, while nevertheless obtaining a protein with like properties.
  • amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e. still obtain a biological functionally equivalent protein.
  • amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophihcity, charge, size, and the like.
  • substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • the antibodies 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 binning.
  • 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).
  • the BIACORE® GE Healthcare, Piscaataway, NJ
  • routine cross-blocking assays such as those described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane, 1988, can be performed.
  • Engineered antibodies of the present 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.
  • Another type of 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 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 hinge region of the antibody of the present invention 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.
  • SpA Staphylococcyl protein A
  • the antibody of the present invention is modified to increase its biological half-life.
  • one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent No.
  • 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.
  • 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.
  • 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).
  • the glycosylation of an antibody is modified.
  • an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation).
  • Glycosylation can be altered to, for example, increase the affinity of the antibody for the antigen.
  • 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.
  • 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 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 fucosyltransf erase.
  • 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 acetylglucosaminyltransferase 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.eurekainc.com/a&boutus/companyoverview.html).
  • the 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 ).
  • 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).
  • 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 present 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.
  • An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody.
  • PEG polyethylene glycol
  • nucleic acid sequence Accordingly, a further object of the invention relates to a nucleic acid sequence encoding an antibody of the present invention.
  • the nucleic acid sequence encodes a heavy chain and/or a light chain of an antibody of the present invention.
  • 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.
  • vector means 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 DNA or RNA sequence e.g. a foreign gene
  • a further object 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.
  • 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.
  • viral vector examples include adenoviral, retroviral, herpes virus and AAV vectors.
  • 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.
  • 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 object of the present 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.
  • transformation means the introduction of a "foreign” (i.e. extrinsic or extracellular) gene, DNA or R A 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 RNA bas been "transformed”.
  • the nucleic acids of the invention may be used to produce an antibody of the present invention in a suitable expression system.
  • 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 Baculo virus 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.G1 1.16Ag.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.
  • Such recombinant host cells can be used for the production of antibodies of the present invention.
  • the method comprises the steps of: (i) culturing the hybridoma FLp26-8.2 under conditions suitable to allow expression of FLp26-8.2 antibody; and (ii) recovering the expressed antibody.
  • 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.
  • 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.
  • 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.
  • 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.
  • the fully human antibody of the present invention may be produced by obtaining nucleic acid sequences encoding CDR domains, as previously described, constructing a humanized antibody expression vector by inserting them into an expression vector for animal cell having genes encoding (i) a heavy chain constant region identical to that of a human antibody and (ii) a light chain constant region identical to that of a human antibody, and expressing the genes by introducing the expression vector into an animal cell.
  • the fully human antibody expression vector may be either of a type in which a gene encoding an antibody heavy chain and a gene encoding an antibody light chain exists on separate vectors or of a type in which both genes exist on the same vector (tandem type).
  • humanized antibody expression vector of the tandem type In respect of easiness of construction of a humanized antibody expression vector, easiness of introduction into animal cells, and balance between the expression levels of antibody H and L chains in animal cells, humanized antibody expression vector of the tandem type is preferred (Shitara K et al. 1994).
  • tandem type humanized antibody expression vector include pKA TEX93 (WO 97/10354), pEE18 and the like.
  • Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska MA. et al. (1994)), and chain shuffling (U.S. Pat. No.5,565,332).
  • the general recombinant DNA technology for preparation of such antibodies is also known (see European Patent Application EP 125023 and International Patent Application WO 96/02576).
  • a further object of the present invention relates to a method of treating cancer in a subject in need thereof comprising administering the subject with a therapeutically effective amount of an antibody of the present invention.
  • treatment refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
  • 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 antibody 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 antibody 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 antibody or antibody portion are outweighed by the therapeutically beneficial effects.
  • the efficient dosages and dosage regimens for the antibody 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 pharmaceutical composition required.
  • a suitable dose of a 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 cancer may, for example, be evaluated in an animal model system predictive of efficacy in human tumors.
  • 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.
  • 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.
  • 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 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
  • Tumors to be treated include primary tumors and metastatic tumors, as well as refractory tumors.
  • Refractory tumors include tumors that fail to respond or are resistant to treatment with chemotherapeutic agents alone, antibodies alone, radiation alone or combinations thereof.
  • Refractory tumors also encompass tumors that appear to be inhibited by treatment with such agents, but recur up to five years, sometimes up to ten years or longer after treatment is discontinued.
  • cancers that may be treated by methods and compositions of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acid
  • the patient suffers from a cancer deriving from epithelial origin.
  • cancer types include, but are not limited to, carcinoma, lymphoma, blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinoma, and islet cell cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies.
  • cancers include squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small-cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer, esophagael cancer, tumors of the biliary tract, as well as head and neck cancer, as well as subtypes of any of such cancers, including, but not limited to chemotherapy
  • SCLC small
  • the present invention relates to a method of treating of preventing weight loss, muscle loss and/or protein blood level decrease in a subject in need thereof comprising administering the subject with a therapeutically effective amount of an antibody of the present invention.
  • the subject is underweight.
  • underweight refers to a subject having a body mass index of below 18.5.
  • body mass index has its general meaning in the art and refers to refers to the ratio which is calculated as body weight per height in meter squared (kg/m 2 ). The BMI provides a simple means of assessing how much an individual's body weight departs from what is normal or desirable for a person of his or her height.
  • BMI braceid — starvation: BMI — less than 15 kg/m 2 ; underweight — BMI less than 18.5 kg/m 2 ; ideal — BMI from 18.5 to 25 kg/m 2 ; overweight — BMI from 25 to 30 kg/m 2 ; obese — BMI from 30 to 40 kg/m 2 ; morbidly obese — BMI greater than 40 kg/m 2 .
  • the method of the present invention is particularly suitable for inhibiting the lipolysis of white adipose tissue, and the loss of skeletal muscle. In some embodiments, the method of the present invention is particularly suitable for stimulating appetite.
  • Underweight may be due to several causes, such as rapid metabolism, poor/inadequate diet or starvation (malnutrition), malabsorption due to defective intestinal function, endocrine disturbances e.g. type I diabetes, psychological problems (such as anorexia nervosa, body dysmorphic disorder, stress and anxiety) and weight loss, due to chronic illnesses and ageing. While in general the underlying cause of the underweight will have to be treated per se, the underweight too may be a health hazard, and as such have to be treated in itself.
  • underweight generally have poor physical stamina, a weakened immune system, as well as being at higher risk of developing diseases such as osteoporosis, heart disease and vascular disease. Additionally, in the female sex, underweight can lead to delayed sexual development, retarded amenorrhoea or complications during pregnancy.
  • the subject suffers from a wasting disorder.
  • wasting disorder has its general meaning in the art and includes but is not limited to anorexia cachexia, anorexia of the aged, anorexia nervosa, cachexia associated with cancer, cachexia associated with AIDS, cachexia associated with heart failure, cachexia associated with cystic fibrosis, cachexia associated with rheumatoid arthritis, cachexia associated with kidney disease, cachexia associated with chronic obstructive pulmonary disease (COPD), cachexia associated with ALS, cachexia associated with renal failure or cachexia associated, and other disorders associated with aberrant appetite, fat mass, energy balance, and/or involuntary weight loss.
  • COPD chronic obstructive pulmonary disease
  • the subject suffers from “cachexia”.
  • cachexia is used for a condition of physical wasting with loss of body fat and muscle mass.
  • cachexia may be associated with and due to conditions such as cancer, required immunodeficiency syndrome (AIDS), cardiac diseases, infectious diseases, shock, burn, endotoxinemia, organ inflammation, surgery, diabetes, collagen diseases, radiotherapy, and chemotherapy.
  • AIDS required immunodeficiency syndrome
  • cardiac diseases infectious diseases, shock, burn, endotoxinemia, organ inflammation, surgery, diabetes, collagen diseases, radiotherapy, and chemotherapy.
  • cachexia may significantly contribute to morbidity or mortality.
  • Another particular group of individuals that are susceptible to developing a cachectic state are those individuals that have undergone a gastrectomy, such as may be practiced on gastric cancer and ulcer patients.
  • the subject suffers from anorexia.
  • anorexia has its general meaning in the art and refers to any eating disorder characterized by markedly reduced appetite or total aversion to food.
  • the subject suffers from anorexia nervosa.
  • subjects suffering from anorexia nervosa have a BMI of less than 17.5 kg/m2.
  • the present invention is drawn to methods of treating a patient exhibiting one or more wasting disorders such as anorexia, cachexia, anorexia of the aged, anorexia nervosa, cachexia associated with cancer, cachexia associated with AIDS, cachexia associated with heart failure, cachexia associated with cystic fibrosis, cachexia associated with rheumatoid arthritis, cachexia associated with kidney disease, cachexia associated with COPD, cachexia associated with ALS, cachexia associated with renal failure or cachexia associated, or hip fracture, and in reducing the mortality and morbidity of critically ill patients, comprising administering to said patient in need of such treatment a therapeutically effective of an inhibitor of NTSR1 activation or expression.
  • wasting disorders such as anorexia, cachexia, anorexia of the aged, anorexia nervosa, cachexia associated with cancer, cachexia associated with AIDS, cachexia associated with heart failure, cachexia associated with cystic fibrosis, cachexia associated with rheumatoid arthritis, cache
  • the present invention also provides for therapeutic applications where an antibody of the present invention is used in combination with at least one further therapeutic agent, e.g. for treating cancer.
  • Such administration may be simultaneous, separate or sequential.
  • the agents may be administered as one composition or as separate compositions, as appropriate.
  • the further therapeutic agent is typically relevant for the disorder to be treated.
  • Exemplary therapeutic agents include other 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 antibody of the present invention is used in combination with a chemotherapeutic agent.
  • a “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer.
  • chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines
  • dynemicin including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYC1NO, morpholino-doxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HC1 liposome injection (DOXIL®) and de
  • the chemotherapeutic agent is chosen from alkilating agent, isotopomerase inhibitors, antifofate, or microtubule disruptor.
  • the chemotherapeutic agent is chosen from the cisplatin and/or paclitaxel, Carboplatin and/or Pemetrexed.
  • the antibody is useful for restore the sensibility of cancer cells to chemotherapeutic agent, such as platinum-based antineoplastic drugs.
  • chemotherapeutic agent such as platinum-based antineoplastic drugs.
  • platinumbased antineoplastic drugs include cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, nedaplatin, triplatin and lipoplatin.
  • the invention relates to a method of treatment for “cold tumor(s)”, ie tumors which are not responding or partially responding to existing treatments, especially chemotherapeutic agents or targeted cancer therapeutics: antibodies of the present invention will turn those “cold tumor(s)” into “hot tumor(s)”, with a synergetic effect with chemotherapeutic agents.
  • the antibody 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. 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.
  • 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-
  • 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 antibody 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.
  • CSF colony stimulating factor
  • 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).
  • G-CSF Neupogen®
  • Amgen Neulasta
  • Leukine GM-CSF
  • Berlex Procrit
  • Procrit erythropoietin
  • Ortho Biotech Epogen
  • Epogen erythropoietin
  • Arnesp erytropoietin
  • Combination compositions and combination administration methods of the present invention may also involve "whole cell” and "adoptive" immunotherapy methods.
  • 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
  • Cellular “vaccines” in clinical trials that may be useful in such aspects include CanvaxinTM, 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
  • adjuvants such as alum
  • the antibody of the present invention is used in combination with an immune checkpoint inhibitor.
  • immune checkpoint inhibitor has its general meaning in the art and refers to any compound inhibiting the function of an immune inhibitory checkpoint protein.
  • immune checkpoint protein has its general meaning in the art and refers to a molecule that is expressed by T cells in that either turn up a signal (stimulatory checkpoint molecules) or turn down a signal (inhibitory checkpoint molecules).
  • Immune checkpoint molecules are recognized in the art to constitute immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see e.g. Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al. , 2011. Nature 480:480- 489).
  • inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD- 1, LAG-3, TIM-3 and VISTA.
  • Inhibition includes reduction of function and full blockade.
  • Preferred immune checkpoint inhibitors are antibodies that specifically recognize immune checkpoint proteins. A number of immune checkpoint inhibitors are known and in analogy of these known immune checkpoint protein inhibitors, alternative immune checkpoint inhibitors may be developed in the (near) future.
  • the immune checkpoint inhibitors include peptides, antibodies, nucleic acid molecules and small molecules. Examples of immune checkpoint inhibitor includes PD-1 antagonist, PD-L1 antagonist, PD-L2 antagonist CTLA-4 antagonist, VISTA antagonist, TIM-3 antagonist, LAG-3 antagonist, IDO antagonist, KIR2D antagonist, A2AR antagonist, B7-H3 antagonist, B7-H4 antagonist, and BTLA antagonist.
  • PD-1 (Programmed Death- 1) axis antagonists include PD-1 antagonist (for example anti-PD-1 antibody), PD-L1 (Programmed Death Ligand-1) antagonist (for example anti-PD-Ll antibody) and PD-L2 (Programmed Death Ligand-2) antagonist (for example anti-PD-L2 antibody).
  • the anti-PD-1 antibody is selected from the group consisting of MDX-1106 (also known as Nivolumab, MDX-1106-04, ONO-4538, BMS-936558, and Opdivo®), Merck 3475 (also known as Pembrolizumab, MK-3475, Lambrolizumab, Keytruda®, and SCH-900475), and CT-011 (also known as Pidilizumab, hBAT, and hBAT-1).
  • the PD-1 binding antagonist is AMP -224 (also known as B7-DCIg).
  • the anti-PD-Ll antibody is selected from the group consisting of YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736.
  • MDX-1105 also known as BMS-936559, is an anti-PD-Ll antibody described in W02007/005874.
  • Antibody YW243.55. S70 is an anti-PD-Ll described in WO 2010/077634
  • AL MEDI4736 is an anti-PD- Ll antibody described in WO2011/066389 and US2013/034559.
  • MDX-1106 also known as MDX-1 106-04, ONO-4538 or BMS-936558, is an anti-PD-1 antibody described in U.S. Pat. No.
  • Merck 3745 also known as MK-3475 or SCH-900475, is an anti-PD-1 antibody described in U.S. Pat. No. 8,345,509 and W02009/114335.
  • CT-011 Panizilumab
  • AMP -224 also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in W02010/027827 and WO2011/066342.
  • Atezolimumab is an anti-PD-Ll antibody described in U.S. Pat. No. 8,217,149.
  • Avelumab is an anti-PD-Ll antibody described in US 20140341917.
  • CA-170 is a PD-1 antagonist described in W02015033301 & WO2015033299.
  • Other anti-PD-1 antibodies are disclosed in U.S. Pat. No. 8,609,089, US 2010028330, and/or US 20120114649.
  • the PD-1 inhibitor is an anti-PD-1 antibody chosen from Nivolumab, Pembrolizumab or Pidilizumab.
  • PD-L1 antagonist is selected from the group comprising of Avelumab, BMS-936559, CA-170, Durvalumab, MCLA-145, SP142, STI-A1011, STIA1012, STI-A1010, STI-A1014, Al 10, KY1003 and Atezolimumab and the preferred one is Avelumab, Durvalumab or Atezolimumab.
  • CTLA-4 Cytotoxic T-Lymphocyte Antigen-4 antagonists are selected from the group consisting of anti-CTLA-4 antibodies, human anti-CTLA-4 antibodies, mouse anti-CTLA-4 antibodies, mammalian anti-CTLA-4 antibodies, humanized anti-CTLA-4 antibodies, monoclonal anti-CTLA-4 antibodies, polyclonal anti-CTLA-4 antibodies, chimeric anti-CTLA-4 antibodies, MDX-010 (Ipilimumab), Tremelimumab, anti-CD28 antibodies, anti- CTLA-4 adnectins, anti-CTLA-4 domain antibodies, single chain anti-CTLA-4 fragments, heavy chain anti-CTLA-4 fragments, light chain anti-CTLA-4 fragments, inhibitors of CTLA- 4 that agonize the co-stimulatory pathway, the antibodies disclosed in PCT Publication No.
  • CTLA-4 antibodies are described in U.S. Pat. Nos. 5,811,097; 5,855,887; 6,051,227; and 6,984,720; in PCT Publication Nos. WO 01/14424 and WO 00/37504; and in U.S. Publication Nos. 2002/0039581 and 2002/086014.
  • Other anti-CTLA-4 antibodies that can be used in a method of the present invention include, for example, those disclosed in: WO 98/42752; U.S. Pat.
  • a preferred clinical CTLA-4 antibody is human monoclonal antibody (also referred to as MDX-010 and Ipilimumab with CAS No.
  • CTLA-4 antagonist antibodies
  • Tremelimumab CP-675,206
  • Ipilimumab Ipilimumab
  • the immunotherapy consists in administering to the subject a combination of a CTLA-4 antagonist and a PD-1 antagonist.
  • immune-checkpoint inhibitors include lymphocyte activation gene-3 (LAG-3) inhibitors, such as IMP321, a soluble Ig fusion protein (Brignone et al., 2007, J. Immunol. 179:4202- 4211).
  • Other immune-checkpoint inhibitors include B7 inhibitors, such as B7-H3 and B7-H4 inhibitors.
  • the anti-B7-H3 antibody MGA271 (Loo et al., 2012, Clin. Cancer Res. July 15 (18) 3834).
  • TIM-3 T-cell immunoglobulin domain and mucin domain 3) inhibitors (Fourcade et al., 2010, J. Exp. Med.
  • TIM-3 has its general meaning in the art and refers to T cell immunoglobulin and mucin domain-containing molecule 3.
  • the natural ligand of TIM-3 is galectin 9 (Gal9).
  • TIM-3 inhibitor refers to a compound, substance or composition that can inhibit the function of TIM-3.
  • the inhibitor can inhibit the expression or activity of TIM-3, modulate or block the TIM-3 signaling pathway and/or block the binding of TIM-3 to galectin-9.
  • Antibodies having specificity for TIM-3 are well known in the art and typically those described in WO201 1155607, W02013006490 and WO2010117057.
  • the immune checkpoint inhibitor is an IDO inhibitor.
  • IDO inhibitors are described in WO 2014150677.
  • IDO inhibitors include without limitation 1-methyl-tryptophan (IMT), P- (3-benzofuranyl)-alanine, P-(3-benzo(b)thienyl)- alanine), 6-nitro-tryptophan, 6- fluoro-tryptophan, 4-methyl-tryptophan, 5 -methyl tryptophan, 6-methyl-tryptophan, 5 -methoxy -tryptophan, 5 -hydroxy-tryptophan, indole 3-carbinol, 3,3'- diindolylmethane, epigallocatechin gallate, 5-Br-4-Cl-indoxyl 1,3 -di acetate, 9- vinylcarbazole, acemetacin, 5 -bromo-tryptophan, 5 -bromoindoxyl diacetate, 3- Amino-naph
  • 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-i l l.
  • the second agent is an agent that induces, via ADCC, the death 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, aVp3, 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, aVp3, 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, aVp3, etc., particularly lymphoma antigens (
  • a further object relates to a method of enhancing NK cell antibody-dependent cellular cytotoxicity (ADCC) of an antibody in a subject in need thereof comprising administering to the subject the antibody, and administering to the subject an antibody of the present invention.
  • ADCC NK cell antibody-dependent cellular cytotoxicity
  • the antibody of the present invention is administered to the subject in the form of a pharmaceutical composition which comprises a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, di sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene- block polymers, polyethylene glycol and wool fat.
  • compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • the used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
  • Sterile injectable forms of the compositions of this invention may be aqueous or an oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include, e.g., lactose.
  • the active ingredient is combined with emulsifying and suspending agents.
  • certain sweetening, flavoring or coloring agents may also be added.
  • the compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug.
  • suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug.
  • Such materials include cocoa butter, beeswax and polyethylene glycols.
  • compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.
  • the compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers.
  • Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.
  • compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers.
  • suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyl dodecanol, benzyl alcohol and water.
  • Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Patches may also be used.
  • the compositions of this invention may also be administered by nasal aerosol or inhalation.
  • compositions are prepared according to techniques well- known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.
  • an antibody present in a pharmaceutical composition of this invention can be supplied at a concentration of 10 mg/mL in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials.
  • the product is formulated for IV administration in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mL polysorbate 80, and Sterile Water for Injection. The pH is adjusted to 6.5.
  • An exemplary suitable dosage range for an antibody in a pharmaceutical composition of this invention may between about 1 mg/m 2 and 500 mg/m 2 .
  • schedules are exemplary and that an optimal schedule and regimen can be adapted taking into account the affinity and tolerability of the particular antibody in the pharmaceutical composition that must be determined in clinical trials.
  • a pharmaceutical composition of the invention for injection e.g., intramuscular, i.v.
  • liposomes and/or nanoparticles are contemplated for the introduction of antibodies 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.
  • SUVs small unilamellar vesicles
  • the physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Kit
  • kits comprising at least one antibody of the invention.
  • Kits containing antibodies of the invention find use in detecting human neurotensin long fragment (LF NTS) or human neuromedin N long fragment (LF anti-NN) expression (increase or decrease), or in therapeutic or diagnostic assays.
  • Kits of the invention can contain an antibody coupled to a solid support, e.g., a tissue culture plate or beads (e.g., sepharose beads).
  • Kits can be provided which contain antibodies for detection and quantification of human neurotensin long fragment (LF NTS) or human neuromedin N long fragment (LF anti-NN) in vitro, e.g. in an ELISA or a Western blot.
  • Such antibody useful for detection may be provided with a label such as a fluorescent or radiolabel.
  • FIGURES are a diagrammatic representation of FIGURES.
  • FIG. 1 LF NTS and LF NN increased tumor growth and induced cachexia.
  • A Schematic representation of NTS precursor maturation. In brain, adrenal, and endocrine N cells of intestine, NTS precursor is cleaved with PCI, PC2, PC5A, to generate two active peptides, NN 6 aa, NTS 13aa, and the Remnant polypeptide 140 aa. Under over production or abnormal production of NTS precursor, Long form of NTS (LF NTS), form of NN (LF NN), and the proform (PF) exhibiting the biological activity similar to the mature peptide, but more stable and more active.
  • LF NTS Long form of NTS
  • LF NN form of NN
  • PF proform
  • NAM02 reduces the tumor growth.
  • A Western blot analysis, 0.5 pg were loaded on the gel for fragments R and PF, and 0.2 pg for FL-NN and FL-NTS and revealed with 3 pg/ml of NAM02.
  • NAM02 improves the response to Lung cancer and neuroblastoma standard of care.
  • A Tumor volume follow up generated by LNM-R cells and treated with US or EU SOC in combination or not with NAM02 (see protocol). The graph represents the percentage of change from DI and show the mean and the individual scores. In two-way ANOVA statistical analysis using Sidak Test for SOC vs SOC + NAM02 p ⁇ O.Olat DI 5, and p ⁇ 0.0001 at, D17 and D19.
  • B Volume of tumors after dissection. In one-way ANOVA, *p ⁇ 0.05,
  • C Fold increase analysis of the tumor growth rate between SOC group and the combination group over time. At each time point the proportion of animals reaching the determined fold increase was calculated. Chi-square statistical analysis *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • D Tumor volume follow up generated by the neuroblastoma cels, SK-NF-I,and treated with SOC in combination or not with NAM02 (see protocol). The graph represents the percentage of change from DI and show the mean and the individual scores.
  • NAM02 downregulates the oncogenic signaling in experimental tumors.
  • the histograms represent the ratio of the phosphorylated form/total form based on band intensity quantification of Western blot.
  • the proteins were extracted from an LNM-R experimental tumor treated three times with NAM02 with 5mg/kg at DI, D5, and D8.
  • NAM02 prevents the emptying of epididymal white adipose tissues.
  • A Size of epidi dymal white adipocytes from mice bearing tumors treated with PBS or NAM02 as describes in figure 2. In t test statistical analysis, **** p ⁇ 0.001.
  • NAM02 counteracts the immune cells depletion induced by LF NN a in tumor microenvironment (TME) and primary and secondary lymphoid organs.
  • TME tumor microenvironment
  • NAM02 up regulate expression of PD-L1 in lung cancer model.
  • the histograms represent quantification of PD-L1 band intensity of Western blot.
  • C The proteins were extracted from LNM-R experimental tumors mice were treated three times with PBS or NAM02 at 5 mg/kg on DI, D5 and D8; *p ⁇ 0.05, **p ⁇ 0.01.
  • the cancer cell lines, LNM-R, NCI-H460., and SK-N-KI were grown in DMEM (Gibco®) supplemented with 10% fetal bovine serum (FBS) (Gibco®) and 2 mM glutamine and grown at 37 °C, in a humidified atmosphere of 5% CO2.
  • Athymic 4-week-old male NMRI-Foxnlnu/nu mice (JanvierTM), were injected with 1 million lung cancer cells for lung cancer cells and 5 million for neuroblastoma cells in 50 % Matrigel (v/v).
  • the ellipsoid formula was used to calculate the tumor volumes. When tumors reached the expected size (see example), groups of mice were randomized. All the procedures were in accordance with the “Guide of the Care and Use of Laboratory Animals”. Institutional Review Board approval was obtained by «Le Comite d'Ethique en 1'Experimentation Animale Charles Darwin # B751201».
  • the primary antibodies anti-p-P38 (1/250) sc-535 anti-P38 (1/250) sc-17852, anti-P-JNK (1/250) sc-6254, anti-JNK (1/250) sc-7345, anti-c-JUN (1/250) Sc-75543, were purchased from Santa Cruz Biotechnology®.
  • the primary antibody anti actin A5442 was provided by Sigma®.
  • Primary antibodies were incubated overnight at 4 °C according to the manufacturer’s instructions.
  • Secondary antibody Anti-Mouse IgG-HRP (1/10 000) A6782 and Anti-rabbit IgG- HRP (1/10000) #7074S were purchased from Sigma and Cell Signaling Technology®, respectively. Secondary antibodies were incubated Ih at RT and visualized by enhanced chemiluminescence (PierceTM ECL 2 Western Blotting Substrate, Thermo ScientificTM).
  • NAM02 mAb was injected i.v. at the dose of 5 mg/kg, once a week.
  • the purified polypeptide LF NTS, LF NN, PF and R were obtained from GenScript. Each polypeptide was injected at the dose of 550 fmol/ml of blood i.v. three times a week, alternatively in the right or the left side of the peritoneum. PBS was used as control therapy.
  • the average volume of blood per mice was estimated at 2.5 ml. This concentration corresponds to three folds of the median of the fourth quartile described by (39). Chemotherapy treatments were injected i.v. at clinically relevant concentration.
  • Adipose tissues were fixed in paraformaldehyde then paraffin embedded. Slides of 5 pm were stained with hematoxylin and eosin. Images were acquired with 1X83 Olympus microscope and ORCA/4 Hamamatsu camera at objective 10 with phase contrast. Adipocyte sizes were obtained after binary transformation with ImageJ 1.53c software (40).
  • spleen, tumors, and lymph nodes were placed in a dissociation buffer composed of DMEM supplemented with 10% FCS (Gibco), 0.24% collagenase A (Roche) and 0.03% DNAse 1 (Roche).
  • FCS Gibco
  • 0.24% collagenase A 0.24% collagenase A
  • DNAse 1 0.03% DNAse 1
  • the spleens and thymus were dissociated in DMEM and supplemented with 10% FCS with a piston syringe and through a 70 pm filter for the spleen. After centrifugation at 700 g for 10 min and 4°C, the cells were dispersed in 5 ml of lysis buffer (ThermoFisher) for 10 min at RT. After adding 10 ml DMEM supplemented with 10% FCS, cells were centrifuged at 700 g for 10 min. The cell pellets were resuspended in PBS containing 5% BSA.
  • lysis buffer ThermoFisher
  • the tumors and lymph nodes were divided into small dices and placed in the Dissociator GentleMACS, (Miltenyi Biotech), for 40 min at 37°C.
  • the cellular suspension was then dispersed through a 70 pm filter. Only the healthy portion of the tumor was dispersed by the dissociator and only the equivalent of 0.5g was loaded on the Ficoll gradient.
  • the 30 ml cell suspension was then loaded on the top of 10 ml Ficoll-Plaque Premium 1084 ficoll. The tube was centrifuged for 25 min at 1025g and 20 °C.
  • the PBMC Peripheral Blood Mononuclear Cells
  • DMEM Peripheral Blood Mononuclear Cells
  • FCS fetal bovine serum
  • the cell pellets were resuspended in PBS containing 5% BSA.
  • cells were distributed at 1.5 xlO 6 cells/tubes in 300 pl PBS and centrifuged at 700 g for 5min.
  • markers panel # 1 Two panels of markers panel # 1 contained (CD45, CD3, CD4, CD8, NK1.1 and CDl lc markers, and Super Bright Buffer), the panel #2 contained (viability,CD19, F4/80, CD206, CD86 markers and Super Bright Buffer).
  • EXAMPLE 1 Effect of LF NTS and LF NN on tumor progression and cachexia, and immune system
  • Neurotensin is produced from a precursor cleaved by convertases to release two peptides, neurotensin (13 aa) and Neuromedin N (6 aa), and a polypeptide the remnant form (140 aa). Under over or abnormal production of NTS precursor, or lack of the appropriate convertases, long active forms of NTS and NN can be released from the cells, LF NTS and LF NN exhibit 163 and 148 aa, respectively.
  • the Proform (PF) (170 aa) is cleaved by the carboxypeptidase E (enkephalin convertase) outside of the cells and is transformed in LF NTS (figure 1A).
  • NTS and NN The human lung cancer cells NCI-H460, from lung pleural effusion, express the high affinity neurotensin receptor (NTSR1) and weakly NTS. Cells were xenografted on nude mice, when the tumor burden reached around 80 mm 3 , groups of 8 animals were formed and treated with 550 fmol/ml of each polypeptide or PBS at DI, D4, D8, DI 1, D15, D18, and D22. The tumor volume was followed over time. All active fragments PF, LF NTS, and LF NN enhanced the tumor growth.
  • Figure IB display the tumor growth rate of each group.
  • the LF NN was the most effective with an increase of tumor growth of 22.7 ⁇ 3.4 folds as compared to 14.8 ⁇ 2.9 folds for control mice, over the 22 days period.
  • LF NTS and PF groups had a tumor growth rate of 20.7 ⁇ 6 and 20.4 ⁇ 6.2 respectively.
  • fragment 24-140 (Remnant) is non-active with a growth rate of 12.1 ⁇ 2.1.
  • Figure 1C shows that the number of mitoses per mm 2 is 60% increased between the PBS or R treated animals and the active forms of NTS (LF NTS, LF NN, and PF).
  • the data presented here confirm the oncogenic effect of LF NTS and LF NN.
  • LF NTS, LF NN and PF treatment induced a drastic decreased epididymal white adipose tissue weight (Figure ID).
  • EXAMPLE 2 Effect of NAM 02 on tumor growth.
  • NAM02 recognized all the form of miss- cleaved, in particular the active form on NTSR1, PF, LF NTS and LF NN ( Figure 2A).
  • the performance of NAM02 on tumor growth was testing on LNM-R cells, a very aggressive and highly metastatic lung cancer cell line overexpressing NTS and NTSR1 (16).
  • the figure 2B represents % of tumor size change from DI and shows the mean and individual scores.
  • the average of burden size was 78 ⁇ 8 and 84 ⁇ 10 mm 3 for the control group and NAM02 treated group.
  • the mice were treated with NAM02 at the dose of 5 mg/kg once a week three times. While in the control group the tumor grows tremendously, in the animals treated with NAM02 the tumor size is reduced by 30% at D 15 and 40% at day 19 (figure 2B).
  • the tumor growth rate and the number of mitoses per mm 2 confirmed loss of aggressiveness with a reduction of 45% and 40% respectively in animals treated with NAM02 (figures 2C).
  • EXAMPLE 3 Effect ofNAM02 on chemotherapy response.
  • a fully human antibody NAM02 has been selected for its ability to improve the response to the US or EU standard of care (SOC) for lung cancer.
  • SOC is the combination carboplatin with paclitaxel
  • UE SOC is the combination cisplatin, with pemetrexed.
  • the rationale for this experiment being the possible future inclusion of the LF-NN/NTS antibody to the standard of care to improve the response.
  • Mice were treated with subtoxic doses of SOC (see methods).
  • the drug response of LNM-R tumor was similar for the SOCs and NAM02 as compared to control, with a tumor size reaching at Day 19, 1625 ⁇ 118 and 1391 ⁇ 156mm 3 , respectively.
  • mice treated with the combination a global shrinkage of the tumor was observed up to D28, whereas in the SOC group a small tumor growth regression was observed, with no real shrinkage. In addition, with time some tumors of the SOC group became out of control. As in the case of lung model, in mice treated with the combination, at the individual level, the tumor size is less dispersed. At D28 some tumor started to growth again, unfortunately this model did not allow to do a second cycle.
  • NTSR1 activation by its cognate agonist induces many oncogenic cellular effects including proliferation, survival, migration, lack of adherence, invasion (13, 17). These effects were associated with several oncogenic pathways, such as PI3K-Akt, MEK-ERK, JNK-c-Jun, FAK- Scr signaling, in different types of cancers (18-24).
  • PI3K-Akt, MEK-ERK, JNK-c-Jun, FAK- Scr signaling in different types of cancers (18-24).
  • NTSR1/NTS is associated with the tumor aggressiveness because under autocrine and/or paracrine regulation NTSR1 is permanently recycled to the cell surface and chronically activated (18, 25, 26). These changes in cellular homeostasis lead to the sustained activation of EGFR, HER2 and HER3 by their own ligands through the activation of metalloproteinases, mimicking simultaneous driving mutation on the three growth factor receptors (17, 27).
  • the stable NTSR1 ligands, LF NTS and LF NN create a permanent tumor cells autocrine and/or paracrine NTSR1 chronic activation causing a continue activation of MEK-ERK, PI3K-Akt, JNK-c-Jun, FAK-Scr, signaling pathways known to promote tumorigenesis and tumor progression.
  • NTS mediates inflammation and cytokines (IL-6, 11-8,..) released by several types of cancers which also directly or indirectly participate in the gravity of the disease and alter the response to treatment (28-30).
  • mice were xenografted with LNM-R cells on both flanks. When burden reached 20 mm 3 mice were treated with PBS or NAM02 at DI, D5 and D8. Mice were killed 24h after the last injection. Tumor protein extract was performed on frozen tissues and analyzed by western blot. The graphs in figure 4 represent the ratio of the band intensity from the phosphorylated protein by the total protein. Actin was used as a loading control. A 50% reduction of AKT and ERK signaling (figures 4A and 4B) was observed in experimental LNM- R tumors under NAM02 exposure in agreement with the decreased tumor growth described in example 2.
  • Example 1 showed that LF NTS is implicated in cachexia induced by cancer. Previous results also indicated that LF NTS murine antibodies prevent or delay the cachexia induced by cancer (8, 31).
  • the epididymal adipocytes size from animals treated with NAM02 is decreased by 20% as compared to control (figure 5A). Size distribution of the adipocytes confirmed this result, with more adipocytes with larger size and less with smaller size in animals treated with NAM02 (figure 5B).
  • EXAMPLE 6 Effect of LF NN and NAM02 on immune system.
  • the study was performed in the syngeneic lung cancer model of Lewis using the mouse lung cancer cells, LLC-1. 500,000 LLC1 cells were injected in the left and right flanks of the 4- week-old C57BL/6J male mice. When the burdens reached 20 to 50 mm3, four groups of mice were randomly made, control, MAN02, LF NN, and the combined group, LF NN plus NAM02. The treatments were injected at day 1, 3, 5, 8, 10 and 12. The last injection was performed 24h before the sacrifice. The treatment was injected in the retro-orbital sinus of the mice in a maximal volume of 100 pl.
  • LF NN synthesized and purified by GenScript were injected at the concentration of 500 pmol/L. This dose refers to 3 times the median of the 4th quartile described by O Melander group (JAMA 2012). The population within this quartile showed a higher risk of breast cancer, diabetes mellitus, cardiovascular disease, and mortality.
  • NAM02 group was treated at the dose of 0.7 mg/kg (55,000 pmol/L). ° This represents a 100-fold ratio (w/w) for NAM02 antibodies and FL NN.
  • FL NN and NAM02 were pre-incubated 2 hours at room temperature on slow-turning wheel before injection.
  • T lymphocytes CD8 +, natural killer cells, dendritic cells and macrophages were strongly reduced in mice treated with LF NN (figures 6A). In all cases this decline was counteracted in the presence of NAM02 (figures 6A).
  • the quantity of tumor macrophages was reduced by 20% in LF NN treated mice as compared to control, but only the differentiation stage (Ml) was altered (figure 6A).
  • Ml differentiation stage
  • NAM02 up regulates PD-L1 in tumor cells.
  • a combination of NAM02, anti PD-L1/PD-1 would i) increase the proportion of responders, ii) provide an alternative in case of resistance, iii) improve tolerability and compliance by adjusting initial doses in induction and maintenance.
  • LF NTS and LF NN can be defined as proactive factors for tumor progression and tumor cell aggressiveness.
  • the NTSR1 gene is normally highly repressed, but de-repressed during the early events of carcinogenesis because of the wnt-Beta-catenin activation pathway and/or epigenetic regulation (32, 33).
  • NTS promoter is very reactive, notably on the proximal CRE/AP-1 responsive element (34).
  • the NTS gene can also be open to the transcription under epigenetic regulation (35, 36).
  • the NTS gene is triggered by several types of stimulus, such as chemotherapy, stress, infection, inflammation, in cancer cells as well as in immune cells (8-12). Therefore, the activation of the receptor localized on the tumor may arrive from immune or tumor cells.
  • NTSR1 When NTSR1 is expressed at the cell surface, its chronic and sustained activation induces auto self-feeding regulation, because the NTSR1 activated the Src and RAS oncogenes, stimulating NTS gene expression (Data not shown .
  • the saturating concentration of NTS agonists induced the activation of NTSR1 gene and a permanent receptor recycling.
  • NTSR1 chronic activation results in the sustained stimulation of several oncogenic pathways (MEK-ERK, PI3K-Akt, JNK-c-Jun, FAK-Scr), which increase aggressiveness of tumor phenotype (24, 37).
  • MEK-ERK PI3K-Akt
  • JNK-c-Jun PI3K-Akt
  • JNK-c-Jun PI3K-Akt
  • JNK-c-Jun a therapeutic tool to reduce NTSR1 activation and expression and restore vulnerability of tumor cells to chemotherapy.
  • our results confirmed that the entire oncogenic signaling induced by the complex LF NTS-NN/NTSR1 are concomitantly down regulated with NAM02, lessening high metastatic process and improving the drug response.
  • the effect of NAM02 on the oncogenic signaling confirm its role as a major oncogenic driver of this complex when this these regulation loops NTRS1/FL NTS/NN take place.
  • Kitabgi P Differential processing of pro-neurotensin/neuromedin N and relationship to pro-hormone convertases. Peptides. 2006;27(10):2508-14.
  • Kisfalvi K Hurd C, Guha S, Rozengurt E. Induced overexpression of protein kinase DI stimulates mitogenic signaling in human pancreatic carcinoma PANC-1 cells. Journal of cellular physiology. 2010;223(2):309-16.

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Abstract

The present invention relates to an antibody, which is capable of binding to the Neuromedin N long fragment, and Neurotensin long fragment with high affinity. The antibody of the present invention neutralises the activity of the Neuromedin N long fragment, and Neurotensin long fragment, in particular their oncogenic activities. In particular, the present invention relates to a neutralising human antibody which binds to the Neuromedin N long fragment, and Neurotensin long fragment, and having a heavy chain variable region which comprises a H-CDR1 region having at least 90% of identity with SEQ ID NO:2, a H- CDR2 region having at least 90% of identify with SEQ ID NO:3 and a H-CDR3 region having at least 90% of identity with SEQ ID NO:4; and a light chain variable region comprising a L-CDR1 region having at least 90% of identity with SEQ ID NO:6, a L-CDR2 having at least 90% of identity with SEQ ID NO:7 and a L-CDR3 region having at least 90% of identity with SEQ ID NO:8.

Description

ANTI-NEUROTENSIN LONG FRAGMENT AND ANTI-NEUROMEDIN N LONG FRAGMENT ANTIBODIES AND USES THEREOF
FIELD OF THE INVENTION:
The present invention relates to a human anti-neurotensin long fragment (LF anti-NTS) and human anti-neuromedin N long fragment (LF anti-NN) antibody and uses thereof.
BACKGROUND OF THE INVENTION:
Neurotensin (NTS) is a 13 amino-acid peptide, discovered by Carraway and Leeman in 1973 (1). Its action as a neuromodulator in the central nervous system has been extensively studied since its discovery and continues to be the focus of many studies. In the periphery, NTS is released from the entero-endocrine N cells of the gastrointestinal tract in response to intraluminal lipid ingestion (2). The peptide predominantly exerts hormonal and neurocrine regulation on the digestive process including the inhibition of small bowel motility and gastric acid secretions, the stimulation of pancreatic and biliary secretions, and the facilitation of fatty acid absorption (3). NTS action is mediated by two different G protein coupled receptors, the high and low affinity neurotensin receptors NTSR1 and NTSR2, respectively, and by a nonspecific single transmembranous sorting receptor encoded by the SORT1 gene, NTSR3/sortiline (4).
Neurotensin (NTS) and Neuromedin N (NN) are peptides generated from a precursor of 170 AA long cleaved by convertases, such as PCI, PC2, PC5A, CPE (5). NTS (13 AA) and Neuromedin N (6 AA) share the same 4 C-terminal AA sequence implicated in the binding and the activation of the high affinity neurotensin receptor 1 (6). Once totally cleaved it remains from the precursor a fragment of 140 amino acids long (7). The inventor’s hypothesis is the following: the NTS gene is abnormally expressed or overexpressed, under physiological or pathological contexts (tumors, inflammation, infection, allergies, and chemotherapy treatment, obesity, ...) (8-12) facilitating the production and the release of the proform (PF), the large form of NTS and/or NN. These three forms, PF, LF NTS, and LF NN are 170, 163 and 148 amino acid long, respectively, bind with a high affinity and activate the high affinity neurotensin receptor 1 (NTSR1). The PF, the LF NTS and the LF NN represent the active form whereas the N terminal fragment of 140 among acid maned R for remnant is inactive on the neurotensinergic system. In the oncology context the high affinity neurotensin receptor 1 (NTSR1) is abnormally expressed or overexpressed in tumor cells of a large number of cancers, especially from epithelial origin (13, 14).
These forms are much more stable than the peptides (Neurotensin has a very short half-life of 1.7 minutes (15)) and are as efficient, or more efficient than the mature peptides. They are, therefore, excellent therapeutic targets.
SUMMARY OF THE INVENTION:
The present invention relates to an antibody, which is capable of binding to the Neuromedin N long fragment, and Neurotensin long fragment with high affinity. The antibody of the present invention neutralises the activity of the Neuromedin N long fragment, and Neurotensin long fragment, in particular their oncogenic activities. In particular, the present invention relates to a neutralising human antibody which binds to the Neuromedin N long fragment, and Neurotensin long fragment, and having a heavy chain variable region which comprises a H- CDR1 region having at least 90% of identity with SEQ ID NO:2, a H- CDR2 region having at least 90% of identify with SEQ ID NO:3 and a H-CDR3 region having at least 90% of identity with SEQ ID NO:4; and a light chain variable region comprising a L-CDR1 region having at least 90% of identity with SEQ ID NO: 6, a L-CDR2 having at least 90% of identity with SEQ ID NO:7 and a L-CDR3 region having at least 90% of identity with SEQ ID NO:8. The present invention also provides the use of such antibodies in the treatment of cancer and cachexia induced by cancer.
DETAILED DESCRIPTION OF THE INVENTION:
In some embodiments, the present invention relates to an antibody having a heavy chain variable region which comprises a H-CDR1 region having at least 90% of identity with SEQ ID NO:2, a H- CDR2 region having at least 90% of identify with SEQ ID NO: 3 and a H-CDR3 region having at least 90% of identity with SEQ ID NO:4; and a light chain variable region comprising a L-CDR1 region having at least 90% of identity with SEQ ID NO:6, a L-CDR2 having at least 90% of identity with SEQ ID NO:7 and a L-CDR3 region having at least 90% of identity with SEQ ID NO: 8.
The present invention provides a human antibody, particularly in a purified form or in an isolated form. In particular, the invention relates to a human antibody comprising:
(a) a heavy chain wherein the variable domain comprises: a H-CDR1 having a sequence set forth as SEQ ID NO: 2, a H-CDR2 having a sequence set forth as SEQ ID NO:3; a H-CDR3 having a sequence set forth as SEQ ID NO: 4;
(b) a light chain wherein the variable domain comprises : a L-CDR1 having a sequence set forth as SEQ ID NO: 6; a L-CDR2 having a sequence set forth as SEQ ID NO: 7; a L-CDR3 having a sequence set forth as SEQ ID NO: 8
In some embodiments, the antibody of the present invention comprises a heavy chain wherein the variable domain comprises at least one CDR having a sequence selected from the group consisting of SEQ ID NO:2 for H-CDR1, SEQ ID NO:3 for H-CDR2 and SEQ ID NON for H- CDR3.
In some embodiments, the antibody of the present invention comprises a light chain wherein the variable domain comprises at least one CDR having a sequence selected from the group consisting of SEQ ID NO:6 for L-CDR1, SEQ ID NO:7 for L-CDR2 and SEQ ID NO:8 for L- CDR3.
In some embodiments, the antibody of the present invention comprises a heavy chain wherein the variable domain comprises at least one CDR having a sequence selected from the group consisting of SEQ ID NO:2 for H-CDR1, SEQ ID NO:3 for H-CDR2 and SEQ ID NON for H- CDR3 and a light chain wherein the variable domain comprises at least one CDR having a sequence selected from the group consisting of SEQ ID NO:6 for L-CDR1, SEQ ID NO:7 for L-CDR2 and SEQ ID NO: 8 for L-CDR3.
In some embodiments, the antibody of the present invention comprises a heavy chain variable region comprising SEQ ID NO:2 in the H-CDR1 region, SEQ ID NON in the H-CDR2 region and SEQ ID NON in the H- CDR3 region ; and a light chain variable region comprising SEQ ID NO:6 in the L-CDR1 region, SEQ ID NON in the L-CDR2 region and SEQ ID NO:8 in the L-CDR3 region. In some embodiments, the antibody of the present invention comprises a heavy chain variable region having at least 70% of identity with SEQ ID NO:1 and/or a light chain variable region having at least 70% of identity with SEQ ID NO:5.
According to the 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.
In some embodiments, the antibody of the present invention comprises a heavy chain variable region of having the amino acid sequence set forth as SEQ ID NO: 1 and/or a light chain variable region having the amino acid sequence set forth as SEQ ID NO: 5.
More particularly the invention relates to an antibody comprising an amino acid sequence having 70% (+ until 99%) of identity with SEQ ID NO:1 or SEQ ID NO:5.
SEQ ID NO:1 Heavy chain variable region: amino acid sequence FR1-CDR1- FR2-CDR2-FR3 -CDR3-FR4
SEQ ID NO:5 Light chain variable region: amino acid sequence FR1-CDR1- FR2-CDR2-FR3 -CDR3-FR4
Therefore, the invention relates to a human antibody comprising: a heavy chain wherein the variable domain has a sequence set forth as SEQ ID NO: 1 a light chain wherein the variable domain has a sequence set forth as SEQ ID NO: 5
The present invention provides a human anti-neurotensin long fragment (LF anti-NTS) antibody or a human anti-neuromedin N long fragment (LF anti-NN) antibody (named NAM02), particularly in a purified form or in an isolated form.
In some embodiments, the present invention relates to a human anti-neurotensin long fragment (LF anti-NTS) antibody or a human anti-neuromedin N long fragment (LF anti-NN) antibody having a heavy chain variable region which comprises a H-CDR1 region having at least 90% of identity with SEQ ID NO:2, a H- CDR2 region having at least 90% of identify with SEQ ID NO:3 and a H-CDR3 region having at least 90% of identity with SEQ ID NO:4; and a light chain variable region comprising a L-CDR1 region having at least 90% of identity with SEQ ID N0:6, a L-CDR2 having at least 90% of identity with SEQ ID NO:7 and a L-CDR3 region having at least 90% of identity with SEQ ID NO: 8.
In particular, the invention relates to a human anti-neurotensin long fragment (LF anti-NTS) antibody or a human anti-neuromedin N long fragment (LF anti-NN) antibody comprising:
(a) a heavy chain wherein the variable domain comprises: a H-CDR1 having a sequence set forth as SEQ ID NO: 2, a H-CDR2 having a sequence set forth as SEQ ID NO:3; a H-CDR3 having a sequence set forth as SEQ ID NO: 4;
(b) a light chain wherein the variable domain comprises : a L-CDR1 having a sequence set forth as SEQ ID NO: 6; a L-CDR2 having a sequence set forth as SEQ ID NO: 7; a L-CDR3 having a sequence set forth as SEQ ID NO: 8
In some embodiments, the human anti-neurotensin long fragment (LF anti-NTS) antibody or the human anti-neuromedin N long fragment (LF anti-NN) antibody of the present invention comprises a heavy chain wherein the variable domain comprises at least one CDR having a sequence selected from the group consisting of SEQ ID NO:2 for H-CDR1, SEQ ID NO:3 for H-CDR2 and SEQ ID NON for H-CDR3.
In some embodiments, the human anti-neurotensin long fragment (LF anti-NTS) antibody or the human anti-neuromedin N long fragment (LF anti-NN) antibody of the present invention comprises a light chain wherein the variable domain comprises at least one CDR having a sequence selected from the group consisting of SEQ ID NO:6 for L-CDR1, SEQ ID NO:7 for L-CDR2 and SEQ ID NO: 8 for L-CDR3.
In some embodiments, the human anti-neurotensin long fragment (LF anti-NTS) antibody or the human anti-neuromedin N long fragment (LF anti-NN) antibody of the present invention comprises a heavy chain wherein the variable domain comprises at least one CDR having a sequence selected from the group consisting of SEQ ID NO:2 for H-CDR1, SEQ ID NON for H-CDR2 and SEQ ID NON for H-CDR3 and a light chain wherein the variable domain comprises at least one CDR having a sequence selected from the group consisting of SEQ ID NO: 6 for L-CDR1, SEQ ID NON for L-CDR2 and SEQ ID NO: 8 for L-CDR3. In some embodiments, the human anti-neurotensin long fragment (LF anti-NTS) antibody or the human anti-neuromedin N long fragment (LF anti-NN) antibody of the present invention comprises a heavy chain variable region comprising SEQ ID NO:2 in the H-CDR1 region, SEQ ID NO:3 in the H-CDR2 region and SEQ ID NO:4 in the H- CDR3 region ; and a light chain variable region comprising SEQ ID NO:6 in the L-CDR1 region, SEQ ID NO:7 in the L-CDR2 region and SEQ ID NO: 8 in the L-CDR3 region.
In some embodiments, the human anti-neurotensin long fragment (LF anti-NTS) antibody or the human anti-neuromedin N long fragment (LF anti-NN) antibody is having at least 70% of identity with SEQ ID NO: 1 and/or a light chain variable region having at least 70% of identity with SEQ ID NO: 5.
According to the 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.
In some embodiments, the human anti-neurotensin long fragment (LF anti-NTS) antibody or the human anti-neuromedin N long fragment (LF anti-NN) antibody comprises a heavy chain variable region of having the amino acid sequence set forth as SEQ ID NO: 1 and/or a light chain variable region having the amino acid sequence set forth as SEQ ID NO: 5.
More particularly the invention relates to a human anti-neurotensin long fragment (LF anti- NTS) antibody or a human anti-neuromedin N long fragment (LF anti-NN) antibody comprising an amino acid sequence having 70% (+ until 99%) of identity with SEQ ID NO:1 or SEQ ID NO:5.
SEQ ID NO:1 Heavy chain variable region: amino acid sequence FR1-CDR1- FR2-CDR2-FR3 -CDR3-FR4
SEQ ID NO:5 Light chain variable region: amino acid sequence FR1-CDR1- FR2-CDR2-FR3 -CDR3-FR4
Therefore, the invention relates to a human anti-neurotensin long fragment (LF anti-NTS) antibody or a human anti-neuromedin N long fragment (LF anti-NN) antibody comprising: a heavy chain wherein the variable domain has a sequence set forth as SEQ ID NO: 1 a light chain wherein the variable domain has a sequence set forth as SEQ ID NO: 5
The sequences of interest in the present application are indicated in the following Table 1 :
Figure imgf000008_0001
According to the present invention, the VH region of the antibody of the present invention consists of the sequence of SEQ ID NO: 1. Accordingly, the H-CDR1 of the antibody of the present invention 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 El- CDR2 of the antibody of the present invention mab is defined by the sequence ranging from the amino acid residue at position 50 to the amino acid residue at position 66 in SEQ ID NO: 1. Accordingly, the H-CDR3 of the antibody of the present invention mab is defined by the sequence ranging from the amino acid residue at position 99 to the amino acid residue at position 112 in SEQ ID NO: 1.
SEQ ID NO: 1 : VH region of the antibody of the present invention FR1-CDR1-FR2-CDR2- FR3-CDR3-FR4 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQASGQGLEWVGWISAYN GDTNYAQKLQGRVTLTTDTSTSTAYMELRSLRSDDTAVYYCASWVAVAGTRYYG MDVWGOGTTVTVS S
According to the present invention, the VL region of the antibody of the present invention consists of the sequence of SEQ ID NO:5. Accordingly, the L-CDR1 of mab is defined by the sequence ranging from the amino acid residue at position 23 to the amino acid residue at position 36 in SEQ ID NO:5. Accordingly, the L-CDR2 of mab is defined by the sequence ranging from the amino acid residue at position 52 to the amino acid residue at position 58 in SEQ ID NO: 5. Accordingly, the L-CDR3 of mab is defined by the sequence ranging from the amino acid residue at position 91 to the amino acid residue at position 100 in SEQ ID NO: 5.
SEQ ID NO: 5: VL region of antibody of the present invention FR1-CDR1-FR2-CDR2-FR3- CDR3-FR4
QSALTQPASVSGSPGQSVTISCTGTSSDIGAYTYVSWYQQHPGKAPKLMIYEVSNRP SGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTTSSTRVFGGGTKLTVL
As used herein the terms "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 nonhypervariable 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. The residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al. This system is set forth in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, 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, while having relatively little detectable reactivity with nonantigen proteins or structures (such as other proteins 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 Neurotensin long fragment and Neuromedin N long fragment).
The term “affinity”, as used herein, means 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.
According to the 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.
“Percent (%) amino acid sequence identity” with respect to a peptide or polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table A below. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code shown in Table A below has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU5 10087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, Calif, or may be compiled from the source code provided in FIG. 8 below. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
The terms "monoclonal antibody", "monoclonal Ab", "monoclonal antibody composition", "mAb", or the like, as used herein 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 invention, the antibody is isolated. An “isolated” antibody is one, which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials, which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
In one embodiment, the invention relates to antibodies that bind to the human neurotensin long fragment (LF NTS) or the human neuromedin N long fragment (LF NN) with a KD of 17.7 nM nM or 2.8 nM respectively. As used herein, the term “KD” is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e. Kd/Ka) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods well established in the Art. One method for determining the KD of an antibody is by using surface Plasmon resonance, or using a biosensor system such as a Biacore® system.
In some embodiments, the invention relates to antibodies restoring responses to treatment, ie tumors which are resistant or partially resistant to existing treatments, especially chemotherapeutic agents, targeted therapeutics into tumor fully or partially responsive to existing treatments in patients with cancer.
In some embodiments, the invention relates to antibodies reestablishing the state of “cold tumor(s)”, ie tumors which have not or poorly been infiltrated with immune cells into “hot tumor(s)”, tumor infiltrated with immune cells rushing to fight the cancerous cells.
In some embodiments, the invention relates to antibodies that restore the immune cells depletion in the primary and secondary immune organs induced by cancer. Antibodies of the invention restore the depletion of immune cells tumor suppressor.
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 site-specific 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 XENOMOUSE™ 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 Boerner 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 phagedisplay 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 antibody of the invention is an antigen binding 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 or SEQ ID NO: 5.
The term “antigen binding fragment” of an antibody, as used herein, refers to one or more fragments of an intact antibody that retain the ability to specifically binds to a given antigen. 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, SMIP, 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).
In some embodiments, the present invention further provides fragments said antibodies which include but are not limited to Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2 and diabodies.
As used herein, the term "Fab" denotes an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, in which about a half of the N-terminal side of H chain and the entire L chain, among fragments obtained by treating IgG with a protease, papaine, are bound together through a disulfide bond.
As used herein, the term "F(ab')2" refers to an antibody fragment having a molecular weight of about 100,000 and antigen binding activity, which is slightly larger than the Fab bound via a disulfide bond of the hinge region, among fragments obtained by treating IgG with a protease, pepsin.
As used herein, the term "Fab"' refers to an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, which is obtained by cutting a disulfide bond of the hinge region of the F(ab')2.
As used herein, a single chain Fv ("scFv") polypeptide is a covalently linked VH::VL heterodimer which is usually expressed from a gene fusion including VH and VL encoding genes linked by a peptide-encoding linker.
As used herein the term "dsFv" refers to 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.
As used herein the term "diabodies" refers to small antibody fragments with two antigenbinding sites, 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.
The Fab of the present invention can be obtained by treating an antibody which specifically reacts with human neurotensin long fragment (LF NTS) or human neuromedin N long fragment (LF anti-NN) 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 human neurotensin long fragment (LF NTS) or human neuromedin N long fragment (LF anti-NN) 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 human neurotensin long fragment (LF NTS) or human neuromedin N long fragment (LF anti- NN) 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).
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.
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. As used herein the term “single domain antibody” has its general meaning in the art and 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 single domain antibody are also called VHH or “nanobody®”. For a general description of (single) domain antibodies, reference is also made to the prior art cited above, as well as to EP 0 368 684, Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), Holt et al., Trends Biotechnol., 2003, 21(11):484- 490; and WO 06/030220, WO 06/003388. The nanobody has a molecular weight approximately one-tenth that of a human IgG molecule, and the protein has a physical diameter of only a few nanometers. One consequence of the small size is the ability of camelid nanobodies to bind to antigenic sites that are functionally invisible to larger antibody proteins, i.e. , camelid nanobodies are useful as reagents to detect antigens that are otherwise cryptic using classical immunological techniques, and as possible therapeutic agents. Thus yet another consequence of small size is that a nanobody can inhibit as a result of binding to a specific site in a groove or narrow cleft of a target protein, and hence can serve in a capacity that more closely resembles the function of a classical low molecular weight drug than that of a classical antibody. The low molecular weight and compact size further result in nanobodies being extremely thermostable, stable to extreme pH and to proteolytic digestion, and poorly antigenic. Another consequence is that nanobodies readily move from the circulatory system into tissues, and even cross the blood-brain barrier and can treat disorders that affect nervous tissue. Nanobodies can further facilitated drug transport across the blood brain barrier. See U.S. patent application 20040161738 published August 19, 2004. These features combined with the low antigenicity to humans indicate great therapeutic potential. The amino acid sequence and structure of a single domain antibody can be considered to be comprised of four framework regions or "FRs" which are referred to in the art and herein as "Framework region 1" or "FR1"; as "Framework region 2" or "FR2"; as "Framework region 3" or "FR3"; and as "Framework region 4" or “FR4” respectively; which framework regions are interrupted by three complementary determining regions or "CDRs", which are referred to in the art as "Complementarity Determining Region for "CDR1”; as "Complementarity Determining Region 2" or "CDR2” and as "Complementarity Determining Region 3" or "CDR3", respectively. Accordingly, the single domain antibody can be defined as an amino acid sequence with the general structure : FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4 respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3. In the context of the invention, the amino acid residues of the single domain antibody are numbered according to the general numbering for VH domains given by the International ImMunoGeneTics information system aminoacid numbering (http://imgt.cines.fr/).
Modifications and changes may be made in the structure of the antibodies of the present invention, and in the DNA sequences encoding them, and still obtain a functional molecule that encodes an antibody with desirable characteristics. In making the changes in the amino sequences, the hydropathic index of amino acids may be considered. 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). A further object of the present invention also encompasses function-conservative variants of the antibodies of the present invention.
"Function-conservative variants" are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). For example, certain amino acids may be substituted by other amino acids in a protein structure without appreciable loss of activity. Since the interactive capacity and nature of a protein define the protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence, and, of course, in its DNA encoding sequence, while nevertheless obtaining a protein with like properties. It is thus contemplated that various changes may be made in the antibodies sequences of the invention, or corresponding DNA sequences which encode said antibodies, without appreciable loss of their biological activity. It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e. still obtain a biological functionally equivalent protein. As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophihcity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
The antibodies 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 binning. 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). For example, the 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. Additionally, routine cross-blocking assays such as those described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane, 1988, can be performed.
Engineered antibodies of the present 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. Another type of 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 addition or alternative to modifications made within the framework or CDR regions, antibodies of the present invention may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half- life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. Furthermore, an antibody of the present invention may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody.
For example, it will be appreciated that the affinity of antibodies provided by the present invention may be altered using any suitable method known in the art. The present invention therefore also relates to variants of the antibody molecules of the present invention, which have an improved affinity for neurotensine. 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.
In some embodiments, 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 some embodiments, the Fc hinge region of the antibody of the present invention 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. In some embodiments, the antibody of the present invention 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.
In some embodiments, 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 CI 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 some embodiments, 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 some embodiments, 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. In yet another 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).
In some embodiments, the glycosylation of an antibody is modified. For example, an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation). 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. 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, EP 1 ,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 one embodiment, the 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 fucosyltransf erase. 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 acetylglucosaminyltransferase 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.eurekainc.com/a&boutus/companyoverview.html). Alternatively, the 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 ).
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 (CI- CIO) alkoxy- or aryloxypoly ethylene glycol or polyethylene glycol-maleimide. In certain embodiments, 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 present invention. See for example, EP O 154 316 by Nishimura et al. and EP 0 401 384 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 present 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 present 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.
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
Nucleic acid sequence Accordingly, a further object of the invention relates to a nucleic acid sequence encoding an antibody of the present invention. In some embodiments, the nucleic acid sequence encodes a heavy chain and/or a light chain of an antibody of the present invention.
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.
So, a further object 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. Host cell
A further object of the present 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.
As used herein, the term "transformation" means the introduction of a "foreign" (i.e. extrinsic or extracellular) gene, DNA or R A 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 RNA bas 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 Baculo virus 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.G1 1.16Ag.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. In another particular embodiment, the method comprises the steps of: (i) culturing the hybridoma FLp26-8.2 under conditions suitable to allow expression of FLp26-8.2 antibody; and (ii) recovering the expressed antibody.
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.
In some embodiments, 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).
The fully human antibody of the present invention may be produced by obtaining nucleic acid sequences encoding CDR domains, as previously described, constructing a humanized antibody expression vector by inserting them into an expression vector for animal cell having genes encoding (i) a heavy chain constant region identical to that of a human antibody and (ii) a light chain constant region identical to that of a human antibody, and expressing the genes by introducing the expression vector into an animal cell. The fully human antibody expression vector may be either of a type in which a gene encoding an antibody heavy chain and a gene encoding an antibody light chain exists on separate vectors or of a type in which both genes exist on the same vector (tandem type). In respect of easiness of construction of a humanized antibody expression vector, easiness of introduction into animal cells, and balance between the expression levels of antibody H and L chains in animal cells, humanized antibody expression vector of the tandem type is preferred (Shitara K et al. 1994). Examples of tandem type humanized antibody expression vector include pKA TEX93 (WO 97/10354), pEE18 and the like.
Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (See, e. g., Riechmann L. et al. 1988; Neuberger MS. et al. 1985). Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska MA. et al. (1994)), and chain shuffling (U.S. Pat. No.5,565,332). The general recombinant DNA technology for preparation of such antibodies is also known (see European Patent Application EP 125023 and International Patent Application WO 96/02576).
Treatment
A further object of the present invention relates to a method of treating cancer in a subject in need thereof comprising administering the subject with a therapeutically effective amount of an antibody of the present invention.
As used herein, the terms "treatment" and "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
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 antibody 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 antibody 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 antibody or antibody portion are outweighed by the therapeutically beneficial effects. The efficient dosages and dosage regimens for the antibody 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 pharmaceutical composition required. For example, the physician could start doses of the antibody 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 a 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, the ability of a compound to inhibit cancer may, for example, be evaluated in an animal model system predictive of efficacy in human tumors. 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 intravenous, 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.
Tumors to be treated include primary tumors and metastatic tumors, as well as refractory tumors. Refractory tumors include tumors that fail to respond or are resistant to treatment with chemotherapeutic agents alone, antibodies alone, radiation alone or combinations thereof. Refractory tumors also encompass tumors that appear to be inhibited by treatment with such agents, but recur up to five years, sometimes up to ten years or longer after treatment is discontinued. Examples of cancers that may be treated by methods and compositions of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified nonHodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.
In some embodiments, the patient suffers from a cancer deriving from epithelial origin. Examples of cancer types include, but are not limited to, carcinoma, lymphoma, blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinoma, and islet cell cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small-cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer, esophagael cancer, tumors of the biliary tract, as well as head and neck cancer, as well as subtypes of any of such cancers, including, but not limited to chemotherapy -resistant, platinum-resistant, advanced, refractory, and/or recurrent types thereof.
In particular, the present invention relates to a method of treating of preventing weight loss, muscle loss and/or protein blood level decrease in a subject in need thereof comprising administering the subject with a therapeutically effective amount of an antibody of the present invention.
Accordingly, in some embodiments, the subject is underweight. As herein, the term “underweight” refers to a subject having a body mass index of below 18.5. As used herein, the term “body mass index” has its general meaning in the art and refers to refers to the ratio which is calculated as body weight per height in meter squared (kg/m2). The BMI provides a simple means of assessing how much an individual's body weight departs from what is normal or desirable for a person of his or her height. Common definitions of BMI categories are as follows: starvation: BMI — less than 15 kg/m2; underweight — BMI less than 18.5 kg/m2; ideal — BMI from 18.5 to 25 kg/m2; overweight — BMI from 25 to 30 kg/m2; obese — BMI from 30 to 40 kg/m2; morbidly obese — BMI greater than 40 kg/m2.
In some embodiments, the method of the present invention is particularly suitable for inhibiting the lipolysis of white adipose tissue, and the loss of skeletal muscle. In some embodiments, the method of the present invention is particularly suitable for stimulating appetite. Underweight may be due to several causes, such as rapid metabolism, poor/inadequate diet or starvation (malnutrition), malabsorption due to defective intestinal function, endocrine disturbances e.g. type I diabetes, psychological problems (such as anorexia nervosa, body dysmorphic disorder, stress and anxiety) and weight loss, due to chronic illnesses and ageing. While in general the underlying cause of the underweight will have to be treated per se, the underweight too may be a health hazard, and as such have to be treated in itself. Indeed, persons suffering from underweight generally have poor physical stamina, a weakened immune system, as well as being at higher risk of developing diseases such as osteoporosis, heart disease and vascular disease. Additionally, in the female sex, underweight can lead to delayed sexual development, retarded amenorrhoea or complications during pregnancy.
In some embodiments, the subject suffers from a wasting disorder. As used herein, the term "wasting disorder" has its general meaning in the art and includes but is not limited to anorexia cachexia, anorexia of the aged, anorexia nervosa, cachexia associated with cancer, cachexia associated with AIDS, cachexia associated with heart failure, cachexia associated with cystic fibrosis, cachexia associated with rheumatoid arthritis, cachexia associated with kidney disease, cachexia associated with chronic obstructive pulmonary disease (COPD), cachexia associated with ALS, cachexia associated with renal failure or cachexia associated, and other disorders associated with aberrant appetite, fat mass, energy balance, and/or involuntary weight loss.
In some embodiments, the subject suffers from “cachexia”. As used herein, the term “cachexia” is used for a condition of physical wasting with loss of body fat and muscle mass. Generally, cachexia may be associated with and due to conditions such as cancer, required immunodeficiency syndrome (AIDS), cardiac diseases, infectious diseases, shock, burn, endotoxinemia, organ inflammation, surgery, diabetes, collagen diseases, radiotherapy, and chemotherapy. In many of these diseases, cachexia may significantly contribute to morbidity or mortality. Another particular group of individuals that are susceptible to developing a cachectic state are those individuals that have undergone a gastrectomy, such as may be practiced on gastric cancer and ulcer patients.
In some embodiments, the subject suffers from anorexia. As used herein, the term "anorexia" has its general meaning in the art and refers to any eating disorder characterized by markedly reduced appetite or total aversion to food. In some embodiments, the subject suffers from anorexia nervosa. In general, subjects suffering from anorexia nervosa have a BMI of less than 17.5 kg/m2.
Accordingly, the present invention is drawn to methods of treating a patient exhibiting one or more wasting disorders such as anorexia, cachexia, anorexia of the aged, anorexia nervosa, cachexia associated with cancer, cachexia associated with AIDS, cachexia associated with heart failure, cachexia associated with cystic fibrosis, cachexia associated with rheumatoid arthritis, cachexia associated with kidney disease, cachexia associated with COPD, cachexia associated with ALS, cachexia associated with renal failure or cachexia associated, or hip fracture, and in reducing the mortality and morbidity of critically ill patients, comprising administering to said patient in need of such treatment a therapeutically effective of an inhibitor of NTSR1 activation or expression.
The present invention also provides for therapeutic applications where an antibody of the present invention is used in combination with at least one further therapeutic agent, e.g. for treating cancer. Such administration may be simultaneous, separate or sequential. For simultaneous administration the agents may be administered as one composition or as separate compositions, as appropriate. The further therapeutic agent is typically relevant for the disorder to be treated. Exemplary therapeutic agents include other 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 antibody of the present invention is used in combination with a chemotherapeutic agent. A “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (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 chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYC1NO, morpholino-doxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HC1 liposome injection (DOXIL®) and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), an epothilone, 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; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2Z ,2ZZ -trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINEO, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); thiotepa; taxoids, e.g., paclitaxel (TAXOL®), albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE), and doxetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; pemetrexed; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluorom ethylornithine (DMFO); retinoids such as retinoic acid; pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU and leucovovin.
In a particular embodiment, the chemotherapeutic agent is chosen from alkilating agent, isotopomerase inhibitors, antifofate, or microtubule disruptor.
In a particular embodiment, the chemotherapeutic agent is chosen from the cisplatin and/or paclitaxel, Carboplatin and/or Pemetrexed.
In some embodiments, the antibody is useful for restore the sensibility of cancer cells to chemotherapeutic agent, such as platinum-based antineoplastic drugs. Example of platinumbased antineoplastic drugs include cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, nedaplatin, triplatin and lipoplatin.
In some embodiments, the invention relates to a method of treatment for “cold tumor(s)”, ie tumors which are not responding or partially responding to existing treatments, especially chemotherapeutic agents or targeted cancer therapeutics: antibodies of the present invention will turn those “cold tumor(s)” into “hot tumor(s)”, with a synergetic effect with chemotherapeutic agents.
In some embodiments, the antibody 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 antibody 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. The inventors show that a combination of the antibody of the present invention and anti PD- Ll/anti-PD-1 would increase the proportion of responders, provide an alternative in case of resistance, improve tolerability and compliance by adjusting initial doses in induction and maintenance.
In some embodiments, the antibody of the present invention is used in combination with an immune checkpoint inhibitor.
As used herein, the term "immune checkpoint inhibitor" has its general meaning in the art and refers to any compound inhibiting the function of an immune inhibitory checkpoint protein.
As used herein the term "immune checkpoint protein" has its general meaning in the art and refers to a molecule that is expressed by T cells in that either turn up a signal (stimulatory checkpoint molecules) or turn down a signal (inhibitory checkpoint molecules). Immune checkpoint molecules are recognized in the art to constitute immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see e.g. Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al. , 2011. Nature 480:480- 489). Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD- 1, LAG-3, TIM-3 and VISTA. Inhibition includes reduction of function and full blockade. Preferred immune checkpoint inhibitors are antibodies that specifically recognize immune checkpoint proteins. A number of immune checkpoint inhibitors are known and in analogy of these known immune checkpoint protein inhibitors, alternative immune checkpoint inhibitors may be developed in the (near) future. The immune checkpoint inhibitors include peptides, antibodies, nucleic acid molecules and small molecules. Examples of immune checkpoint inhibitor includes PD-1 antagonist, PD-L1 antagonist, PD-L2 antagonist CTLA-4 antagonist, VISTA antagonist, TIM-3 antagonist, LAG-3 antagonist, IDO antagonist, KIR2D antagonist, A2AR antagonist, B7-H3 antagonist, B7-H4 antagonist, and BTLA antagonist.
In some embodiments, PD-1 (Programmed Death- 1) axis antagonists include PD-1 antagonist (for example anti-PD-1 antibody), PD-L1 (Programmed Death Ligand-1) antagonist (for example anti-PD-Ll antibody) and PD-L2 (Programmed Death Ligand-2) antagonist (for example anti-PD-L2 antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of MDX-1106 (also known as Nivolumab, MDX-1106-04, ONO-4538, BMS-936558, and Opdivo®), Merck 3475 (also known as Pembrolizumab, MK-3475, Lambrolizumab, Keytruda®, and SCH-900475), and CT-011 (also known as Pidilizumab, hBAT, and hBAT-1). In some embodiments, the PD-1 binding antagonist is AMP -224 (also known as B7-DCIg). In some embodiments, the anti-PD-Ll antibody is selected from the group consisting of YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736. MDX-1105, also known as BMS-936559, is an anti-PD-Ll antibody described in W02007/005874. Antibody YW243.55. S70 is an anti-PD-Ll described in WO 2010/077634 AL MEDI4736 is an anti-PD- Ll antibody described in WO2011/066389 and US2013/034559. MDX-1106, also known as MDX-1 106-04, ONO-4538 or BMS-936558, is an anti-PD-1 antibody described in U.S. Pat. No. 8,008,449 and W02006/121168. Merck 3745, also known as MK-3475 or SCH-900475, is an anti-PD-1 antibody described in U.S. Pat. No. 8,345,509 and W02009/114335. CT-011 (Pidizilumab), also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in W02009/101611. AMP -224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in W02010/027827 and WO2011/066342. Atezolimumab is an anti-PD-Ll antibody described in U.S. Pat. No. 8,217,149. Avelumab is an anti-PD-Ll antibody described in US 20140341917. CA-170 is a PD-1 antagonist described in W02015033301 & WO2015033299. Other anti-PD-1 antibodies are disclosed in U.S. Pat. No. 8,609,089, US 2010028330, and/or US 20120114649. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody chosen from Nivolumab, Pembrolizumab or Pidilizumab. In some embodiments, PD-L1 antagonist is selected from the group comprising of Avelumab, BMS-936559, CA-170, Durvalumab, MCLA-145, SP142, STI-A1011, STIA1012, STI-A1010, STI-A1014, Al 10, KY1003 and Atezolimumab and the preferred one is Avelumab, Durvalumab or Atezolimumab.
In some embodiments, CTLA-4 (Cytotoxic T-Lymphocyte Antigen-4) antagonists are selected from the group consisting of anti-CTLA-4 antibodies, human anti-CTLA-4 antibodies, mouse anti-CTLA-4 antibodies, mammalian anti-CTLA-4 antibodies, humanized anti-CTLA-4 antibodies, monoclonal anti-CTLA-4 antibodies, polyclonal anti-CTLA-4 antibodies, chimeric anti-CTLA-4 antibodies, MDX-010 (Ipilimumab), Tremelimumab, anti-CD28 antibodies, anti- CTLA-4 adnectins, anti-CTLA-4 domain antibodies, single chain anti-CTLA-4 fragments, heavy chain anti-CTLA-4 fragments, light chain anti-CTLA-4 fragments, inhibitors of CTLA- 4 that agonize the co-stimulatory pathway, the antibodies disclosed in PCT Publication No. WO 2001/014424, the antibodies disclosed in PCT Publication No. WO 2004/035607, the antibodies disclosed in U.S. Publication No. 2005/0201994, and the antibodies disclosed in granted European Patent No. EP 1212422 B. Additional CTLA-4 antibodies are described in U.S. Pat. Nos. 5,811,097; 5,855,887; 6,051,227; and 6,984,720; in PCT Publication Nos. WO 01/14424 and WO 00/37504; and in U.S. Publication Nos. 2002/0039581 and 2002/086014. Other anti-CTLA-4 antibodies that can be used in a method of the present invention include, for example, those disclosed in: WO 98/42752; U.S. Pat. Nos. 6,682,736 and 6,207,156; Hurwitz et al., Proc. Natl. Acad. Sci. USA, 95(17): 10067-10071 (1998); Camacho et al., J. Clin: Oncology, 22(145): Abstract No. 2505 (2004) (antibody CP-675206); Mokyr et al., Cancer Res., 58:5301-5304 (1998), and U.S. Pat. Nos. 5,977,318, 6,682,736, 7,109,003, and 7,132,281. A preferred clinical CTLA-4 antibody is human monoclonal antibody (also referred to as MDX-010 and Ipilimumab with CAS No. 477202-00-9 and available from Medarex, Inc., Bloomsbury, N.J.) is disclosed in WO 01/14424. With regard to CTLA-4 antagonist (antibodies), these are known and include Tremelimumab (CP-675,206) and Ipilimumab.
In some embodiments, the immunotherapy consists in administering to the subject a combination of a CTLA-4 antagonist and a PD-1 antagonist.
Other immune-checkpoint inhibitors include lymphocyte activation gene-3 (LAG-3) inhibitors, such as IMP321, a soluble Ig fusion protein (Brignone et al., 2007, J. Immunol. 179:4202- 4211). Other immune-checkpoint inhibitors include B7 inhibitors, such as B7-H3 and B7-H4 inhibitors. In particular, the anti-B7-H3 antibody MGA271 (Loo et al., 2012, Clin. Cancer Res. July 15 (18) 3834). Also included are TIM-3 (T-cell immunoglobulin domain and mucin domain 3) inhibitors (Fourcade et al., 2010, J. Exp. Med. 207:2175-86 and Sakuishi et al., 2010, J. Exp. Med. 207:2187-94). As used herein, the term “TIM-3” has its general meaning in the art and refers to T cell immunoglobulin and mucin domain-containing molecule 3. The natural ligand of TIM-3 is galectin 9 (Gal9). Accordingly, the term “TIM-3 inhibitor” as used herein refers to a compound, substance or composition that can inhibit the function of TIM-3. For example, the inhibitor can inhibit the expression or activity of TIM-3, modulate or block the TIM-3 signaling pathway and/or block the binding of TIM-3 to galectin-9. Antibodies having specificity for TIM-3 are well known in the art and typically those described in WO201 1155607, W02013006490 and WO2010117057.
In some embodiments, the immune checkpoint inhibitor is an IDO inhibitor. Examples of IDO inhibitors are described in WO 2014150677. Examples of IDO inhibitors include without limitation 1-methyl-tryptophan (IMT), P- (3-benzofuranyl)-alanine, P-(3-benzo(b)thienyl)- alanine), 6-nitro-tryptophan, 6- fluoro-tryptophan, 4-methyl-tryptophan, 5 -methyl tryptophan, 6-methyl-tryptophan, 5 -methoxy -tryptophan, 5 -hydroxy-tryptophan, indole 3-carbinol, 3,3'- diindolylmethane, epigallocatechin gallate, 5-Br-4-Cl-indoxyl 1,3 -di acetate, 9- vinylcarbazole, acemetacin, 5 -bromo-tryptophan, 5 -bromoindoxyl diacetate, 3- Amino-naphtoic acid, pyrrolidine dithiocarbamate, 4-phenylimidazole a brassinin derivative, a thiohydantoin derivative, a P-carboline derivative or a brassilexin derivative. Preferably the IDO inhibitor is selected from 1-methyl-tryptophan, P-(3- benzofuranyl)-alanine, 6-nitro-L-tryptophan, 3- Amino-naphtoic acid and P-[3- benzo(b)thienyl] -alanine or a derivative or prodrug thereof
In some embodiments, the antibody 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-i l l.
In some embodiments, the antibody 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-PDl antibodies, anti-PDLl antibodies, anti-TIMP3 antibodies, anti-LAG3 antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies or anti-B7H6 antibodies.
In some embodiments, the second agent is an agent that induces, via ADCC, the death 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, aVp3, 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.
Accordingly, a further object relates to a method of enhancing NK cell antibody-dependent cellular cytotoxicity (ADCC) of an antibody in a subject in need thereof comprising administering to the subject the antibody, and administering to the subject an antibody of the present invention.
Pharmaceutical compositions
Typically, the antibody of the present invention is administered to the subject in the form of a pharmaceutical composition which comprises a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, di sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene- block polymers, polyethylene glycol and wool fat. For use in administration to a patient, the composition will be formulated for administration to the patient. The compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Sterile injectable forms of the compositions of this invention may be aqueous or an oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation. The compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include, e.g., lactose. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added. Alternatively, the compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols. The compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs. For topical applications, the compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyl dodecanol, benzyl alcohol and water. Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Patches may also be used. The compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well- known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents. For example, an antibody present in a pharmaceutical composition of this invention can be supplied at a concentration of 10 mg/mL in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials. The product is formulated for IV administration in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mL polysorbate 80, and Sterile Water for Injection. The pH is adjusted to 6.5. An exemplary suitable dosage range for an antibody in a pharmaceutical composition of this invention may between about 1 mg/m2 and 500 mg/m2. However, it will be appreciated that these schedules are exemplary and that an optimal schedule and regimen can be adapted taking into account the affinity and tolerability of the particular antibody in the pharmaceutical composition that must be determined in clinical trials. A pharmaceutical composition of the invention for injection (e.g., intramuscular, i.v.) could be prepared to contain sterile buffered water (e.g. 1 ml for intramuscular), and between about 1 ng to about 100 mg, e.g. about 50 ng to about 30 mg or more preferably, about 5 mg to about 25 mg, of an antimyosin 18A antibody of the invention.
In certain embodiments, the use of liposomes and/or nanoparticles is contemplated for the introduction of antibodies 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. Kit
Finally, the invention also provides kits comprising at least one antibody of the invention. Kits containing antibodies of the invention find use in detecting human neurotensin long fragment (LF NTS) or human neuromedin N long fragment (LF anti-NN) expression (increase or decrease), or in therapeutic or diagnostic assays. Kits of the invention can contain an antibody coupled to a solid support, e.g., a tissue culture plate or beads (e.g., sepharose beads). Kits can be provided which contain antibodies for detection and quantification of human neurotensin long fragment (LF NTS) or human neuromedin N long fragment (LF anti-NN) in vitro, e.g. in an ELISA or a Western blot. Such antibody useful for detection may be provided with a label such as a fluorescent or radiolabel.
The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.
FIGURES:
Figure 1. LF NTS and LF NN increased tumor growth and induced cachexia. (A) Schematic representation of NTS precursor maturation. In brain, adrenal, and endocrine N cells of intestine, NTS precursor is cleaved with PCI, PC2, PC5A, to generate two active peptides, NN 6 aa, NTS 13aa, and the Remnant polypeptide 140 aa. Under over production or abnormal production of NTS precursor, Long form of NTS (LF NTS), form of NN (LF NN), and the proform (PF) exhibiting the biological activity similar to the mature peptide, but more stable and more active. (B) Follow up during 22 days of tumor growth rate generated by lung tumor cells NCI-H460 xenografted into nude mice. Mice were treated with PBS, or 550 fmol/ml of LF NTS, LF NN, PF, or R at DI, D4, D8, Dl l, D15, D18, and D22. On day one, five groups of 8 mice were randomized by NCLH460 tumor size, reaching between 80 and 110 mm3. Mice were killed 24h to 48h after the last injection. In a two-way ANOVA statistical analysis, *p<0.05, **p<0.01. (C) Number of mitoses counted on paraffin embedded tissue stained with hematoxylin and eosin. In t test statistical analysis, ** p< 0.01, ***p<0.001. (D) LF NTS, LF NN and PF treatment induced a decreased epididymal white adipose tissue weight (n=8). In t test statistical analysis, *p< 0.05.
Figure 2. NAM02 reduces the tumor growth. (A) Western blot analysis, 0.5 pg were loaded on the gel for fragments R and PF, and 0.2 pg for FL-NN and FL-NTS and revealed with 3 pg/ml of NAM02. (B) Tumor volume follow up generated by a lung cancer cell line cells, LNM-R, and treated with PBS, or NAM02, once a week at the dose 5 mg/kg (n=24 for PBS and NAM02 groups). The graph represents the percentage of change from DI and show the mean and the individual scores. In two-way ANOVA statistical analysis using Sidak Test for control vs NAM02 p < O.Olat DIO, and p < 0.0001 at D12, D15, D17, D19. (C) Number of mitoses counted on paraffin-embedded tissue stained with hematoxylin and eosin. In t test statistical analysis, t test ***p<0.001.
Figure 3. NAM02 improves the response to Lung cancer and neuroblastoma standard of care. (A) Tumor volume follow up generated by LNM-R cells and treated with US or EU SOC in combination or not with NAM02 (see protocol). The graph represents the percentage of change from DI and show the mean and the individual scores. In two-way ANOVA statistical analysis using Sidak Test for SOC vs SOC + NAM02 p < O.Olat DI 5, and p < 0.0001 at, D17 and D19. (B) Volume of tumors after dissection. In one-way ANOVA, *p<0.05,
****pO .0001. (C) Fold increase analysis of the tumor growth rate between SOC group and the combination group over time. At each time point the proportion of animals reaching the determined fold increase was calculated. Chi-square statistical analysis *p<0.05, **p<0.01, ***p<0.001. (D) Tumor volume follow up generated by the neuroblastoma cels, SK-NF-I,and treated with SOC in combination or not with NAM02 (see protocol). The graph represents the percentage of change from DI and show the mean and the individual scores. In two-way ANOVA statistical analysis using Sidak Test for SOC vs SOC + NAM02 p < 0.05 at D14 and D16, p < 0.01 at, D18 and D21, p < 0.001 at, D23 and D28.
Figure 4. NAM02 downregulates the oncogenic signaling in experimental tumors. The histograms represent the ratio of the phosphorylated form/total form based on band intensity quantification of Western blot. The proteins were extracted from an LNM-R experimental tumor treated three times with NAM02 with 5mg/kg at DI, D5, and D8. (A) phAKT/AKT, (B) phERK/ERK, (C) phJNK/JNK (D) phC-Jun/c-Jun (E) phSrc/Scr, (F) php38/p38. In t test statistical analysis, *p<0.05, **p<0.01 ***p<0.001.
Figure 5. NAM02 prevents the emptying of epididymal white adipose tissues. (A) Size of epidi dymal white adipocytes from mice bearing tumors treated with PBS or NAM02 as describes in figure 2. In t test statistical analysis, **** p< 0.001. (B) Frequency distribution (percentage) of the epidi dymal white adipocytes from mice bearing tumors treated with PBS or with NAM02 (n=l 1).
Figure 6. NAM02 counteracts the immune cells depletion induced by LF NN a in tumor microenvironment (TME) and primary and secondary lymphoid organs. (A) In TME proportion of CD3, CD8, NK, dendritic cells, and total macrophages and macrophages exhibiting the differentiation Ml or M2 (n=30, 21, 29, 20 for control, NAM02, LF NN, and the combination, respectively). (B) In spleen proportion, NK and dendritic cells, and total macrophages (n=21, 11, 23, 10 for control, NAM02, LFNN, and the combination, respectively) (C) In lymph node proportion of CD8 and dendritic cells (n=15, 9, 17, 10 for control, NAM02, LFNN, and the combination, respectively). (D) In thymus proportion NK cells (n=7, 9, 10, 10 for control, NAM02, LFNN, and the combination, respectively). Animals were treated with PBS, NAM02 alone, LF NN, or with the combination NAM02 and LF NN. In One-way-anova statistical analysis, *p<0.05, **p<0.01 ***p<0.001.
Figure 7. NAM02 up regulate expression of PD-L1 in lung cancer model.
The histograms represent quantification of PD-L1 band intensity of Western blot. (A) LNM-R cells were wild type or silenced for NTSR1 n=3; (B) LNM-R cells were treated for 24 h with 10'6 M SR48692, a specific antagonist of NTSR1 n=3; (C) The proteins were extracted from LNM-R experimental tumors mice were treated three times with PBS or NAM02 at 5 mg/kg on DI, D5 and D8; *p <0.05, **p <0.01.
EXAMPLES
MATERIALS AND METHODS
Cell culture
The cancer cell lines, LNM-R, NCI-H460., and SK-N-KI were grown in DMEM (Gibco®) supplemented with 10% fetal bovine serum (FBS) (Gibco®) and 2 mM glutamine and grown at 37 °C, in a humidified atmosphere of 5% CO2.
Tumor xenografts
Athymic 4-week-old male NMRI-Foxnlnu/nu mice (Janvier™), were injected with 1 million lung cancer cells for lung cancer cells and 5 million for neuroblastoma cells in 50 % Matrigel (v/v). The ellipsoid formula was used to calculate the tumor volumes. When tumors reached the expected size (see example), groups of mice were randomized. All the procedures were in accordance with the “Guide of the Care and Use of Laboratory Animals”. Institutional Review Board approval was obtained by «Le Comite d'Ethique en 1'Experimentation Animale Charles Darwin # B751201».
SDS-PAGE and Western blotting analysis
Western blots were processed as previously described (27). Gels were loaded with 40pg of tumor protein extract, the membranes were revealed with: anti-P-ERK (1/1000) #4695S, anti- ERK (1/1000) #4370S, anti p-SRC (1/1000) #6943S, anti-SRC (1/1000) #2108S, anti-p-AKT (1/1000) #4060S, anti-AKT (1/1000) #4695S, purchased from Cell Signaling Technology®. The primary antibodies anti-p-P38 (1/250) sc-535 anti-P38 (1/250) sc-17852, anti-P-JNK (1/250) sc-6254, anti-JNK (1/250) sc-7345, anti-c-JUN (1/250) Sc-75543, were purchased from Santa Cruz Biotechnology®. The primary antibody anti actin A5442 was provided by Sigma®. Primary antibodies were incubated overnight at 4 °C according to the manufacturer’s instructions. Secondary antibody Anti-Mouse IgG-HRP (1/10 000) A6782 and Anti-rabbit IgG- HRP (1/10000) #7074S were purchased from Sigma and Cell Signaling Technology®, respectively. Secondary antibodies were incubated Ih at RT and visualized by enhanced chemiluminescence (Pierce™ ECL 2 Western Blotting Substrate, Thermo Scientific™).
Mitotic Activity Index
Fragments of the experimental tumors were fixed in paraformaldehyde then paraffin embedded. Slides of 4pm were stained with hematoxylin and eosin. All the slides were scanned with a 3DHISTECH scanner and analyzed with a Panoramic viewer. Early and late metaphase, different forms of anaphase telophase were counted as described in (38). For each specimen mitoses were counted on several fields far from the necrosis area in a total surface of 2-4 mm2.
Treatments
For all in vivo experiments, NAM02 mAb was injected i.v. at the dose of 5 mg/kg, once a week. The purified polypeptide LF NTS, LF NN, PF and R were obtained from GenScript. Each polypeptide was injected at the dose of 550 fmol/ml of blood i.v. three times a week, alternatively in the right or the left side of the peritoneum. PBS was used as control therapy. The average volume of blood per mice was estimated at 2.5 ml. This concentration corresponds to three folds of the median of the fourth quartile described by (39). Chemotherapy treatments were injected i.v. at clinically relevant concentration. For lung tumors models US-SOC 4 mg/kg carboplatin at day 1, 3, 5, and 30 mg/kg pemetrexed (Alimta™) at day 1, 3, 5, and 8, EU-SOC 1 mg/kg cisplatin at day 1, 3, 5, and 10 mg/kg paclitaxel (Taxol ™) at day 1, 3, 5, and 8). For neuroblastoma models SOC was 0.75 mg/kg temozolomide and 7.5 mg/kg irinotecan from day 1 to 5, with or without NAM02 at day 1.
Fat size and distribution
Adipose tissues were fixed in paraformaldehyde then paraffin embedded. Slides of 5 pm were stained with hematoxylin and eosin. Images were acquired with 1X83 Olympus microscope and ORCA/4 Hamamatsu camera at objective 10 with phase contrast. Adipocyte sizes were obtained after binary transformation with ImageJ 1.53c software (40).
Immune cell isolation and purification
After removal, spleen, tumors, and lymph nodes were placed in a dissociation buffer composed of DMEM supplemented with 10% FCS (Gibco), 0.24% collagenase A (Roche) and 0.03% DNAse 1 (Roche). The two axillary and inguinal lymph nodes were analyzed, and most of them macroscopically metastasized.
The spleens and thymus were dissociated in DMEM and supplemented with 10% FCS with a piston syringe and through a 70 pm filter for the spleen. After centrifugation at 700 g for 10 min and 4°C, the cells were dispersed in 5 ml of lysis buffer (ThermoFisher) for 10 min at RT. After adding 10 ml DMEM supplemented with 10% FCS, cells were centrifuged at 700 g for 10 min. The cell pellets were resuspended in PBS containing 5% BSA.
The tumors and lymph nodes were divided into small dices and placed in the Dissociator GentleMACS, (Miltenyi Biotech), for 40 min at 37°C. The cellular suspension was then dispersed through a 70 pm filter. Only the healthy portion of the tumor was dispersed by the dissociator and only the equivalent of 0.5g was loaded on the Ficoll gradient. The 30 ml cell suspension was then loaded on the top of 10 ml Ficoll-Plaque Premium 1084 ficoll. The tube was centrifuged for 25 min at 1025g and 20 °C. The PBMC (Peripheral Blood Mononuclear Cells) are washed in 40 ml of DMEM supplemented with 10% FCS, centrifuged for 10 min at 700 g and 4°C. Cells from the pellet are dispersed in 5 ml of lysis buffer (ThermoFisher) for 15 min at RT to remove the erythrocytes. After adding 20 ml DMEM supplemented with 10% FCS, cells were centrifuged at 700 g for 10 min. The cell pellets were resuspended in PBS containing 5% BSA. For labeling, cells were distributed at 1.5 xlO6 cells/tubes in 300 pl PBS and centrifuged at 700 g for 5min. A mix containing antibodies were added to the cells. All cells are labeled for viability l/1000eme marker LIVE/DEAD Cell Stains Kit (ThermoFisher) for 30 min in the dark. After centrifugation and washing with PBS, cells were fixed with 4% PF A; and analyzed the next day with the flow cytometry cell analyzer Attune NxT. Results were analyzed with FlowJo version 10.7.2, Excel and Prism software.
Two panels of markers panel # 1 contained (CD45, CD3, CD4, CD8, NK1.1 and CDl lc markers, and Super Bright Buffer), the panel #2 contained (viability,CD19, F4/80, CD206, CD86 markers and Super Bright Buffer).
Antibodies Concentration Reterence Supliers anti-CD45 0.125 pg/test #12-0451-81 ThermoFisher anti-CD3 0.5 pg/test #11-0031-82 ThermoFisher anti-CD4 0.5 pg/test #62-0042-80 ThermoFisher anti-CD8 0.125 pg/test #17-0081-81 ThermoFisher anti-CD19 0.5 pg/test #63-0193-80 ThermoFisher anti-CD206 0.25 pg/test #17-2061-80 ThermoFisher anti-CD86 0.25 pg/test #25-0862-80 ThermoFisher anti-NKl.l 1 pg/test #63-5941-82 ThermoFisher anti-F480 1 pg/test #62-4801-82 ThermoFisher anti-CDllc 0.25 pg/test #45-0114-80 ThermoFisher
Super Bright 1 pg/test #SB-4401-42 ThermoFisher
Buffer
EXAMPLE 1: Effect of LF NTS and LF NN on tumor progression and cachexia, and immune system
Neurotensin is produced from a precursor cleaved by convertases to release two peptides, neurotensin (13 aa) and Neuromedin N (6 aa), and a polypeptide the remnant form (140 aa). Under over or abnormal production of NTS precursor, or lack of the appropriate convertases, long active forms of NTS and NN can be released from the cells, LF NTS and LF NN exhibit 163 and 148 aa, respectively. The Proform (PF) (170 aa) is cleaved by the carboxypeptidase E (enkephalin convertase) outside of the cells and is transformed in LF NTS (figure 1A). We first demonstrated the participation of the long active forms of NTS and NN, on tumor progression. The human lung cancer cells NCI-H460, from lung pleural effusion, express the high affinity neurotensin receptor (NTSR1) and weakly NTS. Cells were xenografted on nude mice, when the tumor burden reached around 80 mm3, groups of 8 animals were formed and treated with 550 fmol/ml of each polypeptide or PBS at DI, D4, D8, DI 1, D15, D18, and D22. The tumor volume was followed over time. All active fragments PF, LF NTS, and LF NN enhanced the tumor growth. Figure IB display the tumor growth rate of each group. The LF NN was the most effective with an increase of tumor growth of 22.7 ± 3.4 folds as compared to 14.8 ± 2.9 folds for control mice, over the 22 days period. LF NTS and PF groups had a tumor growth rate of 20.7 ± 6 and 20.4 ± 6.2 respectively. As expected, fragment 24-140 (Remnant) is non-active with a growth rate of 12.1 ± 2.1. To confirm the character more aggressive of the tumors exposed to LF NTS or LF NN the number of mitoses in the healthy part of the tumor was counted. Figure 1C shows that the number of mitoses per mm2 is 60% increased between the PBS or R treated animals and the active forms of NTS (LF NTS, LF NN, and PF). The data presented here confirm the oncogenic effect of LF NTS and LF NN. Moreover, LF NTS, LF NN and PF treatment induced a drastic decreased epididymal white adipose tissue weight (Figure ID).
EXAMPLE 2: Effect of NAM 02 on tumor growth.
A fully human antibody directed against NTS precursor miss cleaved product was selected based on the binding, neutralizing and tumor growth effects. Nam02 showed a KD of 17.7 and 2.8 nM for LF NTS and LF NN, respectively. The IC50 was 0.24 and 0.06 nM for LF NTS and LF NN respectively. Western blot demonstrated that NAM02 recognized all the form of miss- cleaved, in particular the active form on NTSR1, PF, LF NTS and LF NN (Figure 2A). The performance of NAM02 on tumor growth was testing on LNM-R cells, a very aggressive and highly metastatic lung cancer cell line overexpressing NTS and NTSR1 (16). The figure 2B represents % of tumor size change from DI and shows the mean and individual scores. At DI the average of burden size was 78 ± 8 and 84 ± 10 mm3 for the control group and NAM02 treated group. The mice were treated with NAM02 at the dose of 5 mg/kg once a week three times. While in the control group the tumor grows tremendously, in the animals treated with NAM02 the tumor size is reduced by 30% at D 15 and 40% at day 19 (figure 2B). The tumor growth rate and the number of mitoses per mm2 confirmed loss of aggressiveness with a reduction of 45% and 40% respectively in animals treated with NAM02 (figures 2C).
EXAMPLE 3: Effect ofNAM02 on chemotherapy response. A fully human antibody NAM02 has been selected for its ability to improve the response to the US or EU standard of care (SOC) for lung cancer. US SOC is the combination carboplatin with paclitaxel, UE SOC is the combination cisplatin, with pemetrexed. The rationale for this experiment being the possible future inclusion of the LF-NN/NTS antibody to the standard of care to improve the response. Mice were treated with subtoxic doses of SOC (see methods). The drug response of LNM-R tumor was similar for the SOCs and NAM02 as compared to control, with a tumor size reaching at Day 19, 1625 ± 118 and 1391 ± 156mm3, respectively. An additional decrease of 30% was observed when the combination SOC + NAM02 was used with a tumor size reduced to 926 ± 97 mm3 (figure 3A). At the individual level, the tumor size is less dispersed with a slower progression (figure 3A). The tumor size after dissection at D19 showed a similar result with an identical tumor size for SOCs and NAM02 used alone and a decrease of 40% when NAM02 is used in combination (figure 3B). The LNM-R cells model is representative of a progressive disease, therefore the tumor continues to grow regardless of the treatment. Figure 3C shows that the addition of NAM02 to the SOC altered globally the aggressive phenotype of the model with 30% of animals presenting a lower tumor growth rate. The effect of NAM02 is effective at the beginning of the treatment and persistent with time. LNM-R was selected for its extremely aggressive phenotype. Even the SOC or SOC+MAN02 reduced the tumor growth rate, this experimental tumor remains the model of a progressive disease.
In parallel, the effect of NAM02 was tested on the SOC for neuroblastoma, the combination temozolomide with irinotecan. The SKNFI experimental tumors were tested. SKNFI origin was bone marrow. This cell line has a normal chromosomal status, and is non-amplified for MycN. The Figure 3D represents the percentage of tumor size change from DI and shows the mean and individual scores. At DI the average of burden size was 184 ± 14 and 189 ± 29 mm3 for the control group SOC and SOC + NAM02 treated group. The mice were treated the combination from day 1 to 5 with or without NAM02 at DI. In mice treated with the combination a global shrinkage of the tumor was observed up to D28, whereas in the SOC group a small tumor growth regression was observed, with no real shrinkage. In addition, with time some tumors of the SOC group became out of control. As in the case of lung model, in mice treated with the combination, at the individual level, the tumor size is less dispersed. At D28 some tumor started to growth again, unfortunately this model did not allow to do a second cycle.
EXAMPLE 4: Effect ofNAM02 on oncogenic signalization.
NTSR1 activation by its cognate agonist induces many oncogenic cellular effects including proliferation, survival, migration, lack of adherence, invasion (13, 17). These effects were associated with several oncogenic pathways, such as PI3K-Akt, MEK-ERK, JNK-c-Jun, FAK- Scr signaling, in different types of cancers (18-24). In addition, it has been proposed that the complex NTSR1/NTS is associated with the tumor aggressiveness because under autocrine and/or paracrine regulation NTSR1 is permanently recycled to the cell surface and chronically activated (18, 25, 26). These changes in cellular homeostasis lead to the sustained activation of EGFR, HER2 and HER3 by their own ligands through the activation of metalloproteinases, mimicking simultaneous driving mutation on the three growth factor receptors (17, 27).
The stable NTSR1 ligands, LF NTS and LF NN create a permanent tumor cells autocrine and/or paracrine NTSR1 chronic activation causing a continue activation of MEK-ERK, PI3K-Akt, JNK-c-Jun, FAK-Scr, signaling pathways known to promote tumorigenesis and tumor progression.
In parallel, it has also been shown that NTS mediates inflammation and cytokines (IL-6, 11-8,..) released by several types of cancers which also directly or indirectly participate in the gravity of the disease and alter the response to treatment (28-30).
To confirm that NAM02 altered these major oncogenic pathways, we performed the following experiment. Mice were xenografted with LNM-R cells on both flanks. When burden reached 20 mm3 mice were treated with PBS or NAM02 at DI, D5 and D8. Mice were killed 24h after the last injection. Tumor protein extract was performed on frozen tissues and analyzed by western blot. The graphs in figure 4 represent the ratio of the band intensity from the phosphorylated protein by the total protein. Actin was used as a loading control. A 50% reduction of AKT and ERK signaling (figures 4A and 4B) was observed in experimental LNM- R tumors under NAM02 exposure in agreement with the decreased tumor growth described in example 2. As expected JNK-C-Jun signaling was concomitantly down regulated by 50% tumor of NAM02 treated animals (figures 4C and 4D) To confirm the pleiotropic action of NAM02 on oncogenic signaling we evaluated the effect on Scr and P38 signaling and detected a decreased activation of 60% and 30%, respectively (figures 4E and 4F).
EXAMPLE 5: Effect ofNAM02 on cachexia.
Example 1 showed that LF NTS is implicated in cachexia induced by cancer. Previous results also indicated that LF NTS murine antibodies prevent or delay the cachexia induced by cancer (8, 31). The epididymal adipocytes size from animals treated with NAM02 is decreased by 20% as compared to control (figure 5A). Size distribution of the adipocytes confirmed this result, with more adipocytes with larger size and less with smaller size in animals treated with NAM02 (figure 5B).
EXAMPLE 6: Effect of LF NN and NAM02 on immune system. The study was performed in the syngeneic lung cancer model of Lewis using the mouse lung cancer cells, LLC-1. 500,000 LLC1 cells were injected in the left and right flanks of the 4- week-old C57BL/6J male mice. When the burdens reached 20 to 50 mm3, four groups of mice were randomly made, control, MAN02, LF NN, and the combined group, LF NN plus NAM02. The treatments were injected at day 1, 3, 5, 8, 10 and 12. The last injection was performed 24h before the sacrifice. The treatment was injected in the retro-orbital sinus of the mice in a maximal volume of 100 pl.
LF NN synthesized and purified by GenScript were injected at the concentration of 500 pmol/L. This dose refers to 3 times the median of the 4th quartile described by O Melander group (JAMA 2012). The population within this quartile showed a higher risk of breast cancer, diabetes mellitus, cardiovascular disease, and mortality. NAM02 group was treated at the dose of 0.7 mg/kg (55,000 pmol/L). ° This represents a 100-fold ratio (w/w) for NAM02 antibodies and FL NN. For the combined treatment, FL NN and NAM02 were pre-incubated 2 hours at room temperature on slow-turning wheel before injection.
The immune cells from the tumor microenvironement, lymph node, spleen and thymus were isolated, purified, and analyzed by FACS. In tumors microenvironment, T lymphocytes CD8 +, natural killer cells, dendritic cells and macrophages were strongly reduced in mice treated with LF NN (figures 6A). In all cases this decline was counteracted in the presence of NAM02 (figures 6A). The quantity of tumor macrophages was reduced by 20% in LF NN treated mice as compared to control, but only the differentiation stage (Ml) was altered (figure 6A). To be noted a significant increase of NK cells was observed in mice treated with NAM02 alone as compared to control (figure 6A). Also no differences total T lymphocytes (CD3 +) was detected between groups (figure 6A).
In the spleen the percentage of NK and dendritic cells as well as macrophages were also reduced by the LF NN treatment. This reduction is abolished by NAM02 (figure 6B). In Lymph node Lymphocytes T CD8 + and dendritic cells are equally decreased by LF NN. This is not observed in the presence of NAM02 (figure 6C). Finally, in the primary immune organ, thymus the NK cells are strongly reduced again this is abolished by NAM02 (figure 6D),
The data presented here clearly demonstrate that within 15 days of LF NTS exposition, the entire immune system was strongly altered. This consequently diminished tumor immunity, with drastic reduction of NK cells and dendritic reduction in the tumor microenvironment, secondary and primary immune organs. The proportion of macrophages was also reduced in tumors and spleen that emphases the tumor immunity decline. EXAMPLE 7: Activation ofNTSRl downresulates expression ofPD-Ll in tumor cells.
The expression of PD-L1 in cells expressing NTS and NTSR1 compared to cells silenced for NTSR1 showed a drastic increase of PD-L1 expression in silenced cells (figure 7A). We confirmed that PD-L1 upregulation was the result ofNTSRl silencing by treating cells with a specific NTSR1 antagonist, SR 48692. In figure 7B, PD-L1 is upregulated when NTSR1 is blocked. To confirm that NAM02 equally affects this regulation in vivo, mice were xenografted with LNM-R cells and treated with PB S or NAM02 at D 1 , D5 and D8. Tumor protein extraction was performed on frozen tissues. The graphs in figure 7C compare the expression of PD-L in tumors grafted in mice treated with PBS or NAM02.
These data suggest that addition to restore vulnerability of tumor cells to chemotherapy, contract the immune cell depletion in tumor microenvironments, NAM02 up regulates PD-L1 in tumor cells. A combination of NAM02, anti PD-L1/PD-1 would i) increase the proportion of responders, ii) provide an alternative in case of resistance, iii) improve tolerability and compliance by adjusting initial doses in induction and maintenance.
CONCLUSION
LF NTS and LF NN can be defined as proactive factors for tumor progression and tumor cell aggressiveness. The NTSR1 gene is normally highly repressed, but de-repressed during the early events of carcinogenesis because of the wnt-Beta-catenin activation pathway and/or epigenetic regulation (32, 33). On the other hand, NTS promoter is very reactive, notably on the proximal CRE/AP-1 responsive element (34). The NTS gene can also be open to the transcription under epigenetic regulation (35, 36).
The NTS gene is triggered by several types of stimulus, such as chemotherapy, stress, infection, inflammation, in cancer cells as well as in immune cells (8-12). Therefore, the activation of the receptor localized on the tumor may arrive from immune or tumor cells. When NTSR1 is expressed at the cell surface, its chronic and sustained activation induces auto self-feeding regulation, because the NTSR1 activated the Src and RAS oncogenes, stimulating NTS gene expression (Data not shown . In parallel, the saturating concentration of NTS agonists induced the activation of NTSR1 gene and a permanent receptor recycling. NTSR1 chronic activation results in the sustained stimulation of several oncogenic pathways (MEK-ERK, PI3K-Akt, JNK-c-Jun, FAK-Scr), which increase aggressiveness of tumor phenotype (24, 37). In order to diminish this activity, we developed a therapeutic tool to reduce NTSR1 activation and expression and restore vulnerability of tumor cells to chemotherapy. Our results confirmed that the entire oncogenic signaling induced by the complex LF NTS-NN/NTSR1 are concomitantly down regulated with NAM02, lessening high metastatic process and improving the drug response. The effect of NAM02 on the oncogenic signaling confirm its role as a major oncogenic driver of this complex when this these regulation loops NTRS1/FL NTS/NN take place.
REFERENCES:
Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.
1. Carraway R, Leeman SE. The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami. The Journal of biological chemistry. 1973;248(19):6854-61.
2. Rosell S. Substance P and neurotensin in the control of gastrointestinal function. Society of General Physiologists series. 1980;35: 147-62.
3. Rosell S, Al-Saffar A, Thor K. The role of neurotensin in gut motility. Scandinavian journal of gastroenterology Supplement. 1984;96:69-75.
4. Vincent JP, Mazella J, Kitabgi P. Neurotensin and neurotensin receptors. Trends in pharmacological sciences. 1999;20(7):302-9.
5. Kitabgi P. Differential processing of pro-neurotensin/neuromedin N and relationship to pro-hormone convertases. Peptides. 2006;27(10):2508-14.
6. Kitabgi P. Functional domains of the subtype 1 neurotensin receptor (NTS1). Peptides. 2006;27(10):2461-8.
7. Ernst A, Hellmich S, Bergmann A. Proneurotensin 1-117, a stable neurotensin precursor fragment identified in human circulation. Peptides. 2006;27(7): 1787-93.
8. Wu Z, Fournel L, Stadler N, Liu J, Boullier A, Hoyeau N, et al. Modulation of lung cancer cell plasticity and heterogeneity with the restoration of cisplatin sensitivity by neurotensin antibody. Cancer letters. 2019;444: 147-61.
9. Saiyasit N, Chunchai T, Apaijai N, Pratchayasakul W, Sripetchwandee J, Chattipakorn N, et al. Chronic high-fat diet consumption induces an alteration in plasma/brain neurotensin signaling, metabolic disturbance, systemic inflammation/oxidative stress, brain apoptosis, and dendritic spine loss. Neuropeptides. 2020;82: 102047.
10. Theoharides TC. Effect of Stress on Neuroimmune Processes. Clinical therapeutics. 2020;42(6): 1007-14. 11. Piliponsky AM, Chen CC, Nishimura T, Metz M, Rios EJ, Dobner PR, et al. Neurotensin increases mortality and mast cells reduce neurotensin levels in a mouse model of sepsis. Nature medicine. 2008;14(4):392-8.
12. Brun P, Mastrotto C, Beggiao E, Stefani A, Barzon L, Stumiolo GC, et al. Neuropeptide neurotensin stimulates intestinal wound healing following chronic intestinal inflammation. American journal of physiology Gastrointestinal and liver physiology. 2005;288(4):G621-9.
13. Wu Z, Martinez-Fong D, Tredaniel J, Forgez P. Neurotensin and its high affinity receptor 1 as a potential pharmacological target in cancer therapy. Frontiers in endocrinology. 2012;3: 184.
14. Dupouy S, Mourra N, Doan VK, Gompel A, Alifano M, Forgez P. The potential use of the neurotensin high affinity receptor 1 as a biomarker for cancer progression and as a component of personalized medicine in selective cancers. Biochimie. 2011;93(9): 1369-78.
15. Pedersen JH, Andersen HO, Olsen PS, Henriksen JH. Pharmacokinetics and metabolism of neurotensin in man. The Journal of clinical endocrinology and metabolism. 1989;68(2):294- 300.
16. Kozaki K, Miyaishi O, Tsukamoto T, Tatematsu Y, Hida T, Takahashi T, et al. Establishment and characterization of a human lung cancer cell line NCI-H460-LNM35 with consistent lymphogenous metastasis via both subcutaneous and orthotopic propagation. Cancer Res. 2000;60(9):2535-40.
17. Dupouy S, Doan VK, Wu Z, Mourra N, Liu J, De Wever O, et al. Activation of EGFR, HER2 and HER3 by neurotensin/neurotensin receptor 1 renders breast tumors aggressive yet highly responsive to lapatinib and metformin in mice. Oncotarget. 2014;5(l 8):8235-51.
18. Toy -Mi ou-Leong M, Cortes CL, Beaudet A, Rostene W, Forgez P. Receptor trafficking via the perinuclear recycling compartment accompanied by cell division is necessary for permanent neurotensin cell sensitization and leads to chronic mitogen-activated protein kinase activation. The Journal of biological chemistry. 2004;279(13): 12636-46.
19. Zhao D, Bakirtzi K, Zhan Y, Zeng H, Koon HW, Pothoulakis C. Insulin-like growth factor- 1 receptor transactivation modulates the inflammatory and proliferative responses of neurotensin in human colonic epithelial cells. The Journal of biological chemistry. 2011;286(8):6092-9.
20. Moody TW, Lee L, Ramos-Alvarez I, Jensen RT. Neurotensin receptors regulate transactivation of the EGFR and HER2 in a reactive oxygen species-dependent manner. European journal of pharmacology. 2019;865: 172735. 21. Bakirtzi K, Hatziapostolou M, Karagiannides I, Polytarchou C, Jaeger S, Iliopoulos D, et al. Neurotensin signaling activates microRNAs-21 and -155 and Akt, promotes tumor growth in mice, and is increased in human colon tumors. Gastroenterology. 2011; 141(5): 1749-61.el .
22. Lee LF, Guan J, Qiu Y, Kung HJ. Neuropeptide-induced androgen independence in prostate cancer cells: roles of nonreceptor tyrosine kinases Etk/Bmx, Src, and focal adhesion kinase. Molecular and cellular biology. 2001;21(24):8385-97.
23. Hassan S, Dobner PR, Carraway RE. Involvement of MAP -kinase, PI3-kinase and EGF- receptor in the stimulatory effect of Neurotensin on DNA synthesis in PC3 cells. Regulatory peptides. 2004;120(l-3): 155-66.
24. Kisfalvi K, Hurd C, Guha S, Rozengurt E. Induced overexpression of protein kinase DI stimulates mitogenic signaling in human pancreatic carcinoma PANC-1 cells. Journal of cellular physiology. 2010;223(2):309-16.
25. Souaze F, Forgez P. Molecular and cellular regulation of neurotensin receptor under acute and chronic agonist stimulation. Peptides. 2006;27(10):2493-501.
26. Najimi M, Souaze F, Mendez M, Hermans E, Berbar T, Rostene W, et al. Activation of receptor gene transcription is required to maintain cell sensitization after agonist exposure. Study on neurotensin receptor. The Journal of biological chemistry. 1998;273(34):21634-41.
27. Younes M, Wu Z, Dupouy S, Lupo AM, Mourra N, Takahashi T, et al. Neurotensin (NTS) and its receptor (NTSR1) causes EGFR, HER2 and HER3 over-expression and their autocrine/paracrine activation in lung tumors, confirming responsiveness to erlotinib. Oncotarget. 2014;5(18):8252-69.
28. Law IK, Jensen D, Bunnett NW, Pothoulakis C. Neurotensin-induced miR-133alpha expression regulates neurotensin receptor 1 recycling through its downstream target aftiphilin. Scientific reports. 2016;6:22195.
29. Zhao D, Kuhnt-Moore S, Zeng H, Wu JS, Moyer MP, Pothoulakis C. Neurotensin stimulates IL-8 expression in human colonic epithelial cells through Rho GTPase-mediated NF- kappa B pathways. American journal of physiology Cell physiology. 2003;284(6):C1397-404.
30. Koon HW, Kim YS, Xu H, Kumar A, Zhao D, Karagiannides I, et al. Neurotensin induces IL-6 secretion in mouse preadipocytes and adipose tissues during 2,4,6,- trinitrobenzensulphonic acid-induced colitis. Proceedings of the National Academy of Sciences of the United States of America. 2009;106(21):8766-71.
31. Wu Z, Stadler N, Abbaci A, Liu J, Boullier A, Marie N, et al. Effect of Monoclonal Antibody Blockade of Long Fragment Neurotensin on Weight Loss, Behavior, and Metabolic Traits After High-Fat Diet Induced Obesity. Frontiers in endocrinology. 2021;12:739287. 32. Souaze F, Viardot-Foucault V, Roullet N, Toy-Miou-Leong M, Gompel A, Bruyneel E, et al. Neurotensin receptor 1 gene activation by the Tcf/beta-catenin pathway is an early event in human colonic adenomas. Carcinogenesis. 2006;27(4):708-16.
33. Kim JT, Liu C, Zaytseva YY, Weiss HL, Townsend CM, Jr., Evers BM. Neurotensin, a novel target of Wnt/beta-catenin pathway, promotes growth of neuroendocrine tumor cells. International journal of cancer. 2015; 136(6): 1475-81.
34. Dobner PR, Kislauskis E, Bullock BP. Cooperative regulation of neurotensin/neuromedin N gene expression in PC12 cells involves AP-1 transcription factors. Annals of the New York Academy of Sciences. 1992;668: 17-29.
35. Dong Z, Wang X, Evers BM. Site-specific DNA methylation contributes to neurotensin/neuromedin N expression in colon cancers. American journal of physiology Gastrointestinal and liver physiology. 2000;279(6):Gl 139-47.
36. Dong Z, Wang X, Zhao Q, Townsend CM, Jr., Evers BM. DNA methylation contributes to expression of the human neurotensin/neuromedin N gene. The American journal of physiology. 1998;274(3 Pt l):G535-43.
37. Wang Q, Zhou Y, Evers BM. Neurotensin phosphorylates GSK-3alpha/beta through the activation of PKC in human colon cancer cells. Neoplasia (New York, NY). 2006;8(9):781-7.
38. Al-Janabi S, van Slooten HJ, Visser M, van der Ploeg T, van Diest PJ, Jiwa M. Evaluation of mitotic activity index in breast cancer using whole slide digital images. PloS one. 2013;8(12):e82576.
39. Melander O, Maisel AS, Almgren P, Manjer J, Belting M, Hedblad B, et al. Plasma proneurotensin and incidence of diabetes, cardiovascular disease, breast cancer, and mortality. Jama. 2012;308(14): 1469-75.
40. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nature methods. 2012;9(7):676-82.

Claims

CLAIMS:
1. An antibody having a heavy chain variable region which comprises a H-CDR1 region having at least 90% of identity with SEQ ID NO:2, a H- CDR2 region having at least 90% of identify with SEQ ID NO : 3 and a H-CDR3 region having at least 90% of identity with SEQ ID NO:4; and a light chain variable region comprising a L-CDR1 region having at least 90% of identity with SEQ ID NO: 6, a L-CDR2 having at least 90% of identity with SEQ ID NO:7 and a L-CDR3 region having at least 90% of identity with SEQ ID NO:8.
2. The antibody of claim 1 which comprises a heavy chain wherein the variable domain comprises at least one CDR having a sequence selected from the group consisting of SEQ ID NO:2 for H-CDR1, SEQ ID NO:3 for H-CDR2 and SEQ ID NO:4 for H-CDR3.
3. The antibody of claim 1 which comprises a light chain wherein the variable domain comprises at least one CDR having a sequence selected from the group consisting of SEQ ID NO:6 for L-CDR1, SEQ ID NO:7 for L-CDR2 and SEQ ID NO:8 for L-CDR3.
4. The antibody of claim 1 which comprises a heavy chain wherein the variable domain comprises at least one CDR having a sequence selected from the group consisting of SEQ ID NO:2 for H-CDR1, SEQ ID NO:3 for H-CDR2 and SEQ ID NO:4 for H-CDR3 and a light chain wherein the variable domain comprises at least one CDR having a sequence selected from the group consisting of SEQ ID NO:6 for L-CDR1, SEQ ID NO: 7 for L-CDR2 and SEQ ID NO: 8 for L-CDR3.
5. The antibody of claim 1 which comprises a heavy chain variable region comprising SEQ ID NO:2 in the H-CDR1 region, SEQ ID NO:3 in the H-CDR2 region and SEQ ID NO:4 in the H- CDR3 region ; and a light chain variable region comprising SEQ ID NO:6 in the L-CDR1 region, SEQ ID NO: 7 in the L-CDR2 region and SEQ ID NO: 8 in the L- CDR3 region.
6. The antibody of claim 1 which comprises a heavy chain variable region having at least 70% of identity with SEQ ID NO: 1 and/or a light chain variable region having at least
Figure imgf000064_0001
7. The antibody of claim 1 which comprises a heavy chain variable region of having the amino acid sequence set forth as SEQ ID NO:1 and/or a light chain variable region having the amino acid sequence set forth as SEQ ID NO: 5.
8. The antibody of claims 1 to 7 wherein the antibody is a human anti-neurotensin long fragment (LF anti-NTS) antibody or a human anti-neuromedin N long fragment (LF anti -NN) antibody.
9. The antibody of claims 1 to 8 which is selected from the group consisting of Fv, Fab, F(ab’)2, Fab’, dsFv, scFv, sc(Fv)2 and diabodies.
10. A nucleic acid sequence which encodes a heavy chain and/or a light chain of an antibody according to any one of claims 1-9.
11. A vector which comprises the nucleic acid sequence of claim 10.
12. A host cell which has been transfected, infected or transformed by the nucleic acid sequence of claim 10 or the vector of claim 11.
13. A method of treating cancer in a subject in need thereof comprising administering the subject with a therapeutically effective amount of an antibody according to any one of claims 1-9.
14. The method of claim 13 wherein the cancer is selected from the group consisting of lung cancer, neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified nonHodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.
15. The method of claim 13 wherein the antibody is used in combination with a chemotherapeutic agent.
16. The method of claim 13 wherein the antibody is used in synergy with a chemotherapeutic agent, increasing its efficacy before or after the treatment with said antibody.
17. The method of claim 15 wherein the chemotherapeutic agent is chosen from alkilating agent, isotopomerase inhibitors, antifofate, or microtubule disruptor.
18. The method of claim 15 wherein the chemotherapeutic agent is chosen from cisplatin, paclitaxel, Carboplatin and/or Pemetrexed.
19. The method of claim 13 wherein the antibody is used in combination with an immune checkpoint inhibitor.
20. The method of claim 19 wherein the immune checkpoint inhibitor is anti-PD-1 or anti- PD-L1.
21. A pharmaceutical composition which comprises an antibody according to any one of claims 1-9.
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Citations (124)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US244A (en) 1837-06-30 Edward flint
US5204A (en) 1847-07-24 james cantelo
EP0125023A1 (en) 1983-04-08 1984-11-14 Genentech, Inc. Recombinant immunoglobulin preparations, methods for their preparation, DNA sequences, expression vectors and recombinant host cells therefor
EP0154316A2 (en) 1984-03-06 1985-09-11 Takeda Chemical Industries, Ltd. Chemically modified lymphokine and production thereof
EP0173494A2 (en) 1984-08-27 1986-03-05 The Board Of Trustees Of The Leland Stanford Junior University Chimeric receptors by DNA splicing and expression
WO1987002671A1 (en) 1985-11-01 1987-05-07 International Genetic Engineering, Inc. Modular assembly of antibody genes, antibodies prepared thereby and use
EP0239400A2 (en) 1986-03-27 1987-09-30 Medical Research Council Recombinant antibodies and methods for their production
EP0322094A1 (en) 1987-10-30 1989-06-28 Delta Biotechnology Limited N-terminal fragments of human serum albumin
US4861719A (en) 1986-04-25 1989-08-29 Fred Hutchinson Cancer Research Center DNA constructs for retrovirus packaging cell lines
EP0368684A1 (en) 1988-11-11 1990-05-16 Medical Research Council Cloning immunoglobulin variable domain sequences.
EP0401384A1 (en) 1988-12-22 1990-12-12 Kirin-Amgen, Inc. Chemically modified granulocyte colony stimulating factor
WO1991009967A1 (en) 1989-12-21 1991-07-11 Celltech Limited Humanised antibodies
EP0486525A1 (en) 1989-08-01 1992-05-27 Cemu Bioteknik Ab Stabilized protein or peptide conjugates.
EP0519596A1 (en) 1991-05-17 1992-12-23 Merck & Co. Inc. A method for reducing the immunogenicity of antibody variable domains
US5202238A (en) 1987-10-27 1993-04-13 Oncogen Production of chimeric antibodies by homologous recombination
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5278056A (en) 1988-02-05 1994-01-11 The Trustees Of Columbia University In The City Of New York Retroviral packaging cell lines and process of using same
EP0592106A1 (en) 1992-09-09 1994-04-13 Immunogen Inc Resurfacing of rodent antibodies
WO1994019478A1 (en) 1993-02-22 1994-09-01 The Rockefeller University Production of high titer helper-free retroviruses by transient transfection
EP0616640A1 (en) 1991-12-02 1994-09-28 Medical Research Council Production of anti-self antibodies from antibody segment repertoires and displayed on phage
WO1994029351A2 (en) 1993-06-16 1994-12-22 Celltech Limited Antibodies
WO1995014785A1 (en) 1993-11-23 1995-06-01 Rhone-Poulenc Rorer S.A. Composition for the in vivo production of therapeutic products
US5476996A (en) 1988-06-14 1995-12-19 Lidak Pharmaceuticals Human immune system in non-human animal
WO1996002576A1 (en) 1994-07-13 1996-02-01 Chugai Seiyaku Kabushiki Kaisha Reconstituted human antibody against human interleukin-8
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
WO1996022378A1 (en) 1995-01-20 1996-07-25 Rhone-Poulenc Rorer S.A. Cells for the production of recombinant adenoviruses
US5565332A (en) 1991-09-23 1996-10-15 Medical Research Council Production of chimeric antibodies - a combinatorial approach
WO1997010354A1 (en) 1995-09-11 1997-03-20 Kyowa Hakko Kogyo Co., Ltd. ANTIBODY AGAINTS α-CHAIN OF HUMAN INTERLEUKIN 5 RECEPTOR
US5618829A (en) 1993-01-28 1997-04-08 Mitsubishi Chemical Corporation Tyrosine kinase inhibitors and benzoylacrylamide derivatives
US5624821A (en) 1987-03-18 1997-04-29 Scotgen Biopharmaceuticals Incorporated Antibodies with altered effector functions
US5639757A (en) 1995-05-23 1997-06-17 Pfizer Inc. 4-aminopyrrolo[2,3-d]pyrimidines as tyrosine kinase inhibitors
US5641870A (en) 1995-04-20 1997-06-24 Genentech, Inc. Low pH hydrophobic interaction chromatography for antibody purification
US5677425A (en) 1987-09-04 1997-10-14 Celltech Therapeutics Limited Recombinant antibody
US5714350A (en) 1992-03-09 1998-02-03 Protein Design Labs, Inc. Increasing antibody affinity by altering glycosylation in the immunoglobulin variable region
US5728868A (en) 1993-07-15 1998-03-17 Cancer Research Campaign Technology Limited Prodrugs of protein tyrosine kinase inhibitors
US5770429A (en) 1990-08-29 1998-06-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5804396A (en) 1994-10-12 1998-09-08 Sugen, Inc. Assay for agents active in proliferative disorders
US5811097A (en) 1995-07-25 1998-09-22 The Regents Of The University Of California Blockade of T lymphocyte down-regulation associated with CTLA-4 signaling
WO1998042752A1 (en) 1997-03-21 1998-10-01 Brigham And Women's Hospital Inc. Immunotherapeutic ctla-4 binding peptides
WO1998045322A2 (en) 1997-04-10 1998-10-15 Royal Netherlands Academy Of Arts And Sciences Diagnosis method and reagents
US5855887A (en) 1995-07-25 1999-01-05 The Regents Of The University Of California Blockade of lymphocyte down-regulation associated with CTLA-4 signaling
US5859205A (en) 1989-12-21 1999-01-12 Celltech Limited Humanised antibodies
US5869046A (en) 1995-04-14 1999-02-09 Genentech, Inc. Altered polypeptides with increased half-life
WO1999010494A2 (en) 1997-08-25 1999-03-04 Genentech, Inc. Agonist antibodies to the thrombopoietin receptor, and their therapeutic uses
US5882877A (en) 1992-12-03 1999-03-16 Genzyme Corporation Adenoviral vectors for gene therapy containing deletions in the adenoviral genome
WO1999054342A1 (en) 1998-04-20 1999-10-28 Pablo Umana Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity
US5977318A (en) 1991-06-27 1999-11-02 Bristol Myers Squibb Company CTLA4 receptor and uses thereof
US6013516A (en) 1995-10-06 2000-01-11 The Salk Institute For Biological Studies Vector and method of use for nucleic acid delivery to non-dividing cells
US6051227A (en) 1995-07-25 2000-04-18 The Regents Of The University Of California, Office Of Technology Transfer Blockade of T lymphocyte down-regulation associated with CTLA-4 signaling
US6075181A (en) 1990-01-12 2000-06-13 Abgenix, Inc. Human antibodies derived from immunized xenomice
WO2000037504A2 (en) 1998-12-23 2000-06-29 Pfizer Inc. Human monoclonal antibodies to ctla-4
WO2000042072A2 (en) 1999-01-15 2000-07-20 Genentech, Inc. Polypeptide variants with altered effector function
US6100254A (en) 1997-10-10 2000-08-08 Board Of Regents, The University Of Texas System Inhibitors of protein tyrosine kinases
US6121022A (en) 1995-04-14 2000-09-19 Genentech, Inc. Altered polypeptides with increased half-life
US6127374A (en) 1997-07-29 2000-10-03 Warner-Lambert Company Irreversible inhibitors of tyrosine kinases
US6150584A (en) 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
US6165745A (en) 1992-04-24 2000-12-26 Board Of Regents, The University Of Texas System Recombinant production of immunoglobulin-like domains in prokaryotic cells
US6172197B1 (en) 1991-07-10 2001-01-09 Medical Research Council Methods for producing members of specific binding pairs
US6194551B1 (en) 1998-04-02 2001-02-27 Genentech, Inc. Polypeptide variants
WO2001014424A2 (en) 1999-08-24 2001-03-01 Medarex, Inc. Human ctla-4 antibodies and their uses
US6245759B1 (en) 1999-03-11 2001-06-12 Merck & Co., Inc. Tyrosine kinase inhibitors
US6277375B1 (en) 1997-03-03 2001-08-21 Board Of Regents, The University Of Texas System Immunoglobulin-like domains with increased half-lives
US6291158B1 (en) 1989-05-16 2001-09-18 Scripps Research Institute Method for tapping the immunological repertoire
US6306874B1 (en) 1999-10-19 2001-10-23 Merck & Co., Inc. Tyrosine kinase inhibitors
US6313138B1 (en) 2000-02-25 2001-11-06 Merck & Co., Inc. Tyrosine kinase inhibitors
US6316444B1 (en) 1999-06-30 2001-11-13 Merck & Co., Inc. SRC kinase inhibitor compounds
US6329380B1 (en) 1999-06-30 2001-12-11 Merck & Co., Inc. SRC kinase inhibitor compounds
EP1176195A1 (en) 1999-04-09 2002-01-30 Kyowa Hakko Kogyo Co., Ltd. Method for controlling the activity of immunologically functional molecule
US6344459B1 (en) 1996-04-12 2002-02-05 Warner-Lambert Company Irreversible inhibitors of tyrosine kinases
US20020039581A1 (en) 2000-01-27 2002-04-04 Carreno Beatriz M. Antibodies against CTLA4 and uses therefor
US20020086014A1 (en) 1999-08-24 2002-07-04 Korman Alan J. Human CTLA-4 antibodies and their uses
US6420382B2 (en) 2000-02-25 2002-07-16 Merck & Co., Inc. Tyrosine kinase inhibitors
US6479512B1 (en) 1999-10-19 2002-11-12 Merck & Co., Inc. Tyrosine kinase inhibitors
US6498165B1 (en) 1999-06-30 2002-12-24 Merck & Co., Inc. Src kinase inhibitor compounds
WO2003002609A2 (en) 2001-06-28 2003-01-09 Domantis Limited Dual-specific ligand and its use
WO2003035835A2 (en) 2001-10-25 2003-05-01 Genentech, Inc. Glycoprotein compositions
US6582915B1 (en) 1991-12-02 2003-06-24 Medical Research Council Production of anti-self bodies from antibody segment repertories and displayed on phage
US6586423B2 (en) 1999-09-10 2003-07-01 Merck & Co., Inc. Tyrosine kinase inhibitors
US20030153043A1 (en) 1997-05-21 2003-08-14 Biovation Limited Method for the production of non-immunogenic proteins
WO2004003019A2 (en) 2002-06-28 2004-01-08 Domantis Limited Immunoglobin single variant antigen-binding domains and dual-specific constructs
US6682736B1 (en) 1998-12-23 2004-01-27 Abgenix, Inc. Human monoclonal antibodies to CTLA-4
US6696245B2 (en) 1997-10-20 2004-02-24 Domantis Limited Methods for selecting functional polypeptides
WO2004035607A2 (en) 2002-10-17 2004-04-29 Genmab A/S Human monoclonal antibodies against cd20
US6740665B1 (en) 1999-02-10 2004-05-25 Ramachandran Murali Tyrosine kinase inhibitors and methods of using the same
EP1433846A2 (en) 1990-07-10 2004-06-30 Cambridge Antibody Technology LTD Phagemid-based method of producing filamentous bacteriophage particles displaying antibody molecules and the corresponding bacteriophage particles.
WO2004058821A2 (en) 2002-12-27 2004-07-15 Domantis Limited Dual specific single domain antibodies specific for a ligand and for the receptor of the ligand
US20040161738A1 (en) 2000-05-26 2004-08-19 Arumugam Muruganandam Single-domain brain-targeting antibody fragments derived from llama antibodies
US6794393B1 (en) 1999-10-19 2004-09-21 Merck & Co., Inc. Tyrosine kinase inhibitors
WO2004081026A2 (en) 2003-06-30 2004-09-23 Domantis Limited Polypeptides
WO2004101790A1 (en) 2003-05-14 2004-11-25 Domantis Limited A process for recovering polypeptides that unfold reversibly from a polypeptide repertoire
US6875767B2 (en) 2001-06-22 2005-04-05 Merck & Co., Inc. (5-cyano-2-thiazolyl)amino-4-pyridine tyrosine kinase inhibitors
US6927293B2 (en) 2001-08-30 2005-08-09 Merck & Co., Inc. Tyrosine kinase inhibitors
US6958340B2 (en) 2001-08-01 2005-10-25 Merck & Co., Inc. Tyrosine kinase inhibitors
EP1297172B1 (en) 2000-06-28 2005-11-09 Glycofi, Inc. Methods for producing modified glycoproteins
WO2006003388A2 (en) 2004-06-30 2006-01-12 Domantis Limited Compositions and methods for treating inflammatory disorders
WO2006030220A1 (en) 2004-09-17 2006-03-23 Domantis Limited Compositions monovalent for cd40l binding and methods of use
US7041870B2 (en) 2000-11-30 2006-05-09 Medarex, Inc. Transgenic transchromosomal rodents for making human antibodies
US7109003B2 (en) 1998-12-23 2006-09-19 Abgenix, Inc. Methods for expressing and recovering human monoclonal antibodies to CTLA-4
WO2006121168A1 (en) 2005-05-09 2006-11-16 Ono Pharmaceutical Co., Ltd. Human monoclonal antibodies to programmed death 1(pd-1) and methods for treating cancer using anti-pd-1 antibodies alone or in combination with other immunotherapeutics
WO2007005874A2 (en) 2005-07-01 2007-01-11 Medarex, Inc. Human monoclonal antibodies to programmed death ligand 1 (pd-l1)
US7189826B2 (en) 1997-11-24 2007-03-13 Institute For Human Genetics And Biochemistry Monoclonal human natural antibodies
US20070061900A1 (en) 2000-10-31 2007-03-15 Murphy Andrew J Methods of modifying eukaryotic cells
WO2007059782A1 (en) 2005-11-28 2007-05-31 Genmab A/S Recombinant monovalent antibodies and methods for production thereof
US20070254295A1 (en) 2006-03-17 2007-11-01 Prometheus Laboratories Inc. Methods of predicting and monitoring tyrosine kinase inhibitor therapy
WO2009044561A1 (en) * 2007-10-03 2009-04-09 Shizuoka Prefecture ANTI-proNT/NMN MONOCLONAL ANTIBODY
WO2009101611A1 (en) 2008-02-11 2009-08-20 Curetech Ltd. Monoclonal antibodies for tumor treatment
WO2009114335A2 (en) 2008-03-12 2009-09-17 Merck & Co., Inc. Pd-1 binding proteins
US20100028330A1 (en) 2002-12-23 2010-02-04 Medimmune Limited Methods of upmodulating adaptive immune response using anti-pd1 antibodies
WO2010027827A2 (en) 2008-08-25 2010-03-11 Amplimmune, Inc. Targeted costimulatory polypeptides and methods of use to treat cancer
WO2010077634A1 (en) 2008-12-09 2010-07-08 Genentech, Inc. Anti-pd-l1 antibodies and their use to enhance t-cell function
WO2010106180A2 (en) 2009-03-20 2010-09-23 Lfb Biotechnologies Optimized fc variants
WO2010117057A1 (en) 2009-04-10 2010-10-14 協和発酵キリン株式会社 Method for treatment of blood tumor using anti-tim-3 antibody
WO2011066342A2 (en) 2009-11-24 2011-06-03 Amplimmune, Inc. Simultaneous inhibition of pd-l1/pd-l2
WO2011066389A1 (en) 2009-11-24 2011-06-03 Medimmmune, Limited Targeted binding agents against b7-h1
WO2011155607A1 (en) 2010-06-11 2011-12-15 協和発酵キリン株式会社 Anti-tim-3 antibody
US20120114649A1 (en) 2008-08-25 2012-05-10 Amplimmune, Inc. Delaware Compositions of pd-1 antagonists and methods of use
US8345509B2 (en) 2009-04-16 2013-01-01 Chevron U.S.A., Inc. System and method to create three-dimensional images of non-linear acoustic properties in a region remote from a borehole
WO2013006490A2 (en) 2011-07-01 2013-01-10 Cellerant Therapeutics, Inc. Antibodies that specifically bind to tim3
WO2014150677A1 (en) 2013-03-15 2014-09-25 Bristol-Myers Squibb Company Inhibitors of indoleamine 2,3-dioxygenase (ido)
US20140341917A1 (en) 2011-11-28 2014-11-20 Merck Patent Gmbh Anti-pd-l1 antibodies and uses thereof
WO2015033301A1 (en) 2013-09-06 2015-03-12 Aurigene Discovery Technologies Limited 1,3,4-oxadiazole and 1,3,4-thiadiazole derivatives as immunomodulators
WO2015033299A1 (en) 2013-09-06 2015-03-12 Aurigene Discovery Technologies Limited 1,2,4-oxadiazole derivatives as immunomodulators
WO2015185478A1 (en) * 2014-06-02 2015-12-10 INSERM (Institut National de la Santé et de la Recherche Médicale) Anti-neurotensin long fragment antibodies and uses thereof
WO2015185513A1 (en) * 2014-06-02 2015-12-10 INSERM (Institut National de la Santé et de la Recherche Médicale) Anti-neurotensin antibodies and uses thereof

Patent Citations (144)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5204A (en) 1847-07-24 james cantelo
US244A (en) 1837-06-30 Edward flint
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
EP0125023A1 (en) 1983-04-08 1984-11-14 Genentech, Inc. Recombinant immunoglobulin preparations, methods for their preparation, DNA sequences, expression vectors and recombinant host cells therefor
EP0154316A2 (en) 1984-03-06 1985-09-11 Takeda Chemical Industries, Ltd. Chemically modified lymphokine and production thereof
EP0173494A2 (en) 1984-08-27 1986-03-05 The Board Of Trustees Of The Leland Stanford Junior University Chimeric receptors by DNA splicing and expression
WO1987002671A1 (en) 1985-11-01 1987-05-07 International Genetic Engineering, Inc. Modular assembly of antibody genes, antibodies prepared thereby and use
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
EP0239400A2 (en) 1986-03-27 1987-09-30 Medical Research Council Recombinant antibodies and methods for their production
US4861719A (en) 1986-04-25 1989-08-29 Fred Hutchinson Cancer Research Center DNA constructs for retrovirus packaging cell lines
US5648260A (en) 1987-03-18 1997-07-15 Scotgen Biopharmaceuticals Incorporated DNA encoding antibodies with altered effector functions
US5624821A (en) 1987-03-18 1997-04-29 Scotgen Biopharmaceuticals Incorporated Antibodies with altered effector functions
US5677425A (en) 1987-09-04 1997-10-14 Celltech Therapeutics Limited Recombinant antibody
US5202238A (en) 1987-10-27 1993-04-13 Oncogen Production of chimeric antibodies by homologous recombination
EP0322094A1 (en) 1987-10-30 1989-06-28 Delta Biotechnology Limited N-terminal fragments of human serum albumin
US5278056A (en) 1988-02-05 1994-01-11 The Trustees Of Columbia University In The City Of New York Retroviral packaging cell lines and process of using same
US5698767A (en) 1988-06-14 1997-12-16 Lidak Pharmaceuticals Human immune system in non-human animal
US5476996A (en) 1988-06-14 1995-12-19 Lidak Pharmaceuticals Human immune system in non-human animal
US20040110941A2 (en) 1988-11-11 2004-06-10 Medical Research Council Single domain ligands, receptors comprising said ligands, methods for their production, and use of said ligands and receptors
EP0368684A1 (en) 1988-11-11 1990-05-16 Medical Research Council Cloning immunoglobulin variable domain sequences.
EP0401384A1 (en) 1988-12-22 1990-12-12 Kirin-Amgen, Inc. Chemically modified granulocyte colony stimulating factor
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US5585089A (en) 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
US6291158B1 (en) 1989-05-16 2001-09-18 Scripps Research Institute Method for tapping the immunological repertoire
EP0486525A1 (en) 1989-08-01 1992-05-27 Cemu Bioteknik Ab Stabilized protein or peptide conjugates.
WO1991009967A1 (en) 1989-12-21 1991-07-11 Celltech Limited Humanised antibodies
US5859205A (en) 1989-12-21 1999-01-12 Celltech Limited Humanised antibodies
US6150584A (en) 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
US6075181A (en) 1990-01-12 2000-06-13 Abgenix, Inc. Human antibodies derived from immunized xenomice
EP1433846A2 (en) 1990-07-10 2004-06-30 Cambridge Antibody Technology LTD Phagemid-based method of producing filamentous bacteriophage particles displaying antibody molecules and the corresponding bacteriophage particles.
US5770429A (en) 1990-08-29 1998-06-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
EP0519596A1 (en) 1991-05-17 1992-12-23 Merck & Co. Inc. A method for reducing the immunogenicity of antibody variable domains
US5977318A (en) 1991-06-27 1999-11-02 Bristol Myers Squibb Company CTLA4 receptor and uses thereof
US6172197B1 (en) 1991-07-10 2001-01-09 Medical Research Council Methods for producing members of specific binding pairs
US5565332A (en) 1991-09-23 1996-10-15 Medical Research Council Production of chimeric antibodies - a combinatorial approach
US6593081B1 (en) 1991-12-02 2003-07-15 Medical Research Council Production of anti-self antibodies from antibody segment repertoires and displayed on phage
EP0616640A1 (en) 1991-12-02 1994-09-28 Medical Research Council Production of anti-self antibodies from antibody segment repertoires and displayed on phage
US6582915B1 (en) 1991-12-02 2003-06-24 Medical Research Council Production of anti-self bodies from antibody segment repertories and displayed on phage
US6350861B1 (en) 1992-03-09 2002-02-26 Protein Design Labs, Inc. Antibodies with increased binding affinity
US5714350A (en) 1992-03-09 1998-02-03 Protein Design Labs, Inc. Increasing antibody affinity by altering glycosylation in the immunoglobulin variable region
US6165745A (en) 1992-04-24 2000-12-26 Board Of Regents, The University Of Texas System Recombinant production of immunoglobulin-like domains in prokaryotic cells
EP0592106A1 (en) 1992-09-09 1994-04-13 Immunogen Inc Resurfacing of rodent antibodies
US5882877A (en) 1992-12-03 1999-03-16 Genzyme Corporation Adenoviral vectors for gene therapy containing deletions in the adenoviral genome
US5618829A (en) 1993-01-28 1997-04-08 Mitsubishi Chemical Corporation Tyrosine kinase inhibitors and benzoylacrylamide derivatives
WO1994019478A1 (en) 1993-02-22 1994-09-01 The Rockefeller University Production of high titer helper-free retroviruses by transient transfection
WO1994029351A2 (en) 1993-06-16 1994-12-22 Celltech Limited Antibodies
US5728868A (en) 1993-07-15 1998-03-17 Cancer Research Campaign Technology Limited Prodrugs of protein tyrosine kinase inhibitors
WO1995014785A1 (en) 1993-11-23 1995-06-01 Rhone-Poulenc Rorer S.A. Composition for the in vivo production of therapeutic products
WO1996002576A1 (en) 1994-07-13 1996-02-01 Chugai Seiyaku Kabushiki Kaisha Reconstituted human antibody against human interleukin-8
US5804396A (en) 1994-10-12 1998-09-08 Sugen, Inc. Assay for agents active in proliferative disorders
WO1996022378A1 (en) 1995-01-20 1996-07-25 Rhone-Poulenc Rorer S.A. Cells for the production of recombinant adenoviruses
US6121022A (en) 1995-04-14 2000-09-19 Genentech, Inc. Altered polypeptides with increased half-life
US5869046A (en) 1995-04-14 1999-02-09 Genentech, Inc. Altered polypeptides with increased half-life
US5641870A (en) 1995-04-20 1997-06-24 Genentech, Inc. Low pH hydrophobic interaction chromatography for antibody purification
US5639757A (en) 1995-05-23 1997-06-17 Pfizer Inc. 4-aminopyrrolo[2,3-d]pyrimidines as tyrosine kinase inhibitors
US5855887A (en) 1995-07-25 1999-01-05 The Regents Of The University Of California Blockade of lymphocyte down-regulation associated with CTLA-4 signaling
US5811097A (en) 1995-07-25 1998-09-22 The Regents Of The University Of California Blockade of T lymphocyte down-regulation associated with CTLA-4 signaling
US6051227A (en) 1995-07-25 2000-04-18 The Regents Of The University Of California, Office Of Technology Transfer Blockade of T lymphocyte down-regulation associated with CTLA-4 signaling
WO1997010354A1 (en) 1995-09-11 1997-03-20 Kyowa Hakko Kogyo Co., Ltd. ANTIBODY AGAINTS α-CHAIN OF HUMAN INTERLEUKIN 5 RECEPTOR
US6013516A (en) 1995-10-06 2000-01-11 The Salk Institute For Biological Studies Vector and method of use for nucleic acid delivery to non-dividing cells
US6344459B1 (en) 1996-04-12 2002-02-05 Warner-Lambert Company Irreversible inhibitors of tyrosine kinases
US6277375B1 (en) 1997-03-03 2001-08-21 Board Of Regents, The University Of Texas System Immunoglobulin-like domains with increased half-lives
WO1998042752A1 (en) 1997-03-21 1998-10-01 Brigham And Women's Hospital Inc. Immunotherapeutic ctla-4 binding peptides
US6207156B1 (en) 1997-03-21 2001-03-27 Brigham And Women's Hospital, Inc. Specific antibodies and antibody fragments
WO1998045322A2 (en) 1997-04-10 1998-10-15 Royal Netherlands Academy Of Arts And Sciences Diagnosis method and reagents
US20030153043A1 (en) 1997-05-21 2003-08-14 Biovation Limited Method for the production of non-immunogenic proteins
US6562818B1 (en) 1997-07-29 2003-05-13 Warner-Lambert Company Irreversible inhibitors of tyrosine kinases
US6127374A (en) 1997-07-29 2000-10-03 Warner-Lambert Company Irreversible inhibitors of tyrosine kinases
WO1999010494A2 (en) 1997-08-25 1999-03-04 Genentech, Inc. Agonist antibodies to the thrombopoietin receptor, and their therapeutic uses
US6100254A (en) 1997-10-10 2000-08-08 Board Of Regents, The University Of Texas System Inhibitors of protein tyrosine kinases
US6696245B2 (en) 1997-10-20 2004-02-24 Domantis Limited Methods for selecting functional polypeptides
US7189826B2 (en) 1997-11-24 2007-03-13 Institute For Human Genetics And Biochemistry Monoclonal human natural antibodies
US6194551B1 (en) 1998-04-02 2001-02-27 Genentech, Inc. Polypeptide variants
WO1999054342A1 (en) 1998-04-20 1999-10-28 Pablo Umana Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity
US7109003B2 (en) 1998-12-23 2006-09-19 Abgenix, Inc. Methods for expressing and recovering human monoclonal antibodies to CTLA-4
US7132281B2 (en) 1998-12-23 2006-11-07 Amgen Fremont Inc. Methods and host cells for producing human monoclonal antibodies to CTLA-4
US6682736B1 (en) 1998-12-23 2004-01-27 Abgenix, Inc. Human monoclonal antibodies to CTLA-4
WO2000037504A2 (en) 1998-12-23 2000-06-29 Pfizer Inc. Human monoclonal antibodies to ctla-4
WO2000042072A2 (en) 1999-01-15 2000-07-20 Genentech, Inc. Polypeptide variants with altered effector function
US6740665B1 (en) 1999-02-10 2004-05-25 Ramachandran Murali Tyrosine kinase inhibitors and methods of using the same
US6245759B1 (en) 1999-03-11 2001-06-12 Merck & Co., Inc. Tyrosine kinase inhibitors
US6544988B1 (en) 1999-03-11 2003-04-08 Merck & Co., Inc. Tyrosine kinase inhibitors
EP1176195A1 (en) 1999-04-09 2002-01-30 Kyowa Hakko Kogyo Co., Ltd. Method for controlling the activity of immunologically functional molecule
US6329380B1 (en) 1999-06-30 2001-12-11 Merck & Co., Inc. SRC kinase inhibitor compounds
US6316444B1 (en) 1999-06-30 2001-11-13 Merck & Co., Inc. SRC kinase inhibitor compounds
US6498165B1 (en) 1999-06-30 2002-12-24 Merck & Co., Inc. Src kinase inhibitor compounds
US20020086014A1 (en) 1999-08-24 2002-07-04 Korman Alan J. Human CTLA-4 antibodies and their uses
EP1212422A2 (en) 1999-08-24 2002-06-12 Medarex, Inc. Human ctla-4 antibodies and their uses
WO2001014424A2 (en) 1999-08-24 2001-03-01 Medarex, Inc. Human ctla-4 antibodies and their uses
US6984720B1 (en) 1999-08-24 2006-01-10 Medarex, Inc. Human CTLA-4 antibodies
US20050201994A1 (en) 1999-08-24 2005-09-15 Medarex, Inc. Human CTLA-4 antibodies and their uses
US6586424B2 (en) 1999-09-10 2003-07-01 Merck & Co., Inc. Tyrosine kinase inhibitors
US6586423B2 (en) 1999-09-10 2003-07-01 Merck & Co., Inc. Tyrosine kinase inhibitors
US6479512B1 (en) 1999-10-19 2002-11-12 Merck & Co., Inc. Tyrosine kinase inhibitors
US6306874B1 (en) 1999-10-19 2001-10-23 Merck & Co., Inc. Tyrosine kinase inhibitors
US6794393B1 (en) 1999-10-19 2004-09-21 Merck & Co., Inc. Tyrosine kinase inhibitors
US20020039581A1 (en) 2000-01-27 2002-04-04 Carreno Beatriz M. Antibodies against CTLA4 and uses therefor
US6420382B2 (en) 2000-02-25 2002-07-16 Merck & Co., Inc. Tyrosine kinase inhibitors
US6313138B1 (en) 2000-02-25 2001-11-06 Merck & Co., Inc. Tyrosine kinase inhibitors
US20040161738A1 (en) 2000-05-26 2004-08-19 Arumugam Muruganandam Single-domain brain-targeting antibody fragments derived from llama antibodies
EP1297172B1 (en) 2000-06-28 2005-11-09 Glycofi, Inc. Methods for producing modified glycoproteins
US20070061900A1 (en) 2000-10-31 2007-03-15 Murphy Andrew J Methods of modifying eukaryotic cells
US7041870B2 (en) 2000-11-30 2006-05-09 Medarex, Inc. Transgenic transchromosomal rodents for making human antibodies
US6875767B2 (en) 2001-06-22 2005-04-05 Merck & Co., Inc. (5-cyano-2-thiazolyl)amino-4-pyridine tyrosine kinase inhibitors
WO2003002609A2 (en) 2001-06-28 2003-01-09 Domantis Limited Dual-specific ligand and its use
US6958340B2 (en) 2001-08-01 2005-10-25 Merck & Co., Inc. Tyrosine kinase inhibitors
US6927293B2 (en) 2001-08-30 2005-08-09 Merck & Co., Inc. Tyrosine kinase inhibitors
WO2003035835A2 (en) 2001-10-25 2003-05-01 Genentech, Inc. Glycoprotein compositions
WO2004003019A2 (en) 2002-06-28 2004-01-08 Domantis Limited Immunoglobin single variant antigen-binding domains and dual-specific constructs
WO2004035607A2 (en) 2002-10-17 2004-04-29 Genmab A/S Human monoclonal antibodies against cd20
US20100028330A1 (en) 2002-12-23 2010-02-04 Medimmune Limited Methods of upmodulating adaptive immune response using anti-pd1 antibodies
WO2004058821A2 (en) 2002-12-27 2004-07-15 Domantis Limited Dual specific single domain antibodies specific for a ligand and for the receptor of the ligand
WO2004101790A1 (en) 2003-05-14 2004-11-25 Domantis Limited A process for recovering polypeptides that unfold reversibly from a polypeptide repertoire
WO2004081026A2 (en) 2003-06-30 2004-09-23 Domantis Limited Polypeptides
WO2005035572A2 (en) 2003-10-08 2005-04-21 Domantis Limited Antibody compositions and methods
WO2006003388A2 (en) 2004-06-30 2006-01-12 Domantis Limited Compositions and methods for treating inflammatory disorders
WO2006030220A1 (en) 2004-09-17 2006-03-23 Domantis Limited Compositions monovalent for cd40l binding and methods of use
WO2006121168A1 (en) 2005-05-09 2006-11-16 Ono Pharmaceutical Co., Ltd. Human monoclonal antibodies to programmed death 1(pd-1) and methods for treating cancer using anti-pd-1 antibodies alone or in combination with other immunotherapeutics
US8008449B2 (en) 2005-05-09 2011-08-30 Medarex, Inc. Human monoclonal antibodies to programmed death 1 (PD-1) and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics
WO2007005874A2 (en) 2005-07-01 2007-01-11 Medarex, Inc. Human monoclonal antibodies to programmed death ligand 1 (pd-l1)
WO2007059782A1 (en) 2005-11-28 2007-05-31 Genmab A/S Recombinant monovalent antibodies and methods for production thereof
US20070254295A1 (en) 2006-03-17 2007-11-01 Prometheus Laboratories Inc. Methods of predicting and monitoring tyrosine kinase inhibitor therapy
WO2009044561A1 (en) * 2007-10-03 2009-04-09 Shizuoka Prefecture ANTI-proNT/NMN MONOCLONAL ANTIBODY
WO2009101611A1 (en) 2008-02-11 2009-08-20 Curetech Ltd. Monoclonal antibodies for tumor treatment
WO2009114335A2 (en) 2008-03-12 2009-09-17 Merck & Co., Inc. Pd-1 binding proteins
WO2010027827A2 (en) 2008-08-25 2010-03-11 Amplimmune, Inc. Targeted costimulatory polypeptides and methods of use to treat cancer
US8609089B2 (en) 2008-08-25 2013-12-17 Amplimmune, Inc. Compositions of PD-1 antagonists and methods of use
US20120114649A1 (en) 2008-08-25 2012-05-10 Amplimmune, Inc. Delaware Compositions of pd-1 antagonists and methods of use
US8217149B2 (en) 2008-12-09 2012-07-10 Genentech, Inc. Anti-PD-L1 antibodies, compositions and articles of manufacture
WO2010077634A1 (en) 2008-12-09 2010-07-08 Genentech, Inc. Anti-pd-l1 antibodies and their use to enhance t-cell function
WO2010106180A2 (en) 2009-03-20 2010-09-23 Lfb Biotechnologies Optimized fc variants
WO2010117057A1 (en) 2009-04-10 2010-10-14 協和発酵キリン株式会社 Method for treatment of blood tumor using anti-tim-3 antibody
US8345509B2 (en) 2009-04-16 2013-01-01 Chevron U.S.A., Inc. System and method to create three-dimensional images of non-linear acoustic properties in a region remote from a borehole
WO2011066342A2 (en) 2009-11-24 2011-06-03 Amplimmune, Inc. Simultaneous inhibition of pd-l1/pd-l2
WO2011066389A1 (en) 2009-11-24 2011-06-03 Medimmmune, Limited Targeted binding agents against b7-h1
US20130034559A1 (en) 2009-11-24 2013-02-07 Medlmmune Limited Targeted Binding Agents Against B7-H1
WO2011155607A1 (en) 2010-06-11 2011-12-15 協和発酵キリン株式会社 Anti-tim-3 antibody
WO2013006490A2 (en) 2011-07-01 2013-01-10 Cellerant Therapeutics, Inc. Antibodies that specifically bind to tim3
US20140341917A1 (en) 2011-11-28 2014-11-20 Merck Patent Gmbh Anti-pd-l1 antibodies and uses thereof
WO2014150677A1 (en) 2013-03-15 2014-09-25 Bristol-Myers Squibb Company Inhibitors of indoleamine 2,3-dioxygenase (ido)
WO2015033301A1 (en) 2013-09-06 2015-03-12 Aurigene Discovery Technologies Limited 1,3,4-oxadiazole and 1,3,4-thiadiazole derivatives as immunomodulators
WO2015033299A1 (en) 2013-09-06 2015-03-12 Aurigene Discovery Technologies Limited 1,2,4-oxadiazole derivatives as immunomodulators
WO2015185478A1 (en) * 2014-06-02 2015-12-10 INSERM (Institut National de la Santé et de la Recherche Médicale) Anti-neurotensin long fragment antibodies and uses thereof
WO2015185513A1 (en) * 2014-06-02 2015-12-10 INSERM (Institut National de la Santé et de la Recherche Médicale) Anti-neurotensin antibodies and uses thereof

Non-Patent Citations (80)

* Cited by examiner, † Cited by third party
Title
"Antibodies, A Laboratory Manual", 1988, COLD SPRING HARBOR LABORATORY
AGNEW, CHEM. INTL. ED. ENGL., vol. 33, 1994, pages 183 - 186
AL-JANABI S, VAN SLOOTEN HJ, VISSER M, VAN DER PLOEG T, VAN DIEST PJ, JIWA M.: "Evaluation of mitotic activity index in breast cancer using whole slide digital images", PLOS ONE, vol. 8, no. 12, 2013
BAKIRTZI KHATZIAPOSTOLOU MKARAGIANNIDES IPOLYTARCHOU CJAEGER SILIOPOULOS D ET AL.: "Neurotensin signaling activates microRNAs-21 and -155 and Akt, promotes tumor growth in mice, and is increased in human colon tumors", GASTROENTEROLOGY, vol. 141, no. 5, 2011, pages 1749 - 61, XP028325675, DOI: 10.1053/j.gastro.2011.07.038
BIRD ET AL., SCIENCE, vol. 242, 1989, pages 423 - 426
BOERNER ET AL., J. IMMUNOL., vol. 147, 1991, pages 86
BRIGNONE ET AL., J. IMMUNOL., vol. 179, no. 477202-00-9, 2007, pages 4202 - 4211
BRODEUR ET AL.: "Monoclonal Antibody Production Techniques and Applications", 1987, MARCEL DEKKER, INC., pages: 51 - 63
BRUN P, MASTROTTO C, BEGGIAO E, STEFANI A, BARZON L, STURNIOLO GC: "Neuropeptide neurotensin stimulates intestinal wound healing following chronic intestinal inflammation.", AMERICAN JOURNAL OF PHYSIOLOGY GASTROINTESTINAL AND LIVER PHYSIOLOGY, vol. 288, no. 4, 2005, pages G621 - 9
BYSTRYN ET AL., CLINICAL CANCER RESEARCH, vol. 7, July 2001 (2001-07-01), pages 1882 - 1887
CAMACHO ET AL., J. CLIN: ONCOLOGY, vol. 22, no. 145, 2004
CARRAWAY ET AL: "Involvement of neurotensin in cancer growth: Evidence, mechanisms and development of diagnostic tools", PEPTIDES, ELSEVIER, AMSTERDAM, NL, vol. 27, no. 10, 1 October 2006 (2006-10-01), pages 2445 - 2460, XP005661213, ISSN: 0196-9781, DOI: 10.1016/J.PEPTIDES.2006.04.030 *
CARRAWAY RLEEMAN SE: "The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 248, no. 19, 1973, pages 6854 - 61
CRAMERI ET AL., NATURE, vol. 391, 1998, pages 288 - 291
DOBNER PR, KISLAUSKIS E, BULLOCK BP: "Cooperative regulation of neurotensin/neuromedin N gene expression in PC12 cells involves AP-1 transcription factors.", ANNALS OF THE NEW YORK ACADEMY OF SCIENCES., vol. 668, 1992, pages 17 - 29
DONG ZWANG XEVERS BM: "Site-specific DNA methylation contributes to neurotensin/neuromedin N expression in colon cancers", AMERICAN JOURNAL OF PHYSIOLOGY GASTROINTESTINAL AND LIVER PHYSIOLOGY, vol. 279, no. 6, 2000, pages G1139 - 47
DONG ZWANG XZHAO QTOWNSEND CM, JR.EVERS BM: "DNA methylation contributes to expression of the human neurotensin/neuromedin N gene", THE AMERICAN JOURNAL OF PHYSIOLOGY, vol. 274, 1998, pages G535 - 43
DUPOUY SDOAN VKWU ZMOURRA NLIU JDE WEVER O ET AL.: "Activation of EGFR, HER2 and HER3 by neurotensin/neurotensin receptor 1 renders breast tumors aggressive yet highly responsive to lapatinib and metformin in mice", ONCOTARGET, vol. 5, no. 18, 2014, pages 8235 - 51
DUPOUY SMOURRA NDOAN VKGOMPEL AALIFANO MFORGEZ P: "The potential use of the neurotensin high affinity receptor 1 as a biomarker for cancer progression and as a component of personalized medicine in selective cancers", BIOCHIMIE, vol. 93, no. 9, 2011, pages 1369 - 78, XP028245636, DOI: 10.1016/j.biochi.2011.04.024
ERNST AHELLMICH SBERGMANN A: "Proneurotensin 1-117, a stable neurotensin precursor fragment identified in human circulation", PEPTIDES, vol. 27, no. 7, 2006, pages 1787 - 93, XP025067439, DOI: 10.1016/j.peptides.2006.01.021
HASSAN SDOBNER PRCARRAWAY RE: "Involvement of MAP-kinase, PI3-kinase and EGF-receptor in the stimulatory effect of Neurotensin on DNA synthesis in PC3 cells", REGULATORY PEPTIDES, vol. 120, 2004, pages 155 - 66
HOLLINGERHUDSON, NATURE BIOTECHNOLOGY, vol. 23, no. 9, 2005, pages 1126 - 1136
HOLT ET AL., TRENDS BIOTECHNOL., vol. 21, no. 11, 2003, pages 484 - 490
HOOGENBOOM ET AL., J. MOL. BIOL., vol. 222, 1991, pages 581
HURWITZ ET AL., PROC. NATL. ACAD. SCI. USA, vol. 95, no. 17, 1998, pages 10067 - 10071
HUSTON ET AL., PROC. NATL. ACAD. SCI., vol. 85, 1988, pages 5879 - 5883
KIM JT, LIU C, ZAYTSEVA YY, WEISS HL, TOWNSEND CM, JR., EVERS BM: "Neurotensin, a novel target of Wnt/beta-catenin pathway, promotes growth of neuroendocrine tumor cells", INTERNATIONAL JOURNAL OF CANCER, vol. 136, no. 6, 2015, pages 1475 - 81
KISFALVI KHURD CGUHA SROZENGURT E: "Induced overexpression of protein kinase D1 stimulates mitogenic signaling in human pancreatic carcinoma PANC-1 cells", JOURNAL OF CELLULAR PHYSIOLOGY, vol. 223, no. 2, 2010, pages 309 - 16
KITABGI P: "Differential processing of pro-neurotensin/neuromedin N and relationship to pro-hormone convertases", PEPTIDES, vol. 27, no. 10, 2006, pages 2508 - 14, XP025067625, DOI: 10.1016/j.peptides.2006.03.038
KOON HWKIM YSXU HKUMAR AZHAO DKARAGIANNIDES I ET AL.: "Neurotensin induces IL-6 secretion in mouse preadipocytes and adipose tissues during 2,4,6,-trinitrobenzensulphonic acid-induced colitis", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 106, no. 21, 2009, pages 8766 - 71
KOZAKI K, MIYAISHI O, TSUKAMOTO T, TATEMATSU Y, HIDA T, TAKAHASHI T: "Establishment and characterization of a human lung cancer cell line NCI-H460-LNM35 with consistent lymphogenous metastasis via both subcutaneous and orthotopic propagation", CANCER RES., vol. 60, no. 9, 2000, pages 2535 - 40, XP003015335
KOZBOR, J. IMMUNOL., vol. 13, 1984, pages 3001
KYRIAKI BAKIRTZI ET AL: "Neurotensin Signaling Activates MicroRNAs-21 and -155 and Akt, Promotes Tumor Growth in Mice, and Is Increased in Human Colon Tumors", GASTROENTEROLOGY, ELSEVIER INC, US, vol. 141, no. 5, 26 July 2011 (2011-07-26), pages 1749 - 1761.e1, XP028325675, ISSN: 0016-5085, [retrieved on 20110730], DOI: 10.1053/J.GASTRO.2011.07.038 *
LAW IK, JENSEN D, BUNNETT NW, POTHOULAKIS C: "Neurotensin-induced miR-133alpha expression regulates neurotensin receptor 1 recycling through its downstream target aftiphilin ", SCIENTIFIC REPORTS, vol. 6, 2016, pages 22195
LEE LFGUAN JQIU YKUNG HJ: "Neuropeptide-induced androgen independence in prostate cancer cells: roles of nonreceptor tyrosine kinases Etk/Bmx, Src, and focal adhesion kinase", MOLECULAR AND CELLULAR BIOLOGY, vol. 21, no. 24, 2001, pages 8385 - 97
LI ET AL., PROC. NATL. ACAD. SCI. USA, vol. 103, 2006, pages 3557 - 3562
LONBERG, CUR. OPIN.IMMUNOL., vol. 20, 2008, pages 450 - 459
LONBERG, NAT.BIOTECH., vol. 23, 2005, pages 1117 - 1125
LOO ET AL., CLIN. CANCER RES., vol. 15, no. 18, 2012, pages 3834
MARK EVERS ET AL: "Neurotensin and growth of normal and neoplastic tissues", PEPTIDES, ELSEVIER, AMSTERDAM, NL, vol. 27, no. 10, 1 October 2006 (2006-10-01), pages 2424 - 2433, XP027957617, ISSN: 0196-9781, [retrieved on 20061001] *
MARKS ET AL., BIO/TECHNOLOGY, vol. 10, 1992, pages 779 - 783
MELANDER O, MAISEL AS, ALMGREN P, MANJER J, BELTING M, HEDBLAD B: "Plasma proneurotensin and incidence of diabetes, cardiovascular disease, breast cancer, and mortality.", JAMA, vol. 308, no. 14, 2012, pages 1469 - 75, XP009165659, DOI: 10.1001/jama.2012.12998
MELLMAN ET AL., NATURE, vol. 480, 2011, pages 480 - 489
MOKYR ET AL., CANCER RES., vol. 58, 1998, pages 5301 - 5304
MOODY TW, LEE L, RAMOS-ALVAREZ I, JENSEN RT: "Neurotensin receptors regulate transactivation of the EGFR and HER2 in a reactive oxygen species-dependent manner ", EUROPEAN JOURNAL OF PHARMACOLOGY, vol. 865, 2019, pages 172735, XP085938333, DOI: 10.1016/j.ejphar.2019.172735
MULLER, METH. ENZYMOL, vol. 92, 1983, pages 589 - 601
NAJIMI M, SOUAZE F, MENDEZ M, HERMANS E, BERBAR T, ROSTENE W: "Activation of receptor gene transcription is required to maintain cell sensitization after agonist exposure. Study on neurotensin receptor. ", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 273, no. 34, 1998, pages 21634 - 41
NI, XIANDAI MIANYIXUE, vol. 26, no. 4, 2006, pages 265 - 268
PARDOLL, NATURE REV CANCER, vol. 12, 2012, pages 252 - 264
PATTEN ET AL., CURR. OPIN. BIOTECHNOL., vol. 8, 1997, pages 724 - 733
PEDERSEN JHANDERSEN HOOLSEN PSHENRIKSEN JH: "Pharmacokinetics and metabolism of neurotensin in man", THE JOURNAL OF CLINICAL ENDOCRINOLOGY AND METABOLISM, vol. 68, no. 2, 1989, pages 294 - 300
PILIPONSKY AM, CHEN CC, NISHIMURA T, METZ M, RIOS EJ, DOBNER PR: "Neurotensin increases mortality and mast cells reduce neurotensin levels in a mouse model of sepsis", NATURE MEDICINE, vol. 14, no. 4, 2008, pages 392 - 8
ROSELL S: "Substance P and neurotensin in the control of gastrointestinal function", SOCIETY OF GENERAL PHYSIOLOGISTS SERIES, vol. 35, 1980, pages 147 - 62
ROSELL SAL-SAFFAR ATHOR K: "The role of neurotensin in gut motility", SCANDINAVIAN JOURNAL OF GASTROENTEROLOGY SUPPLEMENT, vol. 96, 1984, pages 69 - 75
SAIYASIT NCHUNCHAI TAPAIJAI NPRATCHAYASAKUL WSRIPETCHWANDEE JCHATTIPAKORN N ET AL.: "Chronic high-fat diet consumption induces an alteration in plasma/brain neurotensin signaling, metabolic disturbance, systemic inflammation/oxidative stress, brain apoptosis, and dendritic spine loss", NEUROPEPTIDES, vol. 82, 2020, pages 102047, XP086208492, DOI: 10.1016/j.npep.2020.102047
SAKUISHI ET AL., J. EXP. MED., vol. 207, 2010, pages 2187 - 94
SCHINDELIN J, ARGANDA-CARRERAS I, FRISE E, KAYNIG V, LONGAIR M, PIETZSCH T: "Fiji: an open-source platform for biological-image analysis", NATURE METHODS, vol. 9, no. 7, 2012, pages 676 - 82, XP055343835, DOI: 10.1038/nmeth.2019
SHIELDS, R. L. ET AL., J. BIOL. CHEN., vol. 276, 2001, pages 6591 - 6604
SHIELDS, R.L. ET AL., J. BIOL. CHEM., vol. 277, 2002, pages 26733 - 26740
SOUAZE FFORGEZ P: "Molecular and cellular regulation of neurotensin receptor under acute and chronic agonist stimulation", PEPTIDES, vol. 27, no. 10, 2006, pages 2493 - 501, XP025067623, DOI: 10.1016/j.peptides.2006.04.029
SOUAZE FVIARDOT-FOUCAULT VROULLET NTOY-MIOU-LEONG MGOMPEL ABRUYNEEL E ET AL.: "Neurotensin receptor 1 gene activation by the Tcf/beta-catenin pathway is an early event in human colonic adenomas", CARCINOGENESIS, vol. 27, no. 4, 2006, pages 708 - 16
THEOHARIDES TC: "Effect of Stress on Neuroimmune Processes", CLINICAL THERAPEUTICS., vol. 42, no. 6, 2020, pages 1007 - 14, XP086196210, DOI: 10.1016/j.clinthera.2020.05.002
THOMPSON ET AL., J. MOL. BIOL., vol. 256, 1996, pages 359 - 368
TOY-MIOU-LEONG MCORTES CLBEAUDET AROSTENE WFORGEZ P: "Receptor trafficking via the perinuclear recycling compartment accompanied by cell division is necessary for permanent neurotensin cell sensitization and leads to chronic mitogen-activated protein kinase activation", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 279, no. 13, 2004, pages 12636 - 46
UMANA ET AL., NAT. BIOTECH., vol. 17, 1999, pages 176 - 180
VAN DIJKVAN DE WINKEL, CUR. OPIN. PHARMACOL., vol. 5, 2001, pages 368 - 74
VINCENT JPMAZELLA JKITABGI P: "Neurotensin and neurotensin receptors.", TRENDS IN PHARMACOLOGICAL SCIENCES, vol. 20, no. 7, 1999, pages 302 - 9
VOLLMERSBRANDLEIN, HISTOLOGY AND HISTOPATHOLOGY, vol. 20, no. 3, 2005, pages 927 - 937
VOLLMERSBRANDLEIN, METHODS AND FINDINGS IN EXPERIMENTAL AND CLINICAL PHARMACOLOGY, vol. 27, no. 3, 2005, pages 185 - 91
WANG QZHOU YEVERS BM: "Neurotensin phosphorylates GSK-3alpha/beta through the activation of PKC in human colon cancer cells", NEOPLASIA, vol. 8, no. 9, 2006, pages 781 - 7
WARD ET AL., NATURE, vol. 341, 1989, pages 544 - 546
WARD, NATURE, vol. 341, no. 6242, 12 October 1989 (1989-10-12), pages 544 - 6
WU ZFOURNEL LSTADLER NLIU JBOULLIER AHOYEAU N ET AL.: "Modulation of lung cancer cell plasticity and heterogeneity with the restoration of cisplatin sensitivity by neurotensin antibody", CANCER LETTERS, vol. 444, 2019, pages 147 - 61
WU ZMARTINEZ-FONG DTREDANIEL JFORGEZ P: "Neurotensin and its high affinity receptor 1 as a potential pharmacological target in cancer therapy", FRONTIERS IN ENDOCRINOLOGY, vol. 3, 2012, pages 184
WU ZSTADLER NABBACI ALIU JBOULLIER AMARIE N ET AL.: "Effect of Monoclonal Antibody Blockade of Long Fragment Neurotensin on Weight Loss, Behavior, and Metabolic Traits After High-Fat Diet Induced Obesity", FRONTIERS IN ENDOCRINOLOGY, vol. 12, 2021, pages 739287
YANG ET AL., J. MOL. BIOL., vol. 254, 1995, pages 392 - 403
YOUNES M, WU Z, DUPOUY S, LUPO AM, MOURRA N, TAKAHASHI T: "Neurotensin (NTS) and its receptor (NTSR1) causes EGFR, HER2 and HER3 over-expression and their autocrine/paracrine activation in lung tumors, confirming responsiveness to erlotinib.", ONCOTARGET, vol. 5, no. 18, 2014, pages 8252 - 69
ZAPATA ET AL., PROTEIN ENG, vol. 8, no. 10, 1995, pages 1057 - 1062
ZHAO DBAKIRTZI KZHAN YZENG HKOON HWPOTHOULAKIS C: "Insulin-like growth factor-1 receptor transactivation modulates the inflammatory and proliferative responses of neurotensin in human colonic epithelial cells", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 286, no. 8, 2011, pages 6092 - 9
ZHAO DKUHNT-MOORE SZENG HWU JSMOYER MPPOTHOULAKIS C: "Neurotensin stimulates IL-8 expression in human colonic epithelial cells through Rho GTPase-mediated NF-kappa B pathways", AMERICAN JOURNAL OF PHYSIOLOGY CELL PHYSIOLOGY, vol. 284, no. 6, 2003, pages C1397 - 404

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